1 / 12

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.

iman
Download Presentation

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

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript


  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

More Related