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