1. Vermont Genetics Network Outreach Proteomics Module
Protein Mass Spectrometry:
Theory and Application
Prepared by Bryan A. Ballif, Ph.D.

2. Two Essential Partner Tools in Proteomics
Mass Spectrometry
Gel Electrophoresis

3. First Things First -- Know Your Goal !
● There are nearly as many mass spectrometry
methods as there are mass spectrometry projects.
● Your goal determines your methods which determine
your outcomes.

4. The Four Most Common Protein Mass Spectrometry Projects
(Observe How Each Project Has A Distinct Goal / Method !!)
Where Is this protein
Post-translationally
Modified?
Stimulus
Block
Western
Blot
α-pS
What protein
Changes occur
Following ______?
Stimulus
H2O2
Block
Western
Blot
α-pY
Purify pY Proteins
Identify pY Proteins
Identify pY Sites
Quantify pY Changes
What is this
protein?
What proteins are
present in _____?
Rat
eCSF

5. Typical Protein Mass Spectrometry Work Flow
Protein Preparation /
Analyte Preparation
Junk In …
Junk Out !
Mass Spectrometry
Data Analysis /
Interpretation

6. Most Proteomics Experiments Further Purify the Analyte Using
High Performance Liquid Chromatography (HPLC)
Organic
Solvent
H2O
HPLC
pumps
Mass Spectrometer
Carbon Column
Electric / Magnetic
Fields

7. Two Common Types of Peptide Ionization
Matrix-Assisted Laser
Desorption Ionization (MALDI)
Electrospray Ionization
John Fenn
(2002 Nobel Prize)
Koichi Tanaka
(2002 Nobel Prize)

8. Perhaps the Most Simple Concept for
Mass Measurements in Mass Spectrometer
Remember there are relationships between mass and Energy :
E = mc2 or Ek = ½ mv2
Time of Flight Mass Spectrometry
Electric field gives ions defined kinetic energy.
Image from Kore Technology Limited
Ekinetic = ½ mv2

9. Peptides (Bottom Up Proteomics) Versus Protein (Top Down)
Tryptic Digest
MFSCFLQAGNPQGSRSGFGHNVELVRHASIWVTYHSEEKLLIPYSDEL
MFSCFLQAGNPQGSR
SGFGHNVELVR
HASIWVTYHSEEK
LLIPYSDEL

10. Multiple Peptides may have the same Mass!
Another Problem:
Multiple Peptides may have the same Mass!
NH3
G-F-S-F-P-V-A-T-G-L-M-E-D
COOH
D-G-K-P-R -
NH3
G-F-S-F-P-M-L-G-T-A-V-E-D
COOH
D-G-K-P-R -

11. Basics of Liquid Chromatography (LC)
Tandem Mass Spectrometry (MS/MS) for Peptides
HPLC
pumps
Mass Spectrometer LC
MS1
MS2
Peptide
Ions
Peptide
Fragment
Ions
Relative Abundance
time
organic
concentration in mobile phase
Relative Abundance
m/z
m/z
low
high

12. Knowing your ABC’s and your XYZ’s:
In your “Daughter” or “Fragment” Ions

13. A Closer Look at MS2 (For Peptide Identification)He He 1 2 3 4 5
Y ions 6 7 8 9 10
b ions 1 2 3 4 5
Y ions
NH3
G-F-S-F-P-V-A-T-G-L-M-E-D
G-F-S-F-P-V-A-T-G-L-M-E-D-D-G-K-P-R
COOH
NH3 -
COOH
D-G-K-P-R 1 2 3 4 5 6 7 8 9 10 11 12 13
b ions
Collision-
Induced
Dissociation
( CID )

14. A Closer Look at MS2 (and Phosphorylation )He -
G-F-S-F-P-V-A-T-G-L-M-E-D-D-G-K-P-R
NH3
COOH