Quantitative proteomics
- 2. Introduction of SILAC and its applications • Stable Isotope Labeling of Amino acids in Culture • Develop and promoted by Matthias Mann • Two papers: – Lipid Rafts (PNAS) – Focal Adhesion precursors (Cell under review)
- 3. What does a mass spectrometer do? Precise identification of mass Can trap a single ion species and fragment to get sub fragments (sequence)
- 4. Overview of Biological and Chemical Isotope Labeling Strategies Two complementary samples Labeling Analysis
- 5. SILAC vs. ICAT • Culture system only • De Novo Proteins (no serum contamination) • No optimization • Simplifies MS/MS • More complete peptide coverage • ALL protein samples • Labels selected moieties • Need to optimize labeling • Large linker group. • Reduces complexity
- 6. Stable isotopic amino acids (MW) Cambridge Isotope Catalog Sigma-Aldich Catalog L-Lysine 2HCl (223.13) (4,4,5,5-D4) DLM-2640 - L-Lysine HCl (186.67) (4,4,5,5-D4) - 616192 L-Lysine 2HCl (225.1) (13 C6) CLM-2247 - L-Lysine HCl (188.6) (13 C6) - 643459 L-Lysine 2HCl (227.1) (13 C6, 15 N2) CNLM-291 - L-Lysine HCl (190.59) (13 C6, 15 N2) - 608041 L-Arginine HCl (214.64) (15 N4) NLM-396 600113 L-Arginine HCl (216.62) (13 C6) CLM-2265 643440 L-Arginine HCl (220.59) (13 C6, 15 N4) CNLM-539 608033 L-Arginine HCl (221.6) (15 N4, D7) DNLM-7543 - L-Arginine HCl (227.6) (13 C6, 15 N4, D7) CDNLM-6801 SILAC Labels
- 7. “Light” “Heavy” “Light/heavy” mix How can you distinguish between isotoped species?
- 8. How do you actually get quantitation? 1) Once you’ve identified the complementary peptides, 2) Backtrack and integrate the Xtracted Ion Chromatogram
- 9. I-TRAQ (Isobaric Tag for Relative and Absolute Quantitation
- 10. Experimental Strategy Sample preparation Labeling Chromatographic separations Mass Spectrometry Analysis