Figure 3: PTMScan Direct Ser/Thr Kinases Reagent and Tyr Kinases Reagent targets mapped onto the human kinome tree. Yellow highlighting indicates kinases identified using the reagents. Tyr Kinases Reagent coverage is shown in the inset (“TK”).

Matthew P. Stokes, Jeffrey C. Silva, Charles L. Farnsworth, Xiaoying Jia, Roberto Polakiewicz, Michael J Comb Cell Signaling Technology, Inc., Danvers, MA

Quantitative Profiling of Signaling Pathways Using PTMScan® Direct

A challenge to proteomic studies of post-translational modifications (PTMs) is the ability to focus on proteins of interest rather than randomly identified proteins typified by data dependent analysis. Methods that target specific proteins and pathways known to be critical to cellular signaling are therefore desirable. Cell Signaling Technology (CST) has developed an immunoaffinity-based LC-MS/MS method, called PTMScan® Direct, for identification and quantification of hundreds of peptides from particular signaling pathways or protein types. Cell lines, tissues, or xenografts can be used as starting material. Quantification may be performed using isotopic labeling methods such as SILAC or label-free quantification of chromatographic peak areas. PTMScan Direct is a powerful new method that combines the specificity of antibody-based methods with the sensitivity and high duty cycle of LC-MS/MS analysis allowing thousands of measurements.

1. Multipathway Reagent2. Serine/Threonine Kinases Reagent3. Tyrosine Kinases Reagent4. Akt / PI3K Pathway Reagent5. DNA Damage / Cell Cycle Reagent6. Apoptosis / Autophagy Reagent

PTMScan Direct is a novel method that allows identification and quantification of hundreds of pep-tides from selected protein types or signaling pathways. This allows focus on proteins of interest, bypassing the random sampling of peptides that occurs in traditional data-dependent proteomic analysis. PTMScan Direct is widely applicable in drug development and discovery, as well as in any application where monitoring of known signaling pathways is desired.

1. Rush J. et. al. (2005) Nat. Biotechnol. 23: 94-1012. Lundgren, et. al. (2009) Curr. Protoc. Bioinformatics. 13: Unit 13-33. Stokes M.P. et. al. (2012) Mol. Cell Proteomics. Epub Feb 9th

Figure 1: PTMScan Direct work flow diagram. PTMScan Direct is a published method (Stokes et al, 2012) adapted from the original PhosphoScan® method developed at Cell Signaling Technology (Rush et al, 2005, Patent # 20030044848). Cell lines, tissues, xenografts, or other biological starting materials are lysed under denaturing conditions, digested with trypsin, and desalted over C18 columns. Target peptides are immunoprecipitated using the appropriate PTMScan Direct reagent. Immunoprecipitated peptides are separated on a reversed-phase Magic C18 AQ column and data-dependent MS methods are performed with an LTQ-Orbitrap Velos or Elite mass spectrometer. MS/MS spectra are evaluated using SEQUEST 3G and the SORCERER 2 v4.0 platform from Sage-N Research (Lundgren et al., 2009). Quantification is performed using chromatographic peak apex areas or intensities.

Figure 2: Coverage of selected pathways using the PTMScan Direct Multipathway Reagent. Peptides from purple proteins are identified using the Multipathway Reagent.

InTeRaCTIon MaPFigure 4: PTMScan Direct: akt/PI3K Pathway Reagent interaction map. Interactions were derived from STRING using high confidence scores (>0.700) from experimental, database, and textmining lines of evidence and the substrate search function in PhosphoSitePlus®. Node colors/shapes denote different protein classes. Edge color denotes interaction type.

InTeRaCTIon MaPFigure 6: PTMScan Direct: Dna Damage / Cell Cycle Reagent interaction map. Interactions were derived from STRING using using high confidence scores (>0.700) from experimental, database, and textmining lines of evidence. Node colors/shapes denote different protein classes.

InTeRaCTIon MaPFigure 8: PTMScan Direct: apoptosis / autophagy Reagent interaction map. Interactions were derived from STRING using using high confidence scores (>0.700) from experimental, database, and textmining lines of evidence. Node colors/shapes denote different protein classes.

HeLa -/+ UV DaMageFigure 9: apoptosis / autophagy Reagent: HeLa cell UV damage study. Quantitative data for caspase peptides identified with the Apoptosis/Autophagy Reagent used to probe UV damaged HeLa cells (500 mJ/cm2, harvest 2 hr post treatment). Green indicates increased abundance relative to control.

HeLa -/+ UV DaMageFigure 7: Dna Damage/Cell Cycle Reagent: HeLa cell UV damage study. HeLa cells were untreated or treated with 500 mJ/cm2 UV light and harvested at 2 hr post treatment. Fold changes were calculated from chromatographic peak heights/areas. green cells indicate peptides more abundant with UV damage, Red cells indicate peptides less abundant. Selected DNA damage response proteins are shown in detail with accompanying western blots.

MoUSe TISSUe STUDyFigure 5: akt/PI3K Pathway Reagent mouse tissue study. Heat map of relative intensities for mouse liver, brain, and embryo. Each row represents a different peptide ion and each column represents a different tissue. The max intensity across the three tissues was set to 1 and the other two intensities normalized to the max. Blue indicates higher intensity.

Introduction

PTMScan® Direct Reagents

PTMScan® Direct Method

Multipathway Reagent Selected Targets

Akt / PI3K Pathway Reagent DNA Damage/Cell Cycle Reagent Apoptosis / Autophagy Reagent

Ser/Thr Kinases and Tyr Kinases Reagent Targets

Summary

References

PTP

PIAS

SHP-1

SOCS

TF

TF

p120ras-GAP

SOCS3

Stat3

Erk mTOR

Akt

Shc

GR

B2

Ras

PI3K

SHP-2

Erk

MEK

Raf

Stat3

Stat3

Stat

StatMcl-1

Jak

Jak

Stat3

gp13

0

EGFR

Src

EGF

ErkErk

IL-6

CIS, SOCS, Mcl-1, APPs, TIMP-1, Pim-1, c-Myc, cytokines, TFs, etc.

CrosstalkTumor CellsApoptosis

Renewalof ES Cells

C/EBPβNF-κBAP-1etc.

Nucleus

Cytoplasm

FeedbackInhibition

ISRE/GASAccessoryTF Motif

E-Ras

Tumor-likePropertiesof ES Cells

PI3K

Akt

Jak/Stat Signaling: IL-6 Receptor Family

www.cellsignal.com

© 2002 – 2010 Cell Signaling Technology, Inc. Jak/Stat Signaling: IL-6 Receptor Family • created November 2002 • revised November 2010

SUMO

HistoneH3

CREB SRF

PPARγ

ER Elk-1

Stat1/3 c-Myc/N-Myc

MSK1/2p90RSK

p90RSKp90RSK

Erk1/2

Erk1/2 Erk1/2

MNK1/2

cPLA2

nNos

MEK1/2PP1/

PP2At

B-Raf

B-Raf

PKAc-Raf

c-RafPKC

PLCγ

SrcPYK2 PAK

Spry

Spred

Tpl2/Cot1

PI3K

Pax

FAKFyn

GR

B2

GR

B2

Ras

Rac

Rap1

CR

K

14-3-3

Integrins

MEK1

MP1

Erk1

P14

C-TAK1

IMP

RACK1

ATF1 c-FosEts

Pax6

UBF

MKP-1/2

cdc25

MKP-3

KSR1/2

c-Fos

Fox03 ERα

ATF4

MITF

Mad1

Nur77

ETV1 TIF1A

eIF4B

METTL1

eEF2K GSK-3

rpS6 TCPβ

IκBα

BAD

DAPK

C/EBPβ

Myt1

BUB1

Filamin A

ER81

HMGN1 Ran BP3

TIF1A

p27 KIP1

TSC2PEA-15

Bim

Tal

Shc

FRS2

IRS SOS

SOS

SOS

EPAC

Ion Channels[Ca2+]

RTKs

RTKs

C3GCAS

[cAMP][Ca2+]

TranslationControl

Ion Channels,Receptors

Nucleus

Cytoplasm

Heterodimer

BI-D1870

Transcription

PD98059U0126

Late Endosome

Cell AdhesionCytoplasmic Anchoring

MAPK/Erk in Growth and Differentiation

www.cellsignal.com

© 2003 – 2010 Cell Signaling Technology, Inc. MAPK/Erk in Growth and Differentiation • created January 2003 • revised November 2010

DEPTORRictor

DEPTOR

mTORC2

Akt

Akt

p70 S6K

PI3K

PI3K

Paxi

llin

GSK-3

PHLPP CTMP

LaminA

PP2A

Tpl2

IKKα

eNOS

Bad

Bcl-2

FoxO1

TSC1TSC2

PDCD4

AS160

PFKFB2

PIP5K

S6

p53

MDM2

Bim

Bax p27 Kip

p21 Cip

14-3-3

Cyclin D1

GαGTP

PTEN

PDK1

SykJak1

FAK

ILK

PI3K

PI3K

PI3K

XIAP

Wee1 Myt1

Gab

1G

ab2 IR

S-1

PTEN

Integrin

CytokineReceptor

GlycogenSynthase

PDK1

Gβγ

BCAP

Lyn

Ag

Ataxin-1

Huntingtin

GABAAPRAS40

GPCR

RTK CD19

BCR

PIP3 PIP3

mTOR

Sin1 PRR5

GβL

RaptorGβLmTOR

mTORC1

mTORC1

mTORC2

4E-BP1

ATG13

α/βα/β

BlocksAggregation

PromotesNeuroprotection

SynapticSignaling

mIgmIg

CytosolicSequestration

CardiovascularHomeostasis

NF-κBPathway

Organizationof NuclearProteins

Cell Cycle

Apoptosis

AggregationNeurodegeneration

Inhibits Apoptosis

InhibitsAutophagy

GlycogenSynthesis

Glucose Transport

Glycolysis

Membrane Recruitment

and Activation

Membrane Recruitment

and Activation

Protein Synthesis

Survival

Proliferation

Glucose M

etabo

lism

Neur

oscie

nce

Oth

er

PI3 Kinase Akt Signaling

www.cellsignal.com

© 2003 – 2010 Cell Signaling Technology, Inc. PI3K / Akt Signaling • created January 2003 • revised November 2010

0 1

Kinase

Chromatin or DNA Binding / Repair / Replication

Cell Cycle Regulation

Transcriptional Regulator

Phosphatase

Adhesion or Cytoskeletal Protein

Other

G Protein or Regulator

p38α

p38γ

p38δ

Apoptosis

Protease

Transcription/Translation

Adaptor/Scaffold

Phosphatase

Autophagy

Other

Kinase

RICTOR RAPTOR

RUSC1

ZNF768

XIAP

FYCO1 ATG7

SH3GLB2 ATG12 ATG3

ATG4C

RABGAP1

ATG9A

PTPN11

MYC

NFκB p105NFκB p65

IκBα

JUN

JUNDIκBε

Bcl−11B

PARP1

CASP9

CASP3CASP2 CASP6

CASP8CASP7PRKCA

PRKCH

PRKD3

PKD1

PRKCB

PRKCQ

PKD2

PIK3CB PIP4K2A

PIK3C2A PI4KA

PIK3CA PIK3C3

PI4K2APIK3CD

ERK2

ERK1

ULK4

Nik

IKKβ

ULK1GSK3A

AKT1

AKT3AKT2

DNA−PK

ROCK1

ROCK2mTOR

ANXA5

ARCMIF

CIAPIN1 PDCL3SOD1

PHLDA1

DBC-1DDAH2

API5

p400 NUDT2 PAWR

PDCD10

SH3GLB1

STAG1

LDHA

CLU

CCDC72PDCD5

HELLS APIP CCAR1 GPI

AIFM1BIRC6BIRC5HSP90B1

Bak1

AKT1S1 Bcl-xL

Bcl-9L

BID

BAD

CYC1

CYCS

TNF-R1FAS

Contact InformationMatthew P. Stokes, Cell Signaling Technology, Inc. email: mstokes@cellsignal.com

PTMScan Services Department email: ptmscan@cellsignal.com • web: www.cellsignal.com/servicesCell Signaling Technology®, PhosphoScan®, PhosphoSitePlus®, and PTMScan® are trademarks of Cell Signaling Technology, Inc.