Chemistry 315

Experiment 9:
Mass spectrometry of biomolecules
Required readings:
1. ASMS: Education- What is Mass Spectrometry? http://www.asms.org/docs/what-is-
mass-spec/whatismsposter.pdf (accessed Aug. 26, 2010).
This is a concise summary of mass spectrometry basics and introduction to more
complex subjects.
2. Principles of Instrumental Analysis, 5th Ed., D.A. Skoog, F.J. Holler, T.A. Nieman,
Saunders College Publishing, 1998. Chapter 20: Mass Spectrometry

Focus on understanding the basic mass spectrometer hardware layout, how

ionization occurs and how EI, ESI and MALDI ionize samples.

Recommended further readings:

3. Yamashita, M.; Fenn, J. B. Electrospray Ion Source. Another Variation on the Free-
Jet Theme J. Phys. Chem. 1984, 88, 4451-4459.
4. Fenn, J. B.; Mann, M.; Meng, C. K.; Wong, S. F.; Whitehouse, C. M. Electrospray
Ionization for Mass Spectrometry of Large Biomolecules Science 1989, 246, 64-71.
DOI: 10.1126/science.2675315

Introduction
Protein analysis is a major field of research in both industrial and academic settings.
Identifying unknown proteins in a complex mixture such as blood is critical to
understanding disease states. Confirming that protein bioproducts (such as antibodies) are
pure is very important in pharmaceutical development and production. One of the most
widely used analytical tools for protein analysis is mass spectrometry (MS). MS has been
used effectively in many other fields since its conception by J. J. Thompson circa 1897,
but analysis of large biomolecules was not feasible until the advent of matrix-assisted
laser desorption ionization (MALDI) and electrospray ionization (ESI) ion sources in the
1980s. Whereas previous sources like electron impact (EI) were typically limited to
analysis of volatile substances and/or induced heavy fragmentation in larger molecules,
MALDI and ESI are able to reliably convert large molecules into intact ions for
subsequent identification.

Analyzing biological molecules in by mass spectrometry was previously a problem

because the molecular mass of most proteins and many other analytes is larger than the
mass-to-charge (m/z) limit of quadrupole ion traps, which typically scan to a few

Adapted with permission from J. Chem. Ed. (updated 10/24/10 by Eric Lanni)

1
thousand m/z. ESI overcomes this by multiple protonation, which can convert a
biomolecule M into multiply charged ions with the formula (M + nH)n+ and charge z in
positive ion mode. Because mass spectrometers measure the analyte mass-to-charge ratio
rather than the mass, this allows larger molecules to be observed at a much lower m/z
which is in range for the analyzer. Combined with the (relative) affordability of ion trap
analyzers and electrospray sources, this has made the ESI-trap one of the most widely
used types of instrument for modern biomolecule analysis.

In this experiment, you will first use ESI-Ion Trap MS for mass analysis of a known
peptide, bradykinin. Since the molecular structure of bradykinin is known, you will be
able to predict its spectral features (charge states, isotopic distribution) and compare these
to the experimental data. The data will also be used to evaluate instrument performance
in terms of accuracy and resolution. You will then perform an MS/MS experiment to
fragment bradykinin using collision-induced dissociation (CID) in order to elucidate its
molecular structure. Finally, you will use the same approaches to analyze and identify an
unknown peptide from the list of possibilities shown in Table 2 (below).

Interpreting Mass Spectra

Although MS provides a reliable method for identifying substances and mixtures by their
mass, interpreting mass spectra in practice is often not straightforward; spectral features
such as charge states, isotopic distributions, ion fragments, adduct ions, chemical
interference and background ions must often be sorted out to find useful information.
Ultimately, the goal is to make one or more mass assignments which relate an observed
ion signal (peak) on the mass spectrum to a molecular species. A confident mass
assignment requires: 1) a reasonable theoretical explanation of how the assigned ion may
have formed and 2) a mass error within the expected range of the instrument.
Understanding spectral complications such as those listed above is critical to making
good mass assignments. A partial treatment of this material is provided below; see the
required readings for more information.

2
Isotopic Distributions
Isotopic distributions are clusters (envelopes) of closely-spaced peaks (isotopologues)
arising from variation in a molecules mass due to isotope combinations of the elements
comprising the molecule. Consider, for example, methane:

Figure 1: The molecular structure of methane

In this simple case there are only two elements- carbon and hydrogen- and we can
assume natural isotopic abundances of each. Given knowledge of these abundances
(shown in Table 1, below), how many isotopic combinations are possible in methane?
Several facts are relevant here:

The carbon can be either a 12C or a 13C.

Each H atom can be either a 1H or a 2H (deuterium).
The arrangement of the isotopes within the molecule does not affect total mass.
Each atom in the molecule is considered independently, i.e. the isotope of one atom does
not affect the isotopes of others.

Knowing this, we can determine that there are ten isotopologues of methane:

Figure 2: the isotopologues of methane. Heavier (and less abundant) isotopes are emphasized in red.

Although these ten molecules are all methane they have nominal masses ranging from 16
Daltons (1 Da = 1 gmol-1) to 21 Da due to the additional neutrons included in heavier
isotopes. When the molecule is comprised solely of the most common natural isotopes
(12C and 1H), this is referred to as the monoisotopic mass; for small biomolecules this is
also typically the lightest isotope, though this is not always the case. Isotopes that follow
are termed the M+1, M+2 etc. to indicate the number of additional neutrons present.

3
We have identified ten possible isotopic combinations for methane, but its important to
note that their natural frequency of occurrence (and therefore signal intensity on a mass
spectrum) depends on the probability of their isotopic configuration. To approximate this
we can use an isotopic abundance table, shown below:

By multiplying the number of atoms of a given element by its M+1 Factor and then
summing up this product for all elements present in a molecule, one can obtain the
relative intensity (%) of the M+1 isotopic peak relative to a monoisotopic peak (which is
assumed to be 100% relative abundance); the same can be done using the M+2 Factor
column for the next isotope. (If a given element does not have a factor listed, its
contribution is miniscule and safely ignored for our purposes.) These relative
abundances are referred to as the theoretical isotopic distribution, and they can be useful
in identifying peaks on a mass spectrum. A molecules isotopic distribution is
characteristic of its empirical formula, so when an observed ion is assigned to a molecule
it makes a strong supporting argument to show that beyond the basic mass match,
isotopic distribution is well-matched also.

4
Resolution
In order to observe isotopic distributions (and other closely-spaced features) on a mass
spectrum, the mass spectrometer must provide adequate resolution. Resolution is a
unitless value indicating an instruments ability to resolve two peaks of similar m/z value.
Numerically it is expressed as:

= (1)

Resolution can be defined in several ways, but conveniently we can determine resolution
using only one peak with the full width half maximum (FWHM) method; in this case M is
the m/z value of the single peak and M is the width of that peak at half of its full height.
For unit resolution (resolution sufficient to resolve peaks spaced 1 m/z unit apart, e.g.
isotopic peaks of a singly-charged ion):

= (2)
.

which means that the width of a peak M = 0.5 Da. If the peaks are much broader than
0.5, they will collapse into one broad peak, and only the average mass will be observed.
Note that unit resolution increases proportionally with mass, so more powerful analyzers
are required to resolve similarly-spaced peaks at higher m/z. Ion trap mass spectrometers
normally operate at constant resolving power, though this not always the case. When an
instruments resolution is reported in literature, the m/z value for the peak used for
calculation is generally specified as well (e.g. Resolution was 10,000 at 500 m/z,
calculated by FWHM method).

5
MS/MS
One of the advantages of an ion trap is that once it has trapped a mixture of ions from a
sample, they may be manipulated in a variety of ways before being scanned out for
detection. A common application of this ability is mass spectrometry/mass spectrometry,
(MS/MS or MS2), where a single parent ion is isolated in the trap and then intentionally
fragmented there by collision with helium gas. After fragmentation, the fragment ions are
analyzed and detected as usual. Fragmentation by this method (known as collision-
induced dissociation, or CID) can provide useful additional information about the
structure of an ion.

MS/MS is particularly useful for peptide identification because peptides fragment

predictably along their backbones between amino acid residues, theoretically allowing
the experimenter to sequence the molecule (determine the composition and order of
amino acids present). Each amino acid residue has a unique mass (with the exception of
isomers leucine and isoleucine), so they can be identified by the corresponding mass shift
as they are lost from the peptide parent molecule.

Safety
Skin or eye contact of the peptide solutions should be avoided due to the presence of
acetonitrile (Caution: Avoid breathing vapors. May cause skin irritation) or methanol
(Caution: Poisoning may occur from ingestion, inhalation, or percutaneous absorption);
wear gloves. If concentrated acetic acid is used in solvent preparation, it should be done
in a fume hood to avoid exposure to vapors (Caution: Ingestion may cause severe
corrosion of internal tissues). Safety goggles should be worn at all times. Care should be
exercised while working with syringe (sharp needle, fragile glass body). If skin/eye
contact of any of the chemicals mentioned above does occur, immediately wash with
large amount of water.

Prelab assignment
Note: these will require research on your own! Cite any sources that you use!
1. Find the MSDS for methanol. In a bulleted list, give the following information:
chemical formula, CAS number, boiling point, density, conditions and materials to
avoid, effects of ingestion, and alternate names. (1 point)
2. Draw a diagram of a quadrupole ion trap and briefly explain how it works. Explain
how a quadrupole ion trap differs from a quadrupole mass filter. (3 points)
3. Briefly describe one way in which ions are detected in a mass spectrometer. Why do
samples need to be ionized for detection? (2 points)
4. Explain how electrospray ionization (ESI) generates ions and why ESI works well for
studying proteins. (2 points)
5. Explain the difference between (1 point)
a) Nominal Mass
b) Average mass
c) Monoisotopic mass
6. Calculate or find the average mass and monoisotopic mass of bradykinin. Use this
to predict the m/z values for the following charge states: 3+, 2+, 1+. Assume
protonation as the mechanism of ionization. (2 points)

6
7. A caffeine sample was analyzed by electron ionization (EI) MS and the spectrum
shown below was generated with peaks labeled A, B, and C. The following questions
refer to this spectrum. (2 points)
Which letters represent the (M+2)+, (M+1)+, and M+ peaks in the spectrum?
Which letter represents the base peak in the spectrum?
Does the instrument have adequate resolving power to see caffeine's isotopes?
How do you know?
If the peak at 194.0802 m/z has a width of 0.15 m/z at half-height, what is the
resolution of the spectrum?

A B C

8. Give the m/z value for the following compounds assuming protonation as the
mechanism for ionization (2 points):
a. MeOH+
b. Angiotensin II+2
c. Melittin3+
d. Ubiquitin+5
e. Insulin+5

7
Materials
Equipment
1 ThermoFinnigan LCQ Deca XP Plus mass spectrometer
1 Gas-tight, square-tipped 500 L SGE syringe
2 10 mL beaker

Chemicals
ESI Solution: 50/50 methanol/water + 1% acetic acid (v/v)
Bradykinin (10 M in ESI solution)
Unknown peptide (10 M in ESI solution)

Chemicals for this experiment can be located in the white freezer north of the lab. You
may only use 1 vial of bradykinin and 1 vial of unknown. Dispose of all waste in the
container designated for this experiment. Do not pour waste down the sink!

Procedure
A. Sample Preparation & Setup:
1. In the small white freezer in the room on the north wall of the lab,
a) Find a plastic sample rack labeled Experiment 9. Bradykinin solutions are
prepared in microcentrifuge vials labeled with a B; select one from the rack and
remove it. Leave the rack in the freezer.
b) Find and remove the 100 mL clear glass bottle containing ESI Solution; this
will be used throughout the experiment for rinsing equipment.
2. In the drawers beneath the mass spectrometer find the syringe and 10 mL beakers.
Transfer ~5 mL ESI solution into one of the beakers for use during the lab. Rinse the
syringe with ESI before use. Return the bottle to the freezer before you begin.
3. Fill the syringe with approx. 250 L of ESI solution and make sure there are no
bubbles in the syringe. Connect it securely to the spectrometer's syringe port (see
figure below) and snap the syringe into position on the syringe pump located on the
front face of the spectrometer. See the TA for assistance with this step if you are
unsure of how to secure it. Push the blue button next to the syringe pump once to
begin infusing the ESI solution. The LED above the button will be green when the
pump is running. Allow the ESI solution to run through the instrument for 5 minutes.

Figure 3: Syringe port connection schematic

8
4. Press the blue button to stop the pump, disconnect the syringe, and put the remaining
ESI solution in the waste.
5. Fill the syringe with approx. 250 L of bradykinin solution from the vial and connect
the syringe to the syringe port as before.

B. Instrument Setup & Obtaining the Full Scan Mass Spectrum:

Figure 4: LCQ Tube interface controls with important buttons labeled.

1. Log in to the computer if necessary (login: c1017, pwd: proteomics).

2. Open LCQ Tune from its desktop shortcut if it isn't already open.
3. From the File menu, find ESI Positive Spring 2015 in the list of recent settings
and select it. (This configures the instrument properly for the samples you will be
running.)
4. Switch the instrument from Standby to On. The green box with a triangle (see
figure 4) should appear on the software when the instrument is on.
5. Push the blue button next to the syringe pump once to begin infusing sample. The
LED above the button will be green when the pump is running.
6. Wait a few minutes for a signal to appear and stabilize on the screen. Once the
signal stabilizes, begin averaging spectra by clicking the spectrum averaging
button (see figure above) in the top toolbar of the control window. This will
accumulate and maintain a 10-scan running average. Turn off spectrum
averaging any time you change samples or scan modes to reset the display and
average buffer. Note: if you do not see a strong signal the capillary might not be
attached to the syringe tightly enough try tightening the thread so no solution
escapes!
Once an adequate signal is displayed, print a copy of the spectrum from the File
menu. Zoom in on details (e.g. peaks of interest) by left-clicking and drawing a
horizontal line across the m/z range of interest on the spectrum itself. Print any details
that are relevant to your analysis. To see the entire spectra press the Full Zoom Out
x-axis button (shown in Fig. 4). *Note that this step differs from ZoomScan, and
both are required for your report!*

9
C. Obtaining a ZoomScan Mass Spectrum:
ZoomScan is a special mode of analyzer operation in which trapped ions are ejected
more slowly. This gives improved resolution but reduced sensitivity, and it can only be
used across a narrow mass window (~10 m/z) compared to a full scan.

1. Click the Define Scan button on the top toolbar of the control window. Set Scan Type
to ZoomScan and Center Mass to the m/z value of interest, then click OK.
2. The spectrum view should change to a narrow m/z range around the specified region;
accumulate an average as done previously and then print results.
3. Repeat steps #1-2 for other areas of interest; to return to Full Scan mode open the
Define Scan window and set Scan Type to Full Scan, then click OK.

D. MS/MS (fragmentation) of Bradykinin

1. Click the Define Scan button again and switch to MS/MS mode. Set the following
parameters:
Parent Mass: 1060.5 m/z (bradykinin monoisotopic MH+ ion)
Isolation Width: 2 m/z
Normalized Collision Energy: 0%
Scan Range: m/z 300-1200

2. Click Apply and move the Define Scan window out of the way so you can observe the
spectrum, but dont close it. After 1-2 scans, the spectrum should change dramatically
to show only the parent ion specified; all other ions are being ejected from the trap
prior to detection now. However, there is little or no fragmentation occurring yet
because collision energy is minimized.
3. In the Define Scan window, increase Normalized Collision Energy to 20% and click
Apply. Record your results qualitatively - are there any strong ions aside from the
parent?
4. Increase collision energy by increments of 1% every 10-15 s until you reach 30%
(click Apply after each increment). Record the results qualitatively What happens
to the spectrum as energy is increased? What happens to the parent ion intensity?
5. Turn on Spectrum Averaging and give the program approx. 30 s to average the
spectrum, then print a copy.
6. Deactivate Spectrum Averaging, return to Full Scan and single MS mode in the
Define Scan window; reset scan range to m/z 150-2000. Click OK to close the
window.

10
E. Unknown Analysis:
1. Eject any remaining bradykinin solution from the syringe into the waste container.
Rinse the syringe with ESI. Fill the syringe with 200 L of ESI solution and allow it
to pump through the source for five minutes to help rinse out residual bradykinin.
2. While rinsing return to the freezer and select a vial of unknown peptide solution. The
unknown vials will be labeled with a number on top; there may be multiple unknowns
to choose from, so be sure to record which unknown number you used.
3. Load the syringe with approx. 250 L unknown solution and infuse this into the
instrument as you did with bradykinin. Allow the solution to run through the
instrument until bradykinin is no longer present on the spectra before collecting data.
4. Repeat procedure section B to obtain a full scan spectrum of your unknown.
5. Repeat procedure section C to obtain a ZoomScan of any peaks of interest on the
spectrum.

F. Cleanup & Shutdown

1. Set the instrument back to Full Scan mode (see part D for instructions).
2. Rinse the entire syringe twice ESI solution.
3. Load the syringe with approx. 50 L ESI solution and pump it through the source to
rinse out any remaining sample.
4. Dispose of waste and rinse beaker contents properly, rinse the beakers with deionized
water (dispose of this waste properly too!) and return them to the table.
5. Remove the syringe and place it on the table.
6. Switch the spectrometer from on to standby (see procedure section B). Close out
of all programs.
7. Empty any remaining solution into the waste bottle and then put microcentrifuge
tubes in solid waste container (NOT trash).
8. Clean up any garbage (kimwipes etc.) in your work area!

For your Laboratory Report:

Integrate answers to the Questions below into the Data and Results and Discussion
sections of your lab report.

Questions
1. Bradykinin isotopic distribution analysis:
a) Using the isotopic abundance table provided in these instructions (Table 1),
calculate the expected MH+, (MH+1)+, (MH+2)+ isotopic abundances for the
singly-protonated bradykinin ion. See the Compass site (Exp. 9 Additional
Content) for a guide on using the abundance table.
b) Compare your calculated abundances with those observed on the mass spectrum
for bradykinin; how well do they match? What may cause deviation from theory?
2. Bradykinin fragmentation analysis:
a) Describe the process of the fragmentation experiment and the results observed as
collision energy was increased in the ion trap.
b) The amino acid sequence of bradykinin is (in 3-letter residue abbreviations):

Arg - Pro - Pro - Gly - Phe - Ser - Pro - Phe Arg

11
 Peptides reliably fragment between residues in collision-induced dissociation, so
the mass shift between the parent ion and its fragments indicates which residue(s)
were lost from the ends of the sequence in the process (and therefore provides the
peptide/protein sequence, if thorough fragmentation is performed). Do any of the
fragments observed correspond with portions of this sequence? Note: amino acid
residue masses are easily found online or you can draw the species in ChemDraw.
Simply open the program, type ctrl + shift + n and enter the name of the amino
acid. Then you can highlight the species (just the atoms, not the name) and click
view and show analysis window.
c) Construct a table of all fragment ions observed, along with their mass assignments
(if determined). For ions identified as bradykinin fragments, label them with the
portion of the sequence they correspond to.

3. Unknown peptide identification:

a) Identify the unknown peptide from its spectrum. Explain the approach you took,
and how you ruled out the other possible proteins or peptides on the chart. Tip: If
we select two adjacent charge states n and n + 1 of a biomolecule with molecular
mass M, the m/z, Mn and Mn+1 values are given by (rounding the mass of H to 1):

This relationship allows us to solve for M and n simultaneously.

b) Compile a table of the ions resulting from the unknown peptide. List their
abundance, and whether or not the ions are isotopically resolved.
c) Using the theoretical molecular weight of the unknown from Table 2, calculate
the theoretical m/z for each charge state that was actually observed on the
spectrum. Add this information to the table and calculate the error (m and %) for
each ion.
d) Determine the error (m and %) in the experimentally-determined peptide
molecular mass that was calculated. How does this calculated value compare to
the m/z error of individual ions in magnitude? What is their relationship?

4. Instrument performance analysis:

a) Compare the mass assignment for each observed charge state of bradykinin with
the theoretical values calculated in the prelab assignment. Calculate error (m/z
and %) for each.
b) Select one peak from either of your spectra and use it to estimate the instrument's
resolution in both modes of operation (full scan and ZoomScan) by FWHM
method. How do they compare? (Be sure to use resolved, monoisotopic peaks for
this calculation.)

12
 c) An ion is generally considered significant if it has a signal:noise ratio exceeding
3:1. Identify any significant peaks on your bradykinin spectrum that did not
appear to arise from the ionization of bradykinin. What could account for these
signals? Be specific.

Additional Chemical Engineering Questions

Choose one question and answer it thoroughly in a page or less. Cite your sources (and
you should have several!)

1. The addition of carbohydrates to asparagine, glutamine, serine, or tyrosine residues in

proteins during protein/peptide biosynthesis is a source of protein/peptide
heterogeneity. This is a serious challenge in the manufacture of protein
pharmaceuticals because subtle changes in fermentation conditions can affect the
abundance or these carbohydrate modified variants. Could mass spectrometry be used
to quantify changes in the distribution of these peptide variants? If your answer is yes,
describe the changes that you would expect to see in the mass spectra. Be specific
(give mass shifts etc.)
2. Can mass spectrometry be used to determine the polydispersity of polymers instead of
peptides? What differences are there in detecting polymers and peptides? What
additional challenges would be present in accurately characterizing a polymer such as
polyvinyl chloride? Is this is a reasonable method of quality testing polymers on a
large scale?
3. Mass Spectrometry has been in the news as a new potential method of detecting
variations in the abundance and heterogeneity of blood proteins as a potential cancer
diagnostic. Many scientists are investigating whether mass spectrometry can be used
as a reliable, quick diagnostic tool for the early detection of this disease. Research the
advantages and disadvantages of this technique and application. If you were the CEO
of a large company looking to buy a small startup company that was developing this
detection tool, would you buy it? Why or why not?
4. What are the industrial uses of peptides? List as many companies as you can which
might use peptides in their production processes or during quality control testing.
Assuming you worked for a company which manufactured specific proteins, explain
how mass spectrometry can be used along with Six Sigma product quality targets to
meet or exceed strict FDA standards for quality and consistency of proteins sold.

13