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Jacob D. Jaffe 1 , Michael MacCoss 2 1 Broad Institute, Proteomics Platform, Cambridge MA

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.

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Jacob D. Jaffe 1 , Michael MacCoss 2 1 Broad Institute, Proteomics Platform, Cambridge MA

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  1. Accelerated Protein Signaling Signatures:Highly Multiplexed Assays to Monitor Perturbations of Serine/ThreoninePhosphosignaling Jacob D. Jaffe1, Michael MacCoss2 1Broad Institute, Proteomics Platform, Cambridge MA 2Department of Genetics, University of Washington, Seattle WA

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

  3. Phosphoproteomics: current developments • There are a lot of phosphosites! ( > # genes) • How can we study these systematically?

  4. Interrogation of extant CMAP Data Perturbations ATP-competitive kinase inhibitors staurosporine:MCF7 sanguinarine:MCF7 sanguinarine:HL60 protein protein 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 PPAR agonists PO4 kinase phospha-tase ATP ADP TK inhibitors tyrphostin AG-1478:MCF7 tyrphostin AG-825:MCF7 gefitinib:HL60 imatinib:MCF7 imatinib:PC3 protein HDAC Inhibitors trichostatin A:PC3 trichostatin A:MCF7 valproic acid:MCF7 valproic acid:HL60 valproic acid:PC3 valproic acid:ssMCF7 valproic acid:SKMEL5 • No DNA/RNA involved Kinase/phosphatase genes • Kinases and phosphatases are the key regulators • Therefore, perturbagens that modulate kinase/phosphatase expression or activity should have effects on phosphosignaling

  5. Step 1: Discovery and learning • Cells are colored by isotopic labels (i.e., 13C, 15N, 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

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

  7. Step 2: Equivalent of L1000 – the “P100” • 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

  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. Step 3: Standardize and Disseminate LINCS Repository Other public databases LINCS Member Labs • 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

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