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Vermont Genetics Network Outreach Proteomics Module

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 !.

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Vermont Genetics Network Outreach Proteomics Module

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

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

  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? What protein Changes occur Following ______? What is this protein? What proteins are present in _____? Stimulus H2O2 Stimulus Block Block Western Blot α-pS Western Blot α-pY Purify pY Proteins Identify pY Proteins Identify pY Sites Quantify pY Changes Rat eCSF

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

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

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

  11. organic concentration in mobile phase Relative Abundance Relative Abundance time m/z m/z 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

  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 8 7 6 5 4 3 2 1 Y ions b ions 1 2 3 4 5 6 7 8 9 10 5 4 3 2 1 Y ions 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 NH3 NH3 COOH COOH b ions 1 2 3 4 5 6 7 8 9 10 11 12 13 - Collision- Induced Dissociation ( CID )

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

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