An Introduction to Mass Spectrometry

by
John J. Kelley, III

Overview of Mass
Spectrometry
Sample
Molecule (M)

Ionization

M+/Fragmentation
Mass Analyzer

MassSpectrum

Mechanisms of Sample
Ionization
Protonation: M + H+
MH+
Cationization: M + Cat+
MCat+
Deprotonation: MH
M - + H+
Electron Ejection: M
M+. + e Electron Capture: M + eM -.

Inlet system

Ion Source

The Components of a
Mass Spectrometer
Analyzer

Ion
Detector

m/z

Mass Spectrum

Computer

Types of
Spectrometers

Magnetic

Sector Instruments
Quadrupole Instruments
Time-of -Flight Instruments
Ion Trap
Fourier Transform Ion Cyclotron
Resonance (FT-ICR)

Magnetic Sector Mass

Spectrometer

Quadrupole Mass Spec

Time of Flight Mass Spec

Time-of-Flight Mass
Spectrometry

There are actually two type of mass

analyzers in use for MALDI:
Reflectron time-of-flight mass spectrometer
Linear time-of-flight mass spectrometer

Reflectron & Post Source

Decay

Linear TOF machines- fragmented in flight by only

mass. Ensemble of decay products travel with
parent ion.
If, however, a retarding field is placed in front of the
detector, the extent of fragmentation is easily
measured
The measuring principle uses the fact that the
kinetic energy of a fragment ion is proportional to its
fractional mass. Thus, in the retarding field of the
reflectron, such fragments will turn around earlier
than their respective precursors and will arrive at the
detector sooner.

Post Source Decay (PSD)

fragment ion mass analysis

evaluation

of post source decay following MALDI

R. Kaufmann, J. Biotechnol. 4, 160 (1995).

Post Source Decay

Thus, it came rather as a surprise that roughly

the same kind of information,as in the protein
ladder sequencing, can be obtained as a byproduct in reflectron TOF spectrometers
Simply by the analysis of the abundant post source
decay (PSD) fragments
These fragments are formed in the field-free drift
region after MALDI

Need a reflectron TOF spectrometer and a

precursor ion selector

Application of Mass Spec

to BioMolecules

ESI-MS: Electrospray Ionization Mass

Spectrometry

MALDI-MS: Matrix Assisted Laser

Desorption Ionization Mass Spectrometry

Overview of MALDI-MS:
Instrumentation:

Time-of-flight mass spectrometry

The desorption/ionization process by
nanosecond laser pulses
The principle of matrix assistance
Sample preparation

Block diagram of MALDI- Linear TOF MS

computer

Digital Oscilloscope

Cotter, R.J. In Time-of-Flight Mass Spectrometry; Cotter, R.J., Ed.; American Chemical
Society: Washington, DC, 1994; pp. 41.

Finnigan Corporations Benchtop

LASERMAT 2000

Operation of the LASERMAT 2000 is straightforward, with no need for instrument tuning or
optimization. Samples ranging from small peptides up to large glycoproteins over 200,000
Da can be easily and rapidly measured.

Time-of-Flight Mass
Spectrometry

In time-of-flight (TOF) instruments, positive

ions are produced periodically by
bombardment of the sample with brief
pulses of electrons, secondary ions, or in
this case laser-generated photons.

These laser pulses typically have a

frequency of 10 to 50 kHz and a lifetime of
0.25 s.

Time-of-Flight Mass
Spectrometry

The ions produced by the laser are then

accelerated by an electric field pulse of 103
to 104 V that has the same frequency as,
but lags behind, the ionization pulse

The accelerated particles then pass into a

field-free drift tube. The drift tubes length
can range from 0.5 - 3.0 meters.

Time-of-Flight Mass Spectrometry

All ions entering the tube ideally have the same

kinetic energies, their velocities in the tube must
vary inversely with their masses, with lighter
particles arriving at the detector earlier than the
heavier ones.

t=

m
2zeEs

1/ 2

The ions then drift through a field-free path,

usually 1-3m in length, and are separated in
space and time-of-flight according to the equation:

Mechanisms of matrix assisted laser

desorption/ionization

The matrix serves three major functions:

Isolation of the biomolecules from each other
The

biomolecules are incorporated in a large

excess of matrix molecules, strong
intermolecular forces are thereby reduced (matrix
isolation).
One of the most important conditions for
matrices to work well is an intimate mixing of the
analyte and the matrix.
optimal molar ratios are in the 1:1000 to 1:10000
(analyte to matrix) range. This means that analyte
molecules are singularly and fully surrounded
(solvated) by matrix molecules.

The principle of matrix

assistance

Absorption of energy from the laser light

The

matrix molecules absorb the energy

from the laser light and transfer it into
excitation energy of the solid system.
Thereby inducing an instantaneous phase
transition of a small molecular layers of the
sample into a gaseous species.

The principle of matrix

assistance

Ionization of the biomolecules

An

active role of the matrix in the ionization of

the analyte molecules by photoexcitation or
photoionization of matrix molecules, followed
by proton transfer to the analyte molecules is
likely, though not proven unequivocally to
date.
In practice, success or failure of a matrix
depends on properties other than acting as an
energy transfer mediator
Unfortunately, none of the many theories so
far proposed can fully describe the chain of
events.

The most commonly used

substances
Matrix

Wavelength

Comments

2,5-Dihydroxy benzoic acid (DHB)

337

DHB + 10% 5-methoxy salicylic acid

337

Nicotinic acid

266 nm

4-Hydroxy picolinic acid

337

used for oligonucleotides

Glycerol

2.94m

liquid matrix

used for masses >20 kDa

U. Bahr, M. Karas, F. Hillenkamp, Fresenius J. Anal. Chem. 348, 784 (1994).

Sample Preparation
Simply mix 10 nanomoles of an aqueous
matrix solution with 0.1 picomoles of
analyte solution
Deposit the smallest possible aliquot on
a clean metallic support
This droplet is either air dried or gently
blown dried by means of hairblowers or
radiative heating

Uses of MALDI-TOF in Biomolecule

Analysis
Protein

Analysis

Mass determination of Biomolecules

Maximum size of protein that can be measured,
Human Immunoglobulin
Single Cell analysis of neuropeptides
advantages over classical methods of protein
analysis

Protein Ladder Sequencing

A concatenated set of peptide fragments can be

generated in a controlled fashion

Size Determination of Human immunoglobulin IgM

by MALDI, MW = ~1 MegaDalton

R.W. Nelson et al., Rapid Commun. Mass Spectrom. 9, 625 (1995).

Basic Structure of a Neuron

cell of the nervous system specialized to generate
and transmit nerve impulses

soma

Ventral

Axon

commissure

Axon terminal
dorsal

Peptides used by the Nervous

System

Peptides are widely used by the nervous

system as neurohormones to regulate different
processes such as reproduction, growth,
metabolism and behavior.
The peptides are synthesized in the form of
larger prohormones, from which they are
proteolytically cleaved, further modified, and
stored in secretory granules in axon terminals,
from which they are released in response to
depolarization

Neuropeptide Expression and

Processing Revealed

Peptides in single identified large neurons of

the mollusc Lymnaea stagnalis were
analyzed via MALDI-MS
The samples were placed in matrix
solution and ruptured to allow mixing of
cell contents with the matrix solution
After formation of matrix crystals, the
analytes were analyzed by MALDI-MS

single dorsal CDC cell

single ventral CDC cell

intercerebral commisure

intercerebral
commisure of a
different animal
C.R. Jimenez et al., J. Neurochem. 62, 406

MALDI-MS vs Classical Screening

Methods for mass determination

In many instances, MALDI-MS can give

information of wanted or unwanted targets right
out of the raw or at least crude material
Target protein identification using MALDI-MS
vs SDS-PAGE
SDS-PAGE Sodium Dodecyl sulfonate

polyacrylamide gel electrophoresis

The 0.1%-0.01% accuracy of molecular weight
determination vs 5-10% with SDS-PAGE

MALDI-MS vs Classical Screening

Methods

Short measuring time and negligible

sample consumption
few minutes and subpicomolar amounts

Windfall profits such as information on

microheterogeneities, glycosylation,
phosphorylation

Classical Peptide
Sequencing

The most direct sequence determination

of peptides and proteins is done by an
automated Edman degradation

Edman Degradation
A multi-step

process:

A chemical reaction is used to remove

one amino acid at a time from the amino
terminal
Each amino acid is converted to a stable
form and then identified by analytical
reverse-phase high-performance liquid
chromatography
Currently such sequencing is limited to
less than ~50 residues per day

Using MALDI-TOF to
Sequence Proteins

A new approach to protein sequencing:

Two steps
ladder-generating

chemistry

the controlled generation of a set polypeptide

fragments by wet chemistry, each differing from
the next by one amino acid
data

readout

a one-step readout of the resulting protein

sequencing ladder by matrix-assisted laserdesorption mass spectrometry

A Stepwise Degradation

Carried out with a small amount of

terminating agent present in the coupling
step
A mixture of phenylisothiocyanate (PITC) plus
5% v/v phenlyisocyanate (PIC) is used as the
terminating reagent.

Phenylisocyanate reacts with the NH2

group of a polpeptide chain to yield an Nphenylcarbamyl-peptide, which is stable to
the conditions of degradation

Hydrolysis Step

The trifluoroacetic acid (TFA) is used to

cleave the terminal amino acid (AA) from the
phenylthiocarbamyl (PTC) peptide.

The phenylcarbamyl (PC) peptides formed

are stable to the trifluoroacetic acid used to
cleave the terminal amino acid from the
phenylthiocarbamyl (PTC) peptide.

A Stepwise degradation
AA1-AA2-AA3-AA4-AA5- -AAn
PITC + 5% PIC
PTC-AA1-AA2-AA3-AA4-AA5- -AAn
PC-AA1-AA2-AA3-AA4-AA5- -AAn

Further cycles
(without
separation)

Acid (TFA)
ATZ(AA1) + AA2-AA3-AA4-AA5- -AAn
PC-AA1-AA2-AA3-AA4-AA5- -AAn
after m cycles
PC-AA1-AA2-AA3-AA4-AA5- -AAn
PC- AA2-AA3-AA4-AA5- -AAn
PC-AA3-AA4-AA5- -AAn one-step MALDI-MS
PC-AA4-AA5- -AAn
PC-AAm- -AAn
B. Chait et al., Science 262, 89 (1993).

ladder
sequence
data

One step MALDI-MS readout

PC-AA1-AA2-AA3-AA4-AA5- -AAn

PC- AA2-AA3-AA4-AA5- -AAn

PCH

OH

C
CH2
CH2
N CH C
H
O

m/
z

3*O = 48
5*C = 60
1*N = 14
8*H = 8

HAA2

AAn

________
m =129.7

Protein ladder Sequencing of [Glu1]fibrinopeptide B

B. Chait et al., Science 262, 90 (1993).

A second illustration

Protein Ladder sequencing can distinguish

between the phosphorylated and
unphosphorylated forms of a Serine residue.
Serine has a residue mass of 87.1 daltons;
addition of -PO3H2 in place of a proton results in
an additional mass increment of 80 daltons, for
a phosphorylated Serine residue mass of 167.1
daltons.
this will have important implication in locating
and quantifying phosphorylated residues
because they play a major role in signal
transduction

What is a two dimensional

gel?
Cell extracts are put onto a gel and
individual proteins are separated in the first
dimension by charge and then by size.
The result is a characteristic picture of 500
to 2000 spots, each usually a single protein
Each spot
reveals the amount of protein present
many of the posttranslational modifications
they have undergone

www.expasy.ch

2D-gel of erythroleukemia cell

Goal of the future

the key is to identify a spot on a 2D-gel

Learn something about it that can be

used to search protein databases

Identifying proteins from twodimensional gels

One step toward this goal
was

solved by several groups in the 1980s

who developed methods to transfer spots
from gels onto membranes so they could be
analyzed

Drawback
Identification

of all the resolvable proteins on

a two-dimensional gel by conventional
protein sequencing is a daunting task

Putting it all together:Identifying

proteins from 2D-Gels

Proteins are electroblotted from two-dimensional gels

The spots are excised and digested with trypsin

Trypsin cleaves the C-terminal end of arginine and
lysine producing peptide fragments

Masses are determined at the subpicomole level by

MALDI-MS of the tryptic digest

FRAGFIT, a computer program, then searches the

protein sequence database for multiple peptide
fragments of individual proteins that match the
measured masses.

The FRAGFIT Algorithm

Input for the FRAGFIT program: a list of the peptide

masses, the protease or cleavage reagent, a mass
tolerance, and the number of allowed mis-matches

The program scans the database, generates

sequence fragments based on the specified
protease and computes the theoretical molecular
masses of these fragments

The protein database used

Consist of a combination of several widely available

databases: Swiss-Prot, Protein Identification
Resource, and a translation of GenBank

The resulting database contains 18.8 million

residues, representing over 91,000 entries

The C-language source code for FRAGFIT can be

obtained via E-mail to ckw@gene.com

From Genome to Proteome

It sounds like mission impossible:

Follow the changes taking place inside a cell by
identifying the thousands of different proteins
the cell produces and watching how they
change and flow over time.
To a growing band of researchers, however,
such a mission is becoming more realistic,
thanks to the increasing volume of sequence
data and to improved analytical techniques for
proteins like MALDI-MS

MALDI-TOF Spectra

Tool of the Future

MALDI-MS coupled with this method
facilitates simultaneous processing of a
large number of two-dimensional gel spots
researchers are excited about the
implications:

Drugs induce changes in protein patterns

these changes on the gels should give important

clues about both the drugs desirable effects and its
toxicity

Knock out their favorite gene and see its effect

on the protein pattern