BIOL2226 Learning Resource 2006

CLASS LEARNING RESOURCE
Written by students enrolled in BIOL2226 Protein Technologies
School of Applied Sciences

RMIT University
November 2006
Table of Contents
1. STRUCTURAL ANALYSIS OF PEPTIDES AND PROTEINS
1.1 X-Ray Diffraction

1.2 Nuclear Magnetic Resonance
1.3 Circular Dichroism and Optical Rotatory Dispersion
1.4 Fluorescence spectroscopy of proteins and applications
1.5 Atomic Force Microscopy of Proteins
1.6 Electron Microscopy of Proteins
2. PURIFICATION AND ANALYSIS OF PROTEINS
2.1 Strategies for Protein Purification

2.2 Size Exclusion Chromatography
2.3 Ion Exchange Chromatography
2.4 Affinity Chromatography
2.5 Hydrophobic Interaction Chromatography
2.6 Reversed phase HPLC of peptides and proteins
2.7 Capillary Electrophoresis of Peptides and Proteins
2.8 Purification of membrane proteins
2.9 Industrial Scale Purification of Proteins
2.10 Determination of Protein Concentration and Purity
3. CHEMICAL ANALYSIS OF PROTEINS
3.1 Amino Acid Analysis and sequencing of proteins

3.2 Chemical Modification of Proteins
3.3 Protein Cross-linking methods
4. MASS SPECTROMETRY OF PROTEINS
4.1 MALDI-TOF Mass Spectrometry

4.2 ESI Mass Spectrometry
4.3 Hybrid Mass Spectrometry Techniques
5. PROTEOMICS
5.1 2D gel electrophoresis

5.2 Chromatographic methods for separation of proteomes
5.3 Mass Spectrometry in Proteomics
5.4 Bioinformatics in Proteome Analysis
5.5 Automation and High-Throughput Proteomics
6. PROTEIN-PROTEIN INTERACTIONS
6.1 Chemical methods for study of protein-protein interactions

6.2 Molecular biology approaches for study of protein-protein
interactions
6.3 Biosensor methods for study of protein-protein interactions
6.4 Imaging methods for study of protein-protein interactions
7. SYNTHESIS OF PEPTIDES AND PROTEINS
7.1 t-Boc synthesis and cleavage of peptides

7.2 F-moc synthesis and cleavage of peptides
7.3 Synthesis of proteins using chemoselective ligation methods
7.4 Purification and characterisation of synthetic peptides
7.5 Peptide Libraries and their application
7.6 Applications of synthetic peptides
7.7 Peptide Nucleic Acids and applications
8. PROTEIN ENGINEERING
8.1 Approaches to Protein Engineering

8.2 Antibody Engineering
8.3 Protein Expression systems
8.4 Protein Folding strategies
8.5 Protein Arrays and their application
9. BIOMARKER DISCOVERY
9.1 Biomarker discovery and applications

9.2 SELDI-MS
10 PROTEIN NANOTECHNOLOGY
* Those topics that are in small italics have not been covered in this learning
resource or the reviews have not been submitted to date.
1.1 X RAY DIFFRACTION OF PROTEINS
SAFALYA KUMAR DEEPAK

S 3142939
1.1.1 Introduction.
X rays were discovered by Wilhelm Roentgen in November 1895, while studying

about different types of cathode rays. The unknown rays, which he gave the name “x
rays were capable of producing shadows of his bones on a fluorescent screen. This
discovery soon found uses in medical field. Doctors used X rays to observe the
structure of bones and other internal body parts. In 1912, Von Laue proved that these
rays are waves of light with very small wave length by diffracting them by using a
crystal. When a wave hits an object, some waves are blocked by the object and the
waves that pass the object change their direction of travel. This is called x ray
diffraction. William Henry Bragg and his son Lawrence Bragg were the pioneers to
study the properties of x rays. William Brag developed the x ray Spectrometer in
which the rays fell on a crystal and the reflection of the crystal was measured in an
ionization chamber and the strength and the direction of diffraction was found out.
Lawrence Bragg found out that the reflections of the X rays depend on the spacing of
the atoms in the crystal (Bragg’s law). He found that by analysing the patterns of the
reflections we can determine the arrangement of individual atoms in the crystal. Thus
the “Braggs” created the science of x ray crystallography. [1]
The discovery of x ray crystallography literally changed the scenario of biological

research; and the understanding we had about biological structures and processes.
Structures of Many biological molecules were determined in the first half of the last
century. Bernal and Crowfoot observed the first X-ray diffraction pattern from a
protein in 1934. The same technique was used to determine the double helical
structure of DNA in 1953. In 1959, the structure of Mb to a resolution of 0.6 nm (6 A°)
was established. Since then the detailed structure of many biomolecules have been
determined.
The technology is widely used in many fields of biological research now, especially in
the field of medical and molecular research. The basic technique of x ray
crystallography has been modified many times since it was discovered. The
development of powerful and accurate x ray tubes made it possible to work on small
biological systems like viruses and also helped in determining the structure of many
macromolecules in three dimensions. The x ray imaging technique is used today to
develop 3 D images of proteins and enzymes of importance in molecular biological
research. For example, to see the structure of a particular protein which is produced
by a pathogen inside our body to study its properties in order to develop a drug
against it. The technique is also used to observe the changes inside a biological
system after the administration of a drug or therapy. Proteins do almost all the work
in the living systems. So exploring them more and more in detail enables us to find
solutios for diseases and other biological complexities which were out of our reach
before. [2]
Underlying principle.
To perform their basic biological function, proteins has to assume three dimensional
structures in the living system. These molecules are crystallized and are analysed by
x ray diffraction method. The data from such structural analyses becomes the
foundation stone of all the modern developments in the area of biochemistry,
biophysics, pharmaceutical development and biotechnology. It is the simplest tool
available for biological research to obtain the three dimensional image of protein.
Electrons around the nucleus of an atom are capable of scattering the x rays in
proportion to the density of electrons. The positions of individual atoms or molecules
are determined by analysing the diffraction pattern of the rays. And a three
dimensional image of the biomolecule is made from this.
Fig.1.1 x ray diffraction of a protein crystal.

Source: fig.cox.miami.edu [8]
To get the perfect image of a biomolecule, it should be properly crystallized before

analysing it by x ray diffraction. The shape of the crystal has a lot to do with the
perfection of the image that we get. All biomolecules has water as a major content of
them. Water has a major role in determining the structure of the biomolecules. The
perfect crystal for x ray diffractive analysis of protein is produced by removing the
water content of the biomolecule in a controlled manner. A perfect crystal should be
large and single. It should have the ability to rotate plane polarized light, and with
minimal defects.
There are seven types or classes of crystals. The pictures are given below.
Fig1.2 types of crystals.

Source:
http://blackboard.rmit.edu.au/courses/1/BIOL2226/content/_499118_1/Ramsland_15
Aug06.pdf
The perfect crystal, i.e., the crystal of a protein which gives the perfect diffraction
pattern is prepared by simple controlled vapour diffusion, by using different types of
crystallization matrices. Then the x ray diffraction is carried out and the diffraction
pattern is captured on a film. By using this, a three dimensional image of the protein
is made by using modern computer programs in bioinformatics is made. The overall
process is as follows.
Fig.1.3 structural analysis of protein crystals.

Source:
http://blackboard.rmit.edu.au/courses/1/BIOL2226/content/_499118_1/Ramsland_15
Aug06.pdf
1.1.2 Recent advances.
1.1.2.1 Synchrotrons.
There have been significant advances in recent years in the field of macromolecular
crystallography. Synchrotron rays, CCD detectors and cryocrystallography help the
scientists to make the data collection easy and efficient. We can collect the complete
data of more than one protein in a single day when we use synchrotron rays for
analysis, crystals as small as 60 µm can be used to determine protein structures.
Since most of the protein s form small crystals, this help us to analyse more and
more proteins. We have the technology to analyse more than hundreds protein
crystals per year, using a dedicated beam line on a third-generation synchrotron. The
most time-consuming step is, therefore, likely to be electron density map
interpretation. Methods to automate model building and refinement from electron
density maps are in the early stages of development, and additional support for
research in this area could significantly enhance high-throughput structure
determination.
Synchrotron light is the electromagnetic radiation emitted when charged particles,

usually electrons or positrons, moving at velocities close to the speed of light, are
forced to change direction under the action of a magnetic field. A synchrotron is a
large machine (about the size of a football field) that accelerates electrons to almost
the speed of light. As the electrons are deflected through magnetic fields they create
extremely bright light. The light is channelled down beamlines to experimental
workstations where it is used for research. The development of synchrotrons has
been likened to the invention of the microscope in terms of the revolutionary effect
across a range of sciences and the capacity to delve deeper into the structure of
matter than ever before. [11]
Basic lay out of a synchrotron.
Source: www.synchrotron.vic.gov.au
1.1.2.2 Automation of crystal production.
Structural analysis of biological compounds is now entering a new phase by the

industrialization and automation of the processes. The more sophisticated and
accurate radiation techniques and advances in the purification, crystallization and
expression techniques of proteins have helped a lot in making the time consuming
and tedious process simple.
The crystallization process starts with the preparation of highly purified and soluble
sample. Factors like pH, ionic strength, temperature and concentrations of organic
additives salts and detergents used in the purification have to be considered for this.
A simple sampling technique is more practical than a multidimensional approach.
Due to the impracticability of the multidimensional approach simple and reduced
sampling techniques have been developed. Robots have been developed by certain
companies to make the crystal production automatic. These systems are capable of
performing about more than 100,000 cycles a day. This makes the crystal production
fast and also helps us to store the data for future analysis.
Another two advances in the field are cryo-freezing of the crystals and the Multi
wavelength anomalous diffraction method (MAD). [9]
Cryo-freezing.
The first one, cryo-freezing is the method by which the protein crystals produced is
cooled and stored in liquid nitrogen. This helps to prevent the damage caused to the
crystal by the powerful radiations. [14]
MALDI
In MALD the synchrotron is tuned to different wavelengths. This helps in the

adsorption of the metal ions in the macromolecules. This helps in solving the phase
problem.
MALDI X ray diffraction apparatus.
Source: www.esrf.fr
Apart from this, the advancement in the technologies for the evaluation and
manipulation of the data like modern bioinformatics programs and other
developments in computational chemistry made the usage of this technique less time
laborious.
1.1.3 Evaluation of the technology.
NMR or Nuclear Magnetic Resonance is a technique that is more recent than x ray
crystallography used in the structural analysis of protein and is very widely used now
a days. The basic principle behind this technology is that some atomic nuclei are very
highly magnetic and it can attain different stages of energy in a magnetic field. This
energy variation can be measured to depict a spectrum. The magnetic properties of
the nuclei are affected by the chemical bonds and the short distances between the
molecules. These properties of the molecules are used in the technique of Nuclear
Magnetic Resonance Spectrometry of protein.
Comparison of x ray crystallography and NMR Spectrometry.
NMR can measure distance between atoms without considering the spatial
orientation. This avoids the need to crystallize the protein. So NMR forms a more
straight forward approach than crystallographic techniques.
Normally, NMR is used to study small molecules. But due to the recent advancement
in resolution of the procedure, the upper limit of molecules screened by this
technique came up to 100 daltons from 20 daltons.
The advantages and disadvantages of X ray crystallography and that of NMR

are summarized below.
Advantages of x ray crystallography.

1. Can examine also by this way the solvent effect from different solvents. The
same protein may crystallize into different crystalloid forms.
2. Able to force the protein to another form of crystallization by the change of its
solvent.
3. Could get the whole 3D structure by the systematic analysis of a good

crystallized material.
Disadvantages of x ray crystallography.
1. The crystal structure is necessary only that proteins which can be crystallized
are examinable.
2. Cannot examine solutions and the behaviour of the molecules in solution.
3. This happens when we try to examine powders, gases.
4. Study of motions is not available.
5. Can get only one parameter-set so we are able to observe only one
conformation.
6. No possibility to examine small parts in the molecule.
7. No chance for direct determination of secondary structures and especially

domain movements (big disadvantage against the NMR).
8. The hydrogen in the molecules are not examinable since it has only one
electron.
Advantages of NMR.
1. Several types of information from lots of types of experiments.
2. Obtain angles, distances, coupling constants, chemical shifts, rate constants

etc. These are really molecular parameters which could be examined more
with computers and molecular modelling procedures.
3. have enough strength of the magnetic field (the resolution is the function of
that) than we can handle all of the atoms “personally”
4. With a suitable computer apparatus we can calculate the whole 3D structure.
5. There are lots of possibilities to collect different data-sets from different types
of experiments for the ability to resolve the uncertainties of one type of
measurements.
6. The motion of the segments (domains) can be examined.

7. Capable to lead us for the observation of the chemical kinetics.
8. Can investigate the influence of the dielectric constant, the polarity and any
other properties of the solvent or some added material.
Disadvantages of NMR.
1. Have lots of atoms and a lot of extracted data from a system.
2. This is good for the more accurate determination of the structure, but not for
the availability of higher molecular masses.
3. The resolving power of NMR is less than some other type of experiments
(e.g.: X-ray crystallography) since the information got from the same material
is much more complex.
4. The highest molecular mass which was examined successfully is just a

64kDa protein-complex.
5. There are lots of cases when from a given data-set - a given type of
experiment - we could predict two or more possible conformations, too.
6. Just able to determine the degree of probability of being of the protein

segment in the given conformation.
7. The cost of the experimental implementation is increasing with the higher

strength and the complexity of the determination.
Source: www.cryst.bbk.ac.uk
1.1.4 Application of the technology.
The field of biotechnological research is now focussed on genetics and proteomics.

Proteins are the molecules responsible for all of the biological functions in all
mechanisms of life. The proteins are the functional parts of any living system. So the
knowledge about the structure and function of the protein are necessary for and it is
the next logical step to understanding any biological system. X ray crystallography is
the most powerful tool that is used to generate study and compare the proteins,
enzymes, etc of a living system, their mode of action and the changes that may
cause in a system. Recombinant DNA technology and highly developed X ray
diffraction instruments helps the scientists to obtain solid information from the genes
to the three dimensional images of the proteins that the genes code for. [13]
Most recently, the technology is applied in understanding the sub cellular level and
nucleocytoplasmic transport. [20]
The highly automated analysis methods available make it easy to analyse the vast
bulk of data generated by the genome and proteome analysis. Computational tools
have a major role in that.
1.1.5 Relevant websites.
1. Biological Macromolecular Crystallization Database (BMCD)
2. http://epswww.unm.edu/xrd/resources.htm
3. http://www.bruker-biosciences.com/
4. www.intute.ac.uk/sciences/cgi-
bin/browse.pl?limit=50&id=52&type=%25&sort=record.title
5. http://www.icdd.com/resources/websites.htm
1.1.6 Key industry suppliers.
1. http://www.faxitron.com
2. http://www.bruker-axs.de/
3. www.jyinc.com
4. www.materialsdata.com
5. www.kohzuamrica.com
1.1.7 References
1. www
cambridgephysics.comhttp://wwwoutreach.phy.cam.ac.uk/camphy/xraydiffract
ion/xraydiffraction1_1.htm
2. http://www.phy.princeton.edu/~austin/hf_book/chapter10.pdf.
3. http://wwwoutreach.phy.cam.ac.uk/camphy/xraydiffraction/xraydiffraction12_1
.htm
4. Makin, O.S., Sikorski, P., Serpel, L.C. (2006) Diffraction to study protein and
peptide assemblies, Current Opinion in Clinical Biology,10, 417-422.
5. http://www2.mrc-lmb.cam.ac.uk/mandi.html
6. http://www.brightsurf.com/isearch/index.php
7. http://www.crystalresearch.com/crt/ab34/1120_a.pdf
8. http://fig.cox.miami.edu/~cmallery/150/gene/sf13x1box.jpg
9. http://www.atcg3d.org/PDF/Abola_NSB2000.pdf
10. http://structbio.nature.com
11. http://www.synchrotron.vic.gov.au/content.asp?Document_ID=97
12. http://www.osmic.com/applications_xrd_single_small.asp
13. http://www.nigms.nih.gov/News/Reports/protein_structure.htm
14. http://www-structmed.cimr.cam.ac.uk/Course/Crystals/shooting.html
15. http://www-
structmed.cimr.cam.ac.uk/Course/Crystals/shooting.htmlhttp://www-
structmed.cimr.cam.ac.uk/Course/Crystals/shooting.html
16. http://www.nature.com/horizon/proteinfolding/background/technology.html
17. http://www.cryst.bbk.ac.uk/pps97/assignments/projects/ambrus/html.htm#com
18. Lim, R.Y.H., Fahrenkrog, B. (2006), The nuclear pore complex upclose,
Current Opinion in Cell Biology, 18, 342-347.
19. Scapin, G. (2006) structural biology and drug discovery, Current

pharmaceutical design, 12, 2087-2097.
20. Ng, J.D., Gavira, A.J., Garzia-Ruiz, J.M. (2003), Protein crystallization by
capillary counter diffusion for applied crystallographic structure determination,
Journal of Structural Biology, 142, 218-231.
Chapter 1.2 Nuclear Magnetic Resonance
Yang xi
1.2.1 introduction
The impact of nuclear magnetic resonance spectroscopy on the natural sciences is

critical. The first application of NMR Spectroscopy to a biological sample was
reported by the Jacobson ,Anderson, and Arnold in 1954 [1] . Nowadays NMR
spectroscopy which can be used in the solution and solid state is very useful in
mixtures of analyses; in understanding dynamic effects such as change in
temperature and reaction mechanisms. The most important application of NMR is
that it has been well-established as a excellent way to determine the three-
dimensional structure of proteins . [2]. The fundamental character of magnetic
resonance is the separation between energy levels are quantized which means the
resonance frequency contains plenty and crucial information about the chemical
structure and the local magnetic environment .[3]. However it is essential to retain
information that, with NMR, we are performing experiments on the nuclei of atoms,
not the electrons. And the chemical environment of specific nuclei can be deduced
from information obtained about the nuclei. [4]. In a basic NMR process , a large
magnetic be set up and the energy levels spilt then . Figure 1.2.1 shows the energy-
level diagram for spin system with I=1/2.
Figure 1.2.1 Energy levers for a nucleus with spin quantum number 1/2
Source: http://teaching.shu.ac.uk/hwb/chemistry/tutorials/molspec/nmr1.htm
In such case, these nuclei in low level can be excited into the higher level by the
radiofrequency signals which is determined by the difference in energy between the
energy levels. When radiofrequency disappear , the nuclei in the higher energy state
return to the lower state with emission of radiation which can be pick up by the
receiver. This process is called relaxation process. The chemical shift which defined
as applied magnetic field is the most basic of measurements in NMR. Chemical shift
is caused by the electrons in the molecule produce local magnetic filed .[5] It is
measured relative to a reference compound . For the nuclei1H, 13C, and 29Si, TMS
(tetramethylsilane) is commonly used as a reference.
Proteins are much larger than the small organic molecules such H, C , however the
same NMR theory implicated . The basic 1D spectra become crowded with
overlapping signals to an extent where analysis is impossible as the increased
number of each element present in the protein. So multidimensional (2, 3 or 4D)
experiments have been devised to deal with this problem. To facilitate these
experiments, it is desirable to isotopically label the protein with 13C and 15N as the
predominant naturally-occurring isotope 12C is not NMR-active, whereas the nuclear
quadruple moment of the predominant naturally-occurring 14N isotope prevents high
resolution information to be obtained from this nitrogen isotope. The most important
method used for structure determination of proteins is NOE experiements which
measures distances between pairs of atoms within the molecule. Then the obtained
distances are used to generate a 3D structure of the molecule using a computer
program [6] . NMR spectroscopy can determine structure in several following phases,
each using a separate set of highly specialized techniques. The sample is prepared,
resonances are assigned, restraints are generated and a structure is calculated and
validated.[7] NMR Spectroscopy consists of experiments that try to identify the
physical relationships between atoms in the molecule, such as distances, angles,
and orientations, and use them to search for three dimensional structures that satisfy
the most of the physical constraints
1.2.2 Recent Advances
One of the distinctive ability that NMR spectroscopy has is to regain information
about interactions of proteins with other macromolecules or small molecules, which is
recently increasingly used to determine the 3-D structure of protein . Furthermore
NMR methods have been applied to drug design by the identification and
characterization of small chemicals that restrain protein function. [8]
The high-resolution liquid-state NMR spectroscopy can determine protein structure;

however a large classes of proteins cannot be examined by this method because of
their lack of a sufficiently concentrated solution. Therefore a new method, high-
resolution solid-state NMR has been studied. So far, most studies have worked at
selective isotope labeling of the proteins under investigation. However for practical
purposes, it would be desirable to have tools at hand with which it is possible to
extract structural information directly from uniformly isotope-enriched samples. In the
last few years, a whole arsenal of such tools has been developed by several groups
all over the world.[9] Recent advancements in using multi-dimensional magic-angle-
spinning (MAS) [10] solid-state NMR to study molecular interactions and the
methodological aspects of these experiments are discussed. And interactions
involving polypeptides, such as surface-bound peptides [11]) or nucleotides [12 and
13], provide additional exciting possibilities for ssNMR studies.
As the multi-dimensional NMR-experiments is time-consuming , time-saving NMR

methods has become an important target . The total time necessary for a particular
NMR experiment is determined by the need to achieve a sufficient signal-to-noise
ratio (SNR), the number of repetition cycles for suppressing unwanted signals and
the resolution requirements for indirectly sampled evolution dimensions of multi-
dimensional experiments increase the duration of experiments tremendously beyond
the amount required to achieve sufficient signal-to-noise ratios.[14] . A number of
different approaches that permit for more efficient achievement of multi-dimensional
NMR spectra have been published recently due to the improvements of the
sensitivity of NMR spectrometers. Single scan multi-dimensional spectroscopy
perhaps capitulate the most radical improvement [15]. Approaches involving reduced
dimensionality [16] and projection–reconstruction [17], can be readily implemented
for well known multi-dimensional experiments using current hardware. All of these
techniques can decrease the number of data points required in the evolution
dimensions and can be joint with any coherence selection approach. cogwheel phase
cycling applied by Levitt and co-workers have achieved very considerable reductions
of the number of phase cycle steps in solid state NMR experiments [18].
1.2.3 Evaluation of the Technology
Nowadays, except NMR Spectroscopy, X-Ray Crystallography, which involves using

instruments to detect the electron densities of the molecule crystallized in its native
fold, and thus to locate the atoms in the molecule in the three dimensional space, is
another experimental technology for protein three dimensional structure
determination. Although these two methods base on different principles, both of them
are remain expensive and time-consuming, despite numerous technological
advances.
The ability to characterize protein complexes under physiological conditions at atomic

detail, even if the interactions are weak and transient of NMR spectroscopy makes it
as the unique role in the investigation of protein interactions with small molecules.
Furthermore it also has the unique ability to accurately measure the dynamic
properties of proteins and to probe the process of protein folding [19,20]. In additional
, it does not need crystallization , which may be the most limitation of X- ray
diffraction. This means the structure gotten is much near to that at physiological
state.
However, a major problem of macromolecular NMR is its size limitation caused by

two technical barriers. First, larger molecules have slower tumbling rates and shorter
NMR signal relaxation times, which leads to the reduction of the sensitivity of the
complicated pulse sequences that often use long delays for the necessary coherence
transfer steps. And as more NMR-active nuclei and, therefore, more interactions
among them with the increased molecular weight , more complexity to a given
spectrum introduces . The current size limit of protein NMR is about 40 kDa , which is
much less than that of X – ray diffraction. [21].
1.2.4 Application of the Technology
1.2.4.1 Application of NMR for unfold protein
Some proteins are unstructured by themselves and only fold upon forming specific
complexes with other polypeptides or even small-molecule cofactors , so the
structures of such proteins are not determined just by their amino acid sequence, but
require other molecules in order to adopt a well-defined tertiary structure. Obviously,
NMR can deal with such problem [8].
The example of a protein that folds upon binding is the eukaryotic initiation factor 4G
(eIF4G), which is the core of a multicomponent complex that controls translation
initiation. Among other components, such as the RNA helicase eIF4A and the 40S-
associated eIF3, eIF4G binds the cap-binding protein eIF4E and thus recruits the 5′
end of mRNA to the small ribosomal subunit. It had been reported that the eIF4E-
binding domain of eIF4G is natively unstructured, but folds upon binding to eIF4E
[22]. A recent structural study of the yeast eIF4E–cap–eIF4 complex (Figure1.2.2)
revealed that an 80-residue segment of eIF4G folds upon wrapping around an
otherwise unfolded N-terminal segment of eIF4E [23]. Thus, complex formation
involves a mutually induced folding event.
Figure 1.2.2 Ribbon representation of eIF4E (yellow) in complex with a fragment
ofeIF4G (residues 393–490) (blue). The bound cap analog, m7GDP, is drawn in rod
representation (purple). Upon binding eIF4E, eIF4G folds into a ring-shaped structure
around the N-terminal tail, distal to the cap-binding site.
1.2.4.2. Multi-dimensional NMR to determinate 3-D structure of protein.
The basic of NMR experiments to determine the structure shows as below without
any specific details . Two-dimensional NMR involves using a complex pulse
sequence to disturb the nuclear spins. Typically there are four different periods
involved: preparation, mixing, evolution and detection. The two most common basic
techniques are COSY (homonuclear (J-) correlated spectroscopy) and NOESY
(Nuclear Overhauser Effect spectroscopy). The first gives distances through covalent
bonds, while the latter through space. Different types of pulse sequences are used
for the different types of 2D (and 3D,4D) spectra. The diagonal corresponds to a 1D
spectrum. COSY spectra are the simplest, the cross-peaks arise from Hs on adjacent
Cs or C and N. COSY spectra, are used to determine the amino acid residue identity
NOE interactions are short-range effects and only show atoms closer than 4.5 - 5 A.
Usually they are broken down into three classes, strong NOE's (atoms closer than
2.5A), medium (2.5-3.5A) and longer (3.5-5A). The medium and long range NOEs
are of most value, since the shorter range ones correspond to neighboring covalently
bonded atoms.. [25]
Particular types of secondary structure have characteristic coupling constants and

patterns, which allow assignment of secondary structure as well. NOE's and an
algorithm, such as distance geometry constraints are be applied for the tertiary
structures. Furthermore 3D picture can be determined by the necessary hundreds of
distances determined from the NOEs to be collated . Subsequently various
refinement methods are run on the structure to eliminate errors.
Typically one uses concentrations of protein in the 1-3 mM range (i. e. very
concentrated for protein solutions). 2D spectra may take from ½ to 3 or 4 days to
collect. They are only useful at 500 MHz and higher fields. Today there are a number
of 750 MHz (and even two 800 MHz) instruments in operation. [25]
The example used here is the human ASC PYRIN domain (apoptosis-associated
speck-like protein containing a caspase recruitment domain) which can be
determined according to the basic process , however , with individual details . All the
figures gotten by Fabiola Espejo and Manuel E. Patarroyo of Colombia .[26]
The first indication of secondary structure was obtained by CD spectra. The far-UV
CD spectrum of ACS2 showed two characteristic α-helix minimums at 208 and
224 nm (Fig. 1.2.3).
Fig. 1. 2.3 Far-UV CD spectra of ASC2 doamin. The form of the curve in the samples
shows the typical behaviour of α-helix.
The presence of alpha helix secondary structure elements can be observed.

Negative values (−1 in Fig.1.2.4) indicated the presence of an α-helix.
Fig. 1.2.4. Sequential and medium-range NOEs used to verify the secondary
structure elements derived from NOE analysis. NOEs between amide protons of
consecutive residues and between Hα and the amide proton of subsequent residues
are represented by bars connecting the residues.
Fig. 1.2.4 illustrates NMR-derived data summarizing ASC2 secondary structural

elements. Medium-range NOEs: dNN (i, i + 2), dαN (i, i + 3), dαN (i, i + 4), and dαβ
(i, i + 3), with Cα and Cβ secondary chemical shifts, identify the protein’s five α-
helical regions.
Proton D2O exchange experiments confirmed the presence of α-helix secondary

structure elements (• in Fig 1.2.4) The α-H of amino acids L9, K10, V11, L12, E13, and
L15 were resistant to being substituted, confirming the presence of H bonds in the first
helix. (Fig 1.2.5).
Fig.1.2.5. (A) 500 MHz 1H 15N-HSQC spectrum of recombinant ASC2 obtained at
300 K. Resonance assignments are indicated with the one letter aminoacid code and
residue number. Side-chain amide protons of Asn and Gln are indicated by horizontal
lines. (B) Proton D2O exchange experiments each 20 min confirmed. the NH
implicated in hydrogen bond.
Structure calculation
To weigh that strong NOEs were not observed that form the helix 3 from this protein
when carrying out the structure calculation, one can observe that it is formed a helix
among the amino acids 38 and 43 (Fig 1.2.6).
Fig. 1.2.6. The solution structure of ASC2 domain structure. (A) Superposition of the
backbone atoms in the 25 conformers representing the NMR structure of the ASC2
pyrin domain. (B) A ribbon diagram of the Cα trace of the averaged minimized
structure.
1.2.5 Relevant web site
1. http://matematicas.udea.edu.co/~carlopez/index2a.html
2. http://www.bmrb.wisc.edu/
3. http://www.chemlin.de/chemistry/nmr_spectroscopy.htm
1.2.6 Key Industry Suppliers
1.http://www.oxinst.com/wps/wcm/connect/Oxford+Instruments/Companies/Oxford+
Instruments+Molecular+Biotools/
2. http://www.janis.com/nmr2.html
1.2.7 References
1 Evans, Jeremy N. S.(1995) Biomolecular NMR spectroscopy .Oxford .N Y.
2 Wüthrich, K. (1998) Nat. Struct. Biol. 5, Suppl., 492-495
3 Mirau, P, A .(2002) A practical guide to understanding the NMR of polymers.

Wiley,NY.
4 Sheffield Hallam University (2006)

http://teaching.shu.ac.uk/hwb/chemistry/tutorials/molspec/nmr1.htm
5 Breitmaier, E.(2002) Structure elucidation by NMR in organic chemistry : a practical

guide , 3rd rev. ed. Wiley, England.
6 http://en.wikipedia.org/wiki/NMR_spectroscopy
7 Liu G, Shen Y, Atreya HS, Parish D, Shao Y, Sukumaran DK, Xiao R, Yee A,
Lemak A, Bhattacharya A, Acton TA, Arrowsmith CH, Montelione GT, Szyperski
T.(2005) NMR data collection and analysis protocol for high-throughput protein
structure determination. Proc Natl Acad Sci U S A. 30,10487-92.
8 Koh .T. and Gerhard .W.(2006) NMR studies of protein interactions Current
Opinion in Structural Biology. 16. (1), p 109-117.
9 Marc. B (2006) Molecular interactions investigated by multi-dimensional solid-state

NMR .Current Opinion in Structural Biology. 16. (5), p 613-628.
10 E.R. Andrew, A. Bradbury and R.G. Eades, (1958).Nuclear magnetic resonance

spectra from a crystal rotated at high speed, Nature 182 p. 1659.
11 V. Raghunathan, J.M. Gibson, G. Goobes, J.M. Popham, E.A. Louie, P.S. Stayton
and G.P. Drobny, (2006) Homonuclear and heteronuclear NMR studies of a statherin
fragment bound to hydroxyapatite crystals, J Phys Chem B Condens Matter Mater
Surf Interfaces Biophys 110, pp. 9324–9332.
12 J. Leppert, C.R. Urbinati, S. Hafner, O. Ohlenschlager, M.S. Swanson, M. Gorlach
and R. Ramachandran, (2004) Identification of NH.N hydrogen bonds by magic angle
spinning solid state NMR in a double-stranded RNA associated with myotonic
dystrophy, Nucleic Acids Res 32, pp. 1177–1183.
13 G.L. Olsen, T.E. Edwards, P. Deka, G. Varani, S.T. Sigurdsson and G.P. Drobny,
(2005) Monitoring tat peptide binding to TAR RNA by solid-state 31P-19F REDOR
NMR, Nucleic Acids Res 33, pp. 3447–3454.
14 Gerhard .Z. and Norbert. M.(2006). Cogwheel phase cycling in common triple
resonance NMR experiments for the liquid phase, J Magnetic resonance
181(2),pp244-253.
15 L. Frydman, T. Scherf and A. Lupulescu, (2002) The acquisition of

multidimensional NMR spectra within a single scan, Proc. Natl. Acad. Sci. USA 99
(25), pp. 15858–15862.
16 T. Szyperski, G. Wider, J.H. Bushweller and K. Wüthrich, (1993) Reduced

dimensionality in triple-resonance NMR experiments, J. Am. Chem. Soc. 115, pp.
9307–9308.
17 E. Kupče and R. Freeman, (2003) Reconstruction of the three-dimensional NMR

spectrum of a protein from a set of plane projections, J. Biomol. NMR 27, pp. 383–
387.
18 M.H. Levitt, P.K. Madhu and C.E. Hughes, (2002) Cogwheel phase cycling, J.
Magn. Reson. 155, pp. 300–306.
19 Kay, L. E. (1998) Nat. Struct. Biol. 5, Suppl., 513-517
20 Dobson, C. M. & Hore, P. J. (1998) Nat. Struct. Biol. 5, Suppl., 504-507.
21 Hong,T.Y.(1999) Proc. Natl. Acad. Sci. USA 96, 332-334
22 P.E.C. Hershey, S.M. McWhirter, J.D. Gross, G. Wagner, T. Alber and A.B.
Sachs, (1999) The cap-binding protein eIF4E promotes folding of a functional domain
of yeast translation initiation factor eIF4G1, J Biol Chem 274, pp. 21297–21304
23 J.D. Gross, N.J. Moerke, T. von der Haar, A.A. Lugovskoy, A.B. Sachs, J.E.G.
McCarthy and G. Wagner, (2003) Ribosome loading onto the mRNA cap is driven by
conformational coupling between eIF4G and eIF4E, Cell 115, pp. 739–750.
24 http://www.chemistry.ucsc.edu/~fink/200lecture/8-97.htm
25 Fabiola. E.and Manuel. E. P.(2006) Determining the 3D structure of human ASC2

protein involved in apoptosis and inflammation, j Biochemical and Biophysical
Research Communications 340 (3), pp. 860-864.
Chapter 1.4 Fluorescence Spectroscopy of Proteins and
Applications
M Sabir Patel
1.4.1 Introduction
Fluorescence spectroscopy is a type of electromagnetic spectroscopy used for the

analysis of fluorescence spectra. It is achieved by exciting molecules of certain
compounds using a beam of UV light causing them to emit a light of low energy and
frequency in the form of a generally visible luminescence.
In fluorescence spectroscopy the molecule of the compound absorbs a

photon of light, which excites it from its ground state to one of the higher energy
vibrational states. The molecule loses is vibrational energy upon collision with other
molecules and finally reaches the lowest vibration state of the excited electronic
state. Thereafter the molecule further drops to one of the vibrational states of the
most stable ground electronic state and emits a photon in the process. The photons
thus emitted will vary in their energies and therefore their frequencies. The structures
of the vibrational levels can be determined by analyzing the different frequencies of
lights emitted in fluorescence spectroscopy.
The energy carried by 1 photon is proportional to the frequency of its oscillation and
is given by
E = hν = hc ergs
λ
where ν is the frequency, λ the related wavelength and h = Planck's constant
(6.624 x 10-27 ergs/seconds).
Figure 1 Transitions giving rise to absorption and fluorescence emission

Spectra
(CENTRE for FLUORESCENCE SPECTROSCOPY)
Fluorescence Sensing
Fluorescence promises many potential applications in the field of biotechnology and

clinical chemistry with the use of newer methods of fluorescence sensing together
with newer probes.
Life time based sensing:
Lifetime based sensing which is an active area of research has a major advantage
due to the fact that the fluorescence lifetime is independent of signal intensity due to
the external factors like scattering and absorbtion.
Lifetime based sensing has been recently applied in the high throughput screening
(HTS) mode using two-photon excitation of Calcium Green.
Novel Sensing methods:
The more recent advancements like modulation and polarization sensing have a wide
application in tissue and medical sensing, environmental sensing, bioimaging assays
and HTS.
Modulation sensing transform analyte dependent intensity changes into a change in

the low frequency modulation signal.
Polarization sensing transforms analyte dependent intensity changes into a change

in polarization and/or angle rotation increasing sensitivity and enabling visual
detection.
The methods are calibrated using reference film placed adjacent to the sample.
These sensing methods are generic and can be used with any fluorophore displaying
analyte-dependent change in intensity.
Complex Glucose-Glucokinase
Fluorescence Lifetime Imaging:
Fluorescence Lifetime Imaging Method was developed by CFS. FLIM uses the
fluorescence lifetime at each point in the image and derives an image contrast using
the fluorescence microscope. Certain analytes like Mg2+ , Ca2+ , Cl, K+ or pH etc. alter
the fluorescence lifetimes of many fluorophores. However, there lifetimes are not
dependent on photobleaching and the local probe concentration. Also, FLIM is
independent of the wavelength ratiometric probes. Using visible-wavelength
illumination, FLIM also allows quantitative Ca2+ imaging.
CFS is currently working on the development of a new FLIM instrument and which
will also be available to external users of CFS.
Ionophore and weak base treatments perturbed the cytosolic pH of CHO cells.
Probe Chemistry:
The CFS is focused on the design and synthesis new fluorescent probes, novel
conjugatable emissive transition metal-ligand complexes and lanthanide compounds,
as well as donor-acceptor assemblies to meet the needs for expanding the
applications of fluorescence to not only biochemical and biophysical research, but
also biotechnology, drug discovery and cell biology.
CFS has been successful in introducing a variety of ruthenium(II), rhenium(I) and

Osmium(II) dimine complexes which display lifetimes ranging from 100 nanoseconds
to 1microsecond and these of particular interest to bio-researchers for the study of
the dynamics of biomacromolecules such as proteins and lipids.
Below are some of the projects involving probe chemistry that CFS is currently
working on:
• Studying the enhancement of molecular luminescence near the surface of

silver nanoparticles
• Exploring luminescent iridium(III) polypyridyl complexes
• Developing red emissive energy transfer assemblies for sensing
New Series of Rhenium(I) Complexes have:

• λmax at 610 nm in a buffer with µs-scale lifetime
• MLCT absorption doubled at 400 nm
• Much higher thermal and photochemical stability
• Very high anisotropies
Light Quenching:
A pulse is given to excite the sample for measuring the time resolved fluorescence
which is followed by measurement of time dependent emission. In the case of light
quenching additional pulses follow the excitation pulse in order to modify the excited
state population. This occurs upon illumination with longer wavelength non-absorbing
light which depletes part of the excited population by stimulated emission.
In reality, the fluorphores are not in a quenched state but only appear to be so
because the observation of the residual population was actually made at right angles
to the quenching beam. The "quenched" part of the emission is not observed since it
travels parallel to the quenching beam. Therefore in time resolved light quenching
experiments the emission observed is only before and after the quenching pulse. The
instantaneous change in the intensity and/or anisotropy decays reflects in frequency-
domain as characteristic oscillations.
Light Quenching provides an opportunity to control an excited state population and

orientation of fluorophores. In presence of Light Quenching fluorescence anisotropies
above 0.4 and below -0.2 can be observed.
Based on demonstration Light Quenching can be used to:
• Study the spectral relaxation of solvent-sensitive fluorophores.

• Eliminate unwanted fluorescent species from the mixture of fluorophores.
• Increase the spatial resolution in far-field fluorescence microscopy.
• Quench the fluorescence near the glass surface in total internal reflection
excitation.
Light quenching with parallel excitation and quenching pulses. Excited state
population without light quenching (top), with time-coincident or one-pulse light
quenching (middle) and with time-delayed light quenching (bottom).
Multi-Pulse Fluorescence
Multi-Pulse Fluorescence (MPF) is a new CFS core project, currently under

development.
The basic idea of MPF is to perturb the sample with one light pulse and to start the
time-resolved measurement at a delay time t with a second excitation pulse. The
time-resolved data will be correlated with time-dependent structural changes in the
protein following the perturbation pulse. This approach will be applied to proteins
which undergo conformational changes in response to light, including hempglobin,
rhodopsin and phytochromes. Both steady state and time-resolved measurements
will be performed.
Experimental arrangement for two-pulse fluorescence measurements. L are lenses,

P are polarizers, D are diaphragms, R are polarization rotators, and F are optical
filters. Long delay line DL1 is placed in the UV and a fine delay line DL2 in the visible
beam, DF is a coated optical plate (dichroic filter) and PD is a reference photodiode,
used in time-resolved measurements.
1.4.3 Evaluation of the technology
In Analytical chemistry the filed of atomic spectroscopy employs three most common
techniques. Namely,
• Atomic Absorbtion
• Atomic Emission
• Atomic Fluorescence
Since , atomic fluorescence technique incorporates properties from the other two, we
shall go on to explain this technique and its prime advantage over the other two. The
atoms are excited in the flame source by a beam of light that is focused in to the
atomic vapor. The intensity of this "fluorescence" increases with increasing atom
concentration, providing the basis for quantitative determination.
However, the lamp in this technique is mounted at a specific angel to the that of the
optical system and thus the light detector only sees the fluorescence in the flame and
not the light of the lamp intead. By this arrangement, lamp intensity can be
dramatically increased and in turn raise the number of excited atoms which is a
function of the intensity of the exciting radiation.
In conclusion, even though atomic absorbtion is the mostly widely used of the three,
yet , particular benefits are attained using fluorescence spectroscopy technique.
Fluorescence correlation spectroscopy (FCS) is an analytical process to

measure the fluctuation of fluorescence intensity and which is mainly due to
Brownian motion of the particle. The analysis determines the average number of the
fluorescent particle and average diffusion time, when the particle is passing through
the space. Eventually, both the concentration and size of the particle (molecule) are
can be evaluated. This technique finds a very good application in the fields of
biochemistry, biophysics and analytical chemistry.
Unlike HPLC analysis, this method has no physical separation process and has a
good spatial resolution determined by the optics which is great advantage over
previous methods. This technique enables us to study and gain a better insight into
the nature and function of biochemical pathways in a living cell using fluorescence
tagged molecules.
Radiative Decay Engineering (RDE)
Radiative Decay Engineering(RDE) is more recent of the core projects at CFS and is
currently under the development stage.
Fluorophores when placed close to metal surfaces or colloids, display a varied set of
spectroscopic effects. This effects are due to numerous factors or mechanisms like
quenching with d-3 dependence, enhancement in the local field and radiative decay
rate are responsible for the quantum yield.
For ellipsoidal particles the maximum enhancement in the magnitude of the local field
is about 140. With increase in radiative rate the total radiative decay rate increases
and so does the quantum yield. There are two limiting cases. If the dye has a high
quantum yield (Q0 —>1), the additional radiative decay rate cannot substantially
increase the quantum yield.
In the case of low quantum yield chromophores the enhancement can be as large as
1/Q0. For this reason it is of interest to study fluorophore-metal interactions with low
quantum yield fluorophores. While the actual mechanism is complex, one can
imagine the particles serve as an antenna to radiate faster than knr. This suggests the
emission from weakly fluorescent substances can be increased if they are positioned
at an appropriate distance from a metal surface or colloid.
Non-fluorophore species of chromophores can also become fluorescent with the

ability to increase radiative decay. DNA ,for example, can also become brightly
fluorescent without the use of extrinsic probes. In the case of proteins, tryptophan
residues are quenched by nearby groups like histidine, phenylalanine or disulphide
groups. Desirable mutant proteins can be obtained without site-increased
mutagenesis by increasing the radiative rate and thereby increasing the quantum
yield of the quenched tryptophan residues. Similiarily high quantum yields are
achieved if metal surface are placed close to weakly fluorescent species like bilirubin,
fullerenes, metal-ligand complexes, or porphyrins.
1.4.4 Applications of the Technology
Flouorescence spectroscopy has now found in roads and applications with newer
more rapid and non-destructive methods in the food industry.The emission spectra
can be quite complex, arising from a number of known, as well as unknown
molecules, and the total fluorescence is sensitive to the local environment,
apparently reducing the methods robustness. But with constant improvement and
better sensor technology and excellent optics fluorescence spectroscopy shows
many more promising applications in varied fields of life sciences.
Fluorescence has proved higly effective method in measuring fat and connective
tissue in meat.
New techniques are presently under development for non-destructive measurements

of lipid oxidation based on solid sample fluorescence and the outcome of various
experiments on poultry meat, complex meat products, dairy products and fish have
been rather fruitful.
Diagnostic tools for malignant diseases are being developed using fluorescence
spectroscopy techniques. Autofluorescence emitted by the tissue along with
fluorescence from specially designed probes attached to the target are detected
using spectroscopy. One of the major advantages of using fluorescence
spectroscopy is that it is a non-invasive tool and it can be performed in real-time. The
prime objective is to develop guiding tools for biopsies based on fluorescence
measurements.
Fluorescence emitted from meat

and the fluorophore Rhodamine 6G
ENGINEER RESEARCH and DEVELOPEMENT (ERDC)
The US Army Engineer Research and Developmental Center(ERDC) has the

Fluorescence Spectroscopy Laboratory(FSL) involved in basic and applied research
focused on the development and tesing of fluorophores for recovery by remote
sensing.They are also commited for the testing and detection of living organic and
inorganic materials which may pose as harmful agents or environemental hazards in
relevance to the war fighters.
The FSL boasts of state of the art spectrometers that have the capability to measure
the steady-state and life time decay fluorescence spectra for fluorophores .
The lab uses femtosecond Ti-Sapphire laser that can characterize even the near
infrared fluorophores.
FSL supports imagery based fluorescence measurements using laser-induced

fluorescence and passive fluorescence measurements by Fraunhofer Line
Discrimination.
These measurements are of great importance to defense, intelligence agencies, and

mapping agencies with base line research in polymer detection, backgrounds, and
characterization of flurophores for environmental analysis.
Multi-Photon Excitation
The absorption of two or more long wavelength photons followed by fluorescence

emission from the lowest excited state is termed as Multi-Photon Excitation (MPE).
The primary objective is to study the basic principles and biochemical applications of
MPE.
MPE allows localized excitation in fluorescence microscopy and an enhanced

photoselection in spectroscopy.
The study of MPE has been applied but not limited to
• Spectroscopic properties and images of stained DNA

• Tyrosine, Tryptophon and Proteins
• DPH-labeled membranes
• Detection of anti-cancer drugs in blood
• Ru and Re metal ligand complexes
MPE has also been used to observe a UV fluorescence from saturated

hydrocarbons and cholesterol analogues. CFS also applied MPE in High Throughput
Screening (HTS).
Emission intensity of p-terphenyl with NIR (750 nm), UV (375 nm) and combined (375 and 750 nm) excitation
Emission spectra of p-terphenyl with 250 nm excitation and with 2C2P excitation at 375 and 750 nm.
Microsecond Dynamics of Macromolecules
In general, biological macromolecules are known to have motions ranging from ps to

days or even weeks and these motions are vital for the proper functioning of these
macromolecules. Techniques like fluorescence have made it possible to access time
frames of pico-nanosecond while slower timeframes of ms are possible by NMR and
ESR.With development of stop-flow instrumentation, NMR, temperature jump and
faster correlation spectroscopy it is possible to have measurements in microsecond
window accessible experimentally.
CFS is currently using µs MLC and µs lanthanide probes as donors in FRET

experiments to characterize conformational dynamics in microsecond time window.
The method provides both the KINETICS and CONFORMATIONAL

DISTRIBUTIONS as well as intra and inter molecular diffusion in ms to ms time . It is
an EQUILIBRIUM method which can be applied to very diverse problems.
MAIN AIMS
• Folding/Unfolding of protein secondary structure elements alpha-helix and

beta-hairpin
• Inter-domain motions in histidine and glutamine binding proteins
• Lipid composition and phase state dependence of lateral diffusion in
membranes
• Lateral diffusion in biological membranes and domains such as RAFTs
• Domain motions/ arm flexing in nucleic acid structures such as t-RNA,
ribozyme, junctions and hairpins.
1.4.5 Relevant web sites:
For further reading and learning resources, these are of some of the
web site links which may be useful for further reading.
• http:// www.wikipedia.org.
• http://cfs.umbi.umd.edu/
• http://chemistry.rutgers.edu/grad/chem585/lecture2.html
• http://www.risoe.dk/
• http://www.wam.umd.edu/~toh/models/Fluorescence.html
• http://www.alschemex.com/learnmore/learnmore-techinfo-
principles-analyticalmethodologies.htm#X-
ray%20Fluorescence%20Spectroscopy%20(XRF)
• http://www.umb.no/?viewID=698
Instrumentation:
• CFS Product range can be seen at (http://cfs.umbi.umd.edu/cfs/instr.html)
state-of-the-art time-domain (TD) and frequency-domain (FD) fluorescence

instrumentation for time-resolved studies of biological macromolecules are currently
available at the Center for Fluorescence Spectroscopy (CFS). The excitation sources
are cavity-dumped and frequency-doubled ps dye lasers, or a Ti:Sapphire laser. A
microchannel plate (MCP)-PMT is presently used for Time-correlated single photon
counting (TCSPC) , to provide an instrument response function near 60 ps.
Frequency-domain measurements are possible up to 10 GHz using the Center's FD
instrument, and a high speed MCP-PMT. Available excitation wavelengths range
from UV to NIR. For less demanding applications modulated cw lasers (for FD) is
available. A unique capability of the CFS will be the ability to collect and analyze both
TD and FD data for the same samples, and in the future, simultaneous dual-domain
(DD) analysis of the data. A Ti:Sapphire laser is now available for two- and three-
photon excitation.
Extensive research and development is going on to develop newer abd better

instruments for fluorescence lifetime imaging microscopy (FLIM) and for cell-by-cell
lifetime measurements in flow cytometry. A FLIM instrument with a red sensitive
image intensifier will soon be available for photon migration imaging of tissues and
turbid objects.
INSTRUMENT SPECIFICATIONS
EXCITATION SOURCES (PRIMARY)
• Argon Ion (Coherent) mode locked laser, 1W at 514 nm, fwhm = 120 ps.
• Ti:Sapphire femtosecond laser system (Spectra Physics), 750-920 nm, can
be frequency doubled (375 - 460 nm) or frequency tripled (250 - 310 nm);
fwhm = 90 fs
• Ti:Sapphire, regenerative amplifier, optical parametric amplifier system
(Coherent) 120 fs fwhm; tunable from UV-IR.
• Argon Ion air cooled, CW, 488, 514.5 nm.
• HeCd air cooled, CW, 442 nm (Liconix)
• HeCd air cooled, CW, 325 nm (Liconix)
• Modulated CW laser diodes and light emitting diodes
DYE LASERS
• Rhodamine 6G cavity-dumped dye laser, 560-620 nm, 280-310 nm, after

doubling, fwhm = 5 ps (with saturable absorber if needed)
• Pyridine 1 and pyridine 2 cavity-dumped dye lasers, 680-760 mn, 340-380 nm
after doubling, fwhm = 7 ps
• DCM cavity-dumped dye laser, 620-680 nm, 310-340 nm after doubling, fwhm
= 7 ps
PHOTODETECTORS FOR TCSPC
• MCP-PMT, Hamamatsu R2809, with a red-sensitive photocathode, 60 ps

fwhm
• PMT, Philips XP2020, 500 ps fwhm
PHOTODETECTORS FOR FD
• MCP-PMT, Hamamatsu R2566, 6 µ, for FD measurements to 10 GHz

• PMT, Hamamatsu R928, for FD measurements to 300 MHz
• A red-sensitive 6 µ R2566, for FD measurements to 10 GHz
FLUORESCENCE LIFETIME IMAGING MICROSCOPY (FLIM)

• Zeiss, Axiovert 135 TV inverted fluorescence microscope
• Gain modulated image intensifier operated up to 100 MHz modulation
frequency
• Photometrics PXL-35 CCD frame-shift camera and Innovision software
TIME-RESOLVED MICROSCOPE (TRM)
• Nikon inverted microscope 300

• FD instrumentation 0.4 - 10,000 MHz
EXPERIMENTAL CAPABILITIES
• TCSPC with a MCP-PMT and a ps laser or fs excitation source

• FD up to 10 GHz using the harmonic-content of a ps or fs pulse train
• TCSPC with a laser and fast PMT
• FD from 3 KHz to 400 MHz with a modulated cw source
COMPUTER SPECIFICATIONS
• Three Silicon Graphics Indy workstations running under IRIX 5.3 operation
system
• PC's running under Windows'98, Windows 2000 operating system (see CFS
Network).
• Internet access to all programs. Reflection 4+ for Windows graphics terminal
emulator recommended
• Data transfer between computers possible via Internet (FTP). Data and/or
programs are also available on diskettes (MS-DOS, LS120), or CD-RW
• Terminals adjacent to instrument room
SUPPORTING CAPABILITIES
• Steady state emission spectra (SLM 4800 Spectrofluorometer and SLM AB-2)
• Absorption diode array (190 nm - 1100 nm) spectrophotometer. (Hewlett
Packard)
• Linear dichroism and transition moment determination
• Lab space adjacent to instrument room
• Temperature control, -60 to +90oC
• Ultracentrifuge (Beckman L5-65B), 65,000 rpm
• Gas pressure cell to 100 atm, and a 2 kbar hydrostatic pressure cell
Iridian Spectral Technologies
Iridian Spectral Technologies is a one of the market leaders specialized in the

Fluorescence spectroscopy product line. The detailed information regarding the
product line can be seen at their website
(http://www.iridian.ca/products/category.php?intSearchCategoryID=43)
RISO National Laboratory, Denmark
Website: (http://www.risoe.dk/pol/competence/chemanal/fls920.htm)
Description of Fluorescence Spectroscopy (FLS920)
Apparatus:
A FLS920 from Edinburgh Instruments capable of steady state and time resolved
measurements in the 200nm-900nm range. The system is equipped with a 450W
Xe-lamp for steady state measurements. For time resolved measurements two
different light sources can be used. Short lifetimes (<125ns) can be measured
using a fast 40MHz LED light source (excitation around 378nm, 456nm, 501nm or
598nm). For longer lifetimes (up to 50ms) a 40kHz nanosecond flash lamp can be
used.
Accessories:
• Polarisers to measure orientation.
• A cryostat to measure at low temperature (77K).
Use:
Fluorescence spectroscopy can be used to obtain steady state emission and
excitation spectra (200nm-900 nm) and to measure fluorescence lifetimes (0.1 ns-
50 ms) of fluorescent compounds.
,
Simulation of Fluorescence Spectroscopy
Real-time simulation of a scanning fluorescence spectrofluorometer using the

software.
The software can be downloaded and the instructions and system requirements
are available on the following website :
(http://www.wam.umd.edu/~toh/models/Fluorescence.html)
A wide range of spectroscopy relevant instruments are available at Andor

technologies and orders can be placed for these instruments at their website
(http://www.andor.com/products/)
1.4.7 References
1. Molecular Photoluminescence Spectroscopy(2000). David Harvey. In: Modern

Analytical Chemistry, McGraw Hill, pp.368-380, 423-440
2. Molecular Fluorescence Spectroscopy(2004). Skoog, West, Holler and

Crouch. In: Fundamentals of Analytical Chemistry, Thomson Brooks/Cole.
3. Recent Developments in Fluorescence Spectroscopy (1996). Lakowicz, J.R.,

Terpetschnig, E., Szmacinski, H., Malak, H., Kusba, J. and Gryczynski, I.,. In:
Analytical Use of Fluorescent Probes in Oncology. (Kohen, E., and
Hirschberg, J.G., Eds.), Plenum Press, New York. pp. 65-79.
4. Imaging Applications of Time-Resolved Fluorescence Spectroscopy (1996).

Lakowicz, J.R. and Szmacinski, H. In: Fluorescence Imaging Spectroscopy
and Microscopy. Vol. 137. (X.F. Wang and B. Herman, Eds.), John Wiley &
Sons, Inc. Publishers. pp. 273-311.
5. Emerging Applications of Fluorescence Spectroscopy to Cellular Imaging:

Lifetime Imaging, Metal-Ligand Probes, Multi-Photon Excitation and Light
Quenching (1996). Lakowicz, J. R. In: Scanning Microscopy Supplement Vol.
10. Scanning Microscopy International. pp. 213-224.
6. Fluorescence Spectroscopy of Biomolecules. (1995). In: Molecular Biology
and Biotechnology (R.A. Meyers, Ed.), VCH Publishers, Inc
Chapter 1.5 Atomic Force Microscopy of Proteins
Trusha Jhala
1.5.1 Introduction
The Atomic Force Microscope very much known as the “Eye of Nanotechnology” has
proven to be a powerful tool for biological studies [1]. It is a high resolution imaging
technique for surface morphology in various solutions and gas environments that has
allowed researchers to observe biological processes in real time. [10,11] The Atomic
Force Microscopy(AFM) also known as the Scanning Probe Microscopy, has
revolutionized the field of interfacial surface science by allowing observation at
molecular and atomic levels in the native environment at a single molecule level.
[2,10,11] It was first invented by Gerd Binnig and Christoph Gerber in 1985.[13]The
AFM is used in a wide range of technologies in electronics, telecommunications,
biological, chemical, automotive, aerospace and energy industries. It is used for
studies in abrasion, adhesion, cleaning, corrosion, etching, friction, lubrication and is
used to analyze and investigate on a wide variety of materials.[8,13] It is now used in
many fields of nanoscience and nanotechnology providing better understanding of
events occurring at the molecular level. [11] AFM is widely used because it can be
used for imaging any conducting or non-conducting surface unlike STM which is
limited to conducting surfaces.
1.5.2. Principle
The principle on which the AFM works is by the contact of the cantilever tip and the
sample surface to be imaged. The cantilever tips are made of Si3N4 or Si with a radii
of 4-60nm. The cantilever tip is combined with an ultra-sharp probe. The probe is of
significant importance. The shape of the probe defines the resolution of the AFM and
is made of a sharp tip with a specific spring constant. The surface topography is
measured by keeping the force constant while the tip scans the surface and moves
vertically due to attractive or repulsive interaction forces. The piezo-electric scanners
keep the tip at a constant force when it has to obtain information regarding the
height. It keeps the height constant when it has to obtain information on the force.
Most AFM scanners are in the range of 90 x 90µm in the x-y plane and 5 µm for the z
–direction.[13]The cantilever tip bends upwards due to the ionic repulsive force from
the surface. This bending is measured by a laser beam reflected onto a position-
sensitive photodetector which measures minute sensor deflections. This is used to
calculate the force and allows visualization of the surface topography. [1,13]In a
nanoscope AFM, there is an optical detection system which comprises of the tip
attached to the base of the reflective cantilever. On the back of the cantilever is a
diode laser. When the tip scans the sample surface, the laser beam deflects into a
dual element photodiode which the photodetector measures and then converts to
voltage. The computer software on gaining this input from the photodetector controls
and maintains a constant force or height above the sample surface. [13]
The constant force mode measures the height deviation in real time through the
piezo-electric transducer. The constant height mode measures the deflection force
through the tip to be inserted in the sensitivity of the AFM head. The tip needs
calibration parameters during force calibration of the microscope. Few AFM’s use
200mm wafers, which measure surface roughness of 5nm lateral and 0.01nm vertical
resolution. Scanners measure the local height of the sample by interpreting the
sample under the cantilever or the cantilever over the sample. 3-dimensional
topographical maps can be constructed using the sample height and the probe tip
position. [13,15] The cantilever tip obeys Hooke’s Law and can thus find the
interaction force. The piezo-electric ceramics scanners carry out the movement of the
tip or the sample and can measure resolutions in x-, y- and z-directions.[14]
AFM can be operated in two modes: [14]

• With feedback control
• Without feedback control
With feedback control

When the electronic feedback is switched on, the piezo scanner which is responsible
for the movements of the tip and the scanner reacts to changes and again modifies
the tip-sample separation to the constant pre-determined value to maintain a
constant force. This is known as the height mode.[14]
Figure 1.5.1 - Schematic representation of an atomic force microscopy (AFM). The

cantilever-probe system is deflected by the surface topography of the sample.
Cantilever deflections are detected with a laser beam mirror set-up. The position-
sensitive detector captures normal forces and frictional forces affecting the probe.
Source: http://www.ingentaconnect.com/error/delivery&format=pdf
Without feedback controls:
Here, the electronic feedback is switched off, and is used for imaging very flat
samples. This is known as the constant height or deflection mode. The absence of
electronic feedback may damage the cantilever tip because of the roughness of the
sample or there may be thermal drifting. This can be overcome by keeping electronic
feedback which detects such an error and removes slow variations in topography
whilst highlighting the edges of the features of the sample.[14]
Figure 1.5.2 AFM without feedback controls

Source: http://www.molec.com/what_is_afm.html#
The Cantilever Tip:

As mentioned above, the tip of the cantilever plays a very significant role defining the
resolution of the AFM. For best results, the tip must have a radius of curvature of
around 5nm. The absence of a sharp tip can seriously affect the performance of the
AFM causing broadening, compression, interaction forces or aspect ratio. Tip
broadening occurs when the radius of the tip is greater than the size of the feature
being imaged causing the microscope to respond even before the tip has contacted
the apex. This is known as tip convolution. Compression arises when the material is
particularly soft (DNA) and this may pressurize the surface. The tip has to be
selected according to its material as there are forces due to the chemical nature of
the tip which may affect the outcome.[14]
Figure.1.5.3 Tip convolution

Source:http://www.vpd.ms.northwestern.edu/teaching/AFM_MSc_190_lab.pdf
There are various modes of attraction between the tip and the sample:
Table 1.5.1 Tip-Sample interaction modes

Source: http://www.vpd.ms.northwestern.edu/teaching/AFM_MSc_190_lab.pdf
The common AFM modes of operation are:
Figure 1.5.4 – Three common AFM operation modes.

Source: http://www.ingentaconnect.com/error/delivery&format=pdf
(a) Contact mode: This is the most common operational mode of AFM where the
probe is in permanent contact with the sample surface. In fluids with biological
specimens the probe force is kept below 100 pN. This mode requires minimal sample
preparation and can operate in air and fluid environment. It provides information on
the elasticity, adhesion, hardness, friction of the sample surface. [1, 14]
(b) Tapping mode: This mode uses a probe that oscillates at a constant frequency
and the probe force is dominated by changes in the resonance frequency of the
cantilever. To evade damage to the sample surface, the probe gently taps the
sample surface rather than scraping the surface. The advantage of this mode is that
it can detect surface contaminants that are not seen in height images. [1, 14]
(c) Non-contact mode: This mode is used for samples which are delicate in both air
and fluids and there is no damage to the material because the probe is placed in the
attractive force region. The Van der Vaal forces and electrostatic potentials are used
to measure the force gradients. [1, 14]
AFM has revolutionized the field of interfacial surface science by allowing direct high
resolution visualization of surface topography and its ability to being performed in
various environments. There are a lot of new advances in the instrumentation,
simplifying sample preparation, molecular imaging at a single molecular level has
been achieved from the native environment of the proteins with better resolution that
generates topographs of native proteins with the obtained resolution at nanometer
scale that exhibits the supremacy of AFM. The operational mode of AFM with
advancement of force –spectroscopy is now used extensively by researchers to
study ligand-receptor interactions which aims to measure the forces at single
molecular level. [1,2]
Recent development of the VideoAFMTM has revolutionized the way research is

conducted, allowing researchers to view molecular processes in real-time. This has
opened the door for deeper insight into biological processes at nano level. The
significance of Multimode Scanning Probe Microscope in Bio-nanotechnology
research used alongwith VideoAFM™ has unlocked the various keys to real time
biological processes. The VideoAFM™ imaging rates are 1000 times faster than the
existing AFM’s. It also provides an opportunity visualize molecular interactions and
the forces involved in it. Another positive aspect of this new development is its ability
to smoothly work with conventional AFMs.[15,16]
The use of Mac Mode AFM has opened prospects in drug development, especially in
structural imaging of drug carriers such as liposomes and lactose crystals. Structural
imaging revealing the shape and size of these drug carriers is of peak importance, as
they are used widely in the pharmaceutical research industry using AFM. [2]
The shape and size of dimerystic phosphotydalcholine (DMPC) liposomes in
phosphate buffer can be directly observed using MAC Mode AFM.
Figure 1.5.5 Structural imaging of liposomes

Source: www.molec.com/media/PDFs/B-Biology_App_notes/B2-Bio_Review.pdf
The above image shows round liposomes with their diameter ranging from 50 up to
200nm achieved at a Scan size = 1.15 µm x 1.15 µm
Likewise, surface imaging of the lactose crystal as an inhaled drug carrier in
increasing humidity are shown from 13 to 96%. Mac Mode AFM reveals the crystal
structures melting significantly at 80% humidity. [2]
Figure 1.5.6 Surface imaging of lactose crystals at varied humidity levels achieved at a
scan size of 5 µm x 5 µm.
Source: www.molec.com/media/PDFs/B-Biology_App_notes/B2-Bio_Review.pdf
ADVANTAGES:
Interest in AFM in the study of proteins arose because it could resolve the surface
features of heterogenous samples under different conditions and provide direct
observation of protein complexes in real time. As it could image non-conducting
surfaces, it was be used to analyze a wide variety of biological samples. [1,13]
The main advantage of AFM is that it can provide easily achievable high resolution
and 3-dimensional images of surface topography of biological specimens in various
environments and temperatures. [1,10,11,12,13] Recent advances in this method
have enabled surface imaging at a single molecular level at a resolution all the way
to the nanometer scale. [1] Due to better sample preparation techniques and superior
control of probe-sample interactions it is probable to now analyze protein folding. [1]
AFM methods require little sample preparation. [13] AFM has also been used widely
for imaging individual proteins and other molecules like collagen. Immobilization of
IgG1 antibodies was attained using AFM, where the low affinity of IgG molecules
towards mica was surmounted by cloning a metal-chelating peptide into the carboxy
terminus sequence of IgG. The purified IgG had binded in a regiospecific manner to
the nickel-treated mica. [13] AFM combined with bright-field, fluorescence and other
optical techniques can be used for identification of structures and simultaneously
providing nanometer-resolved images of the sample surface. [13] AFM can measure
intermolecular forces in the nanonewton range in protein synthesis, DNA replication
or drug interaction which enables it to analyze ligand-receptor interactions. [13] An
important part of the study of biological systems is the electrical properties of their
surfaces, where the use of AFM comes in. AFM can image electrical surface charge,
binding forces and electrostatic forces, micromechanical properties, elasticity and
viscosity of live cells and membranes. [13]
On comparing AFM with other microscopy techniques, AFM shows many advantages
over other techniques making it the preferred method used among the researchers.
The table below shows how AFM is advantageous compared to methods like SEM,
TEM and optical microscopy techniques as far as cost, flexibility in various
environments and ease of sample preparation is concerned. [11]
AFM TEM SEM Optical

Max resolution Atomic Atomic 1’s nm 100’s nm
Typical cost
100 – 200 500 or higher 200 – 400 10 – 50
(x $1,000)
Imaging Environment air, fluid, vacuum, special gas vacuum vacuum air, fluid
In-situ Yes No No Yes
In fluid Yes No No Yes
Sample preparation Easy Difficult Easy Easy
Table 1.5.2 Comparison of AFM and other Microscopy Techniques

Source: http://www.molec.com/what_is_afm.html#
AFM versus STM:

The main advantage of AFM over STM is that the latter technique is limited to
imaging conducting samples, while the former can image conductors and insulators.
In AFM, both writing voltage and tip-to-substrate spacing can be individually
controlled whereas in STM, as both are linked, control needs to be adjusted
accordingly. STM has better resolution because the force-distance reliance on tip
shape and contact force is much more complex compared to STM. [13]
AFM versus SEM:

AFM offers better topographic contrast direct height measurements and very clear
views of surface features and doesn’t require coating compared to SEM. [13]
AFM versus TEM:

AFM is less expensive compared to TEM. AFM is also more flexible with various
environments and the sample preparation is easy. [13]
AFM versus Optical Microscope:

Optical Microscopy is comparatively cheaper, requiring less sample preparation.
AFM generates explicit measurements of step heights, shows differences between
materials that are independent of reflectivity. [13]
Limitations:
AFM has enormous applications in the field of Molecular and Microbiology but at the
same time several limitations and difficulties also exists. The most vital aspect of the
technique is sample preparation which can bear the force applied by the scanning
probe for that appropriate solid substrate and well attached sample is needed. When
the sample consists of living cells the immobilization by means of adsorption is not
appropriate because of the limited contact area and as the substrate is very small it
can lead to detachment by the scanning probe. The better alternative developed was
to use porous membranes. This approach can minimize the denaturation but this
method can basically work well for spherical cells not for rod-shaped cells. The other
problem encountered is in relation to the resolution and the image interpretation
which is solely based on the imaging force and probe geometry. Large forces acting
between the sample and the probe during imaging may significantly reduce the
resolution power of the images generated and can also cause molecular damage.
When imaging the samples in the air, a layer of water condensation or other
contaminations cover up both the samples as well as the probe which often leads to
sample damage or makes high resolution images due to strong attractive force. In
order to optimize imaging environment pH and ionic strength are also the factors that
affect image quality. Another difficulty that may arise is due to shadowing or
multiplication of small structures generated due to multiple probe effects generated
from contamination on the probe. [3]
Recent modes of AFM have shown enormous applications in 3-dimensional

structural identification of cells, biomolecules and subcellular entities in various
environments. AFM is also a powerful tool for measure the forces and interactions at
molecular level in real time. AFM has enabled researchers to study processes like
polymerization of fibrinogen and crystal growth and imaging physiochemical
properties living cells.[6]
AFM used to detect electrostatic potential generated by OmpF porin:
AFM was used to reveal structural details of the membrane protein surface and also
the electrostatic potentials generated by the protein. At low electrolyte concentrations
when the charged probes were used, it provided structural imaging of the surface of
membrane protein and also allowed mapping of electrostatic potential of the OmpF
porin. The obtained results corresponded with the electrostatic calculations based on
the atomic OmpF porin in a lipid bilayer at the same concentrations. This method
opens the door to the electrostatic potential of the native protein surfaces with better
resolutions.[5]
AFM used for analyzing the reaction of endothelial cells to histamine treatment:
Atomic force microscopy was used for the investigation of the cellular response
generated to histamine, which is one of the key inflammatory mediators that causes
endothelial hyperpermeability and vascular leakage. The probes used were labeled
with fibronectin and used for measuring the binding strength between 5ß1 integrin
and fibronectin for the quantification of the force needed to break down single
firbonectin-integrin bonds. The cytoskeletal changes, adhesion force and binding
probability on endothelial cells were monitored before and after histamine treatment.
The AFM was used to record changes on live endothelial cells. Cell topography
measurements revealed that histamine provokes cell shrinkage, stiffness and
increases binding probability. To measure stiffness of cell surface, AFM was used in
force mode to measure force adhesion between the AFM tip and the cell surface.[4]
Figure 1.5.7 Contact mode images of endothelial cells before and after histamine
treatment.
Source: http://www.biophysj.org/cgi/content/abstract/89/4/2888
The upper row shows deflection images and the lower images show contrast height
images in contact mode of AFM with the arrows showing cell shrinkage after
histamine treatment. Panels A and B in the graph represent two topographical
profiles.[4]
AFM used to create Nanoscopic Collagen Matrices:
AFM is used to assemble collagen molecules into well-defined 2-dimensional

templates, which may prove to be platforms on non-biological surfaces to direct
molecular and cellular processes. The collagen matrices formed, maintained their
mechanical stability for several months providing more information on the physical
mechanisms through which the biological structures are organized by cells.
The monolayers may be used for directing molecular or the cellular processes.
Nanostructuring the monolayers may offer a mechanism to orient the monolayers to
specific cellular features. The monolayers could also be constructive for storing
information or microarray printing in molecular electronic circuits.[7]
Figure 1.5.8 the AFM mode in contact mode or tapping mode at 100pN is shown.
The applied force was increased to 200pN to adjust collagen fibers as needed. Then
the sample was re-imaged at a force of 100pN
Source: www.doi.wiley.com/10.1002/jemt.20101
Localization of Lipopolysaccharide(LPS) binding-protein by phospholipids

membranes using AFM:
To study the function of the LPS-binding protein in activating immunocompetent cells,
lipid liposomes were adsorbed on mica, and AFM was used to identify the lateral
organization of LBP (LPS-binding protein) in these membranes and its interaction
with LPS aggregates. Cantilever tips were loaded with anti-LBP antibodies.
Membranes were localized with single LBP molecules at low concentration. At high
concentrations, cluster formation of many LBP molecules formed cross-linking of lipid
bilayers. LPS was added to the LBP liposomes. This addition gave rise to LPS
domains, which might be inhibited by anti-LBP antibodies. Thus LBP proves to be
facilitating fusion of lipid membranes and LPS aggregates.[9]
Figure 1.5.9 AFM images of LBP, PS bilayers, and LBP-containing PS bilayers.
(A).Single LBP molecules on mica. Schematic diagram showing the dimensions of a
single LBP molecule.(B). A pure PS bilayer. In a square of 2 _ 2_m, most of the lipid
molecules were scratched off of the mica by the cantilever. (C), image of a PS bilayer
on mica after LBP was added and was adsorbed to the membrane. Schematic
diagram shows single LBP molecules bound to the surface of the membrane.
(D), image of preincubated (PS_LBP) liposomes adsorbed on mica. The schematic
diagram indicates that small domains of LBP molecules were formed [9]
Source: www.biophysj.org/cgi/content/abstract/biophysj.104.057026v1
1.5.5 Relevant Websites
• Agilent Technologies
www.agilent.com/about/newsroom/presrel/2005/29nov-ep05114.html
• IBM Research Press Resources: Atomic force microscopy.

http://domino.watson.ibm.com/comm/pr.nsf/pages/rsc.afm.html
• Laboratory of Biophysics and Surface Analysis

http://pharm6.pharm.nottingham.ac.uk/resource/resource_afm.htm
• Worcester Polytechnic Institute. Nanotechnology Resources

http://www.wpi.edu/Academics/Depts/Physics/AFM/resources.html
• AFM and SPM Laboratories

http://www.mwrn.com/directories/laboratories/afm.aspx
http://www.mwrn.com/microscopy/nano/scanning_microscope.aspx
1.5.6 Key Industry Suppliers of AFM
The list of the key industry suppliers of Atomic Force Microscope’s are as below:[10]
• Asylum Research
www.asylumresearch.com
• BioForce Nanosciences
www.bioforcenano.com
• EXFO Burleigh
www.exfo.com/en/burleigh.asp
• JEOL USA
www.jeol.com
• JPK Instruments
www.jpk.com
• MikroMasch
www.spmtips.com
• Molecular Imaging
www.molec.com
• Nanofactory Instruments
www.nanofactory.com
• Nanoink
www.nanoink.net
• Nanonics Imaging
www.nanonics.co.il
• Nanosurf
www.nanosurf.com
• Nanoworld
www.nanoworld.com
• Novascan Technologies
www.novascan.co
• NT-MDT
www.ntmdt.ru
• Olympus
www.olympus.co.jp/en/insg/probe
• Omicron NanoTechnology
www.omicron.de
• Pacific Nanotechnology
www.pacificnanotech.com
• Photometrics
www.photometrics.net
• Quesant Instrument
www.quesant.com
• RHK Technology
www.rhk-tech.com
• Shimadzu
www.shimadzu.com
• Surface Imaging Systems
www.sis-gmbh.com
• Triple-O Microscopy
www.triple-o.de
• Veeco Instruments
www.veeco.com
REFERENCES:
1. Silva, LP. (2002) Atomic Force Microscopy and Proteins, Protein and Peptide
Letters, Vol.9, No.2, pp.117-125. Bentham Science Publishers Ltd.
2. Zhu, J.Y. (1998) Applications of MAC Mode AFM in Biology, Pharmaceutical

and Other Bio-Related Industries, Application Notes, Molecular Imaging
Corporation
3. Dufrene, Y.F. (October 2002) Atomic Force Microscopy, a Powerful Tool in

Microbiology, Journal of Bacteriology, p.5205-5213, Vol. 184, No.19.
American Society for Microbiology.
4. Trache, A., Trzeciakowski, J.P., Gardiner, L., Sun, Z., Muthuchamy, M., Guo,
M., Yuan, S.Y., Meininger, G. A. (2005) Histamine Effects on Endothelial Cell
Fibronectin Interaction Studied by Atomic Force Microscopy, Biophysical
Journal 89:2888-2898. The Biophysical Society.
5. Philippsen, A., Im, W., Engel, A., Schirmer, T., Roux, B., Muller, D,J.(March
2002) Imaging the electrostatic potential of transmembrane channels: atomic
probe microscopy of OmpF porin. Biophysical Journal 82(3): 1667-1676
6. Lal, R., John, S.A.(1994) Biological Applications of atomic force microscopy.

AJP- Cell Physiology, Vol 266, Issue 1 C1-21, American Physiological
Society.
7. Jiang, F., Khairy, K., Poole, K., Howard, J., Muller, D.J. (2004) Creating
Nanoscopic Collagen Matrices Using Atomic Force Microscopy. Microscopy
Research and Technique 64:435-440
8. Karrasch, S., Hegerl, R., Hoh, J.H., Baumeister, W., Engel A. (1994) Atomic
force microscopy produces faithful high-resolution images of protein surfaces
in an aqueous environment. Proc Nat1 Acad Sci USA. 91(3): 836-838.
9. Roes, S., Mumm, F., Seydel, U., Gutsmann, T., (2006) Localization of the
Lipopolysaccharide-binding Protein in Phospholipid Membranes by Atomic
Force Microscopy. The Journal of Biological Chemistry Vol.281, No.5,
pp.2757-2763. The American Society for Biochemistry and Molecular Biology,
USA.
10. Wright-Smith, C., Smith, C.M.(2001) Atomic Force Microscopy. The Scientist
15.2:p23.
11. Agilent Technologies. (1998-2006), What is AFM?

http://www.molec.com/what_is_afm.html#
12. Smith, A. (May 1999), Atomic Force Microscopy, Microbiology Today

http://www.socgenmicrobiol.org.uk/pubs/micro_today/pdf/smith.pdf
13. Li, H-Q.(1997) General Ideas About AFM

http://www.chembio.uoguelph.ca/educmat/chm729/afm/general.htm
14. Atomic Force Microscopy
http://www.vpd.ms.northwestern.edu/teaching/AFM_MSc_190_lab.pdf
15. Muller, D.J., Aebi, U., Ángel, A. Imaging, measuring and manipulating native
biomolecular systems with the atomic force microscope
www.mih.unibas.ch/Booklet/Booklet96/Chapter3/Chapter3.html
16. Infinitesima announces compatibility with the MultiMode™ Scanning Probe

Microscope from Veeco Corp, March 10th 2006 (Press Release), Oxford, UK
www.infinitesima.com/html/news.html
Chapter 1.6 Electron Microscopy of Proteins
Priyadharshini Sivakumaran
1.6.1 Introduction:
With the relative ease of operation of present day instruments, electron microscopy
has become very popular in the investigation of biologic structures. In many cases,
one can obtain images that are fairly faithful records of the detail in the specimen
within only a few minutes. The two main draw backs of the method are (1) the
relative transparency of proteins to electrons and (2) the disruption of protein
structure as the specimen dry out in the evacuated microscope and are bombarded
by the electron beam. The usual method of increasing the contrast is by adding
heavy metals in one way or another. The simplest way of doing this, the method of
negative staining, is fortunately also the most successful in preserving specimen
order. With the effective resolution limited to 20 Å in electron micrographs of proteins,
little, even in terms of the shape of the molecule, can be deduced from images of
average–sized, isolated monomers. Electron microscopy has therefore been most
successful in the determination of the quaternary structure of assemblies of protein
molecules.
The Transmission Electron Microscope (TEM) is the only instrument that allows the
analysis of biological samples on different scales, ranging from µm down to
Angstrom resolution. Transmission electron microscopy of sections of cells or the
recently developed method of electron tomography allows whole cells or organelles
within cells to be studied. On the other end, electron crystallography employs the
TEM to resolve the atomic details of proteins that are arranged in two-dimensional
crystals.
Most modern transmission electron microscopes can routinely reach a resolution

better than 2Å, at which atoms can be visualized directly. This, however, requires the
sample to be sufficiently resistant to the electron beam and adequately prepared.
Biological samples are usually highly sensitive to the electron beam. High resolution
data of proteins for example can only be recorded at electron doses below 5
electrons/ Å2 with a sample kept at liquid nitrogen temperature, or at doses below 20
electrons/ Å2, when the sample is kept at liquid helium temperature. To record
images with a controlled number of electrons admitted onto the sample, the TEM has
to be operated in a so-called “Low-Dose” mode.
Figure 1.6.1
The electron microscope (EM) focuses on the structure determination of complex

macro-molecular assemblies. Small proteins are studied by means of a technique
known as electron crystallography. Such proteins are purified and two-dimensionally
crystallized, before EM and data processing steps are performed. Larger complexes
are mainly studied by non-crystallographic methods. These proteins are (partially)
purified and their single particle EM images are processed. For these investigations
cryo-electron microscopy is used: objects are embedded in a thin amorphous layer of
ice, which preserves their ultra-structure.
The scanning electron microscopy can be used for mapping the orientation and
organization of protein film adsorbed onto various surfaces at the nano scale. In this
study, the scanning force and electron microscopic visualization of single molecules
of fibronectin either frozen hydrated or adsorbed onto metallic and polymeric surfaces
with different solid surface tensions were presented. The surfaces were characterized
by dynamic contact angle measurements, X-ray photo emission spectroscopy and
scanning force microscopy. The proteins were prepared by fast protein liquid
chromatography (FPLC) and characterized by gel electrophoresis. Protein films on
surfaces were investigated by surface plasmon resonance spectroscopy and directly
imaged by scanning force microscopy. The spreading of the adsorbed fibronectin
revealed dependence on the chemical composition and the solid surface tension.
Structure of fibronectin in solution as well as on solid interface appeared as an
extended straight strand as obtained by imaging with electron and scanning probe
microscopes. Frictional forces during the scan have been of significant contribution in
the imaging mechanism.
1.6.2 Recent Advances:
In recent years, digital image processing has evolved to the point where it is now
possible to more fully exploit the high resolution potential of the transmission electron
microscope (TEM). A system has been developed for semi-automatic specimen
selection and data acquisition for protein electron crystallography, based on a slow-
scan CCD camera connected to a transmission electron microscope and control from
an external computer. The slow-scan CCD camera has been shown to be a valuable
accessory to an electron microscope for direct data acquisition as used in on-line
electron optical adjustments and electron tomographic applications. Use of a slow-
scan CCD camera for the acquisition of diffraction data in an electron crystallographic
application would allow for a fast evaluation and an immediate, subsequent
numerical analysis of the data, contrary to the imaging plate, which too has been
used for the acquisition of electron diffraction intensities. Furthermore, the quality of
the acquired data is higher, since this camera performs better than the photographic
film in terms of linearity, background noise and dynamic range. Its applicability to
protein electron crystallography at 400 kV has resulted from a number of engineering
changes, which were made to the slow-scan CCD camera, such as minimization of
the spurious X-ray signals picked up by the camera.
Areas of interest on the specimen are localised at low magnification and

subsequently imaged on the CCD camera, using a dose which is small compared to
the dose used in the exposure mode. The crystalline quality of the area is evaluated
from the appearance of diffraction peaks in the calculated image Fourier transform. If
the quality is considered good, images can then be recorded in different modes, both
on film and using the CCD camera. Using this system a significant gain, both
quantitatively and qualitatively, can be obtained in acquiring data for electron
crystallography of beam-sensitive materials.
Figure 1.6.2 CCD Camera
Among many scanning probe microscopes, atomic force microscopy (AFM) is a
useful technique to analyse the structure of biological materials because of its
applicability to non-conductors in physiological conditions with high resolution.
However, the resolution has been limited to an inherent property of the technique. To
overcome the problem, a carbon nanotube probe was developed by attaching a
carbon nanotube to a conventional scanning probe under a well-controlled process.
Because of the constant and small radius of the tip (2.5–10 nm) and the high aspect
ratio (1: 100) of the carbon nanotube, the lateral resolution has been much improved.
The carbon nanotube probes also possessed a higher durability than the
conventional probes. These carbon nanotube probes, with high vertical resolution,
enabled to clearly visualize the subunit organization of multi-subunit proteins and to
propose structural models for proliferating cellular proteins. This success in the
application of carbon nanotube probes provides the current AFM technology with an
additional power for the analyses of the detailed structure of biological materials and
the relationship between the structure and function of proteins.
1.6.3 Evaluation of the technology:
Because of the differences in the way TEM and SEM work, each has its own distinct
advantages. With TEM, for instance, it is able to view a sample at a magnification
approximately 10 times that of an SEM (objects as small as three to 10 angstroms for
TEM). Also, because of its ability to transmit through samples, it can not only
characterize particle surfaces, but it can also reveal the sample’s internal structure.
One advantage of SEM is that it provides a better overall visual image of the sample.
This is because as it scans over a sample line by line, it gives the image a depth of
field, almost making the object three-dimensional. In a TEM image, no depth of field
can be seen on the image. Another advantage of SEM is that it is more flexible in the
type of samples it can view because the sample does not need to be nearly as thin
as with TEM. Therefore, SEM can analyse samples such as larger wear debris
particles and distressed machine surfaces.
The advantage of colloidal gold labelling is that the intracellular complexes may be
more precisely located because of the significant improvement in resolution provided
by backscatter electron (BSE) imaging in the SEM. BSE imaging confirmed the
presence and subsarcolemma localization of myoglobin in cardiomyocytes directly
isolated from fresh biopsies.
Electron microscopes are expensive to buy and maintain. As they are sensitive to
vibration and external magnetic fields, suitable facilities are required to house
microscopes aimed at achieving high resolutions. The samples have to be viewed in
vacuum, as the molecules that make up air would scatter the electrons. Recent
advances have allowed hydrated samples to be imaged using an environmental
scanning electron microscope.
Scanning electron microscopes usually image conductive or semi-conductive
materials best. The samples have to be prepared in many ways to give proper detail,
which may result in artefacts purely the result of treatment. This gives the problem of
distinguishing artefacts from material, particularly in biological samples. Scientists
maintain that the results from various preparation techniques have been compared,
and as there is no reason that they should all produce similar artefacts, it is therefore
reasonable to believe that electron microscopy features correlate with living cells.
1.6.4 Application of the technology:
Electron microscopic techniques have been used in studying the structure of many of
the proteins. Some of them are discussed below.
RESPIRATORY CHAIN SUPER COMPLEXES IN MITOCHONDRIAL MEMBRANES
The structure of the individual membrane-bound protein components have been well
characterized, especially by X-ray diffraction studies. During the past years
increasing evidence that the respiratory chain components are organized into
supercomplexes, particularly by the application of Blue-native polyacrylamide gel
electrophoresis. Single particle electron microscopy is used for a structural
characterization of some of the respiratory chain supercomplexes. The study
revealed that the supercomplex composed of monomeric Complex I and dimeric
Complex III from Arabidopsis and more recently the dimeric complex of ATP
synthase. The ATP synthase is substantially kinked in its membrane-embedded
domains and this allows also for the first time to define a functional role for these
supercomplexes. The dimeric ATP synthase complex appears to be responsible for
the folding of the inner mitochondrial membrane.
Figure 1.6.4 ATP synthase
In order to determine the nature of the regulations, structural elucidations of actin

filament-end-binding protein complexes are crucially important. A new procedure has
been developed on the basis of single-particle analysis to determine the structure of
the end of actin filaments from electron micrographs. In these procedures, the
polarity of the actin filament image, as well as the azimuth orientation and the axial
position of each actin protomer within a short stretch near the filament end, was
determined accurately. This improved both the stability and accuracy of the structural
determination dramatically.
Agrin is a large, multidomain heparan sulfate proteoglycan that is associated with

basement membranes of several tissues. Particular splice variants of agrin are
essential for the formation of synaptic structures at the neuromuscular junction.
Electron microscopy was used to determine the structure of agrin and to localize its
binding site in laminin-1. Agrin appears as an approximately 95 nm long particle that
consists of a globular, N-terminal laminin-binding domain, a central rod
predominantly formed by the follistatin-like domains and three globular, C-terminal
laminin G-like domains.
A novel ways to characterise all the larger (membrane) protein complexes from a
specific type of membrane by transmission electron microscopy (EM) in combination
with proteomics was developed. In general, structural studies on proteins are heavily
based on isolated, highly purified protein samples. For X-ray diffraction studies this is
a necessity and for EM, based on crystals, the same is true. Single particle EM is a
well-developed technique to average the individual projections of large protein
complexes. It makes use of sophisticated classification programs to sort out different
projections. It is found that this is also possible in complex mixtures of proteins. In
this way, the projection structures of all larger proteins from disrupted membranes
can be analysed. Proteomics methods can be used to assign the EM projection
structures to specific proteins. This will be achieved by comparing the frequencies of
EM structures with intensities of electrophoresis spots arising from proteins of
complete membranes. As a study object the photosynthetic membrane from
cyanobacteria and the peroxisome membrane from yeast were selected. It is
expected that by combining EM and proteomics major new discoveries can be
achieved because it is a fully novel approach.
1.6.5 Key industry suppliers:
Gatan, Inc. is the world's leading manufacturer of instrumentation and software

used to enhance and extend the operation and performance of electron
microscopes. The Gatan name is recognized and respected throughout the
worldwide scientific community and has been synonymous with high quality
products and the industry's leading technology.
Gatan digital cameras provide the ultimate digital capture and viewing system for the
electron microscopist of today. There are three families in the Gatan range: ES500W
Erlangshen™, the most versatile CCD camera to record TEM images, MultiScan™,
the industry standard TEM digital camera and the UltraScan™ family, the highest
performance imaging system available today. The ES500W is capable of high speed
and high quality imaging with a field of view larger than traditional TEM film. These
advanced features allow the user to image intense electron diffraction patterns
without the “blooming artifact” and observing dynamic events inside a TEM.
'GRACE' is a system for semi-automated specimen selection and data acquisition
using a Gatan slow-scan camera in combination with a Philips CMxx electron
microscope. It was designed for use with specimens of 2-dimensional (2D) protein
crystals, but can also be used for 'single particle' specimens.
With 2D protein crystals it allows the user to mark at low magnification areas in the
specimen that may contain crystalline protein domains or other interesting features,
evaluate the (crystalline) quality of the domains by recording a very low dose image
(at intermediate magnification) in the so called 'Preview Mode' and calculating and
displaying the FFT. If an area is judged 'good' by the operator, a high resolution (low-
dose) image can be recorded on the CCD camera or on film (or both) in the
Exposure Mode. In the case of non-periodic specimens the very-low-dose preview
image can be used itself for selection instead of the FFT. The system can also be
used to just collect images from the positions marked at low magnification, without
any preview.
1.6.6 References:
1. Baldwin, J. & Henderson, R. (1984). Measurement and evaluation of electron
diffraction patterns from two-dimensional crystals. Ultramicroscopy 14, 319-
336.
2. Blundell, T. L. & Johnson, L. N. (1976). Protein Crystallography. New York,

Academic Press.
3. Booy, F. P. & Pawley, J. B. (1993). Cryo-crinkling: what happens to carbon

films on copper grids at low temperature. Ultramicroscopy 48, 273-280
4. Chiu, W. & Jeng, T.-W. (1980). Electron diffraction study of crotoxin complex
at 1.6 Å. In: Electron Microscopy at Molecular Dimensions. State of the Art
and Strategies for the Future. Berlin, Springer-Verlag. 137-142.
5.
Frederic Zenhausern, Marc Adrian and P. Descouts. Solution Structure and
Direct Imaging of Fibronectin Adsorption to Solid Surfaces by Scanning Force
Microscopy and Cryo-electron Microscopy. J. Electron Microscopy 42(6):
378-388 (1993)
6. Ken I. Hohmura, Yutakatti Itokazu, Shige H. Yoshimura, Gaku Mizuguchi, Yu-

suke Masamura, Kunio Takeyasu, Yasushi Shiomi, Toshiki Tsurimoto,
Hidehiro Nishijima, Seiji Akita and Yoshikazu Nakayama2 Atomic force
microscopy with carbon nanotube probe resolves the subunit organization of
protein complexes. J. Electron Microscopy 49(3): 415-421 (2000).
7. Narita A, Maeda Y. Molecular determination by electron microscopy of the

actin filament end structure. J Mol bio. (2006)
8. Denzer AJ, Schulthess T, Fauser C, Schumacher B, Kammerer RA, Engel J,

Ruegg MA. Electron microscopic structure of agrin and mapping of its binding
site in laminin-1. EMBO J. 15; 17(2):335-43. (1998).
9. Electron microscopy of proteins by James R. Harris. London ; New York

Academic Press, 1981-1987
2.2 Size exclusion chromatography
Michelle Hefford
2.2.1 Introduction
Size exclusion chromatography (SEC) is one of a range of chromatographic

techniques. Chromatography is a widely used application and the sale of
chromatographic instruments represents more than half of the total sale of analytical
equipment around the world. [1].
Size exclusion chromatography may also be referred to as Gel Filtration

Chromatography (when using an aqueous mobile phase) or Gel Permeation
Chromatography (when using an organic mobile phase). The term Size exclusion
chromatography covers both these forms and is more widely used and accepted. [2].
All other methods of chromatography involve the sample binding to the support, but
size exclusion chromatography separates different molecules based on their
molecular weight. In size exclusion chromatography, larger molecules are eluted
faster from the column than smaller molecules, giving rise to the name ‘size
exclusion’.
Underlying Principles
The process of size exclusion chromatography is similar to that of other column

chromatography methods, such as affinity chromatography. The sample is injected
into the column via a mobile phase and travels through the column packing or gel
matrix. The column packing consists of permeable pores of various sizes. Depending
on the size of the molecules in the sample, they will permeate the pores. The larger
molecules will not be able to permeate the pores and will elute through the column
faster than the smaller molecules.
As the sample is eluted, one or more detectors are used to measure specific
properties of the mobile phase/sample. This information is displayed via a
chromatogram. [1], [3]
Figure 1.1 Principle of size exclusion chromatography

Source: academic.sun.ac.za/polymer/gpectref.htm
The stationary phase of size exclusion chromatography consists of a column packed

with a matrix (gel) which has pores of different sizes. Columns can be packed with a
gel that will have pores of a set size range depending on the size of the different
molecules in the sample solution. The column packing should be chosen so that the
largest molecule will not permeate through any of the different pore sizes. These
molecules will elute first form the column. The smallest pores in the matrix will only
allow the smallest molecules to permeate through. Therefore the smallest molecules
will spend the most time permeating through the matrix and elute last. [4] [5]
It is important to select the right type of column as the matrix should be inert in
relation to the sample. If the sample reacts with the matrix, this will mean that the
molecules will not be eluting due to their size and this will affect the end results. If the
molecules bind to the matrix, (for example via an ion-exchange interaction), it will
take longer for the molecule to elute from the column, which would indicate that the
molecule is smaller than its actual size. Conversely, if the molecule was repelled by
the matrix, it may be prevented from permeating through the pores (even though it is
small enough to do so). This would mean that the molecule would elute faster than
usual and would be detected as a larger molecule. [5] [6].
The length of the column and the sample size will also affect the resolution of the
results. If the column is longer, it allows more time for better separation of the
‘medium sized’ molecules, which gives better resolution on the chromatogram.
Sometimes a series of columns is needed to get adequate results, but this increases
the time required to run the process. [6]
Prepacked columns are commercially available with different volumes and mediums.
The table below shows some typical examples of medium used in columns for size
exclusion chromatography.
Table 1.1 Properties of typical commercial column packings for size exclusion
chromatography. Source: www.cem.msu.edu/~cem333/week16.pdf
The sample is dissolved in the mobile phase. As the mobile phase elutes through the
column, the molecules within the sample will either permeate through the pores (if
they are able to fit) or travel down the column via the mobile phase solution.
Careful selection of the mobile phase is also very important.
A good mobile phase should have the following properties:

• Dissolve the sample
• Low viscosity
• Not cause the stationary phase to interact with the sample. If anything, the
mobile phase should prevent this.
• Must be compatible with the detector.
Usually more than one type of detector is used with size exclusion chromatography.
The most common detectors used in size exclusion chromatography are UV,
fluorescence, refractive index and evaporative light-scattering detectors (ELSDs).
Size exclusion chromatography systems can also be linked to a Mass Spectrometer
for higher selectivity, but this is quite expensive. [5] [1]
UV detectors can be fixed-wavelength or dual-wavelength. The measurement of

absorbance by the fixed wavelength detector is based on the difference in intensity
between the sample and a reference standard. The dual-wavelength detector
measures the difference in absorbance of two wavelengths of light that are passed
through the sample.
Fluorescence detectors measure the signal emitted by molecules after absorbing

light at a specific wavelength. If the sample does not fluoresce naturally, tags or dyes
can be used to enable detection. The fluorescence signal is linearly proportional to
the sample concentration.
Refractive index detectors measure changes in the refractive index of the sample in
contrast to the mobile phase. This method of detection is sensitive to temperature
changes, and the solvent must remain the same throughout the process.
For use of ELSDs, the liquid sample is converted into a fine spray and then
evaporated to remove any solvent. The remaining sample molecules are then
subjected to a light source and the light scattered by the molecules is detected by a
photodiode. The amount of light scattered is relative to the mass of the sample.
ELSD is commonly used for samples that do not respond well to UV detection.
The table below shows the properties of the chromatography detectors outlined.
RI UV/VIS Fluor MS
Response Universal Selective Selective Selective
Sensitivity 4 microgram 5 nanogram 3 picogram 1 picogram
Linear Range 10 10 10 10
Flow Sensitive Yes No No Yes
Temp. Sensitive Yes No No No
Table 1.2 Properties of SEC detectors.
Source: http://www.waters.com/WatersDivision/ContentD.asp?watersit=JDRS-
5LTGBH#detectors
A chromatogram is a visual presentation of the detection of molecules within the

sample. A high resolution chromatogram shows well defined peaks with a good
baseline. Longer or multiple columns will give better resolution chromatograms, as
the molecules have longer to separate from other molecules of a different size. The
figure below illustrates high and low quality resolution chromatograms.
Figure 1.2 Chromatograms with high resolution and low resolution peaks
Source: [6] Amersham Biosciences, Gel Filtration Principles and Methods
The largest molecules elute in the void volume (vo). These molecules are too large to
permeate the stationary phase. The smallest molecules that permeate the most
pores in the stationary phase are eluted at the total column volume (vt). Molecules of
intermediate molecular weight are eluted at various times depending on their size. Ve
is the elution volume of each molecule.
As shown in the illustration below, the void volume is the volume of the mobile phase,
the total column volume is the volume of the mobile phase plus the stationary phase,
while the volume of the stationary phase is determined as Vt-Vo. [6] [7]
Figure 1.3 Illustration of the different volumes in size exclusion chromatography.

The partition coefficient Kav value can be determined by the equation:
Kav = (Ve-Vo)/(Vt-Vo)
There is a relationship between the Kav value and the logarithm of the molecular
weight of similar molecules.
Selectivity curves are created for stationary phase matrices by plotting the Kav value
of a set of standard proteins against the log of their molecular weight.
Figure 1.4 Selectivity curves of some commercially available gel filtration medium.
Prepacked columns can be purchased and the matrix should be selected carefully so
that the predicted molecular weight of the sample falls within the linear part of the
calibration curve. If the target sample does not have the same molecular shape as
the calibration standards, the result may deviate from the calibration curve. [6] [7]
One of the biggest drawbacks of using SEC is that it can take hours to get results.
Recently there has been a growth in interest in Fast size exclusion chromatography.
Initial studies into Fast-SEC techniques have suggested that reducing the process
time also reduces the resolution to an unacceptable level. Popovici and
Schoenmakers investigated different ways of increasing the speed of size exclusion
chromatography. [8]
The following approaches where considered to reduce the time required:

• Decrease the particle diameter of the stationary phase
• Reduce the column length
• Increase the flow rate
An experiment was performed using columns of different lengths: 50mm x 4.6 i.d.
(internal diameter), 100mm x 4.6mm i.d., 150mm x 4.6mm i.d. A sample with
standards of known molecular weight (1700, 10,900, 117,000, and 1,260,000 Da)
was run through the columns at the flow rates 0.3, 0.6 and 0.9ml/min respectively.
The results are shown in the chromatograms below.
Figure 1.5 Results from various Fast-SEC columns. Source: [8] Fast size exclusion
chromatography – Theoretical and practical considerations.
It was concluded that better resolution can be obtained via increase flow rate and
column length. [8]. Improvements are continually being made by experimenting with
different types of columns and media in order to increase the process time while not
reducing the quality of chromatograms produced.
Large suppliers of SEC column media have developed specific stationary phases
that will maximise the efficiency of the procedure.
2.2.3 Evaluation of the technology
There are many forms of chromatography and a combination of the methods is

usually used in protein preparation and analysis.
Amersham Biosciences describes a Three Phase Strategy for protein purification:

Capture, Intermediate Purification, Final Polishing (CIPP). The right combination of
methods can ensure that the entire process is less costly and time consuming, while
providing pure product. [7]
The aim of the Capture step is to isolate, concentrate and stabilise the protein. Size
exclusion chromatography would not be recommended for this process, as it usually
involves a high volume of sample and would be very time consuming. Ion Exchange
Chromatography (IEX) and Affinity Chromatography are commonly used for this
process as they are faster, higher capacity techniques than size exclusion
chromatography. Column conditions are selected to maximise the binding of the
target protein from the sample and avoid the binding of unwanted contaminants.
Speed is an important issue in this first stage, as the sample often includes impurities
that may cause proteolysis of the target protein, or other denaturing effects.
During the Intermediate Purification, the chosen technique will need to have a high
selectivity for the target protein as the other unwanted components remaining in the
sample after the initial capture stage will be more similar to the target. One or
methods may be used for this process. For each method used a balance must be
found to provide adequate capacity at an acceptable resolution.
The final polishing stage is used to achieve a high purity of the target protein. Size
exclusion chromatography is usually used at this stage as it provides high resolution
results. The volume of the sample is reduced by the first 2 stages, so the process
time of SEC is decreased. [7]
One of the disadvantages of size exclusion chromatography is that it can be slow

process. It can take anywhere from 15 minutes to several hours to perform, with the
typical run time at approximately 30 minutes. [5].
The resolution of the results can be increased by adding more columns, and a series
of columns with different pore sizes is often used. However, this means that the
process run time will increase.
Each different type of chromatography has its advantages and disadvantages. They
can be used in combination, and the materials or procedures altered to gain the best
result in relation to resolution, speed, recovery and capacity. The table below lists the
different protein properties and the techniques that use those properties during
purification.
Figure 1.6 All chromatographic techniques can be optimised to achieve a balance

between resolution, speed, capacity and recovery. Source: [7] Amersham
Biosciences, Protein Purification Handbook
2.2.4 Applications of the technology
The primary application for size exclusion chromatography was for the determination
of molecular weight or molecular weight distribution of polymers. It is now used for a
variety of purposes, including group separation (for example desalting, buffer
exchange), separating monomers from dimers and polymers, determining molecular
weight, final ‘polishing’ purification, and facilitate the refolding of proteins. [6] [5]
Gel filtration is ideal for the cleanup of proteins before purification. Commercially
prepared desalting columns remove the protein from salts and other contaminants of
low molecular weight and can transfer the protein to a new buffer, all in a single
process. Sample volumes can be up to 40% of the column volume.
Table 1.3 Commercially available prepacked columns for sample cleanup
Source: [7] Amersham Biosciences, Protein Purification Handbook
The capacity and speed of this procedure makes it efficient to prepare the sample via
desalting and buffer exchange, in readiness for further purification. The figure below
shows a chromatogram of the desalting of a His6 fusion protein. Using a UV and
conductivity detector facilitates optimisation of the separation. [6].
Figure 1.7 Desalting a His6 protein via size exclusion chromatography

Source: Source: [6] Amersham Biosciences, Gel Filtration Principles and Methods
Size exclusion chromatography is used for profiling protein samples and can be used
for relatively small volumes of sample.
Parini et al used an automated size exclusion chromatography system to analyse
triglyceride and cholesterol content in lipoproteins. [9]
Traditionally lipoprotein separation was done via ultra-centrifugation separation,
which requires large sample volumes.
Samples of 1-10µL were injected into the column and travelled at a flow rate of 40 µL
min-1. The absorption was measure by a UV-VIS detector at 500nm. The run time for
cholesterol analysis was 75 minutes, while the run time for triglyceride analysis was
90 minutes. The chromatogram below shows the cholesterol profiles generated by
running SEC on 5 different sample volumes. The inset shows the lipid content
calculated as a percentage of the peak area. The proportion of VLDL, LDL and HDL
cholesterol remained the same as the sample volume varied.
Figure 1.8 Cholesterol profiles at varied sample volumes. Source: [9] Parini et al,
Lipoprotein profiles in plasma and interstitial fluid analysed with an automated gel
filtration system.
Due to the difficulty in being able to analyse low levels of lipoproteins in small
volumes, the analysis of lipoproteins in Interstitial Fluid has not been widely studied.
Using SEC, Parini et al evaluated the levels of lipoproteins in IF and plasma. The
resulting chromatograms shown below illustrate the levels of cholesterol in plasma
versus IF in 5 healthy volunteers.
Figure 1.9 Cholesterol levels in IF and plasma of 5 healthy individuals. Source: [9]
Parini et al, Lipoprotein profiles in plasma and interstitial fluid analysed with an
automated gel filtration system.
The researchers also applied their method to determining the profile of triglyceride
lipoproteins. Again, injections of increasing volume from 1 to 10µL were run through
the SEC system and the following chromatogram was produced. Once again, the
proportion of the VLDL, LDL and HDL triglyceride remained constant.
Figure 1.10 Triglyceride profiles at varied sample volumes. Source: [9] Parini et al,
Lipoprotein profiles in plasma and interstitial fluid analysed with an automated gel
filtration system.
The fourth peak shown was thought to be free glycerol in plasma. To test this
hypothesis, a reference sample of free glycerol was injected into the SEC system,
along with a plasma sample from a healthy volunteer and from a patient with
hyperglycerolaemia. As is shown in the chromatogram below, the hyperglycerolaemic
patient had a high fourth peak, and this was eluted at the same time as the free
glycerol.
Figure 1.11 Triglyceride profiles of a hyperglycerolaemic patient compared to those

of a healthy subject. Source: [9] Parini et al, Lipoprotein profiles in plasma and
interstitial fluid analysed with an automated gel filtration system.
The SEC method developed by Parini et al has the following significant

improvements on existing methods of lipoprotein analysis.:
• It is able to generate lipoprotein triglyceride profiles
• It is able to determine lipoprotein profiles in Interstitial Fluid
• Components for the system, including reagents are already available.
Analysis of lipoproteins would be of value for the study of some metabolic diseases
such as hyperglycerolaemia and diabetes. [9]
Recently size exclusion chromatography has been used to refold denatured proteins.
Neely et al successfully used SEC for the refolding of the active calcium channel β1b
subunit. [10] Studies of this subunit have been difficult due to its tendency to form
aggregates when expressed in bacteria. Dialysis and fast dilution were initially used
to try and refold the protein via denaturant removal but these techniques failed.
The SEC method was developed which exchanges the buffer, removes aggregates
and promotes refolding of the protein all in the one process. The sample was loaded
onto a column that was calibrated with the refolding buffer and eluted at a rate of
2.5ml/min.
A UV detector was used, and the eluted fractions were further analysed by reducing
SDS-PAGE. The figure below shows the peak at the void volume which was thought
to be aggregates of the subunit. The peaks I and II are thought to have been the β1b
subunit refolded into two different states.
Figure 1.12 Protein refolding of β1b subunit. Source: [10] Neely et al, Folding of active
calcium channel β1b subunit by size exclusion chromatography and its role on
channel function.
The molecules from Peak I were recovered and loaded onto the column again. The
resulting chromatogram showed a high peak again at the void volume, indicating that
the protein had aggregated over time.
The molecules from Peak II were recovered and loaded onto the column again. The
resulting chromatogram (shown below) indicates that the protein has refolded
correctly and remains in a stable condition. The integrity of the Peak II conformation
was confirmed by amino acid analysis.
Figure 1.13 Analysis of Peak II subunit. Source: [10] Neely et al, Folding of active
calcium channel β1b subunit by size exclusion chromatography and its role on
channel function.
The recovery of the β1b subunit using this method averaged at 50%, and the protein
is stable at ph 8-10. [10]
2.2.5 Web Sites
• Amersham Biosciences. Their website has information relating to protein

purification and analysis, as well as products available.
http://www4.amershambiosciences.com
• Wiley Interscience database – relevant journal articles.

http://www3.interscience.wiley.com/cgi-bin/home
• Science Direct database – relevant journal articles.

http://www.sciencedirect.com/
• Access Excellence is a site run by the USA National Health Museum.
http://www.accessexcellence.com/
• Journal of chromatographic science.

http://www.j-chrom-sci.com/index.htm
• Amersham Biosciences - http://www4.amershambiosciences.com

• Sigmaaldrich – http://www.sigmaaldrich.com
• Bio-rad. http://www.bio-rad.com
• Waters - http://www.waters.com
2.2.7 References
1. Rouessae, F. & Rouessae, A. (2000) Chemical Analysis: Modern

Instrumentation Methods and Techniques, John Wiley & Sons, West Sussex,
England.
2. National Health Museum (1999) An Introduction to Chromatography,
http://www.accessexcellence.com/LC/SS/chromatography_background.htm/
3. Hunt, B.J. & Holding, S.R. (1989) Size Exclusion Chromatography, Blackie &
Son Ltd, London
4. Wu, Chi-San (2004) Handbook of Size Exclusion Chromatography and
Related Techniques, 2nd Ed, Marcel Dekker, New York, USA
5. Miller, James M. (2005) Chromatography Concepts and Contrasts, 2nd Ed,
John Wiley & Sons, New Jersey, USA
6. Amersham Biosciences (2002) Gel Filtration Principles and Methods,
http://www5.amershambiosciences.com/aptrix/upp00919.nsf/Content/LabSep
_TechDoc2~handb_pplr
7. Amersham Pharmacia Biotech AB (2001) Protein Purification Handbook,
http://www5.amershambiosciences.com/aptrix/upp00919.nsf/Content/LabSep
_TechDoc2~handb_pplr
8. Parini, P et al (2006) Lipoprotein profiles in plasma and interstitial fluid
analysed with an automated gel-filtration system, European Journal of Clinical
Investigation (2006) 36, 98-104
9. Neely, Alan et al (2004) Folding of Active Calcium Channel β1b-Subunit by
size exclusion chromatography and its role on channel function, Journal of
Biological Chemistry Vol 279, No 21, pp21689-21694
10. Popovici, Simona & Schoenmakers, Peter (2005) Fast size exclusion
chromatography – Theoretical and practical considerations, Journal of
Chromatography A, 1099 (2005), 92-102.
Chapter 2.3 Ion Exchange Chromatography
Ramakrishnan Ramamoorthy
2.3.1 Introduction:
Proteins are considered to be building blocks of all living beings. The word “Protein”
originates from a Greek word Protos which means primary. The meaning indicates
that proteins are an essential component for all living beings. They constitute that
part of the living system without which survival is difficult. . They are molecules which
are formed of a large amino acid groups Proteins are mainly made up of 20 amino
acid groups. They form polymers with a combination of amino acids. [1][2][3] [7]
There are a variety of uses with proteins and hence those applications may utilise a
purified active form of protein in the shortest time possible. The proteins are isolated
from a mixture of proteins or any complex mixture. The basis of purification of
proteins can broadly divided into two major categories.
Proteins Purification
Analytical Purpose Preparative Purpose
The above two purposes for protein purification have a variety of applications in their
own sphere of work.The analytical purpose has an application more in research, to
identify a strand of protein while the preparative method targets the production part,
to produce large proteins for various applications. The biotechnological aspect of
protein is a fast and a rapidly upcoming field. They possess a great diversity in the
drugs and pharmaceutical fields. [4].
The techniques discussed below using the chromatography technique is Ion
Exchange Chromatography. This technique was first adopted for the separation of
proteins by H.A.Sober and E. A.Peterson in the mid 1950’s. [5]
The principle involved in Ion exchange chromatography is utilisation of charge

properties of the molecules for separation. [6]. “The basis for the ion exchange
process is the competitive binding of ions of one kind (proteins) for ions of
another kind, for example other proteins or salt ions of the same charge, to an
oppositely charged chromatographic medium, the ion exchanger.”(Page No
109 Section 4.1)[5] The primary requisite for separation is the matrix within the
column. This is basically a resin which facilitates the separation. Examples of some
resins are agarose, dextran, acryl amide, cellulose etc. (8) there are four steps
adopted in the separations using ion exchange chromatography. They are
a) Equilibration
b) Sample Application
c)Elution
d)Regeneration
The part of the complex mixture which comprises of that portion to be separated is
known as analyte. The analyte reacts with the oppositely charged particles of the
column material..The primary principle applied in the separation of macro molecules
using ion exchange chromatography is isolation based on ionic interaction. [8][6][9a]
Fig 2.3.1 Ion Exchange Chromatography Diagram

Source
:http://images.google.com.au/imgres?imgurl=http://www.chemistry.wustl.edu/~course
s/genchem/Labs/IEcolumn/images/diagram.gif&imgrefurl=http://www.chemistry.wustl.
edu/~courses/genchem/Labs/IEcolumn/diagram.htm&h=410&w=390&sz=43&hl=en&
start=3&tbnid=fx0Pkmqjp46-
oM:&tbnh=125&tbnw=119&prev=/images%3Fq%3Dion%2Bexchange%2Bchromatog
raphy%26svnum%3D10%26hl%3Den%26lr%3D
These further results in subdivisions into:
Ion Exchange Chromatography
Cation Exchange Chromatography Anion Exchange Chromatography

The Cation exchange chromatography uses the positively charged ions as the
column material contains negatively charged particles whereas the anion exchanger
entraps the negatively charged ions using the positively charged ions from the
column material.[9]. Therefore, the binding of a protein in a medium is directly
proportionally to its charge. The proteins are then eluted by either salting out or by
changing the pH [8].
The important factor for any separation using this technique is the selection of resin
and its associated factors. The major factor for resin selection is the overall charge
and the charge distribution of protein. The overall charge is proportional to the pH
and amino acid composition in the protein while the charge distribution varies on how
much is the charge carried by the amino acid. Isoelectric point is also a big
contributor for separation. Generally though, proteins are separated in the pH range
of 4-8, hence a pH range is selected in accordance with the resin and analyte
characteristics for purification or separation [6]
Fig 2.3.2 Ion Exchange Chromatography operation procedure.
Source
http://images.google.com.au/imgres?imgurl=http://www.steve.gb.com/images/scienc
e/ion_exchange_chromatography.png&imgrefurl=http://www.steve.gb.com/science/m
olecular_biology_methods.html&h=283&w=915&sz=15&hl=en&start=32&tbnid=fdEE
amsdanN71M:&tbnh=45&tbnw=147&prev=/images%3Fq%3Dcation%2Bchromatogra
phy%26start%3D18%26ndsp%3D18%26svnum%3D10%26hl%3Den%26lr%3D%26
sa%3
2.3.2: Recent Advancements:

In this period of time, there seems to be a great need and emphasis for cognizance
of new things and new technologies. As required by the society, there are certain
modifications or advancements that are inevitable for better analysis and thesis,
which are updated with more research in a short time period. Hence this section
discusses about the same- the recent advancement in ion exchange
chromatography.
(1) Increasing polymers efficiency for high capacity ion exchange:
Monolith polymers are used for separation as a separating media for a reasonable
period of time. These polymers were initially not very effective with the separations
but, recent studies indicate that monolith polymers, on sulphonation, yield better
efficiency and high capacity. Latex coated monoliths were used initially but yielded
unfavourable results as there was no great efficiency in separation seen.
The polymers were studied by “Joseph Hutchinson and colleagues, from the
University of Tasmania “.Studies were then focused on BuMa-co-EDMA-co-AMPS
(butyl methacrylate-co-ethylene glycol dimethacrylate-co-2-acrylamido-2-methyl-1-
propanesulfonic acid), PS-co-DVB (poly (styrene-co-divinylbenzene)), or GMA-co-
EDMA (poly (glycidyl methacrylate-co-ethylene glycol dimethacrylate)). The initial
analysis was with PS-co-DVB as a polymer to make monoliths, which did not
respond as expected and had a very low efficiency and capacity of separation. With
the second one, GMA-co-EDMA, the research was with three ways to increase the
ion exchange capacity. “The first two methods (involving suflonation with 4-
hydroxybenzenesulfonic acid and thiobenzoic acid respectively), while
increasing capacity, turned out not to be suitable to separating anion mixtures.
The third method, which made use of sodium sulphite, increased capacity of
the monolith by 30-fold and also increased its chromatographic efficiency.”[10]
This research paved way for an effective ion exchange separation. [10],[11]
(2) Use of a an advanced matrix
There have been various evolutions over the past in the various use of the matrix for
ion exchange chromatography. S-Zephyr is found to have a better performance
compared to Mono-S in the cation exchange chromatography for separations. The
conclusions are derived keeping some parameters in view such as the retentivity
time. S-Zephyr shows better results for all types of separations.[12]
(2) Specificity of Adenovirus purification Using anion exchange Chromatography.
In Gene transfer , the choice of vector for the gene transfer to occur ,is recombinent
adenoviruses, wit the the emphasise more the no so popular serotypes and
chimeras- a human engineered protein. With this boom , a purified form of alternative
serotypes is required. Generally , for the purification of adenovirus , the anion
exchange chromatography is the best choice for separation. There may be a change
in the retention times as there are differences to how the capsid proteins are
exposed. With a thorough knowledge of the behaviour of virus , the retention time
can be influenced and an efficient basis for chromatography method can be deduced.
Study reveals , hexon protein was found to be altering the retention difference in the
anion chromatography. An analysis was carried out with with different groups of
steroisotopes. The sterioisotopes were found to bind the anion column well and
Sodium chloride solution was used to elute. The retention times were related with
good accuracy to properties of hexon proteins. Such an analysis helps in establishing
a good chromatography gradients for different serotypes [13]
(4) High Speed Separation at elevated Temperatures:
An application that is sorted for the separation is by high temperature, at about 900C.
A Dionex CS12A column with the help of suppressed conductivity detection is used
for the to separate ions. The temperature of operation being high mitigates the
viscosity and enables to maintain a better flow rate compared to a normal flow rate
utilised and had a better effect on the separations as well. The drawback of such a
method was the retention time was reduced of cations. This led the column being
operated at 600C, which still yielded a better result than the regular methods of
operation. [14]
(5) Application to determine sugars
As the nutritional value plays an important role in the modern world, the detection of
sugars is a great find by ion exchange chromatography. The results that are found to
be obtained out of these results prove genuine and proper. For this analysis, the
exchanger uses a mobile phase which is predominantly a basic material (NaoH), and
the medium or the column which is used is an amine resin column. This analysis,
with pulsed amperometric detection is a big tool in measuring glucose, fructose and
sucrose. The detection was found to be better and also took lesser time compared to
the other method, HPLC. [15]
(6) Rapid Anion Separation:

Separation of anions is faster using Ion exchange chromatography. Such a research
was carried out to facilitate better results in the sphere of food and environment
departments. This method was seen to have a better throughput. Some issues like
hydrophobicity and the interactions between the analytes tends to slow down the
process. Hence a new method is developed for the anion separation in about three
minutes! For this process, a short column was considered and was coated with
cetylpyridinium chloride (CPC), a cationic surfactant, which was found to be doing
really well. The research also revealed that the surfactant was removable by
acetonitrile. Firstly, the test was carried out on nitrite and nitrate, operating at
reasonable flow rates and the separation was successful in less than three minutes.
Later some more samples were tested, which yielded a similar result and also had a
reasonable capacity of recovery. Hence, by a surfactant coating to the column, the
column efficiency is enhanced both in recovery and the time of recovery. [16] [17]
[18]
(7) Separation of ions by switching columns:
A new system is devised for the separation of cations and anions which utilises only
one pump, eluant and detector. The columns are connected to each other side by
side and the columns are utilised by changing the valve, which is useful for
separation in a single run. The two columns do not operate simultaneously. When
one column is in operation , the other column is on the standby for analysis. When
such a system was tested for using 2.4 mM 5-sulfosalicylic acid resonal. This
showed an acceptable separation level. On placing an injection valve, the required
separations of the ions targeted were achieved having better detection and capacity.
[19]
(8) New Column by Grafting:
A recent study by a group of Turkish scientists reveals that a new column can be
developed using particles of monodispersed polymers which can be instrumental
in designing an ion exchanger. The particles, because of their characteristic of
being monodispersable, account for better separations. Latest research reveals a
chromatography column developed using atom transfer radical polymerisation
method.(ATRP) using poly glycidyl methacrylate-co-ethylene dimethacrylate, or
poly(GMA-co-EDM) with ion exchange ligands. The combination was seen to be
a major hit for protein separations. An initiator is used with the poly(GMA-co-
EDM). On further and final analysis of the polymers and when the system was
tested for a run , the conclusions drawn from them was effective separation of
proteins like lysosome, myoglobin etc. The column utilised the optimum column
height and also had very low run to run reproducibility values.[20] [21]
(9) Use of Short columns to separate low membrane proteins:
A monolith known as the Convective interaction media in stationary phase ,which

is to separate macro molecules and operating at a superior flow rate and minimal
pressure drop. They are predominantly focusing on antibody separation, they
have a great scope for separations of biological materials in the shortest time
possible. Monolconal antibodies were bound to rat liver plasma protein and linked
to protein A or protein G. The column seems to have achieved a rapid separation
.The separated antigens were analysed further on Mass spectroscopy. [22]
2.3.3. Evaluation of Technology
Ion exchange chromatography is the most preferred technique for protein separation
as about 57 % proteins separated are via this technique. Large protein volumes are
analysed by this method. The column has a very good efficiency and life period and
can sustain giving a good output. This technique uses nominal chemicals possible
and the samples prepared for analysis need not be more. Basically, with the
presence of the charge in it, this technique has good binding capabilities compared to
the other techniques. A distinctive feature that separates ion exchange
chromatography from the others is it more ions may be evaluated or analysed at
once. The additional advantage that it carries is its reliability, the strength of the
technique and the assurance of results, which make it stand out comparing other
separation methods. They can also be useful for scaling up. [5] [23][24]
Example: Measurement of ammonium nitrite is not simple. Ammonium nitrite
analysis, by other analysis is particularly difficult while ion exchange chromatography
has a distinctive feature of sensitivity towards it. [24]
There are some basic issues regarding ion exchange chromatography. For
separation, setting up the column is an issue as it takes a lot of time doing the same.
Normally the extract out from the surrounding cannot be directly fed into the column.
Generally, separation of materials having different charges is difficult here.
There needs to be a continuous vigil to the column to check if everything is operating
properly and to ensure that everything is alright. It is difficult to optimize.[25]
2.3.4 Applications of the Technology:
Ion exchange Chromatography has gained significant interests in the different fields
from environmental studies, commercial separations , industrial applications etc. The
most utilised area of ion exchange chromatography is the medical, bio molecular
biomedical, lower molecular weight substances and pharmaceutical applications.
(1) IEC technique to purify Monoclonal Antibodies:

Monoclonal antibodies have a great application in the field of medicine for the
treatment of cancer. It is very important that this antibody, prior to its usage , is
very important that it is purified to get the desired protein. This is done using the
ion exchange chromatography method (either by cation or anion exchange). The
isoelectric point of the monoclonal antibodies are from 5-8, hence are bound to
the cation exchangers. The steps where there is no binding taking place is useful
for the reason that the impurities like DNA , viruses and other impure and
unwanted proteins are removed, ensuring a purified antibody.[26] [27]
(2) In the Separation of Ran GDP, Ran GTP , Ran GMPPN
Ran GDP has a big role to play in the transportation of essential substances and
in cell division. This reveals the affinity and the binding of Ran to an ion exchange
column which is very responsive to the concentration of magnesium chloride. At
moderate concentrations of magnesium chloride, Ran was found to elude and
cause an acceptable level of separation. When there was a further reduction the
concentration levels of magnesium chloride, the purity levels of the Ran ,further
seem to enhanced resulting in the further purification , which was confirmed on
testing with a High performance Liquid Chromatography.[28]
(3) To analyse Spermadhesins proteins:

Spermadhesins is an important protein in animals like boar and stallion which play a
big role in capacitaion and sperm egg interactions. There was a detection of a protein
which was similar to Spermadhesins, using the technique of ion exchange
chromatography; the confirmation of the same is sorted. Semen sample was taken
from the object (Bucks), after treatment including dialysis and freeze drying is
injected to the chromatography column. Seminal plasma of the Buck, gave 6.47 +/-
0.3 mg from ion exchange chromatography. Thus, proteins were separated and
analysed .The seminal fluid protein from the buck revealed that proteins were present
that had a similar structure to the Spermadhesins family. [29]
(4)Monolith Application for purification of plasmid DNA.
Plasmid DNA is gaining a great importance for genetic vaccination and gene
therapy. For pDNA to be utilised for the above application, a purified form is very
essential. The crunch step of pDNA production utilises a chromatographic technique.
Monoliths are on the top priority list for separation of pDNA, as they have a very
strong binding affinity towards the pDNA. The best choice of the purification is by
anion exchange chromatography as the polynucleotides are negatively charged and
do not depend upon the buffer levels.. The various natures of ligands, their
respective densities and their optimisation levels were understood. The
scaling up was done using a convective interactive media monoliths operating at low
pressure levels. Hence the they were successful in the production of pDNA (the
intermediate step). Such a technique gained a lot of prominence in the
pharmaceutical industry.[30]
(5)Applications in Food industry
In the baking industry, potassium bromate has a major application as it has a very
good keeping and conditioning property of dough. Potassium bromate but, has a
significant health concerns and hence was banned by the World Health Organisation
as a food additive. Recent studies have shown that this problem can be overcome
using ion exchange chromatography. Initially there were a lot of other techniques that
were tried for detecting the same like HPLC, GC but they were not so successful in
separating bromate. Hence, ion exchange chromatography was considered. The
column used for separating the bromate “Ion Pac AS19”. The separation of bromates
took place with an elusion of potassium hydroxide in a linear column. In order to
calculate the bromate levels, some predominant parameters like the separation time,
the temperature of separation with other parameters were considered. Analysis
showed that after sonication, peaks were formed after 30 mins and there was no
significant contribution by temperature for the separation. The detection limit was
0.5µg/L. When this was actually tested on flour oriented products, the test was
successful had had a very high bromate recovery levels. [31] [32]
2.3.5: Relevant Web Sights

One of the best source of relevant information in the web sight is
www.separationsnow.com. This website has got relevant information for ion
exchange chromatography and all the other techniques as well. Pub Med and
Science direct (RMIT online Library resource) have excellent material for the same
providing a lot of journals articles.
2.3.6: Key Suppliers:

1) Millipore
Billerica, MA 01821
United States of America
http://www.millipore.com
2) Palico Instrument Lab,
Circle Pines. MN 55014-0125, United States of America.
3)Eichrom Technologies, Inc.
4) Thermo Electron and Fischer Scientific

www.thermo.com
5) G.E Health Care

www.gehealthcare.com
2.3.7 Reference:
1) Farlex. Inc (2006) , The free dictionary

http://www.thefreedictionary.com/protein
2) Everything Bio (2005-2006) Protein Definition

http://www.everythingbio.com/glos/definition.php?ID=2259
3)Lehninger, A.L., Cox, M.M.,Dobos,M. and Roche,P.

Lehninger’s Principle of Biochemistry, 4th Edition, Chapter 3(3.2) pp85-88.
4) Virgina Polytechnique Institute and State University,(2005), Learning Resource

http://www.biotech.vt.edu/classes/bion_4784/9-
ProteinPurification/ProteinPurification.html
5) Jan-Christer,J.,Rydern,L (1989) Ion Exchange Chromatography, Protein

Purification: Principles ,High Resolution Methods and applications. Section 4,pp 104-
116
6) Protein Chemist.com , (2003)Tutorial Resource.

http://www.proteinchemist.com/tutorial/iec.html
7) Cornell University learning resource
http://instruct1.cit.cornell.edu/courses/biobm330/ppts/ProtPuroutline_vJB1.ppt
8) Wagening University, (2005) Laboratory of Biochemistry, Department of Agro

Technology and food sciences, Learning resource.
http://gcg.tran.wau.nl/local/Biochem/educatio/Colleges/BSM/lectures/lecture_3/ionex
change.doc
9) Madison Area Technical College,Learning Resource.

http://matcmadison.edu/biotech/resources/proteins/purification/ionEGuide.htm
(9a)Everything Bio,(2005) Definition of Analyte.

http://www.everythingbio.com/glos/definition.php?word=Analyte
10)Secko, D.,(2006), Monolith Polymers reveal High Capacity for Ion Exchange
http://www.separationsnow.com/coi/cda/detail.cda?id=12786&type=Feature&chId=5
&page=1
11)Hutchinson, J.P., Hilder, E.F, Shellie,R.A, Smith, J.A, and Haddad,P.R.(2006)

Towards high capacity latex-coated porous polymer monoliths as ion-exchange
stationary phases. Analyst 131 pp 215-221.
12)Ronger,R.M., Alder,C.M. and Scouten, W.H.(1991) S-Zephyr, a new high

performance ion exchange chromatography column matrix.
Journal of bioseparations 2(5), pp 297-308.
13)Konz,J.O.,Livingood,R.C.,Bett,A.J.,Goerke,A.R.,Laska,M.E.,Sagar,S.L.(2005),Ste
reotype specificity of adenovirus purification using anion exchange chromatography.
Human Gene Therapy 16(11) pp 1346-1353.
14) Chong, J., Hatis,P., and Lucy, C.A.(2003). High-speed ion chromatographic
separation of cations at elevated temperature. Journal of Chromatography.A. 997(1-
2) pp161-169.
15) White, D.R., and Widmer, W.W.(1990) Applications of high performance

chromatography for sugar analysis in citrus juice. Journal of Agricultural food
chemistry 38 pp 1918-1921.
16) Secko, D. (2006) Anion separation in less than three minutes
&page=1
17) Li,J., Zhu, Y., and Guo, Y.(2006) Fast determination of anions on a short coated
column . Journal of Chromatography .A. 1118, pp 46-50.
18)Fritz, J.S, Yan, Z., and Haddad, P.R.(2003) Modification of ion chromatographic
separations by ionic and nonionic surfactants. Journal of Chromatography .A. 997
Pp 21-31.
19)Amin ,M., Lim,L.W and Takeuch, T. (2006) Tunable separation of anions and
cations by column switching in ion chromatography .Journal Article.
http://www.sciencedirect.com/science?_ob=ArticleURL&_udi=B6THP-4KR3J5N-
1&_user=10&_coverDate=08%2F24%2F2006&_alid=472156754&_rdoc=10&_fmt=s
ummary&_orig=search&_cdi=5288&_sort=d&_docanchor=&view=c&_acct=C000050
221&_version=1&_urlVersion=0&_userid=10&md5=8ad429c69a9b17de141228aa6b
15d18
20) Secko, D.(2006) Grafting a new column

&page=1
21)Unsal , E., Elmas, B., Caglayan, B., Tuncel,M., Patir, M and Tuncel, A.(2006)
Preparation of an Ion-Exchange Chromatographic Support by A "Grafting From"
Strategy Based on Atom Transfer Radical Polymerization. Journal of Analytical
Chemistry. 78(16) pp 5868-5875.
22)Rucevic, M., Clifton, J.G., Huang, F., Li,X., Callanan, H., Hixson,D.C and
Josic,D.(2006). Use of short monolithic columns for isolation of low abundance
membrane proteins. Journal of Chromatography A 1123(2) pp 199-204.
23) Portal to Science , Engineering and Technology. Tutorial Source,

Chromatography
http://www.chem.ubc.ca/courseware/121/tutorials/exp3A/columnchrom/
24) Rey ,M.A.,(2001) High-capacity cation-exchange column for enhanced resolution

of adjacent peaks of cations in ion chromatography. Journal of Chromatography
920(1-2) pp 61-68
25)University of British Columbia, Learning Resource
http://www.resonancepub.com/chromtutorial.htm
25 a) Laboratory Talk (2002), Jonathan Bruce.
http://www.laboratorytalk.com/news/mea/mea144.html
[26] Jacob , L., Schluter , H.,(2006) Bio processing and Biopartnering. (article)
[27] Bai,L. , Burman, S., Gledhill L.,(2000) Development of ion exchange

chromatography methods for monoclonal antibodies , J Pharm Biomed Anal :22 pp
605-611.
[28] Bibak, N., Paul, R.M., Freymann, D.M., Yaseen,N.R. (2004)

Purification of RanGDP, RanGTP, and RanGMPPNP by ion exchange
chromatography.Journal of Analytical Biochemistry 333(1) pp 57-64.
[29] Ítalo, D., Teixeira,A., Melo,L.M., Gadelha, C.A.A., Silva da Cunha, R.M., Bloch
Jr,C., Rádis-Baptista,G., Cavada,,B.S.,and José de Figueirêdo Freitas ,V. (2006)
Ion-exchange chromatography used to isolate a spermadhesin-related protein from
domestic goat (Capra hircus) seminal plasma
Journal of Genetics and Molecular Research (Online Journal)
http://www.funpecrp.com.br/gmr/year2006/vol1-5/gmr0193_full_text.htm
Access date : 25th September 2006.
[30] Urthaler, J., Schlegl, R., Podgornik , A., Strancar , A., Jungbauer, A., Necina,
R.(2005) Application of monoliths for plasmid DNA purification development and
transfer to production Journal of Chromatography. A. 1065(1) pp 93-106.
[31] Secko, D.,(2006) IC Traces Bromate in Flour,

&page=1
[32] Shi, Y., Liang, L., Cai, Y., and Mou, S. (2006). Determination of Trace Levels
of Bromate in Flour and Related Foods by Ion Chromatography. Journal of Food
Chemistry 54(15), pp 5217-5219.
Chapter 2.4
Affinity Chromatography
Aekta Chhabra
2.4.1 Introduction
Affinity chromatography can be defined as a method in which biospecific and

reversible interactions can be used as a mode for separation, purification and specific
selection of biologically active material from crude samples The approach was first
introduced in 1968 to purify proteins and today it still represents one of the most
powerful techniques available for purification of biologically active compounds. The
method is an extremely useful tool for studying many biological processes, such as
the enzyme action mechanism, hormones, protein–protein or cell–cell interactions
and other significant biological interactions. It is the specificity of affinity
chromatography, which makes it one of the most powerful forms of chromatography.
Purifications up to several orders of magnitude are obtainable in a single step, and
affinity separations are universally used to remove contaminants that are very difficult
to eliminate using conventional chromatographic techniques. [1]
Affinity chromatography is considered to be a unique technique in purification

technology and finds versatile applications in biochemistry analysis since it is the only
procedure that enables the purification of a biomolecule on the basis of its biological
function or individual chemical structure. Immunoaffinity chromatography is another
form of affinity chromatography, which continues to be a definitive tool for protein
isolation produced by genetic engineering. Time consuming and otherwise
impossible purification and analytical steps are easily achieved by affinity
chromatography. Affinity chromatography is thus the grandfather of most modern
chromatographic techniques, including biosensors, DNA, and protein micro arrays,
along with their varied application in diagnostics particularly protein–protein
interactions and drug screening [1].
Underlying Principle
The principle of affinity chromatography is as follows:
1) The sample is injected into an initially equilibrated affinity chromatography column

2) Substances possessing an affinity for the ligands are retained in the column.
3) Substances with no affinity for the ligands are eluted down from the column.
4) The substances retained in the column can be eluted or recovered later from the
column by alteration of conditions like pH, salt or organic solvent concentration of the
eluent.
Fig 1:Principle of affinity chromatography
Source: http://www.shodex.com/english/da0401.html
The success of entire process rests on specificity based on three aspects—the

matrix, the ligands, and the attachment of the ligands to the matrix. Choosing the
correct ligand is the first important aspect. It is a necessity that the ligand must bind
strongly with the molecule to be recovered. If the ligand chosen possesses the ability
to bind more than one molecule of the sample to be analyzed, then a technique
called negative affinity, which uses ligands to remove everything except the target
molecule from the sample solution, can be used.
The next important step is the matrix selection that marks the success for affinity
chromatography. Matrix materials function to hold the active ligands and provide a
pore structure to increase the surface area thereby increasing the binding capacity of
the molecules. This binding requires that the matrix be activated and then react with
the ligands to bind them tightly onto the matrix. During the entire process, the ligand
must remain active towards the target molecule. Amino, hydroxyl, carbonyl, and thio
groups, are easily activated and can serve as the sites on the matrix to which the
ligands attach. The matrix, in addition to requiring activation, should also be resistant
to contamination when purifying pharmaceutical compounds. Decontamination is
performed by a column rinse with sodium hydroxide or urea. Different matrix
materials have a tendency to be stable in different pH ranges, which essentially
forms the third aspect of selection for affinity chromatography.[2]
Fig 2: The ligand attached to the matrix binds to the protein of interest and is
separated from other proteins.
Source: http://pubs.acs.org/journals/chromatography/chap5.html
At its introduction of the technique in the late ’60s, affinity chromatography was used
to purify classes of proteins which were dependent on their properties or function —
antibody binding, hormone binding, enzyme inhibition, etc. In the last few years
development of affinity chromatography has enabled monoclonal antibody
purification through the special and unique affinity of Protein A (a protein originally
from Staphylococcus aureus), with the constant region of antibody. In the recent
years, computational chemistry, molecular modelling and combinatorial chemistry
have provided opportunity for chromatographic adsorbent development to enable
purification by design. In this particular mode, a specific adsorbent is constructed to
the target biopharmaceutical moiety in a customized program between the
biopharmaceutical company and the adsorbent.[3]
In order to increase the efficacy of purification, the concept of spacers between the
biologically active ligand and the polymer was introduced. 95% of all affinity
purification methods apply the same general principles using spacers.
Immunoaffinity chromatography continues to be instrumental for the isolation of
proteins produced by genetic engineering. Recent years have showed that affinity
columns can be used to remove toxic compounds from blood. Nowadays, chemical
reactions can be combined with the affinity concept to demonstrate and study the
phenomenon of biological recognition. A perfect example of this kind of approach is
affinity labeling, which can identify the residues in the binding-active site of proteins.
The involvement of amino acid residues with active sites of enzymes can be
examined using this technique. Affinity therapy is a biorecognition-based approach,
which selectively delivers a cytotoxic drug or toxin to the targeted or infected cell. The
cell-associated target molecule can be a surface antigen, a surface receptor or any
other type of biomolecule which is specific for a given antibody, hormone, or nutrient.
The cell-associated target molecule can be a cell-surface antigen, a surface receptor
or other type of biomolecule bearing specificity for a given antibody, hormone, or
nutrient. The drug counterpart can comprise the corresponding antibody or hormone
to which a cytotoxic compound (e.g., selected from chemotherapeutic drugs, radio
nucleotides, or toxins from different origins) has been chemically attached in a
synthetic process. This approach was popularly known as affinity therapy, which was
later changed by others to the misnomer, immunotoxins. [1]
Detection of seven specific bands by immunoblot (IB) using glycoproteins (GPs)

purified by lentillectin affinity chromatography has indeed been a remarkable
standard for neurocysticercosis (NCC) serodiagnosis since 1989.This has aided in
the study of certain aspects of the pathological differences between the Asian and
Africa-American types and their respective antigenic components.[4]
Affinity chromatography is undoubtedly the most selective method for protein

purification because it has the required specific purification power to eliminate steps,
increase yields and thereby improve process economics. Some of the most recent
advances in this area have explored the power of practical and combined
approaches for designing highly selective and stable synthetic affinity ligands forming
the pivotal point of the process. Rational molecular design techniques based on
biochemical analysis of protein structures and advanced computational tools, have
made ligand design feasible and faster and much more accurate. Combined
approaches based on peptide and nucleic acid libraries have enabled the rapid
synthesis of new synthetic affinity ligands, which are of potential use in affinity
chromatography. The specificity and versatility of these approaches has made them
dominant methods for designing and selection of multifaceted and novel affinity
ligands with scale-up potential. [5]
The most recent approach involves high throughput screening techniques of libraries
constructed in 96-well plates, containing “microcolumns” of defined affinity
adsorbents. These column libraries are nowadays constructed using parallel
synthesis robotics. Using sensitive protein assays, adsorbents selectively recognizing
the target protein can be identified for further optimization and development.
ProMetic BioSciences Ltd. (Cambridge, U.K.), a subsidiary of ProMetic BioSciences
Inc. (Montreal, QC), uses robust and modern chemical scaffolds primarily based on
triazine derivatized with non-toxic amines. These “ligands” are then immobilized onto
a neutral, beaded agarose support used in a standard packed bed chromatographic
array. This exceptionally intelligent technique has been used to develop high-
performance affinity adsorbents for a plethora of biopharmaceutical targets, including
antibodies, fusion proteins and even proteins present in the blood plasma.
Chromatography will always remain the technology of choice for downstream

processing, despite the many efforts to find new solutions. Affinity chromatography
using custom-designed synthetic ligands is likely to play an ever-increasing role in
the bioseparations which nevertheless form an integral role of all biochemical
analysis. New formats for chromatography will emerge in the next decade and
manufacturers will be challenged with balancing the introduction of cost saving,
higher yielding technology with high levels of specificity.[3]
2.4.3 Evaluation Of Affinity Chromatography process
In protein engineering, the tasks of generating and testing a large number of variants
of a molecule and optimization of expression conditions for one individual molecule
create the need for purification methods possessing ability to handle a large number
of samples simultaneously. A simple affinity chromatography system can be used for
the parallel purification of 24 protein samples, yielding sufficient quantities after
purification to be used for biochemical and functional analysis. This system
employing affinity chromatography has certain advantages over existing systems: the
costs of this system are minimal as compared to other chromatography systems,this
system allows gentler processing of the samples and is therefore beneficial for
proteins prone to be easily damaged. [6]
Affinity chromatography shows its true power in intricate biochemical analysis. The
challenge of bioseparations lies in identifying and extracting the target protein from its
constituent sample mixture. For example, about 300 proteins in blood plasma have
now been identified and separated among thousands of them present at low
concentration. Thus, affinity chromatography combines appropriate selectivity
alongwith high yield.[3]
Affinity chromatography is nevertheless the most selective method for protein

purification because it has the ability to eliminate steps, increase yields and efficiency
and thereby improve process economics. [5]
The affinity chromatography method for the determination of glycosylated

hemoglobin is rapid, specific and precise and offers several advantages over other
methods. Affinity chromatography is relatively insensitive to changes in temperature
and pH and is likely resistant to interferences by other groups. The results are
independent and reproducible over a wide range of hemolysate concentrations. In
addition to this, the method offers good stabilities of specimen, buffers and columns.
The technique allows10-fold reuse and a relatively simple sample preparation without
washing or incubation of erythrocytes. All these aspects make this method technically
less complicated and apt for routine use in a clinical laboratory. [7]
Advantages of affinity chromatography:
• High affinity, thereby ensuring high selectivity
• Binding all or none of the sample molecules, so excellent recovery is possible
• Lots of variations and designs are possible in this method e.g. Usage of tags
according to the desired protocol.
Disadvantages of affinity chromatography:
• Steric hindrance between the molecules tend to lower capacity
• Elution tends to become harsh many a times
• A very specific eluent is required to obtain accurate results
• Limited options available for selection of affinity chromatography beads. [8]
The isolation of proteins and peptides is usually performed using variety of

chromatographic, electrophoretic, dialysis, precipitation and other procedures in the
biotechnology industry; affinity chromatography being one of the most widely used
techniques. Affinity ligand techniques are indeed the most powerful tools available for
downstream processing and possess high values of selectivity and recovery. The
strength of column affinity chromatography is evident from thousands of successful
applications, especially at laboratory and pilot plant stages. However, the
disadvantage of all standard column liquid chromatography procedures still remains
to be the impossibility of the systems available in the market to cope with the
samples containing particulate material thereby making these columns unsuitable for
work in early stages of the isolation/purification process where suspended solid and
fouling components are present in the sample. [9]
2.4.4 Applications of affinity chromatography
Affinity chromatography finds numerous applications in the field of biochemistry.

Early applications included the separation of biomacromolecules from other biological
components of the cell’s machinery. This certainly forms the most important field for
affinity chromatography. In addition to using small ligands to separate large
molecules from a constituent sample mixture, large molecules can now be
immobilized on the matrix and used to separate the contaminating small molecules
adhering to them. Affinity chromatography can also be used to determine dissociation
constants for ligands and molecules. The longer the time for which the molecule
stays on the column, the broader is the chromatographic peak, indicating tight
binding of molecule to the ligand. This quantitative information can further be used for
various biochemical studies. [2]
Immunoaffinity chromatography is used to perform flow-based immunoassays, which

finds versatile applications including affinity-based chiral separations. Affinity
chromatography also serves for the study of drug or hormone interactions with
binding proteins. Some areas of possible future developments like tandem affinity
methods and the use of synthetic dyes, immobilized metal ions, molecular imprints, or
aptamers as affinity ligands for clinical analytes are now being investigated in detail.
[10]
Affinity chromatography is also a powerful protein separation method based on the

principle of specific interaction between immobilized ligands and target proteins.
Peptides can be separated effectively by using this method. Affinity chromatography
on peptide mixtures, used in conjunction with mass spectrometry, has laid down the
basis for study of protein posttranslational modification (PTM) sites and quantitative
proteomics.
Isotope-coded affinity tags have made possible the easy quantitation of proteomes.
Affinity chromatography is becoming a landmark for exploring PTM and protein–
protein interactions, especially with a view towards developing strategies of chemical
derivatization on peptides. With recent advances in technology, more applications of
affinity-based purification are expected in the next few years to come, including
increasing the resolution in 2-DE (2 Dimensional Electrophoresis) and improving the
sensitivity of MS (Mass Spectrometry) quantification. [11]
The most common use of affinity chromatography is to purify recombinant proteins
which is done by tagging the proteins with known affinity to facilitate their
purification.[12] The Two-Step Affinity Purification System launched by Qiagen,
which combines sequential affinity purification steps, is claimed to yield ultra pure
(>98%) protein. BioChrom has also focused on improving column matrices for
affinity chromatography. According to BioChrom's president, Michael Lu, "the
improvement of recoveries of proteins and the speed of protein purification are
still the major issue for most separation media companies." To deal with this
major issue, BioChrom has developed a polymeric material, Hydro cell. The high
level of cross-linking in the polymer beads is designed to give better and faster
and more efficient protein separation compared to conventional silica-based HPLC
columns. Biochrom also claims that their Hydro cell columns are very durable,
capable of tolerating a broader pH range and harsher cleaning regimens.
[13]
Affinity chromatography is now being used as an analytical method to determine the
concentration of the free analyte fractions present in a sample. This method involves
the application of a sample comprising a free and bound analyte to an affinity
column, which is capable of selectively extracting the free portion in the fraction of a
second. The signal generated by the free portion is then subjected to quantification
by standard analytical detection techniques and the concentration of the free portion
is determined by comparison of its signal with that of a calibration curve depicting the
signal of known concentration of the same analyte. [14]
Burkholderia pseudomallei is supposed to be the causative agent of melioidosis, a
disease prevalent in South East Asia and northern Australia. The bacterium is known
to
secrete various extra cellular products possessing direct correlation to its
pathogenesis, e.g. lethal exotoxin, protease and hemolysin. Antibody mediated
affinity chromatography was used to purify the exotoxin as studies have shown that it
exhibits necrotic and cytotoxic activities in addition to inhibiting cellular protein
synthesis This purified protein can aid in understanding the role of virulence factor
and go a long way in facilitating vaccine development to combat melioidosis. [15]
With new drugs rapidly advancing into clinical trials, their intracellular target
identification becomes fundamental for the full understanding of the molecular basis
of their efficacy and toxicity. This is of utmost relevance when the targets belong to a
large family and their inhibitors recognize a conserved site among other different
members of the same class. A typical example is the kinase family, where efforts are
aimed at inhibitor development of distinct kinases for therapeutic applications in
oncology, inflammation and other disease areas. Inhibitor affinity chromatography
can be used as a tool to identify and characterize the intracellular targets of various
kinases. [16]

• Resources, Basic concepts for molecular
techniques, David B. Collinge's Home Page
Molecular Plant Pathology Group
http://www.staff.kvl.dk/~dacoj3/resource/
• Using The Informant for Searching for Analytical Chemistry

Information on the World Wide Web, Stephen R. Heller
http://www.hellers.com/steve/resume/p143.html
• Analytical Sciences Digital Library supported by the NSDL

program of the National Science Foundation
http://www.asdlib.org/list.php?mainCategory=Class%20Materia
l
• Pierce-The protein people

http://www.piercenet.com/Objects/view.cfm?type=Page&ID=83
EFA139-8363-40F8-9F7D-A689125C9EBA
• Nature’s protocol: recipe for researchers,

http://www.nature.com/nprot/journal/v1/n3/full/nprot.2006.127.h
tml
• http://en.wikipedia.org/wiki/Main_Page
• http://www.biopharminternational.com/biopharm/data/articlesta
ndard/biopharm/522003/80026/article.pdf
• Affinity Chromatography: principles and methods

http://uuhsc.utah.edu/coe/hematology/protein/affinity_chromato
graphy.pdf
• Prometic Biosciences
http://wwwold.unict.it/dipchi/05Didattica/Corsionline/Coloranti/15_B
iochimica/affinchrom/affinitychromat.htm
• Thermo Electron Corporation
http://www.thermo.com/com/cda/category/category_lp/0,,15431,00.html?CA=
columns
• http://www.bio.com/store/product.jhtml;jsessionid=HZ5RIFVN2LBXRR3FQLM
SFEWHUWBNQIV0?id=prod1440010
• http://pharmalicensing.com/intelligence/reportsearching.php?action=toc&prod
uctID=854939
• http://www.bioscienceworld.ca/ChromatographyLiquid
• http://www.jenabioscience.com/?gclid=CI-V4I6VnIgCFRw6GAodjDaELw
• http://www.drugdiscoveryonline.com/Content/ProductShowcase/product.asp?
DocID=%7BC840DD69-1408-4BD0-8AC3-
7E7D4B5FD4EC%7D&VNETCOOKIE=NO
• http://www.gmi-inc.com/BioTechLab/perceptivebiocad.htm
• http://www.versamatrix.com/page/en/1007.aspx
• http://wwwold.unict.it/dipchi/05Didattica/Corsionline/Coloranti/15_Biochimica/
affinchrom/affinitychromat.htm
• http://www.iscpubs.com/bg/il/prod/prod1180.html
• Biocentrum Ltd.
http://awe.mol.uj.edu.pl/biocentrum/kolumny_e.pdf
• Prometic Biosciences
http://www.ump.com/prometic.html
• http://www.srlchem.com/products/product_tree.php?entryId=21
• http://www.patricell.com/products.html
Sciences Digital Library

Supported by the NSDL program of the
National Science Foundation Analytical Sciences Digital Library
2.4.7 References
1 Wilchek, M (2004) My life with affinity ,Protein Science, 13:3066-3070
http://www.proteinscience.org
2 Felton, M. & Lesney, M (2006) To Affinity and Beyond: Analytical Alphabet

Soup. In Chromatography: creating a central science, Chapter5,
http://pubs.acs.org/journals/chromatography/chap5.html
3 Curling, J (2006) Bioseparation: The Power of Affinity Purification.In

Bioscience
World
http://www.bioscienceworld.ca/BioseparationThePowerofAffinityPurification
4. Ito. A, etal (2002) Recent advances in basic and applied science for the
control
Of taeniasis/cysticercosis in Asia In The Southeast Asian journal of tropical
medicine and public health, vol. 33, SUP3 (dissem.), pp. 79-82
http://cat.inist.fr/
5 Labrou, N(2003) Design and selection of ligands for affinity

chromatography In Journal of Chromatography B, Volume 790, Issues
1-2 , Pages 67-78 , Preparative Chromatography of Proteins
http://www.gallusimmunotech.com)
6. Gottstein, C& Forde, R(2002) Affinity chromatography system for parallel

purification of recombinant protein samples.In Protein Engineering, Vol. 15,
No. 10, 775-777
http://peds.oxfordjournals.org/cgi/content/full/15/10/775
7. Schmid, G & Vormbrock, R (1984) Chemistry and Materials Science In

Fresenius' Journal of Analytical Chemistry , Vol317,Number6,pp 703-704
http://www.springerlink.com
8.Johannsen, M (2005/06) Fundamentals and applications of

chromatography,
http://www.tu-harburg.de/vt2/chrom/Chromatography05_Part8.pdf
9. Safarik, I & Safarikova, M(2004) Magnetic techniques for the isolation and
purification of proteins and peptides
http://www.pubmedcentral.gov
10.Hage, D (1999) Affinity Chromatography: A Review of Clinical Applications

In Clinical Chemistry 45: 593-615
http://www.clinchem.org
11 .Lee, W & Lee, K (2004) Applications of affinity chromatography in
proteomics In Analytical Biochemistry 324 (2004) 1–10
http://www.leelab.org/research/papers/AnalBiochem324-1.PDF
12.Wikipedia, The free Encyclopedia (2006)
http://en.wikipedia.org/wiki/Affinity_chromatography
13.Smith, C(2005) Striving for purity: advances in protein purification In

Nature Methods 2, 71 – 77
http://www.nature.com
14.Hage, D & Clarke, W (2004) Analysis of free analyte fractions by rapid

affinity chromatography
http://www.patentstorm.us
15. Lim, K., Mohamed, R., Embi, N & Nathan, S. (2005) Mediated Affinity
Chromatography
http://www.msmbb.org.my
16. Valsasina, B., Kalisz, H & Isacchi A(2004) Kinase selectivity profiling
by inhibitor affinity chromatography In Expert Rev Proteomics. Oct ;1:303-
15
http://lib.bioinfo.pl/pmid:15966827

National Science Foundation Analytical Sciences Digital Library
National Science Foundation
Chapter 2.6 Reverse phase HPLC of peptides and proteins
El-Wazani Montaser
2.6.1 Introduction
Reversed –phase HPLC has found a central role in protein studies because
of its versatility, sensitive detection and its ability to work together with
techniques such as mass spectrometry.
Most of all, however, reversed phase HPLC is widely used because of its
ability to separate proteins of
nearly identical structure [1],[2].
Fig.2.6.1.a Fig.2.6.1.b
2.6.1.1 Mechanism of Protein/Peptide Retention
In reversed phase HPLC the particle surface is very hydrophobic due to the
chemical attachment of hydrocarbon groups to the surface. Proteins are
retained by the adsorption of a face of the protein
(Termed the “hydrophobic foot”) to the hydrophobic surface [3].
Fig.2.6.1.1.a Fig.2.6.1.1.b
Fig.2.6.1.1.c
2.6.1.2 Column Characteristics

a) Hydrophobic surface
Fig.2.6.1.2.a
A process called”end-capping” whereby a small organosilane is subsequently

reacted with the silica
surface, further reduces the number of polar silanol groups [4].
b) Column selection and characteristics of sample molecules

The hydrocarbon group forming the hydrophobic phase is usually a linear
aliphatic hydrocarbon of (C18),
(C8) or (C4) carbons. There are guideline as to which phase is likely to be
most effective in separating polypeptides of a given size and hydrophobicity
[5], these are summarized in Figure below.
Fig.2.6.1.2.b
c) Column length
Column length is not important in protein separations and short columns
separate proteins as well
as long columns
Fig.2.6.1.2.C1 Fig.2.6.1.2.C2
d) Column Diameter
See reference [6]
Fig.2.6.1.2.d
2.6.1.3 Mobile Phase

a) Organic Modifier
The purpose of the organic solvent is to desorb polypeptide and protein
molecules from the adsorbent hydrophobic surface. This is typically done by
slowly raising the concentration of organic solvent
(gradient) until the proteins and polypeptides of interest desorb and elute.
Fig.2.6.1.3.a
b) Gradient elution
Proteins and polypeptides are almost always eluted using a solvent gradient
where the relative
concentration of organic solvent is slowly increased during the separation [7].
Fig.2.6.1.3.b1 Fig.2.6.1.3.b2
A reduction of the gradient slope to improve resolution must be tempered with
the need for keeping
analysis time as short as possible. However, adjusting the gradient slope is
important in optimizing
resolution of proteins and peptides [7].
c) Ion-Pairing Reagents
1. Trifluoroacetic acid
TFA added to the mobile phase at concentration of ~0.1% results in good
peak shape on most
columns
Fig.2.6.1.3.C1
2. Alternative ion-pair reagent

Phosphoric acid and heptafluorobutyric acid are some times used in
protein/peptide separation [8]
Fig.2.6.1.3.C2
3. Ion suppression
The major benefit of ion suppression is the elimination of mixed mode
retention effects.
At low pH, carboxylic acid groups will be protonated and only slightly polar.
Increasing the mobile phase
pH to 6-7 will cause the carboxylic acid groups to ionize, making the proteins
and peptide less
hydrophobic. This reduces the retention of all peptides.
Fig.2.6.1.3.C3
2.6.1.4 Flow Rate

Fig.2.6.1.4
2.6.1.5 Temperature
Temperature can have a profound effect on reversed phase chromatography
[9],[10].
Fig.2.6.1.5a Fig.2.6.1.5.b
2.6.1.6 Reversed –phase HPLC- MS
Reference [11],[12].
Fig.2.6.1.6a
Fig.2.6.1.6b
2.6.2 Recent Advanvces
2.6.2.1 Columns
Column developments in HPLC have benefited protein and peptide
separations in a number of ways
a) Atlantis TMdC18 columns
Are a fully LC/MS compatible line of universal C18 columns that offer the
perfect balance of retention for
both polar and non-polar compounds. Atlantis TMdC18 columns are compatible
with aqueous mobile phases, provide enhanced low pH stability and are
available in a wide variety of column configurations ranging from nano-scale
to preparative
Fig2.6.2.1a1 Fig2.6.2.1a2
2.6.3 Evaluation of Technology
a) Comparison between conventional RP-HPLC and Atlantis TMdC18 columns
Conventional RP-HPLC Atlantis TMdC18 columns
Problem Impact Solution and Benefit
Little or no - Re-run using separate - Polar compounds are retained longer with Atlantis
TM
retention of methods for polar dC18 columns
polar compounds - One Atlantis TMdC18 column and method can be us
compounds - Increased method for polar and non-polar compounds
development time and labor - Decreased labor cost
Fig2.6.3.a1
Method requires - Loss of retention is - Atlantis TMdC18 packing material is tested with high
100% aqueous observed polar analytes in 100% aqueous conditions, thereby
mobile phase ensuring its utility in aqueous conditions
for desired
separation
Sudden loss of - Run organic modifier - Atlantis TMdC18 column don’t lose retention in 100%
analyte through column to rewet aqueous mobile phases
retention and regenerate column - Less time spent rewetting columns resulting in low
observed when labor costs.
using highly - Increased labor and - Increased throughput
aqueous mobile solvent costs
phase - Decreased throuput Fig2.6.3.a2
- Reproducibility issues
Short column -High cost due to frequent - The proprietary difunctional bonding chemistry of
life time in column replacement Atlantis TMdC18columns results in low pH stability an
acidic mobile longer column lifetime
phases -Increased instrument - Decreased costs associated with column replacem
downtime and instrument maintenance
- Retention time Fig2.6.3.a3

reproducibility issues
Retaining polar - Multiple columns are - One Atlantis TMdC18column and method can
compounds on required to separate used for polar and non-polar compounds
a conventional analytes with a wide - Easier and faster method development
C18 column range of polarities - Increased throughput
results in - Increased method
increased or development time, Fig2.6.3.a4
infinite retention labor and column
of non-polar costs
compounds - Decreased
throughput
Severe peak - Method fails system - Atlantis TMdC18columns are optimally endcap
tailing for polar suitability guidelines and provide excellent peak shapes using MS
bases is for peak tailing compatible mobile phases
observed - Increased method - Easier and faster method development
development time
Fig2.6.3.a5
Column bleed is - Frequent cleaning of - Atlantis TMdC18columns do not exhibit MS

observed on MS MS source detectable column bleed
- Incorrect or - Decreased instrument downtime and
inconsistent results maintenance costs
Fig2.6.3.a6
Column to - Increased labor - The stringent Atlantis TMdC18 packing materi

column costs due to QC batch test separates highly polar analyte
reproducibility is individual column 100% aqueous mobile phase conditions
inconsistent(e.g. QC testing - Decreased method revalidation and
selectivity, - Revalidate/redevelop development time.
retention,etc) method with each
new batch of Fig2.6.3.a7
columns
www.waters.com (for more information)

2.6.2.1 Columns
b) Monolithic columns (Polymer- based reversed phase chromatography
columns)
These columns not packed with conventional small particles, but rather are
formed as a single rod of very porous material that is in encased in a column
package.This new adsorbent based on porous (100A◦-300A◦ pore diameter),
highly cross linked polystyrene-divinyl benzene spheres [13]. Because of the
high cross linkage, this new adsorbent gives high mechanical stability with a
minimum of shrinking in aqueous and swelling in organic solvents. Its
separation performance for proteins and peptides is demonstrated by
comparing it with silica-based reversed phase columns.
b) Comparison between conventional RP-HPLC and Monolithic columns

(Polymer- based reversed phase chromatography columns)
Test Silica based reversed Polymer-based reversed phase

phase column column
(PLRP-S))
Chemical Stability - The typical silica - No noticeable change in
problems of separation after 400
acidic silanol column-volume wash with
group or other strong base
ionic species, - The strong acid and base
which can wash did not alter
interfere with the chromatographic
separation performance
performance of - pH stable from (1-14)
the matrix and - High TFA concentration
are difficult to will not affect the life of the
remove entirely column
and reproducibly - Not necessitate bonded
by endcapping ligands,
Retentivity - Less surface area - PLRP-S media possesses
(Peptide & protein for retention a much greater surface
separation) area therefore even polar
molecules such as
Parabens may be retained
much longer, resulting in
greater resolution.
Reproducible - Silica-based - Residual monomer and
(Batch-to-Batch) reversed phase surfactant are removed using
materials, where proprietary cleansing
analysis of metal procedures. The result is an
chelate compounds entirely pure reversed phase
can cause poor surface without the possibility
peak shape of leachables or changing
retention characteristics.
- The polymerization
procedure prevents any
possible contamination with
heavy metals.
Scale Up and - The inability to - Separations developed on
sterilization withstand an analytical scale column
extremes of pH can be transferred to a
thus retaining preparative scale column
sanitization and with minimal method re-
sterilization.( will development.
not assist in - The media offers
validation) exceptional loading
capacity due to the high
surface area and the
clean, purely hydrophobic
surface functionality
ensures a very high
recovery.
- The ability of the media to
withstand extremes of pH
thus allowing sanitization
both on-column (clean-in
place or CIP) or on a
batch basis. Ensuring that
the batch is free from
bacterial or similar
contamination will assist in
validation, particularly
under GMP or regulated
procedures.
Lifetimes - Problems in - Polystyrene and
reactive sites divinylbenzene which, in
which often arise addition eliminates all
in silica-based reactive sites
product - Being free from silanols and
- Voids are likely to heavy metal ions the
form from the polymeric nature of PLRP-S
typical silica prevents dissolution of the
problems of stationary phase. Columns
acidic silanol therefore last significantly
group or other longer as voids are unlikely
ionic species to form.
- This feature even allows the
use of high temperature
(>200°C) superheated water
as an eluent without fear of
damage to the stationary
phase
www.polymerlabs.com (for more information)
2.6.2.1 Columns
c) Column packing processes (The future of preparative chromatography)
Introduced by Phenomenex, AXIA™ is an advanced column packing and
hardware design that eliminates
bed collapse as a source of failure in short prep columns.
Using Hydraulic Piston Compression (HPC™) technology patent pending,
several fundamental problems faced daily by preparative chromatographers
have been solved.
Fig.2.6.2.1.c1
c) Comparison between conventional slurry packing and phenomenex, AXIA
Conventional Slurry Packing Phenomenex, AXIA™
- As the - Void formations -Integration of the packing piston so pressure is never

packing as the bed released resulted in extended column lifetime (column
pressure is settles(premature voids are no longer a column failure
released column failure) - Computer control of the entire process to ensure proper
and before sorbent density and uniformity
a -Variability of (density tuned and optimised for different media, )
preparative media bed - Overall column to column consistency is also
column is density from dramatically improved with efficiencies and peak
capped, column to symmetries on par with analytical separations
silica column(degrading -Easily to scale-up
extrudes overall
from the reproducibility) Fig.2.6.2.1.c2
inlet end of
column -Variability of both
efficiency and
- The only peak shape from
options are column to
to scrape column(scale-up
and cap from analytical
more difficult)
-Peak distortion
or asymmetry –
reducing the
return on each
purification cycle
www.axiaprep.com (for more information)
2.6.2.2 UPLC Systems
a) ACQUITY UPLC Systems

ACQUITY UPLC Systems are so much than just fast HPLC. ACQUITY UPLC,
enable to choose the separation that is ideal for the analytical task. No other LC
system on the market today can come close
Fig.2.6.2.2.a
b) Features
Columns for - With BEH Technology TM(bridge ethylsiloxane/silica

high hybrid), ACQUITY UPLC BEH Columns exhibit improved
efficiency efficiency , strength, temperature and pH range the
hallmark of UPLC separation
Fig.2.6.2.2.b1
Versatility - A wide range of complementary chemistries, including

C18,C8, Shield RP18,Phenyl and HILIC
- Sample organizer
- ACQUITY UPLC Guard columns
- Handles multiple sample formats : plates, vials, tubes
- Quick connect fitting
Fig.2.6.2.2.b2
Direct - The superior preparative HPLC separations and hassle-
scalability free scale-up and optimization
and - Column manager with heating/cooling for low dispersion
Flexibility control and precise temperature management
- Optional automated column switching among multiple
columns and a bypass channel
- Sample manager for high capacity processing
- Adjustable FLEXcart for convenient installation and easy
movement between workstations
- Third –party mass spectrometer adaptability
Performance, - Guaranteed performance for column to column and batch
Stability and to batch reproducibility over the life of the column
robustness sensitivity to meet multiple detection requirements( binary
solvent manager, photodiode array detector, evaporative
light scattering detector, UV detector and SQ detector)
Fig.2.6.2.2.b3
Traceability - ACQUITY UPLC console with calculator for eases the
and method development and transfer from HPLC to UPLC
Intelligence - Connections INSIGHT services to monitor system
operational characteristics
- EmpowerTM or MasslynxTM Software for control and data
management
- eCordTM Technology electronically stores all of the
information to track your experiment
d) How do chromatographic technologies compare?
Technology Resolution Sensitivity Speed Scalability Compatibility

to prep- with
Scale MS
HPLC
Waters Excellent Excellent Excellent Excellent Excellent
ACQUITY
UPLC
Conventional Good Good Fair Excellent Good
HPLC
“Ultra-fast Fair Fair Very Good Poor
high-flow” good
HPLC
“High- Very good Good Excellent Good Poor
temperature
high-flow”
HPLC
Monolith Good Fair Excellent Fair Poor
HPLC
www.waters.com ( for more information)
2.6.2.3 NanoACQUITY UPLC System
The waters nanoACQUITY Ultra Performance LCTM system has been designed to
achieve separation at nanoflow rates without flow-splitting, offering significant
improvements in robustness, reproducibility and simplicity of operation over
conventional nanoflow separations technologies. The nanoACQUITY UPLC
system directly benefits from holistic design of ACQUITY UPLC system. The
optimized fluidic design of the nanoACQUITY UPLC System, together with the
proprietary nanoACQUITY 1.7µm BEH chemistry, enables significant
improvement for enhanced analysis of the lowest abundant peptide.
Fig.2.6.2.3

e)
Conventional nanoscale HPLC NanoACQUITY UPLC

- Limitation - Change in - Improve accuracy in qualitative and
of solvent quantitative applications (The
reliability composition nanoACQUITY UPLC System is a direct
and , column nano-flow system that does not require
reproduci back- a flow splitter). The excellent run to run
bility pressure reproducibility is essential for
and flow components to be confidently identified
- Difficult rate may or tracked across sample sets, and for
to result in performing accurate quantitative
precisely poor day to comparison across sample sets
control day and - Increase information content from every
and column to sample. The combination of an
monitorin column optimized fluid path and integrating
g of the retention results in enhanced peak shape and
flow rate time peak capacity, increasing the number of
and reproducibili components that can be detected per
pressure ty. unit time
- Limits Fig.2.6.3.e1
performanc
e in the
HPLC
domain and
places a
greater
burden on
the MS
system
- Comparison
across
samples
problematic
Fig.2.6.3.e2
www.waters.com (for more information)
2.6.2.4 Agilent's new HPLC-Chip technology
Agilent's HPLC-Chip is the first microfluidic chip-based device that can carry
out nanoflow high performance liquid chromatography (HPLC). The center
piece of Agilent's new HPLC-Chip technology is a reusable microfluidic
polymer chip. Smaller than a credit card, the HPLC-chip seamlessly
integrates the sample enrichment and separation -columns of a nanoflow LC
system with the intricate connections and spray tip used in electrospray mass
spectrometry directly on the polymer chip. The technology eliminates 50% of
the traditional fittings and connections typically required in a nanoflow LC/MS
system, dramatically reducing the possibility of leaks and dead volumes and
significantly improving ease of use, sensitivity, productivity and reliability
during analysi
Fig.2.6.2.4.1
The second component of the HPLC-Chip technology is the HPLC-Chip/MS
interface. A chip is inserted into the interface, which mounts on an Agilent mass
spectrometer. The design configuration guarantees that the electrospray tip is in the
optimal position for mass analysis when the chip is inserted. Replacement of the chip
is simple and can be completed in a few seconds as opposed to much longer times
required to change out nanoLC columns. The HPLC-Chip interface will be available
as a standard module within the Agilent 1100 Series LC system.
Fig.2.6.2.4.2
f) The dvantages does the HPLC-Chip offer over conventional technology

Agilent's HPLC-Chip carries out nanoflow HPLC to obtain maximum
sensitivity with minimal sample sizes. The HPLC-Chip integrates sample
preparation, separation, and electrospray tip on a single chip. It significantly
reduces the number of fittings, connections, valves and tubing required for
nanflow HPLC. It also includes a sprayer that interfaces efficiently with a
mass spectrometer, allowing separated compounds such as peptides to then
be identified and quantified via mass spectrometry. This highly integrated,
automated system promises to improve the analysis of complex samples of
unknown composition, increasing productivity and throughput. Compared with
conventional column-based nanoflow HPLC, HPLC-Chip offers unparalleled
ease of use, greater reliability and robustness and higher sensitivity.
Fig.2.6.3.f1
Fig.2.6.3.f2
www.chem.agilent.com (for more information)
2.6.4. Application
1. Expression and purification of a recombinant LL-37 from Escherichia

coli
This study report the first time a method to express in E.coli and purify LL-37.
LL-37 is a 37 residue cationic, amphipathic α-helical peptide. Factor Xa was
used to cleave a 4.5kDa LL-37 from the GST-LL-37 fusion protein and the
peptide was purified using reverse-phase HPLC on a Vydac C18 column with a
final yield of 0.3 mg/ml. The protein purified using HPLC was confirmed to be
LL-37 by the analyses of Westren blot and MALDI-TOF-Mass spectrometry.
The concentration of LL-37 was determined by comparing its peak area with
that of the chemically synthesized LL-37[14].
Fig.2.6.4.1
2. Enabling significant improvements for peptide mapping with UPLCTM
The capabilities of Ultra Performance LCTM technologies make higher

resolution peptide mapping possible [15]. This study demonstrates the
advantages of UPLC for peptide mapping.
Fig.2.6.4.2.a
The separation of a tryptic digest of enolase is shown below. In the UPLC

separation, more peaks are observed. The overall resolution and sensitivity
are higher. In the UPLC map, there are several small peaks that are difficult
to discern with the HPLC run. This result demonstrates that UPLC offers
higher resolution and sensitivity when compared to HPLC under the same
gradient conditions. This suggests that the selectivity of the UPLC column is
suitable for peptide mapping.
Fig.2.6.4.2.b
To show how UPLC can resolve the same number of peaks in a peptide map
as HPLC but in less time, the separation
of an enolase digest was done both with flow rate of 100µl/min. The UPLC
separation shows the same number of peaks and a similar overall elution
pattern as the HPLC separation, but in half the time. UPLC offers the potential
to reduce cycle times for peptide maps.
Fig.2.6.4.2.c
UPLC is particularly important when the peptide map is used to detect
modified peptides. Higher resolution ensures that modified peptides are
resolved from the unmodified form, as well as from other peptides in the
digest. UPLC should be the technique of choice for detecting all the peptides
in a sample.
Fig.2.6.4.2.d
The separation of several peptides with formic acid is compared with TFA on
a UPLC column with MS detection. With formic acid, the peak heights are
about 3-fold higher. This result indicates that the UPLC columns perform
extreme well under conditions that are best for ESI-MS
Fig.2.6.4.2.e
The UPLC-MS separation of a tryptic digest of α-1 acid glycoprotein. The MS

detection was performed with a Q-Tof mass spectrometer, which is well
suited for glycopeptides due to its extended mass range. The glycopeptides
are detected as sharp, symmetrical peaks with UPLC. These characteristics
are important for minimizing spectral overlap of different glycoforms of the
same peptide. UPLC with ESI-TOF mass spectrometry will be a powerful tool
for studying the glycosylation state of proteins.
Fig.2.6.4.2.f
3. Nano-HPLC for determination of proteins in infant formula
To determine the protein content of formula, gel electrophoresis was

performed and the entire protein patterns were analyzed by nano-HPLC-
electrospray tandem mass spectrometry (nano-HPLC/ESI/MS/MS)
The analysis was performed in an LCQ DECA XP ion-trap mass spectrometer
equipped with a nano electrospray ionization source. The electrospray source
was coupled online with an agilent 1100 series capillary HPLC system. Two
microliters of peptide solution in mobile phase was manually loaded into a
capillary column (70mm length x 75µm ID)[16]
Fig.2.6.4.3.1
Fig.2.6.4.3.2
4. HPLC-Chip/MS for analysis of yeast (Saccharomyces cerevisiae)

The following components were integrated onto the HPLC-Chip [17]
1- A 40-nl enrichment column packed with ZORBAX 300SB-C18,5µm particle
size
2- All connections between the two columns and between the analytical
column and the nanoelectrospray tip
3- The nanoelectrospray emitter (10 µm ID)
The HPLC –Chip inserts into the HPLC-Chip/MS interfaces which mounts to
the electrospray source [18]
More identified proteins with the HPLC-Chip/MS
Fig.2.6.4.4.a Fig2.6.4.4.b
Better sequence coverage with the HPLC-Chip/MS provided a higher level of

confidence in the protein identification
Fig.2.6.4.4.c
Excellent reproducibility
Fig.2.6.4.4.d
Fig.2.6.4.4.e
2.6.5 Relevant web sites
www.chem.agilent.com
www.waters.com
www.axiaprep.com
www.polymerlabs.com
14 Expression and purification of a recombinant IL-37 from Escherichia
coli
(online at www.sciencedirect.com (2006))
15 Enabling Significant Improvements for Peptide Mapping with UPLC.
Jeffrey R,Thomas E,Wheat,Beth L.Gillence-Castro, and Ziling Lu.
(www.waters.comWater Corporation,2005).
16 Determination of proteins in infant formula
(online at www.sciencedirect.com(2006))
17 Comparison of HPLC-Chip/MS with conventional nanoflow LC/MS for
proteomic analyses.Martin vollmer, Christine miller and GeorgesL.Gauthier
(online at www.agilent.com/chem/hplc-chip (2005))
Most of the industry suppliers have been mentioned previously on Relevavt

web sites section (2.5.6)
Some additional supplier for Several polymeric columns are now
commercially available from Dionex/LC Packngs, BIA Separations, BioRad,
and Sepragen .
2.6.7 References
1 J.Rivier and R.McClintock., (1983),. Reversed-Phase High Performance

Liquid Chromatography of Insulin from Different Species. J.Chrom.286,112-
119
2 T.Christianson and C.Paech, (1994),. Peptide Mapping of Subtilisins as a
Practical Tool for Locating Protein Sequence Errors during Extensive Protein
Engineering Projects, Anal.Biochem.223,119-129
3 X.Geng and F.E.Regnier,(1984),. Retention Model for Proteins in Reversed-
Phase Liquid Chromatography, ,J.Chrom.296,15-30
4 JD.Pearson, N.T.Lin and F.E.Regnier,(1982),. The importance of Silica Type
for Reverse-Phase Protein Separation, Anal.Biochem.124,217-230
5 P.Tempst,D. Woo,D.B.Teplow,R.Aebersold, L.E.Hood and
S.B.H.Kent,(1986),.Microscale Structure Analysis of a High Molecular Weight
Hydrophobic Membrane Glycoprotein fraction with Platelet-Derived Growth
Factor-Dependent Kinase Activity, J.Chrom.359,403-412
6 M.T.Davis and T.D.Lee, (1992),. Analysis of peptide mixtures by capillary high
performance liquid chromatography: Aparticle guide to small-scale
separations, Protein Science 1, 935-944
7 N.C.Robison, M.D.Dale and L.H.Talbert,(1990)Subunit Analysis of Bovine
Cytochrome c Oxidase by Reverse-Phase High Performance Liquid
Chromatography, Arch. Of Biochem. And Biophys.281(2),239-244
8 M.C.McCroskey,V.E.Groppi and J.D.Pearson, (1987),. Separation and
Purification of S49 Mouse Lymphoma Histones by Reversed-Phase High
Performance Liquid Chromatography Anal.Biochem.169,427-432
9 Y.Chen,C.T.Mant and R.S.Hodges, (2003),. Temperature selectivity effects in
reversed-phase liquid chromatography due to conformation differences
between helical and non-helical peptides, J.Chrom
1010,45-61
10 W.S.Hancock, R.C.Chloupek,J.J.Kirkland and L.R.Snyder, (1994)
Temperature as a variable in reversed-phase high performance liquid
chromatographic separations of peptide and protein samples.I. Optimizing the
separation of a growth hormone tryptic digest, J.Chrom.686,31-43
11 A.Apffel,J.Chakel,S..Udiavar,W.Hancock,C.Souders,E.Pungor,Jr, (1995),.
Application of capillary electrophoresis,high-performance liquid
chromatography,on-line electrospray mass spectrometry and matrix-assisted
laser desorption ionization-time of flight mass spectrometry to the
characterization of single-chain plasminogen activator, J.Chrom.A.717,41-60
12 John Wiley & Sons ,.Overview of Protein and Peptide Analysis by Mass
Spectrometry, Section 16.1.14 in current Protocols in Protein Science.
13 F.Svec and L.Geister, (2006),LCGC 24(S4),22-26
18 ” Fortier, M-H, Bonneil,E.,Goodley,P., and Thibault,P., 2005 ,. Integrated
Microfluidic Device for Mass Spectrometry-Based Proteomics and Its
Application to Biomarker Discovery Programs Anal.Chem.,77(6),1631-1640,
Chapter 2.8 Purification of membrane proteins
Wang Tianshi
2.8.1 Introduction
Membrane proteins are protein molecules that are attached to, or associated with the
membranes of mitochondria, chloroplasts, cells and organs. Membrane proteins have
a crucial role in many cellular and physiological processes. Usually, they are
essential mediators of the transfer of material and information between cells and their
environment and between compartments within cells. [10] Based on their attachment
to the membrane, membrane proteins can be classified into two groups: Integral
membrane proteins and Peripheral membrane proteins. Integral membrane proteins
(IMP), also called intrinsic proteins, is a protein molecule (or assembly of proteins)
that in most cases spans the biological membrane with which it is associated
(especially the plasma membrane) or which, in any case, is sufficiently embedded in
the membrane to remain with it during the initial steps of biochemical purification. In
general, IMPs can be divided into three groups: transmembrane, membrane-
associated and lipid-linked. On the other hand, Peripheral membrane proteins, or
extrinsic proteins, do not interact with the hydrophobic core of the phospholipid
bilayer. Instead they are usually bound to the membrane indirectly by interactions
with integral membrane proteins or directly by interactions with lipid polar head
groups. (Source:
http://www.nlm.nih.gov/cgi/mesh/2006/MB_cgi?mode=&term=Membrane+proteins)
Approximately 40% of the sequenced genes encode for membrane associated

proteins [2]. Many membrane proteins are related to diseases. Using a cost-effective,
simple and rapidly method to purify membrane proteins has become a major
challenge and resolving this problem is also the basis for designing better drugs. The
methods used in membrane protein purification, can roughly be divided into analytical
and preparative methods. The distinction is not exact, but the deciding factor is the
amount of protein, that can practically be purified with that method. Analytical
methods which are usually on a small scale aim to detect and identify a protein in a
mixture, whereas preparative methods are operated on a relatively large scale and
aim to purify the membrane protein complex from membrane fractions while retaining
its native form, mainly to characterize its nature, such as for structural biology and
industrial use. In general, the preparative methods can be used in analytical
applications. The overall steps have been shown in Fig.2.8.1. [5]
Fig.2.8.1 General purification process for membrane protein complexes. [5]
This review will describe some recent advances, compare and evaluate these
techniques and discuss three specific applications.
In recent years, the technologies on purification of membrane proteins were being

improved. This section will describe these progresses on expression of recombinant
protein, choice of detergent and improvement of column chromatography and
analytical method.
With the development of genomic and proteomic technologies, they are opening a
new field of vision on purification of membrane proteins. In an indian study, the gene
encoding an outer membrane protein designated ompTS was amplified by PCR
excluding the region coding for signal peptide, cloned in pQE 30-UA Vector and
expressed using induction with isopropyl thiogalactoside (IPTG) [6]. In a study of the
conformation of the CadF protein which was very hard to purify from Campylobacter
(the most common bacterial agent of gastroenteritis) membranes, Mamelli and his
colleagues developed a novel strategy to produce significant quantities of a
recombinant N-terminal domain of the CadF protein (46.5µg/mg of bacterial dry
weight) and of the native CadF protein (3.5µg/mg of bacterial dry weight).The
nucleotide sequence encoding the N-terminal domain of the CadF protein was cloned
in a pET-based expression vector. The recombinant protein was further produced in
Escherichia coli, purified from inclusion bodies, and refolded. [10] Moreover, recent
studies in this area had also provided new insight into the response of host cells to
membrane protein expression and into the mechanism of membrane insertion [4, 16].
Experimental procedures for handling and isolating integral membrane proteins are
generally more challenging than their soluble counterparts, since the former requires
purification in detergent. A simple and cost-efficient detergent screening strategy is
most important premise of a large-scale protein production and crystallization.
Recently, studies in Sweden were on extracting and purify the recombinant
ammonium/ammonia channel, AmtB, from Escherichia coli. 26 detergents, 4 types of
chromatography columns and various buffer conditions had been screened using a
96-well plate format. Large-scale protein purification and subsequent crystallization
screening resulted in AmtB crystals diffracting to low resolution with three detergents:
UDM, DDM and Cy6. The researches suggested that excluding detergents that were
not useful for high-yield extraction of a specific protein (e.g. due to destabilizing the
protein) might be very helpful to minimize the number of detergents during
crystallization screening. The fact that no crystals of AmtB were grown in the
presence of OG and LDAO might be such an indication. [14]
Column chromatography is the main way to purify the membrane proteins, so it

becomes the key point to improve the technique of chromatography. This technique
contains size exclusion chromatography, ion exchange chromatography, affinity
chromatography, hydrophobic interaction chromatography and reversed phase
chromatography. In a recent report, it suggested that monoclonal antibodies against
several rat liver plasma membrane proteins be bound and cross-linked to protein A or
protein G convective interaction media (CIM) affinity columns with a bed volume of
only 60µL. Because affinity columns had the potential for processing large volumes
of complex biological mixtures within a short time, and CIM could provide a stationary
phase with a high binding capacity for large molecules and be capable of high flow
rates at a very low pressure drop. As a result, antigens recognized by bound
antibodies and co-eluting (interacting) proteins were rapidly isolated in a single step
from either total plasma membrane extracts or subfractions isolated using anion-
exchange CIM disk-shaped columns. [17]
Recently, more novel analytical technologies on membrane proteins were utilized. In

an American study, they found that when wildtype bacteriorhodopsin (bR) and a
labile bR mutant were reconstituted into the phospholipids gel, spectroscopy showed
that the protein was both more stable and has improved conformational homogeneity
as compared to gels formed using monoolein. In addition, they developed a
generally-applicable spectroscopic technique based on the intrinsic fluorescence of
tryptophan residues and this fluorescence assay made possible the rapid evaluation
of lipid gels as media for the crystallization of membrane proteins. [9]
Although it actually has made some progresses to improve the techniques, there still
exits many problems on the purification of membrane proteins to be solved. This part
will mainly discuss the difficulties of membrane protein purification technologies and
how to face those hard problems.
There are several difficulties of techniques on the investigation and separation of
membrane protein complexes originate from their nature as membrane proteins.
Because (1) they are very hydrophobic and have single or several transmembrane
parts, or closely associate with the membrane; (2) in the functional form, many of
them comprise (homologous or heterologous) multi-subunit complexes; (3) such
membrane protein complexes contain many cofactors and, inevitably, lipids; (4) some
membrane protein complexes have several peripheral proteins which are functionally
important but easily detached during the isolation process [5].
The first difficulty is to choose the right detergent for an efficient purification of the
membrane protein of interest due to the hydrophocity of membrane proteins. There
are dozens of different detergents that are commonly used that are less
characterized but still probably useful and many novel detergents under
development. It has also been reported that some compartments of the cell
membrane show resistance towards certain detergents. Moreover, mixtures of
detergents are sometimes used during purification and crystallization. Altogether, the
size of the detergent parameter space becomes very large. On the other hand,
screening fewer detergents may result in poor yield, unstable protein and/or no
protein crystals, and is not recommended. Thus, the presented strategy allows the
screening of tens of detergents, for their efficiency of pure protein production and
crystallization, easily and simultaneously, producing reliable and reproducible results,
at very low cost [14]. For instance, a detergent such as sodium dodecyl sulfate (SDS)
can be used to dissolve cell membranes and keep membrane proteins in solution
during purification; however, because SDS causes denaturation, milder detergents
such as Triton X-100 or CHAPS can be used to retain the protein's native
conformation during purification [20].
Usually a protein purification protocol contains one or more chromatographic steps.

The problem during this process is the complex activities and mechanisms of
membrane protein in the column, and then causes difficulties on obtaining stable and
non-denaturing proteins. Many membrane proteins are glycoproteins and can be
purified by lectin affinity chromatography. Detergent-solubilized proteins can be
allowed to bind to a chromatography resin that has been modified to have a
covalently attached lectin. Some lectins have high affinity binding to oligosaccharides
of glycoproteins that is hard to compete with sugars, and bound glycoproteins need
to be released by denaturing the lectin. RP-HPLC as the first dimension of protein
separation is the opportunities for rapid (ca. 1-min) solubilization with trifluoroacetic
acid (TFA) of the native membrane complex without the need of the time-consuming
extraction or solubilization by detergents [19]. Some additional techniques enable the
facile purification of proteins by chromatography, but they also increase some
uncertain and complex factors [15]. For examples, recombinant integral membrane
proteins are purified by immobilized metal affinity using polyhistidine tag. The effects
of tag length and position in different situations must be considered [13]. Moreover,
Mild detergents that are non-denaturing should be used to allow a partial separation
by column chromatography. However the dual nature of the proteins often leads to
multiple elution points during chromatography depending on which part of the protein
surface has bound to the separation phase.(Source:
http://proteomics.swegene.lu.se/M=research/L=research-membrane)
Up to now, many analytical strategies can be used to separate membrane proteins.

Proteomic approach which aims to detect whole expressed proteins to analyze the
function of such proteins and the functional linkage between them is one of the
important clinical analyses. For this analytical aim, SDS–PAGE and/or 2-dimensional
electrophoresis in conjunction with isoelectric focusing (IEF) or blue native (BN)
electrophoresis are frequently employed. 2D electrophoresis combined with IEF is
widely performed for membrane protein samples. Mass Spectrometry (MS) is the
ultimate technique for the accurate measurement of protein molecular weight (<0.1%
error). In all cases, it can be used to confirm protein identity [18]. However, most
proteome analyses have had to ignore these membrane proteins since most do not
run on 2D gels. If denaturing conditions are used, the hydrophobic regions become
very prone to self-aggregation and this is the major limiting factor in the analysis of
membrane proteins by 2D PAGE. Currently the only viable two-dimensional
separation method is the BAC/SDS diagonal gel method which has a rather low
resolution. Although it has been demonstrated that nearly all membrane proteins can
be separated by one-dimensional SDS-PAGE and identified by mass spectrometry
[7], the disadvantage of this method is that the quantitative aspect is lost and
comparative analyses of protein expression by gel densitometry cannot be carried
out. Therefore, the solutions to analysis of the subunit components in an isolated
membrane complex better are to understand the function of the membrane protein
complex completely and utilize the proper methods and equipments in terms of the
special characters of each membrane protein.
This section will discuss three recent applications; two of them are on the purification
of Gram-negative bacteria’s outer membrane proteins, one is on the prostate-specific
membrane antigen (PSMA).
Beis and his co-workers’ study described the development of a two-step purification
protocol for Escherichia coli outer integral membrane proteins, Wza and Osmoporin
C (OmpC) proteins. In this research, the two very different proteins were purified to
homogeneity by anion exchange and size exclusion chromatography and the purity
of the samples was judged by electrophoretic analysis, mass spectrometry, single
particle analysis, three-dimensional (3D) crystallisation and X-ray diffraction. At first,
Bacterial cells were disrupted and a crude extraction procedure that was the same
for both the Wza and OmpC proteins was fractionated by ultracentrifugation. The
membranes were solubilised overnight at room temperature with rolling and insoluble
materials were then removed by centrifugation. Secondly, initially purification of Wza
was performed using anion exchange chromatography and then Wza was purified
using a linear gradient of 0–100% 1M NaCl elution buffer and size exclusion
chromatography.[2] Finally, the purification of the OmpC protein was carried out
using the same purification protocol as for the Wza [1].
But a problem to separate membrane protein complexes appeared during this

process, size exclusion chromatography failed to separate the Wza from the OmpA.
To avoid this, the co-elution with the Wza protein on the anion exchange
chromatography step is used. The elution protocol was changed to a step gradient
instead of a linear elution and Wza could be eluted with 12% and OmpA with 22% of
1MNaCl elution buffer, respectively. At last, the electron microscopy analysis showed
that the Wza protein can be obtained (Fig. 2.8.4.1b). In addition, X-ray diffraction
studies of the crystals revealed that the two protein batches behave very differently
but in some cases they were in the same shape, an orthorhombic crystal form. For
the detergent choice, extraction of the protein was achieved by using the inexpensive
swittergent SB3-14, and only exchanged to the expensive and more suitable
detergent bOG prior to crystallisation. Again, using a linear gradient resulted in the
elution of OmpC with contaminants. The step elution approach resulted in the
production of highly pure OmpC (Fig. 2.8.4.2a). [2] From this application, it was found
that protein impurity alters the nucleation and/or the growth of the Wza and OmpC
crystals and two or more chromatography steps can be used to make up their
disadvantages each other.
Fig.2.8.4.1 Characterisation of protein impurities and their effects on Wza
crystallisation.
(a) Electrophoretic analysis of Wza. Lane 1 shows the extracted protein, lane 2Wza after anion exchange, lane 3 the
pure Wza after gel filtration and lane 4 shows the Wza without heating to show the multimeric complex. Molecular
weight markers are shown on the left side of the SDS-PAGE in kDa. Electron microscopy analysis of Wza (b) and
Wza–OmpA (d) samples. (c and e) Effect of purification on the growth of well-ordered crystals. Scale bar 100 mm.[2]
Fig.2.8.4.2 (a) SDS-PAGE of OmpC protein. Lane 1 shows the extraction of OmpC from outer membranes, lane
2 the OmpC after the anion exchange chromatography step and lane 3 pure OmpC after size exclusion
chromatography. Molecular weight markers are shown on the left side of the SDS-PAGE in kDa. (b) 3D crystals of
OmpC. Scale bar 100 mm. [2]
Neisserial porins represent more than 60% of outer membrane proteins [12], so the
second application is about the outer membrane protein PorB of Neisseria
meningitides. This protein has been shown to up-regulate the surface expression of
the co-stimulatory molecule CD86 and of MHC class II, be involved in prevention of
apoptosis by modulating the mitochondrial membrane potential and form pores in
eukaryotic cells [11]. As an outer membrane protein, its native trimeric form isolation
is complicated by its insoluble nature and it required the presence of detergent
throughout the whole procedure.
In order to obtain a pure protein it is necessary to analysis the components of the

contaminant and their removing conditions. In this case, DNA, lipooligosaccharide
(LOS) and other debris were the major effects of the impurity of PorB. During this
process, PorB was purified to homogeneity from a mutant meningococcal strain and
the crude outer membrane protein preparation was obtained by extraction with the
zwittergent-Ca2+method [3]. The pellet obtained from this step, described as starting
material (SM), contains all the outer membrane proteins and also residual LOS and
lipoproteins. Initially, two ion exchange columns in tandem were used as the first step
at pH 8.0: a DEAE Sepharose CL-6B column and a CM-Sepharose column. PorB
was recovered almost exclusively in the column flow through (FT), which also
contained some contaminating protein species, LOS and lipoprotein (Fig.2.8.4.3A).
Secondly, size-exclusion chromatography efficiently separated PorB from most of the
residual protein contaminants, but not from LOS or from lipoproteins, such as H.8. A
third step was performed using a resin which does not bind endotoxins, Matrex
Cellufine Sulfate, which bound PorB at pH 7.5 but did not retain LOS or lipoproteins,
allowing their removal with the column flow through. By applying a 0.2–0.5M linear
gradient of NaCl, PorB was eluted between 0.24 and 0.4M NaCl (Fig. 2.8.4.3C) [11,
22]. The analysis demonstrated that LOS and H.8 were absent in the pooled
fractions. These specific contaminants were thus very efficiently removed from the
final product and purified PorB is regained at the end of the purification method by
removing the detergent from the protein solution by extensive dialysis and formation
of protein (Fig. 2.8.4.3D). This case provided several methods to separate the
cofactors that may exit in membrane protein complexes such as changing pH, the
linear gradient of NaCl.
Fig.2.8.4.3 Chromatographic purification of N. meningitidis PorB.
SM, column starting material; FT, column flow through; and pool, PorB-containing fractions. (A) Ion-exchange
chromatography on DEAE/CM columns. The position of PorB and LOS is indicated by the arrows on Coomassie and
silver staining of SDS–PAGE, respectively. (B) Size-exclusion chromatography on Sephacryl S300 column. The
position of PorB, LOS, and H.8 is indicated by the arrows on Coomassie, silver staining, and Western blot with
specific anti-H.8 antibody, respectively. (C) Affinity chromatography on Matrex Cellufine Sulfate column. PorB, LOS,
and H.8 are identified as above. (D) PorB formed into proteosomes free of detergent. The position of PorB and LOS
is indicated by the arrows on Coomassie and silver stain as above. Furthermore, PorB can be detected as a band on
the silver-stained gel, as indicated by the asterisk. [11]
Another case is on PSMA, a membrane protein that has attracted significant attention
as a target for immunioscintigraphic and radioimmunotherapeutic applications for
prostate cancer. For this purification, they optimized the purification of native PSMA
from LNCaP cells using conformational epitope-specific antibody-affinity
chromatography. In contrast to general affinity chromatography, this chromatography
for the purification of native PSMA employs resin bound anti-PSMA monoclonal
antibody 3C6 that reacts with a protein conformational epitope present in the
extracellular portion of human PSMA [21]. As this antibody binds PSMA when
present in a native conformation, only PSMA in a native conformation is retained by
the affinity resin. Then, there are three further methods to purify PSMA and a
comparison of these methods explored is outlined in Table 2.8.4[8]. Western blot
analysis and an HPLC-based enzymatic activity assay were used to compare the
yield and results demonstrated that all three methods provided similar yields of
PSMA. Method A resulted in the least amount of PSMA in a non-native conformation
(0.9%) suggesting that PSMA initially purified by this method was predominately in
an active conformation. These results were consistent with enzymatic activity data
obtained in which PSMA purified by this method exhibited the greatest enzymatic
activity when compared to the other two methods. The ratio of purified PSMA in a
native and active conformation was determined by quantifying the amount of non-
native PSMA not retained in second antibody-affinity isolation. The low amount of
denatured PSMA obtained in Method A when compared to Method B demonstrates
that high pH conditions promote denaturation of PSMA. In comparison to Method B,
Method C confirmed the importance of the essential Zn2+ cofactor during the
desalting step. [8] This result also suggests that Zn2+ may have a stabilizing role in
addition to its functional role in PSMA’s enzymatic activity, In other words, the
addition of both a neutralization step and the inclusion of Zn2+ to the equilibration
buffer in desalting step provides considerable enhancement in the yield of active
PSMA from LNCaP cells.
Table 2.8.4 comparison of methods for the purification of PSMA. [8]
Overall, it is necessary to be considered that not only the methods to separate

common proteins but also the features and environments that membrane proteins
exit. As one coin has two sides, these difficult factors such as hydrophobicity, closely
associate with the membrane, multi-subunit complexes, many cofactors, and several
easily detached peripheral proteins also hint at some solutions to produce membrane
proteins.
1. National Library of Medicine - Medical Subject Headings (2006)

http://www.nlm.nih.gov/cgi/mesh/2006/MB_cgi?mode=&term=Membrane+proteins
2. SWEGENE Proteomics Platform (2002)
http://proteomics.swegene.lu.se/M=research/L=research-membrane
2.8.6 References
1. Baalaji, N.S., Mathew, M.K. & Krishnaswamy, S. (2006) Functional assay of

Salmonella typhi OmpC using reconstituted large unilamellar vesicles: a general
method for characterization of outer membrane proteins. Biochimie. 1-6.
2. Beis, K., Whitfield, C., Booth, I. & Naismith, J.H. (2006) Two-step purification of
outer membrane proteins. International Journal of Biological Macromolecules. 39,
10–14.
3. Blake, M.S. & Gotschlich, E.C. (1984) Purification and partial characterization of
the opacity-associated proteins of Neisseria gonorrhoeae. J. Exp. Med. 159, 452–
462.
4. Jidenko, M., Lenoir, G., Fuentes, J.M., Maire, M. & Jaxel, C. (2006) Expression in
yeast and purification of a membrane protein, SERCA1a, using a biotinylated
acceptor domain. Protein Expression and Purification. 48, 32–42.
5. Kashino, Y. (2003) Separation methods in the analysis of protein membrane
complexes. Journal of Chromatography B. 797, 191–216.
6. Khushiramani, R., Girisha, S.K., Karunasagar, I. & Karunasagar, I. (2006) Cloning
and expression of an outer membrane protein ompTS of Aeromonas hydrophila and
study of immunogenicity in Wsh. Protein Expression and Purification. 4, 1–5.
7. Lin, C., Cotton, F., Boutique, C., Dhermy, D., Vertongen, F. & Gulbis, B. (2000)
Capillary gel electrophoresis: separation of major erythrocyte membrane proteins.
Journal of Chromatography B. 742, 411–419.
8. Liu, T., Toriyabe, Y. & Berkman, C.E. (2006) Purification of prostate-specific
membrane antigen using conformational epitope-specific antibody-affinity
chromatography. Protein Expression and Purification. 1-5.
9. Lunde, C.S., Rouhani, S., Facciotti, M.T. & Glaeser, R.M. (2006) Membrane-
protein stability in a phospholipid-based crystallization medium. Journal of Structural
Biology. 154, 223–231.
10. Mamelli, L., Pagès, J., Konkel, M.E. & Bolla, J. (2006) Expression and
purification of native and truncated forms of CadF, an outer membrane protein of
Campylobacter. International Journal of Biological Macromolecules. 39, 135–140.
11. Massari, P., King, C.A., MacLeod, H. & Wetzler, L.M. (2005) Improved
purification of native meningococcal porin PorB and studies on its structure/function.
Protein Expression and Purification. 44, 136–146.
12. Minetti, C.A., Blake, M.S. & Remeta, D.P. (1998) Characterization of the
structure, function, and conformational stability of PorB class 3 protein from Neisseria
meningitidis. A porin with unusual physicochemical properties. J. Biol. Chem. 273,
25329–25338.
13. Mohanty, A.K. & Wiener, M.C. (2004) Membrane protein expression and
production: effects of polyhistidine tag length and position. Protein Expression and
Purification. 33, 311–325.
14. Niegowski, D., Hedrén, M., Nordlund, P. & Eshaghi, S. (2006) A simple strategy
towards membrane protein purification and crystallization. International Journal of
Biological Macromolecules. 39, 83–87.
15. Raymond, F., Rolland, D., Gauthier, M. & Jolivet, M. (1998) Purification of a
recombinant protein expressed in yeast: optimization of analytical and preparative
chromatography. Journal of Chromatography B. 706, 113–121.
16. Reinhard, G.（2006）Understanding recombinant expression of membrane
proteins. Current Opinion in Biotechnology. 17, 337–340.
17. Rucevic, M., Clifton, J.G., Huang, F., Li, X., Callanan, H., Hixson, D.C.& Josic, D.
(2006) Use of short monolithic columns for isolation of low abundance membrane
proteins. Journal of Chromatography A. 1123, 199–204.
18. Schwabe, T.M.E., Gloddek, K., Schluesener, D. & Kruip, J. (2003) Purification of
recombinant BtpA and Ycf3, proteins involved in membrane protein biogenesis in
Synechocystis PCC 6803. Journal of Chromatography B. 786, 45–59.
19. Sharov, V.S., Galeva, N.A., Knyushko, T.V., Bigelow, D.J., Williams, T.D. &
Schoneich, C. (2002) Two-dimensional separation of the membrane protein
sarcoplasmic reticulum Ca–ATPase for high-performance liquid chromatography–
tandem mass spectrometry analysis of posttranslational protein modifications.
Analytical Biochemistry. 308, 328–335.
20. Tamm, L.K. & Liang, B. (2006) NMR of membrane proteins in solution. Progress
in Nuclear Magnetic Resonance Spectroscopy. 48, 201–210.
21. Tino, W.T., et al. (2000) Isolation and characterization of monoclonal antibodies
specific for protein conformational epitopes present in prostate-specific membrane
antigen (PSMA). Hybridoma 19, 249–257
22. Wetzler, L.M., Blake, M.S. & Gotschlich, E.C. (1988) Characterization and
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.
Charpter 2.9 industrial Scale Purification of Proteins
Guo lu
2.9.1 Introduction
The expansion of techniques and methods for protein purification has been an
indispensable pre-requisite for numerous of the advancements made in
biotechnology of the large scale purification of proteins. Most of the products of
biotechnology are proteins; moreover, these proteins must be arranged in large
volumes in purified form. Generally, if contaminants can be detected, they must be
detached or proven to be harmless. Beside that, the protein must be purified from
other proteins. However, the nucleic acids, carbohydrates, lipids or any other
materials in the sample should not be ignored.
To purify proteins, their inherent similarities and differences should be used. Protein
similarity is applied to purify them away from the other non-protein contaminants.
Alternatively, the differences are applied to purify one protein from another. Proteins
differ from each other in size, shape, charge, hydrophobicity, solubility, and biological
activity. Additionally, the protein product must preserve its biological activity. It is
noticeable that a method which works deftly in a research laboratory may fail
miserably on the industrial production that must be in large scale and reproduced
exactly. The Three Phase Purification Strategy is used as a support to the
development of purification processes for therapeutic proteins in the pharmaceutical
industry. The overall step is shown in figure 2.9.1.[1]
Figure 2.9.1 Preparation and the Three Phase Purification Strategy

Table 2.9.1. Protein properties and their effect on development of purification
strategy
Ion Exchange (IEX) Chromatography
Ion exchange chromatography (IEX or IEC) has become one of the best-known
methods for protein and peptide purification because it offers scalability, high
specificity, and wide choice of column materials. It relies on reversible charge
interactions between a charged biomolecule (such as a protein or nucleic acid) and
an oppositely charged resin-based matrix.
(http://www.biocompare.com/spotlight.asp?id=255)
Hydrophobic Interaction Chromatography(HIC)
With the development of the modern biotechnology industry and its desire for highly
purified pharmaceutical proteins, a advance emphasis has been positioned on entire
processes with admiration to the economy, capacity and the quality of resultant
product. Usually, the separation extend power required is controlled by the need to
resolve the product. It is not only from the background impurities resulting from the
fermentation but also from degradation products and analogues of the drug itself. For
many cases, hydrophobic interaction chromatography (HIC) is an ideal separation
method.
(http://teachline.ls.huji.ac.il/72682/Booklets/AMERSHAM_hydrophobic_interactionMa
nual.pdf)
Alois, Waltraud and Robert believed that the correct folding of solubilized
recombinant proteins is of key importance for their production in industry. Many
recent improvements have been made to the use of of immobilized metal affinity
chromatography and by mimicking the natural folding process with artificial
chaperones. [2]
Affinity Chromatography (AC)
Affinity Chromatography (AC) is a technique that is able to purify a biomolecule with

biological function or individual chemical structure. The material to be purified is
specifically and reversibly, also, it should be adsorbed to a ligand (binding
substance), immobilized by a covalent bond to a chromatographic bed material
(matrix). Changing experimental conditions to favour desorption is the way to achieve
the recovery of molecules. AC media are commonly used for applications. For
example, it is good for purification of fusion proteins, mono- and polyclonal
antibodies, and
glycoproteins.(http://www4.amershambiosciences.com/aptrix/upp00919.nsf/Content/
LabSep_EduC~LC_tech~AC)
Gejing and Gautam pointed out that in the affinity-based approach,compounds are
screened based on their binding affinities to target molecules. The interaction
between targets and compounds can be directly evaluated by monitoring the
formation of non-covalent target–ligand complexes (direct detection) or indirectly
evaluated by detecting the compounds after separating bound compounds from
unbound (indirect detection). Various techniques including high performance liquid
chromatography (HPLC)–MS, size exclusion chromatography (SEC)–MS, frontal
affinity chromatography (FAC)–MS and desorption/ionization on silicon (DIOS)–MS
can be applied. [3]
Gel Filtration (GF)
While possibly the most simple column chromatography technique, gel filtration (GF)
chromatography is one of the most flexible since it can be performed under a
diversity of physical and chemical conditions and normally does not involve an
complicated protocol.1,2 GF (also called size-exclusion chromatography) separates
globular proteins due to their molecular weights. The liquid volume can be "seen" by
the column which consists of a mobile phase and a stationary phase. (http://www.the-
scientist.com/article/display/12820/)
In the recent research by Damien etl., they set up a heterologous expression system
in Sf9 insect cells allowing the expression and production of large amounts of a pure
active human protein and use gel filtration to purify the target protein.[4]
Reversed Phase Chromatography (RPC)
Reversed Phase Chromatography includes any chromatographic method which uses

a non-polar stationary phase. Mathematical and experimental are used in other
chromatographic methods apply. For example, separation resolution is proportional
to the column length and inversely proportional to the column width. It has been
widely used in the pharmaceutical, chemical, and biochemical industry for separating
molecules of small molecular weight. Moreover, in recent years RPC has been
applied to separate larger molecules. (http://en.wikipedia.org/wiki/Reversed-
phase_chromatography)
According to Pattana, Penporn and Aurasorn, Chromatographic separation was

achieved with a reversed-phase Apollo C18 column and a mobile phase of
methanol–acetonitrile-mixed phosphate buffer (pH 2.6; 10 mM) (40:12:48, v/v/v) with
a flow rate of 1.2 ml/min. That proves the RPC is an impact method especially in the
protein purification of pharmaceutical industry. [5]
Expanded Bed Adsorption (EBA)
EBA uses equipment that is known to most users of standard liquid chromatography.
The column has a flow adapter which is located to suit the specific step of resin
preparation or protein purification. Besides that, a series of pumps and valves,
connected through the adapter and bottom of the column is to control the flow rate
and direction of the buffer and sample loading. Therefore, it is possible to perform
initial EBA trials with a little ingenuity and standard chromatographic equipment.
(http://pubs.acs.org/subscribe/journals/mdd/v04/i12/html/12toolbox.html)
Three anion exchanger expanded bed adsorption (EBA) matrices: Streamline DEAE,
Streamline Q XL and Q Hyper Z were evaluated with the aid of EFGP from an
ultrasonic homogenate of Escherichia coli in the research of Cabanne, etl. Based on
the results, the two other matrices gave a good purification of the EGFP (7–15-fold)
but the Q Hyper Z matrix appeared to give the best results. It is composed of little
size and density beads which lead to a higher exchange surface and then a better
mass transfer. [6]
It has been considered to be one of the best-known methods as it is high specificity,

in large scale and has a huge choice of column materials.
Hydrophobic Interaction Chromatography(HIC)
Despite it is used commonly in the industrial scale of purification, it still has some
aspects that influence the product.Based on the inflexibility of the media, the loss of
wall support in a large scale column will have a smaller or greater impact on bed
compression, with associated deterioration of the flow/pressure properties of the
packed bed. The effect of bed compression can be checked by running a
pressure/flow rate curve such as outlined under ‘‘Packing large scale columns’’. Zone
spreading can also be caused by non-column factors such as increased internal
volumes of pumps, valves and monitoring cells and different lengths and diameters of
pipes or tubing. If all the above aspects of scaling up are taken into consideration,
chromatographic variability is normally not a big issue when scaling-up.
In the study of Alois,Christine and Rainer , they considered that HIC exploits the
hydrophobic properties of protein surfaces for separation and purification by
performing interactions with chromatographic sorbents of hydrophobic nature. In
contrast to reversed-phase chromatography, this methodology is less detrimental to
the protein and is therefore more commonly used in industrial scale as well as in
bench scale when the conformational integrity of the protein is important. [7]
Gel Filtration (GF)
There is one factor to consider when choosing a GF medium is the exclusion limit, or
the molecular weight limit, of the pores; proteins over this limit will be completely
excluded from the pores and will not be separated. It is also important to choose the
type of resin. A diversity of resins exists, ranging from silica-based to polymeric.
Some companies recommend cross-linked resins, which are advantageous for high-
pressure purifications as they do not compress and lose porosity under high-pressure
conditions. Nevertheless, non-cross-linked resins are appropriate for routine
purifications.
GF has a number of advantages over other types of liquid chromatography. First,

there is a high upper limit on the size of the proteins which can be purified by means
of this technique. In addition, the pore shape is perfect for separating globular
molecules such as proteins, and the technique does not have need of the use of
protein-denaturing organic solvents. However, this method can be difficult to fine-
tune because protein resolution depends on the sample volume applied to the
column. Therefore, dilute samples are difficult to purify by this technique. In
conclusion, there are two disadvantages for large-scale purification, one is GF media
is expensive;the other is GF chromatography is not directly scalable from an
analytical to a bulk purification level.
It can chose from a variety of column configurations based on the use of the
purification, and the amount of material available. The smaller inner diameter is used
for high resolution separations with very little protein wanted, while large columns
may be demand for industrial protein purifications. Analytical columns with diameters
of up to 5 mm are daily use RPC columns for routine analytical and purification work.
Preparative columns are larger in diameter and can be used for purification of large
quantities of proteins from the range of mg to gram.
The results of the research of Janine, Colin and Robert showed the excellent
potential of one-step RP-HPLC for purification of recombinant proteins from cell
lysates, where high yields of purified product and greater purity are achieved
compared to affinity chromatography. And they suggested that this approach was
also successful in purifying just trace levels (<0.1% of total contents of crude sample)
of TM 1–99 from a cell lysate. [8]
In the more traditional packed-bed methods, the clogging occurs when particulate
matter and cell debris cannot flow around the closely packed resin beads because
the resin is confined between the bottom of the column and the flow adapter.
Compared with the conventional methods, EBA columns are fed from below, and the
adapter is held away from the packed resin level in order to give the resin room to
expand .As a consequence creates spaces between the beads.
IEX separates proteins by the uses of differences in charge to give a very high
resolution separation with high sample loading capacity. It is relay on the reversible
interaction between a charged protein and an oppositely charged chromatographic
medium. Conditions are then altered thus bound substances are eluted differentially.
Increasing in salt concentration or changing in pH can perform this elution. During
binding, target proteins are concentrated and collected in a purified, concentrated
form.
Normally IEX is applied to bind the target molecule, however, it can also be used to
bind impurities if necessary. At different pH values, IEX can be repeated to separate
numerous proteins which have noticeably different charge properties. During a multi-
step purification, this can be used to advantage. [1] In the research of Khandeparkar
and Bhosle, they use this method to Isolation, purification and characterization of the
xylanase and get a expected results. This indicates that this method is effective for
industrial applications. [9]
Hydrophobic Interaction Chromatography (HIC)
HIC separates proteins through differences in hydrophobicity. The technique is ideal

for the capture or intermediate steps in large scale purification. It is based on the
reversible interaction between a protein and the hydrophobic surface of a
Chromatographic medium. High ionic strength buffer, which makes HIC an ideal 'next
step', with ammonium sulphate or elution in high salt, will enhance the interaction
during IEX. Samples in high ionic strength solution attach when they are loaded onto
a column. When conditions are altered, as a result, the bound substances are eluted
differentially.Generally, samples are eluted with a declining gradient of ammonium
sulphate. During binding, target proteins are concentrated and collected in a purified,
concentrated type. [1]
Tony, Lloyd, and Alfons pointed out that they have developed expedient and reliable
methods to isolate cyclosporin synthetase for in vitro biosynthesis of cyclosporins
which is use the Hydrophobic Interaction Chromatography(HIC). It is said that the
industrial implementation of an in vitro biosynthetic approach could potentially prove
useful for the production of important therapeutic cyclosporins which occur as only
minor fermentation by-products.[10]
Figure 2.9.4.1 typical HIC gradient elution
Affinity Chromatography (AC)
Affinity chromatography can be applied to Purify and concentrate a molecule from a

mixture into a buffering solution. It also can reduce the amount of a molecule in a
mixture. Moreover, it can discern the biological compounds bind to a particular
molecule, like drugs.
This method separates proteins on the base of a reversible interaction between a

protein and a specific ligand attached to a chromatographic matrix. It is ideal for a
capture or intermediate step. Also, it can be used while a suitable ligand is available
for the protein of interest. It is high selectivity, hence high resolution, and usually high
capacity for the target protein. The target protein is distinctively and reversibly bound
with a complementary binding substance (ligand). Desorption is performed
particularly, using a competitive ligand, or non-specifically, with changing the pH,
ionic strength or polarity. During binding, samples are concentrated and protein is
collected in purified, concentrated type. The key stages in a separation are shown in
Figure 40. it is also applied to remove specific contaminants, for instance,
Benzamidine Sepharose 6B removes serine proteases. [1]Haijie and Tian had
successfully used this method to purify a calcium-independent lectin (PjLec) from the
haemolymph of the shrimp Penaeus japonicus. This means this method may be
application in the industrial scale purification of calcium-independent lectin.[11]
Figure 2.9.4.2 typical affinity separation
Gel Filtration (GF)
GF separates proteins by means of differences in molecular size. This method is

idyllic for the final polishing steps in purification in the situation that sample volumes
have been reduced. Samples are eluted isocratically while Buffer conditions are
wide-ranging to outfit the sample type or the demand for extra purification, analysis or
storage step, as buffer composition does not directly affect resolution. Then products
are collected in purified form in the chosen buffer.[1] The study of Bernal, Cair o and
Coello Showed that a novel keratinase activity been purified from the bioreaction
broth growing media to apparent homogeneity after single step, (24-fold purification
with a high yield of 54%) using DEAE column chromatography. They believed that all
of the biochemical characteristics, raising the potential use of this enzyme in
numerous industrial applications by using GF method. [12] Also, bata-mannanase
from Trichoderma harzianum strain T4 had been purified by Humberto and Edivaldo,
and they indicated that the thermal stability of the purified Man I make this enzyme
attractive for use in industrial applications by using the GF method to purification in a
large scale[13].
Figure 2.9.4.3 typical GF elution
RPC separates proteins and peptides by means of contrary hydrophobicity relay on

their reversible interaction with the hydrophobic surface of a chromatographic
medium. The binding is usually very strong and requires the use of organic solvents
and other additives (ion pairing agents) for elution because of the nature of the
reversed phase matrices. The key stages in a separation are shown in Figure 43. It is
frequently used in the final polishing of oligonucleotides and peptides and is supreme
for analytical separations, such as peptide mapping. However, RPC is not suggested
for protein purificationin the condition of recovery of activity and return to a correct
tertiary structure are required, because many proteins are denatured in the presence
of organic solvents.[1] Janine, Colin and Robert had developed a one-step facile,
flexible and readily scalable purification method for a recombinant protein, TM 1–99
(113 amino acid residues; 12,837 Da) based on reversed-phase high-performance
liquid chromatography (RP-HPLC) from an E. coli cell lysate.[8]
Figure 2.9.4.4 typical RPC gradient elution
EBA is a single pass operation in which target proteins are purified from crude
sample, without the need for separate clarification, concentration and initial
purification to remove particulate matter. Figure 44a shows the steps involved in an
EBA purification and Figure 44b shows a typical EBA elution pattern. In the research
of Jian-Feng, Guang-Ce and Cheng-Kui, they applied several methods to isolate and
purify large-scale of R-phycoerythrin from red alga Polysiphonia urceolata Grev.
However, the results indicate that using the expanded bed adsorption combined with
ion-exchange chromatography or hydroxyapatite chromatography, R-phycoerythrin
can be puriWed from frozen P. urceolata on large scale. [14]
Figure 2.9.4.5a steps in an EBA purification process
Figure 2.9.4.5b typical EBA elution

http://www.biocompare.com/index.asp
http://www.biocompare.com/spotlight.asp?id=255
http://teachline.ls.huji.ac.il/72682/Booklets/AMERSHAM_hydrophobic_interactionMan
ual.pdf
http://www4.amershambiosciences.com/aptrix/upp00919.nsf/Content/LabSep_EduC
~LC_tech~AC
http://www.the-scientist.com/article/display/12820/
http://en.wikipedia.org/wiki/Reversed-phase_chromatography
http://pubs.acs.org/subscribe/journals/mdd/v04/i12/html/12toolbox.html
2.9.6 Reference
[1]Protein Purification Handbook EditionAB Amersham Pharmacia biotech

https://www4.amershambiosciences.com/aptrix/upp00919.nsf/(FileDownload)?Open
Agent&docid=9C7BA3DA6539F07AC1256EB40044A8B2&file=18113229AC.pdf
[2]Jungbauer, A., W. Kaar, et al. (2004). "Folding and refolding of proteins in

chromatographic beds." Current Opinion in Biotechnology 15(5): 487-494.
[3]Deng, G. and G. Sanyal (2006). "Applications of mass spectrometry in early

stages of target based drug discovery." Journal of Pharmaceutical and Biomedical
Analysis 40(3): 528-538.
[4]Fleury, D., P. Domaingue, et al. "Expression, purification, characterization and

crystallization of a recombinant human cytosolic [beta]-glucosidase produced in
insect cells." Protein Expression and Purification In Press, Corrected Proof.
[5]Sripalakit, P., P. Neamhom, et al. (2006). "High-performance liquid

chromatographic method for the determination of pioglitazone in human plasma
using ultraviolet detection and its application to a pharmacokinetic study." Journal of
Chromatography B 843(2): 164-169.
[6]Cabanne, C., A. M. Noubhani, et al. (2004). "Evaluation of three expanded bed

adsorption anion exchange matrices with the aid of recombinant enhanced green
fluorescent protein overexpressed in Escherichia coli." Journal of Chromatography B
808(1): 91-97.
[7]Jungbauer, A., C. Machold, et al. (2005). "Hydrophobic interaction
chromatography of proteins: III. Unfolding of proteins upon adsorption." Journal of
Chromatography A 1079(1-2): 221-228.
[8]Mills, J. B., C. T. Mant, et al. (2006). "One-step purification of a recombinant

protein from a whole cell extract by reversed-phase high-performance liquid
chromatography." Journal of Chromatography A 1133(1-2): 248-253.
[9]Khandeparkar, R. D. S. and N. B. Bhosle (2006). "Isolation, purification and

characterization of the xylanase produced by Arthrobacter sp. MTCC 5214 when
grown in solid-state fermentation." Enzyme and Microbial Technology 39(4): 732-
742.
[10]Velkov, T., L. G. Singaretnam, et al. (2006). "An improved purification procedure

for cyclosporin synthetase." Protein Expression and Purification 45(2): 275-287.
[11]Yang, H., T. Luo, et al. (2007). "Purification and characterisation of a calcium-

independent lectin (PjLec) from the haemolymph of the shrimp Penaeus japonicus."
Fish & Shellfish Immunology 22(1-2): 88-97.
[12]Bernal, C., J. Cairo, et al. (2006). "Purification and characterization of a novel
exocellular keratinase from Kocuria rosea." Enzyme and Microbial Technology 38(1-
2): 49-54.
[13]Malheiros Ferreira, H. and E. Ximenes Ferreira Filho (2004). "Purification and

characterization of a [beta]-mannanase from Trichoderma harzianum strain T4."
Carbohydrate Polymers 57(1): 23-29.
[14]Niu, J.-F., G.-C. Wang, et al. (2006). "Method for large-scale isolation and
purification of R-phycoerythrin from red alga Polysiphonia urceolata Grev." Protein
Expression and Purification 49(1): 23-31.
Chapter 2.10 Determination of Protein Concentration and
Purity
Xiao zheng Mu
2.10.1 Introduction
Biochemical research often requires the quantitative measurement of protein

concentration and purity in solutions. Many techniques have been developed;
however, most have limitations because either they are not sensitive enough or they
are based on reactions with specific amino acids in the protein. Since the amino acid
content varies from protein to protein, no single assay will be suitable for all proteins.
In this chapter, we will discuss 14 methods which are used in protein concentration
and purity determination area.
In biuret Method, Lowry Method, Bradford Method and BCA Method, chemical
reagents are added to protein solutions to develop a color whose intensity is
measured in a spectrophotometer. A “standard protein” of known concentration is
also treated with the same reagents and a calibration curve is constructed.
Electrophoresis is an analytical tool by which biochemists can examine the
movement of charged molecules in an electric field. There are several
electrophoresis which are very helpful for the analysis of protein concentration and
purity: Polyacrylamide Gel Electrophoreisis (PAGE), Sodium Dodecyl Sulfate-
Polyacrylamide Gel Electrophoresis (SDS-PAGE), Isoelectric Focusing (IEF), Two-
Dimensional Electrophoresis (2-DE), Capillary Electrophoresis (CE) and
Immunoelectrophoresis(IE). The spectrophotometric Method relies on a direct
spectrophotometric measurement. There are two kinds of spectrophotometry which
can be used in protein concentration and purity determination, they are Ultraviolet-
Visible (UV) Absorption Spectrophotometry and Fluorescence Spectrophotometry.
High-performance liquid chromatography (HPLC) is ideally suited for the separation
and identification of amino acids, proteins, nucleic acids and many other biologically
active molecules. The use of nonpolar chemically bonded stationary phases with a
polar mobile phase is referred to as reverse-phase HPLC (RP-HPLC). Amino acid
analysis can also be used to determine amount of protein present by Automated
Edman Degration. None of the methods is perfect because each is dependent on the
amino acid content of the protein. However, each will provide a satisfactory result if
the proper experimental conditions are used and/or a suitable standard protein is
chosen. Other important factors in method selection include the sensitivity and
accuracy desired, the presence of interfering substances, and the time available for
the assay.
In the area of 2-DE, in recent years, some new methods came out such as
fluorescence 2-DE [1, 2]. Through labeling of samples with one of three specially
different fluorescent dyes, Cyanine-2, Cyanine-3 or Cyanine-5, the labeled samples
are then run in one gel and detected individually by scanning the gel at different
wavelengths. After quantitative analysis by the Phoretix/ImageMaster software, the
different expressed proteins can be obtained. Based on the principle of this method,
however, those proteins without lysine residues can not be labeled and lost. At the
same time, the high cost of the whole system prevents it from spreading out [3]. In
2003, Yuan et al. [3] reported a new IPG strip application, called multi-strips on one
gel method. This new method can not only improve the reproducibility and resolution
power of 2-DE pattern, but also achieve high throughput and economical format
which is helpful to automatic proteomic research.
A new technology has been introduced during the past few years that greatly
increased the speed of spectrophotometric measurements. New detectors called
photodiode arrays are being used in modern spectrometers. Photodiodes are
composed of silicon crystals that are sensitive to light in the wavelength range 170-
1100 nm. Upon photon absorption by the diode, a current is generated in the
photodiode that is proportional to the number of photons. Linear arrays of
photodiodes are self-scanning and have response times on the order of 100
milliseconds; hence, an entire UV-VIS spectrum can be obtained with an extremely
brief exposure of the sample to polychromatic light. New spectrometers designed by
Hewlett-Packard and Perkin-Elmer use this technology and can produce a full
spectrum from 190 to 820 nm in one-tenth of a second [4].
2.10.3 Evaluation of the Technologies
The biuret assay has several advantages including speed, similar color development
with different proteins, and few interfering substances. Its primary disadvantage is its
lack of sensitivity [4].
The obvious advantage of the Lowry assay is its sensitivity, which is up to 100 times
greater than that of the biuret assay; however, more time is required for the Lowry
assay. Since proteins have varying contents of tyrosine and tryptophan, the amount
of color development changes with different proteins, including the bovine serum
albumin standard. Because of this, the Lowry protein assay should be used only for
measuring changes in protein concentration, not absolute values of protein
concentration [4]. Specialist literature contains a multitude of modifications for the
Lowry assay. The principal target is to reduce the high susceptibility to interference.
The Lowry method is adversely affected by a wide range of non-proteins. Additives
such as EDTA, ammonia sulfate or Triton X-100 in particular are incompatible with
the test.
The Bradford method is twice as sensitive as the Lowry or BCA test and is thus the
most sensitive quantitative dye assay. It is the easiest to handle and most rapid
method and has the additional advantage that a series of reducing substances (e.g.
DTT and mercaptoethanol), which interfere with the Lowry or BCA test, have no
adverse effect on results. However, it is sensitive to detergents. The main
disadvantage is that identical amounts of different standard proteins can cause
considerable differences in the resulting absorption coefficients. With a microassay
procedure, the Bradford assay can be used to determine proteins in the range of 1 to
20 µg. The Bradford assay shows significant variation with different proteins, but this
also occurs with the Lowry assay. The Bradford method not only is rapid but also has
very few interference by nonprotein components. The only known interfering
substances are detergents, Triton X-100 and sodium dodecyl sulfate. The many
advantages of the Bradford assay have led to its wide adoption in biochemical
research laboratories [4]. In the study of Giraudi et al. [5], they mentioned that a
disadvantage of Bradford assay is the variability of colour development with different
proteins: the absorbance change per unit mass of protein varies with the nature of
the protein assayed. They believe that the Bradford method should give a lower
protein concentration than the real value due to the lower probability of interaction
between dye molecules and free lysine residue.
In the study of Lucarini and Kilikian [6], the methods of Lowry and Bradford were
compared regarding the level of interference of some substances used for
glucoamylase precipitation by ethanol. The method of Bradford suffers no
interference while the method of Lowry showed protein concentration values 20%
increased in the presence of ethanol and Tris. They also mentioned that despite
these interferences, the Lowry method can evaluate more accurately the increase of
purity during fractionation, due to its sensitivity to low molecular weight (below 6 kDa)
proteins and peptides.
The BCA protein assay is based on chemical principles similar to those of the biuret
and Lowry assays. This assay has the same sensitivity level as the Lowry and
Bradford assays. Its main advantages are its simplicity and its usefulness in the
presence of 1% detergents such as Triton or sodium dodecyl sulfate (SDS) [4]. This
test is easier to carry out and sensitivity can be varied using different temperatures.
Furthermore, the dye complex is very stable. However, this test is highly susceptible
to interference, although on the positive side, its insensitivity to detergents is similar
to that of the Lowry method.
Perhaps the most difficult and inconvenient aspect of PAGE is the preparation of
gels. The monomer, acrylamide, is a neurotoxin and a cancer suspect agent; hence,
special handling is required. Other necessary reagents including catalysts and
initiators also require special handling and are unstable. In addition, it is difficult to
make gels that have reproducible thickness and compositions. Many researchers are
now turning to the use of precast polyacrylamide gels. Several manufacturers now
offer gels precast in glass or plastic cassettes. Gels for all experimental operations
are available including single percentage (between 3 and 27%) or gradient gel
concentrations and a variety of sample well configurations and buffer chemistries.
Several modifications of PAGE have greatly increased its versatility and usefulness
as an analytical tool [4].
SDS-PAGE is valuable to estimating the molecular weight of protein subunits. This

modification of gel electrophoresis finds its greatest use in characterizing the sizes
and different types of subunits in oligomeric proteins. SDS-PAGE is limited to a
molecular weight range of 10,000 to 200,000. Gels of less than 2.5% acrylamide
must be used for determining molecular weights above 200,000, but these gels do
not set well and are very fragile because of minimal cross-linking. A modification
using gels of agarose-acrylamide mixtures allows the measurement of molecular
weights above 200,000 [4]. SDS-PAGE is a fundamental method for 2-DE analysis,
since it represents the second dimension run and the run that will finally remain in the
record, since it is at the end of this step that proteins are stained, or blotted and
extracted and further analysed with the powerful tools today available in proteomics
[7].
Modern IEF techniques, both in soluble and immobilised buffers, have much to offer
to users. Adequate solutions exist to the two most noxious impediments to a well
functioning technique, namely lack of flexibility in modulating the slope of the PH
gradient and protein precipitation at the pI value. The solution is use of spacers and
novel mixtures of solubilisers, comprising sugar and high molarities of zwitterions. In
addition, an important spin-off of the IEF know-how seems to be gaining importance
in zone electrophoretic separations: the use of isoelectric buffers. Such buffers allow
delivery of extremely high voltage gradients, permitting separations of the order of a
few minutes, thus favouring very high resolution due to minimum, diffusion-driven,
peak spreading. As an extra bonus, by properly modulating the molarity of the
isoelectric buffer in solution, it is possible to move along the pH scale by as much as
0.3 to 0.4 pH units, thus optimising the pH window for separation [7].
2-DE is a more sensitive analytical method than either electrophoretic method alone
[8]. It is a standard method for judging protein purity. In addition, this technique is
becoming increasingly valuable in developmental biochemistry, where the increase
or decrease in intensity of a spot representing a specific protein can be monitored as
a function of cell growth [4]. However, Classical 2-DE with pH gradient generated by
a carrier ampholyte was limited in its resolution, reproducibility and protein-loading
capacity [9] because of pH-gradient instability with prolonged focusing time: the pH
gradient moves towards the cathode (cathode drift). Detailed comparisons of carrier
ampholyte-based patterns for the same cell material in separate laboratories were
very difficult, furthermore, limiting to establish collective databases of 2-D gel
information [3].
The IEF technique is most useful for the analysis of protein purity, composition, and
antigenic properties. The basic IEF technique allows only qualitative examination of
antigenic proteins. The advanced modifications, rocket IE and two-dimensional IE
should be used to get to quantitative results in the form of protein antigen
concentration [4].
The method sensitivity is a potential disadvantage of the CE technique. However, this

is also a challenge for the HPLC chiral separation since the peak efficiencies for the
commonly used chiral columns are low. An important factor to achieve suitable
method sensitivity is setting the UV wavelength as low as possible whether by HPLC
or CE. Therefore, the UV cut-off of the mobile phase in HPLC and background
electrolyte in CE should be as low as possible. In addition, poor precision is another
disadvantage associated with the use of CE. Since the enantiomeric impurity is
determined based on area percent, this problem is not a major concern. However,
some CE-specific related parameters should be carefully controlled. Migration time
variation is a major concern for the CE separation. But the data shown in the study of
Song et al. [10] indicates this problem can be well controlled as long as the
operational parameters and compositional parameters are optimized.
Although the spectrophotometric assay of protein is fast, relatively sensitive, and

requires only a small sample size, it is still only an estimate of protein concentration.
It has certain advantages over the colorimetric assays in that most buffers and
ammonium sulfate do not interfere and the procedure is nondestructive to protein
samples. The spectrophotometric assay is particularly suited to the rapid
measurement of protein elution from a chromatography column, where only protein
concentration changes are required [4]. The UV Absorption Spectrophotometry
method may be used with in concentrations of up to approximately 4 mg/ml (3.0 A).
This method is simple and rapid, but may be disturbed by the parallel absorption of
non-proteins (e.g. DNA). Unlike the colorimetric process, this method is less sensitive
and requires higher protein concentrations and should thus be used with pure protein
solutions. In addition to the direct absorbance display, evaluation is possible with the
BioPhotometer via the Warburg formula or via standard.
Fluorescence measurements have much greater sensitivity than absorption

measurements. Therefore, the experimenter must take special precautions in making
fluorescence measurements because any contaminant or impurity in the system can
lead to inaccurate results. Preparation of reagents and solutions and control of
temperature must be considered when preparing for a fluorescence experiment [4].
Compare to the classical forms of liquid chromatography (paper, column et al.),

HPLC has several advantages. Firstly, resolution and speed of analysis far exceed
the classical methods. Secondly, HPLC columns can be reused without repacking or
regeneration. On the other hand, reproducibility is greatly improved because the
parameters affecting the efficiency of the separation can be closely controlled.
Furthermore, instrument operation and data analysis are easily automated. Last but
not least, HPLC is adaptable to large-scale, preparative procedures [4].
2.10.4 Applications of the Technologies
Biologists often require certain concentration and purity protein in their researches.
The techniques for protein concentration and purity determination can be used in
many areas. The development of techniques and methods for determination of
protein concentration and purity has been essential for many of the recent
advancements in biotechnology research [11]. The purity of a protein is a pre-
requisite for its structure and function studies or its potential application [12]. New
technologies such as SDS-PAGE is used widely in protein purity and concentration
analysis.
Interferons (IFNs) were originally discovered due to their ability to protect cells
against viral infections [13]. However, IFNs have also potent immunomodulatory
effects and antiproliferative activity against malignant cells [14]. According to the
World Health Organization, potency, purity, identity and stability are the most
important properties for the quality control of these cytokines [15]. In the study of
Ruiz et al. [16], the influence of the protein concentration and a formulation vehicle
on the stability of recombinant human Interferon alpha 2b in solution was evaluated.
RP-HPLC was undertaken on a Vydac wide-pore octyl column. Purity was calculated
as percentage of the main peak divided by the total area. The samples were also
analyzed by SDS-PAGE as described by Laemmli [17]. Increasing therapeutic
applications for recombinant human interferon-γ (rhIFN-γ) has broadened interest in
optimizing methods for its production and purification [18]. In the section of Reversed
phase chromatography in the study of Reddy et al. [18], the major peak was collected
in fractions. The fractions were then analyzed by SDS–PAGE. These fractions
proved to be uncontaminated and were pooled for renaturation of the protein. The
eluted fractions were analyzed by SDS–PAGE. Pure fractions were pooled for
renaturation. In the section of renaturation and gel filtration, the eluted dimer peak
was collected and then both rechromatographed on a Superdex-75 column and
assessed by SDS–PAGE to analyze purity. In the section of Cross-linking analysis,
dimer formation was further confirmed by interchain cross-linking of the monomeric
rhIFN-γ using DSS as a cross-linker [19]. The cross-linked dimer was then analyzed
by SDS–PAGE. A single band corresponding to 33 kDa was confirmed by it.
In the study of micro-scale open-tube capillary separations of functional proteins by

Hanna et al. [20], the demonstration of enhanced purity for functional protein by
open-tube capillary columns as compared with conventional packed-column
approached were performed by analysis of the whole-cell lysate by SDS-PAGE. An
SDS-PAGE Schagger gel of the eluted membrane protein complex indicates that the
two known subunits that make up the octadecameric membrane complex are of
exceptional purity. They also used SDS-PAGE to perform the total elution volume in
each case of a different capillary to demonstrate that by going to increasingly smaller
elution volumes, the tube enrichment factor can be manipulated so as to achieve
increasingly higher final concentration of prepared protein. His-tagged magnesium-
protoporphyrin IX chelatase subunit D and untagged subunit I were expressed and
purified. The fractions eluted from the capillaries were analyzed by SDS-PAGE
following a series of experiments. The HeLa nuclear extracts and samples complex
was eluted from the columns and analyzed by SDS-PAGE, then the individual slices
from the SDS-PAGE gel were digested with trypsin.
The digestibility of novel proteins in simulated gastric fluid is considered to be an

indicator of reduced risk of allergenic potential in food, and estimates of digestibility
for transgenic proteins expressed in crops are required for making a human-health
risk assessment by regulatory authorities [21].In the study of Herman et al. [21],
estimation of digestion efficiency using densitometric measurements of relative
protein concentration based on SDS-PAGE corroborated digestion estimates based
on measurements of dye or fluorescence release from the labeled substrates.
The high resolving power of capillary electrophoresis combined with the specificity of
binding interactions may be used with advantage to characterize the structure-
function relationship of biomolecules, to quantitate specific analytes in complex
sample matrices, and to determine the purity of pharmaceutical and other molecules
[22].
http://matcmadison.edu/biotech/
http://www.proteome.org.au/
http://www.proteome.org/
http://www.ebi.ac.uk/
http://www.proteomesci.com/home/
http://www.biobase-international.com/pages/
http://www.proteome.co.uk/
http://www.hupo.org/
http://au.expasy.org/
http://biotechnology.mq.edu.au/biotechnology.htm
http://www.biology.arizona.edu/
http://www.indstate.edu/thcme/mwking/home.html
http://www.biochemistry.org/
http://www.bioch.ox.ac.uk/
http://www.asbmb.org.au/
http://wbiomed.curtin.edu.au/biochem/
References:
1. Tonge, R., Shaw, J., Middleton, B., Rowlinson, R., Rayner, S., Young, J.,
Pognan, F. et al. (2001). Validation and development of fluorescence two-
dimensional differential gel electrophoresis proteomics technology.
Proteomics 1, 377 396.
2. Zhou, G., Li, H., DeCamp, D., Chen, S., Shu, H., Gong, Y., Flaig, M. et al.
(2002). 2D differential in-gel electrophoresis for the identification of
esophageal scans cell cancer-specific protein markers. Mol Cell Proteomics
1, 117 124.
3. Yuan, Q., An, J., Liu D. G., & Zhao, F. K. (2003). Multi-strips on One Gel
Method to Improve the Reproducibility, Resolution Power and High-
throughput of Two-dimensional Electrophoresis. Acta Biochemica 35, 611-
618.
4. Boyer, R. (2000) Modern Experimental Biochemistry. 3rd edn. Addison Wesley
Longman. 41-43, 116, 121, 130-131, 157.
5. Giraudi, G., Baggiani, C., & Giovannoli, C. (1997). Inaccuracy of the Bradford
method for the determination of protein concentration in steroid-horseradish
peroxidase conjugates. Analytica Chimica Acta 337, 93-97.
6. Lucarini, A. C. & kilikian, B. V. (1999). Comparative study of Lowry and
Bradford methods: interfering substances. Biotech. Tech. 13, 149-154.
7. Righetti, P. G., Stoyanov, A. V. & Zhukov. M. Y. (2001) The proteome
revisited: theory and practice of all relevant electrophoretic steps. Elsevier
Science. Amsterdam. 207, 268, 368.
8. Nelson, D. L., & Cox, M. M. (2003) Lehninger : Principles of Biochemistry. 3rd
edn. New York. Worth Publishers. 115.
9. Klose, J., & Kobalz, U. (1995). Two-dimensional electrophoresis of proteins:
An updated protocol and implications for a functional analysis of the genome.
Electrophoresis 16, 1034 1059.
10. Song, S., Zhou, L., Thompson, R., Yang, M., Ellison, D., & Wyvratt, J. M.
(2002) J. Chromatogr. A. 959, 299-308.
11. Wilchek, M., & Miron, T. (1999). React. Funct. Polym. 41, 263.
12. Altintas, E. B., & Denizli, A. (2006). Monosize poly (glycidyl methacrylate)
beads for dye-affinity purification of lysozyme. Inter. J. Bio. Macromol.. 38, 99-
106.
13. Isaacs, A., & Lindenman, J. (1957). Virus interference-1. The interferons.
Proc R Soc Lond B Biol Sci, 147, 258-267.
14. Bordens, R., Grossberg, S. E., Trotta, P. P. & Nagabhushan, T. L. (1997).
Molecular and biologic characterization of recombinant interferon-alpha2b.
Semin Oncol, 24, S9-41-51.
15. World Health Organizations (1988). Requirements for human interferons
made by recombinant DNA techniques. Technical Series No. 771.
16. Ruiz, L., Reyes, N., Aroche, K., Baez, R., Aldana, R., & Hardy, E. (2006)
Some factors affecting the stability of interferon alpha 2b in solution.
Biologicals 34, 15-19.
17. Laemmli, U. K. (1970) Cleavage of structural proteins during the assembly of
the head of bacteriophage T4. Nature 227, 680-685.
18. Reddy, P. K. et al., (2006) Increased yield of high purity recombinant human
interferon-γ utilizing reversed phase column chromatography, Protein
Expression and Purification, doi:10.1016/j.pep.2006.08.013.
19. Wang, F., Liu, Y., Li, J., Ma, G., & Su, Z. (2006), On-column refolding of
consensus interferon at high concentration with guanidine–hydrochloride and
polyethylene glycol gradients, J. Chromatogr. A. 1115, 72–80.
20. Hanna, C., Gjerde, D., Nguyen, L., Dickman, M, Brown, P. & Hornby, D.
(2006). Micro-scale open-tube capillary separations of functional proteins.
Anal. Biochem. 350, 128-137.
21. Herman, R. A., Korjagin, V. A. & Schafer, B. W. (2005). Quantitative
measurement of protein digestion in simulated gastric fluid. Regu. Toxi.
Phaema. 41, 175-184.
22. Heegaard, N. H., Kennedy, R. T. (1999). Identification, quantitation, and
characterization of biomolecules by capillary electrophoretic analysis of
binding interactions. Electrophoresis 20, 3122-3133.
Chapter 3.1 Amino Acid Analysis and Sequencing of Proteins
Alexander Leahy
Introduction
Understanding proteins and their function is fundamental in the aim of understanding

the biochemistry of living organisms. With this understanding, one can begin to
influence the chemical pathways, contributing to prevention or curing of disease.
Analysis of proteins will usually begin with establishing the primary sequence, or
amino acid sequence, of the protein. There are a number of techniques to achieve
this. One method involves deduction from the corresponding DNA sequence. This
method often needs to be confirmed chemically and so this document will concern
itself only with chemical analysis of the protein.
Amino acid analysis involves the complete hydrolysis of the protein into its
constituent amino acids and then separation of these residues for quantitative
analysis. There are a number of hydrolysis techniques, including acid hydrolysis,
base hydrolysis and enzyme hydrolysis.
Some form of derivatisation, such as with ninhydrin or 6-aminoquinolyl-N-hydroxy-
succinimidyl carbamate is then performed on the amino acids in order to make them
detectable. They are then separated some form of chromatography, such as ion-
exchange or reverse-phase[1]. Capillary electrophoresis is also becoming more and
more common, due to its speed, resolution and sensitivity, and also its ability to
separate enantiomers.[2]
Figure 3.1.1 Perkin Elmer Applied Biosystems Model 420A PTC derivatizer with an
on-line Perkin Elmer Applied Biosystems Model 130A PTC Amino Acid Analyzer.
Source: http://www.biotech.iastate.edu/facilities/protein/aaa.html
N-Terminal and C-Terminal amino acid analysis is a useful procedure as this

information can often assist sequencing of the protein. The N-terminus is a
theoretically simple process which involves reacting the protein with a reagent which
will selectively label the terminal amino acid. The protein is then hydrolysed and
separated. The amino acid can then be identified by the label. The C-terminus,
however, will need to be cleaved by a carboxypeptidase. This continually cleaves the
c-terminal protein over time. By analysing the amino acid composition over time, one
can deduce the C-terminus amino acid.
Sequence analysis is a far more complicated procedure. There are two main
methods commonly used: Edman degradation and mass spectrometry. In both
cases, however, when sequence an entire protein, it is usually necessary to digest
the protein using a protease like trypsin to break the protein into smaller peptides
which can then be sequenced. However these peptide sequences must then be
recombined in order. To achieve this, the process must be repeated but the protein is
digested with a different protease such as pepsin which breaks peptide links at
different amino acids. The resulting sequences are then compared and aligned
where they overlap. Since the peptides will be of differing lengths, the resulting
alignments can be combined together to result in the overall sequence.
Edman degradation can be viewed as an N-terminal sequence analysis. A protein is

adsorbed to a solid phase and is reacted with phenylisothiocyanate. The N-terminal
amino acid is cleaved and then washed from the solid state with a solvent. The
removed amino acid can then be identified through chromatography. By analysing
one amino acid at a time, the sequence can be determined. Unfortunately this
process can only be used for sequences of about 50 amino acids. Each time an
attempt is made to remove the next amino acid, some of the proteins keep the amino
acid and it is removed during the next stage. This introduces some noise into the
results which gets worse with each progressive step. Consequently the protein will
need to by digested with endoproteases and the sequences of the resulting peptides
determined. Overlap of different peptides allow the complete sequence to be
determined.[3]
Figure 3.1.2 Mechanism of Edman Degradation

Source: http://en.wikipedia.org/wiki/Edman_degradation
A more recently developed method of sequencing proteins involves the use of

tandem mass spectrometry(MS/MS) or mass spectrometry with post source decay
(PSD). Essentially, both of these techniques rely on the fragmentation of the peptide
during the flight path of the ion. A common method of fragmentation for MS/MS is
Collision Aided Dissociation. This method involves subjecting the peptide ion
selected from the first MS to collisions with an inert gas, often Argon, which provides
results in enough vibrational energy to break the covalent bonds within the
peptide.[4] Different types of ions are formed, depending on the location of the
fragmentation.
Figure 3.1.3 Sequence ions from fragmentation in mass spectrometry

Source: http://www.ionsource.com/tutorial/DeNovo/full_anno.htm
As seen in Figure 3.1.3, yx and bx ions are formed from fragmentation between the
carbon and nitrogen of the peptide bond. Ideally, a peptide will fragment along the
peptide backbone forming these yx and bx ions. The difference in mass between
these ions will indicate sequence data for the fragment being analysed.
Recent Advances
There have been a number of developments in amino acid analysis in recent years.
With the increase in use of capillary electrophoresis as a method of separation of the
hydrolysed residues[2], there has been increased activity in developing better
detection techniques. A number of detection methods exist for this process, such as
UV detection and Laser Induced Fluorescence (LID).
Mass spectrometry has become the method of choice for sequencing proteins and so
there has been a lot of focus on its development within the last 5 years. In particular,
there has been a lot of attention in the manner ions are fragmented to generate the
mass spectrum from which the sequence is derived.
CAD is one of the more commonly used methods for fragmentation in MS/MS.
However, CAD can sometimes fail to get a complete distribution of fragments from
cleavage along the backbone of the peptide, making sequence determination a
complex task. This is often due to Arg residues preventing random protonation along
the backbone, or post translational modifications that provide a lower energy
cleavage than the backbone. An example of this is phosphorylated residues
becoming the preferred site for cleavage, as shown in Figure 3.1.4. Consequently the
mass spectrum is dominated by a single peak of the peptide without the phosphoric
acid moiety, (Figure 3.1.5 A)
Figure 3.1.4 Fragmentation scheme for loss of phosphoric acid from a multiply
protonated phosphopeptide by CAD.
Source: Syka et. al. (2004), Proc. Natl. Acad. Sci. USA 101:9528-9533[5]
Electron Transfer Dissociation, however, is a technique developed by Syka, et. al. [5]
which transfers an electron to the protonated peptides in the mass spectrometer as
shown in Equation 1.
(Peptide + 3H)3+ + Anion – • Æ (Peptide + 3H)2+• + Anion

[Eq. 1][6]
The electron carrying peptide produces fragments in such a way that does not cleave
any of the chemical modifications from the peptide, but instead promotes cleavage
along the peptide backbone. The diagram below shows mass spectra of a
phosphopeptide. The first spectrum was obtained using the more conventional
collisation activated technique. The spectrum is largely dominated by a single peak
resulting in the loss of the phosphoric acid moiety. Figure x.B shows the mass
spectrum of the same peptide after electron transfer dissociation fragmentation. It is
can be clearly seen that the peaks which result from cleavage along the backbone
make the sequence much more easily determined.
Figure 3.1.5 “Tandem mass spectrometry (MS/MS) spectra obtained from a

phosphopeptide eluted during a nanoflow high-performance liquid
chromatography MS/MS (nHPLC-MS/MS) experiment. (A) The MS/MS spectrum
produced following conventional collisional-activation. Note this spectrum is
dominated by a single mass/charge (m/z) corresponding to loss of a phosphoric
acid moiety. No peptide backbone cleavage is observed. Sequence identification
is, therefore, impossible. (B) The MS/MS spectrum that is produced following
electron transfer dissociation (ETD) fragmentation. Here, every single backbone
cleavage product is observed. The sequence is easily assigned as RKpSILHTIR.
Both panels display single-scan mass spectra.”
Source: Coon, J, et. al (2005), BioTechniques, 38(4), 519 - 523
Chemically Assisted Fragmentation has made significant improvements to the quality
of data obtained from a MALDI-TOF. By sulfonation of the N-terminus of the peptide,
it causes the sequence to become negatively charged. When the peptide is ionised
and fragmented in the spectrometer, both the yx and bx ions pick up protons from the
matrix. This causes the bx ion to become neutral and so will not be detected.
Consequently only the y ion is detected and the sequence is much more easily
determined.
Figure 3.1.6 Peptide sequencing of a peptide containing two phosphorylated

tyrosine residues using Ettan CAF MALDI Sequencing Kit in conjunction with a
MALDI-ToF mass spectrometer in PSD mode. The sequence is ALGADSpYpYTAR
(two fragment peaks are missing from the spectrum, each indicated by an X).
Source:http://www4.amershambiosciences.com/aptrix/upp01077.nsf/Content/Product
s?OpenDocument&parentid=366147&moduleid=165399&zone=Proteomics
In order to obtain a full sequence of a protein, sequenced peptides from different

digestions are usually overlapped, resulting in a complete sequence. A study by
Bandeira, et al has shown that this process can be performed directly on MS/MS
spectra rather than the sequences. And it is the overlapping spectra of peptide
fragment ions which result in the determination of a de novo sequence of a protein.
Each spectra is compared and a multiple pairwise alignment is performed on them,

specifically focussing on bx and yx ions found in the spectra. With an algorithm that
favours multiple alignments over pairwise alignments, a Prefix Residue Mass Spectra
is obtained.
Figure 3.1.7 “Clustering phase. (a) and (b) illustrate our linear representation of
spectra where a dot indicates a peak and the dot size is proportional to the peak
height (used to save space when showing multiple alignments of several spectra). (c)
shows the corresponding PRM spectrum (our preprocessed and scored version of an
MS/MS spectrum). For the convenience of the reader, prefix masses are shown in
green, and suffix masses are shown in red, although this distinction is not known in
advance. Other masses (which do not correspond to prefix or suffix masses) are
shown as black dots. (d) Clustering is then used to take advantage of redundant
information in multiple spectra from the same peptide and (e) obtain a single, more
reliable, consensus PRM spectrum (some of the red dots are hidden by green dots).
All black dots still present in (e) correspond either to neutral losses or to doubly
charged fragments. The increased number and significance of red/green dots in the
consensus PRM spectrum as compared to individual spectra would already yield a
reliable de novo peptide sequence (as illustrated in (f)), although we refrain from
interpreting the spectra until the end of the assembly phase”
Source: Bandeira, et. al (2004), Anal. Chem., 76(24), 7221-7233
The resulting PRM from the alignments of spectra is much simpler to interpret for de
novo sequencing as some of the noise present in individual spectra is removed, with
the added advantage of being able to sequence the complete protein from one
resulting spectra. [8]
Evaluation of the Technology
Hydrolysis of Proteins into Amino Acids for Amino Acid Analysis

Hydrolysis of proteins usually proceed through 1 of 3 different techniques. Acid
hydrolysis is a very harsh method, often using 6N HCl at high temperatures.
Increases in speeds down to 10 minutes can be obtained by heating using
microwaves instead of in an oven. Typically, tryptophan, serine, and threonine are
destroyed by this method. Serine and threonine can be identified using a time course
analysis as they destroy more slowly. The time dependent results allow one to
extrapolate the composition of these amino acids. Tryptophan can be analysed by
hydrolysis with 4N methane sulfonic acid and 4N sodium hydroxide.[9] Enzyme
hydrolysis has advantage of not damaging any of the amino acids, but the enzymes
can also hydrolyse themselves, which affects quantitative measurements. The
enzymes need to be immobilised in a gel in order to avoid this.[8]
Edman Degradation vs Mass Spectrometry Techniques

Edman Degradation had been the method of choice for sequencing proteins for many
years, however mass spectrometry is beginning to become more prominent in this
area. The main advantages of mass spectrometry are its high sensitivity and high
throughput. Mass spectrometry of peptides with modern spectrometers are able to
obtain relevant data about a protein in the femtomole range, and often less. Edman
Degradation sequencers usually require quantites to be in the picomole. The speed
of analysis possible by mass spectrometry makes it a much more appropriate form of
analysis in the proteomic era. With the increasing number of proteins and peptides
requiring identification or characterisation, it is necessary to be able to perform these
analyses quickly, which is easily achievable using mass spectrometry. Software exist
which can analyse mass spectrometry for sequence analysis.
Fragmentation patterns are often very complex in mass spectrometry. The location of
fragmentation of a peptide varies depending on the method used. Ideally a
fragmentation consisting mostly of bx and yx ions would be desirable, but this often is
not the case. Other types of ions, such as those formed from fragmentation of the
side chains and further fragmentation of fragments also add to the peaks in the
spectra observed. As not fragmentation techniques are compatible with all types of
mass spectrometers, this makes it difficult for a laboratory to easily select a method
which would best analyse their protein.
However with so many peptides and proteins already sequenced, it is often not
necessary to sequence an entire peptide to determine its identity. Sequence tags are
short sections of the sequence of a peptide which can be used to identify a peptide
sequence in a database. In fact, frequently the spectrum itself can be used to search
databases for protein identification.
One of the problems with performing sequencing by mass spectrometry is being able
to distinguish between leucine and isoleucine. These amino acids are isomers of
each other and so have identical mass. The only way to distinguish between them
using mass spectrometry techniques is by high energy fragmentation. The ions that
are formed often cleave the side chain of the amino acid, forming d, v and w ions.
These ions can be used to distinguish between these two amino acids.
Applications of the Technology
In 1999 a study was done to sequence neuropeptides present in a tissue sample of

pituitary neurointermediate lobes taken from adult X. laevis toads. The technique
they chose was to use a MALDI-PSD to determine the sequence of the peptides.
Interestingly, the cells were directly mixed with the matrix, 2, 5–dihydroxybenzoic
acid in trifluoroacetic acid, where cell lysis occurred. Once the targets were prepared,
a spot would typically contain femtomoles of a peptide.
The ions generated with the MALDI source were detected using MALDI-TOF MS and
a profile of the peptides present was obtained as shown below.
Figure 3.1.8 Mass profile of peptides in pars intermedia tissue from the amphibian
X. laevis, obtained by direct MALDI TOF MS analysis under delayed extraction
conditions.
Source: Jesperson, S. et al, Anal. Chem., 71(3), 660-666 [11]
The two most prominent ions present at masses of 1050.4u and 1392.7u were
chosen to perform MALDI-PSD analysis. Analysis of the 1050.4 u peak proved
difficult as the peptide had a disulfide link which severely affected the fragmentation
patterns observed. However, identification of the masses of individual amino acids
assisted in identifying the peptide and the peaks matching fragmentation around the
disulfide link supported the conclusion. The figure below shows the identified peptide
and the matching ions from the spectrum.[11]
This example highlights the need for the breakage of disulfide links in peptides before
sequence analysis.via mass spectroscopy.
Figure 3.1.9 Known sequence of the vasotocin peptide (N-terminal end on top),
including an intrinsic disulfide (S-S) bridge between the two cysteine residues.
Indicated are the a-, b-, and y-type ions observed in the MALDI-PSD fragment
spectrum
Source: Jesperson, S. et al, Anal. Chem., 71(3), 660-666 [11]
Sequencing of a novel peptide in the venom of Crotalus durissus collilneatus was

performed on a Micromass Q-TOF Micro mass spectrometer(Waters, USA) in
positive electrospray ionisation mode. This particular instrument is capable of tandem
MS/MS which was utilised to complete the de novo sequencing of the peptide. The
peptide was fragmented with a collision energy fixed at 30 V. The
MassLynks(Waters, USA) software automated the sequencing process from the
mass spectrum obtained. The spectrum yielded the sequence TPPAGPDGGRP
which was supported between the b and y ion series.[12]
.
Figure 3.1.10 MS/MS profile of the fragmentation of the selected ion (511.2 Da
[M + 2H]2+) and the deduced sequence derived from de novo sequencing based on
MassLynks data processing system (Waters, USA). Consistency between the b and
y ion series established the sequence TPPAGPDGGPR.
Source: Higuchi S, et al, Comp. Biochem. Physiol. C. Toxicol. Pharmacol,
144(2):107-21[12]
Relevant Web Sites
Ion Source – Mass Spectrometry and Biotechnology Resource
http://www.ionsource.com/
This site is particularly useful as it provides tutorials on a number of different aspects

of peptide analysis through mass spectrometry. Of particular interest at this site was
the tutorial on de novo peptide sequencing. Along with explanations and exercises of
the techniques, it also contains many references examining the different techniques
and software used in this type of analysis.
Waters is a company that makes analytical instruments for protein analysis. They
have a number of references on their site relevant to the development of amino acid
analysis techniques.
http://www.waters.com/watersdivision/ContentD.asp?watersit=JDRS-
5LTHE6&WT.svl=1
Key Industry Suppliers

Amersham Biosciences, a subsidiary of GE Healthcare Life Sciences, is a major
supplier of the Ettan CAF MALDI Sequencing Kit[13] as well as the Ettan MALDI-
TOF Pro.
Waters is a company from USA which is able to supply many analytical instruments
required for protein analysis. They offer a variety of mass spectrometers, such as
LC/MS, LC/MS/MS, MALDI and GC-MS. They also supply chromatography
instruments, such as the ACQUITY UPLC which can complete an analysis in less
than 30 minutes. They also supply AccQtag which is a derivatisation reagent which
allows amino acid residues to be detected by fluorescence.
Applied Biosystems is a major supplier of a number of different instruments such as

mass spectrometers, chromatography instruments as well as protein sequencers,
such as the Procise cLC Protein Sequencer which uses capillary HPLC to complete
Edman Degradation down to femtomole analysis.[14]
References
1. Iowa State University, (2004) Amino Acid Analysis,
http://www.biotech.iastate.edu/facilities/protein/aaa.html
2. Poinsot V, Lacroix M, Maury D, Chataigne G, Feurer B, Couderc F (2006),
Recent Advances in Amino Acid Analysis by Capillary Electrophoresis,
Electrophoresis, 27, 176-194
3. H. Jakubowski (2006), Biochemistry Online, chapter 2 B
http://employees.csbsju.edu/hjakubowski/classes/ch331/protstructure/olcomp
seqconform.html
4. Hunt, D.F., Yates, J.R., Shabanowitz, J., Winston, S., Hauer, C.R. (1986),
Protein Sequencing by Tandem Mass-Spectrometry.
Proc. Natl. Acad. Sci. USA 83:6233-6237
5. Syka et. al. (2004), Proc. Natl. Acad. Sci. USA 101:9528-9533
6. Coon, J, et. al (2005), BioTechniques, 38(4), 519 - 523
7. Jesperson, S., Chaurand P., van Strien F, Spengler B., van der Greef J,
(1999) Direct Sequencing of Neuropeptides in Biological Tissue by MALDI-
PSD Mass Spectrometry, Anal. Chem., 71(3), 660-666
8. Bandeira N, Tang H, Bafna V, Pevzner P (2004), Shotgun Protein
Sequencing by Tandem Mass Spectra Assembly, Anal. Chem. 76(24), 7221-
7233
9. Karen A West, Jeffrey D Hulmes and John W Crabb (1996), Amino Acid
Analysis Tutorial
http://www.abrf.org/ResearchGroups/AminoAcidAnalysis/EPosters/Archive/4c
.html
10. David E. Metzler, (2001), Biochemistry – The Chemical Reactions of Living
Cells, Harcourt/Academic Press, pp 116
11. Higuchi S, Murayama N, Saguchi K, Ohi H, Fujita Y, da Silva N, Bezerra de
Siqueira R, Kahlou S, Aird S (2006), A novel peptide from the ACEI/BPP-CNP
precursor in the venom of Crotalus durissus collilineatus, Comp. Biochem.
Physiol. C. Toxicol. Pharmacol, 144(2):107-21.
12. Savitski MM, Nielsen ML, Kjeldsen F, Zubarev RA (2005), Proteomics-grade
de novo sequencing approach. J Proteome Res. 4(6):2348-54.
13. GE Healthcare Life Sciences (2002),
http://www4.amershambiosciences.com/aptrix/upp01077.nsf/Content/Product
s?OpenDocument&parentid=366147&moduleid=165399&zone=Proteomics
14. Applied Biosystems (2006),
http://www.appliedbiosystems.com/applications/proteomics/protein_sequenci
ng.cfm
Chapter 3.2 Chemical modifications of proteins
Hellan M Luo
3.2.1 Introduction
Protein chemists have long been interested in altering the chemical, physical and
biological properties of proteins by chemically changing their structure. [1] Almost the
very 1st thing that discovered by scientists is ‘’ Protein can be easily changed upon
treatment with chemical reagents’’. [1] Their liability to chemical reagents and
reaction conditions has been a very serious problem foe many purposes. The
application of modern knowledge of proteins, new chemical reagents and more
sophisticated analytical techniques has made chemical modification of protein
molecules become one of the most useful approaches to study/research of their
properties. [1].
The term “chemical modification" refers to formation or cleavage of covalent bonds,
generally with the side chains, though cleavage of peptide bonds and modification of
the a-amino terminal can formally be included. [3] Most of these methods are not
reversible by dilution, gel filtration or dialysis, however, there are few exceptions==
Schiff base formation with aldehydes and some reactions of Arginine are thus
reversible. [3]. some modifications are reversed by changing the conditions, for
instance lowering the pH, which can be very useful, but most are not and Some
modifications are hydrolyzed in 6N HCl, as do before amino acid analysis; Non-
covalent interactions also have their place - competitive inhibitors which bind at
active sites; low dielectric solvents change the UV absorbance of groups exposed to
solvent, fluorescence-quenching molecules similarly quench the fluorescence of
exposed tryptophan residues.[3][4]
There are 7 main purposes to modify proteins:
Purpose Explanations / main strategies

1. Analysis No. of aa’s present in a protein, done by amino acid
analysis after acid hydrolysis – automated ion exchange
chromatography, detecting peaks of amino acid coming
off a column by reaction with ninhydrin or fluorescamine
2. As an aid in can be done on proteins bound to membranes after
sequence electrophoresis, or even in the gel
analysis
3. To understand What groups are at the site responsible for biological
mechanism of activity? How many? Which ones in the sequence? For
action enzymes we aim to characterize the chemical
mechanism, which requires identifying the participating
groups, eventually to characterize the conformation of the
active site, and beyond that to understand changes in
conformation during catalysis. best done by X-ray
crystallography
4. Physical change in the physical characteristics of the protein,
modification destabilization of complexes, including solubilisation of
hydrophobic proteins
5. Cross-linking insolubilization of protein by intermolecular cross-linking,
and so that for instance insoluble but active trypsin can be
attachment to removed from a digestion by centrifugation
supports
6. Hapten Carrier proteins, to elicit antibodies to the hapten (won’t
attachment get it if it is a free small molecule.)
7. Attachment of sensitive to their environment - chromophores whose
reporter groups absorbance is pH-sensitive,
Table 3.2.1 Severn (7) main purposes of chemical modifications of protein and their
main strategies, summarized from [2] [3]
Modifications occur (1-3)
1. by addition of other groups:
Group Added Target Residue(s) Reversibility

Phosphoryl tyr, ser, thr, his yes
Methyl lys yes
Acetyl lys, N-terminus yes (lys)
Hydroxyl pro, lys no
Carboxyl glu no
ser, thr, hydroxypro,
Sugars no
asn
myristyl cys, his no
glycophospholipid C-terminus no
Prenyl cys no
ADP-ribose ? yes
Dipthamide his no
Ubiquitin lys no
Table 3.2.2 Addiction of other groups, target residue and reversibility for protein
chemical modification, source Molecular Genetics, Protein Modification [2]
2. Or by isomerization of residues.
o L-ala is converted to D-ala in Dermorphin (a peptide of frog skin).

o Cis and Trans proline are interconverted in a reaction important for
protein folding.
o Cysteines are exchanged by a disulfide exchange protein, also
important for folding. [2]
3. During translation (co translational) or after the polypeptide chain has been
completed (post-translational). [2]

Recently, Chemical modification of proteins become more and more popular, one
question about biologically active protein is has been frequently asked ‘’ what is
unique about the structure that accounts for the particular activity?’’ [1] The interests
are mainly focus on the amino acid said chain groups of protein molecule that
participate in the activity or the ‘’ active centre’’ if it happens to be an enzyme [1].
(NOTE, enzyme is a special form of protein and plays very important roles in
biological function).
There are many recent advances developed thanks to the modern protein
technologies, of which, are widely used in Biopharmaceuticals fields; Proteins are
controlled by a vast and dynamic array of post-translational modifications, many of
which create binding sites for specific protein-interaction domains. The proteome can
be linked to post-translational modifications to cellular organization. The most
common strategies are modification-dependent interactions synergize to regulate cell
behavior. Some recent developed technologies include:
1) Site-Specific Chemical Modification of Proteins
Use site-specific chemical modification to study the structure-function

relationships in proteins.[5] This technique is basically prefer to modified specified
site of a protein: such as arginyl residues, Cysteine,lysine residues and other α-
amino groups ,Diethylpyrocarbonate ,Selective reduction of disulfide bonds
,Methionine, Tetranitromethane, tryptophan etc, To modify these specific protein
sites, in combination of relevant reagents, in order for proteins that can be a
better form of membrane transport, better enzymatic activities, and helpful to
study protein structure and function relationship. As well as have better biological
function. [13] [14] [15] [16] [5]
2) Chemical modifications of proteins in Vivo
There are chemical modifications of proteins which control biological activity. The
majority of these modifications are co-translational and post-translational reactions,
some of them are considered to occur in a ‘random’ manner, in such modification,
they can not be catalysed by an enzyme, and /or enzymes or subject to environment
of the proteins.
For examples:
Oxidation of proteins occurs in vivo with generally unfavourable consequences. Nitric
oxide is a potent physiologic agent with diverse systemic effects Peroxynitrite which
is formed from nitric oxide is a mediator of some of these physiologic effects of nitric
oxide.
Glycation is the term used to identify the reaction of reducing sugars with proteins.
This involves the initial formation of a Schiff base followed by rearrangement in the
Maillard reaction eventually resulting in advanced Glycation end (AGE) products
Reaction can occur at lysine and arginine residues with resulting crosslink formation.
[5] [21][22]
3) Chemical Modification and Protein Biopharmaceuticals
The modification of proteins and peptides with poly (ethylene) glycol (PEG) is the
most frequent chemical modification used in the manufacture of
biopharmaceuticals. [5][21][14]
Other recent advances include:
4) A Wiring of the Human Nucleolus

5) In-depth analysis of the membrane and cytosolic proteome of red blood cells.
6) A Mammalian organelle map by protein correlation profiling.
7) Modular stop and go extraction tips with stacked disks for parallel and
multidimensional Peptide fractionation in proteomics.
8) Insulin-dependent Interactions of Proteins with GLUT4 Revealed through

Stable Isotope Labeling by Amino Acids in Cell Culture (SILAC
9) Quantitative proteomic comparison of rat mitochondria from muscle, heart

andActin homolog MreB and RNA polymerase interact and are both required
for chromosome segregation in Escherichia coli.
liver.
Proteins may be modified in vitro so that they may be used for detection,
purification and assay development. In very general terms, protein modification
reagents can be separated into reagents that add, cleave or reduce. Reagents
that add labels are used for immunoassays, flow cytometry, fluorescence-
activated cell sorter (FACS) analysis and molecular structure and function
studies. Addition reagents also include those used to block particular functional
groups. Reagents that enzymatically cleave can be used for removing amino
acids, producing antibody fragments and releasing peptides from fusion proteins.
Reagents that chemically reduce can be used for protein solubilisation and to
facilitate cross-linking. [24]
1) Label Addition Reagents
Reagent Reactivity
Bolton-Hunter Reagent (SHPP) Primary Amines
Water-Soluble Bolton-Hunter Reagent (Sulfo-SHPP) Primary Amines
Succinimidyl-3-(tri-N-butylstannyl) benzoate Primary Amines
Sulfo-SHB Primary Amines
HPPH Carbonyl/Aldehyde
ß-(4-Hydroxyphenyl)ethylmaleimide Sulfhydryl
ß-(4-Hydroxyphenyl)ethyl iodoacetamide Sulfhydryl
Table 3.2.3 Protein modifications reagents can be Iodinate (source: Pierce net
Protein Chemistry) [24]
2) Addition Reagents that Alter or Block Functional Groups

In many applications, especially those involving cross-linking or detection of
specific functional groups, it is sometimes necessary to selectively block one
functional group (e.g., amines) or else add more of one particular functional
group (e.g., add more sulfhydryl groups) to one or more proteins used in an
experiment. Blocking functional groups is also useful for reducing background
detection in certain assays.[24][8][9]
3) Enzymatic Cleavage Reagents

Enzymes may be used as catalysts to affect a wide variety of biochemical
transformations. Whether dissolved in solution or immobilized to an insoluble
support, enzymes can specifically cleave proteins at discrete sites to isolate
fragments of known activity or structure. Such cleavage events also can be a way to
demonstrate protein purity by analysing what peptide fragments result from
proteolysis. Pierce proteolysis products include highly purified enzymes that can be
used in buffered solutions, immobilized enzymes that facilitate simple removal of
protease activity after use, assays to monitor protease activity, and protease
inhibitors to avoid proteolysis in biological samples. Examples are: Factor Xa,
Submaxillaris Protease, TPCK Trypsin [24] [8][9]
4) Reducing Disulfide Bonds
Reduction of disulfide bonds in proteins may be required or beneficial for a

number of reasons:
• Exposure of sulfhydryls for cross-linking

• Protein activation
• Oligomer separation
• Protein denaturation
• Protein solubilization
• Disulfide bond characterization
• Protection of protein thiols from oxidation
The keep reagents that commonly used are: sulfhydryl groups , free thiol groups ,
dithiothreitol (DTT), 2-mercaptoethanol and 2-mercaptoethylamine,
Tris(carboxyethyl), phosphine (TCEP).[24] [8][9]
Some of these will be discussed in part 3.2.4
The same as any technologies, chemical modifications of proteins are also

associated with advantages and disadvantages
The advantages of protein chemical modifications are:
1) For Pharmaceutical proteins are unstable when injected into the blood
circulation. The half life is short, from several minutes to hours. The
consequence is multiple injection and uncomfortable side effects. Chemical
modification is an effective way to increase the longevity and efficacy of the
proteins.
2) Covalent modification is an important strategy for introducing new functions
into proteins.
3) As engineered proteins become more sophisticated, it is often desirable to
introduce multiple, modifications involving several different functionalities in a
site-specific manner.
4) Proteins are the final link of information chain
5) Help understand protein structure and function relationship
6) To perform specific biological function
7) Mildness, high degree of specificity
8) improve biocompatibility and bioactivity
9) Site specific
The disadvantages of Chemical modification of proteins are:
1) Usually not residue specific, other amino acids can be modified too
2) Large amount of reagents are required
3) Expensive
4) Time consuming
5) Large ranges of reagents are available
6) Potential pitfall: Does modifications causes distal conformational changes
rather than specific blocking of active site
7) Need full time qualified staff to perform the modification
8) Need close monitoring during the modification process
Proteins can be easily modified by various methods to alter their structure or

properties. Common modifications include crosslinking, fragmenting, denaturing,
reducing disulfides, and attaching various prosthetic groups (e.g. PEGylation) to a
protein. Protein labelling is another common modification. Proteins can be labelled
with biotin, fluorophores, enzymes or radioiodine. [24]
1) Amino Acid Side Chain Modification Agents [24]

It refers to Reagents used to block amino acid side chains on proteins, change their
charge, or change them to functional groups favourable for cross-linking and
labelling. [24]
One example is: The antitumor protein neocarzinostatin (NCS), isolated from
Streptomyces carzinostaticus, is a single chain polypeptide with 109 amino acid
residues. Complete acylation of the amino groups (alanine-1 and lysine-20) was
observed when NCS was allowed to react with 3-(4-hydroxyphenyl)-propionic acid N-
hydroxysuccinimide ester at pH 8.5. Since bis[(alanine-1, lysine-20)-3-(4-
hydroxyphenyl)]-propionamide NCS was fully active in antibacterial potency and in
the inhibition of growth of leukemic (CCRF-CEM) cells in vitro, it appears that the two
amino groups in the protein are not essential for biological activity.
Radiolabeled NCS was prepared by using a tritiated or 125I-labeled acylating agent.
Since the CD spectra of native and bis(alanine-1, lysine-20)-amino modified NCS
were indistinguishable, there is presumably no change in the native conformation of
the protein due to acylation. Reaction of NCS with ammonium chloride in the
presence of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide at pH 4.75 converted all
the 10 carboxyl groups into carboxamides and produced a protein derivative of basic
character. This modification caused a change in the native conformation of the
protein accompanied by a loss in biological inhibitory activities. [26]
2) Chaotropes, Protein Denaturants [24]

This method is generally refers to Chaotropes such as urea or guanidine disrupts
water interactions and help solubilise hydrophobic proteins and peptides. They also
act as general protein denaturants, unfolding proteins and altering their three-
dimensional structure [24]
3) Cross linking Reagents [24]

It basically means that Chemical crosslinking agents are used to determine near-
neighbour relationships, analyses three-dimensional structures of proteins and
complexes, prepare antibody-enzyme conjugates, immobilize molecules and
conjugate haptens to carrier proteins. [24]
4) PEGylation Reagents [24]

PEGylate peptides and proteins via available amine or sulfhydryl groups. Increase
solubility and stability and reduce immunogenicity of peptides and proteins. [24]
5) Post Translational Modification (PTM) [24]

Isolate post translationally modified proteins such as glycoproteins, ubiquitin-modified
proteins, and phosphopeptides using these simple and efficient kits. [24]
Fig 3.2.1 Post Translational Modification (Source: Wikipedia) [28]
PTMs involving addition include:
• acetylation, the addition of an acetyl group, usually at the N-terminus of the

protein [28]
• alkylation, the addition of an alkyl group (e.g. methyl, ethyl) [28]
o methylation the addition of a methyl group, usually at lysine or arginine
residues.
• biotinylation, acylation of conserved lysine residues with a biotin appendage
• glutamylation, covalent linkage of glutamic acid residues to tubulin and some
other proteins. [28]
• glycylation, covalent linkage of one to more than 40 glycine residues to the
tubulin C-terminal tail [28]
• glycosylation, the addition of a glycosyl group to either asparagine,
hydroxylysine, serine, or threonine, resulting in a glycoprotein
• isoprenylation, the addition of an isoprenoid group (e.g. farnesol and
geranylgeraniol) [28]
• lipoylation, attachment of a lipoate functionality [28]
• phosphopantetheinylation, the addition of a 4'-phosphopantetheinyl moiety
from coenzyme A, as in fatty acid, polyketide, non-ribosomal peptide and
leucine biosynthesis
• phosphorylation, the addition of a phosphate group, usually to serine,
tyrosine, threonine or histidine [28] sulfation, the addition of a sulfate group to
a tyrosine. [28]
As described in Fig 3.2.1, after translation occurs (post translation), folding, oxidation
and signal peptide cleavage takes place, ER export , Golgi transporation as well as
vesicle packing occurs, protease cleavage liberates C peptide, then stimulates insulin
production.
6) Proteases [24]
Proteinases for enzymatic cleavage of proteins to facilitate sequencing, amino acid

analysis, structural analysis and various other applications. We offer proteases
immobilized on agarose for easy removal of the protease following digestion [24]
Certain proteases have been used in food processing for centuries and any record of
the discovery of their activity has been lost in the mists of time. [29] Rennet (mainly
chymosin), obtained from the fourth stomach (abomasum) of unweaned calves has
been used traditionally in the production of cheese. [29]
Proteases may be used at various pH values, and they may be highly specific in their
choice of cleavable peptide links or quite non-specific. Proteolysis generally
increases the solubility of proteins at their isoelectric points.[29]
Fig 3.2.2 From Milk to cheese with Proteases Applications (source:

http://www.lsbu.ac.uk/) [29]
7) Reducing Agents [24]

Solution and solid-phase reducing agents for disulfide-containing peptides and
proteins. [24]
For example: a very strong reducing agent--- Raney nickel, which reduces cysteine
to alanine, and diborane and alkylboranes, which can reduce carboxyl groups to
CH2OH. These then decrease the size of the side chain. Unfortunately the reaction
tends to be rather incomplete, and not commonly used. [3]
3.2.5 Relevant Web Sites
These websites are great for ‘’ Chemical Modification of Proteins’’
1) Some Recent Developments in the Chemical Modification of Proteins with a Note

on Applications to Biopharmaceuticals.
http://www.lundbladbiotech.com/Chemical%20Modification%20of%20Proteins.htm
2) Recent paper
http://www.pil.sdu.dk/Recent_Papers.htm
3) ExPASy Proteomics tools
http://au.expasy.org/tools/
4) Lab velocity
http://researchlink.labvelocity.com/products/index.jhtml?nodeId=2538
5) Biozon
http://biozon.org/people/golan/
6) Better Health--- Health and medical information for consumers, quality assured by the Victorian
government (Australia).
http://www.betterhealth.vic.gov.au/bhcv2/bhcarticles.nsf/pages/Protein?OpenDocum
ent
7) Wikipedia
http://en.wikipedia.org/wiki/Protein
8) Protein Science
http://www.proteinscience.org/
9) Protein Data Bank (PDB)
http://www.rcsb.org/pdb/Welcome.do;jsessionid=mEvHfFKktB0f3mdEYUBdFw**
10) Protein Society
http://www.proteinsociety.org/
11) Organelles proteomics: turning inventories into insights.
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=pubmed&cmd=Retrieve&dopt=Abs
tractPlus&list_uids=16953200&query_hl=1&itool=pubmed_docsum
3.2.6 Key Industry Suppliers (if any)
There are some key industry suppliers, name and website link as below:
(1) Toronto Research Chemicals http://www.trc-canada.com,

(2) Pierce Chemical Company http://www.sigma-aldrich.com
(3) Sigma-Aldrich http://www.labguideonline.com
(4) Pierce net Protein Chemistry
http://www.piercenet.com/Products/
(5) Protein Tech group http://www.ptglab.com/
(6) Invitrogen (Australia)
http://www.invitrogen.com/getgid.cfm?gid=168&returnURL=http://probes.invitrogen.c
om/handbook/sections/0502.html
(7) Biotechnique
http://www.biotechniques.com/default.asp?page=marketplace&subsection=companyl
isting&id=301
3.2.7 References
1. Means, Gary. E, Feeney, Robert. E, Chemical Modification of proteins (1971).

Holden-Day Press, San Francisco, Ca, USA
2. Molecular Genetics, Protein Modification (2006)
http://opbs.okstate.edu/~melcher/mg/MGW2/MG252.html
3. Protein chemical Modification
Http://www.cook.rutgers.edu/~dbm/chemmods.htm
4. Protein Chemistry
http://www.bi.umist.ac.uk/users/mjfajdg/2PAB/default.asp
5. Ralph A. Bradshaw Some Recent Developments in the Chemical Modification of
Proteins with a Note on Applications to Biopharmaceuticals
http://www.lundbladbiotech.com/Chemical%20Modification%20of%20Proteins.htm
6. Irwin, W.A., Gaspers, L.D., and Thomas, J.A. (2002), Inhibition of the
Mitochondrial Permeability Transition by Aldehydes. Biochem.Biophys.Res.Commun.
291, 215-219
7. Involvement of conserved histidine, lysine and tyrosine residues in the mechanism
of DNA cleavage by the capase-3 activated DNase CAD, Nucleic Acids Research 30,
1325-1332
8. Methods in Molecular Biology: Protein Stability and Folding, Theory and Practice.
Vol. 40, Bret A. Shirley, ed. 1995.
9. Slopes, R.K. 1982, pp. 185-193, Protein Purification: Principles and Practice,
Springer-Verlag, New York
10. Toronto Research Chemicals http://www.trc-canada.com,
11. Pierce Chemical Company http://www.sigma-aldrich.com
12. Sigma-Aldrich http://www.labguideonline.com
13.. Wu, X., Chen, S.G., Petrash, J.M., and Monnier, V.M. (2002), Alteration of
Substrate Selectivity through Mutation of Two Arginine Residues in the Binding Site
of Amadoriase II from Aspergillus sp. Biochemistry 41, 4453-4458.
14.. Gärtner, E.M., Liebold, K., Legrum, B., Fasold, H., and Passow, H. (1997), Three
different actions of phenylglyoxal on band 3 protein-mediated anion transport across
the red cell membrane. Biochimica et Biophysica Acta 1323, 208-222.
15. Irwin, W.A., Gaspers, L.D., and Thomas, J.A. (2002), Inhibition of the
Mitochondrial Permeability Transition by Aldehydes. Biochem.Biophys.Res.Commun.
291, 215-219.
16. Kučera, I. (2003), Passive penetration of nitrate through the plasma membrane of
Paracoccus denitrificans and its potentiation by the lipophilic
tetraphenylphosphonium cation. Biochim. Biophys. Acta 1557, 119-124.
17. Lundblad, R.L. (1994), Techniques in Protein Modification, Chapter 6, The
Modification of Cystine, CRC Press, Boca Raton, Florida, USA, pps. 91-96.
18. Yano, H., Kuroda, S., and Buchanan, B.B. (2002), Disulfide proteome in the
analysis of protein function and structure. Proteomics 2, 1090-1096.
19. Loo, T.W., Bartlett, M.C., and Clarke, D.M. (2003), Substrate-induced
conformational changes in the transmembrane segments of human p-glycoprotein.
Direct evidence for the substrate-induced fit mechanism for drug binding. J. Biol.
Chem. 278, 13603-13606.
20. van der Sluis, E.O., Nouwen, N., and Driessen, A.J.M. (2002), SecY-SecY and
SecY-SecG contacts revealed by site-specific crosslinking, FEBS Letters 527, 159-
165
21. Dage, J.L., Sun, H., and Halsall, H.B. (1998), Determination of
diethylpyrocarbonate-modified amino residues in α1-acid glycoprotein by high-
performance liquid chromatography electrospray ionization-mass spectrometry and
matrix-assisted laser desorption/ionization time-of-flight-mass spectrometry.
Analytical Biochemistry 257, 176-185.
22. . Alderton, A.L., Faustman, C., Liebler, D.C., and Hill, D.W. (2003), Induction of
redox instability of bovine myoglobin by adduction with 4-hydroxy-2-nonenal.
Biochemistry 42, 4398-4405.
23. http://www.pil.sdu.dk/Recent_Papers.htm
24. Pierce net Protein Chemistry
http://www.piercenet.com/Products/Browse.cfm?fldID=6C6EDE13-F974-4D12-
A929-34E275A1D2EB
25. J. Jefferson Smith, David W. Conrad, Matthew J. Cuneo and Homme W. Hellinga
(2005) Orthogonal site-specific protein modification by engineering reversible thiol
protection mechanisms, Protein Science (2005), 14:64-73
26. Samy TS. Neocarzinostatin: effect of modification of side chain amino and
carboxyl groups on chemical and biological properties. : Biochemistry. 1977 Dec
13;16(25):5573-8.
27. Frisch B, Boeckler C, Schuber F.,
Synthesis of short polyoxyethylene-based heterobifunctional cross-linking reagents.
Application to the coupling of peptides to liposomes. Bioconjug Chem. 1996 Mar-Apr;
7(2):180-6.
28.Wikidia
http://en.wikipedia.org/wiki/Post-translational_modification
29. Application of Proteases in food industry (2006)
http://www.lsbu.ac.uk/biology/enztech/proteases.html
Chapter 4.1 MALDI-TOF Mass Spectrometry
Michael Christopher
4.1.1 Introduction
Mass spectrometry is the ultimate technique for the accurate determination of the
molecular weight of a molecule by measuring its mass-to-charge (m/z) ratio. Ionized
molecules are generated by inducing either the loss or gain of a charge from a
neutral species [1]. Once formed, ionized molecules are electrostatically directed into
a mass analyzer where they are separated according to their m/z ratio, and finally
detected. Matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS)
was first introduced in 1988 by Tanaka et al. [2], and Karas and Hillenkamp [3]. It has
since become a widespread analytical tool for the identification of peptides, proteins,
glycoproteins, and other biomolecules (carbohydrates, lipids, oligonucleotides, and
natural products) [1]. The efficient and directed transfer during a matrix-assisted
laser-induced desorption event provides high ion yields of the intact analyte with sub-
picomole sensitivity, and enables the mass analysis of complex biological samples
such as proteolytic digests (Table 4.1.1). MALDI allows for easy preparation and
rapid analysis of multiple samples at the same time (using a direct insertion multi-
sample plate), and sample amounts in the femtomole to picomole range. MALDI
predominantly generates ions that are singly charged, making it easier to identify
intact molecules[1].
Advantages Disadvantages
Practical mass range of up to 300 kDa. Matrix background, which can be a problem
Species of much greater mass have been for compounds below a mass of 700 Da.
observed using a high current detector This background interference is highly
dependent on the matrix material
Typical sensitivity in the order of low Possibility of photodegradation by laser
femtomole to low picomole. Attomole desorption/ionization
sensitivity is possible
Soft ionization with little to no fragmentation Acidic matrix used in MALDI may cause
observed degradation on some compounds
Tolerance of salts in millimolar
concentrations
Suitable for the analysis of complex mixtures
Table 4.1.1 Advantages and disadvantages of MALDI

Source: Siuzdak, G. (2003) The Expanding Role of Mass Spectrometry in
Biotechnology, p. 26. MCC Press, San Diego, CA
It is generally believed that MALDI causes the ionization and transfer of a sample
from the condensed phase to the gas phase via laser excitation and vaporization of
the solid sample matrix (Figure 4.1.1). In MALDI analysis, the analyte is first co-
crystallized with a large molar excess of a matrix compound, usually a UV-absorbing
weak (nonvolatile) solid organic acid, such as α -cyano-4-hydroxycinnamic acid
(CHCA) [1]. The biomolecules are isolated from each other within the matrix, which
results in a reduction of the strong intermolecular forces that exist between them.
Irradiation of this analyte-matrix mixture UV-laser light (usually 337 nm) results in the
vaporization of the matrix (i.e. the matrix molecules absorb laser light (photon)
energy, and convert it into excitational (vibrational) energy). This vibrational energy is
then transferred to the co-crystallized biomolecules, causing them to vaporize.
However, since the biomolecules are physically shielded by the matrix molecules,
they do not directly absorb energy from the laser, thereby minimising sample damage
due to irradiation. The photoexcitation/photoionization of the matrix molecules results
in proton transfer to the analyte molecules. Once in the gas phase, the desorbed
charged molecules are the directed electrostatically (by high voltage ~20-25 kV) from
the MALDI ionization source into the mass analyzer [1].
Figure 4.1.1 MALDI-TOF Mass Spectrometer Sample Ionization

Source: http://www.psrc.usm.edu/mauritz/maldi.html
Linear time-of-flight (TOF) mass analyzers measure the precise time it takes for an
accelerated ion to traverse a high vacuum (~10-6 torr) field-free drift zone to a time-of-
flight detector (Figure 4.1.2). The pulsed nature of MALDI (ions are generated in
short, nanosecond pulses) is well suited to TOF analyzers since the ion’s initial time
of flight can be started with each pulse of the laser and completed when the ion
reaches the detector [1]. With MALDI-TOF mass analyzers, all the ions are given the
same amount of energy through an accelerating potential [1]. Because the ions have
the same energy, but a different mass, the lighter ions reach the detector first
because of their greater velocity, while the heavier ions take longer due to their
heavier masses and lower velocity. In essence, the time an ionized molecule takes to
arrive at the detector depends on the mass, charge, and kinetic energy of the ion [1].
Linear MALDI-TOF mass analyzers can routinely analyze intact proteins and large
peptides from a mass range of 0.7 to 200 kDa, with an an accuracy range of 0.01-
0.1%.
Figure 4.1.2 Schematic of a linear MALDI-TOF Mass spectrometer

Source: http://www.psrc.usm.edu/mauritz/maldi.html
The MALDI-TOF reflectron mass analyzer combines linear TOF technology with an
electrostatic mirror (reflectron) [1]. The ions enter the source region and are
accelerated toward the reflectron. The ions separate in time based on their relative
m/z ratio, reverse their path in the reflectron, and impact the time-of-flight reflectron
detector. The reflectron offers higher resolution over a linear TOF mass analyzer by
increasing the amount of time that ions take to reach the detector while reducing
(focussing) their kinetic energy distribution (i.e. equalising the different kinetic
energies of ions that arise during ionisation and acceleration). MALDI-TOF reflectron
mass analyzers can analyze small proteins, peptides, hormones and small molecules
up to 10 kDa, can distinguish monoisotopic masses, and have an accuracy range of
1-10 ppm [1].
MALDI-TOF (for proteins) and MALDI-TOF reflectron (for peptides) mass analyzers
have resolving power capabilities in the order of 400 to 10000 respectively. The
resolution and accuracy also depends on the presence of an internal standard, the
size/type of peptide/protein, sample purity and preparation, and the selection of
matrix material [1]. Even though MALDI is known to be more tolerant of salts, buffers,
and impurities, sample cleanup procedures (e.g. ZipTipTM or cold water washing) are
still useful [1]. The features of a peptide that affect peptide detection by MALDI-TOF
MS include its hydrophobicity (tends to adhere to a solid matrix), ionization efficiency,
mass, and basicity (arginine-containing peptides generally produce signals that are 2
to 20-fold stronger than lysine-containing peptides).
A type of tandem analysis is also possible with MALDI-TOF reflectron mass

analyzers. Tandem analysis (MS/MS) is the ability of the mass analyzer to separate
an ion, generate fragment ions from the original (selected) ion, and then analyze the
fragmentation ions [1]. MS/MS is accomplished by taking advantage of MALDI
fragmentation that occurs following ionization, known as post-source decay (PSD).
Post-ionization fragment ions from the same precursor ion (peptide) have different
kinetic energies, and the reflectron separates them based on this property, thereby
producing a fragment ion spectrum [1]. The fragmentation patterns obtained from
MALDI-TOF reflectron tandem MS experiments of proteolytically digested or
chemically cleaved target protein(s) yield specific partial sequence information (i.e.
two peptides with identical amino acid contents but different sequences will exhibit
different fragmentation patterns) and structural information (by performing successive
MS experiments on a number of generations of fragment ions) [1].
The design of modern mass analyzers have changed significantly in the last five
years to interface with MALDI and electrospray ionization (ESI), now offering much
higher accuracy and resolution, increased sensitivity, broader mass range and the
ability to give structural information. This has revolutionized biomolecular analyses,
allowing a measure of a wide range of biomolecular ions with ppm mass accuracy
and subfemtomole sensitivity [1]. An innovation that has had a dramatic effect on
increasing the resolving power of MALDI-TOF instruments has been delayed
extraction (DE), the process of cooling and focussing the ions immediately after the
MALDI ionization event [1]. In traditional MALDI instruments, the ions were
accelerated out of the ionization source immediately as they were formed
(continuous extraction). However, with DE, the ions are allowed to “cool” for ~150
nanoseconds before being accelerated to the analyzer. This cooling period generates
a set of ions with much smaller kinetic energy distribution, thereby resulting in a
dramatic improvement in resolution and accuracy for biomolecules less than 30 kDa
[1].
In addition, the automation of multisample probe preparation and sample analyses

with MALDI mass analyzers is becoming increasingly important in proteomics and
combinatorial chemistry [1]. Automation has made it possible to rapidly identify
species of interest by profiling changes in the expression levels of thousands of
proteins. Automated liquid handling robots have been developed that perform all the
sample preparation steps for peptide mapping experiments, including gel destaining,
alkylation/reduction, in gel digestion, peptide extraction, and MALDI target plating [1].
Commercial MALDI-TOF systems are currently available that can perform over 1,000
mapping experiments in just 12 hours. These systems are able to perform automated
calibrations, vary laser position energies, and adjust laser firing location to maximize
signal intensity [1]. Similarly, automated data processing systems can recognize
suitable signals, identify monoisotopic peaks, and submit summary peak lists directly
to a search engine for rapid identification.
The state-of-the-art ultraflex III MALDI-TOF and MALDI-TOF/TOF MS (produced by

Bruker Daltonics in 2006) incorporates next-generation technology, including
smartbeamTM laser technology for MALDI in vitro molecular imaging of peptide and
protein biomarker distributions in tissue sections; panoramic PANTM technology for
high mass resolution across a broad mass range; unique T3-sequencing capabilities,
allowing top-down sequence analysis on many intact proteins; tunable laser speed
from 1-200 Hz and adjustable focus size from 10 to 80 µm for high-throughput
analysis and sensitive detection of labile protein modifications; fully integrated
workflow for liquid-chromatography (LC)-MALDI experiments; and the MALDI-
TOF/TOF provides two MS/MS methods for de novo sequencing (LID-LIFT TOF/TOF
MS for highest specificity; and high-energy collision-induced decomposition (CID) for
in-depth analysis of selected proteins) [4].
MALDI and ESI have clearly evolved to be the ionization sources of choice when it
comes to biomolecular analysis [1]. MALDI has the ability to analyze complex
mixtures, with less suppression signal than ESI, making it extremely useful for
analyzing biological samples such as protein digests (see Table 4.1.2) [1].
Ionization Typical Matrix Degradation Complex LC/MS Sensitivity

source mass range interference mixtures amenable
(Da)
Electrospray 70000 none none somewhat excellent high
ionization limited femtomole
(ESI) to low
picomole
Comments Excellent LC/MS tool; low salt tolerance (low millimolar); multiple charging useful,
but significant suppression with mixtures occurs; low tolerance of mixtures; soft
ionization (little fragmentation observed)
NanoESI 70000 none none somewhat OK but high
limited but low flow zeptomole
better rates can to low
than ESI present a femtomole
problem
Comments Very sensitive and very low flow rates; applicable to to LC/MS; but low flow rates
require specialized systems; has reasonable salt tolerance (low millimolar); multiple
charging useful but significant suppression can occur with mixtures; reasonable
tolerance of mixtures; soft ionization (little fragmentation observed)
MALDI 300000 yes photo good for possible low to high
degradation complex femtomole
and matrix mixtures
reactions
Comments Somewhat tolerant of salts; excellent sensitivity; matrix background can be a
problem for low mass ions; soft ionization (little fragmentation observed);
photodegradation possible; suitable for complex mixtures and small molecules
Desorption/ 3000 none photo good for possible low to high
Ionization on degradation complex femtomole
silicon (DIOS) mixtures
Comments Somewhat tolerant of salts; excellent sensitivity; soft ionization (little fragmentation
observed); photodegradation possible; suitable for complex mixtures and small
molecules
Table 4.1.2 General comparison of Ionization sources
Biotechnology, pp. 35-36. MCC Press, San Diego, CA
However, complete and routine sequence determination through mass analysis of

intact proteins and complex biological samples has yet to be realized due to
significant signal suppression [1] . Even the tryptic digestion of a mixture containing
3-5 proteins will result in a peptide mixture complex enough to cause considerable
ionization signal suppression [1]. Thus, biological samples of proteins (or peptides in
a proteolytic digest) must first be separated by gel electrophoresis (SDS PAGE
separation on a 1D gel or 2D gel) or liquid chromatography or capillary
electrophoresis prior to mass analysis. At present, the method of choice for preparing
a biological sample for mass analysis on a MALDI-TOF reflectron is 1D or 2D gel
electrophoresis (which separate intact proteins), followed by proteolytic digestion [1].
By contrast, liquid chromatography (LC)-based methodologies (e.g. HPLC)
fractionate the peptide mixtures before analysis, thus decreasing signal suppression
and improving the analysis of any given peptide [1]. One of the most popular means
of performing peptide LC-MS/MS involves the direct coupling of the LC to an ion trap
MS through an ESI interface The advantages and disadvantages of protein
identification with MALDI-TOF reflectron and LC-MS/MS are shown in Table 4.1.3.
MALDI-TOF reflectron LC-MS/MS with an ion trap analyzer

Advantages Very fast In addition to molecular mass data,
tandem MS measurements are performed
in real time
Widely available MS/MS information adds additional levels
of confirmation
Easy to perform analysis Multiple proteins can be analyzed
simultaneously with simple reversed-phase
LC run
High accuracy (10-50 ppm) adds Useful for PTM identification
reliability to data
Useful for wide range of proteins High coverage of proteins (30% to 90%)
depending on the protein
Disadvantages Problematic for mixtures of proteins Computationally intensive; large database
searches can take hours to days; relatively
slow
Typically less coverage than LC-
MS/MS approach
Table 4.1.3 Protein Identification with MALDI and LC-MS/MS

Biotechnology, p. 131. MCC Press, San Diego, CA
At the Australian Proteome Analysis Facility (APAF), the identification of protein(s)

involves digesting the protein with trypsin, and analysing the digested protein with a
MALDI-TOF MS to produce a peptide mass fingerprint [5]. The experimentally
measured monoisotopic masses of the peptides seen in the mass spectrum are
selected using software, and then compared to all the theoretically predicted trypsin
peptide digests from a database containing hundreds of thousands of proteins [6,7]
using computer search programs such as Mascot, MS-Fit, Aldente and Profound
(see section 4.1.5) [1, 5]. This application is recommended for the identification of
known purified proteins, and has the advantages of rapid analysis, high sensitivity,
and being suitable for large numbers of samples. However, the protein of interest
must already be in the protein database, is generally not suitable for proteins < 15
kDa or for the identification of post-translational modifications, and the match is
based on peptide masses, not sequence information [5].
Once a peptide mass fingerprint is generated via MALDI-TOF MS as described

above, the most abundant peptide ions can then be subjected to MALDI-TOF/TOF
analysis, providing added information that can be used to determine the protein
sequence [5]. The results of both types of analyses are combined and search
engines such as Mascot are used against protein, DNA or EST databases to identify
the protein of interest (1,5]. This application is recommended for the identification of
known purified proteins requiring a higher level of confidence than with MALDI-TOF
alone, and is able to identify 2-3 proteins in the same spot and allow the identification
of small proteins < 15 kDa based on sequence information [5].
Other major applications for MALDI-TOF technology, with specific examples, include:
1) the identification of distinct protein profiles that contribute to subtypes and

facilitate classification of certain cancers (e.g. acute leukemia) for future
treatment and prognosis [8]. In this study, the proteins of leukemic cells from 61
cases of acute leukemia characterized by French-American-British (FAB)
classification were separated by 2-D gel electrophoresis, and the differentially
expressed protein spots were identified by MALDI-TOF MS. Distinct protein
profiles of acute leukemia FAB types or subtypes, including acute myeloid
leukemia (AML) and its subtypes M2, M3 and M5, and acute lymphoid leukemia
(ALL), were identified. Myeloid-related proteins 8 and 14 (involved in AML
differentiation) were highly expressed in M2 and M3 subtypes, and heat shock 27
kDa protein 1 and other proteins were highly expressed in ALL, making it
possible to clinically distinguish AML from ALL [8].
2) the identification of a complete proteomic profile of snake venom (a complex

mixture of proteins and peptides) for potential medical treatments [9]. In this
study, the venom proteomic profiles from two snakes common to southern China,
the cobra and viper, were assessed using four different approaches, including gel
filtration and 2D gel electrophoresis plus MALDI-TOF MS. The novel identification
of 124 and 74 proteins and peptides in the cobra and viper venom respectively
was reported. Functional analyses revealed that cobra venom has a high
abundance of cardio- and neurotoxins, whereas viper venom contains a
significant amount of haemotoxins and metalloproteinases. Only 50% of gel spots
were confirmed to be venom proteins, probably due to incomplete protein
databases, which suggests that post-translational modifications may be a
significant characteristic of venomous proteins [9].
3) the accurate, sensitive and rapid identification of multiple bacterial strains

relevant to public health and food safety [10]. In this study, multiple strains of six
species of Campylobacter coli isolated from animal, clinical, or food samples
were analyzed by MALDI-TOF MS. Whole bacterial cells were harvested from
colonies or confluent growth, transferred directly into solvent and then onto a spot
of dried 3-methoxy-4-hydroxycinnamic acid (matrix). “Species-identifying”
biomarker ions (SIBI) were evident from analyses of multiple reference strains for
each of the six species. MALDI-TOF MS analysis of 75 additional Campylobacter
strains isolated from humans, poultry, swine, dogs and cats revealed I)
associations of SIBI with source, (ii) strains previously speciated incorrectly, and
(iii) “strains” composed of more than one species [10].
The following is a list of Australia’s leading research/medical Mass Spectrometry

Facilities (with their relevant websites) that use MALDI-TOF and MALDI-TOF TOF
mass analyzers in areas including protein identification, the investigation of protein
expression, protein or peptide biomarker discovery in various illnesses, and potential
therapeutic discovery from both plants and animals.
1) The Australian Proteome Analysis Facility (APAF Ltd)

(http://www.proteome.org..au/);
2) The Australian National University (ANU) Research School of Biological Sciences
(http://www.rsbs.anu.edu.au/);
3) The University of Queensland Cellular Proteomics Mass Spectrometry Facility
(MCPMSF) within the Institute for Molecular Bioscience (IMB)
(http://www.imb.uq.edu.au/);
4) The RMIT School of Applied Sciences Mass Spectrometer Facility
(http://minyos.its.rmit.edu.au/); and
5) The Ludwig Institute for Cancer Research (LICR) (http://www.ludwig.edu.au/).
Some useful learning web resources linked to these Mass Spectrometer Facilities
and MALDI-TOF and MALDI-TOF TOF mass analyzers include:
1) Wikipedia, the free encyclopedia (http://en.wikipedia.org/);

2) http://I-mass.com/ for general articles, tutorials, MS tools, and links to MS
literature;
3) http://www.spectroscopynow.com/Spy/basehtml/SpyH; and
4) Bioscienceworld, insights for the life sciences industry
(http://www.bioscienceworld.ca/)
In addition, some computer search engines used to search MALDI-TOF peptide

mass fingerprint data against protein databases for the identification of proteins
include:
1) MASCOT (http://www.matrixscience.com);
2) MS-Fit (http://prospector.ucsf.edu);
3) ALDENTE (http://au.expasy.org/tools/aldente/); and
4) ProFound (http://129.85.19.192/profound_bin/Web/ProFound.exe).
The following is a list of the major suppliers (and their website home pages) of MS
instruments, particularly MALDI-TOF and MALDI-TOF TOF mass analyzers:
Bruker Daltonics (http://www.bruker-biospin.com.au/)
Applied Biosystems (http://www.appliedbiosystems.com.au/)

Shimadzu Biotech (http://www.shimadzu.com/)
Agilent Technologies (http://www.agilent.com/)
Waters (http://www.waters.com/)
Thermo Electron Corporation (http://www.thermo.com/)
4.1.7 References
1. Siuzdak, G. (2003) The Expanding role of Mass Spectrometry in Biotechnology,

MCC
Press, San Diego, CA.
2. Tanaka, K., Waki, H., Ido, Y., Akita, S., Yoshida, Y., Yoshida, T. (1988) Protein
and polymer analysis up to m/z 100.000 by laser desorption time-of-flight mass
spectrometry. Rapid Commun Mass Spectom. 2, 151-153.
3. Karas, M. & Hillencamp, F. (1988) Laser desorption ionization of proteins with

molecular masses exceeding 10.000 daltons. Anal Chem. 60, 2299-2301.
4. Bruker Daltonics. (2006) Ultraflex III & ultraflex III TOF/TOF. Ultimate
Performance
MALDI-TOF & TOF/TOF Systems.
http://www.bdal.de/modux3/modux3.php?pid=105,001,003,01,01,006,009,0&rid=105,
001
5. The Australian Proteome Analysis Facility (2006) MALDI MS Analysis for Protein
Identifications. Protein Identification by MALDI-TOF (PMF).
http://www.proteome.org.au/MALDI-MS/default.aspx
6. The NCBInr protein database. (2006) A non-redundant database compiled by the

National Center for Biotechnology (NCBI) at the National Institute of Health (United
States).
http://www.ncbi.nlm.nih.gov/
7. The SWISS-PROT protein database. (2006) The Australian mirror of the curated
protein sequence database compiled by the Swiss Institute of Bioinformatics and the
European Bioinformatics Institute.
http://au.expasy.org/sprot/
8. Cui, J., Wang, J., He, K., Jin, B., Wang, H., Li, W., Kang, L., Hu, M., Li, H., Yu,
M., Shen,
B., Wang, G., Zhang, X. (2004) Proteomic analysis of human acute leukemia cells:
insight into their classification. Clin Cancer Res. 10, 6887-6896.
9. Li, S., Wang, J., Zhang, X., Ren, Y., Wang, N., Zhao, K., Chen, X., Zhao, C., Li,
X.,
Shao, J., Yin, J., West, M., Xu, N., Liu, S. (2004) Proteomic characterization of two
snake venoms: Naja naja atra and Agkistrodon halys. Biochem J. 384, 119-127.
10. Mandrell, R., Harden, L., Bates, A., Miller, W., Haddon, W., Fagerquist, C. (2005)
Speciation of Campylobacter coli, C. jejuni, C. helveticus, C. lari, C. sputorum, and C.
upsaliensis by matrix-assisted laser desorption ionization-time of flight mass
spectrometry. Appl Environ Microbiol. 71, 6292-6307.
Chapter 5.1 2D Gel Electrophoresis
Paolo Dominic Navidad
5.1.1 Introduction
Two-dimensional (2D) gel electrophoresis is a commonly used method for analyzing

complex protein mixtures. It is used to separate a large number of proteins based on
two independent biological properties – the isoelectric point and the molecular weight
[1]. The sample is displaced into two dimensions perpendicular to each other, with
each dimension corresponding to either biological property. The basic steps of 2D
gel electrophoresis are [2]:
• sample preparation
• first dimension isoelectric focusing
• second dimension gel electrophoresis
• staining
• image analysis
Sample preparation involves the solubilization and denaturation of the sample

proteins. A successful preparation should not cause aggregation and chemical
modification during the run [3]. Solubilization separates the proteins into their
individual components and involves the use of a solubilization/denaturation (SD)
buffer. The main components of the buffer are a chaotrophe, such as urea and
thiourea, to disrupt hydrogen and hydrophobic bonds; a reductant, such as beta-
mercaptoethanol, to break disulphide bonds; a detergent, such as TRITON X-100, to
disrupt membranes and to solubilize lipids; ampholytes, to aid protein solubilization
and nucleic acid precipitation; and protease inhibitors [4]. The first dimension of
the 2D procedure is isoelectric focusing (IEF), where proteins are separated based
on their isoelectric points (pI) due to the establishment of a pH gradient in a
polyacrylamide gel [1]. The pH gradient is formed by either the addition of carrier
ampholytes, through the use of an immobilized pH gradient (IPG) strip, or a
combination of the two [4]. Ampholytes are added to the gel and must be pre-
focused to produce a gradient, while IPG strips are separately prepared from the
polyacrylamide gel. An advantage of using IPG’s is that reproducibility is improved
due to improved mechanical stability and a pH gradient that doesn’t drift during a run
[3]. Because of this, IPG-2D gel electrophoresis (IPG-Dalt) is more commonly
performed as a flexible method for proteomic analysis [5]. In IPG-Dalt, the samples
are first run on IPG strips (Figure 5.1.1), after which the strip is equilibrated with SDS
to ensure the proteins all have a negative charge, DTT to reduce disulphide bridges
that may have reformed, and iodoacetamide for protein alkylation and to remove
excess DTT [3]. The samples are now separated according to their respective pI.
Figure 5.1.1 Individual IPG strips in an electrophoresis unit
Source: http://www.weihenstephan.de/blm/deg/manual/manfrm.htm
The second dimension SDS-PAGE can be run on either a vertical or a horizontal set-
up [6]. In a horizontal system, the equilibrated IPG strips are placed gel side down
on the surface of the stacking gel without any embedding procedure, while a vertical
system involves embedding IPG strips in agarose on top of the vertical second
dimension gel (Figure 5.1.2) [7]. An electrical charge is then applied to separate the
proteins according to their size. Similar to what happens in an ordinary SDS-PAGE
procedure, the molecules migrate across the gel at different speeds, with larger
molecules moving more slowly than smaller molecules. Horizontal gels produce
sharper spots because these are half as thick as vertical gels, but vertical systems
allow for simultaneous running of multiple gels in a single electrophoresis tank [5].
Figure 5.1.2 Loading an equilibrated IPG strip on top of a polyacrylamide gel

Source: http://www.weihenstephan.de/blm/deg/manual/manfrm.htm
After the separation of the samples, individual spots may be visualized by staining
the gel. A variety of different types of staining procedures – some colorimetric, some
fluorescent – may be used in this procedure, each with its own advantages and
disadvantages. Silver staining is a highly sensitive procedure and the end results
can be mass-spectrophotometry compatible, but it is time-consuming, labor-
intensive, and causes poor staining with some proteins [5]. Also, spot intensity does
not necessarily correlate to protein concentration. Zinc or copper staining produces a
negative stain (gel is stained white, proteins are unstained) because zinc/copper
does not stain SDS, which coats the proteins [4]. Zinc/copper staining is rapid and
inexpensive but cannot be used on thin gels, which provide poor contrast with this
procedure. Coomassie Blue is the most commonly used stain for acrylamide gels,
and is expensive and easy to use. However, a destaining step is required and non-
specific staining, particularly with polysaccharides, sometimes occurs [3].
Fluorescent dyes bind to the SDS that coats the proteins. Fluorescent staining may
be done before IEF, after IEF but before SDS-PAGE, or after SDS-PAGE [5]. There
are many different kinds of fluorescent stains. Generally speaking, they are highly
sensitive, quick and easy to use, and can be used for quantitative purposes.
However, some fluorescent dyes are quite expensive and may be incompatible with
some plastic-backed gels [3]. After staining, the finals step is identification and
characterization of the protein spots. Gels are scanned and image analysis can be
performed to determine statistically and scientifically significant spots [2]. Image
analysis is conducted using specialized software that have the ability to perform spot
detection, spot matching between gels, and spot quantification and comparison [8].
These spots can then be excised, characterized, and identified by MS. An example
of a 2D gel is shown below (Figure 5.1.3).
Figure 5.1.3 Silver stained sample of human embryonic kidney cells (hek293 cell
line)
Source: http://gelbank.anl.gov/cgi-
bin/2dgels/gel_display_with_list.pl?NameOfGel=221
IPG strips have led to improved visualization of low-abundance proteins and to

further resolve protein species in the gel. In a protocol proposed by Herbert and
Righetti wherein they used a multicompartment electrolyzer (MCE), load ability,
detection, and sensitivity were enhanced through sample prefractionation [9]. An
MCE is an apparatus with chambers of different pH ranges that were separated by
membranes (Figure 5.1.4).
Figure 5.1.4 Schematic diagram of an MCE

Source: Herbert, Righetti. 2000. Electrophoresis
Proteins with similar pI migrate to the same chamber. After prefractionation, 2D

PAGE was performed on the samples (E. coli, human plasma extracts). The authors
observed that protein precipitation and smearing was reduced. Also, because a
larger amount of prefractionated proteins can be loaded onto the gel, a larger number
of spots can be observed.
Membrane proteins are difficult to recover with 2D gel electrophoresis because they
are hardly soluble in buffers used in IEF [10]. As a result, some species are partially
or completely absent in the final gel. Santoni et al. performed prefractionation of
Arabidopsis proteins using treatment of Triton-X114, carbonate, and
chloroform/methanol, and solubilization with a set of zwitterionic detergents [11]. The
authors observed improved isolation of membrane proteins for 2D gel
electrophoresis.
Fluorescent stains have several advantages over more traditional silver stains. They
are more sensitive, easy to use, and have a better dynamic range [10]. Spyro post-
electrophoretic fluorescent stains, in particular Spyro Ruby (Molecular Probes,
Eugene, OR), are some of the more recent stains to be developed. Spyro Ruby, in
addition to being highly sensitive, is also compatible with subsequent protein analysis
such as Edman sequencing and MS [12]. Since Spyro Ruby is expensive, Rabilloud
et al. have developed a protocol to produce an alternate stain that performs similar to
Spyro Ruby [13].
Image analysis is often laborious and slow, and difficulties are encountered
especially when performing spot boundary assignment, normalization of the gel
background and intensity variation, and spot matching between gels. There are
various commercial software packages that perform these tasks, but these still
require manual users, leading to a problem in reproducibility [10]. Smilansky
developed a system called Z3 that performed image analysis with improved speed
and precision. The algorithm that was developed relied on “computation of the
registration directly from the raw images, region-based matching, and
complementary pseudocolor display” [14].
Westbrook et al. performed 2D gel electrophoresis using combinations of very

narrow-range IPG strips, and compared the results to broad-range and narrow-range
IPGs [15]. They observed that very narrow-range IPGs have the ability to separate
different protein species and isoforms. With very narrow-range IPGs, they also
observed that the number of co-migrating protein species was reduced, leading to
more reliable database searches compared with broad- or narrow-range IPGs.
Reproducibility of 2D gel electrophoresis can be improved by using differential in-gel

electrophoresis (DIGE) [16]. This technique involves two pools of protein extracts
labeled with Cy3 and Cy5. These are then mixed and separated on a 2D gel. The
patterns are visualized by the excitation of the dyes and comparison of the resulting
images allows quantitation of the spots. Proteins that exist in both pools would
migrate to the same spot. DIGE can be a useful tool in comparative proteomics,
such as analyzing protein level differences caused by a disease state, drug
treatment, or life cycle stage [17].
Laser capture microdissection (LCM) can be used in combination with 2D gel

electrophoresis to produce more specific results [18]. LCM is a technique that can be
used to separate cells in heterogenous mixtures. A transfer film is placed on a tissue
sample or heterogenous mixture. A laser beam activates an area in the film where
the cells of interest are located, and these cells attach to the film [19]. Because the
subsequent sample is more or less homogenous, protein analysis can be improved.
2D-GE in its basic form has been in use for quite some time and is usually
categorized as part of classical proteomics. Classical proteomics involves a
separation step (2D-GE) followed by an identification step, usually MS. 2D-GE is a
relatively simple method for mapping differences in protein expression, and is
currently the most rapid method for direct targeting of protein expression differences
[20]. With some recent advances, 2D-GE has progressed since it was first described
by O’Farrell in 1975 [21]. IPGs have improved the resolutions of gels, while
fluorescent dyes provided sensitivity in visualization [22]. Computer software are
continuously being developed and improved. The use of DIGE has overcome some
problems with reproducibility. Because of its simplicity, versatility, and relatively easy
visualization, 2D-GE is still in use in spite of being a relatively old technology. In fact,
“no other technology allows the ready separation and quantitation and identification
of complex protein mixtures” [21]. However, there still remain several disadvantages
to this technique.
A major limitation of this technique is problems with reproducibility. Because no two

gels run in the exact same manner, comparisons between images will produce some
discrepancies. Even the use of DIGE cannot completely bypass the problem. The
issues of gel-to-gel variation and intrinsic biological variations must be addressed
[20]. Gel replication in triplicates or more may be performed, but this would entail
laborious image analysis.
2D-GE has limited sensitivity. One hundred fifty µg of protein from a total cell extract
generally generates around 2000-3500 identifiable spots depending on the dye used,
and around 10,000 proteins using narrow pH gradients [21,23]. The total number of
proteins in a cell is estimated to be around 30,000. Low-abundance, very high or low
molecular weight, very acidic or basic, and/or hydrophobic species are often
underrepresented in a 2D gel. Besides the use of sensitive stains, other methods to
bypass the problem are sample fractionation, selective extraction of high abundance
proteins, and loading large sample sizes in a large gel [21]. However, other factors
such as resolution and ease of image analysis, labor-wise, will be sacrificed for
sensitivity.
Other limitations for this technique include limited loading capacity of IPG strips,
relatively low throughput, and low linear range of visualization procedures [20]. In
addition, image analysis is still a labor-intensive step in 2D-GE. Despite advanced
computer programs that aid in this task, full automation has not yet been achieved
and manual involvement is still required. This, in addition to supplying extra work,
may also lead to loss in reproducibility [10].
There are alternative methods that may be used, but these are generally not as
efficient as 2D-GE. Liquid chromatography-mass spectrometry (LC-MS) is a tandem
procedure wherein a protein mixture is separated by LC, and “pure” compounds are
introduced directly to the mass spectrometer [24]. Therefore, proteins with similar
retention characteristics can be differentiated via their mass spectra. However, LC-
MS is much less versatile than 2D-GE, and does not have the ability to provide
quantitative data [10]. Using LC-MS also presents difficulties in performing
differential display analysis [20]. The use of stable isotope-coded affinity tags (ICAT)
for protein extraction and tagging is commonly done in quantitative proteomics.
Samples are labeled and identified using LC/MS/MS [21]. This technique can be
used in differential expression studies of whole proteomes, and its main strength is in
its ability to provide quantitative data. A major limitation of this technique is that it is
unable to label proteins that do not contain cysteine [25]. Since protein digestion is
involved, it is difficult to establish the association of post-modified forms of peptides
and their assembly into modified proteins [23]. Also, due to non-denaturing
conditions, the source of fluorescence must be checked to determine that it is due to
the target protein and not from an unwanted protein complex [26].
Still, 2D-GE is considered the gold standard as a protein separation method prior to
MS because it is a proven research tool due to its simplicity and ability to quantify
and visualize a range of samples greater than what other techniques can currently
achieve.
Proteomics, in general, has a wide range of applications. For 2D-GE specifically, its
main applications are protein identification and differential expression [4]. Coupled
with MS, it is used for large-scale identification of proteins in a sample. In one study,
Xu et al. used 2D-GE and MS to identify soybean leaf proteins [27]. They analyzed
260 spots and compared these against various databases. They discovered that
majority of the identified leaf proteins were involved in energy metabolism. There are
clinical applications for this technique, as well. Human myocardial proteins were
identified using 2D-GE [28]. Using 2D-GE/MS, proteomic profiles of various cells can
be generated to provide not only information about protein content, but also on
protein activity, interactions, and localization. Hoffrogge et al. performed proteomic
analysis on neuronal stem cells, Lominadze et al. analyzed neutrophils, and
Martinez-Hereida et al. did work on sperm cells [29,30,31]. Proteomic analysis using
2D-GE can also be used on pathogens. Pereira et al. looked at proteomic profiles of
the bacterium H. pylori to investigate possible pathogenic factors [32].
The other major application for 2D gel electrophoresis is in differential proteomics,

which compares distinct proteomes such as normal versus diseased cells or
diseased versus treated cells [20]. With the advent of DIGE, reproducibility has
improved since multiple protein mixtures can be run on a single gel. 2D-GE may be
used for identification of biomarkers in drug treatment. Discovery of novel protein
biomarkers would aid in drug development and enable detection of disease at an
early stage, measurement of disease progress, and monitoring of treatment efficacy
[33]. There have been several studies that used 2D-GE that have shown promising
results. Torres-Cabala et al. used the technique as one of the steps in identifying
possible biomarkers that may distinguish between malignant and benign thyroid
lesions [34]. Roessler et al. also used 2D-GE in identifying PSME3 as a possible
tumor marker for colorectal cancer, while Li et al. looked for possible breast cancer
metastatic markers [35,36]. Differential proteomics using 2D-GE can be used in
studying pathogens. For example, a proteome profile of a resistant organism may be
compared with that of a susceptible organism. Pieper et al. utilized this method to
study vancomycin resistant S. aureus strains and discovered an enzyme that was
involved in an altered cell wall turnover rate and altered peptidoglycan structure [37].
Foucher et al. performed proteomic analysis on arsenic resistant T. brucei, which
causes sleeping sickness, and discovered a protein that was present in both resistant
and susceptible organisms but at different pI [38].
While non-gel-based methods are also being used for quantitative differential
proteomics, 2D-GE methods, with complementary technology that improve sensitivity
and expand analytical range, are still considered highly useful and informative tools
in proteomic analysis.
These are some websites relevant to 2D gel electrophoresis. There are various
databases that provide data on proteins and various 2D-PAGE reference maps.
Some of these are:
• ExPASy - SWISS-2DPAGE (human, mouse, E. coli, yeast)
http://au.expasy.org/ch2d/
• Max Planck Institute for Infection Biology (mostly microbial organisms)
http://web.mpiib-berlin.mpg.de/cgi-bin/pdbs/2d-page/extern/index.cgi
• Argonne National Lab, Protein Mapping Group
http://gelbank.anl.gov/2dgels/index.asp
• Joint ProteomicS Laboratory
http://www.ludwig.edu.au/jpsl/Databases.asp
• Danish Centre for Translational Breast Cancer Research
http://proteomics.cancer.dk/cgi-bin/CelisWeb.exe?MsetList.htm
• Siena-2DPAGE
http://www.bio-mol.unisi.it/2d/2d.html
• World-2DPAGE Portal (a portal for 2D PAGE databases)
http://au.expasy.org/world-2dpage/
A tutorial on 2D-GE by Dr. James R. Jefferies can be accessed on the University of

Wales at Aberystwyth on the link below. Biocompare has also produced an easy-to-
understand tutorial that covers the basic concepts of the technique.
• Jefferies tutorial
http://gelbank.anl.gov/2dgels/index.asp
• Biocompare 2D-GE tutorial
http://www.biocompare.com/tutorials/2DE_tutorial/html/proc_overview.asp
A manual on 2D-GE using IPGs by Angelika Görg can be located on the website
below. It covers the basic steps of the procedure and possible variations in the
protocol.
http://www.weihenstephan.de/blm/deg/manual/manfrm.htm
Technical information on 2D-PAGE can be obtained at the ExPASy site. It contains

protocols and a list of chemicals used in the procedure.
http://us.expasy.org/ch2d/protocols/
A good journal that covers the advances, mainly technical, in the field of 2D-GE is
Electrophoresis, while many applications of the technology are published by
Proteomics. Issues and articles from both journals are available online.
• Electrophoresis home page
http://www3.interscience.wiley.com/cgi-bin/jhome/10008330
• Proteomics home page
http://www3.interscience.wiley.com/cgi-bin/jhome/76510741/
Companies that are involved in the 2D-GE industry have two main groups of
products: the reagents that are used in sample preparation, gel running, etc, and the
various image analysis software. Some companies offer both types of products but
there are several bioinformatics companies that develop specialized software.
Invitrogen offers 2D-GE systems and components such as IPG strips, ampholytes,
and gels. They also produce Spyro Ruby fluorescent gel stains and Spyro photo
filters.
• Invitrogen 2D PAGE
http://www.invitrogen.com/content.cfm?pageid=10830
• Protein stains for 2D gels
http://probes.invitrogen.com/servlets/directory?id1=8&id2=78&id3=335
Companies that provide 2D-GE reagents, gels, and equipment include GE
Healthcare (formerly Amersham Biosciences), NextGen Sciences, and Bio-Rad
Laboratories. Sigma-Aldrich has various stains and IPG strips for use in 2D-GE.,
while Genetix has the GelPix instrument that can perform spot excision, data
tracking, and onboard imaging.
• GE Healthcare 2D Electrophoresis
http://www4.amershambiosciences.com/aptrix/upp01077.nsf/Content/2d_elec
trophoresis
• NextGen Sciences 2D Electrophoresis
http://www.nextgensciences.com/NewWebsite/Products/2DElectrophoresis.ht
m
• Bio-Rad Laboratories home page
http://www.biorad.com
• Sigma-Aldrich Protein Electrophoresis
http://www.sigmaaldrich.com/Area_of_Interest/Life_Science/Proteomics_and_
Protein_Expr_/Protein_Analysis/Protein_Electrophoresis.html
• Genetix GelPix
http://www.genetix.com/instr/gelpix.asp
Various 2D-GE programs and software are offered by companies such as GE

Healthcare and Bio-Rad Laboratories. There are also companies that specialize
exclusively in image analysis software. Shimadzu Biotech offers software such as
the Phoretix 2D. Syngene is a well-known company that produces gel-
documentation and analysis systems. Genomics Solutions has gel imaging and
picking systems in addition to image analysis software. The GELLAB II+ is a
comprehensive program for gel analysis and is available from Scanalytics Inc. (now
part of BD Biosciences).
• Shimadzu Biotech home page
http://www.shimadzu-biotech.net/
• Syngene home page
http://www.syngene.com/
• Genomic Solutions Proteomics
http://www.genomicsolutions.com/showPage.php?cachevar=&menuID=361
• Scanalytics GELLAB II+
http://www.scanalytics.com/product/gellab/index.shtml
5.1.7 References
1. The Maiman Institute for Proteome Research, Tel Aviv University (2006) Two-
Dimensional Gel Electrophoresis,
http://www.tau.ac.il/lifesci/units/proteomics/2dimgel.html
2. Molecular Structure Facility, University of California at Davis (2006) 2-D Gel
Electrophoresis,
http://msf.ucdavis.edu/2-d_gel.html
3. Biocompare (2006) 2D Gel Electrophoresis Tutorial,
http://www.biocompare.com/tutorials/2DE_tutorial/html/background.asp
4. Jefferies, J.R. (2005) 2D Gel Electrophoresis for Proteomics Tutorial,
http://www.aber.ac.uk/parasitology/Proteome/Tut_2D.html
5. Görg, A., Obermaier, C., Boguth, G., Harder, A., Scheibe, B., Wildgruber, R.
& Weiss, W. (2000) The current state of two-dimensional electrophoresis with
immobilized pH gradients. Electrophoresis. 21, 1037-1053.
6. Carrette, O., Burkhard, P.R., Sanchez, J. & Hochstrasser, D.F. (2006) State-
of-the-art two-dimensional gel electrophoresis: a key tool of proteomics
research. Nat. Protocols. 1, 812-823.
7. Görg, A., Boguth, G., Harder, A., Obermaier, C., Scheibe, B., Wildgruber, R.
& Weiss, W. (1998) Two-Dimensional Electrophoresis of Proteins Using
Immobilized pH Gradients,
http://www.weihenstephan.de/blm/deg/manual/manfrm.htm
8. Mathematical and Information Sciences, Commonwealth Scientific and
Industrial Research Organisation (2005) 2D Gel Image Analysis,
http://www.cmis.csiro.au/iap/RecentProjects/protein.htm
9. Herbert, B. & Righetti, P.G. (2000) A turning point in proteome analysis:
sample prefractionation via multicompartment electrolyzers with isoelectric
membranes. Electrophoresis. 21, 3639-3648.
10. Lilley, K.S., Razzaq, A. & Dupree, P. (2001) Two-dimensional gel
electrophoresis: recent advances in sample preparation, detection and
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11. Santoni, V., Kieffer, S., Desclaux, D., Masson, F. & Rabilloud, T. (2000)
Membrane proteomics: use of additive main effects with multiplicative
interaction model to classify plasma membrane proteins according to their
solubility and electrophoretic properties. Electrophoresis. 21, 3329-3344.
12. Molecular Probes (2005) SPYRO® Ruby Protein Gel Stain,
http://probes.invitrogen.com/media/pis/mp12000.pdf
13. Rabilloud, T., Strub, J., Luche, S., van Dorsselaer, A. & Lunardi, J. (2001) A
comparison between Spyro Ruby and ruthenium II tris (bathophenanthroline
disulfonate) as fluorescent stains for protein detection in gels. Proteomics. 1,
699-704.
14. Smilansky, Z. (2001) Automatic registration for images of two-dimensional
protein gels. Electrophoresis. 22, 1616-1626.
15. Westbrook, J.A., Yan, J.X., Wait, R., Welson, S.Y. & Dunn, M.J. (2001)
Zooming-in on the proteome: very narrow-range immobilised pH gradients
reveal more protein species and isoforms. Electrophoresis. 22, 2865-2871.
16. Zhou, G., Li, H., DeCamp, D., Chen, S., Shu, H., Gong, Y., Flaig, M.,
Gillespie, J.W., Hu, N., Taylor, P.R., Emmert-Buck, M.R., Liotta, L.A.,
Petricoin III, E.F. & Zhao, Y. (2002) 2D differential in-gel electrophoresis for
the identification of esophageal scans cell cancer-specific protein markers.
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17. GE Healthcare (2005) Ettan DIGE System User Manual,
http://www6.amershambiosciences.com/aptrix/upp00919.nsf/(FileDownload)?
OpenAgent&docid=ABD636471F4EFF17C12571C000813047&file=18117317
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18. Craven, R.A., Totty, N., Harnden, P., Selby, P.J. & Banks, R.E. (2002) Laser
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proteomics methods to the study of breast cancer. J. Mammary Gland Biol. 7,
385-393.
22. Görg, A., Postel, W. & Günther, S. (1988) The current state of two-
dimensional electrophoresis with immobilized pH gradients. Electrophoresis.
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23. Hitt, E. (2006) Separation of complex protein samples prior to mass
spectrometry remains a bottleneck in the rapid resolution of proteomes,
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24. Ardrey, R.E. (2003) Introduction. In Liquid Chromatography-Mass
Spectrometry, pp. 1-5. J. Wiley, Chichester, UK.
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26. SWEGENE Proteomics Lund (2003) Peptide-based Approaches to
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Separation and identification of soybean leaf proteins by two-dimensional gel
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29. Hoffrogge, R., Beyer, S., Volker, U., Uhrmacher, A.M. & Rolfs, A. (2006) 2-DE
proteomic profiling of neuronal stem cells. Neurodegener. Dis. 3, 112-121.
30. Lominadze, G., Ward, R.A., Klein, J.B. & McLeish, K.R. (2006) Proteomic
analysis of human neutrophils. Methods Mol. Biol. 332, 343-356.
31. Martinez-Heredia, J., Estanyol, J.M., Ballesca, J.L. & Oliva, R. (2006)
Proteomic identification of human sperm proteins. Proteomics. 6, 4356-4369.
32. Pereira, D.R., Martins, D., Winck, F.V., Smolka, M.B., Nishimura, N.F.,
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Berndt, P., Engel, A.M., Pfeffer, M., Karl, J., Bodenmueller, H., Ruschoff, J.,
Henkel, T., Rohr, G., Rossol, S., Rosch, W., Langen, H., Zolg, W. & Tacke,
M. (2006) Identification of PSME3 as a novel serum tumor marker for
colorectal cancer by combining two-dimensional polyacrylamide gel
electrophoresis with a strictly mass spectrometry-based approach for data
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Lu, J.S., Di, G.H., Ou, Z.L., Xia, Q.C., Shen, Z.Z. & Shao, Z.M. (2006)
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chromatography-ion trap-mass spectrometry. Proteomics. 6, 3352-3368.
37. Pieper, R., Gatlin-Bunai, C.L., Mongodin, E.F., Parmar, P.P., Huang, S.T.,
Clark, D.J., Fleischmann, R.D., Gill, S.R. & Peterson, S.N. (2006)
Comparative proteomic analysis of Staphylococcus aureus strains with
differences in resistance to the cell wall-targeting antibiotic vancomycin.
Proteomics. 6, 4246-4258.
38. Foucher, A.L., McIntosh, A., Douce, G., Wastling, J., Tait, A. & Turner, C.M.
(2006) A proteomic analysis of arsenical drug resistance in Trypanosoma
brucei. Proteomics. 6, 2726-2732.
Chapter 5.2 Chromatographic Methods for the Separation of
proteomes
5.2.1 Introduction
The term proteome is referred as the complement and collection of proteins in a

biological system. Proteomics, the study of proteomes, has been defined widely
as the link between proteins and genomes. The major distinguishable difference
between genomes and proteomes are, genome is defined by the sequence of
nucleotides, where as proteome requires the knowledge of structure of proteins.
In a biological system proteomes are larger than genomes, this is due to alternative
Splicing of genes and posttranslational modifications like phosphorylation.
Proteomics
Provides a detailed description of protein analysis of different protein mixtures and
their properties, interactions and modifications. This method also provides
information of
changes in protein expression levels, which are associated with drug treatment,
genetic manipulation or changes in metabolism. Different proteome molecules are
mixed together in a solution. There are different chromatographic techniques to
extract and evaluate the selected proteomes from the sample. SDS-PAGE (Sodium
dodecyl sulphate – polyacrylamide gel electrophoresis) and Mass Spectroscopy are
two important chromatographic techniques, which are used commonly in biochemical
test to separate and identify the proteomes.
Electrophoresis is the process in which migration of charged molecules takes place

in response to the electric field. The rate of migration of charged molecules depends
on the size, shape and charge of the molecules. The basic principle in this technique
depends on the charge of proteins. Sodium dodecyl sulfate is an anionic detergent,
which binds strongly to proteins and all proteins acquires an negative charge,
irrespective of their native charges. Heating in a buffer containing reducing agents
like 2-mercaptoethanol and SDS denatures proteins and disrupts all intra and
intermolecular protein interactions. These denatured proteins are resolved on the
basis of size in a buffered gel. These SDS-PAGE gel systems are very useful in
analysis of separation of proteome mixtures.
The above photograph is an example of separation proteins on the basis of charge
by SDS- PAGE electrophoresis. The dotted spots on the photograph indicate the
protein molecules. Extraction and separation of proteins is based on length of the
dotted spots on the gel.
The other chromatographic technique used in separation of proteomes is Mass

Spectroscopy. In this technique the desired proteomes are extracted on the basis of
their mass. The desired protein to be extracted is treated with the enzyme trypsin and
this sample is passed into a mass spectrometer and the substance is combined with
an electron beam. This electron beam has sufficient energy to separate the
molecules based on their mass. The positive cation molecules produced are
accelerated in a vacuum through a magnetic field and are separated on the basis of
mass to charge ratio. The value of mass to charge ratio is equal to the molecular
weight of the proteome. There is a simple diagrammatic representation of mass
spectroscopy is shown below. The molecular fragment whose mass is to be
determined is passed into an electron beam and ion accelerating array and then it is
passed into magnetic field bends, in this charged particles are present. The
molecules pass through this magnetic field and collected at the final stage.
There has been great development in chromatographic techniques in last 5years.

Especially in the medical field, the scope of chromatographic techniques has been
Used in various drug formulations. Some of the examples of aspects in which these
Techniques used in last five years include:
Use of recombinant antibodies in proteomics Use of recombinant antibodies in

proteomics is increasing day to day, using this techniques large phage antibody
libraries are developed, which binds to target protein.
These antibody libraries are used in analysis of proteomes.
Arrays for protein expression profiling
Protein expression patterns in a cell or tissue of an organism is measured by using

Two-dimensional gel electrophoresis. By using this technique analysis of proteins of
interest is extracted from a multiple sample containing different proteins, this is an
Important tool for analysis of protein expression, which is an key component in the
Field of proteomics. These separation techniques are useful to human society in
various
Medical fields and acts as a platform for diagnostic and prognostic monitoring of
diseases.
Proteomics in early detection of cancer:
The developments in mass spectroscopy and two-dimensional electrophoresis has

contributed to development of biomarker research. Chip based techniques like
surface enhanced laser desorption and emerging methods like antibody arrays are
recent developments, using spectroscopy. By using the biomarkers one can detect
and prevent the cause of cancer. Biomarkers detect the status of the infected cancer
cells in the body and also detect the distinct changes that occur in the cells, which
transforms from nondiseased to neoplastic.
Determining the structure of protein
There are more than 10,000 protein folds in existence in human beings. Proteins
sequences are distributed among these folds, which are non-homogenous and most
of them are rare. The distribution of proteins in these folds follows the asymptotic
power laws, which are identified in different biological and physical systems of the
body and are associated with the scale free networks. By using chromatographic
techniques we can identify the proteins that are distributed in these folds.
Techniques useful in diagnosis and treatment of cancer
Using the new techniques like biomarkers for early detection and diagnosis of
dangerous diseases like cancer was in wide use in early 2003. Two-dimensional gel
electrophoresis (2D-PAGE) is the foundation of many discovery-based proteomics
studies. Technologies such as laser capture micro dissection (LCM) and highly
sensitive MS methods are widely used methods to recognize the protein that are
articulated between distinct cell populations. Technologies such as reverse phase
protein arrays will facilitate the recognition of target pathways in small biopsy
specimens. The other technique used to analyse the classification of lysates from
body fluids which suites for the diagnosis and tratment of the disease is Surface-
enhanced laser desorption/ionization time-of-flight (SELDI-TOF). Application of gel
based proteomic techniques also enables to discover the drugs and prevention for
lung and bladder cancer by using the technique of tissue biopsies. Most of the
eukaryotic protein activity is altered by post-translational modifications. To record the
modification sites in detail, techniques like novel mass spectrometric peptide
sequencing and analysis technologies are widely used to study the chemistry of
modifications.
Recent advances in mass spectroscopy-based proteomics is an indispensable tool

In the fields of molecular and cell biology. By using these techniques study of protein-
Protein interactions on small scale and proteome interactions on the large scale are
possible. The most important aspect of this technique is identifying the genome and
proteome of malaria parasites. The important ability of mass spectroscopy is to
identify and isolate the specific protein from complex samples, which is an important
aspect in the fields of Medicine and biology.
The chromatographic techniques like gel electrophoresis, which is followed by

blotting to PVDF membrane for N terminal sequencing to produce peptides that can
separated by HPLC. These techniques are used to identify the proteins that are
articulated in the cell and available in the low micrograms or even less. The structural
analysis of proteins is done by mass spectroscopy (MS), which is the new technique
compared to Edman degradation which is the older method used to remove the N
terminal of amino acids. But this is an older and time-consuming method, so this
method is replaced by mass spectroscopy. Mass spectroscopy is a fast and method
of choice for sensitive analysis and used in large scale industrial applications. The
ionization methods like MALDI (matrix assisted laser desorption and ionization) and
ESI (electrospray ionization) are commonly used to produce proteins for MS analysis.
By using these techniques proteins of choice are identified by peptide mass finger
printing or by using sequence tags.
The newly developed techniques like MALDI and Bioinformatics has a wide spread in
the fields of medicine, chemistry and proteome data. MALDI (matrix-assisted laser
desorption/ionization) is used in mass spectroscopy to analyse the biomolecules,
Oligonucleotides and proteins by sensitivity and MS inherent accuracy. MALDI also
has the impact on nucleotide polymorphism and challenges of post-genome area.
Bioinformatics is a new technique in proteomic technologies, which is introducing the
new algorithms to handle the various data sets. New algorithms for image analysis of
two-dimensional gels have been developed within the last five years by
bioinformatics.
The recent advances in mass spectroscopy has brought the analysis of protein into
Lime light of cancer research. By using these techniques in cancer research, one can
Find different issues of proteomics in cancer research like profiling of tumour cells,
tumour fluids and protein microarrays and pharmacoproteomics and mapping of
cancer signaling pathways and the role of biomarkers in diagnosis of cancer disease
and monitoring of the disease and therapeutic and immune response to the cancer.
The above-mentioned are the benefits of the development of the techniques like
functional protein arrays and data handling. Cancer is a dysregulstion in the network
of the intracellular and extracellular signaling system of the body. Molecular therapy
given to the cancer patient is to target the effected signaling system of human body.
The rising technology in proteomic techniques used with genomic analysis fulfils this
need and bring the scientific approval for molecular stratification. Proteomic
technology offers the state of kinase pathways, and provides post-translational
phosphorylation data, which is not accessible by gene arrays. Such techniques
provides a new pathways for developing new clinical therapy in curing the disease.
5.2.3 Evaluation of the Technique
Advantages of the Techniques
These chromatographic techniques play an important role in the field of extraction

and purification of proteomics. SDS-PAGE is the most popular protein separation
technique which remains unrivalled in its capability to separate the complex mixtures
of proteins. No other technique can be compared to SDS PAGE in the terms of
resolution and sensitivity in the extraction of large mixture of protein samples. This
separation technique involves two different electrophoretic techniques. In the first
phase it separates the proteins according to their isoelectric point and in the second
phase it separates according to their molecular weights. By conducting this technique
the protein molecules are detected as spots according to their isoelectric point and
molecular weights.
Mass spectrometry plays an important role in separation of proteomes from a

complex mixtures of the sample. Especially in the field of medicine and molecular
biology, mass spectrometry involves in the analysis of human plasma proteome. The
mobility of ions in gases is greater than condensed phases, by using the technique of
mass spectroscopy one can handle the complexity of the samples. The other
advantages of this technique include the early detection of cancer cells in the body.
Multi lectin affinity chromatography is used to isolate the glycoprotein part of the
serum samples collected from the patients suffering from breast cancer. The
peptides present in the serum sample of the cancer patients are isolated by the mass
spectrometer.
Disadvantages of the Techniques
There are also some disadvantages in the SDS PAGE electrophoresis. The bands
produced by SDS-PAGE are sharper, which affects the protein shape and charge.
Sodium dodecyl sulfate is an anionic detergent present in the SDS PAGE
electrophoresis disrupts protein quaternary, tertiary and secondary structural levels
And renders all proteins highly negative charge. There are also some problems with
protein weight estimated by SDS PAGE. Joule heating is the other disadvantage of
SDS PAGE in which high power electrophoretic separations run out and leads to
unintentional consequences.
In mass spectroscopy the mass spectral information of the sample is lost due to
the limit of detection of the sample is lowered. This is not much enhanced than a
gas chromatography with flame ionization detector. By this technique the problem
arises like chemical noise due to any component containing an ion with the same
mass as that of the component which is scanned. In this methods some compounds
cannot accept hydrocarbons easily. No information of fragments is produced by the
tandem mass spectrometry on cationized molecules. During the process of electron
ejection or electron capture more amount of fragmentation is generated. In MALDI
Mass spectroscopy the background matrix, which is used as a stationary phase
creates a problem for molecular samples with a mass below 700Da. By using acidic
matrix in the MALDI mass spectroscopy also causes degradation in some
compounds.
The major applications of the SDS PAGE electrophoresis include the implementation
of on chip preconcentration of proteins. In this technique two membranes of different
sizes are used for photopolymerization. Proteins are trapped on the membrane of
SDS PAGE and eluted out by reversing the electric field. By using this technique
proteins with low concentrations are also detectable with in a time span of 30 min of
preconcentration time. Preconcentration analysis is also used in DNA analysis and
clinical diagnostics.
An application of proteomics in the aquaculture field leads to the development of new

farming techniques. SDS PAGE separation technique is used to analyse the fish
muscle protein content, by collecting the information on physiology of fish muscle. By
using SDS PAGE technique in situ protein hydrolysis and de novo sequencing of
peptides by MALDI and capillary electrophoresis in determination of fatty acids and
metal ion content of the fish muscle protein.
These days monoclonal antibody therapies are used to cure various human
diseases, which has led to development of different analytical methods to detect and
quantitate different size variants. In the place of SDS CE using gel based polymer for
separation of different proteins, these days SDS PAGE electrophoresis is used due
to its various advantages. SDS PAGE technique utilizes non-gel polymer solutions
for separation. The other important solutions like dextrans are also used as
separation matrices. These dextrans are used as matrices in bare fused silica
capillary, which is the advanced technology used in the analysis of separation
technique.
Mass spectroscopy is an important tool used for differentiation of cancerous (HOC

313 and HSC 3) and noncancerous cells (HaCat). These two cells can be
differentiated due to overexpression of EGFR on the surface of cancer cells. In
photothermal cancer therapy nanonanospheres and nanorods conjugated with anti-
epidermal growth factor receptor (anti-EGFR) antibodies that specifically target
EGFR on the cell surface are used in cancer diagnostics and therapy. The use of
nanorods allow for in vivo therapy and the catalytic effect of gold nanoparticles on
oxidation of NADH to NAD+ is investigated. This conversion of NADH to NAD+ is
supported by nuclear resonance and mass spectroscopy.
In United States of America an annual rate of 2.2 million tons of phenolic resins and
Phenol formaldehyde polymers are produced. Previously these polymers are thought
to be nonbiodegradable and are produced for many industrial and commercial
purpose. After conducting various experiments using gas chromatography and mass
spectroscopy it was founded that a degradation product named 13C labeled phenol
was detected. The main fungus responsible for this biodegradation was white-rot
fungus Phanerochaete chryso- sporium. This was the basic platform for analysis of
bioremediation and biorecycling of phenolic resins.
Mass spectroscopy is also used in the field of security applications in military

services due to its detectable capacity for chemical and biological weapons in the
society. So various government agencies today are funding to buy these instruments
in order to protect the general public from the attack of these weapons. Mass
spectrometry also is beginning to enter into the airport security market, where
explosives detection is the primary need. Recent advances in the technology are
bringing down their size, for allowing them to fit better in military vehicles and ships.
There are also some benchtop mass spectroscopy instruments are available, which
are employed in laboratories.
References:
http://www.healthtech.com/2005/bmks/msb.asp(access date
25sep2006)
http://www.mrc-dunn.cam.ac.uk/research/proteomes.html(access date
25sep2006)
http://www.chem.uic.edu/web1/ocol/spec/MS1.htm(access date
30sep2006)
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views.asp(access date 10oct2006)
http://masspec.scripps.edu/MSHistory/whatisms.php(access date
18oct2006)
Anson V Hatch, Amy E Herr, Daniel J Throckmorton, James S

Brennan, Anup K Singh. (Jul 15, 2006) Analytical Chemistry.
Washington: Vol.78, Iss. 14; pg. 4976. (access date 28oct2006)
Caesar, Kwadwo O., (2006) Analysis of a recombinant monoclonal

antibody by molecular sieving capillary electrophoresis by M.S., Hood
College, 64 pages; AAT 1434028 (access date 24oct2006)
Cole R (Editor)(1997). Electrospray Ionization Mass Spectrometry:

Fundamentals, Instrumentation, and Applications. New York: Wiley and
Sons, (access date 28oct2006)
Gianluca Monti, Lorenzo De Napoli, Pietro Mainolfi, Roberto Barone, et al.

(Apr 15, 2005) Analytical Chemistry. Washington: Vol.77, Iss. 8; pg. 2587
Holt LJ, Enever C, de Wildt RM, Tomlinson IM. (2000 Oct; 11) Curr
Opin Biotechnol. (5): 445-9. (access date 24oct2006)
Righetti, PG, Castagna, A, Antonioli, P, et al. Prefractionation

techniques in proteome analysis:( JAN 2005) the mining tools of the
third millennium ELECTROPHORESIS 26 (2): 297-319 (access date
15sep2006)
White-Rot Fungi Demonstrate First Biodegradation of Phenolic Resin

Adam C Gusse, Paul D Miller, Thomas J Volk. (Jul 1, 2006.)
Environmental Science & Technology. Easton: Vol. 40, Iss. 13; p.
4196 (access date 21oct2006)
Chapter 5.3 Mass Spectrometry in Proteomics
Amit Joglekar
5.3.1. Introduction:
Proteomics is the study of the function of all expressed proteins. Tremendous

progress has been made in the past few years in generating large-scale data sets for
protein–protein interactions, organelle composition, protein activity patterns and
protein profiles in cancer patients [1]. In general it deals with the large-scale
determination of gene and cellular function directly at the protein level [2]. The term
proteome was first coined to describe the set of proteins encoded by the genome [3]
and the term was coined to make an analogy with the term genomics [4]. Proteomics
now evokes all the proteins in any given cell. It also analyses the set of all protein
isoforms and modifications, the interactions between them, the structural description
of proteins. In short it analyses everything that is 'post-genomic' [1]. Mass
spectrometry (MS) has increasingly become the method of choice for analysis of
complex protein samples. MS-based proteomics is the technique made possible by
the availability of gene and genome sequence databases and technical and
conceptual advances in many areas most important being the discovery and
development of protein ionization methods, as recognized by the 2002 Nobel prize in
chemistry [2].
Early proteomics study relied on protein separation by two-dimensional gel

electrophoresis, with subsequent mass spectrometric identification of protein spots.
The ability of mass spectrometry to identify very small amounts of protein from
complex mixtures is a primary driving force in proteomics [1]. Proteomics has been
possible only because of the previous achievements of genomic studies, which
provided the 'blueprint' of possible gene products that are the focal point of
proteomics studies [1]. So far, protein analysis such as the identification of primary
sequence, post-translational modifications (PTMs) or protein–protein interactions by
MS has been most successful when applied to small sets of proteins. The systematic
analysis of the much larger number of proteins expressed in a cell is now also rapidly
advancing mainly due to the development of new experimental approaches [2]. To
analyse such huge number of proteins is an explicit goal of proteomics. To catalog all
human proteins and ascertain their functions and interactions presents a daunting
challenge for scientists. An international collaboration to achieve these goals is being
co-ordinated by the Human Proteome Organization [4].
5.3.2 Principles and instrumentation:
The following block diagram shows the components of a mass spectrometer-

Fig 5.3.1: Components of a Mass Spectrometer
Source: (http://www.asms.org/whatisms/p4.html)
Mass spectrometric measurements are carried out in the gas phase on ionized
analytes. By definition, a mass spectrometer consists of an ion source, a mass
analyser that measures the mass-to-charge ratio (m/z) of the ionized analytes, and a
detector that registers the number of ions at each m/z value.
Electrospray ionization (ESI) and matrix-assisted laser desorption/ionization

(MALDI) are the two techniques most commonly used to volatize and ionize the
proteins or peptides for mass spectrometric analysis [5] [6]. ESI ionizes the analytes
out of a solution and is therefore readily coupled to liquid-based separation tools
such as for chromatographic and electrophoretic technique as shown in fig 5.3.2[2].
MALDI sublimates and ionizes the samples out of a dry, crystalline matrix via laser
pulses. MALDI-MS is normally used to analyse relatively simple peptide mixtures,
whereas integrated liquid-chromatography ESI-MS systems (LC-MS) are preferred
for the analysis of complex samples [7].
The mass analyser is the main and the central component of this technology. In
the context of proteomics, its key parameters are sensitivity, resolution, mass
accuracy and the ability to generate information-rich ion mass spectra from peptide
fragments (tandem mass or MS/MS spectra) (Fig 5.3.3)[8-10]. There are four basic
types of mass analyser currently used in proteomics research. These are the ion
trap, time-of-flight (TOF), quadrupole and Fourier transform ion cyclotron (FT-MS)
analysers. They are very different in design and performance, each with its own
strength and weakness. These analysers can be used separately or, in some cases,
put together in tandem to take advantage of the strengths of each.[2]
Fig 5.3.2: Use of ESI and SDS-Page and HPLC technique for a proteomic
experiment.
The typical proteomics experiment consists of five stages. In stage 1, the proteins to
be analysed are isolated from cell lysate or tissues by biochemical fractionation or
affinity selection. This often includes a final step of one-dimensional gel
electrophoresis. MS of whole proteins is less sensitive than peptide MS and the
mass of the intact protein by itself is insufficient for identification. Therefore, proteins
are degraded enzymatically to peptides in stage 2, usually by trypsin, leading to
peptides with C-terminally protonated amino acids, providing an advantage in
subsequent peptide sequencing. In stage 3, the peptides are separated by one or
more steps of high-pressure liquid chromatography in very fine capillaries and
eluted into an electrospray ion source where they are nebulized in small, highly
charged droplets. After evaporation, multiply protonated peptides enter the mass
spectrometer and, in stage 4, a mass spectrum of the peptides eluting at this time
point is taken (MS1 spectrum, or 'normal mass spectrum'). The computer generates a
prioritized list of these peptides for fragmentation and a series of tandem mass
spectrometric or 'MS/MS' experiments ensues (stage 5). These consist of isolation of
a given peptide ion, fragmentation by energetic collision with gas, and recording of
the tandem or MS/MS spectrum. The MS and MS/MS spectra are typically acquired
for about one second each and stored for matching against protein sequence
databases. The outcome of the experiment is the identity of the peptides and
therefore the proteins making up the purified protein population.
Source:
(http://www.nature.com/nature/journal/v422/n6928/fig_tab/nature01511_F1.html)
Fig 5.3.3: Stages in MS/MS experiment
The left and right upper panels depict the ionization and sample introduction process in
electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI). The
different instrumental configurations (a−f) are shown with their typical ion source. a, In
reflector time-of-flight (TOF) instruments, the ions are accelerated to high kinetic energy and
are separated along a flight tube as a result of their different velocities. The ions are turned
around in a reflector, which compensates for slight differences in kinetic energy, and then
impinge on a detector that amplifies and counts arriving ions. b, The TOF-TOF instrument
incorporates a collision cell between two TOF sections. Ions of one mass-to-charge (m/z)
ratio are selected in the first TOF section, fragmented in the collision cell, and the masses of
the fragments are separated in the second TOF section. c, Quadrupole mass spectrometers
select by time-varying electric fields between four rods, which permit a stable trajectory only
for ions of a particular desired m/z. Again, ions of a particular m/z are selected in a first
section (Q1), fragmented in a collision cell (q2), and the fragments separated in Q3. In the
linear ion trap, ions are captured in a quadruple section, depicted by the red dot in Q3. They
are then excited via resonant electric field and the fragments are scanned out, creating the
tandem mass spectrum. d, The quadrupole TOF instrument combines the front part of a triple
quadruple instrument with a reflector TOF section for measuring the mass of the ions. e, The
(three-dimensional) ion trap captures the ions as in the case of the linear ion trap, fragments
ions of a particular m/z, and then scans out the fragments to generate the tandem mass
spectrum. f, The FT-MS instrument also traps the ions, but does so with the help of strong
magnetic fields. The figure shows the combination of FT-MS with the linear ion trap for
efficient isolation, fragmentation and fragment detection in the FT-MS section.
Source: (http://www.nature.com/nature/journal/v422/n6928/fig_tab/nature01511_F2.html)
5.3.2.1 Ion source
The first very important component in a mass spectrometer is the ion source
through which various ions are created after which they are passed into the mass
analyzer. There are various types of ion sources through which ions can be
generated. The choice of ionic source depends on the experiment and the
compounds to be analysed. The various ion sources are listed below[11]-
• Electron impact ionization

• Chemical ionization
• Field ionization
o Desoprptive ionization
Fast atom bombardment ionisation
Field desorption ionisation
Plasma desorption ionisation
Laser desorption ionisation
• Matrix assistsed laser desorptive ionisation (MALDI)
• Surface enhanced laser desorption ionisation (SELDI)
• Electrospray ionisation (ESI)
Generally in proteomics laser desorption method is the preferred choice of ionisation.

MALDI is used in lot of circumstances although SELDI is also used. In the laser
desorption method a laser is used to deliver large density of energy into a small
space. The laser beam is focussed onto a matrix, which has an absorption band that
closely matches the energy of laser radiation. Once the beam hits this matrix the
material has a lot of energy and will desorb and ionize. This matrix substance is
mixed with the substance to be examined. The details of the method are explained in
chapters 4.1.
ESI is also used in many cases where the sample is present in the electrospray
capillary of small internal diameter. The analyte is forced into this capillary at a
particular flow rate and the resulting electrospray plume nebulizes the sample. The
details are present in chapter 4.2.
5.3.2.2 Mass Analyzers
The basic types of mass analyzers used in mass spectrometry are summarized
below:
Type Acronym Principle
Time-of-flight TOF Time dispersion of a pulsed beam; separation by
time-of-flight
Magnetic sector B Deflection of a continuos beam; separation by
momentum in magnetic field
Linear Quadrupole Q Continuous ion beam in linear radio frequency
quadrupole field; separation due to stability of
trajectories
Linear Quadrupole ion LIT Continuous ion beam and trapped ions; storage
trap and eventually separation in linear radio
frequency quadrupole field due to stability of
trajectories
Quadrupole Ion trap QIT Trapped ions; separation in three dimensional
radio frequency quadrupole field due to stability of
trajectories
Ion cyclotron ICR Trapped ions; separation by cyclotron frequency
resonance (Lorentz force) in magnetic field
Table 5.3.1 Basic types of mass analysers
Source: Mass Spectromerty: A Textbook, 2004
5.3.2.2.1 Time-of-Flight analyzer
The principle of TOF is simple in the sense that ions of different mass: charge ratio
i.e. m/z are dispersed in time during their flight along a field-free drift path of known
length. Provided all the ions start their journey at the same time or within a sufficiently
short time interval, the lighter ions will arrive earlier at the detector than the heavier
ones.[12]
Fig 5.3.3 Time-of-Flight Ion Charge State Analyzer

Source: (http://htx.pppl.gov/assets/tof.gif)
5.3.2.2.2 Magnetic sector analyzer
These are comparatively large devices capable of high resolution and accurate
mass determination suited for wide variety of ionization methods. It is basically a
momentum analyzer rather than a direct mass analyzer as commonly assumed.[12]
Fig 5.3.4 Path of magnetic sector analyzer
Source: (http://www.chem.ox.ac.uk/spectroscopy/mass-
spec/Lecture/oxweb%20sector%202.jpg)
5.3.2.2.3 Linear Quadrupole analyzer
A linear quadrupole mass analyzer consists of four hyperbolically or cylindrically

shaped rod electrodes extending in the z-direction and mounted in a square
configuration. The pairs of opposite rods are each held at the same potential which is
composed of a DC and an AC component.[12]
5.3.2.3 Detectors
The final element of the mass spectrometer is the detector. The detector records
the charge induced or current produced when an ion passes by or hits a surface. In a
scanning instrument the signal produced in the detector during the course of the scan
versus where the instrument is in the scan (at what m/z) will produce a mass
spectrum, a record of ions as a function of m/z[13].
The simplest of the detector is a Faraday cup i.e. an electrode where the ions
deposit their charge. They are still in use to measure abundance ratios with high
accuracy in isotope ratio mass spectrometry [12]. Other type of detector includes the
electron multiplier, where, the ions when bombarded on metal (or PbO coated
surface) induce emission of electrons. [13].They became predominant with the
advent of scanning mass spectrometers. Progress has also been made to employ
cryogenic detectors, a rather special type of an ion counting detector for high-mass
ions in TOF-MS.[12]
Sequencing of the human genome and numerous pathogens [14] was a great
outbreak for overall biological sciences and opened new gates for active research in
the field of molecular biology. As a result it also opened gates for proteomics [14].
Interest has been developed in applying proteomics to understand the process of
disease development, develop new biomarkers for the purpose of diagnosis and
hence early detection of the disease [14]. Once biomarkers are developed further
work can be done in developing drug delivery systems.
Studies have identified disease related changes in the protein expression using 2D
gels and mass spectrometry. Studies on the diseases of heart [15] have gathered a
set of pathological conditions with acute onset and some with slow progression of
diseases and some with chronic progression. Changes in the myocardial proteins
associated with the heart failure have been found out in relevant animal models such
as that in rat myocytes [15]. Altered overall levels of specific proteins or altered post-
translational modifications of proteins such as myosin light chain 2 have been
reported in heart failure [16].
Mass spectrometry has been applied to the in situ proteomic analysis of tissues.
This approach allows imaging of protein expression in normal and disease tissues
[17]. In this method the frozen tissue is sliced and sections are applied on the MALDI
plate, which are then analysed at regular intervals. The mass spectra obtained at
each interval are then compared yielding a spatial distribution of individual masses
across the tissue section [14]. Tumour analyses using this approach have shown
differences in protein expression between normal and tumour tissues that may have
specificity for different tumour types [17].
Disease biomarkers play a significant role in terms of diagnosis of a disease. There

is substantial interest in applying proteomics to the identification of disease markers.
These include the comparative analysis of op protein expression on a diseased and a
normal tissue, analysis of secreted proteins in cell lines and many such applications
[14]. Serum analysis is done by Surface Enhanced Laser Desorption Ionisation
(SELDI) method [18]. The mass spectrum patterns obtained for different samples
reflect the protein and peptide contents of these samples. Patterns that distinguish
between cancer patients and normal subjects with remarkable accuracy have been
reported for several types of cancer [18]. Very often the masses observed match
precisely to the predicted masses of the proteins; and this was observed in a study
where the proteins secreted by the CD8 T cells was identified to be -defensin 1, 2
and 3 as contributing to the anti-HIV-1 activity of CD8 antiviral factor [19].
Proteomics has also contributed to the studies of pathogens such as Plasmodium

falciparum, the malarial parasite. After the genome of this parasite was revealed,
comparative proteomic studies have been done which lead to the identification of a
potential drug and vaccine target [20, 21]. Aside from comprehensive identification of
microbial proteins, proteomics is relevant to numerous aspects of microbial disease
pathogenesis and treatment [14]
Finally there have also been developments in the instrumentation of this technique.
In the case of LC–MS, the last two decades have seen some significant
developments and improvements in instrumentation design, especially the
introduction of robust, user-friendly interfaces such as those based on atmospheric
pressure ionisation techniques, e.g. electrospray (ESI) and atmospheric pressure
chemical ionisation (APCI)[22].
5.3.4 Evaluation of the technology:
Mass spectrometry has come of age in this decade and has proved a powerful
technique to unleash the unknown. Many proteins have been identified till date by
using this instrument. As mentioned earlier, the proteins are digested with trypsin and
then analysed by mass spectrometer. The masses are then submitted to various
protein databases where the protein is theoretically digested and then compared with
proteins that are experimentally generated by the database. The match is scored on
number of factors, depending on the search program utilised.
This technique has certain advantages and disadvantages associated with it. It is
rapid with a low turn around time. It is highly sensitive even with low amounts of
sample. It measures masses of molecules accurately and hence it is used to
determine the number of subunits in different olygomers, which cannot be
determined accurately by other methods like size exclusion, analytical
ultracentrifugation or cryo electron microscopy [23]. Though accurate, this technique
can’t be used efficiently enough for non-covalent compounds [23]. Before entering
the mass analysers, the sample undergoes ionisation and not all molecules ionize
well during the ionization process; dissociation of the complexes could also occur
during ionization. One of the important requirements of this technique is that once the
masses are submitted to the databases, the protein should be present in the
database list (databases websites given at the end). Proteins that are less than
15kDa are not suitable for MALDI-TOF mass spectrometry [24].
5.3.5 Applications:
In recent years there has been development of powerful technology that has given
a boom for biological sciences. Mass spectrometry is one of them which has been
recently been used extensively in the new field of proteomics. Protein analysis is
done by mass spectrometry routinely these days. It finds applications in the medical
field for the diagnosis of diseases. New biomarkers are being identified by using
mass spectrometric analysis, which would improve tuberculosis diagnosis [25].
It finds application in forensic sciences. The ability of mass spectrometry to extract

chemical fingerprints from microscopic levels of analyte is invaluable enabling the
legally defensible identification and quantification of a wide range of compounds [22].
Determining the use of chemical warfare agents (CWAs) in times of war or in acts of
terrorism requires rapid and reliable methods. Nerve agents are extremely potent
Organo phosphorus compounds that cause biological effects by irreversibly inhibiting
the enzyme acetylcholinesterase (AChE). To confirm exposure, biological samples,
e.g. urine, can be analysed for the agents [22]. Depending on the compounds to be
detected, GC-MS or LC-MS is routinely used.
Screening of illicit drugs by mass spectrometry is one of the common applications.

Drug abuse during pregnancy is a major problem and has been associated with
prenatal complications and high morbidity and mortality rates of newborns. Meconium
is the first faecal matter produced by the neonate typically within the first 5 days of
birth. Use of this specimen can extend the window of drug detection considerably, i.e.
to approximately the last 20 weeks of pregnancy [22].
Recently, a quantitative determination of azithromycin in human plasma was done

by HPLC-MS [26]. Azithromycin is a semisynthetic macrolide antibiotic of the
erythromycin group. It has been used to treat infections caused by respiratory
pathogens, including Legionella pneumophila, Haemophilus influenzae and
Branhamella catarrhalis [26]. The determination of antibiotics is carried out by
microbiological assays, but, they tend to lack specificity and their use involves
difficulty in confirming what kinds of drugs remain in biological sample [26]. Thus,
liquid chromatography–mass spectrometry (LC–MS) and liquid chromatography–
tandem mass spectrometry (LC–MS/MS) seem to be the most promising technique
for separation and quantitative analysis of drugs and have recently been used in the
determination of azithromycin[26].
Such and many more applications such as the identification of new biomarkers in
thyroid cytology [27], proteomic strategies to identify urinary biomarkers for prostate
cancer [28], to understand the meat quality through proteomic approach [29] have
proven to be fruitful. There are many more applications of mass spectrometry in
many fields, but is beyond the scope to list them all.
5.3.5 Relevant WebPages
The following pages for Programs used to search MALDI-TOF Peptide Mass
Fingerprint data:
• MASCOT: http://www.matrixscience.com/
• MS-Fit: http://prospector.ucsf.edu/
• ALDENTE: http://au.expasy.org/tools/aldente/
• ProFound: http://129.85.19.192/profound_bin/WebProFound.exe
5.3.6 References:
1. Tyers, M. M., M. (2003) From genomics to proteomics, Nature.
2. Mann, R. A. a. M. (2003). Mass spectrometry-based proteomics. Paper presented

at the Nature.
3. Wilkins, M. R., Pasquali,C, Appel, R.D, Ou, K, Golaz ,O, Sanchez, J.C, Yan, J.X,
Gooley, A.A, Hughes,G, Humphery-Smith, I., Williams, K.L & Hochstrasser, D.F.
(1996) From proteins to proteomes: large scale protein identification by two-
dimensional electrophoresis and amino acid analysis., Biotechnology, 61-65.
4. http://en.wikipedia.org/wiki/Proteomics
5. Fenn, J. B., Mann, M., Meng, C. K., Wong, S. F. & Whitehouse, C. M. (1989)
Electrospray ionization for the mass spectrometry of large biomolecules., Science.
246, 64-71.
6. Karas, M. H., F. (1988) Laser desorption ionization of proteins with molecular

mass exceeding 10000 daltons, Analytical Chemistry. 60, 2299-2301.
7. Aebersold, R. G., D. R. (2001) Mass spectrometry in proteomics, Chemical

Review. 101, 512-526.
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9. Aebersold, R. G., D. R. (2001) Mass spectrometry in proteomics, Chemistry

Review. 101, 269-295.
10. Mann, M., Hendrickson, R. C. & Pandey, A. (2001) Analysis of proteins and
proteomes by mass spectrometry, Annual Review of Biochemistry. 70, 437-473.
11. Herbert, C. G. J., R.A. W. (2003) Mass Spectrometry Basics, CRC PRESS,
United States of America.
12. Gross, J. H. (2004) Mass Spectrometry: A Textook, Springer-Verlag Berlin

Heidelberg, Germany.
13. http://en.wikipedia.org/wiki/Mass_spectrometry#Detector
14. Hanash, S. (2003) Disease proteomics, Nature. 422, 226-232.
15. Van Eyk, J. E. (2001) Proteomics: unraveling the complexity of heart disease
and striving to change cardiology, Current opinion in Molecular Therapeutics. 3, 546-
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16. van Der Velden, J. e. a. (2001) Effects of calcium, inorganic, phosphate, and pH
on isometric force in single skinned cardiomyocytes from donor and failing human
hearts, Circulation. 104, 1140-1146.
17. Stoeckli, M., Chaurand, P., Hallahan, D. E. & Caprioli, R. M. (2001) Imaging
mass spectrometry: a new technology for the analysis of protein expression in
mammalian tissues, Nature Medicine. 7, 493-496.
18. Petricoin, E. F., Zoon, K. C., Kohn, E. C., Barrett, J. C. & Liotta, L. A. (2001)
Clinical proteomics: translating benchside promise into bedside reality, Nature
Reviews Drug Discovery. 1, 683-695.
19. Zhang, L. e. a. (2002) Contribution of human -defensin 1, 2 and 3 to the anti-

HIV-1 activity of CD8 antiviral factor, Science. 298, 995-1000.
20. Lasonder, E., Ishihama,Y., Andersen,J. S., Adriaan, M. W., Vermunt, A. P.,
Sauerwein,R.W., Wijnand, M. C., Eling, N. H., Waters,A. P., Stunnenberg,H.G. &
Mann,M. (2002) Analysis of the Plasmodium falciparum proteome by high-accuracy
mass spectrometry, Nature. 419, 537-542.
21. Florens, L., Washburn,M. P., Raine,J.D., Anthony,R.M., Grainger,M., Haynes,J.

D., Moch,J. K., Muster,N., Sacci,J. B., Tabb,D. L., Witney,A.A., Wolters,D., Yimin
Wu, Gardner,M.J., Holder,A. A., Sinden,R.E., Yates,J.R. & Carucci,D.J. (2002)
Analysis of the Plasmodium falciparum proteome by high-accuracy mass
spectrometry, Nature. 419, 537-542.
22. Wood, M., Laloup, M., Samyn, N., del Mar Ramirez Fernandez, M., de Bruijn, E.
A., Maes, R. A. A. & De Boeck, G. (2006) Recent applications of liquid
chromatography-mass spectrometry in forensic science, Journal of Chromatography
A. 1130, 3-15.
23. Poliakov, A. (2006) Mass spectrometry on non-covalent macromolecular

complexes in
24. Australian Proteome Analysis Facility:MALDI MS Analysis for Protein

Identifications.
http://www.proteome.org.au/MALDI-MS/default.aspx
25. Agranoff, D., Fernandez-Reyes, D., Papadopoulos, M. C., Rojas, S. A., Herbster,
M., Loosemore, A., Tarelli, E., Sheldon, J., Schwenk, A. & Pollok et, al. (2006)
Identification of diagnostic markers for tuberculosis by proteomic fingerprinting of
serum, Lancet. 368, 1012-1021.
26. Chen, B.-M., Liang, Y.-Z., Chen, X., Liu, S.-G., Deng, F.-L. & Zhou, P. (2006)
Quantitative determination of azithromycin in human plasma by liquid
chromatography-mass spectrometry and its application in a bioequivalence study,
Journal of Pharmaceutical and Biomedical Analysis. 42, 480-487.
27. Torres-Cabala, C., Bibbo, M., Panizo-Santos, A., Barazi, H., Krutzsch, H.,
Roberts, D. D. & Merino, M. J. Proteomic identification of new biomarkers and
application in thyroid cytology, Acta Cytologica. 50, 518-528.
28. Downes, M. R., Byrne, J. C., Dunn, M. J., Fitzpatrick, J. M., Watson, R. W. G. &
Pennington, S. R. Application of proteomic strategies to the identification of urinary
biomarkers for prostate cancer: A review, Biomarkers: Biochemical Indicators Of
Exposure, Response, And Susceptibility To Chemicals. 11, 406-416.
29. Mullen, A. M., P.C. Stapleton, D. Corcoran, R.M. Hamill and A. White. (2006)
Understanding meat quality through the application of genomic and proteomic
approaches, Meat Science. 74, 3-16.
Chapter 5.4 Bioinformatics in proteome analysis
Supriya Narayanan
(s3119801)
5.4.1 Introduction
The complement of proteins found in a single cell in a particular environment is called

the proteome. (derived from PROTEin complement to a genOME) [1, 3]. The term
was coined by Mark Wilkins in 1995 [4]. Proteome can also be used to refer to all the
proteins present in a simple organism such as yeast as shown in the diagram. The
study of the proteome is called proteomics. It is a study of not only all the proteins in
the cell but also the way they interact, the changes they undergo and the effects they
have within the organism.[2] Proteomics can be defined as the qualitative and
quantitative comparison of proteomes under different conditions to further unravel
biological processes[1]. There are various tools available for carrying out analysis of
the proteome such as electrophoresis, chromatography, X-ray crystallography, NMR
and mass spectrometry. However, proteomics data is under continuous
improvements and new technologies are emerging for generating high throughput
results [6]. It is difficult to handle this large influx of information using traditional
methods. Bioinformatics is emerging as a good means to handle and interpret this
data.
Yeast proteome
http://fig.cox.miami.edu/~cmallery/150/gene/yeast_proteome2.jpg
Bioinformatics is defined by the, BISTIC Definition Committee chaired by Dr. Michael

Huerta of the National Institute of Mental Health on July 17, 2000 as follows : “ the
research, development, or application of computational tools and approaches for
expanding the use of biological, medical, behavioral or health data, including those to
acquire, store, organize, archive, analyze, or visualize such data.” [5]
The use of computational technology has also enabled a high degree of

reproducibility and sensitivity in the technologies used to study the proteome [10].
Bioinformatics software concentrates on three main areas

♦ Interpreting mass spectrometry information.
♦ Computing the structure of a protein
♦ Sequence comparison [13]
A brief description of the informatics requirements of various processes of biological

analysis is shown in the diagram
Figure 1 : A brief description of the informatics requirements

Source: Scott D. Patterson & Ruedi H. Aebersold, (2003) Proteomics: the first
decade and beyond, Nature Genetics 33, 311 - 323
Proteomics is a relatively new field developing only in the last 10 years. Based on 2D
gel electrophoresis protein profiles, ideas were proposed in 1970s and 1980s to build
protein databases such as the human protein index.
In the late 1980s and early 1990s, there were only small sequence databases.
However, through the 90s, along with genomic data, protein data was also gathered
and the databases began to grow. With complete libraries at hand, rapid identification
of proteins was only limited by the ability to extract their sequence information. This
gap was rapidly filled by mass spectrometry techniques and database search
algorithms.
In 1993, five independent reports were published that described the implementation
of this insight in database search algorithms. These algorithms, together with MALDI-
TOF mass spectrometry peptide analysis, constituted a 'protein identification' method
that is now known as peptide mass mapping (or peptide mass fingerprinting). In this
type of analysis, the collected 'MS spectra' are used to generate a list of proteolytic
(peptide) fragment masses, which are then matched to the masses calculated from
the same proteo-lytic digestion of each entry in a sequence database, resulting in
identification of the target protein.
Algorithms that match MS/MS spectra to sequence databases have greatly facilitated
mass spectrometric protein identification by this approach. MS/MS spectra are also
ideally suited to search translated EST and other sequence databases containing
incomplete sequences.
The next development was of the gel-independent approach to proteomics using LC-
MS/MS systems. The combination of LC-MS/MS and sequence database searching
has been widely adopted for the analysis of complex peptide mixtures generated
from the proteolysis of samples containing several proteins. This approach is often
referred to as 'shotgun' proteomics and has the ability to catalog hundreds, or even
thousands, of components contained in samples isolated from very different sources.
Specific examples include the identification of proteins in the periplasmic space of
bacteria, yeast ribosomal complexes, murine nuclear interchromatin granule clusters
(nuclear speckles), murine mitochondrial soluble intermembrane proteins, human
urinary proteins, yeast TFIID-associated proteins, proteasomal proteins, human
microsomal proteins, human membrane proteins and yeast nuclear pore proteins
pre-fractionated by SDS polyacrylamide gel electrophoresis, and proteins from yeast
lysates.[26]
Some of the aspects of bioinformatics in proteomics which have shown development

in the last year include:
♦ An increasing number of bioinformatics databases provide graph views of

interconnected components of a database. The collection of possibly
interconnected maps are called atlases. So far though, there have been no
tools developed to interpret these results. e.g., BacMap [27, 28]
♦ In recent times, data is being organised using an emerging field called
systems biology. This involves the breaking down of biological systems into
its component parts which are linked to each other. This helps in
understanding the system as a whole yet preventing redundancy of data and
better storage
♦ A recent development is the integration of time into simulation models being
generated. Simulations are being made which reproduce and thereby quantify
the behaviour of a system over a period of time. The yield of a reaction, the
steps of a molecular pathway, up to the full network of interacting entities that
characterise a cellular activity can be modelled. The behaviour of the resulting
systems is tested in response to defined perturbations. [27, 28].
♦ Data can be organised based on the use of ‘synthetic biology’ which involves
the artificial assembly of natural parts. [27, 28] This can then be organised as
networks which can act as both a representation as well as simulation of
interacting proteins.
♦ Last but not the least is the development of a standard for all proteomics
formats. Several working groups, all launched through the Proteomics
Standards Initiative (PSI) of HUPO (Human Proteome Organisation), are
currently in charge of defining standardised general proteomics formats. Such
standards are expected to facilitate data comparison, exchange and
verification. Besides an involvement in setting a standardised general
proteomics format, PSI supports other working groups in key areas of
proteomics, i.e., gel electrophoresis, mass spectrometry and protein-protein
interaction data.
From its inception to the present day, proteomics has evolved substantially.
Conceptually, proteomics has become a biological assay for the quantitative and
qualitative analysis of complex protein samples. Technologically, proteomics has
become a combination of relatively mature tools that support protein cataloguing and
quantitative proteome measurements reliably, sensitively and at high throughput.[26]
This is an invaluable tool for various reasons. Firstly, global data sets are rich in
information but difficult to analyze using traditional knowledge-based interpretation.
Secondly, the more the data the better it is. That is, it is much more informative to
collect several global data sets on the same system, and to use mathematical tools
such as cluster analysis to extract biological insights or to formulate hypotheses.
Thirdly, It is expected that additional systematic proteomic data, including activity
profiles, interaction maps and profiles of (regulatory) modifications, will provide
further insights into the structure, function and control of biological systems.
The use of bioinformatics in proteome analysis has made the handling of data much
easier.
Before computer processing comes into the picture, extensive data, particularly
through crystallography and NMR, are required for the study of any protein. With the
development of bioinformatics tools and databases, the structure and its relationship
to function of newly discovered proteins can be understood in a very short time.
The use of high throughput platforms and parallel processing in proteomics has
resulted in a huge surge in the amount of data being generated throughout the world.
However, the large scale analysis of proteins results in data analysis problems [14].
This is because of the nature of the two main technologies being used to study the
structure of the protein:
♦ Systems based on gel electrophoresis
♦ Non-gel based methods.
Software written for analysis of gel data has been improved over the years but still
requires manual intervention.
On the other hand, data from non-gel based methods such as mass spectrometry
consists of not only the peptides being analyzed but also the noise in the system. As
the vast majority of the data is noise, there is a huge wastage of computer resources
and time in trying to analyze this data. Also, algorithms written to analyze the MS
data only indicate the significance of the matches and not the actual value. Hence
there are a huge number of false positive results. The analysis of these results by
untrained or inexperienced personnel can lead to acceptance of false matches and
the faulty identification of the protein.
The wide adoption of proteomics approaches into biological research will require
several developments to combat data overload and ensure data quality. First, tools
must be readily available to de-select MS/MS spectra from search routines that are
unlikely to yield a match because of poor quality. Second, search algorithms require
further refinement to diminish the false positives and false negatives (merely setting
scores high to diminish false positives is counter to the aim of the experiment); this
problem is beginning to be addressed through the development of true probability-
based scores that are akin to the assignment of quality scores to each base in DNA
sequencing. Third, spectral matching algorithms for peptide MS/MS spectra need to
be made commercially available. And fourth, a database of truly nonredundant
transcripts of the organism under study is required, together with an extensive
relational database that can acquire data from the diverse range of instruments
involved in each stage of the proteomics experiments
The ability to generate information has now outstripped the ability to analyze the
information being generated. This is because the amount of information being
entered into the databases in increasing by geometric succession. However, the rate
of increase of computing power is based on Moores law.
The ability to collect large proteomic data sets already outstrips the ability to validate,
to interpret and to integrate such data for the purpose of creating biological
knowledge. Therefore, software tools will be developed to help manage, interpret,
integrate and understand proteomic data. The lack of suitable software tools currently
limits essentially all areas of proteomic data analysis, from database searching using
MS/MS spectra to the assembly of large data sets containing different types of data
in relational databases. To derive value from the data that goes beyond an initial
scan for 'interesting observations' and to make data portable and comparable, it will
be necessary to develop algorithms that assign a score to each data point that
estimates the probability that the observation is correct. Just as the assignment of
quality scores to each base in DNA sequencing using the algorithm Phred was
essential for the success of genome sequencing programs, it can be expected that
probability-based scores calculated for proteomic data will have a similar impact on
proteomics.
More recent initiatives have shown that quantification of information is the next
hurdle.
Every data involved in bioinformatics analysis has to be weighted with an appropriate
number in order to give it the proper significance. These weights express the relative
importance of each component of the entity or each event of a process.
A recent development or branch of bioinformatics is systems biology. This field has

the advantages of portraying the actual relationship between proteins. As it is stored
as components, redundancy is avoided and time taken for downloading is minimised.
Some challenges involved in this technology are
♦ Enormous complexity of proteome The detection, and particularly the
molecular analysis of this complexity, remains an unmatched task.
♦ The second challenge is the need for a general technology for the targeted
manipulation of gene expression in eukaryotic cells. An approach that has
proved successful for the systematic analysis of biological systems relies on
iterative cycles of targeted perturbations of the system under study and the
systematic analysis of the consequences of each perturbation. Although
recent advances in using RNA interference in higher eukaryotic cells open up
exciting possibilities, the general targeted manipulation of biological systems
in these species remains unsolved.
♦ The third challenge is the limited throughput of today's proteomic platforms:
iterative, systematic measurements on differentially perturbed systems
demand a sample throughput that is not matched by current proteomic
platforms.
♦ The fourth challenge is the lack of a general technique for the absolute
quantification of proteins. The ability to quantify proteins absolutely, thereby
eliminating the need for a reference sample, would have far-reaching
implications for proteomics—from the determination of the stoichiometry of
protein complexes to the design of clinical studies aimed at discovering
diagnostic markers.
Studies have also highlighted the limitations of shotgun proteomics, including the
difficulty of detecting and analyzing by collision-induced dissociation (CID) mass
spectrometry all of the peptides in a sample, the qualitative nature of data-dependent
experiments, and the challenge of processing the tens of thousands of CID spectra
generated in a typical experiment—one of the many informatics challenges that still
faces scientists in this field. On average, a protein digested with trypsin will generate
30−50 different peptides. A tryptic digest of the proteome of a typical human cell will
therefore generate a peptide mixture containing at least hundreds of thousands of
peptides. Even the most advanced LC-MS/MS systems cannot resolve and analyze
such complexity in a reasonable amount of time.
[26,27,28]

Some of the major applications of bioinformatics in proteomics are
.
Structural analysis
♦ High throughput data from gel electrophoresis

New algorithms for image analysis of two dimensional gels have been
developed within the last five years[15]
♦ Mass spectroscopy data analysis

Within mass spectrometry data analysis algorithms for peptide mass
fingerprinting (PMF) and peptide fragmentation fingerprinting (PFF) have
been developed[6, 9,16,17].
♦ Prediction of protein 3-d structures

Various tools have been developed to predict 3D structure of protein by
either comparing with existing structures or ab initio.[18]
♦ Structural analysis of receptors, molecules involved in cell signalling

Analysis of the various structures and how they function together can be
visualised as simulations.[18]
Functional analysis
♦ Understanding protein-protein interactions

The interaction between various proteins can be studied using systems
biology.[23, 25]
♦ Sequence comparison
Sequence comparison helps identification of individual proteins and
understanding the difference between normal and abnormal cell
proteomes[7].
♦ Sequence to structure information

The relationship between structure and sequence in proteins can be
studied using bioinformatics tools[11,26]
Evolutionary analysis
♦ Tracing ancestral connections

Construction of phylogenetic trees and multiple sequence alignment help
trace the evolutionary relationship between proteins.[24,25]
Biomedical applications
♦ Drug discovery
Proteomics has a major role to play in drug discovery as simulations can

be done of potential drug targets, such as for cancer therapy, and
tested.[20, 21]
♦ Biomarkers development
Proteomics is very useful as it can compare a whole proteome or sub-

proteome at a time and can thus help design biomarkers. [ 14}
♦ Proteomic profiling
Proteomic profiling helps determine the differences in protein expression
patterns between cells or with a cell at different times.[22]
Others
♦ Analysis of microarrays
Chips containing arrays can be automated and connected to software for
analysis.[19]
Apart from analysis, bioinformatics is also used to store information in databases

which can also function as knowledge resources for scientists throughout the
world[8].
Include useful leaning sites, reference/leading laboratory sites, resource sites e.g.
Databases
Protein sequence databases
Swiss-prot (http://www.expasy.com/swiss_prot )
PDB (http://www.rcsb.org/pdb/home/home.do)
Protein structure databases
CATH (http://www.cathdb.info/latest/index.html)
SCOP (http://scop.mrc-lmb.cam.ac.uk/scop/)
Bioinformatics tools
Various bioinformatics tools and what they are used for is as given below.
A. Predict the protein sequence from a given nucleotide sequence by finding the
most probable open reading frame.
1. By direct translation of sequences without introns
ExPASy Translation Tool (at Swiss Institute of Bioinformatics)

http://www.expasy.ch/tools/dna.html
NCBI ORF Finder
http://www.ncbi.nlm.nih.gov/gorf/gorf.html
2. By predicting promoters, splice sites, termination sites, etc
GENSCAN (http://genes.mit.edu/GENSCAN.html)
BCM SearchLauncher (http://dot.imgen.bcm.tmc.edu:933l/)
Grail (http://compbio.ornl.gov/Grail-1.3/)
FGENEH(http://genomic.sanger.ac.uk/gf/gf.shtml)
Genmark(http://genemark.biology.gatech.edu/GeneMark/webgenemark.html)
B. Identification of protein based on sequence
AACompIdent(http://www.expasy.ch/tools/aacomp/)
AACompSim
http://www.expasy.ch/tools/aacsim/
TagIdent
http://www.expasy.ch/tools/tagident.html
C. Identification of physical properties based on sequence
compute pI/MW
http://www.expasy.ch/tools/pi_tool.html
peptideMass
http://www.expasy.ch/tools/peptide-mass.html
SAPS
http://www.isrec.isb-sib.ch/software/SAPS_form.html
D. Identification of similar proteins from databases
BLAST
FASTA
GenQuest Q Server 9http://www.bis.med.jhmi.edu/Dan/gq/gq.form_rm.html
E. Align multiple sequences to identify patterns between proteins of various species

or proteins having evolutionary significance.
ClustalW
http://www.ebi.ac.uk/clustalw/
BCM Search Launcher

http://dot.imgen.bcm.tmc.edu:9331/multi-align/multi-align.html
Identification of specific domains or motifs can be done using
Pfam
http://www.sanger.ac.uk/Pfam/
SCOP - Structural Classification of Proteins

http://scop.mrc-lmb.cam.ac.uk/scop/
PROSITE
http://www.expasy.ch/prosite/
A database of regular expression-like patterns (motifs)
PRINTS - a diagnostic collection of protein fingerprints

http://www.biochem.ucl.ac.uk/bsm/dbbrowser/PRINTS/PRINTS.html
fingerPRINTScan
http://bioinf.man.ac.uk/fingerPRINTScan/
BLOCKS
http://www.blocks.fhcrc.org/
BLOCKS are ungapped MSA representing conserved protein

regions
G. Structure prediction tools – fold and secondary structure prediction
PredictProtein Server
http://www.embl-heidelberg.de/Services/sander/predictprotein/
Meta PP
http://dodo.cpmc.columbia.edu/predictprotein/submit_meta.html
Transmembrane region prediction
TMHMM
http://www.cbs.dtu.dk/services/TMHMM-1.0/
TOPPRED
http://www.biokemi.su.se/~server/toppred2/
Coiled coil region prediction
MultiCoil
http://gaiberg.wi.mit.edu/cgi-bin/multicoil.pl
The MultiCoil program predicts the location of coiled-coil regions in amino
acid sequences and classifies the predictions as dimeric or trimeric. The
method is based on the PairCoil algorithm
Tertiary structure prediction by homology modeling
Swiss-Model - Automated Protein Modeling Server

http://www.expasy.ch/swissmod/SWISS-MODEL.html
ModBase - Database of comparative protein structure models

http://pipe.rockefeller.edu/modbase/
Align your protein structure vs other protein structures
DALI Server - Automated Protein Structure Alignment

http://www.ebi.ac.uk/dali/
[12]
5.4.7 References
1. Swiss-Prot database, definitions

http://au.expasy.org/proteomics_def.html
2. J. S. Petersen , Cinjecture corporation

http://www.wisegeek.com/what-is-proteomics.htm
3. Rediscovering biology, online text book

http://www.learner.org/channel/courses/biology/textbook/proteo/proteo_1.html
4. Bioinformatics definition committee, July17, 2000,

http://www.bisti.nih.gov/CompuBioDef.pdf
5. Blueggel M, Chamrad D, Meyer HE.(2004) Bioinformatics in proteomics Curr

Pharm Biotechnol, 5(1):79-88.
6. Haoudi A, Bensmail H. (2006 Jun) Bioinformatics and data mining in

proteomics, Expert Rev Proteomics.;3(3):333-43
7. Kremer A, Schneider R, Terstappen GC. (2005 Feb-Apr) A bioinformatics

perspective on proteomics: data storage, analysis, and integration, Biosci
Rep.;25(1-2):95-106.
8. Kearney P, Thibault P. (2003 Apr) Bioinformatics meets proteomics--bridging

the gap between mass spectrometry data analysis and cell biology. J
Bioinform Comput Biol.;1(1):183-200.
9. Stephens AN, Quach P, Harry EJ. (2005 Apr) A streamlined approach to

high-throughput proteomics. Expert Rev Proteomics.;2(2):173-85
10. . Yee A, Pardee K, Christendat D, Savchenko A, Edwards AM, Arrowsmith

CH, Structural proteomics: toward high-throughput structural biology as a tool
in functional genomics.
11. Online Bioinformatics courses and lectures

http://www.bioinformaticscourses.com/bioinform_old/proteinAnalysis1.
html
12. Englbrecht CC, Facius A. (2005 Dec) Bioinformatics challenges in proteomics

Comb Chem High Throughput Screen.;8(8):705-15
13. Scott D. Patterson, Ruedi H. Aebersold, (2003) Proteomics: the first decade
and beyond, Nature Genetics 33, 311 - 323
14. He QY, Chiu JF.(2003 Aug), Proteomics in biomarker discovery and drug
development , J Cell Biochem. 1;89(5):868-86.
15. Dowsey AW, Dunn MJ, Yang GZ, (2004 Dec), ProteomeGRID: towards a
high-throughput proteomics pipeline through opportunistic cluster image
computing for two-dimensional gel electrophoresis., Proteomics.;4(12):3800-
12.
16. Chamrad DC, Korting G, Stuhler K, Meyer HE, Klose J, Bluggel M ( 2004
Mar). , Evaluation of algorithms for protein identification from sequence
databases using mass spectrometry data, Proteomics.;4(3):619-28.
17. Cristoni S, Bernardi LR. (2004 Dec), Bioinformatics in mass spectrometry

data analysis for proteomics studies. Expert Rev Proteomics.;1(4):469-83.
18. Lundgren DH, Eng J, Wright ME, Han DK. (2003 Nov) PROTEOME-3D: an
interactive bioinformatics tool for large-scale data exploration and knowledge
discovery. Mol Cell Proteomics.;2(11):1164-76..
19. Boguski MS, McIntosh MW.(2003 Mar), Biomedical informatics for

proteomics. Nature. 13;422(6928):233-7.
20. Rai AJ, Chan DW. .(2003 Mar), Cancer proteomics: Serum diagnostics for
tumor marker discovery.. Nature. 13;422(6928):233-7.
21. Burbaum J, Tobal GM. ( 2002 Aug) Proteomics in drug discovery. Curr Opin
Chem Biol.;6(4):427-33.
22. White CN, Chan DW, Zhang Z. (2004 Jul), Bioinformatics strategies for
proteomic profiling. Clin Biochem. 37(7):636-41.
23. MacCoss MJ (2005 Feb) Computational analysis of shotgun proteomics data.

Curr Opin Chem Biol.;9(1):88-94.
24. Cannataro M, Cuda G, Veltri P.( 2005;) Modeling and designing a proteomics
application on PROTEUS. Methods Inf Med44(2):221-6.
25. Lester PJ, Hubbard SJ. (2002 Oct), Comparative bioinformatic analysis of
complete proteomes and protein parameters for cross-species identification in
proteomics. Proteomics.;2(10):1392-405.
26. Martens L, Hermjakob H, Jones P, Adamski M, Taylor C, States D, Gevaert

K, Vandekerckhove J, Apweiler R, 2005 Aug, PRIDE: the proteomics
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27. Patricia M. Palagi, Patricia Hernandez, DanielWalther and Ron D.

Appel,(2006), Proteome informatics I: Bioinformatics tools for processing
experimental data, Proteomics, 6, 5435–5444
28. Frédérique Lisacek, Sarah Cohen-Boulakia and Ron D. Appel,(2006)

Proteome informatics II: Bioinformatics for comparative proteomics,
Proteomics , 6, 5445–5466
Chapter 5.5 Automation and High-Throughput Proteomics
Mohammad Tariq Sadat Hai
5.5.1 Introduction
Proteomics is the study the structure, function and interactions of the total encoded
proteins (proteome) as well as their isoforms and modifications in a particular cell
line, tissue, or a whole organism of interest. There are three main approaches in
proteomics: expression proteomics, cell-map proteomics, and structural proteomics.
Expression proteomics (also called “differential expression proteomics”) is the study
of the set of all protein isoforms and their cell to cell differences and modifications.
Cell-map proteomics deals with protein–protein interactions within the cells, tissues
and so on. Structural proteomics includes the determination of three-dimensional
protein structures as well as their higher order complexes on a genome wide scale
[1, 2, 3].
DNA and mRNA, being physico-chemically homogeneous and amplifiable by PCR

methods, are amenable to automation. Proteome analysis, in contrast to analysis of
DNA or mRNA, has been limited by a number of factors: (i) the level of protein
expression cannot be predicted from the level of mRNA; (ii) proteins undergo many
post-translational modifications resulting in different conformations/components with
distinct functionalities; and (iii) protein maturation and degradation are dynamic
processes altering the final amount of active protein independent of mRNA level.
Moreover, as the proteome dataset are growing larger and becoming more complex
everyday it is becoming more difficult to archive and analyze the dataset manually.
Thus, proteome analysis requires a higher degree of throughput and automation to
allow: (i) the extraction and high-resolution separation of all protein components,
including membrane proteins and proteins having low copy number; (ii) the
identification and quantification of each protein component; and (iii) the comparison,
analysis, and visualization of complex changes in expression patterns [4, 5].
Figure 5.5.1 Automated robot used to mount and align

protein crystals at Berkeley Lab Advanced Light Source [3]
Today the attainment of high-throughput proteomics has been possible due to the
modern technological supports such as the highest sensitivity of current
instrumentations related to proteome analysis, the most sophisticated protein
separation technologies and the highest precision in computational data analysis
methods [6]. A number of automated technologies (robotics and intelligent systems
technologies) have made it possible to capture a higher quality snapshot of the large
and complex proteome and analyse their activities [3].
Automated and high-throughput technologies are functional in three areas of

proteomics: (i) 2-D Gel Electrophoresis (2-DE) and Mass Spectroscopy (MS) are
used to separate, identify and characterize the set of proteins within the proteome; (ii)
protein microarray technology is used to monitor differential expression and
interactions of the proteins; and (iii) structure and imaging techniques are used for
the determination of protein 3-D structure protein localization and quantitative
analysis of protein-protein interactions [3].
Automation of DNA sequencing technologies has contributed to the acceleration of

human genome project which was initially a laborious, expensive and personnel-
intensive task. Similarly, automation is changing the field of proteomics today while
saving both cost and time of experiments [3].
Traditionally 2-DE has been used for obtaining the global picture of the expression
levels of a proteome under various conditions. In recent years, MS technologies have
evolved as a versatile tool for examining the simultaneous expression of more than
1000 proteins and the identification and mapping of posttranslational modifications.
High-throughput methods performed in an array format have emerged enabling
large-scale projects for the characterization of protein localization, protein-protein
interactions, and the biochemical analysis of protein function [7].
Automation of 2-D Gel Electrophoresis (2-DE) and Mass Spectroscopy (MS)
While 2-DE is extremely useful, it has some technical limitations. These include the
manual handling of gels which are cumbersome to run, have limits in sample
capacity, have poor dynamic range (has low sensitivity for very acidic or basic
proteins), and are biased toward abundant and soluble proteins (cannot detect low-
abundance proteins in absence of additional sample enrichment techniques). The
resolution of 2-DE is not sufficient compared to the enormous diversity of cellular
proteins, and there may be co-migrating proteins in the same spot. In addition, 2-DE
is time consuming and it may take days to run and analyze a single gel [3, 7, 8, 9,
10].
To overcome these limitations, several approaches have emerged. In one approach,

a number of 2-DE products have been developed that support automated gel
processing systems.
The a2DEoptimizer by NextGen Sciences features automated gel casting that can
cast multiple gels simultaneously being controlled and monitored by computer. It also
has the ability to create user-defined gradient gels which can be difficult to create
manually. Large Scale Biology, under their subsidiary, Predictive Diagnostics, has
released BAMF (Biomarker Amplification Filter), a computer platform combining 2-
DE, NMR, MS, and biomarkers to identify individual proteins [3].
Several features that are commonly offered by many of the newer automated gel
processing systems include ‘the ability to: (i) import and export gels into standard bit-
mapped graphics formats; (ii) manipulate, preprocess, filter, and organize gel
bitmaps; (iii) visualize and compare gels; (iv) create, queue, and monitor
computational analysis tasks; and (v) present results (e.g., peptide matches in an
excised, digested protein spot)’ [3].
To overcome the calculation-intensive process of image analysis of 2-DE gels,

ProteomeGRID, a high-throughput 2-DE image analysis computing platform, has
been developed that utilizes a gel matching algorithm to overcome the bottleneck of
spot matching. It builds on the proTurbo cluster image computing engine with Grid-
enabled versions of automatic 2-DE and MS analysis algorithms. Specific emphasis
is placed on the integrated development of a HUPO PSI GPS (Human Proteome
Organization Proteomics Standards Initiative General Proteomics Standards) object
model and ontology for statistical Image mining of 2-DE gels. The PSI will also drive
the automated spot cutting, protein digestion and MS between the 2-DE and MS
bioinformatics pipelines [9].
Taking into account the limitations of 2-DE, a number of alternatives to 2-DE has also
emerged in recent years. Non-2-DE-based protein and peptide separation methods
combined with new protein chemistries and enrichment methods and highly
automated MS instrumentation have been developed that provide new tools for
analyzing the properties of proteomes with increased sensitivity and throughput [10].
Non-2-DE-based methods include Time-Of-Flight (TOF) MS and the ‘soft ionization’

methods, namely Matrix-Assisted Laser Desorption Ionization (MALDI), ElectroSpray
Ionization (ESI) and Surface-Enhanced Laser Desorption Ionization (SELDI) [7, 11].
These ionization sources are often coupled to TOF, ion trap or quadrupole analyzers
or combinations of these analyzers. Using MS, peptides and proteins are identified
by their mass-to-charge (m/z) ratio, which correlates with the molecular mass. MS
can also be applied to sequence peptides and proteins. Post-translational
modifications also change the m/z-ratio [11].
Using liquid chromatography with various columns and pressure conditions or by bait
molecules mounted on a fixed surface, provided on small chips, proteins can be
sorted based on their various biochemical characteristics, such as hydrophobicity,
anion-, cation- or metal ion-binding capabilities, or specific protein–protein
interactions, e.g. receptor–ligand or antibody–antigen interactions. The small chip
procedure has pushed proteomics studies into high-throughput application on a
large-scale [11].
These ProteinChips® (developed by Ciphergen Biosystems Inc., USA) can also be

coated with specific antibodies enabling the study of the variety of specific gene
products. This technique has sensitivity can be in the picomole-to-femtomole range
and is able to detect low abundant peptide and proteins in biological material [11].
SELDI TOF/MS is most effective at profiling low molecular weight proteins (<20 kDa).
The application of small sample volumes (µl-range) and the detection of between
15,500 (low resolution SELDI-TOF) and >400,000m/z-ratios (high resolution SELDI-
TOF) makes proteomic profiling of diverse biological samples possible [11]. Figure
5.5.2 below illustrates a typical SELDI-TOF technology.
Figure 5.5.2 Surface-Enhanced Laser Desorption and Ionization (SELDI) technology. Using a
robotic sample dispenser, 1 µL of serum is applied to the surface of a protein-binding chip. A
subset of the proteins in the sample binds to the surface of the chip. The bound proteins are
treated with a matrix-assisted laser desorption ionization matrix and are washed and dried. The
chip, which contains multiple patient samples, is inserted into a vacuum chamber where it is
irradiated with a laser. The laser desorbs the adherent proteins and causes them to be
launched as ions. The time of flight (TOF) of the ion before detection by an electrode is a
measure of the mass-to-charge (m/z) value of the ion. The ion spectra can be analysed by
computer-assisted tools that classify a subset of the spectra by characteristic patterns of
relative intensity [12].
The MALDI-TOF instruments usually perform protein mass fingerprinting (PMF) [13].
Compared with MALDI, ESI has a significant advantage in that in can be easily
coupled to separation techniques such as liquid chromatography (LC) and HPLC,
allowing high-throughput and on-line analysis of peptide or protein mixtures.
Typically, proteins in a complex mixture are separated by ionic or reverse phase
column chromatography and then subjected to tandem MS (MS/MS) analysis via
online ESI [7, 10]. However, more recently offline spotting of peptides onto sample
targets and use of MALDI instruments capable of high throughput MS/MS analysis
has also become an option [10].
The development of multidimensional liquid chromatography (MDLC)-based

detection and quantification of multiplexed isotopic iTRAQ-labeled proteins has
enabled the global analysis of complex biological system and has permitted the
simultaneous comparison of samples of different cell states and disease specimens.
Fully automated liquid chromatography-tandem mass spectrometry (LC-MS/MS)
usually generates tens of thousands of MS/MS spectra. Many thousands of peptides
can be collectively analyzed by multiple LC-MS/MS runs in each proteomic
experiment such that thousands of proteins can be identified. Automated data
processing software package has been developed (e.g. Multi-Q) which uses iTRAQ
labeling for multiplex protein quantitation [8].
Figure 5.5.3 Multi-Q quantitation system. Multi-Q package accepts two data inputs: raw data
from mass spectra and protein identification results from search engines such as MASCOT,
SEQUEST, and X!Tandem. Data then undergo data conversion, peptide ratio determination,
and protein ratio determination, and final protein relative abundance ratios are produced [8].
As shown in Figure 5.5.3 above, Multi-Q provides a data converter for handling
spectral raw data and can also accept search results from various search engines,
including SEQUEST, X!Tandem and MASCOT. After automatic filtering of non-
iTRAQ-labeled peptides, the ratio of all peptides with unambiguous identification is
determined [8].
The Computational Proteomics Analysis System (CPAS) is an open-source, web-

based analysis platform that contains an entire data analysis and management
pipeline for Liquid Chromatography Tandem Mass Spectrometry (LC-MS/MS)
proteomics, including experiment annotation, protein database searching and
sequence management, and mining LC-MS/MS peptide and protein identifications
[14].
Automation of Protein Microarray
Automation has been achieved in different categories of protein microarray

technology. The ProteinChip technology described above works in the microarray
format and methodology. It is a parallelized approach that provides information on
protein structure, character, and PTMs. Also, other commercially available protein
microarray kits has their own features For example, Whatman FAST® Quant system
performs parallel processing and can perform over 500 measurements from 56
samples; Panorama™ Ab Microarray Cell Signaling Kit by Sigma-Aldrich can detect
minute quantities of protein – as low as a few nanograms per milliliter; BD Lyoplate™
Technology by BD Biosciences can allow microarray plate design flexibility.
Using microfluidics technology, chips can be etched with microscopic channels in

which miniature assays are performed. Caliper Technologies has developed one
such device called the LabChip® 3000 Drug Discovery System [3].
Automation of Protein Structure and Imaging
The determination of protein 3-D quaternary structures has greatly increased in the
past few years. The most common techniques for structural analysis of protein
include NMR, X-ray crystallography, structure prediction methodology, and also MS
to a certain degree.
Imaging techniques can detect protein–protein interactions and protein localization.

Such techniques include transfected cell arrays, Green Fluorescent Protein-based
(GFP) labeling, and Fluorescence Resonance Energy Transfer (FRET).
Multiplexed Surface Plasmon Resonance (SPR) [34, 35] is a automated approach to

the quantitative analysis of protein interactions. Advantages of SPR include its low
target consumption and freedom from radioactive labeling
VEGA ZZ, a molecular modeling software package, can analyze protein structures. It
has an extensive list of features including multiple file format support, atomic
potential attribution, 3-D molecular editor, and a protein–protein docking system [3].
5.5.3 Evaluation of High-Throughput Proteomics
The gaol of high-throughput proteome analysis is to catalog and quantify proteins

that a whole organism or specific tissue or cellular compartment expresses under
certain conditions. To achieve such a daunting goal, two critical factors need to be
addressed: first, it is required to demonstrate that the methods and technology of
high-throughput proteomics generate valid, reproducible, and reliable results, and
second, the gap between the high-throughput data and biological discovery must be
bridged. The intrinsic complexity of the biology, multiplied by the enormous volumes
of data generated, make the analysis and interpretation of high-throughput
proteomics experiments extremely difficult [15].
Protein microarrays are representing the first new technology that can profile the
state of a signaling pathway target even after the cell is lysed. The reverse-phase
protein microarray (see Figure 5.5.4 below) has a unique ability to analyze signaling
pathways using small numbers of human tissue cells that were microdissected from
biopsy specimens procured during clinical trials. These arrays can be manufactured
in a sectored array format where dozens of analytes can be queried simultaneously
on one slide, which thereby increases the throughput and facile data analysis more
readily [12].
FIGURE 5.5.4 Reverse-phase protein arrays. Reverse-phase array immobilizes the

cellular lysate sample to be analyzed. Lysates are prepared from cultured cells or
microdissected tissues and are arrayed in miniature dilution curves. The analyte molecule
contained in the sample is then detected by a separate labeled probe (e.g., antibody) that
is applied to the surface of the array [12].
Reverse protein microarrays do not require direct tagging of the protein as a readout
for the assay, which yields dramatic improvement in reproducibility, sensitivity and
robustness of the assay over other techniques [12].
Though ProteinChip® technology has proved to be promising in disease biomarker

detection, it has some key limitations. For example, very little is known about the true
nature of the m/z-ratios. The vast majority of the peaks obtained in the MS analysis
are unknown and it requires a tremendous amount of work and scrutiny to
characterize all these peaks. Many of these peaks will be in the range of pico- to
femtomole. Presently, there is no technique available that can amplify peptides and
proteins quickly and reliably as the PCR does with DNA [11].
Figure 5.5.5 Four functional areas of the ProteusLIMS system (GenoLogics)
illustrating the typical proteomics workflow [3].
Many current automated procedures in proteomics generate large quantities of

unwieldy data. Researchers are therefore relying on Laboratory Information
Management Systems (LIMS) to assist them with the extraction of useful information.
ProteusLIMS system of GenoLogics is comprised of four capabilities: lab
management, instrument and data integration, bioinformatics and data management,
and analytics and reporting (see Figure 5.5.5 above). Modas, a current LIMS project
under development by nonlinear dynamics, supports 2-DE and MS, integrating
analysis with LIMS into one package. The LIMS software will be the key to being able
to take full advantage of automation advancements [3].
5.5.4 Applications of High-Throughput Proteomics
Modern technological capabilities have allowed the identification and quantification of

most of the large number of proteins in complex biological samples such as whole
cell lysates, tissues, blood etc. and this has allowed the use of proteomics in
understanding complex disease processes, early detection of the disease using
proteomic patterns of body fluid samples [12], developing new diagnosis for them
and improving the drug development process [6].
Protein microarray holds great promise in the identification of drug and drug targets,
as well as in basic proteomic research such as determining protein-protein
interaction, protein-lipid, and enzyme-substrate interactions and also in clinical
diagnosis. The reduced sample consumption in the microarray format is crucial in
proteome analysis since only minimal amounts of samples are available. Other
promises of protein microarray include real-time patient monitoring during disease
treatment and therapy [7]. In future, blood tests may be performed by providing fewer
drops of blood onto a chip with specific protein markers, providing valuable
diagnostic and real-time prognostic information [3].
In the last few years, ProteinChip® technology has been applied in the detection of
cancers such as cancers of the head and neck, lung, breast, pancreas, kidney,
bladder, prostate and ovary through detection of biomarkers in serum, urine,
pancreatic juice, nipple aspirate fluid or tissue homogenates. Besides early detection
of cancer, systematic analysis of the serum, tissue, cellular, and subcellular
proteome may help to find novel biomarkers that uncover transplant rejection, drug
toxicity, and chronic inflammatory or cardiovascular diseases [11].
The virulent and the attenuated strains of M. tuberculosis and Heliobacter pylori were
studied by high-resolution 2-DE and MALDI mass fingerprinting for potential DNA
vaccines and candidate antigens for serological detection. Marker proteins were
selected for the development of potential vaccines that show the high success of
proteomics studies [16].
MS/MS combined with ESI or MALDI has been developed as a potential tool for
identification of targeted microorganisms through analysis of peptides generated from
cellular proteins. Acid extraction of bacterial spore proteins followed by peptide
sequencing using MALDI MS/MS has been used to discriminate species from the
genus Bacillus in spore mixtures. LC-ESI MS/MS has been used in bacterial
classification based on the peptide sequence information generated from LC-ESI
MS/MS analysis of a bacterial protein digest. This method can be a strong
complement to the alternative approaches of comparing microbial genomes based on
DNA sequencing or microarray hybridization techniques [17].
In recent years, proteomics studies were performed in order to identify the

proteomics signature for personalized medicine that best targets the patient's entire
disease-specific protein network [12, 16]. Due to the diversity of cancer development
process with many unexpected subtypes, many different responses to treatment are
observed and establishment of an individualized therapy is required. The diverse
functions of proteins and their appearance in various species of different
modifications dictate their functional investigations and determine the type of cancer
phenotype which is not possible on the level of DNA or RNA [16].
Details about CPAS-assisted interactive data analysis, CPAS architecture and

implementation, and sample data loaded into CPAS are available at
http://proteomics.fhcrc.org/CPAS.
The international website of HUPO (Human Proteome Organisation) is

http://www.hupo.org/. HUPO is an international consortium of national proteomics
research associations, government researchers, academic institutions, and industry
partners. HUPO promotes the development and awareness of proteomics research,
advocates on behalf of proteomics researchers throughout the world, and facilitates
scientific collaborations between HUPO members and Initiatives. Presently, there are
seven HUPO-sponsored Scientific Initiatives –
1. Human Liver Proteome Project (http://www.hupo.org/research/hlpp/)

2. Human Brain Proteome Project (http://www.hupo.org/research/hbpp/)
3. Proteomics Standards Initiative (http://www.hupo.org/research/psi/)
4. Human Antibody Initiative (http://www.hupo.org/research/hai/)
5. Plasma Proteome Project (http://www.hupo.org/research/hppp/)
6. Mouse Models of Human Disease (http://www.hupo.org/research/mmhd/)
7. Human Disease Glycomics/Proteome Initiative
(http://www.hupo.org/research/hgpi/)
The human protein atlas (created to show the expression and localization of proteins
in a large variety of normal human tissues and cancer cells) is available at
http://www.proteinatlas.org/
The ‘Journal of proteome research’ publishes scientific articles on the recent

advances of proteomics. The journal can be accessed at
http://pubs.acs.org/journals/jprobs/index.html.
The journal ‘Drug Discovery Today’ publishes many articles focusing on the
application of proteomics technology (e.g. protein microarray) in the field of drug
development. The journal can be accessed at http://www.drugdiscoverytoday.com/.
The journal ‘Proteomics’ is a key resource for information on proteomics. The journal
is available at http://www3.interscience.wiley.com/cgi-
bin/jhome/76510741?CRETRY=1&SRETRY=0
A comprehensive list of available current LIMS packages may be found at the
LIMSource (www.limsource.com).
Many other publications related to proteomics can be found at http://pubs.acs.org.
The following table lists some Protein Interaction Resources and some other
resources of Interest [18]:
Database of interacting proteins (DIP) http://dip.doe-mbi.ucla.edu
BIND http://www.bind.ca
MIPS http://mips.gsf.de
Protein–protein interaction database http://www.ebi.ac.uk/intact

(IntAct)
Human Protein Reference Database http://www.hprd.org/
(HPRD)
Human Protein Interaction Database http://www.hpid.org
(HPID)
HUPO PSI http://psidev.sourceforge.net/
http://psidev.sf.net/
Index site: http://www.expasy.ch/ch2d/2d-index.html
The Proteome Analysis DB http://www.ebi.ac.uk/proteome/
Swiss 2DPAGE http://www.expasy.ch/ch2d/
2DWG Image Meta-database http://www-lecb.ncifcrf.gov/2dwgDB/
PEDRo http://sourceforge.net/projects/pedro
http://pedro.man.ac.uk/
Open Proteomics Database http://bioinformatics.icmb.utexas.edu/OPD/
Systems Biology Institute http://www.systemsbiology.org/
SBEAMS http://www.sbeams.org/
The Pathway Resource List (PRL) http://www.cbio.mskcc.org/prl/index.php
An extensive list of commercially available automated and high-throughput proteomic

products and their industrial sources and price is available at the website of
Biocompare® http://www.biocompare.com/jump/256/Lab-Automation.html.
The following table lists some proteomics software systems and their resources [6]:
Software Focus URL
Mascot MS–MS search engine www.matrixscience.com
Mascot Integra Data management for proteomics www.thermo.com
Sequest MS–MS search engine www.proxeon.com
EPICenter Data management and validation for www.agilent.com
peptide ID data
Spectrum Mill MS–MS search engine environment www.genologics.com
Proteus LIMS Proteomics data management LIMS www.proteomecenter.org
Peptide Prophet High-throughput validation of peptide www.proteomecenter.org
identifications
Protein Prophet Protein identification (statistical) www.thegpm.org
X!Tandem Open source search engine for MS–MS www.thegpm.org
GPM Public database of identified peptides www.proteomecenter.org
XPRESS Quantitative differential analysis for ICAT www.proteomecenter.org
SBEAMS Systems biology experiments analysis and http://ncrr.pnl.gov/prism
management
PRISM High-throughput proteomics information www.proteomesoftware.com
management system
Scaffold Protein identification automation software www.phenyx-ms.com
Phenyx Protein identification and validation http://proteome.nih.gov
platform
DBParser Protein identification and validation http://mzmine.sourcefourge.net
platform
MZmine Differential LC-MS analysis of http://www.cebitec.uni-bielefeld.de
metabolomics data
ProDB Storage and analysis of identification www.proteios.org
proteomics experiments
PROTEIOS Storage, analysis and annotation of www.proteomesystems.com
proteomics experiments
ProteomIQ Integrated proteomics data management http://genome.ucsc.edu
platform
Proteome Browser Protein sequence annotation http://www-helix.inrialpes.fr
PepLine Software pipeline for protein identification www.waters.com
Protein Expression Quantitative and qualitative proteomics http://bio.mki.co.jp/product/xome
System analysis
Xome Quantitative and qualitative proteomics www.caprion.com
analysis
CellCarta Integrated suite for quantitative proteomics www.proteomecenter.org
analysis
mzXML File format (standard) for mass spectra www.proteomecenter.org
data
Trans-Pproteomic XML-based analysis pipeline for www.appliedbiosystems.com
Pipeline proteomics data
Software Focus URL
ProICAT Protein quantitation and identification for www.appliedbiosystems.com
ICAT
ProQuant Protein quantitation and for iTRAQ www.amershambiosciences.com
DeCyder MS Identification and quantitation analysis www.bioinformaticssolutions.com
platform
MS peaks De novo protein identification http://ca.expasy.org
Expasy proteomics Protein sequence analysis tools and www.ionsource.com
server databases
Ion Source On line resource of mass spectrometry www.matrixscience.com
methods
5.5.7 References
1 Lee, W.C. and Lee, K.H. (2004) Applications of affinity chromatography in

proteomics, Analytical Biochemistry Volume 324, Issue 1 , 1 January 2004, Pages 1-
10
2 Tyers, M. & Mann, M. (2003) From genomics to proteomics, Nature 422, 13 March
2003, 193-197
3 Alterovitz, G., Liu J., Chow J. and Ramoni, M.F. (2006) Automation, parallelism,
and robotics for proteomics, Proteomics 2006, 6, 4016-4022
4 James, P. and Quadroni, M. (1999) Proteomics and automation, Electrophoresis

1999, 20, 664-677
5 Wilke, A., Rückert, C., Bartels, D., Dondrup, M., Goesmann, A., Hüser, A.T.,
Kespohl, S., Linke, B., Mahne, M., McHardy, A., Pühler A. and Meyer, F. (2003)
Bioinformatics support for high-throughput proteomics, Journal of Biotechnology
Volume 106, Issues 2-3, 19 December 2003, Pages 147-156
6 Topaloglou, T. (2006) Informatics solutions for high-throughput proteomics, Drug

Discovery Today Volume 11, Issues 11-12, June 2006, Pages 509-516
7 Zhu, H., Bilgin, M. and Michael Snyder, M. (2003) Proteomics, Annual Review of
Biochemistry 2003, 72: 783-812
8 Lin, W.T., Hung, W.N., Yian, Y.H., Wu, K.P., Han, C.L., Chen, Y.R., Chen, Y.J.,
Sung, T.Y. and Hsu, W.L. (2006) Multi-Q: A Fully Automated Tool for Multiplexed
Protein Quantitation, Journal of Proteome Research 2006, 5(9), 2328-2338
9 Dowsey, A.W., Dunn, M.J. and Yang, G.Z. (2004) ProteomeGRID: towards a high-
throughput proteomics pipeline through opportunistic cluster image computing for
two-dimensional gel electrophoresis, Proteomics 2004, 4, 3800–3812
10 Roe, M.R. and Griffin, T.J. (2006) Gel-free mass spectrometry-based high
throughput proteomics: Tools for studying biological response of proteins and
proteomes, Proteomics 2006, 6, 4678–4687
11 Rocken, C., Ebert, M.P.A. and Roessner, A. (2004) Proteomics in pathology,

research and practice, Pathology – Research and Practice 200 (2004) 69–82
12 Petricoin, E.F. and Lance A. Liotta, L.A. (2003) Clinical Applications of
Proteomics, The journal of Nutrition 133:2476S-2484S, July 2003 (Supplement:
Nutritional Genomics and Proteomics in Cancer Prevention)
13 Arrigoni, G., Fernandez, C., Holm, C., Scigelova, M. and James, P. (2006)
Comparison of MS/MS Methods for Protein Identification from 2D-PAGE, Journal of
Proteome Research 2006, 5(9), 2294-2300
14 Rauch, A., Bellew, M., Eng, J., Fitzgibbon, M., Holzman, T., Hussey, P., Igra, M.,
Maclean, B., Lin, C.W., Detter, A., Fang, R., Faca, V., Gafken, P., Zhang, H.,
Whitaker, J., States, D., Hanash, S., Paulovich, A. and McIntosh, M.W. (2006)
Computational Proteomics Analysis System (CPAS): An Extensible, Open-Source
Analytic System for Evaluating and Publishing Proteomic Data and High Throughput
Biological Experiments, Journal of Proteome Research 2006, 5(1), 112-121
15 Hogan, J. M., Higdon, R. and Kolker, E. (2006) Experimental Standards for High-
Throughput Proteomics, OMICS A Journal of Integrative Biology, Volume 10,
Number 2, 2006, 152-157
16 Liebold, B.W., Graack, H.R. and Pohl, T. (2006) Two-dimensional gel

electrophoresis as tool for proteomics studies in combination with protein
identification by mass spectrometry, Proteomics 2006, 6, 4688–4703
17 Dworzanski, J.P., Deshpande, S.V., Chen, R., Jabbour, R.E., Snyder, A.P., Wick,
C.H. and Li, L. (2006) Mass Spectrometry-Based Proteomics Combined with
Bioinformatic Tools for Bacterial Classification, Journal of Proteome Research 2006,
5(1), 76-87
18 Kremer, A., Schneider, R. and Terstappen, G.C. (2005) A Bioinformatics

Perspective on Proteomics: Data Storage, Analysis, and Integration, Bioscience
Reports, Vol. 25, Nos. 1/2, February/April 2005, 95-106
Chapter 6.2 Molecular Biology Approaches for Study of
Protein-Protein Interactions
Natalia Samosir
6.2.1 Introduction
Protein-protein interactions are an essential key in all biological processes, from

replication and expression of genes to the morphogenesis of organisms. The
standard molecular biology approach to study protein-protein interactions is the
yeast two-hybrid technique. The yeast two-hybrid system was first described
1989 by S. Fields and O-K. Song. The system is based on the reconstitution of a
transcriptional activator. Upon protein-protein interaction of two fusion proteins, a
functional activator is obtained, resulting in the activation of a reporter gene[1].
Figure 1. Yeast two-hybrid transcription

Source: http://www.scq.ubc.ca/?p=246
Firstly, two fusion proteins are created: the protein of interest (X), which is
constructed to have a DNA binding domain (BD) attached to its N-terminus, and its
potential binding partner (Y), which is fused to an activation domain (AD). If protein X
and protein Y interact, then their DNA-BD and AD will combine to form a functional
transcription activator (TA). This newly formed TA will then go on to transcribe a
reporter gene, which is simply a gene whose protein product can be easily detected
and measured. In this way, the amount of the reporter produced can be used as a
measure of interaction between the protein of interest and its potential partner[1, 2].
The Underlying Principle

First, it is necessary to construct the ‘bait’ and ‘hunter’ fusion proteins. The ‘bait’
fusion protein is the protein of interest linked to the GAL4 binding domain (GAL4 BD).
This is done by inserting the segment of DNA encoding the bait into a plasmid. This
plasmid will also have inserted in it a segment of Gal4 BD DNA next to the site of bait
DNA insertion[3, 4]. Therefore, when the DNA from the plasmid is transcribed and
converted to protein, the bait will now have a binding domain attached to its end. The
same procedure is used to construct the ‘hunter’ protein, where the potential binding
partner is fused to the GAL4 AD[2, 4].
Figure 2. Plasmid construction of ‘bait’ and ‘hunter’ proteins
In addition to having the fusion proteins encoded for, these plasmids will also contain
selection genes, or genes encoding proteins that contribute to a cell’s survival in a
particular environment. An example of a selection gene is one encoding antibiotic
resistance; when antibiotics are introduced, only cells with the antibiotic resistance
gene will survive. Yeast two-hybrid assays typically use selection genes encoding
proteins capable of synthesizing amino acids such as histidine, leucine and
tryptophan[2].
Once the plasmids have been constructed, they must next be introduced into a host
yeast cell by a process called transfection. In this process, the outer-membrane of a
yeast cell is disturbed by a physical method, such as sonification or chemical
disruption. This disruption produces holes that are large enough for the plasmid to
enter, and in this way, the plasmids can cross the membrane and enter the cell[3, 4].
Figure 3. Transfection process

Once the cells have been transfected, it is necessary to isolate colonies that have
both ‘bait’ and ‘hunter’ plasmids. This is because not every cell will have both
plasmids cross their plasma membrane; some will have only one plasmid, while
others will have none. Isolation of transfected cells involves identifying cells
containing plasmids by virtue of their expressing the selection genes mentioned
previously[4].
After the cells have been transfected and allowed to recover for several days, they
are then plated on minimal media, or media that is lacking one essential nutrient,
such as tryptophan. The cells used for transfection are called auxotrophic mutants;
these cells are deficient in producing nutrients required for their growth. By supplying
the gene for the deficient nutrient in the ‘bait’ or ‘hunter’ plasmid, cells containing the
plasmid are able to survive on the minimal media, whereas untransfected cells
cannot. Selection in this way occurs in two rounds: first on one minimal media plate,
to select for the ‘bait’ plasmid, and then on another minimal media plate, to select for
the ‘hunter’[3, 4].
Once inside the cell, if binding occurs between the hunter and the bait, transcriptional
activity will be restored and will produce normal Gal4 activity. The reporter gene most
commonly used in the Gal4 system is LacZ, an E. coli gene whose transcription
causes cells to turn blue. In this yeast system, the LacZ gene is inserted in the yeast
DNA immediately after the Gal4 promoter, so that if binding occurs, LacZ is
produced. Therefore, detecting interactions between bait and hunter simply requires
identifying blue versus non-blue[2, 4].
Variation to the Two Hybrid System

Reverse two-hybrid and split hybrid system
The “reverse” two-hybrid system has been invented to select for disrupted two-hybrid
interactions e.g. by mutations, drugs or competing proteins. In this system the
interaction of X and Y proteins induces the transcription of a reporter gene that
confers toxicity to the yeast[5].
Three-hybrid System
In this yeast two-hybrid variation a third protein (Z) is expressed along with the DNA-
BD and AD fusions. Expression of the reporter gene is used to select for interactions
that occur only in the presence of this protein. Three-hybrid systemwas developed to
detect and analyse RNA-protein interactions in which the binding of a bifunctional
RNA molecule links the DNA-BD and AD hybrid-proteins and activates transcription
of the reporter gene. This system is known as RNA three-hybrid system[5].
SOS Recruitment system (SRS)

This membrane-associated two-hybrid system make use of the Ras pathway in
yeast. When localized at the plasma membrane, the yeast Ras guanyl nucleotide
exchange factor (RGEF) cdc25 stimulates GDP/GTP exchange on Ras and
promotes downstream signalling events that ultimately lead to the cell growth. A
mutant yeast strain harbouring the temperature sensitive cdc25-2 allele is still able to
grow at 25°C but fails to grow at 36°C. However, the human RGEF (hSOS) when
targeted to the plasma membrane efficiently complements the mutation, leading to
cell growth at 36°C. In the SRS the translocation of hSOS is dependent on a protein-
protein interaction: the bait X is fused to C-terminally truncated hSOS, which is active
but unable to target to the plasma membrane. The bait is co-expressed with a prey Y,
which can either be an integral membrane protein or a soluble protein that is
anchored to the membrane by means of a myristoylation signal[5].
Prokaryotes Two Hybrid System
In recent years, there has been a growing interest in the development of prokaryotic
two-hybrid. A large number of prokaryotes two hybrid systems have been described,
but despite their many advantages, the prokaryotes two hybrid systems still have not
been widely implemented for large-scale or proteomic scale protein interacting
mapping in the same way as the yeast two hybrid system[6, 7].
In order to detect protein–protein interactions using the prokaryotes two hybrid

systems described to date, both the Prey and Bait vectors for the systems have to be
introduced into an appropriate E.coli reporter strain by bacterial transformation[6].
The resulting transformants are then assayed for reporter gene activation/repression
or a reconstituted enzyme activity in order to detect protein–protein interactions. It is
proposed that bacterial conjugation could be exploited as a technically simplified and
more efficient means of introducing plasmids in combination to test for protein–
protein interactions[6, 7].
Prokaryotes two hybrid systems present many advantages over yeast-based

technologies, which largely derive from the ease with which E.coli can be genetically
manipulated, the lack of cellular compartmentalisation, its faster growth rate and the
higher transformation efficiencies that are attainable permitting rapid and more
efficient screening of complex libraries[6]. The systems permit the investigation of
prokaryotic protein–protein interactions in a prokaryotic genetic background, but they
can also be used for the analysis of eukaryotic proteins. This may be particularly
desirable in circumstances where homologous yeast proteins interfere with an
interaction by interacting with and sequestering one of the interacting partners
leading to false negatives, or by acting as a bridge between two proteins leading to
false positives. The absence of such homologous eukaryotic proteins in E.coli may
result in the observation of less false positives and negatives[6, 7].
Mammalian Two Hybrid System

Like the yeast two-hybrid system, this is a genetic, in vivo assay based on the
reconstitution of the function of a transcriptional activator. In this system, one protein
of interest is expressed as a fusion to the Gal4 DNA-binding domain and another
protein is expressed as a fusion to the activation domain of the VP16 protein of the
herpes simplex virus[8, 9]. The vectors that express these fusion proteins are
cotransfected with a reporter chloramphenicol acetyltransferase (CAT) vector into a
mammalian cell line. The reporter plasmid contains a cat gene under the control of
five consensus Gal4 binding sites. If the two fusion proteins interact, there will be a
significant increase in expression of the cat reporter gene[8]. Previously, it was
reported that mouse p53 antitumor protein and simian virus 40 large T antigen
interact in a yeast two-hybrid system. Using a mammalian two-hybrid system, it was
able to independently confirm this interaction[8].
The mammalian two-hybrid system can be used as a complementary approach to
verify protein-protein interactions detected by a yeast two-hybrid system screening.
In addition, the mammalian two-hybrid system has two main advantages; assay
results can be obtained within 48 hours of transfection, and protein interactions in
mammalian cells may better mimic actual in vivo interactions[8, 9].
Far Western Analysis

Far-Western blotting was originally developed to screen protein expression libraries
with 32P-labeled glutathione S-transferase (GST)-fusion protein[10, 11]. Far-Western
blotting is now used to identify protein-protein interactions. In recent years, far-
Western blotting has been used to determine receptor-ligand interactions and to
screen libraries for interacting proteins[10]. With this method of analysis it is possible
to study the effect of post-translational modifications on protein-protein interactions,
examine interaction sequences using synthetic peptides as probes, and identify
protein-protein interactions without using antigen-specific antibodies[10, 12].
The far-Western blotting technique is quite similar to Western blotting[10]. In a
Western blot, an antibody is used to detect the corresponding antigen on a
membrane. In a classical far-Western analysis, a labelled or antibody-detectable
“bait” protein is used to probe and detect the target “prey” protein on the
membrane[10, 12]. The sample (usually a lysate) containing the unknown prey
protein is separated by SDS-PAGE or native PAGE and then transferred to the
surface of the membrane, the prey protein becomes accessible to probing[10]. After
transfer, the membrane is blocked and then probed with a known bait protein, which
usually is applied in pure form. Following binding of the bait protein with the prey
protein, a detection system specific for the bait protein is used to identify the
corresponding band[10-12].
Depending on the presence of a label or tag on the bait protein, one of four detection
methods is used to detect far-Western blot protein-protein interactions[10]:
• Direct detection of prey protein with a radioactive bait protein
• Indirect detection with antibody to the bait protein
• Indirect detection with antibody to the tag of a fusion-tagged bait protein
• Detection with biotinylated bait protein and enzyme (HRP/AP) labeled with
avidin or streptavidin
The yeast two-hybrid system became one of the most popular technologies for the
detection of protein-protein interactions because it is fairly simple, rapid and
inexpensive (avoids the costly protein purification and antibody development needed
in the traditional biochemical methods). No previous knowledge about the interacting
proteins is necessary for a screen to be performed[5].
Limitations
Some classes of proteins are not suitable to analysis by the yeast two-hybrid system.
For example, transcriptional activators may activate transcription without any
interaction. Another class of troublesome proteins are those containing hydrophobic
transmembrane domains which may prevent the proteins from reaching the
nucleus[13]. To overcome this limitation one of the alternative membrane-associated
two-hybrid systems may be used. Other proteins may require modification by
cytoplasmic or membrane associated enzymes in order to interact with binding
partners[5, 13].
False Positives and False Negatives

The two-hybrid system has a tendency to produce false positive, that is reporter gene
activity where no protein-protein interaction is involved. Frequently, such false
positives are caused by bait proteins that act as transcriptional activators[13]. Other
false positives may be caused by proteins that lead to non-specific interactions for
largely unknown reasons. Some bait or prey proteins may affect general colony
viability and hence allow a cell to grow under selective conditions and activate
reporter gene activation. Mutations or other random events of unknown nature may
be invoked as potential explanations as well. Overall, extremely few cases of false
positives can be explained mechanistically[5, 13].
Comparison to Advantages Disadvantages
other methods
Two-Hybrid Simple and inexpensive Significant risk of false positives
Coverage of low abundant
proteins
Mass Spectrometry Identification of protein Expensive and time consuming
complexes Purification required
In vitro binding Defined conditions Potentially non-physiological
conditions
Protein Chips Defined conditions Requires purification of proteins
Potentially highly parallel
Table 1. Brief comparison of Two Hybrid System with other technology to analyse
protein-protein interaction
(Source: http://itgmv1.fzk.de/www/itg/uetz/publications/Vollert2003-2H.pdf)
Drug discovery is a lengthy and costly process which involves target identification
and validation, drug screening and safety assays, development of animal models and
ultimately, testing of potential drug candidates in clinical trials[14]. The development
of a novel drug may take 10-15 years, with cost estimates of ~800 million US$. It is a
complex process that includes the identification of biological targets as well as the
identification leads that aim at altering or inhibiting the function of a particular
target[14].
The yeast Saccharomyces cerevisiae has long been recognised as a valuable model
organism for studies of eukaryotic cells. Auerbach et al highlighted emerging yeast
based functional genomic and proteonomic technologies in their paper, using yeast
as a model organism in drug discovery processes. These approaches include the
utilisation of variations of the yeast two hybrid systems. With regard to screening for
novel drugs, the yeast two hybrid system can be broadly applied to two areas:
identification of target and its validation and screening for compounds that inhibit the
interaction between two therapeutic target proteins[14, 15].
As a genetic assay, the yeast two-hybrid system is perfectly suitable for high-
throughput studies. This has been used successfully to create so-called “whole
genome interaction networks” where all proteins of a given organism are
systematically tested against each other using high-throughput yeast two-hybrid
strategies[14, 16].
As many proteins that play important roles in human disease have orthologues in
lower eukaryotes such as yeast, D. melanogaster or C. elegans, a potential route
towards identification of novel targets is to create interactions networks in these
model organisms and then try to transfer the insights gained to the human situation.
Comprehensive protein interaction maps have been created for yeast and recently
also for D. melanogaster and C.
Elegans. As an example, the D. melanogaster interaction map identified a total of 4,
780 high-confidence interactions, involving 4,679 proteins[14, 17]. The authors then
used the Homophila database which lists all proteins in D. melanogaster that have
orthologues implicated in disease pathways in human in order to integrate their
protein interaction data with already known disease pathways in human[15, 17]. As
an example of this approach, they demonstrate that a previously known transcription
factor involved in human B cell non-Hodgkin’s lymphoma is connected to two
calcium-dependent phosphatases, a finding that suggests calcineurin phosphatases
may be valid drug targets for treating lymphomas and other cancer types[14].
The two-hybrid system has also been adapted further to study drug-protein
interactions[18]. This technique, termed the yeast "three-hybrid system", uses a
synthetic heterodimer consisting of two different organic ligands to bring into
proximity the DNA-binding domain fused to the receptor of one ligand and the
activation domain fused to the receptor for the second ligand[5]. The feasibility of this
system was demonstrated by using as the hybrid ligand a heretodimer of covalently
linked dexamethasone and FK506. The receptor for dexamethasone was fused to
the LexA DNA binding domain and a Jurkat cDNA library fused to a transcriptional
activation domain was screened; three overlapping clones of FKBP12, the human
FK506 binding protein, were isolated[5, 18].
Reverse two-hybrid systems that can be used to select small molecules that inhibit
protein-protein interactions also have been demonstrated[18]. It is described that
expression of proteins that interact through the two-hybrid system is controlled by the
GAL promoter. Following galactose induction, the two interacting proteins are
synthesized and their association induces the synthesis of a toxic gene. Only cells
where a small molecule inhibits the protein-protein interaction survive. Using this
system, nanomolar concentrations of FK506 were shown to disrupt the association of
FKBP12 with R1 of the transforming growth factor β receptor family[18].
Structure Factory in Germany

http://www.proteinstrukturfabrik.de/
Harvard Institute for Proteomics

www.hip.harvard.edu/
Several collaborative Structural Genomics Centers funded by the NIH

(www.nigms.nih.gov/funding/psi.html)
http://www.promega.com/
http://www.stratagene.com/
http://www.clontech.com/
6.2.7 References
1. Fields, S. & Song, O. K. (1989) A Novel Genetic System to Detect Protein-Protein

Interactions, Nature. 340, 245-256.
2. Sobhanifar, S. (2003) The Yeast Two-Hybrid Assay: An Exercise In Experimental

Eloquence in Science Creative Quaterly
source: http://www.scq.ubc.ca/?p=246
3. Phizicky, E. M. & Fields, S. (1995) Protein-Protein Interactions: Methods for

Detection and Analysis, Microbiological Reviews. 59, 94-123.
4. Criekinge, W. V. & Beyaert, R. (1999) Yeast Two-Hybrid: State of the Art,

Biological Procedures Online. 2, 1.
5. Vollert, C. & Uetz, P. (2003) The Two Hybrid System in Encyclopedic Reference
of Genomics and Proteonomics in Molecular Medicine
Source: http://itgmv1.fzk.de/www/itg/uetz/publications/Vollert2003-2H.pdf
6. Clarke, P., Cuív, P. Ó. & O'Connell, M. (2005) Novel mobilizable prokaryotic two-
hybrid system vectors for high-throughput protein interaction mapping in Escherichia
coli by bacterial conjugation, Nucleic Acid Research. 33, e18.
7. Serebriiskii, I. G., Fang, R., Latypova, E., Hopkins, R., Vinson, C., Young, J. K. &
Golemis, E. A. (2005) A Combined Yeast/Bacteria Two Hybrid System, Molecular
and Cellular Proteonomics. 4.6, 819-826.
8. Luo, Y., Batalao, A., Zhou, H. & Zhu, L. (1997) Mammalian two-hybrid system: a
complementary approach to the yeast two-hybrid system, Biotechniques. 22, 350-
352.
9. Schenborn, E., deBerg, L. & Brondyk, W. (1998) The CheckMateTM Mammalian
Two-Hybrid System, Promega Notes. 66, 2-8.
10. Hall, R. A. (2004) Studying protein-protein interactions via blot overlay or Far
Western blot, Molecular Biology Methods. 261, 167-174.
11. Einarson, M. B. & Orlinick, J. R. (2002) Identification of Protein-Protein

Interactions with Glutathione-S-Transferase Fusion Proteins in Protein-Protein
Interactions pp. 37-57, Cold Spring Harbor Laboratory Press
12. Mahlknecht, U., Ottmann, O. & Hoelzer, D. (2001) Far-Western based protein-
protein interaction screening of high-density protein filter arrays, Journal of
Biotechnology. 88, 89-94.
13. McAlister-Henn, L., Gibson, N. & Panisko, E. (1999) Applications of the Yeast
Two-Hybrid System, Method. 19, 330-337.
14. Auerbach, D., Arnoldo, A., Bogdan, B., Fetchko, M. & Stagljar, I. (2005) Drug
Discovery Using Yeast as a Model System: A Functional Genomic and Proteonomic
View, Current Proteonomics. 2, 1-13.
15. Edwards, A. M., Arrowsmith, C. H. & Pallieres, B. d. (2000) Proteonomics: New

tools for a new era in Modern Drug Discovery pp. 34
16. Gwynne, P. & Heebner, G. (2003) Drug Discovery and Biotechnology Trends –
Proteomics I: In Pursuit of Proteins in Science
17. Stanyon, C. A., Liu, G., Mangiola, B. A., Patel, N., Giot, L., Kuang, B., Zhang, H.,
Zhong, J. & Jr, R. L. F. (2004) A Drosophila protein-interaction map centered on cell-
cycle regulators Genome Biology. 5, R96.
source: http://genomebiology.com/2004/5/12/R96
18. Parsons, A. B., Geyer, R., Hughes, T. R. & Boone, C. (2003) Yeast genomics
and proteonomics in drug discovery and target validation, Progress in Cell Cycle
Research. 5, 159-166.
Chapter 6.3 Biosensor methods for study of protein-protein
interactions
Yung Chih Chen
6.3.1 Introduction
A biosensor is a device that uses biological molecules to detect chemicals or other

biological molecules. Biosensor usually consists of three components, a biological
detection system, a transducer and an output system. Typically, biological detection
system can be enzyme, antibody, micro-organism and cell, which are immobilized on
the surface of a signal transducer. Two types of transducers, optical and
piezoelectric, are commonly implementation in biosensor. Surface Plasmon
Resonance is using optical transducer. Quartz Crystal Microbalance (QCM) is using
piezoelectric transducer. The most important system in biosensor methods for study
of protein-protein interaction is called Surface Plasmon Resonance (SPR). SPR has
been demonstrated in the past decade to be an outstanding sensitivity and extreme
powerful probe of the interaction of a variety of biopolymers with various ligands. A
number of applications such as protein-protein, protein-DNA, protein-ligand, and
protein-membrane have been developed. SPR provides a means not only for real-
time identifying these interactions but also for building a variety of assays.
In a typical SPR biosensing experiment, one of the interacting pair is immobilized on

an SPR-active gold –coated glass slide. The other interactant is prepaid in an
aqueous buffer solution. A glass slide with a thin gold coating is mounted on a prism.
When light passes through the prism and slide, reflects off the gold and passes back
through the prism to a detector. The reflectivity is subject to the index of refraction,
which is determined by the total mass of the thin gold. In other words, changes in
reflectivity versus angle give a resonance signal that is proportional to the volume of
biopolymer bound near this surface (Fig 6.3.1.1). A typically readout is indicated as
Fig 6.3.1.2. At time 0, there is no interaction. At time 100s, solution contain the other
interactant is introduced, therefore the association effect begin. At time 300s, the
maximum association happen and pure buffer start to flush into flow channel. At time
420-520 s, the starting surface is regenerated with a sequence of reagents. Related
techniques include plasmon waveguide, QCM, Dual Polarisation Interferometry, and
Surface Enhanced Laser Desorption Ionization (SELDI). This chapter will discuss the
pros and cons of SPR and introduce a little bit about SELDI. Then present the
current application of SPR.
Fig 6.3.1.1 Basic components of an instrument for SPR biosensor
Source: www.fz-juelich.de/ibi/ibi-1/protein-protein_interaction
Fig 6.3.1.2 A typical Surface Plasmon Resonance Readout

Source: www.cpac.washington.edu/~campbell/projects/plasmon.pdf
6.3.2 Recent Advanced

6.3.2.1 From MS to MALDI and then SELDI
A Mass Spectrometer is a device that measures the mass to charge ratio and
typically consist of three parts; an ion source, a mass analyser and a detector
system. Aebersold (2003) have identified the ionization process in MS may
deteriorate the characteristics of protein and peptides[1]. The classic 2D gel
electrophoresis combined with mass spectrometry cannot accurately measure small
difference in concentration[2]. Instead of technical advances on 2D PAGE, it still
regards as time consuming and not suitable to examine large number of samples.
Matriz-assisted Laser/Desorption ionisation time of flight mass spectrometry (MALDI-
TOP MS) offer a more sensible of identifying small volumes of protein and this
technique rapidly take over the previous MS following its discovery. MALDI can
quickly recognize masses of peptides generated from a pure fragmented protein and
it provide highly reliable datas. SELDI offer a similar measurement of analysing
biological mixtures. The main differences between MALDI and SELDI is that in
MALDI the surface or beads are passive probes and do not contribute to the reaction
whereas in SELDI, protein are immobilized on one of a variety of chip surfaces, all
with different binding specificities (Refer to chapter 4.1 and chapter 5.3 for more
details).
6.3.2.2 Quality Control and Quality Assessment for SELDI
SELDI coupled with protein chip is an effective tool for simultaneous detection of a
variety of relevant protein expression under different condition. Differences in protein
expression level can then be used to identify disease, differentiate different stage of a
disease, or different time point following by toxicant treatment. Therefore, analyses of
SELDI data play a pivotal role in developing SELDI technology. Many researches
have been conduct under rigorous procedures to detect and discard low quality
spectra prior data analysis in SELDI. Hong et al (2005) presented a novel 144
spectrum as a correlation matrix to measure and detect low quality data[3] (Refer to
chapter 9.2 for more details).
6.3.2.3 SPR&MS
Larsericsdotter et al (2006) have optimized SPR and MS interface by hybrid their

functionalities. They called this system ligand fishing process to characterize
biomolecular interaction and identify the interaction partner inside cell proteomics. A
general variation in chip based affinity separation system may cause by surface to
volume ratio of the fluidic system. They have investigated low molecular weight
molecules with optimized protocol to avoid non-specific signal in the final MS
analysis[4].
6.3.2.4 Develop high throughput SPR Biosensing by using SPR Microscope
Campell et al (2004) develop novel SPR biosensing array measurement that can
measure 120 interactions simultaneously and a computer interfaced video camera to
probe the interactions (Figure6.3.2.2) By implanting array method in SPR, it offer at
least two advantages: measurement smaller protein and calculating Kinetic and
concentration in parallel[5].
Fig 6.3.2.4 Design of SPR microscope

Source: www.cpac.washington.edu/~campbell/projects/plasmon.pdf
6.3.2.5 SPR&AFM
Another advanced of SPR biosensor is that the method may be combined with AFM
to obtain additional information such as identification of surface topology of bound
proteins and thickness of protein layers immobilized on the surfaces of protein
arrays.

6.3.3.1 Advantage of SPR
6.3.3.1.1 Evaluation of macromolecules
Recombinant protein is a paramount technique for most laboratories studying

biological problems. It is pivotal to be able to show the recombinant protein has the
same characteristics at its native counterpart. With regards to this point, scholars
have been proved the availability and reliability of using SPR by confirming that the
protein binds its natural ligands. Due to post translation modification has been raised
the importance in proteome study, the binding interaction require a correctly folded
protein instead of only amino acid sequence match. The SPR is particularly well
suited to demonstrating the binding of macromolecules. Setting up an assay for any
interaction is very fast and the date generated is reliable.
6.3.3.1.2 Kinetic & Equilibrium measurements
The fact that SPR generates real-time binding data make it well suited to the analysis
of equilibrium measurement and kinetic measurement. With respect to equilibrium
analysis, it is very time-consuming and required several injection of different
concentration. In general, the time of equilibrium is determined primarily by the
dissociation rate constant K-off. Slow K-off values are usually resulting from high
affinity interaction (KD<10 nM). In contrast, weak (KD>100 nM) interaction has fast
K-off value and tends to be easily studied. SPR could generate highly reproducible
data. With respect to kinetic measurement, SPR is easy to use and the analysis
software is available, it is quite a common practice of generating kinetic data.
6.3.3.1.3 Lack of labelling requirement
Due to labelling protein or other detection probes may result in loss of biological
activity, non-labelling methods are preference. The current non-labelling methods
include SELDI, AFM and SPR[6].
6.3.3.1.4 Operated in situ
In addition to the use of unlabeled protein, SPR offer the advantage that it operated
in situ. In other words, there is no requirement of substrate to rinsed or dried the
sample before analyse. This feature is extremely useful for quantifying low affinity
protein-protein interaction[7].
6.3.3.2 Disadvantage of SPR

6.3.3.2.1 High Throughput Assays
SPR is not suitable for high throughput assays; due to average analysis take
approximately 10 mins. Common conundrums include surface deteriorate over time
and air bubble may blockage micro fluidic system. Many research have been conduct
to change this disadvantage.
6.3.3.2.2 Concentration Assays
SPR are limited to observing several binding interaction simultaneously, therefore it

is not suitable for concentration measurements.
6.3.3.2.3 Protein denaturation
During the immobilizing procedure, protein may undergo denaturation and show non-
specific binding in the analysis of protein-protein interaction.
6.3.4 Application of SPR
There are currently two types of SPR sensors, angular and spectral SPR biosensors,
widely be used in proteome researches. Angular SPR biosensors are based on the
angular interrogation and analyse protein interaction by scanning incidence angles at
a constant wavelength. On the other hand, spectral SPR biosensors are based on
the wavelength interrogation and scan wavelengths at a constant incidence angle to
analyse biomolecular interactions.
6.3.4.1 SPR imaging
SPR imaging measured the intensity change of refractive index on the gold surface.
Jung’s group (2004) have applied this technology into monitoring protein expression
by E. Coli. In short, they immobilized three proteins; maltose-binding protein tagged
human interleukin-6, hexahistidne-tagger human growth factors and glutathione S-
transferase-tagged human interleukin-6, onto gold-coated slides pre-treated with
cyclodextran, Ni-iminodiacetate and glutathione, respectively. They found the
conundrum of SPR imaging was the SPR spectrum may not horizontally move, which
results in the fluctuation of SPR imaging[8].
6.3.4.2 SPR spectroscopic imaging
The main difference between SPR spectroscopic imaging and SPR imaging is that
the former detected the shift of SPR wavelength, but the later detected the SPR
intensity. The SPR spectroscopic imaging was combined position automation and
SPR spectroscope into one technology. There are two modes of SPR spectroscopic
imaging: line-scanning modes and whole scanning modes. A typical experiment of
antibody-antigen interaction of SPR spectroscopic imaging is that tthree G-proteins
such as rhoA, rhoA N19, rac1, and C-reactive protein were immobilized onto a protein
array modified with a mixed thiol layer of 11-mercaptoundecanoic acid and
mercaptohexanol. Then, incubated the array with two monoclonal antibodies against
rhoA and rac1. Afterwards, the array were analysed by SPR spectroscopic imaging
by whole scanning mode (Fig. 6.3.4.2)[9].
Fig.6.3.4.2 SPR spectroscopic imaging by whole scanning mode

(Source: www.e-emm.org/article/article_files/EMM037-01-01.pdf)
The whole scanning method can provide detailed information and cross reaction of
antigen and antibody. In the figure 6.3.4.2, it shows anti-rac1 have a strong binding to
rac1. However, anti-rhoA has cross reactivity with rhoA N19. The drawbacks of whole
scanning methods is that it takes a long time to finish scanning due to the whole
protein array must be scanned by graduate moving to all spots. Yuk et al (2005) have
demonstrated the analysis of antibody-antigen interaction, by line scanning mode.
They scanned protein arrays along with the central lines and present the data by
color spectrum. In their experiment, 7 different proteins, such as tissue
transglutaminase (TGase), hemoglobin, haptaglobin, rhoA, rac1, GST and rhoA N19,
were immobilized and then their interaction with three antibodies, anti-rhoA, anti-
rac1, and anti-haptoglobin were analysed by the line-scanning mode of the SPR
biosensor. In summary, the sensitivity of an in situ spectral SPR biosensor was not
as good as an ex situ spectral SPR biosensor[9].
6.3.4.3 Investigate mutate protein by SPR
SPR is a wonderful tool to investigate the effects of amino acid substitution on

binding properties and interacting characteristics. Bozzi et al (2005) analyse
dystroglycan by SPR. Dystroglycan is comprised by two subunits α-dystroglycan and
β-dystroglycan, plays a pivotal role in muscle stability and neuromuscular disorders.
α-dystroglycan tend to interact with and β-dystroglycan by non covalent bond. Many
researches have proved that α-dystroglycan interact with extracellular matrix protein.
β-dystroglycan plays as a transmembrane protein. It binds dystrophin and other
cytosolic protein such as Grb2. It is believed that α and β subunits intertwine their
functionality affecting the stability of the entire network. By immobilizing different
direction of α and β-dstroglycan, Bozzi and his group have identify that the specific
aromatic residues between C-terminal region of the α-dystroglycan and the N-
terminal ectodomain of the β-dystroglycan play a crucial role in inter-subunit
interaction[10].
6.3.4.4 Kinetic analysis between β-dystroglycan and Grb2
Grb-2 is an adaptor molecule regulates signal transduction and cytoskeleton

organization. Grb2 is consist of one SH2 (Sre homology 2) domain and flanked by
two SH3 domain. Grb2 binds to the cytoplasmic domain of β-dystroglycan, which
contains several proline-rich sequenced. Therefore,Torreri et al (2005) performed
SPR analysis: “1) to determine which of the two Grb2-SH3 domains, the N-terminal
or the C-terminal, binds to β-dystroglycan and 2) to identify the binding site on β-
dystroglycan by comparing the kinetics parameters of β-dystroglycan fragments
containing one (amino acids 876-895) or two (amino acids 821-895) proline rich
consensus sequences for the Grb2-SH3 interaction[11]”. In conclusion, they have
found that both Grb2-SH3 domains bind β-dystroglycan, but C-terminal SH3 domain
has lower affinity than N-terminal Sh3 domain. They also identified amino acid (876-
895) of β-dystroglycan plays a crucial role in the determination of binding affinity.
6.3.5 Relevant websites
Corn’s research group - University of California, Irvine

http://corninfo.ps.uci.edu/
Campbell research group- University of Washington, Department of

Chemistry
http://www.cpac.washington.edu/~campbell/
Dr.Zhou’s research
http://www.calstatela.edu/dept/chem/Zhou/research.html
Wikipedia
http://en.wikipedia.org/wiki/Surface_plasmon_resonance
Introduction of SPR
http://www.uksaf.org/tech/spr.html
More detail about SPR

http://home.hccnet.nl/ja.marquart/BasicSPR/BasicSpr01.htm
Company System Website

Biacore AB (Sweden) Biacore www.biacore.com
Affinity Sensors IAsys www.affinity-sensors.com
IBIS Technologies IBIS www.ibis-spr.nl
Jandratek GmbH Plasmonic www.plasmonic.de
GWC instrument FT-SPR 100 www.gwcinstruments.com
Texas Instrument TI-SPR www.ti.com/spreeta
BioTuL Bio Instruments Kinetics Instrument www.biotul.com
(Germany)
Nippon Laser Electronics SPR-670 www.rikei.com
(Japan)
Artificial Sensing OWLS www.microvacuum.com
Instruments (Switzerland)
Aviv (NJ) PWR-400 www.avivinst.com
Quantech Ltd (MN) FasTraQ www.quantechltd.com
6.3.7 References:
1. Aebersold, R. & Mann, M. (2003) Mass Spectrometry-based proteomics, Nature.

422, 198-207.
2. Rodland, K. D. (2004) Proteomics and cancer diagnosis: the protential of mass
spectrometry, Clin Biochem. 37, 579-583.
3. Henderson, N. A. & Steele, R. J. C. (2005) SELDI-TOF proteomic analysis and
cancer detection, Journal of the Royal Colleges of Surgeons of Edinburgh &Ireland.
3, 383-390.
4. Larsericsdotter, H., Jansson, O., Zhukov, A., Areskoug, D., Oscarsson, S. & Buijs,
J. (2006) Optimizing the surface plasmon resonance/mass spectrometry interface for
functional proteomics applications: How to avoid and utilize nonspecific adsorption,
Proteomics. 6, 2355-2364.
5. Shumaker-Parry, J. & Campbell, C. T. Quantitative Methods for Spatially-
Resolved Adsorption/Desorption Measurement in Real Time by SPR Microscope,
Anal. Chem. subm.
6. Zhu, H. & Snyder, M. (2003) Protein Chip Technology, Curr Opin Chem Biol. 7,
55-63.
7. Young-Sam, L. & Milan, M. (2002) Protein chips:from concept to practice, Trends
in Biotechnology. 20.
8. Jung, J. M., Shin, Y. B., Kim, M. G., Ro, H. S., Jung, H. T. & Chung, B. H. (2004)
A fusion protein expression analysis using surface plasmon resonance imaging, Anal
Biochem. 330, 251-6.
9. Yuk, J. S. & Ha, K. S. (2005) Proteomic applications of surface plasmon
resonance biosensors: analysis of protein arrays, Exp Mol Med. 37, 1-10.
10. Bozzi, M., Sciandra, F., Pavoni, E., Ferri, L., Torreri, P. & Petrucci, T. (2005).
Analysis at the molecular level of the interaction between alpha-dystroglycan and
beta-dystroglycan. Paper presented at the GGPO30332, Telethon meeting.
11. Torreri, P., Ceccarini, M., Macioce, P. & Petrucci, T. C. (2005) Biomolecular
interactions by Surface Plasmon Resonance technology, Ann Ist Super Sanita. 41,
437-41.
Chapter 7.1 t-Boc synthesis and cleavage of peptides
Rajesh Ramanathan
7.1.1 Introduction
Proteins play a crucial role in many of the physical and chemical processes in the
living cell [1]. These ubiquitous molecules have a highly organized three dimensional
structure in solutions. Peptides are smaller version of proteins which generally are
less organized in solution state [2]. There is no clear dividing line between proteins
and peptides but acceptable distinctions being proteins are larger peptides. With the
completion f the human genome project the study of proteins have increased
considerably, also the use of proteins as target molecules for synthesis of drugs has
increased the need for artificial synthesis of peptides [3].
The possibility of synthesizing organic compounds was considered seriously only

after Friedrich Wohler synthesized urea, also called carbamide in the lab in 1828.
Prior to this, the theory of ‘vitalism’ held sway. According to the tenets of vitalism, the
compounds required for life were far more complicated than the compounds which
constituted inanimate matter and had the ability to self-propagate due to their
possession of a ‘vital force’ or spark. Thus these ‘organic’ compounds were above
the laws of physics and chemistry and were not worth studying as they were by
definition, inexplicable. Certainly, (it was believed) they could not be synthesized.
Wohler’s synthesis of urea, an organic compound, invalidated vitalism and inspired
chemists to try and synthesize other organic compounds. Proteins came to the
limelight when James Sumner demonstrated that urease was a protein. And finally,
between the years 1899 and 1908, Emil Fischer, while working with proteins,
discovered the nature of the amide or peptide bond between amino acids. With this
important discovery, Fischer in collaboration with Fourneau was able to synthesize
the first peptides, starting with dipeptides [glycyl-glycine] in 1901 and working his way
up to an 18 amino acid peptide in 1907, consisting mostly of glycine and leucine
residues. This monumental achievement laid the foundations for the science of
peptide synthesis. A series of important discoveries following this which led to
peptide synthesis as we know it today, are given below [4]-
1] Bergmann and Zervas, 1932- The discovery of the carbobenzoxy group which
could be used as an easily removable functional group for polyfunctional amino
acids.
2] Theodor Curtius- The discovery of the Curtius rearrangement which could be

carried out with benzyl alcohol to give carbobenzoxy-protected amine groups.
3] Sifferd and du Vigneaud, 1935- Successfully synthesized carnosine, one of the

first times a small naturally occurring peptide was synthesized.
4] Harrington and Mead, 1935- Successfully synthesized glutathione.

5] du Vigneaud et al., 1953- Successfully synthesized the active peptide hormone,
oxytocin.
6] Schwyzer and Sieber, 1963- Successful synthesis of porcine adrenocorticotrophic

hormone.
7] Bodzansky et al, 1967- Synthesis of the peptide, secretin by solution phase

synthesis.
8] Bruce Merrifield, 1963- Invention of the solid phase synthesis method.
As we shall see further on, this last discovery was perhaps the most important one; it
allowed for the increased use of synthetic peptides in experiments and has mostly
replaced solution phase peptide synthesis. Bacteria and viruses were and are still
used for the production of proteins by means of expression systems. But with the
advent of solid phase and solution phase peptide synthesis it was possible to mass
produce peptide sequences.
7.1.2 Principle
The main principle behind solid phase peptide synthesis can be summed up as
follows ‘if a peptide is bound to solid insoluble matrix then the reagents that are
unreacted in the production phase of any synthetic step can be washed, decreasing
the time required for chemical synthesis, decreasing the chances of side reactions.’
The T-Boc synthesis by Merrifield uses an acid labile temporary protecting group
while the F-Moc synthesis developed by Sheppard uses a basic labile temporary
protecting side group.
Both methods of solid phase peptide synthesis are carried out from C terminal to N
terminal, with the N terminal end being protected by a removable/temporary group to
which the next amino acid in line is attached. The process is reversed in nature,
where the cell’s ribosomes synthesize proteins from the N terminal to the C terminal.
Solution Phase Peptide Synthesis [4]- Although this method has mostly been
replaced in labs by the solid phase synthesis method, it is still used for large scale
production of synthetic peptides. It is often referred to as the classical method of
peptide synthesis. The N-α-protected amino acids are added in a stepwise manner to
a growing chain which starts from the C-terminal amino acid. Each step of the
coupling process is brought about by one of given methods as follows-
1] Carbodiimide Method [4]- It involves using dicyclohexylcarbodiimide [DCC] as the

coupling agent to couple two amino acid residues.
2] Mixed Carbonic Anhydride Method [4] - It is popular because of its effectiveness at

low temperatures as well as the high yield and purity percentage of the final product.
The first step activation carboxyl group is using an alkyl chlorocarbonate to form
carbonic anhydride. In the second step, carbonic anhydride is added in a known
excess to react with the free amino group of the growing peptide chain.
Solid Phase Peptide Synthesis [SPPS] [5]- This is the preferred method of peptide
synthesis, especially for small labs that require high quality synthetic peptides in
small amounts. In this technique, a solid matrix is placed in a reaction vessel. This
resin has a reactive group which forms the base for the growing peptide chain and
which can bind to the carboxyl end of the N-protected amino acids. A single cycle
consists of de-protecting the temporary reactive group, draining the de-protecting
agent, and adding another amino acid to the growing chain. At the end of each cycle,
the peptide is tested for purity. When the desired sequence has been achieved, the
completed peptide is removed by another cleavage agent, which targets the bond
between the C terminal amino acid and the support. The peptide is then freeze dried
and analyzed by means of high performance liquid chromatography [HPLC] and
mass spectrometry to characterize it.
Two kinds of protecting groups are present on amino acids. The ‘temporary’
protecting groups protect the amino end of the amino acids and are removed at the
beginning of each cycle. These are the N-α-tert.-butyloxycarbonyl [Boc] group and
the N-α-fluorenylmethyloxycarbonyl [Fmoc] groups. The differences in the two
methods of SPPS are based on the chemistry associated with these two groups, the
former being an acid labile group and the latter being a base labile group. Therefore,
we have the Boc synthesis technique and the Fmoc synthesis technique. The
‘permanent’ groups are present to protect the side chains from reacting with each
other and are usually similar for both methods. These are usually ether, ester or
urethane derivatives of benzyl alcohol. In Boc chemistry, the permanent groups are
often modified with the addition of electron donating methyl/methoxy groups or
electron withdrawing halogenic groups. The permanent groups are removed in the
final step of cleavage.
Boc Chemistry [6]- Boc chemistry is the classical method of solid phase peptide
synthesis, developed by Merrifield. The temporary group Boc group is introduced on
to amino acids using either di-tert-butyl dicarbonate or 2, 2-phenylacetonitrile, in
aqueous 1, 4-dioxanee containing NaOH [7]. This Boc group cannot be removed by
alkali and neucleophiles, but can be removed rapidly by inorganic and organic acids.
This is one of the main steps in the Boc chemistry. The initial Boc protected amino
acid is attached to the resin by an HF cleavable bond. Other Boc protected amino
acids are added in successive cycles. A single cycle can be outlined as follows-
Deprotection- The protecting, acid labile group is removed using neat trifluoroacetic
acid [TFA].
Activation- Meanwhile, the amino acid is activated using O-(1H-benzotriazole-1-yl)-

N,N,N',N'-tetramethyluronium hexafluorophosphate [HBTU] in DMF or a combination
of HBTU and 1-hydroxybenzotriazole [HOBT] in DMF for the amino acid residues,
arginine, glutamine and asparagine.
Addition- Dimethylformamide [DMF] is used to neutralize and wash away the residual
TFA and the activated amino acid is added to the reaction vessel. Confirmation of the
binding is obtained by carrying out a ninhydrin test on a small sample of the resin and
measuring the absorbance of the ninhydrin solution against the amount of resin taken
in the sample. In case, the percentage of binding is relatively low, the entire cycle is
repeated again, with the same amino acid. This process is referred to as double
coupling. The result after double coupling is usually accepted as final, no matter what
it is. The entire cycle is then repeated again with the next amino acid until the desired
sequence has been achieved.
Removal- After the desired peptide sequence is complete; the peptide is removed
from the resin using anhydrous hydrogen fluoride [HF].
Thiol compounds are often added to protect the peptide from the carbocations that
are generated in the process.
Figure 7.1.4 - Summary of the Boc chemistry.
Reference: http://en.wikipedia.org/wiki/Peptide_synthesis
The use of Fluorous tags in conjunction with solid phase peptide synthesis has been
recently discovered. Taking into consideration the unique property of Fluorous
compounds, they were used to tag the growing peptide and were de-tagged at the
end.
The unique property of fluorous compounds was used to tag in conjunction with
SPPS as described by [8]
Fig 7.1.2 – Fluorous Peptide Synthesis
Reference: www.fluorous.com/peptides.html
The level of sophistication is set to increase with the improvement in linker design,
rapid on and off bead technology as well as automated synthesis equipment.
Advanced Automation Peptide Protein Technologies Apex 396 for multiple peptides,
Model 90 for small and mid scale production and Model 400 for large scale
production are the examples of automated systems. Matrix 384 automated
synthesizers can produce up to 384 peptides simultaneously. Although there is
extensive use of high throughout techniques like automated synthesizers, most
organizations prefer to have knowledge of manual peptide synthesis.
The transition from t-Boc to F-Moc was one the most notable advancement in Solid
Phase Peptide Synthesis. The major change in F-Moc was the use of mild basic
condition (piperidine) for the cleavage of peptide in the place of HF cleavage in the
classical method.
7.1.4 Evaluation of technology
Boc chemistry uses an acid labile temporary protecting group while Fmoc chemistry
uses a mild base labile group as the temporary protecting group. Although the
chemistry and the reagents required are different, the cycle is essentially similar to
Boc chemistry. Deprotection of the amino acids is achieved by 20% piperidine in
DMF. Deprotection is much slower than deprotection by TFA in Boc chemistry
because the reaction kinetics is influenced by the formation of an anionic nitrogen
group.
The main advantage of Fmoc chemistry is that it does not involve the use of HF to
remove the permanent protecting groups or for cleavage from the solid support. TFA
is used instead. The use of HF is dangerous, harsh on the peptide and is difficult to
handle.
Also when peptides are evaluated by HPLC, Mass Spectroscopy, Sequence
Analysis, it has been found that synthesis by Fmoc chemistry is more reliable and
accurate. This was true as Fmoc uses less harsh conditions for cleavage of peptide
from resin and removal of permanent protecting groups. G.B. Fields et al. compared
the two techniques and indicated that Fmoc chemistry is more reliable and tends to
give desired peptides in better purities [9].
7.1.5 Application of technology
Advances in the development of methods for the design and synthesis of peptides,
pseudo-peptides and related compounds helps in the understanding of protein
structure, domain structure and fold, topology, and dynamics. Peptide therapy has
enormous potential in diverse areas as growth control, blood pressure,
neurotransmission, hormone action, pain, digestion, reproduction.
Some of the medical and biological applications for synthetic peptides are
Structure and function studies – Design of synthetic peptides in better

understanding ligand-receptor/acceptor interactions through structure and function
studies. It is essential to know the complementary binding ligand for binding,
transduction and release of a receptor/ acceptor [4]. We lack methods to design
ligands with specific properties. The use synthetic peptides with specific activities are
used for the study of structure-function relationships.
Enzyme inhibitors - Inhibition of enzymatic systems is on eof the prime targets for
regulation and control of diseases. Current targets include cancer cells, bacteria,
humans and many other systems example 5-flurouracil is used as chemotherapeutic
agent, penicillin are considered as first line antimicrobials, while lovastatin is used as
a cholesterol reducing agent. Recently peptides have been used as therapeutic
agents for targeting specific enzymes or systems. For example hirudin peptides have
helped to understand the mechanism of thrombin-hirudin coagulation pathway.
Therapeutic agents – development of peptide therapeutic agents has become very

popular area of research [10]. For example; cardiovascular agents like angiotension
converting enzyme inhibitors, immunological agents like cyclosporine have made
transplants possible.
Receptor Studies – the use of peptides in the study of receptor types and subtypes
and providing analogues for in vivo studies [4].
Antigenic and Immunogenic uses – synthetic peptides are used in the study of
immune responses due to its ability to mimic similar antigenic sequences in proteins.
The ability to design novel analogues which mimic certain properties of the original
compound present in the body has been used to study peptide hormones,
neurotransmitters, enzyme inhibitors and so on [11].
Peptide synthesizer by http://www.huck.psu.edu/stf/mcf/home.html

Anaspec: http://www.anaspec.com/
Wikipedia: http://en.wikipedia.org/wiki/Peptide_synthesis
Large scale production: www.pharmaceutical-
technology.com/contractors/contract/polypeptide/
7.1.7 Key suppliers
Auspep – The name on the world’s finest peptides

http://www.auspep.com.au/products_services/products_services.htm
GenScript Corporation
http://www.genscript.com/peptide.html
Sigma Aldrich
http://www.sigmaaldrich.com/Brands/Fluka___Riedel_Home/Organic___Synthetic/Pe
ptide_Synthesis.html
Biocompare provides a comparison between many major companies that are

involved in peptide synthesis.
http://www.biocompare.com/matrix/118/Peptide-Synthesis.html
7.1.8 References:
1. Ashely, M.J., V.J. Hruby, and J.-P. Meyer, Bioorganic Chemistry Introduction
to Peptides and Proteins, ed. S.M. Hecht. 1998: Oxford University Press.
2. Lyod-Williams, P., F. Albericio, and E. Giralt, Chemical approaches to the
Synthesis of Peptides and Proteins. 1997: University of Barcelona, Barcelona,
Spain.
3. AnaSpec. 2006 [cited 2006 24 October]; Available from:
http://www.anaspec.com/.
4. Grant, G.A., Synthetic Peptides: Beginning the Twenty-first Century. 2nd ed.
2002: Oxford University Press.
5. Ermolat’ev, D.S. and E.V. Babae, Solid-phase synthesis of N-(pyrimidin-2-
yl)amino acid amides. Chmestry Department, Moscow State University,
Moscow, Russia, 2005: p. 172-178.
6. Merrifield, B., Solid Phase Peptide Synthesis - The synthesis of Tetrapeptide.
Journal of the American Chemical Society, 1963. 85: p. 2149.
7. Bodanszky, M. and A. Bodanszky, The Practice of Peptide Synthesis. 1984,
Berlin, Springer-Verlag.
8. Zhang, W., Fluorous tagging strategy for solution-phase synthesis of small
molecules, peptides and oligosaccharides. Curr. Opin. Drug Discovery
Development, 2004. 7(6): p. pp. 784-797.
9. Fields, G.B., et al., Evaluation of Peptide Svnthesis As Practiced in 53
Different Laboratories.
10. Beeley, N. and A. Berger, A Revolution in Drug Discovery. BJM, 2000. 321: p.
pp. 581-582.
11. Hurby, V.J., F. al-Obeidi, and W.Kazmierski, Emerging approaches in the
molecular design of receptor selective peptide ligands - Conformational,
topographical and dynamic consideraions. Journal of Biochemistry, 1990.
268: p. pp. 249-262.
Chapter 7.4 Purification and characterization of synthetic
peptides
Sandip D Kamath
7.4.1 Introduction
Peptides can be defined as long chains of amino acids, where the amino acids are
linked together by peptide bonds. Peptides and proteins are complex biomolecular
structures which form the basic building blocks of life. It is the final product or the
expression of the vast genetic information. In the biological process, peptides are
produced by the addition of amino acids, one after another through the formation of
peptide bonds, in a sequence that is dictated by the RNA sequence, which in turn is
decided by the DNA material of the cell. This is called the primary structure of the
peptide thus formed. The folding of the structure then takes place to give a final
functional protein. The size, shape, structure and function of the protein are decided
by the primary amino acid sequence of that protein. In nature, the assembly of amino
acid starts from the amino acid terminal and it proceeds towards the carboxy terminal
of the last amino acid. Peptides can also be synthesized in the lab artificially, which
are called synthetic peptides.[1] Synthetic peptides can be made by a process called
“Solid Phase” chemical synthesis. In this process, the synthesis of the peptide, starts
from the carboxy terminal and makes its way to the amino terminus, in contrast to the
biosynthetic pathway. The amino acid that constitutes the carboxy terminal of the
proposed peptide is attached to a solid surface, i.e. a resin. Subsequent amino acid
molecules are linked to the previous amino acid at its carboxy terminal forming a
peptide bond. This is not a straight forward reaction; there are other reactive groups
on the amino acid that can react with the forming chain and form secondary
structures. To avoid this, blocking agents are used to protect other reactive groups
and expose only the required group. t-BOC and f-MOC are two such agents that are
widely used in preparation of synthetic peptides.[2] After the linking process of the
amino acid is complete, the blocking agent is removed and the amino group is
exposed for the next amino acid to react with it. This cycle is continued until the
required peptide is formed. Peptides containing up to 20 amino acids can be easily
formed with the above technique. It is a bit more difficult to synthesize peptides
containing up to 100 amino acids, though they have been synthesized successfully.
[2]
The making of synthetic peptides is not as easy as it seems. It is always haunted by

several problems right from the synthesis stage till the final pure peptide is obtained.
The synthesis of peptides by the above mentioned technique is not perfect. This
process results in contaminants that are nearly similar to the required peptide. Such
contaminants include amino acid deletions, truncated amino acid sequences, and
peptides with altered amino acid sequences. Small organic molecules such as
phenols and thiols are also present due to the removal of the synthesized peptide
from the solid support. Such products act as impurities in the final product, which are
non-functional. Such impurities interfere with the function of the original peptide and
gives unwanted results. Hence it is of utmost importance that the synthetic peptides
formed are purified to the desired level of purity before it is used in different
applications.
Before the actual purification of the synthetic peptide is performed, it is necessary to
know each and every aspect of the required peptide. Knowledge about the protein is
necessary, because it helps in deciding, which kind of process is to be used for the
purification, and to fine tune the settings of that process. This is where the
characterization of the peptide comes into the picture. Characterization is a process
where in details of the peptide like molecular weight, amino acid sequence,
isoelectric pH, and shape are elucidated. Various techniques are employed for the
characterization. Sequencing maybe done by Edman’s method, molecular weight
determination can be done by gel filtration or mass spectroscopy.[2]
After the characterization of the required peptide, the next step is its purification.
Different chromatographic techniques are employed in the purification step such as
preparative reversed phase high performance liquid chromatography (RP-HPLC), Ion
exchange chromatography (IEC), Size exclusion chromatography (SEC) and affinity
chromatography.[3] The first step in the purification of any peptide is to describe the
optimal conditions of the process. Specific information about the peptide and related
topics should be gathered; as in the intended use of the product, availability of the
starting material and its handling, intended use of the final product, contaminants that
have to be removed completely, the level of purity required in the final product, and
the equipment and resources available.[4] The time frame to complete the work and
the economical constraints should be defined before finalizing the purification
layout.[5] Information regarding the purity and contaminants will help in designing the
purification protocol. Purification of proteins is a complex procedure. Most of the
times it is not a one step process. Depending on the level of purity required for the
final peptide, the process of purification of peptides is designed in a multi-step
manner. The initial steps help in the removal of impurities that are easily detectable
and separable, and the final step is used for removal of contaminants that are nearly
similar to the original peptide in terms of size and composition.[4] Each added step in
the process will lead to some loss of the final product. Better purity might be achieved
by more number of steps, but it results in lower yields of the final product due to the
loss associated with each step. Hence it becomes necessary to use fewer, optimum
number of steps in purification to get a good yield of the final product with acceptable
level of purity. Different chromatographic techniques must be engaged in a logical
sequence so as to avoid the need of any additional processing steps and to keep the
steps to a minimum.
Figure 7.4.1 The Three phase purification Strategy

(www.biochem.uiowa.edu/donelson/Database%20items/protein_purification_handbo
ok.pdf)
The purification sequence can be broadly divided into 3 stages. The first stage is
called capture, wherein the main aim is to isolate, concentrate and stabilize the main
peptide. [6] At this step, one cannot expect to obtain a highly pure target protein as
the outcome. The main goal of this stage is to separate the target peptide from
contaminants that are very dissimilar to it, and from impurities that might destabilize
the target peptide [6]. RP-HPLC is a good technique to isolate the protein, but
clogging might occur due to the large impurities present in the crude sample and
hence not recommended. Techniques widely used for carrying out this stage are Ion
exchange chromatography and hydrophobic interaction chromatography [6]. The
second step is called the intermediate stage. In this stage the major bulk impurities
are removed. The resolution of the separation in this stage is required to be a little
higher because the contaminants are such that it more or less resembles the target
peptide in terms of structural properties. RP-HPLC is a suitable technique that can be
used at this stage of purification. The final stage is the Polishing stage [6]. The aim of
this step is to remove trace impurities from the sample. These impurities are very
similar to the target peptide in terms of structure, and it is difficult to separate them
using a low resolution separation technique. The aim of the polishing stage is to
obtain 100% pure target peptide. Size exclusion chromatography maybe used for this
stage, but for separating structural variants that differ very slightly from the target
peptide, RP-HPLC might be used owing to its excellent resolution power.[6]
Figure 7.4.2 Interaction of a solute with a typical reversed phase medium. Water adjacent to
hydrophobic regions is postulated to be more highly ordered than the bulk water. Part of this
‘structured’ water is displaced when the hydrophobic regions interact leading to an increase in
the
overall entropy of the system.
(teachline.ls.huji.ac.il/72682/Booklets/AmershamRPCManual.pdf)
Large scale preparative purification of the synthetic peptides requires both high
resolution separation and the ability of the technique to be scaled up. When the RP-
HPLC technique is used, the purification of the synthetic peptide is optimized using
small particle reversed phase medium and then scaled-up accordingly keeping the
selectivity same, and by increasing the particle size.

In the past several years, there have been quite a few technological advances in the
field of chromatography. Apart from the automated purification systems that are
increasingly being used, there have been innovative changes made in the basic
principles on which the purification of synthetic polymers are based on. The
purification technique is becoming more and more customized in regards to the
target peptide, thus enabling faster purification process and better purity level of the
final product.
A recent chromatographic technique that has been proposed is the novel mixed-
mode reversed phase weak-anion-exchange type stationary phase. The separation
material contains two distinct binding domains in a single chromatographic ligand.
First is the lipophilic alkyl chain for the hydrophobic interactions, similar to what we
see in reversed-phase chromatography, and second a cationic site for anionic
exchange chromatography for interaction with an oppositely charged solute.[7] The
purified final product was analyzed HPLC-UV and HPLC-ESI-MS and it was found
that all the major impurities had been removed in a single run, employing the HPLC-
WAX stationary phase. IN comparison to RP-HPLC this technique has improved
productivity, and the yield of the pure peptide per run using the HPLC/WAX method
was found to be 15 times higher as compared to the standard gradient elution RP-
purification.[7]
Polymeric particles have been long used in the purification of biomolecules. These
are generally semi-rigid porous particles with limited mechanical stability and are
used in low pressure, low flow conditions. This limits its use to the capture phase of
purification and it is unsuitable for the high pressure polishing stage of the purification
protocol. In the large-scale preparative and process scale purification of synthetic
peptides, new polymers which make the stationary phase have been developed. The
rigid copolymers of styrene and divinylbenzene have been developed for the same
purpose.[8] These polymeric particles have high mechanical stability and are able to
operate at high linear velocities. The pore size and morphology are optimized so as
to facilitate unhindered solute diffusion and to provide maximum surface area to
enhance the loading capacity. A pore size of 100 Angstorm is developed for the
purification of synthetic peptides. The stability of the polymers enables cleaning with
sodium hydroxide without any particle deterioration or any snags in the selectivity.[8]
One of the main problem encountered in the purification of synthetic peptides is the
separation of contaminants that are similar in shape and structural properties to the
target peptide. These impurities are closely eluted near the target peptide which
makes it difficult to separate. This problem can be taken care of with the use of
fluoros-based separation.[9] There are two different methods that can be followed.
Either the final target peptide is tagged with an appropriate fluoros protecting group
or the impurity that resembles the target peptides or to the intermediate unreacted
product. Then with the use of Affinity chromatography, the fluoros tagged entity can
be easily separated from the rest. It has been demonstrated that the fluoros tagged
impurities can be easily separated from the target peptide using Fluoros-HPLC.[9]
Reversed phase chromatography plays a central role among the cluster of

technologies used for the characterization and purification of peptides. It has found
both analytical and preparative applications in the area of biochemical separation and
purification. It plays a lead role in the high sensitivity protein characterization. This
role involves the micropurification of peptide and subsequent identification of the
isolated peptide fragments. The general procedure is that the peptide sample is
subjected to micro-scale reversed phase chromatography and subsequently passed
on to analysis by techniques such as Edman sequencing or Mass spectrometry.
Mass spectrometry is performed by using a flow splitter, which allows a fraction of the
product eluent to the ESI-MS (electrospray ionization mass spectrometer). Another
method is the offline analysis with the use of MALDI-TOF MS (Matrix-assisted laser-
desorption Time of flight mass spectrometer). The data collected through these steps
ultimately leads to the peptide characterization.
RP-HPLC is mainly used for the purification of synthetic peptides. The main
advantage of this technique is that it can used for the separation of proteins that are
available in microquantities and at the same time, be used for large scale purification
of peptides. Also it is generally coupled with other chromatographic techniques in a
logical sequence to get good yield of the final product and perform this task in a
suitable time frame. Biomolecules with hydrophobic character can be easily
separated using reversed phase chromatography with excellent recovery and
resolution. But there are some problems associated with the use of RPC. One of the
main problems is the buildup of contaminants in the reversed phase columns. The
contaminants have lesser retention power and are eluted in the void volume.[10]
These undesired impurities might be interpreted by the detector as chromatographic
peaks, baseline upsets or negative peaks. If the contaminants are retained on the
column and if the mobile phase is not strong enough to elute them, then they start
accumulating near the column heads, after many injections. If the contaminant build
up is too high in the RP column, then they start to act like a stationary phase, and
analytes interact with these modified phase to give an altered separation patern.
Retention times can shift and tailing occurs [10]. If there is too much contamination in
the column, the pressure on the pump increases to a high level and can cause the
column to settle and create a void. The best method to prevent this is specific
washing techniques after a number of runs on that column.[10]
Perhaps the area that has been affected by this technology the most is the
Biopharmaceutical sector. In this sector, it is of utmost importance that the peptides
used as therapeutic agents are free of any sort of impurities as it might lead to the
deterioration of the therapeutic agent. Apart from its applications in the medical field,
it has wide ranging uses in other fields as well.
Affinity chromatography has gained popularity in recent years, due to the high
specificity in the separation of the target peptide. The selection of peptide ligand is
the most important step, which can enable the separation of the target molecule in a
single step. Any alterations in it can lead to unwanted results. Therefore it is
necessary to obtain a pure sample of the peptide ligand, and it can be done by using
the chromatographic purification techniques. For example the PY574 alpha is an 18
amino acid peptide constituting part of the intracellular domain of the PDGF α-
receptor. This peptide was synthesized and used as a ligand for subsequent affinity
purification of transduction signal protein form the cell lysates. The purification was
achieved in a single step using reversed chromatographic technique. [12]
Another application, where a synthetic peptide was used as a ligand, was in the one
step affinity purification of the recombinant urokinase type plasminogen activator
receptor. In this experiment the high affinity synthetic peptide antagonist (AE152)
was synthesized and purified to be used as a ligand for performing a affinity
purification.[11]
Protein purification laboratory research- GE Healthcare

www.chromatography.amershambiosciences.com
Genetic engineering news

www.genengnews.com
Reversed Phase HPLC basics for LC/MS

www.ionsource.com/tutorial/chromatography
Handbook of Analysis and Purification of peptides and proteins by RP-HPLC

http://www.vydac.com/vydacpubs/brindex.html
Applied Biosystems.
http://products.appliedbiosystems.com
ROHM HAAS Advanced Biosciences

www.rohmhaas.com/ionexchange
Viscotek
http://viscotek.com
7.4.7 References
1. Nelson, D. L. & Cox, M. M. (2000) Lehninger Principles of Biochemistry, 3 edn,

Worth Publishers, New York.
2. Sheppard, R. C. (1975) Amino-acids, Peptides and Proteins in pp. Vol 9 29-33,
The Chemical Society, London.
3. Pasch, H. & Trathnigg, B. (1999) HPLC of Polymers, Springer Verlag, New York.
4. Reversed Phase Chromatography Principles and methods in, Amersham
Pharmacia Biotech,
5. Bailey, P. D. (1990) An Introduction to Peptide Chemistry, John Wiley and sons,
New York.
6. Protein Purification Handbook in, Amersham Pharmacia Biotech,
7. Nogueira, R., Lammerhofer, M. & Lindner, W. (2005) Alternative high-
performance liquid chromatographic peptide separation and purification concept
using a new mixed-mode reversed-phase/weak anion-exchange type stationary
phase, Journal of Chromatography A. 1089, 158-169.
8. Lloyd, L. L., Millichip, M. I. & Watkins, J. M. (2002) Reversed-phase poly(styrene-
divinylbenzene) materials optimised for large scale preparative and process
purification of synthetic peptides and recombinant proteins, Journal of
Chromatography A. 944, 169-177.
9. de Visser, P. C., van Helden, M., Filippov, D. V., van der Marel, G. A., Drijfhout, J.
W., van Boom, J. H., Noort, D. & Overkleeft, H. S. (2003) A novel, base-labile
fluorous amine protecting group: synthesis and use as a tag in the purification of
synthetic peptides, Tetrahedron Letters. 44, 9013-9016.
10. Majors, R. E. The Cleaning and Regeneration of Reversed Phase HPLC
Columns in, Agilent Technologies, Delaware USA.
11. Jacobsen, B., Gardsvoll, H., Juhl Funch, G., Ostergaard, S., Barkholt, V. &
Ploug, M. One-step affinity purification of recombinant urokinase-type plasminogen
activator receptor using a synthetic peptide developed by combinatorial chemistry,
Protein Expression and Purification. In Press, Corrected Proof.
12. Purification of a phosphorylated PDGF a-receptor derived peptide at high pH
using a polymer stationary phase. Amersham Biosciences , Application
Note 18-1132-63.
Chapter 7.5 Peptide library and its application
Patel Saravatichandra
7.5.1 Introduction:
In the field molecular biology there is great discovery of the DNA structure by Watson
and crick reveals the most of secret of the biological system up to sequencing of the
gene. The sequencing of the gene is really very helpful to make the RNA and protein
because the final product of DNA metabolism is the protein. In the transcription the
RNA formed is not same as the revers of the amino acids sequence as per the gene
sequence. In the post transcriptional modification the some nucleotide are spliced
and just useful are interconnected and give protein. In this recent time the large
amount of sequencing data is available because of human genome project, but due
to improper decoding techniques we are helpless to find perfect gene sequences. It
is very difficult to find gene without any intron sequence nucleotide give directly
proteins. To solve this we have some idea like to find the entire factor for the invitro
post transcriptional modification, but it is too hard to decode large number of
searched sequence. The use of this DNA and RNA sequence to cure disease is very
tedious method and time consuming.
The decoding of proteins from its amino acid sequence and use whole group of small
peptide is more convenient and easy. The use of different specific known protein
particle will give us sure result what we want. “Peptide library is a systematic
combination of different peptides in large number. It is a powerful tool for drug
discovery, structural studies and other applications. Solid phase peptide
synthesis, along with other methods, has been successfully used to prepare
peptide libraries."[1].The basic principle of the peptide library is expression of
libraries of peptides in mammalian cells to select for trans-dominant effects on
intracellular signalling systems. [3] Peptide library is the use of peptide to find
signalling pathways of the cell. These peptides which we will design as per
expectation interfere with intracellular signalling. This will be understood by the use of
reagent we used. These reveals of this signalling pathway are helpful to cure and find
a drug for that decease. When we carry the peptide library into the cell due their
small size we can infrequently scan that peptide which binds to the intracellular
protein or surface protein. The peptides are as affinity reagents so we can isolate
their target protein complex and distinguished their mechanism. We will create this as
therapeutics model for drug discovery. [2]
Fig-1 general mechanism of peptide library
Source: http://www.stanford.edu/group/nolan/library_systems/peptide_systems.html
7.5.2 Recent advancement in the peptide library:

In this recent time, advancements in bioinformatics lead to the great research in this
field. There is great advancement in the peptide library technology. There are so
many group, laboratories and scientist are now working in this area. Each and every
day new peptide and their associate system are searched. Some of the
advancements are stated below.
(1)Treatment of cocaine addiction:
The cocaine abuse is the very worst social problem. Government and social group
trying to reduce this abuse but it can be curable now. In recent time some of the
researchers use intranasal managed filamentous bacteriophage which will produce
cocaine sequestering antibody on their cell surface. These antibodies bind to the
cocaine molecule and stop their psychological effect in the rodent model.
Fig 2 reaction of catalytic antibody with cocaine

Source: 10.1208/aapsj070359
This antibodies block cocaine molecule and therefore cocaine disable to bind
agonistically to that nerve receptor. So there is no psychological effect on central
nervous system. To develop this system they use three approach “(1) Those
compounds that can be used in a substitution-based treatment as a cross-tolerant
stimulant; (2) medications that serve as antagonists by blocking the binding of
cocaine to its cognate receptors; and (3) compounds that function by acting at other
sites distinct from the cocaine site of action but functionally antagonize the effects of
cocaine. Several biopsychological models have been proposed and evaluated to
address addiction and relapse prevention. [5] “Unquestionably, an improved
pharmacotherapy would increase the effectiveness of such programs and alternative
strategies for treating cocaine addiction are needed if progress is to be made.”[4]
Among this approaches the second approach is good to cure the effect of cocaine on
central nervous system.
(2)Epitope mapping using m RNA display:
To map the epitope or to discover the specific part of the antigen we have to depend
on the antibody, which is protein -protein interaction process. Here antibodies are
used as molecular reagent .in the traditional method. We have to depend on the
expression of the different overlapped poly peptide .This is achieved very fine and
correct sequence by using the antibody probing reactivity. This method is very
tedious, time consuming and costly.
In this display technique each expressing vector contain multiple copy of each single
polypeptide sequence on its surface. The revivals of the active peptide are by the
affinity of that peptide by the florescence- activated cell sorting or by biopanning. In
short the peptide is fused with the mRNA at the 3' end with the help of tethered
puromycin compound. This fusion is now selected by the RT-PCR amplification for
the sequencing. [6]
Fig-2 out line of the whole procedure of epitope mapping using mRNA display
Source: http://peds.oxfordjournals.org/cgi/content/full/18/7/309
(3)Cloning at molecular level for the expression of the insect FMRF amide receptor:
FMRF amide and its compound in nervous system are found in very wide amount in
all invertebrates which has very useful function. Here we use the Chinese hamster
ovary cell to express the cloned Drosophila orphan receptor. The screening of
peptide library discloses that the receptor reacted with very high affinity FMRF amide.
This affinity is very high than the drosophila’s FMRF receptor.
The peptide library is tested on the drosophila and other insect and many peptide
hormones. “Addition of 10–5 or 10–6 M of these peptides to the pre-treated CHO cells
gave negative results for many of these peptides, but peptides resembling FMRF
amide at their C termini, and FMRF amide itself, gave clear bioluminescence
responses, Because FMRF amide was the most potent peptide in inducing the
bioluminescence response.”[7] For the testing they prepare eight of the peptide which
is associated of to FMRF amide peptide of drosophila. It is assumed that this peptide
is the found in the prepohormone. After testing all the peptide, from the result it is
found that the FMRF amide-6 ea the most potent intrinsic protein. [7]
7.5.3 Advantages of the peptide library
There are many advantages of the peptide library techniques but the main
advantages are as per the following
(1)With the use of genetic engineering we can identification of the gene coded for the
surface protein but this gene is for the all the surface protein so it is random peptide.
The main advantage of this peptide library is an affinity of the peptide towards its
target protein. [8]
(2) Display avoids the problems of cytotoxicity, soluble protein expression and
secretion bias in cell-based systems; it could be an ideal means by which to display
functional (single chain) proteins for applications such as target discovery and
functional identification. [9]
(3) Para tope-specific purification of antibodies has distinct advantages over

conventional methods of antibody purification with respect to its capacity to isolate
product of high purity and immunoreactivity. [10]
(4) This library offers the advantage of multimeric binding (several hundred copies of
the peptide are displayed on each phage), and the peptide sequences identified from
the binding phases often reveal sequence themes that make it easier to narrow in on
the consensus sequences that bind your target[11]
(5) “By delivering libraries of peptides to cells we can scan for rare peptides that bind
to and induce function of intracellular signalling proteins and surface molecules.
Using these peptides as affinity reagents we can isolate their target protein
complexes and discern mechanism. Finally, we are using them to create therapeutic
modalities from either their targets, the peptides themselves, or by some better
understanding of the biological process that is uncovered.” [2]
(6) Up to this time there is no dependable, predictive and generally applicable

method to determine the interference of signalling of the cell invivo, to discover tissue
differentiation, abnormal or disease development and organ development. This
discovery leads to the use of this isolated signalling pathway for the future. [12]
Limitation of the peptide library technique:
The main limitation of the peptide library technique is in the discovery of the epitope.
When we searching the antigen related to the diagnostic purpose that microbial
sequence database is not complete. Therefore searching of similarity will fail to
notice any antigens which are not presented in the available database. [13] “The use
of longer peptides cannot overcome a primary limitation of peptide libraries, the
inability to represent conformational epitopes comprising amino acids from distant
positions along the polypeptide sequence.”[14] In the random peptide display in
identifying actual epitope is very time required to improve specific phage clones
because three or more round for the panning require getting consensus amino acid
sequence ant find epitope. This will take 3-4 week. This technique is costly and
labour précising. “Additionally, a random peptide library, despite a theoretical size of
>1012 peptides, may not include all possible amino acid combinations and therefore
may not contain specific binding-peptide motifs.”[15]
Another limitation of the peptide library technique is the folding of engineered protein
to their natural form. [16]Owing to the poor sequence diversity some shapes may be
incomplete with the target .This incompleteness is because of electrostatic charges
repulsion .Hence these peptides are not selected to their target protein complex. [17]
“One limitation of synthetic peptide libraries is the lack of specific criteria for deciding
whether a detected binding activity is genuine or is due to non-specific
interactions.”[18] More over the problem of coupling of amino acids mixture for the
preparation of peptide library. The coupled peptide like di, tri, and tetra are not found
as they initially formed. [19]
7.5.4 Application of the peptide library technology:
(1) Combinatorial peptide library methods for immunobiology research.
This technology is used widely in the research of the immunological pathways.

Peptide library is very useful to determine the epitopes on the surface of the antigen
which will useful for the different diagnostic kits of the immunoassay. This epitopes
are useful for the vaccination. “In recent immuno biological applications, peptide
libraries have proven monumental in the definition of MHC anchor residues, in
lymphocyte epitope mapping, and in the development of peptide vaccines. Peptides
identified from such libraries, when presented in a chemical micro array format, may
prove useful in immunodiagnostics. Combinatorial peptide libraries offer a high-
throughput approach to study limitless biological targets. Peptides discovered from
such studies may be therapeutically and diagnostically useful agents.”[20]
(2) Phage display using peptide library:
The phage presentation of the peptide library is a powerful tool for the recognition of
compound and to distinguish and bind to that target. Phage system is used for the
detection of protein ligand dealings for the binding affinity improvement. The varieties
of display methodology that survive offer a variety of capability in panning for
attraction interaction. This display system offer a opportunity to address issue of
expression prejudice and discover the possibility of creating low molecular
compounds which have drug like property. Phage display using peptide library will be
used as a biomarker for the disease like cancer cardiovascular disease and
angiogenesis. “The peptides are capable of homing to specific pathways and targets
within those pathways. By generating the proteins expressed from cDNA libraries,
display methodologies can provide a direct link between phenotype and
genotype.”[21]
(3)Peptide library for the protein- protein interaction:
In the cell the signal transduction process, the interaction of protein is the major
reaction. We want to determine that how this protein interact and with signalling
complex and produce a signal. Some of the peptide library developed which is useful
for the study of protein-protein interaction. This all approach basically depends on
peptide or the DNA sequence of peptide to identify the motif for the binding. An
oriented peptide array library. (OPAL) approach that will ease high through place
proteomics investigation. “OPAL integrates the principles of both the oriented peptide
libraries and array technologies. Hundreds of pools of oriented peptide libraries are
synthesized as amino acid scan arrays. We demonstrate that these arrays can be
used to map the specificities of a variety of interactions, including antibodies, protein
domains such Src homology 2 domains, and protein kinases.”[22]
(4) Exploring Biochemistry and Cellular Biology with Protein Libraries:
Peptide library express a broad network for essential enzymes and binding protein
specificity. Furthermore to introduction rules for the molecular level recognition, the
binding preferences and tolerance from such library ca n reveals the mechanism of
biochemical and cellular procedures. The peptide obtained from protein library will
also useful for the pharmaceutical compounds and even reagents to further discovery
of cell biology. [23]
7.5.5 Relevant websites:
(1) http://www.stanford.edu/group/nolan/library_systems/library_systems.html
(2) http://www.ncbi.nlm.nih.gov
(3) http://peds.oxfordjournals.org/cgi/content/full/19/5/211
(4) http://www.healthtech.com/2003/pgd/index.asp
(5) http://www.uvminnovations.com/biomed.html
7.5.6 Key industrial suppliers
(1) AUSPEP-private limited 2002
Website: http://www.auspep.com.au/products_services/custom%20services.htm
(2) Chemical Synthesis Services, Elvingston Science Centre.
: +44(0)2838332200 F: +44(0)1875408151 Email:albachemservices@css-almac.com
Website: http://www.albachem.co.uk
(3) Genzyme Pharmaceuticals, 675 West Kendall Street, Cambridge, MA 02142, USA Tel:
617-374-7248, Fax: 617-768-6433
Website: http://www.genzymepharmaceuticals.com
7.5.7
Reference:
[1]Peptide library, Princeton Bimolecule Corporation, i, Corporation
http://www.pbcpeptide.com/Peptide%20Library.htm access date (23/09/06)
[2]http://www.stanford.edu/group/nolan/library_systems/library_systems.html access
date (23/09/06)
[3] Dominant effector genetics in mammalian cells, Rigel, Inc., San Francisco,
California, USA.
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=
11137994&dopt=Abstract
[4] Dickerson, T.J., Janda, K.D., Recent Advances for the Treatment of Cocaine
Abuse: Central Nervous System Immunopharmacotherapy. AAPS Journal.
2005; 07(03): E579-E586. DOI: 10.1208/aapsj070359
[5]Hoffman, J.A., III, Caudill, B.D., III, Koman, J.J, III, Luckey, J.W., Flynn, P.M.,
Hubbard, R.L. Comparative cocaine abuse treatment strategies: enhancing client
retention and treatment exposure. J Addict Dis. 1994; 13:115-128.
PubMed DOI: 10.1300/J069v13n04_01
[6]William, W.J., Olsen, B.N., Roberts, R.W., Epitope mapping using mRNA display
and a unidirectional nested deletion library
http://peds.oxfordjournals.org/cgi/content/full/18/7/309 access date(23/09/06)
[7] Cazzamali, G., Grimmelikhuijzen, C. J. P., Molecular cloning and functional

expression of the first insect FMRF amide receptor,
http://www.pnas.org/cgi/content/full/99/19/12073#F4 Access date (24/09/06)
[8] Lunder, M., Bratkovic, T., Doljak, B., Kreft, S., Urleb, U., Strukelj, B., Plazar, N.
Comparison of bacterial and phage display Peptide libraries in search of target-
binding motif. http://lib.bioinfo.pl/pmid:16258189 Access date (24/09/06)
[9] He, M., Taussig, M. J., Ribosome displays: Cell-free protein display technology,
http://www.discerna.co.uk/technique%20review.pdf access date (24/09/06)
[10] Murray A, Smith R.G., Brady K, Williams S, Badley, R.A., Price M.R.
Cancer Research Laboratories, University of Nottingham, Nottingham, NG7 2RD,
United Kingdom. Generation and refinement of peptide mimetic ligands for Para
tope-specific purification of monoclonal antibodies.
http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=Retrieve&db=PubMed&list_uids=
11520027&dopt=Abstract , Access date (24/09/06)
[11]Phage display screening, http://www.schafer-n.com/phagedisplay.htm Access

date (24/09/06)
[12]Wesley, C.S., methods for determining notch signalling and uses

http://www.uvminnovations.com/biomed.html Access date (24/09/06)
[13] Hamby, C. V., Llibre, M., Utpat, S. Wormser, G. P., Use of Peptide Library
Screening To Detect a Previously Unknown Linear Diagnostic Epitope: Proof of
Principle by Use of Lyme Disease Sera, Department of Microbiology and
Immunology, Division of Infectious Diseases, Department of Medicine of New York
Medical College, Valhalla, New York, http://cvi.asm.org/cgi/content/full/12/7/801
Access date (25/09/06)
[14] Sims, K. L., Schryvers, A. B., Peptide-Peptide Interactions between Human

Transferrin and Transferrin-Binding Protein B from Moraxella catarrhalis, Department
of Microbiology and Infectious Diseases, University of Calgary, Calgary, Alberta,
Canada,
http://www.pubmedcentral.nih.gov/articlerender.fcgi?artid=152632&tools=bot Access
date (25/09/06)
[15] Holzem, A, Nähring, J. M. , Fischer, R. ,Rapid identification of a tobacco mosaic

virus epitope by using a coat protein gene-fragment–pVIII fusion library , Institute for
Biology I (Botanik/Molekularbiologie), RWTH Aachen, Worringerweg 1, D-52074
Aachen, Germany
Fraunhofer Department for Molecular Biotechnology, IUCT, Grafschaft, Auf dem
Aberg 1, D-57392 Schmallenberg, Germany,
http://vir.sgmjournals.org/cgi/content/full/82/1/9 Access date (25/09/06)
[16] Park, S., Xu, Y., Stowell, X. F., Gai, F., Saven, J. G. , Boder , E. T., Limitations of
yeast surface display in engineering proteins of high thermostability, Department of
Chemistry, University of Pennsylvania, Philadelphia, PA 19104, Department of
Chemical and Bimolecular Engineering, University of Pennsylvania, Philadelphia, PA
19104 and Laboratory for Research on the Structure of Matter, University of
Pennsylvania, Philadelphia, PA 19104, USA Present address: Department of
Biology, Massachusetts Institute of Technology, Cambridge, MA 02139, USA,
http://peds.oxfordjournals.org/cgi/content/full/19/5/211 Access date (25/09/06)
[17] Palmer, S. J., Redfern, M. R., Smith, G.C., Cox, J. P. L, Sticky Egyptians: a
technique for assembling genes encoding constrained peptides of variable length,
Department of Chemistry and 1Department of Mathematical Sciences, University of
Bath, Bath BA2 7AY, UK, http://nar.oxfordjournals.org/cgi/content/full/26/11/2560
Access date (25/09/06)
[18] Sims, K. L., Schryvers, A. B.. Peptide-Peptide Interactions between Human

Transferrin and Transferrin-Binding Protein B from Moraxella catarrhalis, Department
of Microbiology and Infectious Diseases, University of Calgary, Calgary, Alberta,
Canada, http://jb.asm.org/cgi/content/full/185/8/2603 Access date(25/09/06)
[19] Boutin, J. A., Gesson, Henlin, J. M., Bertin, S., Lambert, P.H., Volland, J.P.,
Fauchère, J.L., Limitations of the coupling of amino acid mixtures for the preparation
of equi molar peptide libraries, a Department of Peptide and Combinatorial Chemistry
and b Department of Analytical and Physical Chemistry, Institut de Recherches
SERVIER, 11 Rue des Moulineaux, F92150 Suresnes, France.
http://www.5z.com/moldiv/volume3/pp43- 60.pdf Access date (25/09/06)
[20] Liu, R, Enstrom, A.M., Lam, K.S. Combinatorial peptide library methods for
immunobiology research, UC Davis Cancer Centre, Division of
Haematology/Oncology, and Department of Internal Medicine, University of California
Davis, Sacramento, CA, USA.
www.ncbi.nlm.nih.gov/entrez/query.fcgi?cmd=
http://Retrieve&db=PubMed&dopt=Abstract&list_uids=12543103 Access date
(25/09/06)
[21] Molecular display, http://www.healthtech.com/2003/pgd/index.asp Access date

(25/09/06)
[22] Rodriguez, M., Shawn S.,-C. Li, Harper, J. W. Songyang, Z, An Oriented

Peptide Array Library (OPAL) Strategy to Study Protein-Protein Interactions, Verna
and Marrs McLean Department of Biochemistry and Molecular Biology, Baylor
College of Medicine, Houston, Texas 77030 and the Department of Biochemistry,
Faculty of Medicine and Dentistry, University of Western Ontario, London, Ontario
N6A 5C1, Canada, http://www.jbc.org/cgi/content/full/jbc;279/10/8802 Access
date(25/09/06)
[23] Diaz, J. E., Howard, B. E., Neubauer, M.S., Exploring Biochemistry and Cellular
Biology with Protein Libraries, Allison Olszewski1 and Gregory A. Weiss, Department
of Chemistry and 2Department of Molecular Biology and Biochemistry, University of
California, Irvine Irvine, CA 92697-2025, USA.http://chem.ps.uci.edu/~gweiss/Diaz-
CIMB-03.pdf Access Date (25/09/06)
Chapter 7.6 Applications of Synthetic Peptides
Shruti Saptarshi
7.6.1 Introduction
Proteins are complex organic molecules made up of carbon, nitrogen,
oxygen, hydrogen and sulphur. These are abundantly found in nature in the form of
enzymes, hormones, antibodies, and thus play an important role in the overall
metabolism of living organisms. Proteins are made up of twenty different types of
basic structural units called the amino acids. The amino acids found in all the living
beings are the same. Every amino acid consists of a chiral carbon atom, an amino
group, a carboxy terminal and a replaceable /reactive R group. A condensation
reaction between the carboxy and amino terminals respectively of two amino acids
leads to the formation of a Peptide Bond. A couple of amino acids linked by the
peptide bond form a dipeptide. Peptides are essentially small chains of up to 20 such
α amino acids linked by the peptide bond. These are basically small fragments of the
protein molecule that are functional. Under natural circumstances peptides are
synthesized by the process of m RNA translation.
As mentioned above proteins play a pivotal role in function and maintenance

of the cell structure. Hence proteins as chemical moieties have a vast array of
industrial, therapeutic, diagnostic applications. However it is not always feasible to
obtain the required protein mainly because of the minimal amounts available,
complexity involved in its extraction, or most importantly the expenditure involved in
the process. Peptides on the other hand are specific functional sub units of proteins.
Lately production of peptides on large scale has received prominence because of
their pharmacological importance.
Peptides can be obtained directly from the tissue by the process of

purification, use of recombinant DNA technology [1]. However the best means of
obtaining peptides on large scale is by the use of chemical synthesis.
The basic idea of chemically synthesizing peptides is to obtain maximum

number of amino acid residues in the peptide. Three approaches are used in
synthesizing peptides namely, Solution phase synthesis, reverse proteolysis, solid
phase synthesis. The method commonly used is SPPS. It was developed by the
pioneering efforts of R. Bruce Merrifield in the year 1962.
As the name suggests SPPS involves synthesizing the peptides while it is still
attached to a solid phase. The solid phase used is an insoluble polymer contained in
a column usually a resin. The artificial synthesis of the peptide occurs from the
carboxy terminal unlike the amino terminal in case of natural synthesis.
Fig 7.6.1 Synthesis of peptides schematic representation.
Source: http://courses.cm.utexas.edu/emarcotte/ch339k/fall2005/Lecture-Ch3-
2/Slide22.JPG
The first step involves coupling of the amino acid to the ligands present on the
resin. This is made possible by the means of the chloromethyl linkers. The main
criterion of this step is to achieve a stable bond between the amino acid and the
polymer support. An alternative resin used now a days is the PAM i.e.
phenylacetamidomethyl resin. It helps form a much stable bond between the amino
acid and the resin as compared to the ones traditionally used.
Once the amino group is firmly attached to the polymer support, its reactive
functional group and the R group has to be protected so as to prevent the formation
of complex secondary structures. Typical labile groups used for protecting the alpha
amino groups include the t-Boc (tertbutyloxycarbonyl) and the F-moc (9-
flourenylmethloxycarbonyl). Yet another important characteristic of these groups is
easy removal so as to add a new amino group.t-boc is a stable labile group and can
be easily removed by using TFA (trifluroacetic acid) and dichloromethane. Whereas
F-moc can be removed by using concentrated amine solutions such as piperidine in
N- methylpyrrolidone. The labile groups used to protect the R group remains
attached during the entire process of synthesis.
For the formation of the peptide bonds during the elongation of the peptide it
is essential to activate the carboxyl groups. This is achieved by using symmetric
anhydrides, carbodiimides, phosphonium and uronium salts. This process is
repeated until the desired peptide length is achieved. Once ready the peptide is
cleaved from the polymer support to produce a free peptide which then subjected to
characterization and purification, by using techniques such as reverse phase HPLC,
mass spectroscopy, ion exchange chromatography etc. The newly synthesized
peptide is then lyophilized.
Now a day’s peptide synthesis can also be achieved by using automated

means. A vast number of automated instruments are available which carry out the
coupling, activation, deprotection of the peptides all at the same time with higher
efficiency in terms of purity. Common examples of such automated systems include
MilliGen, Applied biosystems, Advanced chemtech etc. As a result it is now possible
to obtain synthetic peptides of length from 2 to 80 amino acids with a purity of about
99%.
The latest technology has made it possible to manufacture complex peptides.

Ones consisting of long chains of hydrophobic amino acids, modified amino acids
such as phosphotyrosine, phosphoserine, phosphothreonine etc. It is also possible to
synthesize peptides containing other chemical compounds such as dyes and
disulphide bonds. Modifications such as biotinylation (process of tagging a molecule with
biotin), acylation (process of introducing an acyl group in a molecule), and nitro nation too
can be introduced. Peptides are commonly used as antigens for immunization
purposes. This requires conjugation of the peptide to a carrier protein. Such a
conjugation can be brought about at the C or N terminal of the amino acid by making
use of a pre activated carrier protein. Carbodiimide hydrochloride is an example of
such a cross linker that binds to Asp, Glu residue of an amino acid sequence.
Peptide sequences are specific and this makes the peptides unique with
regards to its chemical and physical properties. Peptides are manufactures for
myriad reasons. Peptides being specific in nature have a wide use in therapeutics,
protein engineering, in studying structure – function relationships,
immunodiagnostics. Novel molecules such as oligonucleotide-peptides, PNA (peptide
nucleic acids), peptoids, mimetics, MAPs, antibodies etc. are all essentially derived
from peptides.
7.6.4 Applications
MULTIPLE ANTIGENIC PEPTIDE SYSTEMS:

Fig 7.6.2 Structure of a MAP system.
Source: http://www.tjhecheng.com/uploadfile/200451033879.jpg
Antibodies are now days increasingly used for therapeutic reasons.

Antibodies can be artificially synthesized for a large number of proteins (antigenic
moieties). These proteins can be the ones separated by electrophoresis, present
natively, inactivated pathogens such as viruses or peptides artificially synthesized. As
compared to all the forms of antigens mentioned above synthetic peptides are
preferred as they help in targeting specific epitopes, are pure, and low on
expenditure in terms of their manufacturing. Moreover an antigenic peptide can be
introduced in any species so as to obtain antibodies. Also different antibodies can be
made against different epitopes on the same antigenic peptide. Antibodies can be
raised against specially modifies for eg. Phosphorylated, biotinylated, peptide
sequences. Such state specific antibodies have been used in studying cell signaling
pathways. However the specificity of the synthetic peptides can be a problem at
times. The small size can be a limitation in eliciting an immune response. Hence the
need of conjugating the peptide with a specific antigen carrier system. The MAP i.e.
is the multiple antigen peptide system provides the answer. This was first describes
by Dr. James Tam. It makes use of a peptidyl core of three or seven branched lysine
residues upon which the antigenic sequences of interest can be built using the above
stated spps method. The basic advantage of using such a system is the high molar
ratio of the peptide antigen to the core molecule. Hence there is no further need of
conjugation to a carrier protein. Immunogenic properties of multiple antigenic peptide
systems containing defined T and B epitopes have been aptly described [2].
They brought about an immunization with chemically defined synthetic

polymers, MAP system consisting of T and B epitopes of the circumsporozoit protein
of P.bergehi and found an induction of a significant immune response with high levels
of circulating antibodies. A secondary immune response against Map system showed
increased levels of circulating IgG. This study proved that MAP systems are potent
immunogens and are also capable of inducing immunologic memory.
Synthesis and bioactivities of two antigenic peptides 26-43(P26) and 116-

131(P116) derived from 28 kDa glutathione S- transferase of S. mansoni (Sm28
GST), two multiple antigenic peptides (P26)4 – MAP and (P116)-4MAP with an
oligomeric lysine core were synthesized by solid phase peptide synthesis method.
This experimentation was carried out on laboratory animals. Positive dot ELISA
results were obtained for the same. As mentioned by[3]
“A new method of synthesis of Multiple Antigen Peptide System, wherein the

carrier is a core matrix with branched lysine and β-alanine residues, is now n use.
The antigenic peptide is separately synthesized in a protected form and coupled to
the core with diisopropylcarbodiimide (DIPCDI) in presence of 1-hydroxy-
benzotriazole (HOBt). This procedure has two major advantages: firstly, it allows an
independent characterization (and purification) of the core and of the peptides;
secondly, it allows a possible further coupling to a protein carrier, after an
intermediate addition of Cys to the N-terminal amino acid.”
PEPTOIDS
Fig 7.6.3 Structure of a typical Peptoid

Source: http://barronlab.chem-eng.northwestern.edu/Images/Peptoid.jpg
Peptoids or peptidomimetics are molecules like peptides. However they differ

in terms of properties. Peptoids are oligomers of N- substituted glycines rather than α
carbon substituted glycine units. These are basically designed to mimic the complex
biological properties of the parent peptide. Peptoids are protease resistant and hence
are not easily broken down in the body like peptides. Peptoids are synthesized using
solid phase sub monomer method and a peptide synthesizer. This particular
characteristic of peptoids is now being further explores in the field of biomedicine. As
mentioned in one of the recent papers, scientists are designing peptoids with
selectively positioned reactive groups along the molecule backbone. This was done
by developing the backbone peptoid on a solid support, and then attaching the
bioreactive groups to it by using copper catalyzed reaction between an azide and
alkyne. In this method the azide or the alkyne unit is first attached to the backbone,
then the bioreactive molecule already containing a coupling moiety is added which
results in instant ligation. Such a type of peptoid can also find applications in energy
transduction as well as in information storage.[4]
Peptoids have also found potential application as Fibrin mimics for surgical
glue as stated in the journal article by [5]. New substrates for transglutaminases are
being developed with peptidomimetic backbones. These substrates are then
attached to polymers such as PEG to form polymeric substrates. One of the
examples is of a hydrogel formulation that contains calcium loaded liposomes,
hrFactorXIII, thrombin, enzymatic substrate, all based upon a PEG-peptide conjugate
containing four arms. Each of these four arms ends with a peptidomimetic aping the
gamma chain of the protein fibrin. This material can be used in wound healing, tissue
scaffolding and drug delivery.
Yet another application that is being explored for its potential clinical benefits
is the use of Peptide Mimetics as vaccines for cancer. The basic principle on which
the cancer vaccines work is basically inducing an immune response against the
tumor antigen by the means of whole tumor cell vaccines, dendritic cell vaccines,
adjuvant vaccines, DNA, viral vector vaccines etc. Most of the tumor antigens
exhibited by the tumor cells are also expressed by normal cells. Hence it is difficult to
induce a strong humoral immune response against the self antigens. Peptides
mimetic are artificially designed specific proteins. Thus if such antigenic determinants
mimicking the tumor antigens are introduced into the body then it will be possible to
induce a stronger immune response. However the only ambiguity associated with this
form of immunotherapy is the risk of initiating an autoimmune response. A recent
report stated that immunizing mice with a peptide that mimics MUC1 is able to
generate a strong cellular and protective response against MUC1 as well as lysing
human breast cancer cell lines [6]
This idea is still in its infancy and demands conducting many clinical trials so
as to device a new strategy that can be useful for humans. Peptoids are also being
characterized for their antimicrobial properties. One of the research papers reported
the antibiotic activity of a peptoid CHIR29498 and some of its analogues against a
host of gram positive as well as gram negative bacteria. Destruction of the bacterial
cell wall was found to be the mode of action for the peptoid [7].
PEPTIDE NUCLEIC ACIDS:
Fig 7.6.4 Diagrammatic representation of Peptide Nucleic Acid

Source: http://tesla.jci.tju.edu/pics/pna.jpg
Peptide nucleic acids are achiral DNA/RNA analogs. Unlike the sugar
phosphate backbone present in the nucleic acids these contain pseudo peptide
namely N– (2-amino-ethyl--glycine units. This skeleton also it is not charged. PNAs
have a unique ability to bind to DNA as well as RNA thereby forming hybrids that are
much more stable. They also exhibit complementarity like their natural counterparts
thus obeying the Watson – Crick hydrogen bonding scheme. These are synthesized
by using solid phase peptide synthesis. Moreover peptide nucleic acids are relatively
resistant to attack by proteases and possess thermal stability along with ionic
strength. Hence they fine wide applications in the fields of chemistry, medicine, and
genetic diagnostics. PNAs are used extensively in anti gene, anti sense studies as it
is able to inhibit translation and transcription of the gene towards which it is targeted.
They can be used as markers in DNA mapping projects. PNAs labeled with biotin,
fluorescent dyes, reporter enzymes are commonly used in hybridization experiments
such as DNA arrays, Northern, Southern blots, and detection of point mutations.
PNAs are now used as tools for biosensors which help in detecting specific DNA
sequences in test samples. It involves immobilization of a single – stranded nucleic
acid probe onto an optical transducer over which the sample is passed. Any kind of
mismatch is conveyed in the form of an electrical signal which is then detected. Thus
PNAs can be used in place of synthetic DNA or RNA but with added benefits as
mentioned above.
PEPTIDES AS VACCINES:
One of the most critical goals of vaccine development is to trim down the
structurally complex molecules to smaller ones which are high on resolution, can be
rapidly modified, exhibit specificity in terms of the antigenic property. Use of synthetic
peptides is hence the best option. Synthetic peptides are commonly used as
immunizing agents. They are the simplest alternatives to vaccine development. The
synthetic methods described above have made it possible to synthesize peptides
corresponding to specific antigenic epitopes present on an infectious agent.
Currently lots of effort is being put in finding peptide based vaccines against cancer
and AIDS. One such report stated synthesis of conformationally constrained peptides
that bind tightly to 2F5monoclonal antibody (specific against a recognition epitope on
the HIV envelope glycoprotein gp 41). This peptide is to be conjugated with a carrier
protein and then used as a vaccine so as to elicit an immune response against HIV
neutralization. This idea has potential therapeutic benefits.[8]
Antigenic and immunogenic properties of totally synthetic peptide based

antifertility vaccines too have been reported in one of the research papers. It is
observed that vaccine containing synthetic peptide made up of 15 residue –defined
cell epitope and 10 residue LHRH i.e. (leuteinizing hormone releasing hormone)
epitope induces an explicit antibody formation and then upon sterility in mice. Such a
type of vaccine too can be of therapeutic importance. [9]
Yet another report stated synthesis of a peptide vaccine against influenza

AchiH3N2 virus. It was prepared by introducing residues of the influenza virus
haemagglutinin in to a frame component residue which included the agretopes (site
of contact between the antigen and the MHC complex) of the antibody. It was
observed that this vaccine induced a characteristic T cell response which had a
neutralizing effect on the virus.[10]
OTHER EXAMPLES OF APPLICATIONS OF SYNTHETIC PEPTIDES:

Apart from the examples described above, peptides have been excessively
used in synthesis of a large number of hormones for example: LHRH, Insulin,
Calcitonin, gonadotropins, other pituitary hormones etc.
Synthetic peptides are also now used as regents for immunodiagnosis, also
as inhibitors and for protein engineering purposes. Application of synthetic peptides
in structure function studies is of great importance.
The relevant websites pertaining to this topic are as follows:
• Activotech – Peptide synthesis Research and development

www.activotech.com/products/research.htm
• Peptide Synthesis
www.learner.ccf.org/services/molecbiotech/peptide.php
• Biomimetic oligomers
http://barronlab.chem-eng.northwestern.edu/Project1.html
• http://www.chem.qmul.ac.uk/iubmb/misc/phorm.html
Key industrial suppliers of synthetic peptides in their various forms include the
following:
• United Biomedical Inc.
• GL Biochem (Shanghai) Ltd.
• Invitrogen Corporation
• CSBio Inc.
• Biopeptide Co., Inc.
• EZBiolab Inc.
7.6.7 References
1. Nelson, D.L. and M.M. Cox, Principles of Biochemistry. 3 ed. 2000,

Hampshire: MacMillan Press.
2. Chai, S.K., et al., Immunogenic properties of multiple antigen peptide systems
containing defined T and B epitopes. J Immunol, 1992. 149(7): p. 2385-2390.
3. Huang, H.-Q., et al., Synthesis and bioactivities of two multiple antigen
peptides as potential vaccine against schistosoma. Bioorganic & Medicinal
Chemistry Letters, 2005. 15(9): p. 2415-2419.
4. Holub, J.M., H. Jang, and K. Kirshenbaum, Clickity-click: highly functionalized
peptoid oligomers generated by sequential conjugation reactions on solid-
phase support. Org. Biomol. Chem, 2006. 4: p. 1497-1502.
5. Nathan Brown, N.C., James Patch, Shannon Seurynck, Yu Zhang,
Biomaterials, 2001.
6. V. Apostolopoulos., J.M., M. Plebanski and T. Mavromoustakos, "Applications
of peptide mimetics in Cancer." Current Medicinal Chemistry, 2002. 9: p. 411
- 420.
7. Goodson, B., et al., Characterization of Novel Antimicrobial Peptoids.
Antimicrob. Agents Chemother., 1999. 43(6): p. 1429-1434.
8. Taylor, J. 2004 [cited 28-10-2006]; Available from:
http://rutchem.rutgers.edu/content_dynamic/faculty/john_taylor.html.
9. Ghosh, S. and D.C. Jackson, Antigenic and immunogenic properties of totally
synthetic peptide-based anti-fertility vaccines. Int. Immunol., 1999. 11(7): p.
1103-1110.
10. K Ogasawara, H.N., Y Itoh, T Gotohda, J Arikawa, H Kida, R A Good, and K
Onoé, A strategy for making synthetic peptide vaccines. Pub med central,
1992. 19: p. 8995-8999.
7.7 Peptide Nucleic Acids
Desai Urvi
7.7.1 Introduction
Fig: PNA having peptide backbone.

Source: http://en.wikipedia.org/wiki/PNA
DNA and RNA are the two forms of genetic materials present in nature from long
ago. Scientists are trying to synthesis DNA and RNA in lab and have got a little
success too. But now they have discovered a new form of Nucleic acid called as
Peptide Nucleic Acid (PNA).
PNA, Peptide Nucleic Acid was synthesis by Neilsen, Egholm, Berg and Buchardt in
1991.DNA and RNA consist of sugar backbone and phosphate and nitrogen bases
attached to back bone. PNA is an artificially synthesised analogue of DNA, having
pseudo peptide in place of sugar phosphate backbone in DNA. PNA has a backbone
of repeated units of N-(2-aminoethyl)-glycine units linked by peptide bonds. Which
are attached to four bases (adenine, guanine, thymine or cytosine) found in DNA. It
also has amino and carboxyl terminals like amino acids. It has N terminus at first
position and it corresponds to 3’ end of DNA or RNA. I.e. 5’ end of PNA binds to
complementary single stranded DNA. This means that PNA sequences are written
from 3’ to 5’. Unique feature of PNA is that it is shows resistance to nucleases and
proteases. [4]
Fig: PNA having peptide backbone. Fig: comparison of DNA and PNA
Source: http://en.wikipedia.org/wiki/PNA Source:
http://www.biosyn.com/new_popup.htm
PNA has attracted major attention in fields of chemistry and biology because of its
attractive chemical, physical, and biological properties and its chances to be very
useful as an active component for diagnostic as well as pharmaceutical applications.
PNA has behaviour like DNA; it binds to the complementary nucleic acids strands.
Also as the backbone of PNA is not nucleic acid it is neutral in nature and results in
stronger binding between PNA/DNA then between PNA/PNA and results in better
specificity. They are also resistant to hydrolytic (reactions) cleavage and so are not
easily degraded inside the living cell. Also PNA is proficient of sequence specific
recognizing DNA and RNA obeying Watson and Crick hydrogen bonding scheme.
The hybrid form by hydrogen bonding is having too great thermal stability and unique
ionic strength effects. It forms stable triplex of PNA-DNA-PNA by identifying duplex
homopurine sequence of DNA and binds to it by strand invasion, which has a looped
out DNA strand from duplex DNA which it invaded. [2] It is experimented that the
melting temperature (Tm) of a normal dA10-dT10 DNA hybrid is 23°C while the Tm of a
similar dA10-dT10 DNA/PNA hybrid is 86°C1.
PNA was designed originally to recognize double strand DNA. IT was thought to form
oligonucleotides that can base pair to double strand of DNA via. Hoogsteen base
paring in major groove. In this way there are nitrogen base pairs of DNA in PNA but
the sugar backbone is replaced by a pseudo peptide. It was believed that if this
peptide would be of neutral nature it will recover triplex binding capability of ligand.
The PNA designed in this way mimic the single strand nucleic acid. Many structures
were taken into consideration during early stags but later on when criteria’s like water
solubility, rigidity and least chemical accessibility the final structure which was
accepted was of PNA. [3]
It is been thought that this novel discovery of PNA will be useful in lot of ways. PNA
can be used as a probe molecule as it is having unique stability and other
biochemical properties. It is having more enzymatic and chemical stability than
nucleic acid can hybridize very well too.
It has better chances of having new ways of detection using PNA as it has unique
molecular structure. [5]
PNA is now been used as gene targeting drug, where in PNA’s directed towards
double stranded DNA exhibit antigene properties where as targeting of single
stranded RNA leads to antisense effects. As a result PNA’s are used as bactericidal
antibiotics for regulating splice site selection and as telomerase inhibitor.
As PNA’s are neutral in nature they cannot be easily delivered as they do not have
charge. Many new methods have been used to overcome this problem. Incorporating
positive charge to PNA so that it can be easily transported has been tried, for which
lysine and arginine has been used. Also ligands are attached e.g. short peptide
sequences like antibodies or steroids for delivery of PNA’s. [14]
Antisense PNA targeting was used to identify critical HIV gene segment inside gag-
pol encoding gene. Here translation of mRNA of HIV gag-pol was disrupted with
antisense targeting stopping the production of virion from cells infected cronically with
HIV. [16]
Light up probes are developed using PNA. There is asymmetric cyanine dye thiazole
orange (TO) attach to it, and when PNA binds to target DNA, dye binds to DNA and
gives flurosense. [17]
It is been mentioned earlier that the basic properties of PNA are as such much more
superior. Like specific binding, neutral nature, protection against hydrolytic cleavage
and many more. It is seen that PNA-DNA duplex is very stable than corresponding
DNA-DNA or DNA-RNA duplexes. [3] There is formation of D-loop in PNA generation
which is used to study mechanism of transcription and inducing gene expression.The
PNA are synthesised in laboratory which is big advantage and are easily synthesised
by tBoc or fMoc chemistry. PNA-DNA/mRNA hybrids recognized with high efficiency
by enzyme RNase H which reflects its ability to act as a substrate to identify cellular
enzyme as in DNA. Also there are more betterment done in the PNA itself so as to
increase its efficiency. Like at Oxford research lab researchers have developed
nucleo-amino acid derived from proline and spacer amino acid, they provide
conformational constraint to the PNA. Spacer amino acid is at n terminus and it
provide selective binding to either RNA or DNA. The chiral PNA so form can be used
in quality control as it forms single enantiomer product. The spacer presents help to
modify properties of PNA like adding charge to backbone or increasing
hydrophobicity. [15]
PNA has low cell membrane permeability which result in negligible intracellular
concentrations. As it has resistance to water solubility it has less bioavability. They
cannot be delivered to conventional cationic formulations eg. Liposome and micro
spheres as they are neutral in nature. To increase the uptake of PNA because of its
low solubility it is used in covalent bounded DNA-PNA hybrid form. It is seen that
PNA’s are expensive in aspect of production as compared to other artificially
synthesised peptides. [14]
7.7.4 Application of Technology
PNA was originally designed as a ligand for the recognition of double stranded DNA.
Later on it was found out that peptide nucleic acids are very stable, specific in their
nature, water resistant and hydrolytic cleavage resistant , and as a result it has a
broad spectrum of uses now.
The specificity of PNA to bind to a chosen target is of major interest and is there for
used in medical and biotechnological context. They show a new scope for
development in field of gene therapeutic agent, diagnostic devices for genetic
analysis and molecular tools for nucleic acids manipulation.
They are also used for antisense and antigene therapy in eukaryotic nerve cells and
even in rat’s brain. This activity is also shown in E.coli. PNA binds to complementary
sequences on DNA and thus can inhibit transcription of that gene that is antigene
strategy and for antisense strategy an analogs can be designed which will bind to
complementary sequences in mRNA after recognizing it, and in this way it inhibits the
translation of that gene. [2]
Fig: Antigene and Antisense strategy.

Source: http://www.fasebj.org/cgi/content/full/14/9/1041
Detection of SNP; PNA are now used to detect SNP’s which in turn is used to detect
neurodegenerative disease identification. In this detection method PNA is used as a
probe along with S1 nuclease enzyme and amplifying conjugated polymer (CP). PNA
is flurolabelled and it hybridizes to DNA at sequence of interest and thus helps in
recognization of SNP’s. [6]
Detection of Transcription Factors: A photo Functionalised PNA ongomer was
designed, which was used to detect transcription factor. [7]
Chromosomal Identification: Centromeric PNA probe specific to chromosomes 1, 4,
9, 16, X and Yare used to detect oocytes, blastomeres and polar bodies. The
identification method used was FISH.[8]
PNA Blocker Probes Enhance Specificity in Probe Assays: Mismatch hybridization of

Labelled probes to non-target sequence can be prevented by non-labelled PNA
known as ‘blocker’. This prevents unwanted hybridization without harming the
sensitivity of detection. Also they improve PCR amplification by providing high noise
ratios. It was possible to identify a single base mutation in the K-ras gene at levels of
only 1.5 copies per 100 copies of wild type DNA. [9]
PNA directed genome rare cutting: Usual restriction enzyme is converted into
infrequent genome cutter using PARC i.e. PNA-assisted rare cleavage based on
Achilles’ heel’ cleavage strategy. A sequence specific complex of double stranded
genomic DNA and bis-PNA is treated with DNA methyl transferase also known as
methylase. Bis-PNA is removed and sample is treated with restriction enzyme which
recognizes same methylase sight and thus cannot cleave them. There are few
methylation sights which were protected from methylation by bis-PNA binding to
them which will be identified by restriction enzyme and will cleave them after bis-PNA
are removed. Bis-PNAs with various combination and with different
methylation/restriction enzymatic pairs generates a new class of genome rare cutters
[10]
Purification of Nucleic acids: PNA hybridize to nucleic acid in two different ways.
Sequence specific method and generic method. Sequence specific method is a
selective method which requires sequence information on the target and synthesis of
dedicated PNA which bind to target DNA. The generic method does not require
sequence of target DNA and uses triplex form of PNA. It is capable of bulk
purification. [11]
-http://www.horizonpress.com/hsp/books/pna.html
-www.springerlink.com/index/D5078873017U6131.pdf
-http://www.eurogentec.com/module/FileLib/oligo_16.pdf
-http://www.fasebj.org/cgi/reprint/14/9/1041.pdf
- http://www.abrf.org/ABRFNews/1993/September1993/sep93impepna.html
7.7.6 Key industries suppliers
1) Monomer Sciences Inc. United States. Phone: 256-379-5279. Fax: 256-379-5282

E-mail: information@monomer.com
2) Eurogentec North America, San Diego, CA.Phone: 858-793-2661 Fax: 858-793-
2666
Contact email: info.usa@eurogentec.com Web site: http://www.eurogentec.com
3) Bio-Synthesis, Inc., Texas. Phone: 972-420-8505 Fax: 972-420-0442

Email: biosyn@biosyn.com
7.7.7 References
[1] Peptide Nucleic Acids: Protocols and Applications. Horizon Printing Press. (1999)
http://www.horizonpress.com/hsp/books/pna.html Access Dt.:26/10/2006
[2] Ray, A., Nordén, B. Peptide nucleic acid (PNA): its medical and biotechnical
applications and promise for the future. Department of Physical Chemistry, Chalmers
University of Technology, S 412 96, Gothenburg, Sweden.
http://www.fasebj.org/cgi/content/full/14/9/1041 Access Dt.: 27/10/2006
[3] Peptide Nucleic Acids. , Horizon Scientific Press.

http://www.horizonpress.com/gateway/pna.html , Access DT.: 26/10/2006
[4] Matsudaira, P., Coull, J., Peptide Nucleic Acids: A New Nucleic Acid Analog.
Whitehead Institute for Biochemical Research, Dept. of Biology. Massachusetts
Institute of Technology, Cambridge and Millipore Corporation, Core R& D, Specialty
Chemistry Group, Bedford, MA 01 730
http://www.abrf.org/ABRFNews/1993/September1993/sep93impepna.html
[5]Brandt, O., Hoheisel, J., Peptide nucleic acids on micro arrays and other
http://www.moltools.org/pooled/articles/BF_SITEART/view.asp?Q=BF_SITEART_13
9244 Access Dt.: 27/10/2006
[6] Gaylord, B. S, Massie. M. R., Feinstein, S. C., Bazan, G. C., SNP detection using
peptide nucleic acid probes and conjugated polymers: Applications in
neurodegenerative disease identification.(2004). Materials Department and Institute
for Polymers and Organic Solids and Neuroscience Research Institute, University of
California, Santa Barbara, CA 93106.
http://www.pnas.org/cgi/content/abstract/102/1/34. Access Dt.: 27/10/2006
[7] Fujimori, F., Kitagawa, F., Abe, Y., Ohori, Y., Kiyota,R., Nakamura,Y., Ikeda, H.
and Murakami, Y., Application of peptide nucleic acid (PNA) for detection of
transcription factors binding probes. Department of Biological Science & Technology,
Faculty of Industrial Science & Technology, Tokyo University of Science, Yamazaki,
Noda-Shi, Chiba, 278-8510, Japan.
http://nass.oxfordjournals.org/cgi/content/abstract/2/1/69 . Access Dt.:27/10/2006
[8] Paulasova, P., Andréo, B., Diblik, J., Macek, M. and Pellestor, F. The peptide
nucleic acids as probes for chromosomal analysis: application to human oocytes,
polar bodies and preimplantation embryos.(2004) Laboratory of Assisted
Reproduction, Motol Hospital, Vuvalu 84, 150 06 Praha 5, Czech Republic and
CNRS UPR 1142, Institut de Génétique Humaine, 141 rue de la Cardonille, F-34396
Montpellier Cedex 5, France http://molehr.oxfordjournals.org/cgi/content/full/10/6/467
Access Dt.:27/10/2006
[9] Fiandaca, M. J., Hyldig-Nielsen, J.J. and Coull, J.M. PNA Blocker Probes
Enhance Specificity in Probe Assays.(2004)
http://www.horizonpress.com/hsp/abs/abspna.html Access Dt.:28/10/2006
[10] Demidov, V.V., Frank-Kamenetskii , M. D., PNA directed genome rare cutting.
http://www.horizonpress.com/hsp/abs/abspna.html Access Dt.:28/10/2006
[11] Orum, H., Purification of nucleic acids by hybridisation to affinity tagged PNA
probes. http://www.horizonpress.com/hsp/abs/abspna.html Access Dt.:28/10/2006
[12]Fujimori, F., Kitagawa, F., Abe, Y., Nakamura, Y., Ikeda, H. and Murakami, Y.,
Design of the photosensitized peptide nucleic acids for the analysis of geno-typing.
Department of Biological Science & Technology, Faculty of Industrial Science &
Technology, Tokyo University of Science, Yamazaki, Noda-Shi, Chiba, 278-8510,
Japan. http://nass.oxfordjournals.org/cgi/reprint/1/1/177.pdf Access Dt.:28/10/2006
[13] Marin V. L., Roy S, Armitage BA., Recent advances in the development of
peptide nucleic acid as a gene-targeted drug. Department of Chemistry, Carnegie
Mellon University, 4400 Fifth Avenue, Pittsburgh, PA 15213-3890, USA.
[14] Wang, G., XU, X. S., Peptide nucleic acid (PNA) binding-mediated gene
regulation. 1Institute of Environmental Health Sciences, Wayne State University, 2727
Second Avenue, Detroit. http://www.nature.com/cr/journal/v14/n2/full/7290209a.html .
Access Dt.: 29/10/2006
[15] New Peptide Nucleic Acids, Technology transfer from University of Oxford.
http://www.isis-innovation.com/licensing/1332.html . Access Dt.:30/10/2006.
[16] Peptide nucleic acids as epigenetic inhibitors of HIV-1. International journal of
Peptide Research and Therapeutics, September 21, 2006. Pages 269-286.
www.springerlink.com/index/D5078873017U6131.pdf Access Dt.: 30/10/2006
[17] Snvik, N., Westman, G., Wang, D. and Kubusta, M. Light up probes: Thiozole
Orange conjugated Peptide Nucleic Acid for detection of Target Nucleic Acid in
Homologous Solution. Department of Molecular Biology. Lundberg Institute.S-413 90.
Goteburg, Sweden. Department of Organic Chemistry. Chalmers University of
Technology, S 412 96, Gothenburg,Sweden.
http://www.molbiotech.chalmers.se/research/mk/lightup/Anal%20Biochem%20281,%
2026%20(2000).pdf Access Dt.:30/10/2006
Chapter 8.2 Antibody Engineering
Rushabh Gohil
8.2.1 Introduction
A ntibodies represent the most diverse and important class of recognition

molecules known. The ability of antibodies to recognize target molecules with
exquisite affinity and specificity is widely exploited for diagnostic and therapeutic
purposes, with over 10 antibody-based drugs currently available to treat conditions
ranging from cancer to autoimmunity and organ rejection. In antibody engineering,
molecular biology approaches are used to improve the function of antibody
molecules by altering their amino acid sequence. The affinity and specificity with
which antibodies recognize antigens, their stability in various environmental
conditions, their therapeutic efficacy, and their detection in diagnostic applications
may thus be enhanced[1].
Fig.8.2.1 Antibody Specificity for Antigens (Source:

http://content.answers.com/main/content/wp/en/thumb/1/16/180px-Antibody.png)
Antibodies are used widely in medicine and science as indicator

molecules. The specific binding properties are used in countless clinical diagnostic
tests, and for the identification and quantification of antigens under study in the
laboratory, where techniques as immunoblotting, immunoprecipitation, enzyme-linked
Immunosorbent assay (ELISA) and radioimmunoassay (RIA) are indispensable.
Antibodies are also increasingly used for other applications such as purification of
bio-molecules (immunoaffinity chromatography), for both diagnostic (imaging) and
therapeutic applications in vivo, and even for catalysis of chemical reactions.
Different antibody derived constructs are rapidly advancing as putative tools for
treatment of malignant diseases. Antibody engineering has added significant new
technologies to modify size, affinities, solubility, stability and biodistribution properties
for immunoconjugates[2].
Underlying Principles for the Production of Monoclonal Antibodies
Antibody Engineering is described as the means for modification of

antibodies to their increase affinity and specificity, in order to make them apt to aid
the various protein technologies as mentioned above. Antibody engineering is
basically a tool used to construct hybrid forms of the prevalent antibodies
(immunoglobulins) known as Monoclonal Antibodies. Structurally, these monoclonal
antibodies are induced with a variation in their variable or Fab region (i.e. the antigen
binding site) in order to create specificity to a particular antigen[3].
Fig.8.2.2 A Monoclonal Antibody showing the desired modification in the Fab region
to create specificity in its binding site to a particular antigen.
(Source: http://www.immuno-precise.com/images/idio-1.gif)
Monoclonal antibodies can be produced in specialized cells through a

technique now popularly known as Hybridoma Technology. This technology was
discovered in 1975 by two scientists, Georges Kohler of West Germany and Cesal
Milstein of Argentina who jointly with Niels Jerne of Denmark were awarded the 1984
Nobel Prize for Physiology and Medicine.
The term hybridoma is myeloma cell culture applied to fused cells

resulting due to fusion of following two types of cells: (i) an antibody producing
lymphocyte cell (e.g. a spleen cell of mouse immunized with red blood cells from
sheep), and (ii) a single myeloma cell (e.g. bone marrow tumor cell) which is capable
of multiplying indefinitely. These fused hybrid cells or hybridoma have the antibody
producing capability inherited from lymphocytes and have the ability to grow
continuously and are hence referred to as immortal[4].
Antibodies are mass-produced in the laboratory by fusing a myeloma

cell from a mouse with a mouse B-cell that makes a specific antibody called
‘hybridoma’. The hybridoma cell is an antibody-producing factory, as it is a
combination of a B-cell that recognizes a particular antigen and a myeloma cell that
exists indefinitely. These multiple clones are called monoclonal antibodies (MAbs), as
they are derived from a single hybridoma cell.
The production of monoclonal antibodies using hybridoma technology

in the laboratory involves a series of carefully designed steps, which is essential to
synthesize these MAbs for their specificity of the purpose for which they are required.
Antigens specific to the process are repeatedly injected to the mice for
the production of specific antibody, facilitated due to proliferation of the desired B
cells. These mice then produce the desired sets of lymphocytes in reaction to the
antigens.
The spleen cells (rich in B cells and T cells) are separately cultured.
These spleen cells produce antibodies specific to the antigens that were injected.
The myeloma cells producing the effect of the antigens in the mice are cultured
separately. The myeloma cell line used for the purpose of synthesizing a Hybridoma
should be peculiar for two important characteristics. One, it should have stopped
synthesizing antibodies; and two; it should be a mutant that can not synthesize the
enzyme hypoxanthine guanine phosphoribosyl transferase (HGPRT).
Fig.8.2.3 Proposed structure of HGPRT with a proposed transition state inhibitor

ImmGP (Source:
http://www.nature.com/nsmb/journal/v6/n6/images/nsb0699_588.gif)
Fusion of spleen cells to myeloma cells is induced, using polyethylene

glycol (PEG), to produce the hybridoma. The hybrid cells are grown in selective
hypoxanthine aminopterin thymidine (HAT) medium. HAT medium contains a drug
aminopterin, which blocks one pathway for nucleotide synthesis, making the cells
dependent on another pathway that needs HGPRT enzyme, which is absent in
myeloma cells. Therefore, myeloma cells that do not fuse with B cells will die. Also,
the HGPRT-.B cells will die because they lack the property of immortal growth.
Therefore the HAT medium allows the selection of hybridoma cells, which inherit the
HGPRT gene from B cells and the property of immortal growth from myeloma cells.
The desired hybridoma is selected for cloning and antibody

production. This is facilitated by preparing single cell colonies that will grow and can
be used for screening of antibody producing hybridomas. Only one in several
hundred cell hybrids will produce antibodies of the desired specificity. The selected
hybridoma cells are cultured for the production of monoclonal antibodies in large
quantities. These hybridoma cells may be frozen for future use and may also be
injected in the body of an animal so as to produce antibodies in the body, which can
be recovered later from the body fluid.
Fig.8.2.3 Production of Monoclonal Antibodies using Hybridoma Technology

(Source:
http://www.aecom.yu.edu/cancer/new/Assetts/images/new%20navbar/cores/image1.j
pg)
Considerable efforts during the last 10-15 years have been made to
improve the yield of monoclonal antibodies using hybridoma technology. Also,
consistent efforts are being made to develop newer methods for the production of
monoclonal antibodies using antibody engineering as a key tool. One such
commendable effort in the recent times has been the use of Phage Display to
produce monoclonal antibodies using recombinant phages.
Both conventional hybridoma and phage display antibody production
exploit the vast diversity of the mammalian antibody repertoire. The fundamental
difference is that with hybridoma antibody production, this diversity is harnessed by
the immortalization of antibody producing B-cells, while with phage display it is the
genes that encode antibody variable regions (V-genes) that that are immortalized.
Thus, the sacrificing of animals like mice is overcome by the use of phase display
technique to create hybridomas. Also, a further major advantage of antibody
production by phage display is that in many cases the whole process can be
performed in vitro, thereby negating the requirement for target antigens to be
immunogenic[5].
There have also been quite a few advancements in the process of using
hybridomas to create monoclonal antibodies. One such advancement is that the cell
fusions are facilitated through the use of polyethylene glycol (PEG). This reagent is
used to assist the fusion of the myeloma and spleen cells and also helps in
preserving the hybridomas. A second advancement is the use of continuous cell lines
as fusion partners for the antibodies producing B cells. Feeder layers consisting of
extra cells to feed newly formed hybridomas are used for optimal growth and
hybridoma production. The most common feeder layers consist of murine peritoneal
cells, marcrophages derived from mouse, rat or guinea pigs and extra non
immunized spleen cells.
Human fibroblasts, human peripheral blood monocytes or thymus cells are

also used as feeder cells, but had some limitations like depletion of nutrients meant
for hybridoma and contamination. As a reason, other sources of hybridoma growth
factors (HGF) like interleukins derived from human cells are being used in their
place[5].
Another major advancement in the field of synthesizing monoclonal

antibodies using Hybridoma technology is the generation of humanized monoclonal
antibodies. This technique overcomes the shortcoming of using rodent antibodies,
wherein the rodent antibodies are perceived as foreign materials by the human
lymphocytes and are hence rendered inefficient. Humanized monoclonal antibodies
being produced recently consist of only the antigen binding complementarity
determining regions (CDRs) from the rodent in association with human framework
regions have been produced. This is achieved by either the fusion of mouse
myeloma cells with human lymphocytes (blast cells in peripheral blood lymphocytes),
or by the immortalization of human cells by Epstein Barr virus. In another approach,
transgenic mice carrying human genes for V, D, J and C regions have been
produced. These can be used for the production of human antibodies directly by
hyperimmunization[6].
Fig.8.2.5 Example of a Humanized Antibody made up of Mouse Complementarity
Determining Regions embedded in the Human Antibody Framework. (Source:
http://www.hopkins-arthritis.som.jhmi.edu/edu/acr/images/mab_humanized_ab.jpg)
In recent years, techniques have been developed wherein, antibody genes

can be isolated from lymphocytes of immunized animals and then cloned and
expressed in bacteria. The antibodies produced in bacteria under the control of
cloned genes can be screened for binding to specific antigens. Thus, while
hybridoma technology can immortalize antibody producing cells, gene technology
immortalizes antibody by producing genes[7].
Computer graphic techniques are also being used to build specific antigen
binding sites in antibodies. Using this approach some designer antibodies of practical
value has already been produced. In this strategy, genes are not really cloned from
lymphocytes, but are instead designed from a repertoire of antibody genes available
in a collection.
Recent patent applications related to antibodies

Table 8.2.1 Recent patent applications related to antibodies
Priority
Publication
Patent # Subject Assignee Inventor(s) application
date
date
Enriching for nucleic acids
encoding multimeric
antibodies having a biological Carter P,
function; comprises Cosman DJ,
Amgen (Thousand
WO 200563817 transfecting mammalian cells Martin FH, Shen 8/31/2004 7/14/2005
Oaks, CA, USA)
with polynucleotides W, Yan W, Zhou
containing a library of nucleic C, Zhou H
acids encoding multimeric
antibodies and a vector.
An antibody directed
specifically against des-
Lys58-beta2-microglobulin or
its fragment, and capable of Statens Serum Institute Corlin DB,
WO 200563335 12/30/2003 7/14/2005
forming a complex with des- (Copenhagen) Heegaard NHH
Lys58-beta2-microglobulin
present in a nonimmobilized
form, or present in solution.
Treating or preventing a
bone-related or cartilage-
Amgen (Thousand Pisegna M,
WO 200563292 related disease, condition or 12/22/2003 7/14/2005
Oaks, CA, USA) Simonet WS
disorder, or modulating bone
mineral density or bone
strength in an individual,
comprising administering an
amount of an antibody or its
fragment that specifically
binds a polypeptide
comprising fully defined
442−amino acid sequences.
An agonist anti-trkC antibody
comprising a heavy-chain
complementarity determining
region (CDR), and/or a light-
Rinat Neuroscience (S.
chain CDR; useful for treating
WO 200562955 San Francisco, CA, Pons J 12/23/2003 7/14/2005
and/or preventing
USA)
neuropathies including Taxol-
induced, cisplatin-induced
and pyridoxine-induced
sensory neuropathy.
Preventing and/or treating
Type 1 diabetes mellitus in a
prediabetic human subject or
in a human subject suffering ILEX Products (San
WO 200562893 Arthaud L 12/22/2003 7/14/2005
from the disease, comprising Antonio, TX, USA)
administering an amount of
an anti-CD52 antibody, that
is, Campath-1H.
Treating diabetic retinopathy
in a patient by administering
an amount of an antibody to
gamma interferon and/or an
amount of an antibody to
CD20; antibodies are also Advanced Biotherapy
Skurkovich B,
US 20050152902 useful for treating (Woodland Hills, CA, 6/5/2001 7/14/2005
Skurkovich S
hyperimmune reactions, USA)
including transplant rejection,
autoimmune diseases of the
eye and ocular disorders
incidental or connected with
autoimmune diseases.
Treating autoimmune disease
such as rheumatoid arthritis,
psoriasis, inflammatory bowel
disease, Crohn’s disease,
ulcerative colitis, eczema,
asthma, lupus,
atherosclerosis and diabetes,
comprising detecting CD20 or Genentech (S. San
WO 200560999 Brunetta PG 12/19/2003 7/7/2005
CD20-positive B cells in a Francisco, CA, USA)
sample from the patient, and
where CD20 or CD20-positive
B cells is detected in the
sample, administering a
CD20 antagonist or antibody
to treat the autoimmune
disease.
Amplifying nucleic acid
encoding a portion of an
antibody; useful for producing
an antibody library and for
identifying an antibody having Bowdish KS,
Alexion
a desired binding specificity. Frederickson S,
WO 200560641 Pharmaceuticals 12/15/2003 7/7/2005
The method enables Lin Y, Maruyama
(Cheshire, CT, USA)
improved nucleic acid T, Renshaw M
amplification with decreased
mispriming and amplification
of sequences other than the
target sequence.
Detecting the presence or
absence of or determining
whether a patient is at risk for
Bangur CS,
developing a cervical cancer, Corixa (Seattle, WA,
US 20050142620 Zehentner- 8/19/2004 6/30/2005
comprising contacting a test USA)
Wilkinson BK
cervical tissue sample with an
antibody that binds to a
polypeptide and detecting the
amount of the antibody that
binds to the polypeptide.
An immunoassay method
useful for detecting antigens
such as hepatitis B virus
surface antigen. The method Toa Iyo Denshi (Kobe,
JP 2005172546 Kawade Y 12/10/2003 6/30/2005
enables easy, convenient and Japan)
simultaneous detection of the
antigen and the antibody of
the same pathogen.
A novel antibody that is
immunoreactive with a Beckmann MP,
mammalian interleukin-4 Cosman DJ,
Immunex (Seattle, WA,
US 20050118176 receptor (IL-4R), chosen from Idzerda R, March 2/14/1990 6/2/2005
USA)
murine IL-4R and human IL- CJ, Mosley BA,
4R; useful for inhibiting Park LS
binding of IL-4 to IL-4R.
Source: Thomson Scientific Search Service (formerly Derwent). The status of each
application is slightly different from country to country. For further details, contact
Thomson Scientific, 1725 Duke Street, Suite 250, Alexandria, Virginia 22314, USA. Tel:
1 (800) DERWENT (http://www.thomson.com/scientific).
8.2.3 Evaluation of Antibody Engineering
Although, being touted as the most efficient and revered technologies, the
use of monoclonal antibodies obtained by the process of antibody engineering has its
fair share of advantages and disadvantages. Continuous efforts are being made by
the protein technology industry to overcome the major shortcomings of this
engineering technology[8].
Major advantages of the use of monoclonal antibodies through antibody

engineering principles are:
1. Monoclonal antibodies represent single antibody molecules that bind to

antigens with the same affinity and promote the same effector functions. This
confers homogeneity to the monoclonal antibodies so produced.
2. The product of a single hybridoma reacts with the same epitope on antigens,
thus providing specificity to the monoclonal antibody.
3. The Immunizing Antigen, used as the raw material in this technique need not be
pure or characterized and is ultimately not needed in large amounts to produce
large quantities of the monoclonal antibody.
4. It is possible to select for specific epitope specificities and generate antibodies
against a wider range of antigenic determinants.
5. Unlimited quantities of a single well-defined mono-specific reagent can be used
for antibody production.
Inspite of having such great advantages, the technique of antibody does

have a few disadvantages which are in the process of being eliminated.
1. Because the antibody is monoclonal, it may not produce the desired biologic
response thus providing insufficient effector response.
2. Monoclonal antibodies against conformational epitopes on native proteins may
lose reactivity with antigens that have been minimally perturbed. This could
threaten their specificity for the antigens.
3. Antibodies sometimes display unexpected cross-reactions with unrelated
antigens.
4. The production of monoclonal antibodies through antibody engineering requires
a very large commitment in terms of time, effort as well as the capital
expenditure it incurs!
8.2.4 Applications of Antibody Engineering
The Antibody Engineering Technology used to produce monoclonal

antibodies has a variety of academic, medical and commercial uses. The applications
of the Engineered Antibodies can be broadly divided into three classes, viz.,
Diagnosis, Immunopurification and Immunotherapy.
Diagnosis
Antibodies are used in several diagnostic tests to detect small amounts of

drugs, toxins or hormones. In diagnosis, one of the major applications of antibody
engineering is in the detection of pregnancy by assaying of hormones such as the
Human Chorionic Gonadotropin (HCG) with monoclonal antibodies. Once
monoclonal antibodies for a given substance have been produced, they can be used
to detect the presence and quantity of this substance, for instance in a Western blot
test (to detect a protein on a membrane) or an immunofluorescence test (to detect a
substance in a cell). Another diagnostic use of antibodies is the diagnosis of AIDS by
the ELISA test[1]. They are also very useful in immunohistochemistry which detect
antigen in fixed tissue sections. Radioimmunodetection is also widely used. (Fab') 2
and Fab fragments are preferred for imaging, because both targetting and blood
clearance are rapid. Tumours as small as 0.5 cm, which are missed by other
radiological methods, can be detected by radiolabelled antibodies or Fab fragments.
The technique of ELISA (enzyme linked immunosorbant assay), utilizing monoclonal
antibodies has also been used for cytogenetic analysis in wheat[5].
Immunopurification
Immunopurification involves separation of one substance from a mixture of

very similar molecules. Monoclonal antibodies can also be used to purify a substance
with techniques called immunoprecipitation, radioimmunoassay and affinity
chromatography. For instance, individual interferons could be purified using
monoclonal antibodies and could be used for inactivating T-lymphocytes responsible
for rejection of organ transplants. Researchers use monoclonal antibodies to identify
and to trace specific cells or molecules in an organism, for example, developmental
biologists at the University of Oregon use monoclonal antibodies to find out which
proteins are responsible for cell differentiation in the respiratory system. Antibodies
have been used for the purification and quantification of certain molecules present in
trace amounts, such as hormones, cyclic nucleotides, polypeptides, enzymes,
antigens, etc. The assay is extremely sensitive and quantities as low as nano or pico
molar concentrations can be detected in small volumes (l ml) of body fluids such as
plasma, urine or cerebrospinal fluid[5].
In the process of immunoprecipitation, when correct conditions are

employed, antigen and antibody react to form a precipitate. This precipitation
phenomenon has been used to separate and purify enzymes and other antigenic
substances using monoclonal antibodies in a variety of ways. One application of
immunoprecipitation is immunoelectrophoresis. This is the most sensitive method for
the detection of enzymes and antigens in a mixture. It involves combination of
elcctrophoresis and gel diffusion[1].
In affinity chromatography, the monoclonal antibody is generally
immobilized on an insoluble matrix and packed in a Column. Protein solution
containing the specific enzyme is applied over these immobilized antibodies. The
specific enzyme binds with the antibody while other components do not, and thus the
specific enzyme is separated. Antigens can also be purified using affinity
chromatography with monoclonal antibodies[1, 5].
Immunotherapy
Immunotherapy (also known as biologic therapy) is a treatment that uses

certain parts of the immune system to fight diseases. For therapeutic uses,
monoclonal antibodies are so designed that they will neutralize the reaction or
response by one defined antigen, but still preserve the reaction of all other antigens.
Several antigens of T cell receptor complex, including CD3, CD4 and CD8 have been
the targets of specific antibodies for therapy. Most widely used monoclonal antibody
is OKT-I, which has been licensed for clinical use, particularly for the treatment of
acute renal allograft rejections. Monoclonal antibodies have also been used for
treatment of patients with malignant leukemic cells, B cell lymphomas, and a variety
of allograft rejections after transplantation[1]. Antibodies are used in the
radioimmunodetection and radioimmunotherapy of cancer, and some new methods
can even target only the cell membranes of cancerous cells. A new cancer drug
based on antibody engineering technology is Rituxan, approved by the FDA in
November 1997[5]. Immunotherapy for various immune diseases like the infectious
diseases and the autoimmune diseases also are currently being treated by various
monoclonal antibodies designed by the technique of antibody engineering.
Monoclonal antibodies can be used to treat viral diseases, traditionally considered
untreatable. In fact, there is some evidence to suggest that antibodies may lead to a
cure for AIDS[5].
1. Thomson Scientific Search Service (2006)

http://www.thomson.com/scientific
2. Marasco, W.A. (2006) NCFR Centre for Therapeutic Antibody Engineering

http://research.dfci.harvard.edu/nfcr-ctae/
3. Ludwig Institute For Cancer Research (2006) Antibody Engineering Home

Page
http://www.licr.org/D_programs/d4c2_AbEngineering.php
1. Abbott Laboratories
The diverse family of pharmaceutical, medical, and nutritional products

from Abbott Laboratories includes a broad range of specialized medicines; medical
diagnostic instruments and tests; minimally invasive surgical devices; a spectrum of
nutritional supplements for infants, children and adults; and products for veterinary
care. (http://www.abbott.com/global/url/home/en_US)
2. Alexion
Alexion Pharmaceuticals is a leading American biotechnology company
that is preparing to launch its first commercial product, eculizumab in 2007 and
another product pexelizumab being studied in a large Phase III trial.
(http://www.alexionpharm.com/)
3. Biogen IDEC
Biogen IDEC has a strong track record of success in the development,

manufacture and commercialization of novel, first-in-class products that significantly
improve human healthcare with products like Rituxan®, Zevalin® and Avonex®
already reaching phase IV and many more products in clinical trials.
(http://www.biogenidec.com/index.html)
4. Cambridge Antibody Technology
CAT is committed to the development of human monoclonal antibodies as

new treatments for important human diseases to improve patients' lives. HUMIRA® is
already approved and marketed in 57 countries worldwide and nine further human
monoclonal antibodies originating from CAT are in clinical trials.
(http://www.cambridgeantibody.com/html/home)
5. Genentech
Genentech manufactures and commercializes multiple protein-based

biotherapeutics for serious or life-threatening medical conditions. Herceptin®,
Avastin®, Rituxan® are few of the leading products of this company
(http://www.gene.com/gene/index.jsp)
6. Genmab
Genmab A/S is a biotechnology company that creates and develops

human antibodies for the treatment of life-threatening and debilitating diseases.
Genmab has numerous products in development to treat cancer, infectious disease,
rheumatoid arthritis and other inflammatory conditions. Genmab is an international
company with operations in Europe and the United States. (http://www.genmab.com/)
7. ImClone Systems
The only patented drug of this company is Erbitux® (Cetuximab). ImClone

Systems has launched LORHAN (Longitudinal Oncology Registry of Head and Neck
carcinoma), an independent national registry of patients with head and neck cancer.
LORHAN is non-drug specific and provides detailed longitudinal treatment data on
patients managed in all practice settings. (http://www.imclone.com/)
8. Medarex
Medarex is a biopharmaceutical company focused on the discovery,

development, and potential commercialization of fully human antibody-based
therapeutics to treat life- threatening and debilitating diseases, including cancer,
inflammation, autoimmune and infectious diseases. Medarex applies its UltiMAb®
technology and product development and clinical manufacturing experience to
generate, support and potentially commercialize a broad range of fully human
antibody products for itself and its partners.
(http://www.medarex.com/index_flash.htm)
9. MedImmune
The company's marketed products include Synagis® (palivizumab),

Ethyol (amifostine), FluMist® (Influenza Virus Vaccine Live, Intranasal), and
®
CytoGam® (cytomegalovirus immune globulin intravenous (human)), with additional

products in clinical testing. (http://www.medimmune.com/)
10. UCB
UCB is a leading global biopharmaceutical company dedicated to the

research, development and commercialization of innovative pharmaceutical and
biotechnology products in the fields of central nervous system disorders,
allergy/respiratory diseases, immune and inflammatory disorders and oncology -
UCB focuses on securing a leading position in severe disease categories.
(http://www.ucb-group.com/)
8.2.7 References
1. Goding, J. W. (1983) Monoclonal antibodies : principles and practice : production

and application of monoclonal antibodies in cell biology, biochemistry, and
immunology, Academic Press, New York.
2. Roitt, I. M. (2001) Roitt's Essential Immunology, 9 edn, Blackwell Science, Oxford.
3. Barrett, J. T. (1976) Basic immunology and its medical application, Saint Louis
:Mosby.
4. Kuby, J., Kindt, T. & Osbourne, B. (2000) Kuby Immunology, 4 edn, W H

Freeman, New York.
5. Maynard, J. A. & Georgiou, G. (2000) Antibody Engineering, Annual Rev.

Biomedical Engineering. 2, 339-376.
6. Hayhurst, A. & Georgiou, G. (2001) High Throughput Antibody Isolation, Curr
Opin Chemical Biology. 5, 683-689.
7. Baker, M. (2005) Upping the Ante on Antibodies, Nature Biotechnology. 23, 1065-
1072.
8. Stites, D. P., Terr, A. I. & Parslow, T. G. (1997) Medical Immunology, 9 edn,

Appleton & Lange, Stamford, Connecticut.
Chapter 8.3 Protein expression systems
Krutika Wikhe
8.3.1 Introduction
Proteins which are composed of amino acids form the building blocks of the human
body. Their immense importance and role in biological pathways has lead to an
explosion in protein studies. As researchers started to delve into proteins, their
structures and their functions, the need to produce them aroused. This in its turn led
to the search for a proper biological system to produce the proteins such that their
integrity, quality and quantity could validate such a study. Well it didn’t turn out to be
an easy task and I m sure many protein scientists of that era would agree that just
finding such a system and then optimizing that system for a protein proved to be a
daunting task. And we, who do produce proteins on a regular scale for studying their
effects, toxicity, functions, structures and many other uses too ought to be thankful
for those protein scientist who did the major chunk of the work by finding such
biological systems for us.
Once scientists came up with the first biological expression system, viz. E.coli, they
thought that all the problems associated with protein production are history. But then
other problems cropped up like inclusion body formation, intracellular secretion of
proteins, and purification and separation of the desired protein from the rest of the
cellular mass. This lead to a search for a better and more high through put
expression system and from that time till now there has been continuous research for
a more efficient and high throughput protein expressing system. We have come a
long way from using E.coli, although it is still the preferred system over others. Over
the course of time yeast, mammalian cells, Baculovirus and the most recent cell free
protein expression systems have been exploited.
8.3.2 Evaluation of the technology and its applications:

The above mentioned expression systems along with their pros and cons will be
discussed as follows:
¾ E.coli: The genetics and biochemistry of E.coli are probably the best
understood of any known organism. The knowledge gained in studying E.coli
biology has been applied to the development of many molecular cloning
techniques. Most cloning vectors and methods utilize E.coli as a preferred
host, primarily because of the ease with which it can be grown and
manipulated. It is also suitable for expressing proteins because of its rapid
doubling time and its ability to grow on a wide range of nutrient media. It also
provides numerous transcriptional and translational control elements that can
be applied to the expression of foreign genes. The steps involved in foreign
gene expression are :
1) Insertion of the gene into an expression vector, mostly plasmid.
2) Transforming a suitable E.coli host strain with the plasmid for example
by electroporation.
3) Evaluation of protein stability and expression.
4) Once small scale experiments have verified the expression and stability
then large scale production of the protein can be started in fermentation
system.
5) Production is followed by purification and characterization of the
protein.
This flow chart explains the above steps in a diagrammatic fashion:
Fig 8.3.1 A flow diagram for a typical E.coli based expression system.
Ref: http://silver-server.dur.ac.uk/Teaching/Expression_Systems/2_E_Coli/index.htm
The important elements in a typical E.coli expression vector are:

Selectable marker: Expression plasmids should have a sequence encoding
a selectable marker to ensure maintenance of the vector and for
identification of transformed cells.
Origin of replication (ori): Replication of plasmid as an independent
extrachromosomal element is controlled by its origin of replication.
Promoters: A controllable transcriptional promoter is helpful in expression
system so as to control the induction and direct the production of mRNA
from the cloned gene. The most commonly used is the lac promoter which
utilizes the β-galactosidase gene. An example of a commercially available
lac promoter vector is pBluescript [1,2]
The most common expression strategies in E.coli include direct intracellular
expression, secretion and fusion proteins. Incase of intracellular expression
recombinant proteins often form dense insoluble aggregates called
inclusion bodies. Expression of cloned gene products as secreted proteins
has been developed as an alternative to intracellular expression. This can
be achieved by adding an N-terminal signal sequence that can be cleaved
afterwards. But even in secretion techniques, the yield of the protein is
often low and inconsistent. To tackle this problem of inconsistent yields and
protein insolubility fusion proteins were brought into picture. Fusion
proteins are created by expression of the target protein in frame with a
highly expressed protein partner or carrier protein[3]. The most successful
fusion systems use Maltose Binding protein (MBP) and Glutathione-S-
Transferase (GST)[3]. One of the manufacturers of commercial expression
systems is Invitrogen which has developed a new range of systems using
Gateway technology[4,5]. The name of the system is Champion™ pET
Expression System with Lumio™ Technology. Stratagene has developed
the VariFlex™ Bacterial Expression Systems [6].
Although E.coli is the first choice of expression system for any protein
researcher, you really can’t expect that organism to satisfy everyone’s
needs. What I mean by that is, for researchers interested in eukaryotic
proteins which need to be post-translationally modified, a eukaryotic
system must be used. Furthermore, if the protein is expressed in an
insoluble state in E.coli, one way to circumvent this problem is to express
the protein in eukaryotic system.
¾ Yeast: For more than a decade the yeast Saccharomyces cerevisiae has
been used extensively utilized or the production of foreign proteins. One of
the reason for choosing Saccharomyces as a system for expression is the
vast knowledge base about the organism and presence of eukaryotic post-
translational modification pathways. The other positive aspect towards using
yeast as a system for production of proteins is that it has been approved as a
safe organism by the FDA and hence it can be used for production of
biologically important proteins on a commercial scale[7]. The goals achieved
in expression of foreign proteins in S.cerevisiae are achievement of desired
yield, production of proteins with desired post translational modifications and
secretion to the extra cellular medium. Through continuous research in this
field, there are now many vectors and host strains available to direct gene
expression in S.cerevisiae. A variety of choices are now available with
respect to specific elements used to direct expression and secretion like the
promoters used, the signal sequence for secretion, selectable marker and
even the mechanism of replication[7].
Early on in the 1970s, the methylotropic yeast Pichia pastoris was developed
to convert methanol into a high quality protein. By early 1980s the focus
shifted from Saccharomyces to Pichia as a eukaryotic microbial system to
produce large quantities of heterologous protein of interest to protein
researchers[7]. The transformation methods developed for Saccharomyces
work well even with Pichia. We can also produce either intracellular or
secreted protein with Pichia. Secretion requires the presence of a signal
peptide on the expressed protein to target it towards the secretory
pathway[8]. Currently Invitrogen has the exclusive rights for the distribution of
Pichia expression technology[9].
The preferred reason for using Pichia is it gives a much higher yield of
desired protein than Saccharomyces. Another reason for expression in Pichia
is it secretes very low levels of its native proteins and so it can be easily
purified from the medium.
The major producers of commercial yeast systems are Invitrogen, BD
Biosciences. BD Biosciences Clontech offers the YEASTMAKER™ Yeast
Transformation System and the YEASTMAKER™ Yeast Plasmid Isolation Kit,
as well as many types of yeast media and MATCHMAKER Yeast Two-Hybrid
System[10]
But as with all other systems, there are some problematic issues concerning
protein production in yeast too. Briefly the following problems arise:
1. Genetic instability of the transformed yeast particularly during scale-up.
2. Inability to produce toxic proteins.
3. Inefficient secretion of larger (>30kDa) proteins.
4. Proteolysis of secreted proteins.
¾ Baculovirus:
Baculovirus has emerged out as a popular system for overproducing
recombinant proteins in eukaryotic cells. The main difference in Baculovirus
expression system and that of yeast is that in Baculovirus we can use a
helper-independent virus that can be propagated to increase titers in insect
cells. This in turn would result in high protein production which after all is one
of the aims during protein production. Another positive aspect of using
Baculovirus is it has a large genome and hence can take in large inserts of
foreign DNA. Finally Baculovirus being non-infectious to humans they give a
possible advantage when expressing oncogenes or toxic proteins[11].
Currently the most widely used system employs the lytic virus Autographa
californica. The basic methodology of expressing foreign proteins in this
system involves the following steps:
1. Gene is first cloned into a transfer vector
2. The recombinant vector is transfected into insect cells.
3. In a homologous recombination event, the foreign gene is inserted into
the viral genome.
4. Recombinant viruses can be identified by DNA hybridization or PCR
technique.
The general advantages and disadvantages of the system are as follows:

Table 8.3.1 Advantages and disadvantages of Baculovirus system. Ref: [8]
Some commercially available Baculovirus systems are by BaculoDirect™ by

invitrogen and BD BaculoGold™ by BD Biosciences[12,13]. The following is a
diagrammatic representation of the Baculovirus expression system in
BaculoGold™
Fig 8.3.2 Baculovirus expression in BaculoGold™
Ref: http://www.bdbiosciences.ca/image_library/baculogold-system.jpg
¾ Mammalian cells:
In recent years, mammalian cells have been used vastly for production of
recombinant proteins mainly those requiring post-translational modifications.
They also serve as means for examining aspects of gene replication,
transcription and translation. Although there is a wide range of mammalian
cells available for expression only few have emerged as systems of choice for
protein production. The common features of mammalian cell lines desired
are, they should be capable of continuous growth and can be grown in
suspensions, they should have low risk of infection by viruses and can be
characterized easily with respect to morphology and gene copy number[14].
The general procedure for mammalian cell expression is as follows [8]:
Fig 8.3.3 Protocol for mammalian cell expression.
Mammalian cells generally are preferred when expressing proteins for human
applications. The following are the typical uses of mammalian expression
systems:
1. Verification of cloned gene product.
2. Production and isolation of genes from cDNA libraries.
3. Production of correctly folded and glycosylated proteins
4. Production of clinically active viral surface antigens and monoclonal
antibodies.
Mammalian produced cells are quality controlled through a process whereby
incompletely folded and unassembled proteins into the secretory pathway are
selectively inhibited.
Even the use of human cell line is not perfect, since transformation required
to produce a stable cell line might in turn result in altered glycosylation.
Moreover mammalian expression techniques are time consuming, difficult on
a larger scale and costly. Mostly optimal techniques are a compromise
between transfection efficiency and post treatment viability of cells and
regulating this factor is generally troublesome. Complex nutrient requirement
and low product concentration also means that the end product should
warranty this approach to be commercially viable.
One of the major sources of commercially available mammalian expression
vectors are Stratagene[15] and BioLabs[16].
¾ Cell-Free protein synthesis:

Over four decades ago two scientists, Nirenberg and Matthaei did
revolutionary studies in cell free protein synthesis[17]. From then till now it
has covered lots of research ground and has come up as a valuable tool for
understanding how mRNAs are translated into functional polypeptides. Cell
free expression system is based on the early demonstration that cell viability
is not required for protein synthesis to occur. Translation can occur even by
using a crude lysate from any given organism which provides all the required
translational machinery, enzymes, tRNA along with exogenously supplied
RNA template amino acids and an energy source. Although any organism can
be used for the preparation of a cell free protein extract the most popular
ones are E.coli, wheat germ, and rabbit reticulocytes[17]. A key objective for
cell translation systems is to synthesize biologically active proteins. Cell free
systems offer many advantages over traditional cell based expression
methods. We can easily modify reaction conditions to favor protein folding,
decrease cell sensitivity towards product toxicity. It is also suitable for high
through-put reaction systems because of reduced reaction volumes and
process time[17,18].
We have seen that each system has some unique features and qualities
which allow it to be used for certain kinds of proteins. Most protein
researchers base their choice of expression system on the kind of protein
they have to express and the extent of purification, yield and structural and
functional conformation they desire. To give an overall comparison of all the
above mentioned expression systems the following diagrammatic
representation would prove helpful:
Fig 8.3.4 Comparison of protein expression systems.
Ref: http://www.proteinsciences.com/technology/technology_why.htm

Due to an increasing interest in protein studies there has been an increased demand
for recombinant protein production. Lawrence Livermore National Laboratory and
Onyx Pharmaceuticals, Inc. have successfully produced an automated system foe
expression of large number of proteins encoded by cDNA clones from IMAGE
(Integrated Molecular Analysis of Genomes and their Expression)[19]. Baculovirus is
the system of choice in this technique of converting cDNAs to proteins. This system
was developed for the analysis of human proteome. Functional and structural
analysis of novel gene products identified by Expressed Sequence Tag (EST) is also
possible by this system.
A technique called SPEX, Surface Protein Expression Vector using gram positive
bacteria Streptococcus gordonii was developed by Myscofsky et.al[20]. They
demonstrated that exogenous DNA sequences can be fused to sequences specifying
surface expression resulting in the production and secretion of heterologous proteins.
Advantages of using this system is it can produce proteins on a larger scale by taking
advantage of natural pathway that is designed to export proteins of varying size and
structures to the outside of the bacterial cell[20]. The expressed protein can either be
anchored on the surface or secreted in the media thus avoiding the formation of
inclusion bodies.
To allow recombinant proteins to play a role in large industrial applications a more
robust and efficient production technology is required. With this aim in mind,
Srinivasan et.al[21] developed a novel prokaryotic system based on a nonpathogenic
organism Ralstonia eutropha. This systems permits high cell density culture in a
defined minimal media and does not require antibiotics for protein stability or IPTG
for protein induction. Srinivasan et.al used the strain NCIMB 40124 of R.eutropha in
their experiments. They tried to express a protein (organophosphohydrolase[OPH]
from Pseudomonas diminuta MG) which is difficult to obtain in soluble form from
E.coli. they achieved cell densities of 150g/l which in turn would give rise to higher
protein yields and without any inclusion body formation.
Another innovative method developed to overcome the problems faced in traditional
protein expressing system was the use of actinomycete[22]. N. Nakashima and t.
Tamura[22] tested the norcardioform actinomycete Rhodococcus erythropolis which
grows at temperatures ranging from 40 C to 350C as an expression host system. The
expression was controlled by using the antibiotic Thiostrepton. It is a known fact that
protein are better expressed and produced by the host cell at lower temperatures.
Incase of R.erythropolis few proteins were found to be expressed at much higher
levels at low temperatures. DNaseI which is expressed as an insoluble protein in
E.coli was expressed as a soluble protein at 40C in R.erythropolis. moreover proteins
derived from cold-adapted organisms require expression at lower temperatures as
they have low stability at higher temperatures[22].
8.3.4 Key industry suppliers:

NextGen sciences (http://nextgensciences.com ) have developed the
Expressionfactory™, an instrument that automates the cloning, expression and
purification within a single stage. When combined with its corresponding software it
enables the parallel exploration of different constructs, host cells and growth
conditions[23]. Another cost effective approach to high through put protein production
mainly for structural analysis purposes has been provided by PSF (Protein Structure
Fabrik in Berlin, http://proteinstructurefabrik.de ). It involves the parallel expression
and purification of recombinant proteins with His tag (Histidine) or GST tag
(Glutathione S-Transferase) from bacterial expression systems. Invitrogen
(http://www.invitrogen.com ) has presented the Gateway system for E.coli extracts
whereas Roche Applied Science (http://roche-appliedscience.com ) has produced the
Rapid Translation System for cell-free expression using E.coli and wheat-germ
extracts[23].
8.3.5 References:
[1] Stratagene (2006) Stratagene.

[2] Stratagene (2006) in:
http://www.stratagene.com/products/displayProduct.aspx?pid=267.
[3] Esposito, D. and Chatterjee, D.K. (2006) Current Opinion in Biotechnology
17, 353-358.
[4] Invitrogen (2006) in:
https://catalog.invitrogen.com/index.cfm?fuseaction=viewCatalog.viewProduct
Details&sku=&productDescription=1031&ref=http%3A%2F%2Fwww%2Egoo
gle%2Ecom%2Eau%2Fsearch%3Fnum%3D20%26hl%3Den%26safe%3Doff
%26q%3DE%2Ecoli%2Bexpression%2Bsystem%252Cinvitrogen%26btnG%3
DSearch%26meta%3D.
[5] Invitrogen (2006) in: http://www.invitrogen.com/content.cfm?pageid=9608.
[7] Romanos, M.A., Scorer, C.A. and Clare, J.J. (1992) Yeast 8, 423-488.
[8] Wiley, J. (1997) Current Protocols in Protein Science 5.
[9] Invitrogen (2006) in:
http://www.invitrogen.com/content.cfm?pageid=10838&CID=KNC-
GOOGLE&s_kwcid=yeast%20expression%20system|527213053.
[10] Biosciences, B. (2006) in:
http://www.clontech.com/clontech/proteomics/pdf/ProteinExpression.pdf BD
Biosciences.
[11] Sridhar, P. et al. (1994) Journal of Biosciences 19, 603-614.
[12] Invitrogen (2006) in: http://www.invitrogen.com/content.cfm?pageid=9981.
[13] Biosciences, B. (2006) in:
http://www.bdbiosciences.com/pharmingen/products/display_product.php?key
ID=72.
[14] Rai, M. and Padh, H. (2001) Current Science 80, 1121-1128.
[16] BioLabs (2006) in:
http://www.neb.com/nebecomm/products_intl/productE3000.asp.
[17] Katzen, F., Chang, G. and Kudlicki, W. (2005) Trends in Biotechnology 23,
150-156.
[18] Stevens, R.C. (2000) Structure 8, R177-R185.
[19] Joanna S. Albala, K.F., Ian R. McConnell, Karen L. Pak, Peg A. Folta, Brian
Karlak, Bonnee Rubinfeld, Anthony H. Davies, Gregory G. Lennon, Robin
Clark, (2001) Journal of Cellular Biochemistry 80, 187-191.
[20] Myscofski, D.M. and Hruby, D.E. (1998) Protein Expression and Purification
14, 409-417.
[21] Srinivasan, S., Barnard, G.C. and Gerngross, T.U. (2002) Appl. Envir.
Microbiol. 68, 5925-5932.
[22] Nakashima, N. and Tamura, T. (2004) Biotechnology and Bioengineering 86,
136-148.
[23] Dale, G.E. (2004) Drug Discovery Today 9, 783-784.
PROTEIN FOLDING STRATEGIES
STUDENT NAME: SYED SHABANA AMEER

STUDENT NUMBER:s3147908
Introduction:
Proteins are composed of 20 basic units called amino acids which consist of a central
carbon atom (the alpha-carbon) bound to an amino group (NH2), a carboxyl group
(COOH), a hydrogen atom, and one of 20 different R groups. The alpha-carboxyl
group of one amino acid is joined to the alpha-amino group of the next by a peptide
bond to form chains of amino acid residues (polypeptide chains). Proteins are
functional polypeptide chains. The unbranched chain of amino acid residues has
direction, beginning at the amino end, and the chain of regularly repeating peptide
bonds is called the backbone, while the R groups projecting from the backbone are
known as side chains. The peptide bonds of the backbone are rigid and planar due to
their partial double bond character.
There are an unlimited number of conformations that proteins could adapt, but most
fold spontaneously into one particular stable shape. This particular shape occurs
because backbone groups and side chains interact with each other, and water, so that
particular conformations have more stabilising interactions than others. Randomly
coiled protein is void of its activity, and that isolated proteins in solution can revert to
their original active conformation after denaturing conditions are removed. It was
concluded, therefore, that the information needed to refold that protein into its native
form must be inherent in the amino acid sequence and that a protein’s sequence
specifies its conformation.
Globular proteins fold into a compact globular shape. In contrast, fibrous proteins do
not fold into a compact shape as their function lies in their fibrous nature. The
hydrophobic nature of certain amino acids is the main driving force in the adoption of
these compact structures. The side chains of amino acids can be polar or non-polar.
Non-polar residues are hydrophobic and pack together in the interior of the globular
protein structure to avoid contact with water, whereas polar residues, and the polar
groups in the backbone, are hydrophilic and form hydrogen bonds with each other and
water. These hydrogen bonds form a major part of protein structure stability, and are
formed when a hydrogen atom is shared between a hydrogen donor and a hydrogen
acceptor. When hydrogen bonds form between backbone groups in proteins the alpha-
amide group is the donor and the alpha-carbonyl group the acceptor.
The folding of a protein can not be a random search through all possible structures as
this would take far longer than the observed time, of approximately one second, for
typical proteins to fold from random to their folded state. Among the millions of
possible folding patterns, proteins take up one working, native, structure. Proteins are
thought to initially fold rapidly into a structure in which most of the final secondary
structure elements have formed and are aligned in roughly the correct way. This is an
open and flexible conformation, called a molten globule, and is the starting point for a
relatively slow process in which the side chains are repeatedly adjusted to form the
correct tertiary structure. This second stage is thought to have a variety of correct
pathways to the final conformation. This process can be summarised as local folding,
formation of long range interactions, then local rearrangements to give the final most
stable folded state. This model has the assumption that, although hydrophobic
residues direct the initial folding, they also direct the slower tertiary folding, and
allows rearrangements to be made.
In aqueous environments protein folding is driven by the tendency of hydrophobic

residues to be excluded from water. This is only possible because polar residues and
backbone groups can interact with the water at the protein exterior and with
themselves in the non-polar environment of the protein interior. This conformation is
further stabilised by electrostatic and Vander Waals bonds. The conformational
folding of proteins destined for non-aqueous environments, such as spanning the cell
membrane, differs because the non-polar residues no longer need to pack into a
hydrophobic core.
The forces and interactions described above fold a protein into a particular 3D
conformation, and this apparently complex structure of proteins is in fact governed by
a set of relatively simple principles. These principles are explained by splitting the
conformation into various levels which build on each other to produce the entire
protein shape. To ensure proper folding, cells have evolved a sophisticated and
essential machinery of proteins called molecular chaperones that assist the folding of
newly made polypeptides. The importance of proper protein folding is underscored by
the fact that a number of diseases, including Alzheimer's and those involving
infectious proteins (prions), result from protein-misfolding events.
Molecular chaperones have an essential role in the regulation of protein conformation
states -- the process during which transient or stable interactions with client proteins
affects their conformation and activity. Chaperones capture unfolded polypeptides,
stabilize intermediates, and prevent misfolded species from accumulating in stressed
cells. The capacity of the Hsp70 and Hsp90 chaperones to regulate these processes
involves a constellation of positive and negative co-chaperones that function in
various combinations to interact with chaperones to release folded proteins, to
facilitate the assembly or disassembly or chaperone-containing heteromeric
complexes, to confer substrate specificities, and to affect subcellular trafficking.
All the information needed to specify the three dimensional conformation of protein is
encoded by proteins amino acid sequence. In vitro protein folding is studied primarily
using small model proteins consisting of fewer amino acids. These small model
proteins can be unfolded and they spontaneously fold back into native structure upon
removal of denaturant. Several features define the basic differences between folding
of proteins in vivo and in vitro.Firstly the cytosol is an extremely crowded
environment with more macromolecular concentrations. Such macromolecular
crowding leads to excluded volume effects which strongly affect biochemical rates by
increasing protein association constants and thereby increasing intermolecular
interactions including aggregation. Secondly about one third of newly synthesized
proteins must be targeted to an organelle or are secreted to an extra cellular
compartment where their functions are fulfilled. These proteins are targeted as nascent
or loosely folded polypeptides to their translocation machinery where they fold to
their native states. Thirdly all proteins are synthesized by the ribosome from N- to C-
terminus, implying that as long as polypeptide synthesis proceeds, the folding
information is incomplete. Three different models explain how and when a folding of
a polypeptide chain to its structure is achieved in living cells. The first model suggests
that folding of a growing polypeptide chain is postponed by chaperone binding until
its synthesis is completed. Here folding is initiated only upon release of protein from
ribosome. The second model suggests that formation of secondary and tertiary
structures begins as soon as polypeptide chain emerges from ribosomal exit. The third
model proposes a step wise folding where initial folding is initially delayed and is
allowed to proceed only when sufficient sequence information is available for the
generation of folded domain.
Recent Advances:
Recent adavances in genetic engineering have heightened the interest in research

related to predicting native protein folding and docking conformations. The ability to
predict these structures would greatly increase our understanding of hereditary and
infectious diseases and aid in interpretation of genomic data.Also the ability to
understand peptide docking would revolutionize the process of denovo drug design.
Also the recent advances in genetic engineering, high powered computing and global
optimization continue to stimulate interest in the area of molecular modeling and
protein structure prediction. The goal of these efforts is the ability to correctly predict
native protein conformations and the binding interactions of macromolecules. These
two problems currently dominate the field of computational chemistry and, through
the use of detailed molecular models; they have also greatly influenced research.
Cellular activities are carried out by interactions among many proteins and genes. Set
of coordinated interactions involved in a certain cell process is referred to as
pathways, for example, wnt pathway. Understanding and finding these organized
interactions is important. Due to recent advances in biotechnology, the amount of
partial data related to interactions, proteins, genes etc. is increasing tremendously.
Using this fragmented data to infer knowledge on cellular networks is an important
and active research area. For example, micro array expression profile data gives us
what genes are expressed together and how much. Expression profile data can be used
by itself for the purpose of understanding pathways up to a point. However, if we can
combine expression profile data with protein interaction data (there are several
databases of known interactions), we can infer new knowledge more effectively. The
reason is that, most probably, proteins acting in a pathway are expressed together
(similar expression profile) and most probably these proteins interact with each other.
Various methods from graph based modelling to association rule mining can be used
to bring together interaction data and expression profile and infer new knowledge.
Feeling the molecular forces through a haptic device (i.e. force reflecting robotic
device) while visualizing and manipulating them in 3D virtual environments will be
an invaluable tool for engineers and scientists in almost every field. The haptic
feedback will allow finer control of atoms during manipulation and provide a gateway
between our world and the “nano” world. The role of force feedback in molecular
simulations with applications to protein-ligand docking are investigated. Protein-
ligand interactions in biochemical applications determine phenomena ranging from
sensory perception to enzyme catalysis Computationally fast models are developed
for simulating molecular interactions and then use the haptic device during the
simulations to guide a ligand into a receptor site while reflecting the forces acting on
the ligand to the user in real-time. Presence of a haptic interface will accelerate the
binding process and reduce the development time involved in scientific analysis.
Proteins need to be flexible in order to perform their cellular functions. For example,
most, if not all, biological processes are regulated through association and
dissociation of protein molecules. Thus, the elucidation of the mechanism governing
flexibility is critically important in biology and health sciences for the ultimate goal of
controlling the functions of proteins and designing new proteins (protein engineering)
and new drugs. In many proteins, large conformational transitions involve relative
movements of almost rigid structural units. The vibrational motions are indicative of
the large amplitude motions. The largest amplitude motions obtained by normal mode
analysis will be compared with the conformational changes of the proteins observed
by the crystallographers upon a substrate (such as a drug) binding. In this study a set
of proteins with both open and closed (with and without a ligand) conformations will
be extracted from the protein data bank. These conformations will be analyzed and a
computational tool will be developed to model these proteins' motions. An
optimization algorithm will be used to find the combinations of these modes in order
to have a trajectory between the open and closed conformations.
Advantages And Disadvantages:
Understanding how proteins fold not only is one of the most interesting theoretical
problems in molecular biophysics but also has far-reaching medical and
biotechnological consequencesThe great advantage of a really simple model is that
you can solve it exactly, at least for short chains of amino acids. You can examine
every possible folding of every possible sequence, picking out the ones of interest.
You can know with certainty which configurations have the most favorable
properties. Another advantage of a simple model is that you don't have to be an expert
in protein chemistry or molecular dynamics to play with it. Determining the process
by which proteins fold into particular shapes, characteristic of their amino acid
sequence, is commonly called the protein folding problem. The Protein Folding
Problem refers to the combinatorial problems involved in enumerating the
conformations of a given Protein molecule. Let each amino-acid residue in a 100
residue protein have 6 possible conformations, this leads to 6^100 possible
conformations available for this protein, this calculation does not include side chain
conformations which will increase the number of degrees of freedom further. The
question is now how does the protein fold given this large number of possible
conformations. These simple calculations urge the development of new efficient and
accurate search methods. Solving the folding problem has enormous implications:
exact drugs can be designed theoretically on a computer without a great deal of
experimentation. Genetic engineering experiments to improve the function of
particular proteins will be possible. Simulating protein folding can allow us to go
forward with the modelling of the cell. Protein folding can go wrong for many
reasons. When an egg is boiled, the proteins in the white unfold and misfold into a
solid mass of protein that will not refold or redissolve. In a similar way, irreversibly
misfolded proteins form insoluble protein aggregates found in certain tissues that are
characteristic of some diseases, such as Alzheimer's Disease.
Applications:
Main application of protein design is to understand why proteins fold and why they
misfold and aggregate. Understanding protein folding is important due to its
applications in the field of biomedicine (drug design, Mad Cow disease cause, etc.,)
and nanotechnology (self-assembly of nanomachines).The shape of a protein is the
principal determinant of its function. Arbitrary strings of amino acids do not, in
general, fold into a well-defined three-dimensional structure, but evolution has
selected out the proteins used in biological processes for their ability to fold
reproducibly into a particular three-dimensional structure within a relatively short
time. Some diseases are actually caused by slight misfoldings of a particular protein.
Understanding the mechanisms that cause a string of amino acids to fold into a
specific three-dimensional structure is an outstanding scientific challenge.
Appropriate use of large scale biomolecular simulation to study protein folding is
expected to shed significant light into this process. The level of performance
provided by Blue Gene (sufficient to simulate the folding of a small protein in a year
of running time) is expected to enable a tremendous increase in the scale of
simulations that can be carried out as compared with existing supercomputers.
Immunological therapeutic approaches are currently being developed for a number of

conformational neurodegenerative conditions, including Alzheimer's disease (AD)
and prion disease. Passive immunization is now in clinical trial for the treatment of
AD. In wild-type prion model mice active and passive immunization can produce
resistance to prion infection with prolongation of the incubation period. Mucosal
prion vaccines which can prevent infection in a proportion of animals later exposed to
the prion agent orally. These studies can be used to develop active vaccines for
livestock and eventually in humans who are at risk for developing prion infection.
Mutants of the gonadotropin releasing hormone receptor are frequently misrouted

within the cell, but otherwise competent proteins. "Pharmacoperones," small
molecules that penetrate cells and correct folding,and protect such defective
molecules, allowing the misfolded mutants to escape retention by the cell's quality
control apparatus and route correctly to the plasma membrane. This observation and
the indication that disease is a frequent corollary of protein misfolding/misrouting
suggests that protein rescue may be an under-appreciated therapeutic approach. The
observation that a percentage of wild-type proteins may also be misrouted suggests
that this is a novel form of post-translational regulation associated with normal
function that can also be therapeutically exploited.
Key industrial suppliers(key to engineering designs) Los Alamos National

Laboratory:
Protein production can be regulated by transcription factors that bind to specific DNA
sites thus regulating the transcription rate of proximal genes. Finding these sites
known as Transcription Factor Binding Sites is fundamental to understanding gene
expression regulation. Professor Uri Keich in computer science works on this motif
finding problem. To find the most pronounced motifs in input sequences and to
analyse their significance to determine if they are artefacts of the size of data. This
work led to innovative significance evaluation of other statistical tests that is
especially important for analysing large datasets.
Many neurodegenerative diseases such as Alzheimers disease and prion diseases

cannot be definitely diagnosed in a person.Only autopsy can confirm the disease. In
chemical and Biomolecular engineering, Professor Kelvin Lee achieved a proof of
concept for using spinal fluid in an objective molecular based test for Alzheimers
disease,ante-mortem.Dozen of the 2000 or more proteins in the cerebrospinal fluid of
patients are expressed differentially resulting in identifiable protein bar codes. These
barcodes are useful in diagnosis and result in improved treatments.
Proteins carry out the cells work acting as catalysts and controllers for numerous
chemical reactions and helping to give organs and tissues their shape. Studies of a
protein molecules intricately folded,3D structure are important for medical research,
drug development, chemical industrial processes and basic research because a
proteins structure determines its function. Crystals grown from dissolved protein are
useful to know about protein structure and are useful in research. This is a new
technology called rapid assay of protein folding which uses folding of a green
fluorescent protein to monitor folding and solubility of a test protein. This is done by
linking the two proteins in a hybrid molecule. When the hybrid is synthesized in the
host cell, the green fluorescent protein achieves its fluorescence if proper folding took
place. Folding assay can be used to treat incurable diseases like Alzheimers and
Huntingtons.
References:
http://www.crbmb.com/cgi/reprint/39/5-
6/261.pdf#search=%22role%20of%20chaperones%20in%20folding%20of%20protein
%22 role of
access date 15/09/06.
http://ccbb.ku.edu.tr/Teaching/ms_projects_offered.htm. access date 24/09/06.
Cyrus Levinthal
Are there pathways in protein folding ?
J. Chim. Phys. (1968), vol 65, pp 44-45 access date 10/10/06.
http://gams.cam.nist.gov/hpcc/cstl/proj-2.html access date 27/09/06.
C. Clementi, H. Nymeyer and J.N. Onuchic. (2000). Topological and energetic

factors: what determines the structural details of the transition state ensemble and 'on-
route' intermediates for protein folding? An investigation for small globular proteins.
Journal of Molecular Biology, 298: 937-953. access date 19/10/06.
protein folding disorders http://www.healthtech.com/2006/pfd/index.asp access date

22/10/06.
http://www.lanl.gov/news/dateline/Dateline0999.pdf
Moody PCE, and AJ Wilkinson, Protein Engineering, IRL press Oxford,1990.

Chapter 8.5 Protein Arrays and their Applications
Huong Nguyen
8.5.1 Introduction
The transition from genomics to proteomics for studying protein interactions has
brought the field of protein arrays into the limelight [1,2]. In allowing the fast and
miniaturised parallel analysis of massive numbers of diagnostic markers in complex
samples within a single experiment, high-throughput (HT) protein arrays are
promising tools for discovery and validation of biomarkers, drug screening,
diagnostics and clinical assays [3-8]. The technological feasibility of protein arrays
depends on the different factors that enable the arrayed proteins to recognise
molecular partners and on the specificity of the interactions involved [1].
There are two general types of protein arrays. Firstly, analytical arrays utilise
antibodies, antibody mimics or other proteins to measure the presence and
concentrations of proteins in complex mixtures. Analytical arrays can be subdivided
into forward- and reverse-phase protein arrays [9]. Forward-phase protein arrays
immobilise a single test sample containing several different target analytes on the
substratum. Reverse-phase arrays consist of multiple, different samples that are
immobilised on a chip. Each spot represents an individual test sample [10]. This
format allows multiple samples to be analysed under the same experimental
conditions for any given analyte [10,11]. Secondly, functional protein arrays assess a
collection of target proteins or even an entire proteome for a wide range of
interactions and biochemical activities [8,12,13].
Various types of HT protein array techniques include protein microarrays, antibody

microarrays, aptamer microarrays, protein nanoarrays and microfluidic arrays [5]. All
arrays are essentially bait-and-capture assays. Bait molecules are immobilised on a
substratum as homogeneous or heterogeneous spots. These molecules can be
aptamers, antibodies, cell lysates, phage or recombinant protein/peptide, a nucleic
acid or tissue. The capture molecule can be a complex biologic mixture such as
serum or a cell lysate, antibody or ligand [10]. After washing and blocking surface
unreacted sites, the immobilised molecules (probe) interrogate the sample applied to
the array (analyte). Thus the specific presence or absence (and sometimes quantity)
of targets are uncovered. By scanning the entire array a large number of binding
events are detected in parallel [11] (Fig 1).
Fig. 1 Typical protein array experiment [11]
Proteins can be arrayed either on flat solid phases or in capillary systems. Preferred
solid phases are modified glass or filter membranes because of their low-
fluorescence background. Binding can be covalent or non-covalent. Parameters such
as charge, viscosity, membrane pore size, pH, binding capacity and non-specific
binding play important roles in the generation of protein arrays [5].
Protein arrays are usually printed (gridded/spotted) and imaged using the same
arrayers and scanners as for DNA arrays. Arraying devices are largely pin-based
systems that transfer nanoliter amounts of liquid either on the outside of solid pins or
inside a split- or ring-shaped reservoir. Current detection strategies are classified as
label-free methods and labelled probe methods. Label-free methods include mass
spectrometry (utilises a protein-selective surface for immobilisation of a complex
protein solution) [5] and surface plasmon resonance (SPR) (optical biosensors for
monitoring biomolecular interactions) [4]. Imaging has also been based on either
direct fluorescent labelling of antigens, indirect labelling of antibodies or sandwich
assays using secondary antibodies or specific antibody-binding reagents [5,11] (Fig
2). Different fluorophores allow multiplexing and differential protein expression
profiling [5].
Fig. 2 Representation of labelled probe methods used in protein array detection [11]
Ordered protein spots are arranged in either planar macroarray or microarray

formats. The format utilised reflects the relative size and number of spots per square
centimetre to be studied. Macroarrays are ideal for the study of dozens of proteins,
meanwhile microarrays are ideal for the large-scale analysis of proteomes [1]. As a
result of miniaturisation, microarrays can analyse many parameters in parallel and
only require minimal amounts of reagents and sample [14].
As the potential of protein arrays garners increasing attention, researchers are

investing more effort into cultivating the technology. As a result, many advances in
the field have arisen in the last five years in the areas of surface chemistry, capture
molecule attachment, protein labelling and detection methods and HT protein
production.
Surface Chemistry
Considerable effort has been made in the last years to improve immobilisation of
proteins on modified glass surfaces. Examples of recent advances include:
Introducing functional groups on the surface by glass modification with

organosilanes such as 3-glycidoxypropyltrimethoxysilane (GOPS) or 3-
aminopropyltriethoxsilane (APTES). Organosilanes can directly provide the
functional groups for ligand attachment (GOPS) or react with a bifunctional ligand
bearing the desired reactive group [11].
A microarray surface has been developed with ProLinkerTM, a calixcrown

derivative with a bifunctional coupling property that permits efficient
immobilisation or capture proteins on solid matrices such as gold films or
animated glass slides [11].
Gels such as polyacrylamide or agarose are being used to physically entrap of
proteins in. The 3D structure of these substrates increases the loading capacity
and does not disturb the potential functional sites or regulatory domains of the
protein. The aqueous environment of the gel also reduces protein denaturation
[11].
Some researchers are implementing microfluidic printing of arrayed chemistries

on individual protein spots blotted onto membranes. Other researchers are using
in-jet printing technology to create protein microarrays on chips [15].
Capture Molecule Attachment
The selection and production of the capture agents are the most critical points in
protein-detecting arrays. They must be highly specific for the protein of interest and
with an affinity sufficient to capture even proteins at very low concentration [11].
Antibodies are ideal capture molecules. Advances in antibody microarrays have

established methods for selection, production and purification of antibodies with high
affinity and reduced cross reactivity. In addition, antibody microarrays containing
more than 200 polyclonal and monoclonal antibodies specific for cell cycle, apoptosis
and nuclear signalling proteins are now commercially available. This allows for the
analysis of protein abundance change in biological samples in a very large dynamic
range using a two-colour approach [11].
Two array technologies quickly being adopted by researchers are 2D arrays and
bead arrays. In 2D arrays the capture agents are arrayed into planar substrates such
as polystyrene, glass or silicon. These arrays utilise fluorescently labelled reporter
molecules and are analysed using microarray scanners. In contrast, bead arrays
immobilise capture agents onto beads containing an integrated reporter dye. The dye
encodes for the identity of the capture agent on the bead. These arrays are read
using a fluorescent particle counter [12].
Protein Labelling and Detection Methods
Advancements in protein labelling and detection methods include:
Atomic force microscopy (AFM) has been developed for detection at the singular
molecular level in protein nanoarrays which exhibit almost no detectable non-
specific protein blinding [10]. It is also currently widely used for surface
characterisation in protein microarrays. AFM reveals the change in height of an
immobilised protein upon binding with its cognate molecule [11].
Detection methods developed for microarrays, due to miniaturised format, are

required to provide high sensitivity (high signal to noise ratio) and HT. The use of
probes and signal amplication techniques with chromogenic or fluorescent probes
has led to the attainment such criteria [11].
Surface enhanced laser desorption ionisation (SELDI) is a reverse screening

technology suited for the detection of small proteins and peptides. SELDI
technology is easy to handle and suitable for the rapid detection of differences in
total protein content of different samples. This approach is a rapid screening
platform for any unknown protein biomarker [9].
Semiconductor quantum dots have been applied as labelling techniques. They

are brighter and more stable than organic dyes. Researchers have successfully
applied such quantum dots for the labelling of proteins on cells and within the
cytoplasm and nucleus [16].
HT Protein Production
Manufacturing HT protein arrays requires the efficient production of large numbers of

proteins. This calls for highly parallel and automated recombinant expression
systems. HT subcloning of human open reading frames have been illustrated.
Recombination-based cloning allows a rapid exchange of vectors and expression
systems. A recent development, the pTriEx vector (Novagen, USA), can be used for
expression in E. coli, baculovirus-infected insect cells and vertebrate cells [5].
Multidimensional chromatography of cell and tissue lysates or cellular subfractions

could become an important alternative to recombinant protein expression. By
consecutive chromatography and isoelectric focusing, cellular protein extracts have
been directly fractionated, arrayed and detected with antibodies [5].
Cell-free expression has become an alternative to cell-based systems for HT

applications. Proteins are expressed from cDNA templates in cell-free systems which
can easily be generated by PCR and stored [16].
The production of proteins using cDNA libraries in E. coli with subsequent purification
remains the gold standard. The procedures were adapted to HT expression in fully
automated systems. The purification is mainly based on short affinity tags to either
the N or C terminus of recombinant proteins and involves immobilised metal affinity
chromatography to affinity media. Alternate hosts such as Pichia pastoria and
Saccharomyces cerevisiae have been tested for HT protein expression [16].
Protein arrays are an evolution of previous technologies such as DNA arrays.

Consequently, protein array technologies exhibit some of the advantages and
disadvantages of these approaches. But protein arrays also present a number of
advantages and disadvantages due to the unique physical and chemical
characteristics of proteins.
Advantages
The key advantages of protein arrays over other techniques are based its capacity to
characterise a huge number of ordered protein spots simultaneously, thus replacing
numerous individual binding parameters in parallel assays with different probes [1].
Another advantage is that protein arrays, derived from DNA arrays, can be
manufactured using technologies adapted from DNA microarray production [17,18].
DNA arrays have been successful in gene expression profiling and mutation
mapping. As the focus shifts from genomics to proteomics, protocols are needed to
study the activity of encoded proteins that directly manifest gene function [13].
Protein arrays can accomplish this providing the proteins’ natural shape and
functionality are maintained [5].
Protein array technologies benefit from current techniques requiring higher volumes
of capture reagents and being less sensitive. For example, ELISA experiments
require nanogram or microlitre amounts of capture reagents and display picomolar
sensitivity. In contrast, protein microarrays require picogram or picolitre amounts of
capture agents and display femtomolar sensitivity. Protein microarrays also enable
20,000 spots per substrate.
Disadvantages
Protein array technology is not as straightforward as DNA arrays due to the complex
structure of proteins [4]. The complexity of proteins has proven to be a bottleneck in
the progression of protein arrays but the disadvantages will lessen as scientists work
to gain a better understanding of proteins.
Examples of disadvantages include:
Proteins demonstrate a staggering variety of chemistries, affinities and

specificities. Proteins may require more multimerisation, partnership with other
proteins or post-translational modification to demonstrate activity or binding
[4,5,9,12,13].
Manufacturing HT protein arrays requires the efficient production of large

numbers of proteins. There is no equivalent amplification process like PCR that
can generate large quantities of protein [4,5,12,14].
Expression and purification of proteins is a tedious task and does not guarantee
the functional integrity of the protein [19,20]. Preserving the native characteristics
of proteins is essential for downstream analysis.
Many proteins are notoriously unstable, which raises concerns about microarray
shelf life [12,14].
The application of protein arrays appear to be feasible in areas such as target

identification and characterisation, target validation, diagnostic marker identification
and validation, pre-clinical study monitoring and patient typing. The variety of
applications highlights the potential of the technology. Below are examples of recent
major applications of protein arrays.
Autoimmune Profiling
The diversity of the autoimmune response is a great challenge for the development
of antigen-specific tolerising therapies. In the area of autoimmune profiling,
researchers fabricated assays containing 196 distinct biomolecules, comprising
proteins, peptides, enzyme complexes, ribonucleoprotein complexes, DNA and post-
translationally modified antigens. The assays included sera from eight human
autoimmune diseases including systemic lupus erythematosus and rheumatoid
arthritis [16]. To identify new autoantigens that may act as tolerising vaccines, an
antigen microarray consisting of 232 proteins were identified as potential targets of
autoimmune response in chronic experimental autoimmune encephalomyelitis, a
mouse model for multiple sclerosis. Such analysis of immune response can also be
applied to other diseases [6].
Target Identification and Validation

Drug companies today must discover new disease targets rapidly and develop drugs
that are highly specific for their targets to minimise harmful side-effects [21].
Recombinant protein arrays can potentially assist in both areas. Researchers are
investigating whether specific disease markers could be screened against high-
density (proteome-wide) and/or low-density recombinant protein arrays to identify
proteins that interact with the markers. These interacting proteins could then be
investigated further to differentiate those proteins that show promise as new drug
targets for combating the disease of interest [17,22].
Additionally, existing drug candidates could be screened against recombinant protein

arrays to measure the specificity of the drug molecules. For example, obesity can
result in metabolic syndrome X, type 2 diabetes, cardiovascular disease and other
disorders. Scientific studies have recently indicated that peroxisome proliferator-
activated nuclear receptor δ (PPARδ) can activate fat metabolism, potentially
providing a means of treating or even preventing obesity. PPARδ is usually activated
through the binding of a specific ligand or hormone. Drug screening candidates that
mimic this specific ligand against a recombinant protein array composed of other
closely related nuclear receptors would rapidly identify those mimics that are highly
specific for PPARδ and that do not interact significantly with the other nuclear
receptors. This application could potentially provide a method for eliminating drug
candidates that can cause harmful side-effects through interaction with non-target
molecules [17].
Protein arrays have also been applied in identifying new kinases and their substrates.
Phosphorylation of proteins by protein kinases plays a central role in regulating
cellular processes and may contribute to many diseases, including diabetes,
inflammation, and cancer. Therefore, kinases are an important class of drug targets.
Different enzyme activities including phosphatases, peroxidases, galactosidases,
restriction enzymes and protein kinases have been analysed on protein, peptide, and
nanowell microarrays. In one study, a total of 119 known or predicted protein kinases
were expressed, purified as GST fusion proteins, arrayed and cross-linked on a
protein chip and assayed with 17 different substrates for auto-phosphorylation by
treatment with radio-labelled ATP. New activities were found. For example, 27
kinases showed phosporylation activity of poly-Glu-Tyr. This indicates that many
kinases are capable of phosphorylating tyrosine even if they are members of the
serine/threonine family on the basis of sequence comparison [6].
Clinical Applications in Cancer Research
Cancer research is currently one of the largest areas of application for protein arrays.
Cancer proteomics encompasses the identification and quantitative analysis of
differentially expressed proteins from normal tissue, premalignant and malignant
tissues [10,23]. Serum screening was performed in several studies to characterise
the serum and plasma of patients suffering from diverse cancers, such as colon, lung
or nasopharyngeal cancer. All studies demonstrated the applicability of arrays to this
field and led to the identification of known of new potential biomarkers [16].
In another applicaton, mutations and polymorphisms of p53 were functionally

characterised with regard to their DNA-binding capacity on protein microarrays.
Organ- and disease-specific microarrays have been created using reverse-phase
protein arrays, which were created by immobilisation of the whole repertoire of
patient proteins. Such arrays were then applied to quantify the phosphorylated status
of signal proteins and to monitor cancer progression from histologically normal
prostate epithelium to prostate intra-epithelial neoplasia and invasive prostate cancer
[16].
Researchers have also immobilised anti vascular endothelial growth factor (VEGF)
antibodies to ProteinChip arrays to analyse the expression of VEGF protein isoforms
in lung tumors and normal lung tissue. VEGF plays an important role in the
development and metastasis of tumors and is therefore, an important target in novel
anti-cancer treatments. The lung tumors that were analysed expressed a wide variety
of VEGF isoforms, while normal lung tissues only expressed low amounts of the
smallest VEGF isoform [10].
Proteomics
Proteomics is promising for the early detection of disease using proteomic patterns of
body fluid samples. Proteome analysis may also be important to make individualised
selection of therapeutic combinations that best target the entire disease-specific
protein network. Investigation of the proteome may also give a real-time assessment
of therapeutic efficacy and toxicity. Identifying changes in the diseased protein
network associated with drug resistance will make it possible to adjust the therapy
[10].
For example, reverse-phase protein arrays have been used to study the fluctuating
state of the proteome in minute cell quantities. The activation status of cell signalling
pathways controls cellular fate. Deregulation of these pathways can lead to
carcinogenesis. Reverse-phase protein arrays have used to analyse the status of key
points in cell signalling involved in prosurvival, mitogenic, apoptotic and growth
regulation pathways in the progression from normal prostate epithelium to invasive
prostate cancer. Focused analysis of phospospecific target proteins revealed
changes in cellular signalling events through disease progression and between
patients. Gene expression alone cannot determine the activation (ie.
phosphorylation) state of in vivo signal pathways checkpoints [9].
Food Safety
Food safety is a top priority for many countries. As a result, great effort has been put
into developing technologies to ensure food products meet safety standards. A
particularly interesting application is the xMAP (www.luminexcorp.com) system.
This assay uses colour-coded microspheres to attach capture molecules. The beads
are sorted by flow cytometry. Each type of bead is identified according to its
fluorescence label and the quantity of the captured target on each bead. This
approach has been shown capable of performing 100 different assay types
simultaneously and is used for the detection of bacterial pathogens in food [17].
The following websites are useful resources for learning more about protein arrays:
www.lab-on-a-chip.com
News resource for microarray and microfluidic applications
www.functionalgenomics.org.uk
Extensive resource for both genomic and proteomics-based applications
www.bioarraynews.com
Weekly news resource of developments in the microarrays sector
http://arrayit.com/
Commercial supplier (Telechem) with useful web resources
Examples of commercially available planar protein arrays:
Company Product Application/Kits Weblink

Schleicher & ProVisionTM HCA Cytokine www.schleicher-
Schuell Profiling schuell.de/bioscience
Bioscience
Zyomyx Inc. Zyomix Protein Cytokine www.zyomyx.com
Profiling Biochip Profiling
System
Pierce SearchLightTM Cytokine www.searchlightonline.com
Biotechnology Arrays Profiling
Inc.
RayBiotech Inc. RayBioTM Cytokine and www.raybiotech.com
Cytokine Arrays protein profiling
BD Biosciences BD ClontechTM Comparative www.bdbiosciences.com
Ab Microarray protein analysis
SIGMA- PanoramaTM Ab Comparative www.sigmaaldrich.com
ALDRICH Co. Microarray Cell protein analysis
Signalling Kit
Protometrix Inc. The Yeast Services protein www.protometrix.com

ProtoArrayTM interaction
studies
Molecular Rolling Circle Multiplexed www.molecularstaging.com
Staging Inc. amplification protein profiling
technology
(RCATTM)
Zeptosens AG ZeptoMARTM Reverse www.zeptosens.com
CeLyA Cell Screening
Lysate Arrays
Ciphergen SELDI Protein Reverse www.ciphergen.com
Biosystems Inc. Chip Screening
Examples of commercially available bead-based systems:
Company/Technology Applications/Kits Weblink

Luminex Corporation xMAP, Luminex 100TM www.luminexcorp.com
BD Biosciences BDTM cytometric bead www.bdbiosciences.com
array (CBA)
8.5.7 References
1. Sakanyan, V. (2005) High-throughput and multiplexed protein array

technology: protein-DNA and protein-protein interactions, J Chromatog B,
815, 77-95.
2. Witte, K. & Nock, S. (2004) Recent applications of protein arrays in target

identification and disease monitoring, Drug Discov Today: Technol, vol. 1, no.
1, pp. 35-40.
3. Arenkov, P., Kukhtin, A., Gemmell, A., Voloshchuk, S., Chupeeva, V. &
Mirzabekov, A. (2000) Protein microchips: use for immunoassay and
enzymatic reactions, Anal Biochem, 278, 123-131.
4. Espina, V., Woodhouse, E., Wulfkuhle, J., Asmussen, H., Petricoin, E. &
Liotta, L. (2004) Protein microarray detection strategies: focus on direct
detection technologies, J Immunol Methods, 290, 121-133.
5. Walter, G., Bussow, K., Lueking, A. & Glokler, J. (2002) High-throughput

protein arrays: prospects for molecular diagnostics, Trends Mol Med, vol. 8,
no. 6, pp. 250-253.
6. Lueking, A., Cahill, D. & Mullner, S. (2005) Protein biochips: a new and
versatile platform technology for molecular medicine, Drug Discov Today:
Targets, vol. 10, no. 11, pp. 789-794.
7. Barton, S. (2005) The promise of biomarkers: research and applications, Drug

Discov Today, vol. 10, no. 9, p. 615-616.
8. Unwin, R., Evans, C. & Whetton, A. (2006) Relative quantification in

proteomics: new approaches for biochemistry, Trends Biochem Sci, vol. 31,
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9. Poetz, O., Schwenk, J., Kramer, S., Stoll, D., Templin, M. & Joos, T. (2004)
Protein microarrays: catching the proteome, Mech Ageing and Dev, 126, 161-
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10. Hoeben, A., Landuyt, B., Botrus, G., De Boeck, G., Guetens, G., Highly, M.,
van Oosterom, A. & de Bruijn, E. (2006) Proteomics in cancer research:
methods and application of array-based protein profiling technologies, Anal
Chimica Acta, 564, 19-33.
11. Cretich, M., Damin, F., Pirri, G. & Chiari, M. (2006) Protein and peptide
arrays: recent trends and new directions, Biomol Eng 23, 77-88.
12. LaBaer, J. & Ramachandran, N. (2005) Protein microarrays as tools for

functional genomics, Curr Opin Chem Biol, 9, 14-19.
13. Zhu, H. & Snyder, M. (2003) Protein chip technology, Current Opinion in
Chemical Biology, 7, 55-63.
14. Stoll, D., Bachmann, J., Templin, M. & Joos, T. (2004) Microarray technology:
an increasing variety of screening tools for proteomic research, Drug
Discovery Today: Targets, vol. 3, no. 1, pp. 24-31.
15. Lopez, MF & Pluskal, MG (2003) Protein micro- and macroarrays: digitising
the proteome, J Chromatogr B, 787, 19-27.
16. Angenendt, P. (2005) Progress in protein and antibody microarray

technology, Drug Discovery Today: Targets, vol. 10, no. 7, pp. 503-511.
17. Schofield, M., Sharma, N. & Ge, H. (2004) The recombinant protein array:
use in target identification and validation, Drug Discov Today: Targets, vol. 3,
no. 6, pp. 246-252.
18. Lal, SP, Christopherson, RI & dos Remedios, CG (2002) Antibody arrays: an
embryonic but rapidly growing technology, Drug Discov Today, vol. 7, no. 18,
pp.143-149.
19. Binder, S., Hixson, C. & Glossenger, J. (2006) Protein arrays and pattern
recognition: new tools to assist in the identification and management of
autoimmune disease, Autoimm Reviews, 5, 234-241.
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Biotechnol, 22, 630-637.
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functional protein microarrays, Curr Opin Mol Ther, 5, 271-277.
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technology, Drug Discov Today, 10, 503-511.
Chapter 9.1 Biomarkers: Discovery and Applications
Aditya Paranjape
9.1.1 Introduction
Biomarker is a characteristic that can be objectively measured and elevated as an

indicator of normal biological processes, pathogenic processes or pharmacological
responses to a therapeutic intervention [4].
These characteristics include: serum, plasma, urine, CSF, saliva, tissues etc.
Biomarkers provide us a dynamic and powerful approach to understanding the wide

spectrum of neurological disease with application in observational and analytical
epidemiology, randomized clinical trials, screening and diagnosis and prognosis.
These markers offer the means for homogenous classification of diseases and risk
factors and thus they can extend our base information about the underlying
pathogenesis of diseases. Markers are also able to reflect the entire journey of
disease from the earliest manifestation to the terminal stages. Careful assessment of
validity of markers is required with respect to the stage of the disease. In practice
Biomarkers include tools and technologies that can aid in understanding the
prediction, cause, diagnosis, progression, regression or outcome of treatment of
disease. Biomarkers are becoming widely used in many aspects of drug discovery
and drug development such as dosing, efficacy and as surrogates for clinical
outcome [6]. They are now also being utilized for drug development to curtail
expensive trials and are being used to make critical decisions about whether a drug
should be continued beyond the clinical stage of development Additionally;
biomarkers are increasingly being used for patient selection with respect to
responses to targeted therapies, cancer research and research for various disease
states.
Biomarkers have been classified by (Perera and Weinstein) based on the sequence
of events from exposure to disease. Though biomarkers readily lend themselves to
epidemiological investigations, they are also useful in the investigation of the natural
history and prognosis of a disease. In addition to delineating the events between
exposure and disease, biomarkers have the potential to identify the earliest events in
the natural history, reducing the degree of misclassification of both disease and
exposure, opening a window to potential mechanisms related to the disease
pathogenesis, accounting for some of the variability and effect modification of risk
prediction. The recent interest in biomarker discovery is because of new molecular
biologic techniques that promise to find relevant markers rapidly, without detailed
insight into mechanisms of disease [4]. By screening many possible biomolecules at
a time, a parallel approach can be tested. Genomics and proteomics are some
technologies that are used in this process. There is considerable interest in
biomarker discovery from the pharmaceutical industry. Blood test or other biomarkers
could serve as intermediate markers of disease in clinical trials, and also be possible
drug targets.
Fig.9.1.1 Disease pathway and potential impact of biomarkers (Journal for American
Society for Experimental Neuro Therapeutics, Inc.)
Biomarkers can also be substances used as indicators of a biologic state. It can be

any kind of molecule indicating the existence (past or present) of living organisms. In
particular, in the fields of geology and astrobiology biomarkers are also known as bio
- signatures. The term is also used to describe biological involvement in the
generation of petroleum.
The study of biomarkers, or chromosomal abnormalities that can possibly predict

how a person’s disease may progress or respond to treatment, falls under the
category of “chemoprevention” as scientists hope that the end result of these studies
will provide an aid in early detection and screening, which could hopefully make a
dent in the statistics representation of bladder cancer specific deaths [2]. The
biomarker must be expressed differently in normal and high-risk tissue, with clear
evidence of progression from normal tissue to biomarker to cancer and, ideally,
should appear early in carcinogenesis. If the use of biomarkers proves to be a tool for
achieving successful preventive intervention, this would support the possibility that a
preventive agent that could reverse the molecular events (or suppress their
consequences) for one tumor site may be effective in preventing a variety of tumors
[7]. The development and validation of biomarkers are important to the success of
testing chemo preventive agents .Examples of biomarkers include genetic markers
(eg, nuclear aberrations [such as micronuclei], gene amplification, and mutation),
cellular markers (eg, differentiation markers and measures of proliferation, such as
thymidine labeling index), histologic markers (eg, premalignant lesions, such as
leukoplakia and colonic polyps), and biochemical and pharmacologic markers (eg,
ornithine decarboxylase activity).
During the Clinical Biomarker Summit March 2006, it was contemplated that as the
field matures, biomarkers were making their way into clinical trials. Faced with
relative lack of experience in implementing biomarkers in clinical trials, many
researchers and clinicians were facing similar challenges in modifying trial design
and defining the right control population, validating biomarker assays from the
biological and analytical perspective, and using biomarker data as a guideline for
decision making. The Clinical Biomarkers Summit also addressed biomarker
translation from pre-clinical to clinical studies and a variety of biomarker applications
in clinical trials, including patient selection, monitoring clinical efficacy and safety, and
clinical pharmacology. The Summit also takes note of the bridging gap between the
pharmaceutical and diagnostics industries and the potential of companion
diagnostics. Specific case studies of leveraging biomarkers in accelerating and
streamlining clinical trials will offer a steady status report, no hype! The Clinical
Biomarkers Summit, is built on a solid three-year track record of Cambridge Health
tech Institute’s Biomarker Series, is the first meeting in the Series to focus exclusively
on clinical applications of biomarkers.
At the Biomarker World Congress 2005, Pennsylvania over 500 thought leaders from
260+ organizations, representing 20 countries, had gathered to discuss biomarker
implementation in drug and diagnostic development. A year later, the largest meeting
of its kind, The Biomarker World Congress 2006 is dedicated to all areas of
biomarker research spanning the pharmaceutical and diagnostic pipeline. The
meeting brought together a unique and international mix of large and medium
pharmaceutical, biotech and diagnostics companies, leading universities and clinical
research institutions, government and national labs, CROs, emerging companies and
tool providers-making the Congress a perfect meeting-place to share experience,
foster collaborations across industry and academia, and evaluate emerging
technologies. The Congress also offers a balance of scientific sessions covering the
latest research and strategic presentations and brainstorming sessions for the
decision makers.
Since the start of the 21st century there has been a lot of research work on there has
been a lot of research worldwide for biomarkers three prominent of them are noted
below,
Many recent investigations have been conducted to determine whether new

biological markers will help predict disease progression and potential clinical
applications of these tumor markers are under active investigation. Recent attention
has focused on which tumor markers may predict the responsiveness of a particular
bladder cancer to systemic chemotherapy. Some of these new predictive and
prognostic markers include DNA ploidy, S-phase, p53, p21, the retinoblastoma (Rb)
gene, MDR-1, bcl-2, cell adhesion molecules, blood group antigens, tumor
associated antigens, proliferating antigens, oncogenes, peptide growth factors and
their receptors, tumor angiogenesis and angiogenesis inhibitors, and cell cycle
regulatory proteins. Beta human chorionic gonadotropin (ß-hCG), carcinoembryonic
antigen, CA-125, CA 19-9, and others have been evaluated and shown to correlate
with clinical response to chemotherapy. G Actin and Ki67 have indicated response to
BCG and radiation.
The most discussed chromosomal biomarker of all is the p53 protein, a tumor
suppressor gene on chromosome 17p. (Between 2003 and 2005) over 4,00 articles
on this subject were published. A wealth of studies seems to confirm that the p53
protein, if mutated and over expressed in cancerous cells, is an indication of a
potentially aggressive condition. It’s been shown that both grade and stage of
(invasive) bladder cancer are related to p53 alterations. Mutations in the p53 gene
can be detected in tissue sections by immuno histochemistry. Since the wild-type p53
gene has a short life, immuno staining of normal urothelium with p53 monoclonal
antibodies is negative. When mutations in the p53 gene occur, the mutated proteins
aggregate in tetrameric and pentameric macromolecules of longer life. The result is
an accumulation of p53 protein that provides a positive immuno staining reaction.
The reaction is observed in the nuclei of tumor cells affected by these events. The
function of p53 is critical to the way that many cancer treatments kill cells since
radiotherapy and chemotherapy act in part by triggering cell suicide in response to
DNA damage [11]. This successful response to therapy is greatly reduced in tumors
where p53 is mutant so these tumors are often particularly difficult to treat. It is
hoped that better understanding of p53 can guide the development of new treatments
for cancer. Scientists are beginning to unravel some of the mysteries and in the test
tube at least, are beginning to find ways to make these damaged p53s work again.
Such discoveries could potentially offer a powerful and selective new way of treating
cancer.
Now considering another biomarker, the p21 gene, is showing that people with p21-
positive tumors have a decreased probability of tumor recurrence. An article from
Journal of the National Cancer Institute, July 15, 1998, discusses a multi-centre,
randomized clinical trial using p53-status of tumor cells and other molecular markers
like p21 to guide treatment decisions in bladder cancer patients, one of the first of its
kind. [6]
The research team from (Norris Comprehensive Cancer Center) conducted a study
on 242 patients with locally confined bladder tumors who were followed for an
average of 8.5 years. Analysis was done of of the p21 protein and it’s interaction with
the p53 protein. Results of the study indicated that patients with p21-positive tumors
survived disease-free significantly longer than those patients with p21-negative
tumors. Furthermore, it was shown that the way the p21 and p53 proteins interact
with each other can give a very good indication of which patients must be considered
at high risk for recurrence. The article also stated that p53 is known to be a primary
regulator of p21, since genetic changes in p53 may lead to loss of p21 expression
and function. This in turn leads to unregulated cell growth, and is thought to
contribute to the aggressive behavior of some tumors. There hypothesis seemed
confirmed by the study was what the scientist there proclaimed. Patients with p53-
altered/p21-negative tumors demonstrated a higher rate of recurrence and worse
survival compared with those with p53-altered/p21-positive tumors. Patients with
p53-altered/p21-positive tumors demonstrated a similar rate of recurrence and
survival as those with p53-wild type tumors.
At the Centre for Translational Cancer Research, efforts being done to focus on
three types of biomarker discovery protein biomarkers in tissues and cells, bodily
fluid (blood, urine, and other fluids) protein biomarkers, and transcriptome (RNA)
biomarkers. These approaches together facilitate translational cancer research by
providing new tools to the clinician for the enhanced diagnosis, follow-up and
screening of cancer patients.
Tissue Proteins:
Cell and tissue analysis of cancer specimens allows

researchers to assess the exact site and expression level of
protein cancer biomarkers in architecturally preserved or
homogenized tissues. Analysis can include histo
morphological examination in collaboration with a pathologist,
use of immuno histochemical staining with specific antibodies
on cell and whole mount tissue specimens, and Western blot
analysis of cell or tissue homogenates. (Picture showing
Tissue specimen)
Fluid Proteins:
The emerging field of proteomics provides new tools for the early detection of cancer
from human serum, cerebral spinal fluid, urine and other complex samples.
Proteomic research provides information regarding the proteome’s dynamic and
rapid changes which result from exogenous exposure or endogenous factors. The
CTCR Core offers a proteomic analysis based on the patented Surface Enhanced
Laser desorption/Ionization (SELDI) technology. Assays using SELDI time-of-flight
mass spectrometry (TOF-MS) to provide a means to identify new candidate
biomarker proteins because of their ability to detect and quantify multiple post-
translationally modified and processed protein forms in a single assay.
RNA Molecules:
New methods to analyze gene expression or the

“transcriptome” of cancer cells for comparison to the patterns
of normal cells provides a powerful new means of identifying
RNA biomarkers for specific cancers. Several research
projects are underway using the core facilities available to
CTCR researchers. These projects seek to analyze total
expressed RNA using microarrays, microRNAs that function
as regulators of protein expression, quantitative analysis of
single genes using Q-PCR, and to develop new methods to
analyze RNA expression in archived cancer tissue
specimens.
Mapping of human genome leads to the Discovery of new biomarkers, currently,

scientists are striving to find proteins (biomarkers) that are specific to various
disorders. More and more biomarkers are being identified with the help of
sophisticated enabling instruments and technologies such as mass spectrometers
and protein microarrays. These novel biomarkers are likely to aid researchers in
developing precise clinical diagnostics and drugs that are capable of detecting and
curing fatal diseases. The early detection and diagnosis of fatal diseases –
particularly cancer, Alzheimer’s, and Parkinson’s – enhances the prospects of curing
the affected patients. This would encourage physicians to recommend patients to
undertake biomarker-based screening tests that can predict these deadly diseases in
the early stages. These tests are also likely to help physicians in prescribing
personalized medication to patients after pinpointing the particular pathway that is
playing an active role in the disease progression. This would not only lead to
reduction in side-effects but also ensure that patients are administered the right
medication.
There are various production approaches used for biomarkers; it is essential that the
most rewarding and least time consuming procedure of them is selected which yields
the best suited biomarker that incorporate many capabilities.
Capabilities of Biomarkers,
• Delineation of events between exposure and disease

• Establishment of dose-response
• Identification of early events in the natural history
• Identification of mechanisms by which exposure and disease are related
• Reduction in misclassification of exposures or risk factors and disease
• Establishment of variability and effect modification
• Enhanced individual and group risk assessments
TABLE 9.1.3,
To summarize advantages and disadvantages of Biomarkers,
Advantages Disadvantages
Objective assessment Timing is critical
Precision of measurement Expensive (costs for analyses)
Reliable; validity can be established Storage (longevity of samples)
Less biased than questionnaires Laboratory errors
Disease mechanisms often studied Normal range difficult to establish
Homogeneity of risk or disease Ethical responsibility
[The American Society for Experimental NeuroTherapeutics, Inc. NeuroRx. 2004

April; 1(2): 182–188]
9.1.4 Applications of Biomarkers
The advancement in the biomarker discovery has led to the use of Markers in most of
the therapeutic treatment or clinical trials, thus applications of these are many and a
few are listed below as per there success rate. The uses of the biomarkers are as
given,
As Cancer Markers
The increase in the use of tumor markers as screening and diagnostic tools has
generated much hope for the identification of broadly reacting or pan-cancer
antibodies. Upstate/Chemicon provides a large variety of many well characterized
cancer antibodies for breast cancer detection, chronic lymphatic leukemia (CCL), and
other cancers.
As Neurodegenerative Markers
Chemicon’s line of neurological antibodies has well-characterized neurodegenerative
antibodies and assays for researching Alzheimer’s, Fragile X Syndrome, Parkinson’s
& Huntington’s and other related biomarkers.
As Metabolic Markers
Biomarkers of metabolic processes are emerging as key tools in diagnosing

dysfunction and disease states. This is becoming increasing important due to the
near epidemic surge in metabolic syndrome and resultant cardiovascular disease
and Type 2 diabetes.
As Drug Response Markers
Inflammation, oxidative stress and reactive species are continuously being monitored
as drug response markers. Chemicon now has available a monoclonal antibody to
RAGE, a member of the immunoglobulin super family of cell surface molecules that
bind molecules that have been irreversibly modified by non enzymatic glycation and
oxidation.
Biomarkers are also verified in multiple applications like,
• Immuno histochemistry
• Western Blotting
• Immuno precipitation
• Flow cytometric Analysis
• ELISA
9.1.5 Relevant Web sites
1. http://www.healthsystem.virginia.edu/internet/biomolec/biomarkers.cf
m
(University of Virginia Health System)
2. http://www.udel.edu/ctcr/research/biomarkers_discovery.htm
(Centre for Translational Cancer Research)
3. http://www.frost.com/prod/servlet/report-brochure.pag?id=D301-01-00-
00
(Frost and Sullivan Research Services)
4.
http://www.mdsps.com/over/MarketingMaterials/BiomarkersDiscovery.pdf
(MDS Pharma Services)
5. http://innovationwell.net/COMTY_biomarkers?h2ls
The shift in focus from discovering genomic biomarkers to protein biomarkers is

driving demand for robust research instruments that enable multiplexing and reduce
manual steps such as sample preparation. Technological developments in terms of
automation and increase in throughput are enabling researchers to solve the riddle of
the human proteome – which is much more complex than the human genome.
Companies that manufacture protein chips containing high-density arrays of
functional proteins and micro fluidics-based platforms are likely to cash in on these
novel research efforts aimed at discovering new biomarkers. The Companies mostly
into Biomarker discoveries are given below,
Chemicon/Upstate is responding to this growing market need by offering high

quality, well characterized antibodies many of which have been validated in a
variety of applications. Chemicon/Upstate’s innovative partnership has brought
together a large line of biomarkers for various disease states such as specific
cancers, neurodegenerative, cardiovascular and metabolic diseases.
Together Chemicon/Upstate brings researchers and drug development
companies access to innovative assays and platform technologies for biomarker
discovery and drug development.
Developed and manufactured under GMP conditions, our antibodies, kits and
reagents provide consistent high quality products for all of your research, drug
discovery and drug development needs. We offer many well-characterized
antibodies for research and discovery to numerous disease and disease related
targets. (www.chemicon.com)
Millipore Serological Corporation, Life Sciences is the parent company for most of
the biomarker producing companies which have their acquisition in most of the
companies mentioned below, which mostly work in collaboration with Millipore
they are,
1. LINCO (http://www.lincoresearch.com)
2. Celliance (www.celliancecorp.com)
3. MDS Pharma Services
4. Frost & Sullivan Research and Partnership Services (GPS)
5. Biomarker Pharmaceuticals
Many studies using biomarkers in the above mentioned industries are going on but it
never achieves its full potential because of the failure to adhere to the same rules
that would apply for the use of variables that are not biological. The development of
any biomarker should precede or go in parallel with the standard design of any
epidemiological project or clinical trial. In forming the laboratory component, pilot
studies must be completed to determine accuracy, reliability, interpretability, and
feasibility. The investigator must establish “normal” distributions by important
variables such as age and gender. The investigator will also want to establish the
extent of intra individual variation, tissue localization, and persistence of the
biomarker. Moreover, he or she will need to determine the extent of inter individual
variation attributable to acquired or genetic susceptibility. Most, if not all of these
issues can be resolved in pilot studies preceding the formal investigation.
9.1.7 Reference
1. Galasko D. New approaches to diagnose and treat Alzheimer’s disease: a

glimpse of the future. Clinical Geriatr Med 17: 393–410, 2001.
2. Gordis L. Epidemiology and public policy. In: Epidemiology (Gordis L, ed), pp

247–256. Philadelphia: W.B. Saunders, 1996.
3. Hulka BS. Overview of biological markers. In: Biological markers in

epidemiology (Hulka BS, Griffith JD, Wilcosky TC, eds), pp 3–15. New York:
Oxford University Press, 1990.
4. IIyin et al, Trends in Biotechnology, 22, 411 – 416, 2004
5. Merikangas K. Genetic epidemiology: bringing genetics to the population-the

NAPE Lecture 2001. Acta Psychiatr Scand 105: 3–13, 2002.
6. Naylor S. Biomarkers: current perspectives and future prospects. Expert Rev

Mol Diagn 3: 525–529, 2003
7. Perera FP, Weinstein IB. Molecular epidemiology: recent advances and

future directions. Carcinogenesis 21: 517–524, 2000.
8. Reiber H, Peter JB. Cerebrospinal fluid analysis: disease-related data

patterns and evaluation programs. Journal Neurol Science 184: 101–122,
2001.
9. Rohlff C. Proteomics in neuropsychiatric disorders. Int J

Neuropsychopharmacol 4: 93–102, 2001.
10. Schulte PA. A conceptual and historical framework for molecular

epidemiology. In: Molecular epidemiology: principles and practices (Schulte
PA, Perera FP, eds), pp 3–44. San Diego: Academic Press, 1993.
11. Verbeek MM, De Jong D, Kremer HP. Brain-specific proteins in cerebrospinal

fluid for the diagnosis of neurodegenerative diseases. Ann Clin Biochem 40:
25–40, 2003
9.2. SELDI - MS (SURFACE ENHANCED LASER DESORPTION
IONIZATION – MASS SPECTROMETRY)
Bharathirajan Panneerselvam
9.2.1Introduction:
SELDI-MS (Surface Enhanced Laser Desorption Ionization – Mass Spectrometry) is

a powerful technique that combines chromatography and mass spectrometry. This
technique was invented by Tai-Tung yip and T.William Hutchens in the early 1990’s
at the Baylor College of medicine, and then afterwards gained in popularity as a
powerful tool for protein analysis. [1]
SELDI encompasses two major subsets of MS (Mass Spectrometry) Technology

1. SEND –Surface Enhanced Neat Desorption and
2. SEAC –Surface Enhanced Affinity Capture.
The underlying principle in SELDI is surface-enhanced affinity capture through the
use of specific probe surfaces. Once captured on the SELDI protein chip array,
proteins are detected by TOF MS.
Figure: 1 Schematic diagram comparing the configuration of PBS-II TOF MS.
Source: http://home.ccr.cancer.gov/ncifdaproteomics/pdf/ERMD.pdf [2]
“SELDI employs aluminum chips 1-2 mm in diameter, spotted with protein capture
bait such as a chemical affinity resin, small molecule, antibody, DNA or enzyme.
Users apply a crude sample to the chip, allow proteins with affinities to capture
molecules to bind to the surfaces and then wash away any impurities or loosely-
bound proteins. Analytes are laser desorbed directly from the chip, ionized and
analysed by mass spectroscopy; A SELDI experiment produces a mass spectral
fingerprint that can distinguish differences in protein expression levels between
diseased and normal samples”.Giannoula Klemant,Pediatric Oncologist, Dana Farber
Cancer Institute and Children’s Hospital, Boston ,says,” The main reason why I
became really interested in this technology is that for a biotechnologist, it is often
quite important to analyze groups of treated and untreated animals or people.
Traditional Mass Spectrometry analysis or permits the analysis of sample pairs,(i.e.)
a comparison of a single treated and untreated sample, which not only takes really
long hours, but also prevents any statistical analysis”[1, 3]
Figure: 2 Diagrammatic representation of overall Principle behind SELDI operation.

Source: Judith Y.M.N. Engwegen, Marie-Christine W. Gast, Jan H.M. Schellens and
Jos H. Beijnen, (2006) Clinical proteomics: searching for better tumour markers with
SELDI-TOF mass spectrometry, TRENDS in Pharmacological Sciences 27: 251-259
The above diagram shows the Principles of SELDI-TOF MS using the ProteinChip
System.
(a) Protein profiling.
(i) The application of microliters of sample, for example, diseased and
healthy persons to an eight-spot array with hydrophilic, hydrophobic, cationic, anionic
or immobilized-metal affinity capture chromatography surface. [4] (Figure 3, 4&5)
Figure: 3 Two ProteinChip Arrays with 8 spots.
Source: http://www.proteomicsnijmegen.nl/Seldi_pages/selditof.htm
Figure: 4 Variety of ProteinChip arrays available for sample preparation.

Source: Haleem J. Issaq, , Timothy D. Veenstra, Thomas P. Conrads, and Donna
Felschow The SELDI-TOF MS Approach to Proteomics: Protein Profiling and
Biomarker Identification Biochemical and Biophysical Research Communications
292, 587–592 (2002)
Figure: 5 Bioprocessor for liquid handling procedures. The arrays are put into the
processor
Source: http://www.proteomicsnijmegen.nl/Seldi_pages/selditof.htm
(ii) Shows the addition of an appropriate binding buffer.

(iii) Shows on-chip sample purification using one or more wash buffers.
(iv) “Shows the application of energy-absorbing matrix (e.g. sinapinic acid) for the
absorption of laser energy. Thus the Laser irradiation desorbs bound proteins and
positively ionizes them. Owing to the electric field, they migrate in the mass analyser
(TOF MS): small (diamond) and multiply charged proteins (oval) faster than large and
single-charged ones (triangle). Thus, the proteins are separated. Time of flight (t) is
proportional to protein mass per charge: m/zZconstant x t2”. [4]
(b) (Figure shows a comparative study of the results from SELDI result and Gel
result.)
“SELDI-TOF mass spectrum and the spectrum depicted in gel view. The protein m/z
is displayed on the x-axis and the protein abundance is depicted on the y-axis.
Spectra are searched for differentially expressed protein m/z values using the
ProteinChip bioinformatics software or other suitable statistics or bioinformatics.
Computational algorithms are used to build models for the classification of diseased
and healthy samples with the discriminating m/z values (e.g. a classification tree).
Here, expression differences are visible between ovarian cancer and control
samples. (i) A peak at 9.2 kDa (haptoglobin fragment) is over expressed in ovarian
cancer. Other unregulated peaks are visible at (ii) 4.1 kDa and (iii) 4.5 kDa, and (iv) a
down regulated peak is seen at 2.7 kDa.”[4, 6]
The most significant features of SELDI are its ability to provide a rapid protein
expression profile from different types of clinical and biological samples. Researchers
proved its effectiveness in biomarker discovery and identification. It also plays a vital
role in the study of protein - protein and protein-DNA interaction.SELDI proves to be
highly versatile by its success in the study of identification of potential diagnostic
markers for breast, bladder, prostate and ovarian cancers and Alzheimer’s diseases
to the study of biomolecular interactions and characterization of posttranslational
modification.Infact, this technique was first used in early-stage ovarian cancer
detection. This technique enables application to easily accessible body fluids such as
serum.
[4, 5] .In this minireview SELDI’s advantage and disadvantage, key industry suppliers
and its various applications along with few learning resources sites are discussed in
details.
Towards the end of 1993, this technology was introduced, its high potentiality and
versatile function attracted researchers for more studies on SELDI which lead to the
development of SELDI at commercial level in Ciphergen Biosystems, Palo Alto, CA,
USA, and this organisation appreciated more research that solved a number of
medical and basic research problems. [6-10]
Merchant and Weinberger’s review published in 2000 seems to be the first review
that states a brief and precise description of SELDI technology with its advantages in
biomedical research. But with in the next four years diversity of ProteinChip arrays
with improved array surface characteristics has increased with the availability of
SEND Technology. There has been continuous research publication that states the
enhancement in SELDI instrument performance, added automation tools, improved
experimental protocols and fine-turned software for biomarker pattern data analysis.
Today it is simple to investigate biomarker candidates that are characterized by
identifying PTM (Post Translational Modification). [10]
Recent Inventions by CIPHERGEN
1. “The new ProteinChip System Series 4000 for the rapid translation of SELDI
biomarker discoveries into assays”. [1]
”Invention of ProteinChip system Series 4000 by Ciphergen was specifically

designed to enable and ease the Pattern Track process of biomarker discovery to
assay. The basic idea behind it started, when the process begins with novel
biomarker discovery, which aimed for differential protein expression between sets of
biological samples using Ciphergen ProteinChip technology. Moreover validation
studies with larger sample sets select the biomarkers with the highest predictive
value. Another issue is the validation of the biomarkers, only the validated marker
proteins are enriched and identified using peptide mass fingerprinting. Hence inorder
to resolve this, Chromatographic or antibody based biomarker assays are designed
which is implemented in the final step of the Pattern Track process”. [1]
Significance of this series 4000

Sensitivity, reproducibility and dynamic range of the Series 4000 highly favours
discovery of biomarkers and assays directly on a single platform.
Validating markers up-front saves the valuable time of the researchers and their
effort when compared to those methods that favours discovery of whether the
markers stand up in larger sample populations after assay development. [1]
2. Automated SELDI for biomarker identification
Using SELDI, Center for Orthopaedic Research in collaboration with the Arkansas
Breast Cancer Research Program has developed a core facility for protein biomarker
identification which was manufactured by Ciphergen. The focus of the UAMS SELDI
facility is biomarker identification in cancer. [22]
The SELDI resource is capable of:
• Clinical and Research Proteomics identification.

• Biomarker identification and validation.
• Protein discovery.
• Protein characterization.
Figure 6 Protein Identification by Automated SELDI

Source: www.cor.uams.edu/services.asp
3. “Ciphergen’s BioSepra chromatography products and services have been

combined with SELDI ProteinChip technology for a new approach to protein
purification called Process Proteomics. Ciphergen’s on-spot methodology
dramatically accelerates and simplifies purification development and analysis”. [14]
Advanced SPME/SELDI Fiber Introduction to IMS and MS [11]
“The application of polypyrrole (PPY) solid phase micro extraction (SPME) coatings
as both an extraction phase and a surface to enhance laser desorption and ionization
(SELDI) analyte is introduced in here. This SPME/SELDI fiber integrates sample
preparation and sample introduction on the tip of a coated optical fiber, as well as the
transmission media for the UV laser light. Using ion mobility spectrometry (IMS)
detection, the signal intensity was examined as function of extraction surface area
and concentration of analyte. The linear relationship between concentration and
signal intensity shows potential applicability of this detection method for quantitative
analysis. Extraction time profiles for the fiber, using tetraoctylammonium bromide
(TOAB), illustrated that equilibrium can be reached in less than one minute. To
investigate the performance of the PPY coating, the laser desorption profile was
studied. The fiber was also tested using a Q-TOF MS instrument with leucine
enkephalin as a test analyte. Since no matrix was used, mass spectra free from
matrix background were obtained. This novel SPME/SELDI fiber is easy to
manufacture, and is suitable for studying low mass analytes because of the inherent
low background. These findings suggest that other types of conductive polymers
could also be used as an extraction phase and surface to enhance laser
desorption/ionization in mass spectrometry”. [11]
9.2.3 Evaluation of technology.
Advantages:
Technology is easy to use and technically simple.

Easy to use hardware/software integration with advanced display and
analysis tools.
Its potentiality enables biomarker discovery, validation, identification and the
ability to create an assay on the one platform.
Validating markers up-front saves the valuable time of the researchers and
their effort.
Easily automated.
Simple sample preparation.
High throughput sample preparation using "Deep proteome" tools, in order to
control pre-analytical parameters.
Reduction of sample complexity.
Reduced sample needs - Typically 1-2 µl of crude sample per analysis.
Direct application of whole sample (fast on-chip sample clean up)
High-throughput that allows analysing large number of samples statistically.
[1]
Rapid throughput: Hundreds of samples can be analyzed by a single user in a
matter of days.
Suitability for low abundance proteins (e.g., transcription factors and a
majority of
Cellular proteins).
Rapid protein profiling.
Improved discovery of protein binding partners through protein-affinity
interactions.
High throughput (upto 96 samples per bioprocessor)
Enable access to PTM (Post Translational Modification)[13.14,15]
John Semmes, Director, Center for Biomedical Proteomics at Eastern Virginia
Medical School, says,”SELDI seems to attract proteomics researchers
because of its ability to reduce sample complexity such as affinity purification
and concentration and it also applies them directly to the chip surface, this
action saves time which in turn increases the reproducibility since there is no
much variability in the process”. [1]
Not only qualitative detection of peptides and proteins (including Isoforms) is
performed, but quantitative detection is also achieved.
On-chip characterization identification using proteolytic digestion of the target
protein and subsequent identification through peptide mapping.
Disadvantages:
This technology is currently applicable only to those proteins with maximum

molecular weight <20 kDa and provides relatively lower mass accuracy than
the 2D-PAGE MS method.[13]
Unsuitable for high molecular weight proteins (>100kDa)
Limited to detection of bound proteins.
Lower resolution and mass accuracy than MALDI-TOF[14]
Uses mild ionisation procedure.
Difficult to match protein ID to pattern feature.
Quantitative consistency questionable.
Reproducibility questionable.[15]
9.2.4 Application
“Based on patented Surface Enhanced Laser Desorption/Ionization (SELDI)

technology,
Ciphergen’s ProteinChip Systems offer a single, unified platform for a multitude of
Proteomics research applications.”[24]
SELDI’s various application from discovery to development.
Biomarker discovery
Interaction Difference Mapping
Expression Difference Mapping
DNA-protein interaction
Protein-protein interaction
Receptor-ligand interaction
Antibody-antigen interaction
Protein purification
Clinical trials stratification
Phosphorylation/signal transduction
Glycosylation analysis
Epitope mapping
Protein ID/peptide mapping
Toxicity markers
Biomarker discovery
The alternations by SELDI in the detection of cancer proteome, biomarkers and

pattern of biomarkers lead to improvement in early detection, diagnosis and
treatment monitoring in cancer. [14, 16].this technique was first used for early-stage
ovarian cancer detection. [5]
Figure 7 Shows biomarker discovery process.
Source: http://ciphergen.com/doclib/docFiles/262.pdf
PATTERN RECOGNITION
“Single protein biomarkers with clinically relevant predictive power are quite rare.
Multiple marker panels generated by SELDI are beginning to deliver on the promise
of high-confidence clinical stratification. Ciphergen’s Biomarker Patterns Software
enables rapid sample stratification with an easy-to-interpret, tree-based classification
output as shown in the figure –8“ .[17]
Figure 8 Pattern recognition

VALIDATION
To verify the reliability of potential biomarkers, high numbers of samples need to be
comparatively analyzed to further substantiate their usefulness. A single user can
manually prepare and analyze up to 200 samples per day.[17]
Figure 9 validation profile maintenance.
PROTEOMICS APPLICATIONS
“SELDI enable clinical researchers to rapidly discover, characterize, and validate

predictive protein biomarkers and biomarker patterns, right in their lab. Technology
also enables a wide variety of Interaction Difference Mapping applications in basic
biochemistry and drug discovery. Quadrupole tandem MS extends the application
range to “on-spot” epitope mapping and peptide sequencing capabilities”. [14]
Source:
Emanuel F Petricoin and Lance A Liotta, (2004), SELDI-TOF-based serum proteomic
pattern diagnostics for early detection of cancer, Current Opinion in Biotechnology.
15:24–30
“Biomarker amplification and harvesting by carrier molecules. Low molecular weight

peptide fragments, produced within the unique tissue microenvironment and
generated as a consequence of the disease process, permeate through the
endothelial cell wall barrier and trickle into the circulation. Here, these fragments are
immediately bound with circulating high-abundance carrier proteins, such as albumin,
and protected from Kidney clearance. The resultant amplification of the biomarker
fragments enables these low abundance entities to be seen by MS-based detection
and profiling. In the future, harvesting nanoparticles, engineered with high affinity for
binding, can be instilled into the collected body fluids or injected directly into the
circulation to bind with the disease- and toxicity-related information archive. These
nanoparticles and their bound diagnostic cargo can then be directly collected, filtered
over engineered filters and queried by high-resolution MS. A ‘look up table’, where
the exact identities of each of the peaks will be compared against the accurate mass
tag of each of the peaks within the spectra, will enable the simultaneous identification
of each entity within the pattern as well as allowing the discovery of the diagnostic
pattern itself”.[18 ]
POST-TRANSLATIONAL MODIFICATIONS
“Because the SELDI ProteinChip platform delivers the exact molecular weights of the
molecules present in a given sample, a wide variety of covalent modifications
(phosphorylation, acetylations, oxidations, Glycosylation and others) can be
analyzed.
For the detection of phosphorylation, an IMAC array loaded with Gallium enables
specific enrichment and detection of phospho-peptides. This also provides a powerful
approach to the quantitation of kinase activity”. [17, 14]
9.2.5 Relevant Web-sites

1. http://www.biocompare.com/index.asp The Website name is Biocompare - a
guide for Life Scientist.
2. http://www3.interscience.wiley.com/cgi-bin/home The Website name is Wiley

interscience.
3. http://www.biomedcentral.com/home/ The Website name is Biomed central -

the open access publisher
4. http://www.dissectmedicine.com/ The Website name is Dissect medicine –

collaborative medical news.
5. http://www.medscape.com/viewarticle/538192 The Website name is

Medscape today – medical news website.
6. www.expasy.org The Website name is ExPASy (Expert Protein Analysis

System) proteomics server of the Swiss Institute of Bioinformatics (SIB) is
dedicated to the analysis of protein sequences and structures as well as 2-D
PAGE
7. www.matrixscience.com This Website features Mascot, a powerful search

engine that uses mass spectrometry data to identify proteins from primary
sequence databases. To assist you, Mascot forms a substantial knowledge
base concerning protein identification by MS.
8. www.ncbi.nlm.nih.gov The Website name is NCBI (National Centre for

Biotechnology Information) creates public databases, conducts research in
computational biology, develops software tools for analyzing genome data,
and disseminates biomedical information - all for the better understanding of
molecular processes affecting human health and disease.
9. www.nen.com The Website name is PerkinElmer Life Science.
10. prospector.ucsf.edu This Website features Proteomics tools for mining

sequence databases in conjunction with Mass Spectrometry experiments
11. www.proteome.com The Website name is BIOBASE is the leading content

provider of biological databases, knowledge tools and software for the life
science industry
12. Prowl.rockefeller.edu This Website contains various tools for protein studies.
13. www.blackwellpublishing.com the website name is FEBS (Federation of

European Biochemical Societies) Journal search engine.
9.2.6 Key industry suppliers. [19]
Major instrument vendors
www.advion.com
www.agilent.com
www.appliedbiosystems.com
www.bdal.com
www.ionics.ca
www.mds-sciex.com
www.packardinst.com
www.perkinelmer.com
www.shimadzu-biotech.net
www.thermo.com
www.waters.com
Major companies involved in Protein chips
www.affibody.com
www.affymetrix.com
www.bdbiosciences.com
www.biochem.roche.com
www.cambridgeantibody.com/
www.ciphergen.com
www.clontech.com
www.geneprot.com
www.morphosys.com
www.procognia.com
www.prolinx.com
www.sigmaaldrich.com
www.somalogic.com
www.zeptosens.com
www.zyomyx.com
Major Companies exploiting proteomics
www.abbottdiagnostics.com
www.astrazeneca.com
www.atheris.ch
www.bayerdiag.com
www.cellzome.com
www.chiron.com
www.Digene.com
www.incyte.com
Vendors supplying miscellaneous proteomics products
www.amershambioscience.com
www.genomicsolutions.com
www.probes.com
www.proxeon.com
Major companies dealing with protein products.

Source: http://www.tekes.fi/eng/publications/proteomics.pdf
Conclusion:
SELDI enables researchers gain a better understanding of biological functions at the

protein levels, as tool provides a direct approach to understanding the role of proteins
in the biology of disease, monitoring of disease progression and in the therapeutic
affects of drugs. [15, 16]Thus in this review the overall advancement of SELDI
technology and its various application along with the principles, advantages and
disadvantages are discussed in details. These types of review papers drive the
creation of such technologies and advancement in them. Because on looking from
the last decade there has been an exponential growth in the collective understanding
and the utility of this technique which could be picturized from the evolution of SELDI
from an inherently evolution of seldi from an inherently challenging research project
into a useful tool for biomedical research.[16]”Finally, as SELDI ProteinChip
technology is applied more frequently on a large scale, automation of sample
preparation, chip reading and data analysis will become imperative”.[15 ]
9.2.7 References.
1. Biocompare - a guide for Life Scientist web site (15/10/06) (update Feb 16
‘05) http://www.biocompare.com/spotlight.asp?id=316
2. Thomas P Conrads, Ming Zhou, Emmanuel F Petricoin, Lance Liotta and

Timothy D Veenstra, (2003) Cancer diagnosis using proteomic patterns,
Expert Rev. Mol. Diagn. 4: 411-420.
3. Semmes et al., (2005). Evaluation of Serum Protein Profiling by Surface-
Enhanced Laser Desorption/Ionization Time of Flight Mass Spectrometry for
the Detection of Prostate Cancer Assessment of Platform Reproducibility,
Clinical Chemistry. 51: 102-112.
4. Judith Y.M.N. Engwegen, Marie-Christine W. Gast, Jan H.M. Schellens and

Jos H. Beijnen, (2006) Clinical proteomics: searching for better tumour
markers
with SELDI-TOF mass spectrometry, TRENDS in Pharmacological Sciences
.27:
251-259.
5. Petricoin, E.F. et al. (2002) Use of proteomic patterns in serum to identify

ovarian
cancer. Lancet. 359, 572–577.
6. Frears E.R., Stephens D.J., Walters C.E., Davies H. , Austen B.M, (1999) The
role of cholesterol in the biosynthesis of β-amyloid, NeuroRepor., 10 : 1699-
1705.
7. Shane Beck, (1998) Ciphergen's ProteinChip Arrays , The Scientist. 12: 15-
17.
8. Glaser .V, (1998) Gen.Engineer News, 18: 1
9. Glaser.V, Rosetta, (1998) Polymeric arrays and methods for their use in
binding assays, Nature biotechnology.15: 937-938.
10. Strauss, E., (1998) , After the genome IV meeting : News Ways to probes the
molecule of life, Science. 282: 1406-1407.
11. Engwegen, J.Y.M.N. et al. (2006) Identification of serum proteins

discriminating colorectal cancer patients and healthy controls using surface
enhanced laser desorption ionisation-time of flight mass spectrometry
(SELDI-TOF MS). World J. Gastroenterol.12:1536–1544.
12. Dr. Benjamin Reed, An Introduction to SELDI ProteinChip® Proteomics and

related Applications to Pharmacoproteomics and Clinical Biology, University
of water loo,Ontario, Canada, Faculty of science website.
www.science.uwaterloo.ca/.../spmeseldi.htm
13. David W Speicher,The Wistar Institute Porteomics short course lecture2,

Protein profiling,Biological applications and Human disease.(2004)
http://bioinformatics.biomed.drexel.edu/CfIB_Resources_files/Week5/Day1H2
.pdf
14. Wiley interscience. http://www3.interscience.wiley.com/cgi-bin/home

15. Weinberger S.R., Merchant.M, (2000) Recent advancement in surface-
enhanced laser desorption / ionization time of flight – Mass spectrometry,
Electrophoresis, 21: 1164-1177.
markers
with SELDI-TOF mass spectrometry, TRENDS in Pharmacological Sciences.
251-259.
17. Ciphergen website (2006) http://ciphergen.com/doclib/docFiles/262.pdf
18. Harri siitari and Heini koivistoinen (2004) Proteomics – challenges and
possibilities in Finland http://www.tekes.fi/eng/publications/proteomics.pdf
19. Emanuel F Petricoin, and Lance A Liotta, (2004) SELDI-TOF-based serum

proteomic pattern diagnostics for early detection of cancer, Current Opinion in
Biotechnology. 15:24–30.
20. Ning Tang, Pete Tornatore, and Scot R. Weinberger , (2004)Current

Developments in SELDI Affinity Technology, Mass Spectrometry Reviews. 23
: 34–44.
21. Sonja V,Steve C,Scot R W & Andreas W (2005) Protien quantification by the
SELDI-TOF-MS based Protienchip system Nature Methods .2 : 393-395.
marker
with SELDI-TOF mass spectrometry. Trends in Pharmacological Sciences
.27:
251-259.
23. Yue Hu, Suzhan Zhang_, Jiekai Yu, Jian Liu, Shu Zheng, (2005) SELDI-TOF-
MS: the proteomics and bioinformatics approaches in the diagnosis of breast
cancer, The Breast , 14 : 250–255.
24. Haleem J. Issaq,, Timothy D. Veenstra, Thomas P. Conrads, and Donna

Felschow (2002) The SELDI-TOF MS Approach to Proteomics: Protein
Profiling and Biomarker Identification,Biochemical and Biophysical Research
Communications, 292, 587–592.
25. S.R. Weinberger , E. Boschettib, P. Santambienb, V. Brenacb, (2002)

Surface-enhanced laser desorption–ionization retentate chromatography_
mass spectrometry (SELDI–RC–MS): a new method for rapid development of
process chromatography Conditions, Journal of Chromatography B. 782 :
307–316.
26. Dan Agranoff, August Stich, Paulo Abel and Sanjeev Krishna, (2005)
Proteomic fingerprinting for the diagnosis of human African trypanosomiasis,
Trends in Parasitology. 21:154-157.
27. University of Arkansas for Medical Sciences, Center for Orthopaedic Research,
Orthopaedic Surgery Department. www.cor.uams.edu/services.asp
28. Emanuel F Petricoin1and Lance A Liotta, (2004), SELDI-TOF-based serum

proteomic pattern diagnostics for early detection of cancer, Current Opinion in
Biotechnology. 15:24–30.
29. Junjun Wang,y Defa Li, Lawrence J. Dangott,z and Guoyao Wuy (2006)
Proteomics and Its Role in Nutrition Research,Journal of nutrition,1759-1762.
30. Eastern Virginia Medical School website

http://www.evms.edu/vpc/seldi/seldiprocess/index.html
Protein Nanotechnology
By Payal Patel
S3120173
10.1 Inroduction
The branch of engineering that deals with manipulating or constructing things smaller
than 100 nanometres is called as Nanotechnology. In healthcare, diagnosis takes
and important part to treat a disease. Identification and quantification of proteins and
their folding mechanism are very important in diagnosis of diseases. Small quantities
of proteins, which generally escape from detection and are responsible for the
diseases, now, can be quantified by protein nanotechniques. Proteins are long
stringy molecules that fold up into complex and useful shapes due to very subtle
interactions between their component parts. Proteins do most of the molecular
manipulation work in our bodies, joining and splitting molecules, moving things as
small as atoms and as large as cellular organelles from one place to another, and
making cellular metabolism work.
The combination of nanotechnology and molecular biology has led to a new

generation of nanoscale-based devices and methods for probing the cell machinery
and elucidating intimate life processes occurring at the molecular level that were
invisible to human inquiry.
After using all this advanced technology scientists were able to say that they can
actually switch on and off protein because they take a control on specific protein and
works on it. Protein Nanotechniques have given better understanding of
biotechnology processes.
10.2 Recent Advances
The worldwide emergence of nanoscale science and engineering was marked by the
announcement of the National Nanotechnology Initiative (NNI) in January 2000.
Recent research on biosystems at the nanoscale has created one of the most
dynamic science and technology domains at the confluence of physical sciences,
molecular engineering, biology,
biotechnology and medicine. Nanotechnology is beginning to allow scientists,
engineers, and physicians to work at the cellular and molecular levels to produce
major benefits to life sciences and healthcare. In the next century, the emerging field
of nanotechnology will lead to new biotechnology based industries and novel
approaches in medicine.
The recent developments in nanotechnologies such as protein microarrays,

biosensors etc. and their application in diagnosis of diseases at proteomics level
have also been seen. Major advances in the last several years in scanning probe and
scanning optical analytical methods permit viewing the vital chemical processes and
microscopic structures in biological systems with unprecedented resolution. These
new analytical probes reveal a detailed picture of the microscopic structure of living
cells and a view of chemical processes at the molecular scale.
The atomic force microscope, for example, can locate and measure the
extraordinarily small forces associated with receptor-ligand binding on cell surfaces.
Microscopic electrical probes can detect a living cell’s exchange of ions with its
environment or the propagation of electrical signals in nerves. The optical
instruments combined with chemically selective light –emitting fluorescent probes,
can follow chemical processes on the surfaces of and inside a living cell. This
capability allows observation of the biochemical process and interactions of cells in
living systems. In human body cells contain naturally occurring molecular motors. For
eg. F1-ATPase which is part of the large, membrane-embedded complex that
synthesizes ATP within mitochondria (Figure). This structure is only about 10 nm in
size, is a robust, fully functional rotating motor that is powered by natural biochemical
processes.
Figure: The molecular motor protein F1-ATPase. An actin filament is attached to a
motor protein to provide load to and allow visualization of the motor rotation
www.wtec.org
During the last few years, scientists have developed the technology for rapidly
mapping the genetic information in DNA and RNA molecules, including detection of
mutations and measurement of expression levels. This technology uses DNA
microchip arrays that adapt some of the lithographic patterning technologies of the
integrated circuit industry. Miniaturization of allied analytical processes such as
electrophoresis will lead to increases in throughput and reduced cost for other
important methods of analysis such as DNA sequencing and fingerprinting. For
example, new research is aimed at replacing the tedious, slow, and expensive
process of DNA sequencing in slab gels with miniaturized integrated micro fabricated
analytical systems (Figure).
Figure: Photo mosaic of a DNA separation chip.
www.wtec.org
The image is pieced together from twelve optical micrographs. The inset shows a
small region 0.8 mm long containing dense pillars that act as a molecular sieve to
separate DNA molecules according to size. Conventional gel electrophoresis works
essentially the same way, and for this reason these nanofabricated structures are
called “artificial gels.” This technology has the potential to revolutionize DNA
separation techniques by providing an inexpensive, durable, and reproducible
medium for DNA electrophoresis
To deliver drugs and genes into cells nanoparticles considerably smaller than one
micron in diameter have been used. The particles can be combined with chemical
compounds that are ordinarily insoluble and difficult for cells to internalize. These
particles can then be introduced into the bloodstream with little possibility of clogging
the capillaries and other small blood vessels. The efficacy and speed of drug action
in the human body can thereby
be dramatically enhanced. As part of new technology nanoparticles can be used in
similar ways, by carrying DNA fragments they can be used to incorporate specific
genes into target cells (Figure).
Figure: The “Gene Gun,” a system that uses nanoparticles to deliver genetic material
to transfect plant and animal cells.
www.wtec.org
In this system, submicron gold particles coated with DNA are accelerated with a
supersonic expansion of helium gas. The particles leave the front of the device at
high velocity and penetrate the cell membrane and nuclear membrane, thus
delivering the genetic material to the nucleus.
A biosensor is an analytical device which converts a biological response into an

electrical signal (Figure). The term 'biosensor' is often used to cover sensor devices
used in order to determine the concentration of substances and other parameters of
biological interest even where they do not utilise a biological system directly.
Figure: Schematic diagram showing the main components of a biosensor. The

biocatalyst (a) converts the substrate to product. This reaction is determined by
the transducer (b) which converts it to an electrical signal. The output from the
transducer is amplified (c), processed (d) and displayed (e).
www.wtec.org
In last 5 years NASA Ames has been a leader in nanotechnology. In this last 5 years
they have done so many projects to use this useful technique in bioscience and
medicine. So, they are important part of advances in protein nanotechnology and
their application in field.
10.3 Evaluation of Technology
As all other technology protein nanotechnology also have some disadvantage with
plenty of advantages.
As described in recent advances and application of nanotechnologies in medicine

and biology it is having so many advantages to people. The major one is it helps in
diagnosis of disease. It makes process simple and short. By using microarray
techniques we can find out cause of disease soon and can treat a person soon.
Biosensors, gene gun, flurecent biological labels are few examples that are covered
here.
There are few disadvanges of this technique that makes scientists think sometimes
while using it. It requires infrastructure for nanobiology. It is similar to those for other
fields: multi-user facilities to provide access to specialized technologies, funding
mechanisms and organization structures that encourage and support
multidisciplinary teams and are responsive to rapid technological change, and
training of a new generation of scientists and engineers who are prepared to
maximally exploit this new knowledge are required. It is very costly so that is big
disadvantage.
10.4 Application of Technology
The protein nanotechnology has application in different fields. In life science and
medicine, engineering, physics etc.
DNA detector arrays that today operate in the micron size range provide the potential
to
do thousands of experiments simultaneously with very small amounts of material.
Figure shows an image of a chip with 6,400 microdots, each containing a small
amount of a different gene in the yeast genome and capable of determining how
active that gene is in yeast. Yeast cells were grown under various conditions; the
amount of red or yellow light represents the level of RNA produced from the DNA in
that gene, under those conditions. Similar experiments using this or related
technologies can now be performed with tens or hundreds of thousands of human
genes. By comparing the pattern of gene expression of normal tissue with cancerous
tissues, scientists can discover which few genes are being activated or inhibited
during a specific disease. This information is critical to both the scientific and clinical
communities in helping to discover new drugs that inhibit cancer-causing genes. The
important point is that these technologies allow physiological changes in yeast or
humans to be characterized, molecule by molecule, in just a few hours. Five years
ago, an experiment like this would have taken dozens of scientists months to
complete.
Figure: The full yeast genome in a microarray chip.
www.wtec.org
For more than a decade there has been an intensive effort to prepare high-quality
nanometer-size colloidal crystals of many common semiconductors. At the onset, this
effort had a strong focus on fundamental studies of scaling laws, in this case,
quantum confinement of electrons and holes. Over this decade, tremendous
advances occurred in both the spectroscopy and the fabrication methods. This
yielded a new class of very robust macromolecules with readily tunable emission
energy. To the extent that applications of this technology were envisioned at the
onset, they were focused in the domain of optoelectronics. Yet quite unexpectedly, it
turns out that these colloidal nanocrystals can be used as fluorescent labels for
biological tagging experiments. Biological tagging is one of the most widely employed
techniques for diagnostics and visualization. As shown in Figure it appears as though
for many applications, the colloidal nanocrystals are advantageous as labels. This
has led to rapid commercialization of the new nanotechnology.
It has significant advantages over conventional dyes

¾ 4Reduced photo bleaching
¾ 4Multi-color labelling, parallel screening
¾ 4Infrared labels, blood diagnostics
¾ 4Molecular size nanocrystals are bio-compatible, with many other possible
applications.
Figure: Semiconductor nanocrystals as fluorescent biological labels
www.wtec.org
Nanotechnology also has revolutionary advances in military capability. For instance,

the confluence of biology, chemistry, and physics at the nanometre scale is enabling
significant advances in military sensors for biological and chemical warfare agents.
Civilian disaster response teams and commercial medicine will benefit as well. We
cannot afford to respond to a nerve gas attack. Defence research and development
programs are pursuing many sensor options; two related technologies are nearing
fruition and will have medical applications as well.
One is a colorimetric sensor that can selectively detect biological agent DNA; it is in
commercial development with successful tests against anthrax and tuberculosis
(Mirkin 1999). Compared to present technology, the sensor is simpler, less expensive
(by about a factor of 10), and more selective—it can differentiate one nucleotide
mismatch in a sequence of 24, where 17 constitutes a statistically unique
identification.
Figure: Anthrax detection: when the anthrax target is present, pairs of nanoparticles
assemble together via the DNA filaments and change the color of the respective
suspension
www.wtec.org
10.5 References
1. Baselt, D.R., G.U. Lee, and R.J. Colton. 1996. Biosensor based on force
microscope technology. Journal of Vacuum Science and Technology B 14(2):789-
793.
2. Brown, P. 1999. http://cmgm.stanford.edu/pbrown/yeastchip.html.
3. Bruchez, M. Jr., M. Moronne, P. Gin, S. Weiss, and A.P. Alivisatos. 1998.

Semiconductor nanocrystals as fluorescent biological labels. Science 281:2013-
2016.
4. Chan, W.C.W., and S.M. Nie. 1998. Quantum dot bioconjugates for ultrasensitive
nonisotopic detection.
Science 281:2016-2018.
5. Colton, R. 1999. (Chemistry Division, Naval Research Laboratory -- private

communication).
6. Jelinski, L. 1999. Biologically related aspects of nanoparticles, nanostructured

materials, and nanodevices.
In Nanostructure science and technology. NTSC Report, ed. R.W. Siegel, E. Hu, and
M.C. Roco.
Baltimore: World Technology Evaluation Center (WTEC). Web site
http://itri.loyola.edu/nano/IWGN.Worldwide.Study/. Also published by Kluwer
Academic Publishers.
7. Hameroff, S., et al. 1990. Scanning tunneling microscopy of cytoskeletal proteins:

Microtubules and intermediate filaments. J. Vac. Sci. and Tech. A 8:687-691.
8. Lysaght, M.J. 1998. An economic survey of the emerging tissue engineering

industry. Tissue Eng. 4:231- 238.
9. Mirkin, C. 1999. (Department of Chemistry, Northwestern University – private

communication).
10. Noji, H. 1998. The rotary enzyme of the cell: The rotation of F1-ATPase. Science
282: 1844-1845.
11. Odde, D.J. and M.J. Renn. 1998. Laser-based direct-write lithography of cells.
Ann. Biomed. Eng. 26:S- 141.
12. Renn, M.J., et al. 1999. Laser guidance and trapping of mesoscale particles in
hollow-core optical fibers. Phys. Rev. Lett. 82:1574-1577.
13. Santini, J.T. Jr., M.J. Cima and R. Langer. 1999. A controlled-release chip.
Nature 397:335-338.
14. Shi, H., et al. 1999. Template-imprinted nanostructured surfaces for protein
recognition. Nature 398:593- 597.
15. Indian Journal of Clinical Biochemistry, 2005, 20 (2) 48-53

www.medind.nic.in
16. Barry, R. and Ivanov, D. (2004) Microfluidic in biotechnology. J. Nanotechnol. 2,
1-5.
17. U.R. Muller, D.V. Nicolau (Eds), Microarray Technology and Its Applilcations
18. Biosensors, www.lsbu.ac.uk
19. www.comspacewatch.com/news
20. J Nanosci Nanotechnol. 2006 Sep-Oct;6(9-10):2736-53.
21. www.pubmed.com

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CLASS LEARNING RESOURCE

Written by students enrolled in BIOL2226 Protein Technologies

School of Applied Sciences

1. STRUCTURAL ANALYSIS OF PEPTIDES AND PROTEINS

1.1 X-Ray Diffraction

2. PURIFICATION AND ANALYSIS OF PROTEINS

2.1 Strategies for Protein Purification

3. CHEMICAL ANALYSIS OF PROTEINS

3.1 Amino Acid Analysis and sequencing of proteins

4. MASS SPECTROMETRY OF PROTEINS

4.1 MALDI-TOF Mass Spectrometry

5.1 2D gel electrophoresis

6.1 Chemical methods for study of protein-protein interactions

7.1 t-Boc synthesis and cleavage of peptides

8.1 Approaches to Protein Engineering

9.1 Biomarker discovery and applications

SAFALYA KUMAR DEEPAK

X rays were discovered by Wilhelm Roentgen in November 1895, while studying

The discovery of x ray crystallography literally changed the scenario of biological

Fig.1.1 x ray diffraction of a protein crystal.

To get the perfect image of a biomolecule, it should be properly crystallized before

Fig1.2 types of crystals.

Fig.1.3 structural analysis of protein crystals.

1.1.2 Recent advances.

Synchrotron light is the electromagnetic radiation emitted when charged particles,

1.1.2.2 Automation of crystal production.

Structural analysis of biological compounds is now entering a new phase by the

In MALD the synchrotron is tuned to different wavelengths. This helps in the

1.1.3 Evaluation of the technology.

Comparison of x ray crystallography and NMR Spectrometry.

The advantages and disadvantages of X ray crystallography and that of NMR

Advantages of x ray crystallography.

3. Could get the whole 3D structure by the systematic analysis of a good

Disadvantages of x ray crystallography.

2. Cannot examine solutions and the behaviour of the molecules in solution.

3. This happens when we try to examine powders, gases.

4. Study of motions is not available.

6. No possibility to examine small parts in the molecule.

7. No chance for direct determination of secondary structures and especially

1. Several types of information from lots of types of experiments.

2. Obtain angles, distances, coupling constants, chemical shifts, rate constants

4. With a suitable computer apparatus we can calculate the whole 3D structure.

6. The motion of the segments (domains) can be examined.

1. Have lots of atoms and a lot of extracted data from a system.

4. The highest molecular mass which was examined successfully is just a

6. Just able to determine the degree of probability of being of the protein

7. The cost of the experimental implementation is increasing with the higher

1.1.4 Application of the technology.

The field of biotechnological research is now focussed on genetics and proteomics.

1.1.5 Relevant websites.

1. Biological Macromolecular Crystallization Database (BMCD)

1.1.6 Key industry suppliers.

19. Scapin, G. (2006) structural biology and drug discovery, Current

The impact of nuclear magnetic resonance spectroscopy on the natural sciences is

1.2.2 Recent Advances

The high-resolution liquid-state NMR spectroscopy can determine protein structure;

As the multi-dimensional NMR-experiments is time-consuming , time-saving NMR

1.2.3 Evaluation of the Technology

Nowadays, except NMR Spectroscopy, X-Ray Crystallography, which involves using

The ability to characterize protein complexes under physiological conditions at atomic

However, a major problem of macromolecular NMR is its size limitation caused by

1.2.4 Application of the Technology