Klarisa Rikova, Ailan Guo, Kimberly A. Lee, Anthony Possemato, Yi Wang, Sean A. Beausoleil, Daniel E. Mulhern, Jian Min Ren, Michael J. CombCell Signaling Technology, Inc., Danvers, MA

Immunoaffinity Enrichment and Quantitative Profiling of Protein Phosphorylation, Methylation, and Acetylation in Receptor Tyrosine Kinase Pathway-addicted Cancer Cell Lines

Somatic mutation and altered protein expression patterns cause inappropriate activation and deactivation of key players in cellular signaling, result-

ing in the oncogenic phenotype. Phosphorylation patterns of oncogenes and other signaling proteins have been studied extensively in the last sev-

eral years, resulting in significant advances in our understanding of cancer and our ability to identify and therapeutically target tumor drivers.

Here we take the next step in this process, expanding the enriched, identified, and quantified post-translational modification (PTM) space to include not only

phosphopeptides but also acetylated, methylated, and ubiquitinated peptides, resulting in a multifaceted analysis of cellular protein modification to better under-

stand the complex signaling events of cancer biology.

Variation in cellular signaling measured by ten different candidate motif antibodies was evaluated by performing western blot analysis of cell lysates from 22 dif-

ferent lung cancer cell lines. This screening tool allowed us to select those antibodies with the most diverse staining patterns across the cell lines for global pro-

teomic PTM profiling. Phosphotyrosine, acetyl-lysine, methyl-arginine, ATM/ATR substrate (s/tQ), and AGC/CAMK/STE family kinase motif, which specifically

enriches close to 100 active kinase phosphopeptides, were all selected for use in peptide immunoprecipitation, allowing us to characterize and quantify a broad

PTM space.

Initial method development and PTM profiling were performed using a set of six lung cancer cell lines (A549, H1650, H1703, H3122, H3255, and MKN45) that

have different signaling pathways driving their growth and transformation. Phosphotyrosine profiling of these cell lines with tandem mass tag (TMT) quantifica-

tion revealed clear quantitative differences in phosphorylation, successfully identifying their activated signaling pathways.

The established method of TMT labeling coupled with serial peptide immunoprecipitation was applied to profile drug-treated and untreated RTK pathway-addicted

cancer cell lines, with quantitiative analysis of the effects of SU11274 on the Met-addicted MKN45 cell line, Crizotinib on the Alk-addicted H3122 cell line,

Gleevec on the PDGFRa-addicted H1703 cell line, and Iressa on the EGFR-addicted H3255 cell line. These samples were combined in 6-plex groups for TMT

analysis with the A549 cell line serving as a non-responsive control. Serial fractionation and LCMS/MS analysis resulted in identification and quantification of

hundreds of sites of phosphorylation, arginine methylation, and lysine acetylation. Those sites found to be drug-sensitive, including all PTM types, were used for

pathway analysis of signaling downstream of each receptor tyrosine kinase (RTK) disease driver.

Figure 1. Western blots used to select motif antibodies for phosphopeptide immunoprecipitation.

Variation in cell signaling measured by ten different candidate motif antibodies was evaluated by performing Western blots of cell lysates from 22 different

lung cancer cell lines. This screening tool allowed us to select those antibodies with the most diverse staining patterns across the cell lines for global pro-

teomic PTM profiling. Phosphotyrosine, ATM/ATR substrate (s/tQ), acetyl-lysine, and methyl-arginine were all selected for use in peptide immunoprecipitation,

allowing us to characterize and quantify a broad PTM space. In contrast, the CK2 substrate antibody showed less diverse staining and was not used in the

cell line profiling studies.

Method oF quAntiFicAtion

Figure 4. Receptor tyrosine kinase disease drivers identified in appropriate cell lines using tMt hcd only method. The expected pat-

terns of expressed and phosphorylated RTKs is observed, with PDGFRa in H1703, ALK in H3122, EGFR overexpressed in H3255, and Met in MKN45.

EGFR is expressed in all of these immortal cell lines at some level, and phosphorylation of some sites (Y1110 for example) is detected in all cases. GSK3B

is also constitutively expressed and phosphorylated. MKN45 has an extremely high level of tyrosine phosphorylation, so some interference is detected in

the TMT channel associated with that cell line.

Figure 5. experimental flowchart. Cell lysates were prepared from RTK inhibitor treated and untreated cell lines, proteins were digested with trypsin,

peptides were TMT-labeled, and samples were mixed for 6-plex analysis. The peptide mixtures were serially fractionated through immunoprecipitation with

antibodies specific for phosphotyrosine, ATM/ATR substrate motif, AGC/CAMK/STE family kinase motif, acetyl-lysine, and methyl-arginine, though any

motif-recognizing antibody could be used with this protocol. The enriched peptides from each immunoprecipitation were analyzed by LCMS/MS using an

LTQ® Orbitrap® Elite, with quantification of the TMT labels enabled by HCD fragmentation.

Figure 7. example drug-responsive modification sites.

Figure 8. Pathway diagrams for each cell line were generated using cytoscape®. These diagrams include all proteins with modification sites

increasing or decreasing at least 2x upon relevant drug treatment. Proteins with PTM site peptides decreasing upon treatment (dark green protein names) are

distinguished from those with sites increasing upon drug treatment (red protein names). Proteins identified in different PTM classes (phosphotyrosine, ATM/

ATR substrate phosphorylation, AGC/CAMK/STE kinase family motif phosphorylation, lysine acetylation, and arginine methylation) are distinguished by differ-

ent colored protein symbols. Shapes of protein symbols signify protein functionality (cytoskeletal, adaptor, kinase, adhesion protein, etc.). First order interacting

partners with the driving RTK are presented in bright yellow.

table 1. number of modification sites identified and quantified for each PtM type analyzed. The 6-plex sample components are as follows:

Sample Mix 1: A549 untreated, A549+Crizotinib, A549+Gleevec, A549+Iressa, A549+SU11274.

Sample Mix 2: A549 untreated, A549+Crizotinib, H3122 untreated, H3122+Crizotinib, MKN45 untreated, MKN45+SU11274.

Sample Mix 3: A549 untreated, A549+Iressa, H1703 untreated, H1703+Gleevec, H3255 untreated, H3255+Iressa.

Figure 6. Percent of sites responsive for each PtM motif studied. At least a two-fold change in reporter ion signal/noise was required to be

classified as responding. As expected, phosphotyrosine sites are the most responsive to RTK inhibition, but many other phosphorylation, acetylation, and

methylation responses were also observed.

coMbining iMMobiLized MetAL AFFinity chRoMAtogRAPhy (iMAc) With PhosPhoPePtide iMMunoPReciPitAtion

:: establish method for quantitative analysis of multiple classes of post-translational modifications from a single sample

:: Apply method to investigate signaling downstream of several different oncogenic receptor tyrosine kinases

:: intelligent fractionation through sequential immunoprecipitation can be used to probe numerous PtM spaces, allowing in-depth

pathway mapping of valuable samples

:: quantification and sample multiplexing with tMt labeling increases annotation rate over label-free quantification with lower

sample analysis time

:: deep data sets generated through enrichment of different types of modified peptides allow elucidation of novel biological

relationships

Abstract

Antibody Selection

Evaluating LCMS methods

Validating methods

Results

Objectives

Summary

0

500

1000

1500

2000

2500

3000

label free (6 analyses) TMT - HCD only TMT - MS3

Nu

mb

er id

enti

fied

Quantification method selection: K-Ac

redundant peptides

unique peptides

unique sites

redundant peptides

unique peptides

unique sites

0

200

400

600

800

1000

1200

1400

1600

label free (5 analyses) TMT - HCD only TMT - MS3

Nu

mb

er id

enti

fied

Quantification method selection: R-Me

0

500

1000

1500

2000

2500

3000

3500

4000

4500

5000

control 1 control 2 IMAC 1 IMAC 2 control 1 control 2 IMAC 1 IMAC 2

H3122 MKN45

Un

iqu

e p

epti

des

iden

tifi

ed

Coupling IMAC with phosphopeptide IP

total peptides

phosphopeptides

0

0.2

0.4

0.6

0.8

1

1.2

Y768 Y1507 Y1197 Y1252 Y216 Y1110

PDGFRα ALK EGFR Met GSK3B EGFR

Rel

ativ

e re

po

rter

ion

sig

nal

/no

ise

A549

H1650

H1703

H3122

H3255

MKN45

Cell line 1

Cell line 2

Cell line 3

IP ATM/ATR substrate peptides

TMT labeling

IP AGC kinase motif peptides

IP acetyl-lysine peptides

IP methyl-arginine peptides

IP phosphotyrosine peptides

Proteolytic digestion

Mix samples

LCMS/MS analysis

tnauq editpep TMT

LCMS/MS analysis

tnauq editpep TMT

LCMS/MS analysis

tnauq editpep TMT

LCMS/MS analysis

tnauq editpep TMT

LCMS/MS analysis

tnauq editpep TMT

AGC/CAMK/STE kinase (107 sites total)

Phosphotyrosine (532 sites total)

phosphotyrosine

ATM/ATR substrate (96 sites total)

Acetyl-lysine (2514 sites total)

Methyl-arginine (899 sites total)

AGC/CAMK/STE kinase (102 sites total)

phosphotyrosine

Phosphotyrosine (566 sites total)

ATM/ATR substrate (294 sites total)

Acetyl-lysine (1999 sites total)

Methyl-arginine (975 sites total)

AGC/CAMK/STE kinase (102 sites total)

phosphotyrosine

Phosphotyrosine (1063 sites total)

ATM/ATR substrate (329 sites total)

Acetyl-lysine (1997 sites total)

Methyl-arginine (998 sites total)

AGC/CAMK/STE kinase (70 sites total)

Acetyl-lysine (2508 sites total)

phosphotyrosine

Phosphotyrosine (364 sites total)

ATM/ATR substrate (44 sites total)

Methyl-arginine (868 sites total)

unchanged up 2x down 2x

unchanged up 2x down 2x

unchanged up 2x down 2x

unchanged up 2x down 2x

h1703 cell line treated with PdgFRa inhibitor gleevecMKn45 cell line treated with Met inhibitor su11274

h3122 treated with ALK inhibitor crizotinibh3255 treated with egFR inhibitor iressa

RTK

CTK

AGC/CAMK/STE Family

AGC/CAMK/STE Family

Acetylation

Acetylation

PY

PY

Methylation

Methylation

G protein

Surface

Surface

AdaptorScaffold

AdaptorScaffold

Adhesion Extracellular Matrix

Adhesion Extracellular Matrix

CytoskeletalProtein

CytoskeletalProtein

RNAProcessing

RNAProcessing

ATM/ATR Substrate

S/T Kinase

RTK

RTK

RTK

S/T Kinase

S/T Kinase

S/T Kinase

AGC/CAMK/STE Family

AGC/CAMK/STE Family

Acetylation

Acetylation

PY

PY

Translation

Methylation

Methylation

G protein

G protein

G protein

TranscriptionalRegulator

TranscriptionalRegulator

Surface

Surface

UnknownFunction

AdaptorScaffold

AdaptorScaffold

Adhesion Extracellular Matrix

Adhesion Extracellular Matrix

CytoskeletalProtein

CytoskeletalProtein

ATM/ATR

ATM/ATR

ATM/ATR

CTK

CTK

CTK

ALK driven signaling h3122 +/- crizotinib

PdgFRa driven signaling h1703 +/- gleevec

Met driven signaling MKn45 +/- su11274

egFR driven signaling h3255 +/- iressa

Contact InformationKimberly A. Lee, Cell Signaling Technology, Inc. email: klee@cellsignal.com

PTMScan Services Department email: ptmscan@cellsignal.com • web: www.cellsignal.com/servicesCell Signaling Technology® and PTMScan® are trademarks of Cell Signaling Technology, Inc. / LTQ® and Orbitrap® are registered trademarks of Thermo Finnigan, LLC. / Cytoscape® is a registered trademark of The Cytoscape Consortium.

sample Mix 1 sample Mix 2 sample Mix 3 total sites (nonredundant)

phosphotyrosine 236 1111 572 1369K-Ac 1928 1988 2492 3679R-Me 757 986 900 1329ATM/ATR substrate (pSQ) 264 384 135 574ACG kinase motif 137 173 160 302

0

0.2

0.4

0.6

0.8

1

1.2

K37 K57 K348 K362 K19 K24

AL

DH

1A1

AL

DH

1A1

AL

DH

1A1

AL

DH

1A1

HIS

T2H

3D

HIS

T2H

3D

Rel

ativ

e ab

un

dan

ce

Acetyl-lysine

0

0.2

0.4

0.6

0.8

1

1.2

S836 S1610 S265

MC

EF

AP

C

MA

GE

-D2

Rel

ativ

e ab

un

dan

ce

ATM/ATR substrate

A549

A549+Crizotinib

H3122

H3122+Crizotinib

MKN45

MKN45+SU11274

A549

A549+Crizotinib

H3122

H3122+Crizotinib

MKN45

MKN45+SU11274

0

0.2

0.4

0.6

0.8

1

1.2

K37 K57 K348 K6 K9 K13 K17

AL

DH

1A1

AL

DH

1A1

AL

DH

1A1

HIS

T1H

4J

HIS

T1H

4J

HIS

T1H

4J

HIS

T1H

4J

Rel

ativ

e ab

un

dan

ce

Acetyl-lysine

A549

A549+Iressa

H1703

H1703+Gleevec

H3255

H3255+Iressa

0

0.2

0.4

0.6

0.8

1

1.2

Y265 Y1367 Y68

EM

L4

Met

cofi

lin 1

Rel

ativ

e ab

un

dan

ce

Phosphotyrosine

0

0.2

0.4

0.6

0.8

1

1.2

Y226 T231 S216 S684

RS

K2

RS

K2

AH

NA

K

ER

K3

Rel

ativ

e ab

un

dan

ce

AGC/CAMK/STE family kinase motif

A549

A549+Iressa

H1703

H1703+Gleevec

H3255

H3255+Iressa

0

0.2

0.4

0.6

0.8

1

1.2

Y849 Y1172 Y68

PD

GF

Rα

EG

FR

cofi

lin 1

Rel

ativ

e ab

un

dan

ce

Phosphotyrosine

A549

A549+Iressa

H1703

H1703+Gleevec

H3255

H3255+Iressa

A549

A549+Crizotinib

H3122

H3122+Crizotinib

MKN45

MKN45+SU11274

0

0.2

0.4

0.6

0.8

1

1.2

Y34 T41 S216 S684

4E-B

P1

4E-B

P1

AH

NA

K

ER

K3

Rel

ativ

e ab

un

dan

ce

AGC/CAMK/STE family kinase motif

A549

A549+Crizotinib

H3122

H3122+Crizotinib

MKN45

MKN45+SU11274

0

0.2

0.4

0.6

0.8

1

1.2

R64 R69 R108 R108

vim

enti

n

vim

enti

n

BA

G4

SL

C37

A2

Rel

ativ

e ab

un

dan

ce

Methyl-arginine

A549

A549+Iressa

H1703

H1703+Gleevec

H3255

H3255+Iressa

Figure 2. qualitative comparison of tMt labeling quantified by Ms2 or Ms3 with label-free quantification. Six cell lines (A549, H1650,

H1703, H3122, H3255, and MKN45) either were processed individually and analyzed separately using 45 minute LCMS gradients and CID fragmentation

(label-free), or were TMT-labeled and combined for immunoprecipitation. The TMT-labeled samples were analyzed using 150 minute LCMS gradients, either

using HCD MS2 for both peptide identification and quantification in a single step (TMT-HCD only), or using CID MS2 for peptide identification with HCD

MS3 for TMT quantification (TMT-MS3). The number of modification sites, unique peptides, and total redundant peptides identified using each method is

displayed for acetyl-lysine and methyl-arginine immunoprecipitation samples. In all cases, the TMT method with HCD peptide identification and quantifica-

tion resulted in discovery of the highest number of PTM sites, and this method was selected for further studies.

Figure 3. iMAc sample clean-up step reduces background of non-phosphorylated peptides and allows identification of more

phosphopeptides. Identical tyrosine phosphopeptide immunoprecipitations were performed and analyzed with or without subsequent IMAC to further

enrich phosphopeptides over background unphosphorylated peptides. IMAC was found to significantly reduce levels of contaminating unphosphorylated

peptides and thus increase the number of phosphopeptides identified.