This document provides an overview of biological mass spectrometry techniques. It discusses matrix assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI) as two common ionization sources. It also describes different mass analyzer types including time-of-flight (TOF), quadrupoles, and Fourier transform ion cyclotron resonance (FT-ICR). Additionally, it covers fragmentation methods and applications of mass spectrometry in protein identification, modification analysis, and bacteria identification.

1. Biological Mass Spectrometry
A mini course

2. Biological Mass Spectrometry
General Mass Spectrometry
The General mass spectrometer
Analyzers types
Calibrations and mass Accuracy
Detection
Matrix Assisted Laser Desorption (MALDI)
TOF
Electrospray Ionization (ESI)
Quadrupoles, Ion Traps, FTMS, and O-TOF
Fragmentation
Post source dissociation (PSD) (with MALDI)
In source dissociation (with ESI)
Collision induced dissociation CID
IRMPD and ESI

3. The General Mass Spectrometry
M a s s S p e c tro m e te r
Io n S o u rc e A n a ly z e r D e te c to r
M ALDI TO F
S p ra y S o u rc e s Q u a d r u p o le s
T r a p p in g in s t r u m e n t s

4. Analyzers
• Time of Flights mass spectrometer
• Quadrupole mass spectrometer
• Quadrupole Ion traps
• FT-MS, FT-ICR

5. Time-of-flight Mass
Spectrometers
• Basically a tube that ions travel through
with lower mass ions traveling quicker
through the tube.
• Reflectron increases resolution by
compensating for energy and increasing
path length.
• Delayed extraction increases resolution.

6. O-TOF
Pulser
Ion Beam
Grid
T T
o OF

7. Quadrupole Mass Spectrometer
- Rods
+
+ - - +
+ Ions
-

8. Quadrupole Ion Traps
-
End Caps
+
+ - - +
Ring Ions
+
-

9. FT-MS, FT-ICR
+
+ B

10. Calibration
• All Mass Spectrometers MUST BE CALIBRATED. This adjust
parameters in the mass assignment of the ions.
• In general, the closer in space and time the calibration is the more
accurate the calibration.
• Default calibrations are usually set up on instruments for nominal
mass accuracy.
• Calibrations can be done with any compound with a known elemental
formula. The best calibration compounds are similar type of
compounds to the type you are going to measure accurately.
• It is best to have calibration compounds above and below the
compound of interest

11. Mass Accuracy
• Higher resolution allows for higher mass accuracy
in two ways
– Resolves stardards and sample from background
– Better centroiding of peaks
• Use of internal standards with a high resolution
(>10,000) is the “Gold standard” for mass
accuracy.
• Mass Accuracy needed is dependent on use and
available information.

12. Ion Detec tion
R Volta ge ac ross resistor

13. WHY MALDI?
• Less Sensitive to Salts
• Lower PRACTICAL detection limits
• Easier to interpret spectra (less multiple
charges)
• Quick and easy
• Higher mass detection
• Higher Throughput (>1000 samples per
hour)

14. Matrix
• Needs to be involatile (most are solids at
room temperature)
• Needs to absorb the laser wavelength that
you are using. (most cases 337 nm)
• Preferably dissolves in same solvent as the
sample
• Typically, the matrices are acidic.

15. HOT and Cold Matrices
• DHB is a cold matrix, the samples are not
as likely to be fragmented, may not ionize
some molecules.
• Alpha-cyano dihydroxybenzoic acid is
considered a hot matrix. More likely to
fragment the molecules. Can produce
multiply charged proteins.

16. Sample preparation
• Dried Droplet- mix sample with matrix and
drop on plate at the same time. –Easy to do.
• Layered method- put matrix on plate then
dry before adding sample. Good for low
concentrations, more difficult.
• Want high Matrix-Analyte ratio.

17. The MALDI Instrument
T im e - o f F lig h t
M a s s S p e c tro m e te r
S o u rc e A n a ly z e r D e te c to r
D e la y e d E x t r a c t io n R e fle c t r o n W h y d e fle c t t h e io n s ?
PSD

18. Basic Physics
• Kinetic Energy= ½ mv2
• Potential Energy of charged particle=qV
• Potential Energy +Kinetic Energy=Constant

19. Delayed Extraction
• Kinetic Energy of ions leaving the surface is not
constant. Also, their could be a time delay, in
addition.
• Delayed Extraction is a method used to
compensate for the spread of kinetic energy of the
ions leaving the surface.
• Delayed Extraction is mass dependent.
• Delayed Extraction will also compensate for some
time delay of ions coming off the surface.

21. M1 kE < M2 kE

24. T t4
=
Sam ple plate
M1 kE< M2 kE
E= kE + PE
Potential Voltage
M1 kE > M2 kE
Detector
M1
M2

25. Reflectron
• The reflectron is another method used to
compensate for the spread in Kinetic
energies in the time of flight.
• Reflectron does not compensate for any
time delay coming off the surface.
• Three types of reflectrons ( single stage,
two stage, and curved field)

26. KE M1 > KE M2
M1
M2

27. KE M1 > KE M2
T t2
=
M1
M2

28. KE M2= KE M1= 0
KE M1 > KE M2 M1
T t3
= M2

30. T 5
=

31. Why Deflect the ions?
• Detector is made up of multiple detectors similar
to the electron multiplier called “channels”.
• When an ion strikes the detector the “channel” is
dead for milliseconds.
• When to many matrix ions hit the detector, it kills
the signal.
• Linear detector is specially design to compensate
for larger number of ions, but is not perfect.

32. Why Electrospray Ionization?
• Electrospray Ionization can be easily
interfaced to LC.
• Absolute signals from Electrospray are
more easily reproduced, therefore, better
quantitation.
• Mass Accuracy is considered better.
• Multiple charging is more common then
MALDI.

33. Detection of Peptides
MALDI
Detec ted
Peptid es

34. Advantages of Multiple Charging
• Can use instruments with lower maximum
m/z (i.e., Quadrupoles, ion traps, FTMS)
• For FTMS, the resolution is better at lower
m/z values, therefore, ESI helps one obtain
better resolution at higher m/z values.
• Multiply charge ions tend to fragment easier
then singly charge ions.

35. Basic ESI Source
- 3kV
T Pum ping
o

36. Needle options
• High flow needles (for connection to
regular LC columns(up to 1ml/min))
• Nanospray needles (high sensitivity work)
• Microspray needles (connected to micro LC
columns)
• Atmospheric Pressure Chemical Ionization
(for non-polar molecules)
• Multiple needles setups

37. Why Fragment the ions?
• Molecular weight is only one piece of data
the instrument can provide
• Data base searches on tryptic peptides do
not always reveal the protein.
• Sequence and modification information can
be obtained by fragmentation

38. Ways to Induce Fragmentation
• Post Source Decay-MALDI
• In source Dissociation-ESI
• Low Energy –High Energy Collision
induced dissociation
• Inferred multi-photon dissociation (in a
trap)
• Electron Capture dissociation (in a trap)

39. Tandem Mass Spectrometers
• Traps
• Methods using single MS’s
• Triple Quadrupoles
• Q-Tofs
• TOF-TOF

40. PSD
• Easily done on simple MALDI with
reflectron
• Might not produce enough fragments
• Relatively low precursor ion selection

41. Physics Reminder
• Conservation of Energy assures that the
kinetic energy of fragments is determined
by the relative masses of the products.
• KE=1/2mv2
• KEprecursor=KEproduct ion+KEproduct neutral
• vprecursor=vproduct ion
• KEproduct ion=KEprecursor*(mproduct ion/mprecursor)

42. M > M2
K > K2
1

43. M > M2
K > K2
1

44. M > M2
K > K2
1
Velocity in Field Free
Region on the same
for both fragments

45. Types of Reflectrons
• Two stage- Give best resolution on single MS
mode, requires doing PSD in Stages.
• Single Stage-Large Reflector takes a significant
amount of space. PSD can be done in single stage,
however, low mass product ions have poor
resolution.
• Curved field-Another Large Reflector that takes
up space. Allows PSD to be obtained in single
stage with good resolution throughout PSD, but
single MS mode does not have as good a
resolution.

46. In Source Dissociation
• This is done by increasing the voltage between the
entrance to the sample orifice and the first
skimmer.
• Can be done on simple electrospray instrument.
• Produces results similar to low energy CID
• No mass selection, therefore, requires sample to be
extremely pure.
• Can be used to produce multiple MS on triple and
hybrid instruments.

47. CID
• CID is divided into low energy (<100 eV) and
high energy(>1000 eV) based on the collision
energy.
• High energy CID produces more fragments, but is
more complicated to interpret.
• Low energy CID has a limit on the m/z it can
dissociate of approximately 1000.
• High energy CID produces charge remote
fragmentation that can be used to distinguish
leucine and isoleucine.

48. IRMPD and ECD
• Complimentary techniques
• Can only be performed in trapping
instruments-Mostly FTMS.
• ECD requires a multiply charged ion
• Used for dissociating high molecular weight
fragments

49. Interpretation of Tandem Mass
Spectra of Peptides
• Known Sequence- Calculate expected fragments
and compare to tandem spectra to see match
• Modified Sequence-Calculate unmodified
sequence compare to tandem spectra to see
difference where modification occurs.
• Unknown Sequence- Check Database to see if it is
a match
• Unknown Sequence not in Database- Manual
Interpretation (Practice!Practice!Practice!)

50. Peptide fragmentation
a b c
O
Nterm -RCH-C-NH-CHR-Cterm
x y z

51. Manual Interpretation
• Goal-Assign as many abundant fragments as
possible to a spectra
• Remember Cysteine modifications
• Know type of fragments that are typically
observed by dissociation method.
– Low Energy (b and y, loss of neutrals from these
fragments)
– High Energy (x,y,z, a,b,c, v, d,w)
Reference: Ioannis A Papayannopoulos, Mass Spec.
Rev.,1995, vol. 14, 49-73.

52. Steps in Manual Interpretation
• Choose a large peak
• Look for ions that are different in mass from the
fragment by a specific amino acid mass. If there is
look for an other fragment that differs by a
specific amino acid mass to build a partial
sequence.
• Decide the type of fragments that corresponds to
the above sequence by either mass difference to
the precursor or complimentary fragments.
• Look for other complimentary fragments and low
mass ions to confirm the partial sequence.
• Choose another unassigned peak and repeat
procedures until sequence is determined.

53. Peptide modifications to help
Interpretation
• O18 water digestion- all C terminal
fragments will have unique Isotope pattern
• N-terminal or C-terminal modifications-
results in distinct fragmentation patterns.

54. LC Interfaces
• Direct LC with Electrospray.
– Direct connection to mass spectrometer
– Real Time monitoring
• Vacuum deposition for MALDI
– Indirect conection to mass spectrometer
– Not Real Time, allows one to make decisions
later. Multiple MS/MS on single LC peaks.

55. LC detection methods
• Simple acquiring of spectra.
• Precursor and product ion scans
• Data Dependent scans
• Selective Ion Monitoring
• Selective Reaction monitoring

56. Applications
• Protein Identifications
• Protein Modifications
• Bacteria Identifications
• SNP’s

57. Protein Identification
• Identify Protein mass
• Digest Protein
• Analyze by MALDI
• Data Base Search
• If negative results, go to Tandem Mass
Spectrometry
• Data Base Search on Tandem Mass
Spectrum

58. Protein Modifications
• Tandem Mass Spectrometry is very important
• Can obtain full mass of proteins to help determine
mass and number of modifications
• Digest proteins and run mass spectrometry.
• Use Tandem to determine the exact position
• Use of unmodified protein or peptide can be useful
in the interpretation

59. Bacteria Identification
• Uses MALDI of Bacteria to Identify
Proteins produced by bacteria
• Data base search of MALDI spectra
identifies bacteria
• At this point, no large consistent Data Base
is available.