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Accelerated Protein Signaling Signatures:Highly Multiplexed Assays to Monitor Perturbations of
Serine/Threonine Phosphosignaling
Jacob D. Jaffe1, Michael MacCoss2
1Broad Institute, Proteomics Platform, Cambridge MA2Department of Genetics, University of Washington, Seattle WA
Gene Expression
Phospho-signaling
q
• q is large (hopefully)
• Phospho-signaling is inaccessible through expression profiling
• Phospho-signaling can be acute or sustained
Phosphosite.org database (CST)Non-redundant sites: 97,222Non-redundant proteins: 13,384Sites curated from literature: 94,031Sites 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?
kinase
ATPADP
PO4
phospha-
tase
prot
ein
prot
ein
prot
ein
• 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:HL60valproic 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
PPARagonists
ATP-competitive kinase inhibitors staurosporine:MCF7 sanguinarine:MCF7 sanguinarine:HL60
Kina
se/p
hosp
hata
se g
enes
Perturbations
Interrogation of extant CMAP Data
‘Light’ Cells
‘Heavy’ Cells Mass Spectrometry
‘Medium’ Cells
Control Tx
Tx 1
Tx 2
Mix
Digest
Fractionate
PhosphopeptideEnrichment
2.8x ↑
2.4x ↓
• 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
Step 1: Discovery and learning
Cell Lines/Conditions
Phos
posit
es
-5
-4
-3
-2
-1
0
1
2
3
4
5
Conditions
Example Coherent Cluster
log 2
fold
cha
nge
Cluster avg.
SelectRepresentative
Member(s)
FNHM(pS)QQGPRLLWIDA(pT)AGGNK
...
ExpertCriteria
A. C.B.
We propose to do for phosphosignaling what the Broad LINCS group has done for gene expression
• Natural synergy between projects
• Exploit existing robust methods
Phos
phos
ites
• 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
Sign
al
Assay time
Step 2: Equivalent of L1000 – the “P100”
What should we see? Protein
Copy #/cell
Phosphorylation Stoichiometry
# cells req to see phospho
(250 amol)
# cells req to see protein (250 amol)
1,000 1% 1.51E+07 1.51E+05 1,000 10% 1.51E+06 1.51E+05 1,000 50% 3.01E+05 1.51E+05 10,000 1% 1.51E+06 1.51E+04 10,000 10% 1.51E+05 1.51E+04 10,000 50% 3.01E+04 1.51E+04
100,000 1% 1.51E+05 1.51E+03 100,000 10% 1.51E+04 1.51E+03 100,000 50% 3.01E+03 1.51E+03
• 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
• ~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
LINCS Repository
Other public databases
LINCS Member Labs
Step 3: Standardize and Disseminate
• Standard software platform (MacCoss Lab, U. Wash.)• Cross-laboratory reproducibility
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
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