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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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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




Gene Expression

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

Phosphoproteomics: current developments

  • There are a lot of phosphosites! ( > # genes)
  • How can we study these systematically?

Interrogation of extant CMAP Data


ATP-competitive kinase inhibitors staurosporine:MCF7 sanguinarine:MCF7 sanguinarine:HL60



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


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

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

We propose to do for phosphosignaling what the Broad LINCS group has done for gene expression


  • Natural synergy between projects
  • Exploit existing robust methods

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


Assay time


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.
end result
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

Step 3: Standardize and Disseminate

LINCS Repository

Other public databases

LINCS Member Labs

  • Standard software platform (MacCoss Lab, U. Wash.)
  • Cross-laboratory reproducibility
call for nominations
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
  • 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