1. Accelerated Protein Signaling Signatures: Highly Multiplexed Assays to Monitor Perturbations of Serine/Threonine Phosphosignaling Jacob D. Jaffe 1, Michael MacCoss 2 1 Broad Institute, Proteomics Platform, Cambridge MA 2 Department of Genetics, University of Washington, Seattle WA

2. Gene Expression Phospho-signaling   is large (hopefully) Phospho-signaling is inaccessible through expression profiling Phospho-signaling can be acute or sustained

3. Phosphosite.org database (CST) Non-redundant sites:97,222 Non-redundant proteins:13,384 Sites curated from literature:94,031 Sites using site-specific (SS) methods: 10,006 Sites using only discovery- mode MS (MS) methods: 86,378 Sites using both SS and MS methods: 4,773 Phosphoproteomics: current developments There are a lot of phosphosites! ( > # genes) How can we study these systematically?

4. kinase ATP ADP PO 4 phospha- tase protein No DNA/RNA involved Kinases and phosphatases are the key regulators Therefore, perturbagens that modulate kinase/phosphatase expression or activity should have effects on phosphosignaling HDAC Inhibitors trichostatin A:PC3 trichostatin A:MCF7 valproic acid:MCF7 valproic acid:HL60 valproic acid:PC3 valproic acid:ssMCF7 valproic acid:SKMEL5 cardiovascular agents digitoxigenin:HL60 digitoxigenin:MCF7 digitoxigenin:PC3 digoxigenin:HL60 digoxigenin:MCF7 digoxin:HL60 digoxin:MCF7 helveticoside:HL60 helveticoside:MCF7 helveticoside:PC3 lanatoside C:HL60 lanatoside C:MCF7 TK inhibitors tyrphostin AG- 1478:MCF7 tyrphostin AG- 825:MCF7 gefitinib:HL60 imatinib:MCF7 imatinib:PC3 PPAR agonists ATP-competitive kinase inhibitors staurosporine:MCF7 sanguinarine:MCF7 sanguinarine:HL60 Kinase/phosphatase genes Perturbations Interrogation of extant CMAP Data

5. Cells are colored by isotopic labels (i.e., 13 C, 15 N, but not radioactive) Generic technology enriches all phosphopeptides However, most phosphosites are Ser or Thr and NOT Tyr Ser/Thr phosphorylation is low hanging fruit Mass Spec provides both identification AND quantification Step 1: Discovery and learning

6. We propose to do for phosphosignaling what the Broad LINCS group has done for gene expression Natural synergy between projects Exploit existing robust methods Phosphosites

7. Use synthetic peptide internal standards for better quantification and proof of ID LOD/LOQ /copies per cell When you want to guarantee you measure it each and every time! Next-gen instruments will make this even more selective May enable us to skip phosphopeptide enrichment altogether Signal Assay time Step 2: Equivalent of L1000 – the “P100”

8. What should we see? Assays will be constructed such that we will always monitor the phospho- and non- phospho-states of the site as well as a different peptide to serve a surrogate for total protein levels.

9. End result ~100-plex phosphosite MRM-MS assay – 60-90 minutes/sample – $100-200/sample Reduced representation suitable for signature generation Requirements compatible will low cell numbers or tissue samples Absolute stoichiometry on every site, every time

10. LINCS Repository Other public databases LINCS Member Labs Step 3: Standardize and Disseminate Standard software platform (MacCoss Lab, U. Wash.) Cross-laboratory reproducibility

11. Call for nominations! Perturbations – Exploit extant CMAP data – Look for kinase and phosphatase modulators – Can be small molecule, shRNA, or “other” Systems – Relevant cell lines / disease models – Should cover “signaling space” Cancer signaling Immune Signaling Cell cycle

12. Acknowledgements LINCS Program and Program Officers – U01 CA164186-01/Jaffe MacCoss Lab, Univ. of Washington – Brendan MacLean Broad Institute Proteomics Platform – Philipp Mertins – Steve Carr Broad Institute LINCS Centers – Todd Golub – Aravind Subramanian