1. 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)

2. What does a mass spectrometer do? Precise identification of mass Can trap a single ion species and fragment to get subfragments (sequence)

3. Theoretically (database), know what the fragments should be (Trypsin digest) Purify over monomeric avidin column (This is a selection/simplification scheme) Ideally, should see ratios between state1 and state2 (they will have the same retention time). Check to see if the mass is correct. Reminder of ICAT technology

4. Expect to see a ratio between the peak sizes.

5. SCX profile LC profile of a single fraction

6. Composite of SCX separation.

7. Overview of Biological and Chemical Isotope Labeling Strategis Two complementary samples Labeling Analysis

8. Example: Have large analysis space

9. 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

10. “Light” “Heavy” “Light/heavy” mix How can you distinguish between isotoped species?

11. Time-course of deuterated isoleucine incorporation. Also gives you an indication of the robustness of the assay. Can compare the peak strength.

12. How do you actually get quantitation? 1)Once you’ve identified the complementary peptides, 2)Backtrack and integrate the Xtracted Ion Chromatogram

13. Two papers 1) Unbiased Quantitative Proteomics of Lipid Rafts Reveals High Specificity for Signaling Factors Foster LJ, de Hoog CL, Mann M PNAS, 2003 2) RNA and RNA-binding proteins participate in early stages of cell spreading through Spreading Initiation Centers, sites of nascent focal adhesion formation. De Hoog CL, Foster LJ, Nielsen, MM, Mann M (Cell), pending

14. Experimental Strategy: 1)Grow cells with “heavy” or “light” until saturation (5 divisions) 2)Add cholesterol depleting drugs (3 conditions) 1)Methyl-beta cyclodextrin 2)Filipin 3)nystatin

15. Biochemistry outline 1)Lyse cells by either TX-100 or pH11 buffer. 2)Determine protein concentration and mix samples. 3)Purify lipid rafts. 4)Trypsinize 5)MS/MS analysis

17. Example of 2 proteins: Doublet recognition Identity Abundance ratios EnrichedNon-enriched

18. Global analysis of raft protein data: M  CD is most efficient at extracting cholesterol Ratio between experimental/control population Detergeant extractions: 1)M  CD 2)Filipin 3)Nystatin 4)Average pH extration See sharp Cutoffs

19. This is the AMAZING table: Clear demarcation of Raft proteins Associated Proteins Non-specific proteins ratios close to 1 (mostly high abundance cellular proteins (biochemical background)) Serum proteins (not shown) Raft proteins (1TM or fatty acid mod) Associated proteins (no TM, but associate) Positives and Negatives all make sense!!

20. Conclusions (from this paper and others): Rafts enrich >10-fold for signalling proteins. Rafts bind not just tyrosine kinases but also serine/threonine kinases and G proteins (more ubiquitous than thought)

21. Focal adhesion protocol overview. Sample difference is attachment Then IP with talin, vinculin, paxillin. Isotoped peptide identification Isotoped peptide characterization (talin) Peptide quantification (.83)

22. Protein results summary: 304 proteins identified: Only a small number of proteins were attachment dependent. (from SILAC quantitation data). There are many problems holes in their quantitation data. I’m not up to giving them a hard time today. Cell biology…. Studied RACK1 Come back to what I think SILAC/ICAT criteria is…

23. -RACK1/vinculin interaction is greater in adherent cells (2.06). -In spread cells, they don’t colocalize. However, when cells are DETACHED (1hr), then allowed to SPREAD, they begin to colocalize(2µm). (stage 1) Focal adhesion begin to appear under the colocalized spots (stage2) The cell spreads and colocalization disappears (stage3) Similar results for paxillin, talin, and FAK

24. Actin forms a ring around this initial complex (SIC or spreading initiation complex). This ringstructure is transient and gone by stage 3.

25. RNA binding proteins were found reproducibly in all 3 Ips. The RNA-binding proteins hnRNP K, FUS/TLS, hnRNP E1, SmB, SmD, but NOT snRNP U1 were found in SICs.

26. SICs stain for nucleic acids. This is Rnase I sensitive. In fully spread cells, don’t see splicesome components (part C).

27. 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

28. Which method would you use? -SILAC can only be used in tissue culture. If you are doing this, it simplifies the question (vs. ICAT) GREATLY. -Cell lines vs. tumors. Are the phenotypes and profiles from a tumor reflected on a dish? Can solve lack of antibody problem temporarily… -3-dimensional growth. SILAC can eliminate background completely! -I believe this is the major advantage of SILAC (even without quantitation) -Criteria before proteomics Proteomics is trivial/non-trivial!!! Just a glorified Western. Information overload. QUICK, CLEAN secondary screen. Otherwise, proteomics is useless. Just come up with interesting, unverifiable data!!!

29. Matrigel is useful and simple/complex (~10mg/ml protein)