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 Mass Spectrometry Gel Electrophoresis

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 !!) 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

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

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 Matrix-Assisted Laser Desorption Ionization (MALDI) Electrospray Ionization John Fenn (2002 Nobel Prize) 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 Time of Flight Mass Spectrometry Electric field gives ions defined kinetic energy. Image from Kore Technology Limited Ekinetic = ½ mv2

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

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 -

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

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

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 )

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