Vermont Genetics Network Outreach Proteomics Module

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Vermont Genetics Network OutreachProteomics
Module
Protein Mass Spectrometry Theory and
Application Prepared by Bryan A. Ballif, Ph.D.
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Two Essential Partner Tools in Proteomics
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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.
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The Four Most Common Protein Mass Spectrometry
Projects (Observe How Each Project Has A Distinct
Goal / Method !!)
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Typical Protein Mass Spectrometry Work Flow
Protein Preparation / Analyte Preparation
Mass Spectrometry
Data Analysis / Interpretation
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Most Proteomics Experiments Further Purify the
Analyte Using High Performance Liquid
Chromatography (HPLC)
Organic Solvent
H2O
HPLC pumps
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Two Common Types of Peptide Ionization
Matrix-Assisted Laser Desorption Ionization
(MALDI)
Electrospray Ionization
John Fenn (2002 Nobel Prize)
Koichi Tanaka (2002 Nobel Prize)
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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
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Peptides (Bottom Up Proteomics) Versus Protein
(Top Down)
Tryptic Digest MFSCFLQAGNPQGSRSGFGHNVELVRHASIWVTYH
SEEKLLIPYSDEL MFSCFLQAGNPQGSR SGFGHNVELVR HASIWVT
YHSEEK LLIPYSDEL
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Another Problem Multiple Peptides may have the
same Mass!
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Basics of Liquid Chromatography (LC) Tandem Mass
Spectrometry (MS/MS) for Peptides
HPLC pumps
Mass Spectrometer
LC
MS2
MS1
Peptide Fragment Ions
Peptide Ions
low
high
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Knowing your ABCs and your XYZs In your
Daughter or Fragment Ions
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A Closer Look at MS2 (For Peptide Identification)
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Collision- Induced Dissociation ( CID )
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A Closer Look at MS2 (and Phosphorylation )
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G-F-S-F-P-V-A-T-G-L-M-E-D-D-G-K-P-R
NH3
COOH