Vermont Genetics Network Outreach Proteomics Module Protein Mass Spectrometry: Theory and Application

Vermont Genetics Network Outreach
Proteomics Module
Protein Mass Spectrometry:
Theory and Application
Prepared by Bryan A. Ballif, Ph.D.
Two Essential Partner Tools in Proteomics
Gel Electrophoresis
Mass Spectrometry
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.
The Four Most Common Protein Mass Spectrometry Projects
(Observe How Each Project Has A Distinct Goal / Method !!)
What is this
protein?
What proteins are
present in _____?
Where Is this protein
Post-translationally
Modified?
What protein
Changes occur
Following ______?
Stimulus
Stimulus
H2O2
Block
Block
Western
Blot
α-pS
Western
Blot
α-pY
Rat
eCSF
Purify pY Proteins
Identify pY Proteins
Identify pY Sites
Quantify pY Changes
Typical Protein Mass Spectrometry Work Flow
Protein Preparation /
Analyte Preparation
Junk In …
Mass Spectrometry
Data Analysis /
Interpretation
Junk Out !
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
Two Common Types of Peptide Ionization
Electrospray Ionization
John Fenn
(2002 Nobel Prize)
Matrix-Assisted Laser
Desorption Ionization (MALDI)
Koichi Tanaka
(2002 Nobel Prize)
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
Carbon Column
Time of Flight Mass Spectrometry
Electric field gives ions defined kinetic energy.
Image from Kore Technology Limited
Ekinetic = ½ mv2
Mass Spectrometer
Electric / Magnetic
Fields
Peptides (Bottom Up Proteomics)
Versus Protein (Top Down)
Tryptic Digest
MFSCFLQAGNPQGSRSGFGHNVELVRHASIWVTYHSEEKLLIPYSDEL
MFSCFLQAGNPQGSR
SGFGHNVELVR
HASIWVTYHSEEK
LLIPYSDEL
Another Problem:
Multiple Peptides may have the same Mass!
NH3
G-F-S-F-P-V-A-T-G-L-M-E-D -D-G-K-P-R
COOH
NH3
G-F-S-F-P-M-L-G-T-A-V-E-D -D-G-K-P-R
COOH
Basics of Liquid Chromatography (LC)
Tandem Mass Spectrometry (MS/MS) for Peptides
HPLC
pumps
Mass Spectrometer
Relative Abundance
organic
concentration in
mobile phase
low
m/z
high
time
MS1
MS2
Peptide
Ions
Peptide
Fragment
Ions
Relative Abundance
LC
m/z
Knowing your ABC’s and your XYZ’s:
In your “Daughter” or “Fragment” Ions
A Closer Look at MS2 (For Peptide Identification)
He
He
8
NH3
b ions
7
6 5 4 3 2 1
G-F-S-F-P-V-A-T-G-L-M-E-D-D-G-K-P-R
G-F-S-F-P-V-A-T-G-L-M-E-D -D-G-K-P-R
1 2
CollisionInduced
Dissociation
( CID )
3 4 5
6 7 8 9 10 11 12 13
Y ions
COOH
A Closer Look at MS2 (and Phosphorylation )
P
He
NH3
-
G-F-S-F-P-V-A-T-G-L-M-E-D-D-G-K-P-RCOOH