1. Phosphoproteomics and motif mining Martin Miller Ph.d. student CBS DTU miller@cbs.dtu.dk

2. Outline MS-based phosphoproteomics substrate-motifs in intracellular signalling research project: motif decomposition of the phosphotyrosine proteome

3. Mass spectrometry-based proteomics Aebersold & Mann, Nature 422: 198-207, 2004. Select one peptide species Collide Separate fragments Y3Y3 Y4Y4 Y5Y5 Y6Y6 Y7Y7

4. Mass spectrometry-based proteomics

5. PTM detection using MS

6. Quantitative phosphoproteomics using SILAC

7. Growth media lacking the SILAC labeling amino acid (e.g. Arg) Stable Isotope Labeled Amino Acids: Δm=6 DaΔm=10 Da Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC)

9. State AState B Light IsotopeHeavy Isotope Mix 1:1 Optional Protein Fractionation Digest with Trypsin Protein Identification and Quantitation by LC-MS Ong et al., Mol. Cell. Proteomics 1, 2002. Typical SILAC experiment workflow Upregulated protein - Peptide ratio >1 Background protein - Peptide ratio 1:1

10. SILAC labeling for quantitation Convenient No extra step introduced to experiment, just slightly different growth medium All identified proteins are - in principle – quantifyable Quantitation of proteins affected by different stimuli, disruption of genes, etc. Quantitation of post-translational modifications (phosphorylation, etc.)

11. Fishing for modification- dependent interactors using a bait sequence Asp-Ser-Trp-Ala-Arg-Leu-His-Gly-Tyr-Met-Ile-Met-Glu-Pro-Lys solid support Asp-Ser-Trp-Ala-Arg-Leu-His-Gly-Tyr-Met-Ile-Met-Glu-Pro-Lys solid support P

12. phosphorylation specific pull-down experiments Schulze W and Mann M. (2004) JBC 2004

13. Advantages of the SILAC pull- down method No overexpression – no tagging Straightforward separation between specific interactors and background binders Detection of low abundance and moderate affinity interactors Especially suited for PTM interaction studies Determination of the exact interaction site within the protein Important protein-protein interactions in cell signaling are frequently mediated by short, unstructured sequences – linear motifs

14. sequence motifs in intracellular signalling linear peptides sequence motifs guide signalling kinases phosphorylation- dependent interaction domains (SH2, PTB, 14- 3-3 etc.) directionality and specificity

15. Kinome tree and kinase substrates Linding et al, Cell, accepted

16. SH2 domain tree

17. A specific branch of the SH2 domain tree

18. The Phospho.ELM database currently contains 13614 phosphorylation sites in 4421 eukaryotic proteins. However, only ~23% of have know function. Thus there is a unique opportunity to mine for novel phosphorylation motifs “The Widening Gap”

19. Research protect motif decomposition of the phosphotyrosine proteome A new method for clustering uncharacterized phosphopeptides and mining for novel phosphorylation motifs following slides are erased because the data is confidential since results are not published yet