Simplifying Proteomics
PTMScan®
PTMScan Products
Products andand Services
ServicesA limited number of genes can generate a tremendous levelof complexity at the protein level due to processes such asalternative splicing and post-translational modification (PTM).
PTMs are essential for many cellular functions such as protein
activity, subcellular localization, degradation, and proteinprotein interactions. Proteomic methods that profile PTMs
provide insight into both normal and disease biology that is
not feasible at the genetic level.
There are many types of PTMs including:» Methylation» Phosphorylation» Succinylation» Acetylation» Proteolytic cleavage» Ubiquitination

GENOME
Thousands of genes

TRANSCRIPTOME
Hundreds of thousands of transcripts

PROTEOMEMillions of proteoforms

PTMScan®: Antibody enrichmentof modified peptides for mass
spectrometry-based proteomics
Cell Signaling Technology (CST ) has established PTMScan®
technology, a proprietary proteomic method that employs
validated PTM- and motif-specific antibodies developedby CST to enrich PTM-containing peptides prior to liquid
chromatography tandem mass spectrometry (LC-MS/MS).
PTMScan technology allows identification and quantification of hundreds to thousands of even low abundance PTM
sites, which can then be narrowed down to the most relevant
actionable targets. PTMScan technology uses a more focused
approach to PTM-peptide enrichment than other strategies
such as immobilized metal affinity chromatography (IMAC).
TM

PTMScan can be used to:• Determine novel PTM sites that are phosphorylated,ubiquitinated, acetylated, etc.
• Identify and validate drug targets• Discover biomarkers• Elucidate off-target drug effects• Explore the mechanism of action of drugs/chemical modulators

IP with PTM/Motif Abs

Contents

3 Discovery vs. Direct
4 PTMScan® Kits vs. Services
5 PTMScan® Discovery and Direct Services
6 Complementary Services
7 PTMScan® Discovery Products and Services
12 PTMScan® Direct Services
13 From Bench to Bedside
14 Support for Drug Discovery

Mass Spectrometry
Data
Analysis

Candidates for further analysis

INCREASING COMPLEXITY

Simplifying
Proteomics

Key to Success: PTMScan® Antibodies
The antibodies used to enrich PTM-containing peptides are key to the success of PTMScan technology. They are:
• Designed and produced in-house• Rigorously tested for specificity, sensitivity, and lot-to-lot consistency• Specially formulated for immunoaffinity enrichment

The table below outlines the three types of antibodies for PTMScan technology.
Antibody type

Recognizes

ExampleStandard site-specific
PTM antibody

Modified amino acid in thecontext of a specific sequenceof amino acids surrounding it.

A CSTTM antibody to Akt1 phosphorylated at serine 473 only recognizes
that particular phosphoserine and thesurrounding amino acids.

Modified amino acid within acertain motif.

The Akt substrate motif antibody willrecognize the sequence RXRXXS*
in any protein only when the serine
residue is phosphorylated (where X
can be any amino acid).

Motif antibody

PTM-specific antibody(PTM-antibody)

Any peptide with the PTM ofinterest.

A CST acetyl-lysine antibody willrecognize all acetylation sites
independent of flanking amino acidsequences.

MultiMab Antibody Mixtures
for Broader Coverage
TM

MultiMab™ rabbit monoclonal antibody mixtures combine individual rabbit
monoclonal clones in optimized ratios for broadest possible coverageof the motif/PTM of interest. MultiMab mixtures are used in PTMScan
Services and Kits and are available off-the-shelf for other applications.

For full list of MultiMab antibody mixtures visit:www.cellsignal.com/MultiMab

Serum-starved

H2O2 TreatedP
FPQFSYSASPXRXRXXSXXAcXXXXKXXXX

H2O2 + λ phosphatase

Phospho-Tyrosine (P-Tyr-1000) MultiMab™ Rabbit mAb mix #8954: Confocal
IF analysis of C2C12 cells, serum-starved (left), treated with H2O2 (2 mM, 10 min;middle), or treated with H2O2 followed by λ phosphatase (right), using #8954 (green).
Actin filaments were labeled with DyLightTM 554 Phalloidin #13054 (red). Blue pseudocolor = DRAQ5® #4084 (fluorescent DNA dye).

www.cellsignal.com/Proteomics

2

Discovery vs. Direct
Which option is right for your research?
PTMScan® Discovery

PTMScan® Direct(PTM/motif-based enrichment)(mass spectrometry-based antibody array)

PTM/motif
Abs

Is a specific pathway targeted?

84Is antibody enrichment performed?

4

4

Is LC-MS/MS performed?

4

4What type of antibodies are used?

PTM or motif antibodies to undefined targets.

Standard site-specific antibodies to definedtargets within the known pathway(s) of interest.

What is the bead format?

Antibodies against one PTM or motif on eachbead.

Antibodies against many targets on each bead(a bead-based multiplex assay).

Which species can you use?

Can be used on samples from many different
species including, but not limited to, human,
mouse, rat, drosophila, and arabidopsis.

Validated for human and mouse.
(Contact us for other species.)

Case Study“Deep, quantitative coverage of the acetylome
using novel anti-acetyl-lysine antibodies and an
optimized proteomic workflow.”“PTMScan Direct: identification and quantificationof peptides from critical signaling proteins by immunoaffinity enrichment coupled with LC-MS/MS.”

Svinkina, T., et al (2015) Mol. Cell. Proteomics 14(9):2429–40.

Stokes, M., et al (2012) Mol Cell Proteomics. 11(5):187–201.

Use PTMScan Discovery to find new information with quantitative analysis of PTMs.

Use PTMScan Direct to quantitatively assay
the activity of components of known signaling pathways across cell lines or treatments.

Summary3

Site-specific Abs
to defined targets

For Research Use Only. Not For Use in Diagnostic Procedures.

PTMScan® Kits vs. Services
PTMScan® Kits
With PTMScan® Antibody Kits you can perform your own enrichment
and LC-MS/MS analysis. They provide the antibody reagents for
10* peptide enrichment experiments and the detailed protocols
needed to discover new sites of post-translational modification.§*A smaller format for certain kits (3 assays instead of 10) allows investigators to run pilot studies.
§
Kits also contain a limited use license.

PTMScan® Services
CST scientists work with you from project planning to delivery of a comprehensive data package that includes:• Qualitative/quantitative tables• Informatics tables• Microsoft® PowerPoint® summary• Microsoft® Word® guide to prioritizing follow-up candidates

PTMScan®
Service Workflow
Discovery, Direct,
or KinomeView®
Project Discussion
Experimental DesignStatement of
Work/Quotation

Submission of Samples

Project Execution

Data Package Delivery
and Consultationwww.cellsignal.com/Proteomics 4

PTMScan® Discovery and Direct Services
Workflow
Protease-digested
SamplePTMScan® Reagent Immobilized
to Protein-A or -G Agarose

Analyze Eluted Peptide
Fraction by LC-MS/MS

Relative Quantification/Data Analysis

Assign Sequences to MS/MS
Spectra with SEQUEST®

Cytoskeletal
2%

Transcription
Regulation
2%

Other
10%

Enzyme26%

Chaperone
2%
Translation
3%
Adhesion/
Extracellular Matrix
6%
Receptor/
Transporter
10%

Mitochondrial
23%
Chromatin/
Epigenetics
16%

PTMScan® Service Data
The data set generated by a PTMScan®
service experiment
includes quantification of PTM changes,
the identity of eachprotein, and specificlocation of each
modification site.

Normalized Fold Change
SU11274 vs.
StaurosporineDMSO Control vs. DMSO Control

-5.0
-13.6
-158.0
-3.4
-13.3
-7.0
2.6
-7.0
-5.3
-21.3
-2.1
-10.3
2.7
4.8

-4.6
-2.1
-7.2
1.8
-29.4
-24.5
1.1
-9.4
-N.D.
-3.0
-2.9
-1.8
2.5
2.5

ProteinName

Site-7/+ 7 Sequence

PeptideUpstream Kinase

EphA2

897 RVSIRLPsTSGSEGVLPS*T*SGSEGVPFR

Akt1

FOXO1A

319 TFRPRTSsNASTISGTSS*NASTISGR

Akt1

FOXO4

32 QSRPRSCtWPLPRPE

SCT*WPLPRPEIANQPSEPPEVEPDLGEK

Akt1

QIK

358 DGRQRRPsTIAEQTV

RPS*TIAEQTVAK

Akt1, Akt2

S6235, 236, 240 IAKRRRLsSLRASTSRLS*S*LRAS*TSK

Akt1, Akt2, P70S6Kβ, PKACα, PKCα, PKCδS6236, 240 AKRRRLSsLRASTSKRLSS*LRAS*TSK

Akt1, Akt2, P70S6Kβ, PKACα, PKCα, PKCδBRAF

365 GQRDRSSsAPNVHINSSS*APNVHINTIEPVNIDDLIR

Akt1, Akt3

GSK3β

9 SGRPRTTsFAESCKPTTS*FAESCKPVQQPSAFGSMK

Akt1, AurA, CAMK2β, GSK3β, KHS1, PKACα, PKCαGSK3β9, 21 SGRPRTTsFAESCKPTTS*FAESCKPVQQPS*AFGSMK

Akt1, AurA, CAMK2B, GSK3β, KHS1, PKACα, PKCαPEA-15116 KDIIRQPsEEEIIKLDIIRQPS*EEEIIK

Akt1, CAMK2α, CK2α1GSK3α21 SGRARTSsFAEPGGGTSS*FAEPGGGGGGGGGGPGGSASGPGGTGGGK

Akt1, CAMK2β, PKACA, PKCα, PKCβRANBP3126 VKRERTSsLTQFPPSTSS*LTQFPPSQSEER

Akt1, ERK1, RSK2, p90RSK

eIF4B

422 RERSRTGsESSQTGTTGS*ESSQTGTSTTSSR

Akt1, p70S6K, p90RSK

eIF4B

422, 425 RERSRTGsESSQTGTTGS*ESS*QTGTSTTSSR

Akt1, p70S6K, p90RSK

Table view presentation of data from PTMScan® analysis of MKN-45 cells treated with SU11274 or staurosporine. Shown are representative data for thebasophilic Akt substrate motifs RXRXX(s/t) and RXX(s/t). Relative abundance changes of 2.5-fold or greater (treated versus control) for phosphorylated peptides areindicated by green (increase) or red (reduction) highlighting.

5

For Research Use Only. Not For Use in Diagnostic Procedures.

Complementary Services
KinomeView® Western Blot Profiling
KinomeView® profiling is a western blot prescreen
using the same PTM and motif antibodies available for
PTMScan analysis.

pY
pS/pT PDK1 Docking Motif F/YpS/pTF/Y• Observe global signaling changes acrossmultiple samples• Optimize experimental conditions (treatment dose, time point,cell line or tissue, etc.)
The KinomeView® Profiling Kit #9812 or Service
The kit provides motif antibodies for a kinome-wide view of cellular phosphorylation for customers who want to perform the experiment themselves.
The KinomeView® Profiling Service also includes motif antibodies for studying
changes in additional PTMs. The service is performed by CST scientists andincludes data analysis and expert project consultation.

TKL

pT MAPK Motif PXpTP• Select the best antibodies for use in subsequent
PTMScan Discovery experiments

pT CK Motif pS/pTD/EXD/E

TK

STE

pS MAPK Motif PXpSP

CMGC

pS/pT PKA Motif K/RK/RXpS/pT

CK1

pS CDK Motif K/RpSPXK/R

pS/pT Akt Motif (R)XRXXpS/pT

pT Motif pTP

AGC

Atypical Protein Kinases
ABC1

PDHK

Alpha

PIKK

Brd

RIO

pS/T Akt Motif RXRXXpS/pT

CAMK

TIF1

pT Motif pTXR

pS/pT ATM Motif pS/pTQG
pS/T PKD Motif LXRXXpS/pT
pS/pT ATM Motif pS/pTQ

pS PKC Motif K/RXpSXK/R

Immobilized Metal Affinity Chromatography (IMAC) Service
IMAC is another strategy for enriching phosphorylated peptides before LC-MS/MS. It captures negatively charged
phosphopeptides using positively charged metal ions.

Global Proteome Profiling Service
Quantification of protein levels across cell lines or treatments without enrichment for PTM-containing peptides.

Ingenuity® Pathway Analysis
Mapping of PTMScan® results onto canonical pathways and new interaction networks using Qiagen’s Ingenuity®
Pathway Analysis (IPA) software.

www.cellsignal.com/Proteomics 6

PTMScan® Discovery Products and Services
UbiScan® (Ubiquitination Proteomics)=Trypsin Cleavage Site

UbiScan® proteomics uses a proprietary antibody against the di-glycine remnant(K-ε-GG) left on ubiquitinated lysine residues after trypsin digestion. This ubiquitin
remnant motif antibody is used to enrich ubiquitinated peptides from a trypsin digested
sample prior to LC-MS/MS analysis.

UbiScan® – Ubiquitination Proteomics

Trypsin

GG

K-ε-GG Antibody

QGAVKLVGAR

VLRLRGGKQGAVKLVGAR

Protein ubiquitination is involved in many cellular processes including proteasomal
degradation, endocytosis, DNA repair, cell cycle regulation, and gene expression.
Abnormal ubiquitination is involved in diseases such as cancer, metabolic syndrome,and neurodegenerative diseases.

Ubiquitin Remnant
Motif (K-ε-GG):
The Ubiquitin Remnant
Motif (K-ε-GG) antibody
is used to enrich K-ε-GG
containing peptides
from complex mixtures.

Ubiquitin

Substrate Protein

Antibody‡

KinomeView®

PTMScan®

Target

Motif

No.

Kit

Service

Kit #9812

ServiceUbiquitin Remnant

K-ε-GG–#14482/#5562

4

–

4

Select UbiScan® Publications:Sook-Young, J. et al. (2015) Proteomic Analysis of UbiquitinLike Posttranslational Modifications Induced by the Adenovirus
E4-ORF3 Protein. Journal of Virology 89(3) 1744-55.

Kronke, J. et al. (2014) Lenalidomide causes selective degradation of IKZF1 and IKZF3 in multiple myeloma cells. Science343(6168), 301–305.

Mathew, R. et al. (2012) BTB-ZF factors recruit the E3 ligase
cullin 3 to regulate lymphoid effector programs. Nature
491(7425), 618–621.

Kronke, J. et al. (2015) Lenalidomide induces ubiquitinationand degradation of CK1α in del(5q) MDS. Nature 523(75599),
183–188.

Theurillat, J.P. et al. (2014) Ubiquitylome analysis identifies
dysregulation of effector substrates in SPOP-mutant prostatecancer. Science 346(6205), 85–89.

Emanuele, M.J. et al. (2011) Global identification of modular
Cullin-RING ligase substrates. Cell 147(2), 459–474.
Kim, W. et al. (2011) Systematic and quantitative assessmentof the ubiquitin-modified proteome. Mol. Cell 44(2), 325–340.

PhosphoScan® (Phosphorylation Proteomics)
Complementary Phosphopeptide Enrichment Strategies
Comparison of PTMScan® and IMAC enrichment methods:
PTM profiling of human gastric carcinoma cells.

A

P-Tyr

Phosphopeptides were enriched using various classes of motif/PTM antibodies (tyrosine, proline,
serine/threonine, and basophilic), or IMAC.

• Over 20,000 phosphorylation sites were identified across all samples.
• There was minimal overlap between phosphopeptides enriched using
antibody-based versus IMAC methods.
• Antibody-based methods reduce sample complexity for a more focused
approach to phosphopeptide enrichment.

(5,341)

3.6%

Baso

(2,607)

6.9%

IMAC

(12,625)

Antibody-based and IMAC enrichment strategies arehighly complementary and both should be used to
get the broadest coverage of the phosphoproteome.

11.2%
Area proportional Venn diagram showing the overlap between motif antibody and IMAC enrichment of phosphopeptides
from MKN-45 cells (A). Comparisons were made on unique proteins/sites. MKN-45 cells were treated and profiled as
outlined (B). Combinations of motif antibodies were used to enrich phosphopeptides as indicated (C).

For Research Use Only. Not For Use in Diagnostic Procedures.

(2,646)

16.2%
Ser/Thr(4,245)View more data showing complementarity of PTMScan Discovery and IMAC at: www.cellsignal.com/PTMScanIMAC

7

ProlineB

C

MKN-45 Cells

DMSO(control)SU11274

Staurosporine(Met inhibitor)

IMAC(PKC inhibitor)

pY-1000

Basophilic

Ser/Thr Motif

Proline-directed

Antibody

Motif

pY

PhosphotyrosineY

Akt Substrate

RXX(S/T)•

Akt Substrate

RXRXX(S/T)

AMPK/PKD Substrate

LXRXX(S/T)

CDK Substrate(K/R)(S/T)PX(K/R)

PKA Substrate(K/R)(K/R)X(S/T)

PKC Substrate(K/R)X(S/T)(K/R)

MAPK Substrate

PX(S/T)PPLK Binding Motif

S(S/T)PtP Motif(S/T)PtPE Motif(S/T)PEtXR/tPR Motif(S/T)(X/P)R

14-3-3 Binding Motif(R/K)XX(S/T)XPATM/ATR Substrate(S/T)Q

ATM/ATR Substrate(S/T)QGCK Substrate(S/T)(D/E)X(D/E)

Baso

•
•
•
•
•
•

Pro

Ser/Thr

•
•
•
•
•
•
•
•
•
•
•
•
•
•
•

•
•
•
•
•
•

Note: Custom mixes of Ser/Thr motif antibodies can be made for service projects. Please inquire.

KinomeView®

PhosphoScan® – Phosphorylation Proteomics Antibody‡

PTMScan®

Target

Motif

No.

Kit

Service

Kit #9812

14-3-3 Binding Motif

R/KXXSXP

#9601

–

4

–

4

Akt Substrate
Akt Substrate(Ser/Thr) AMPK Substrate(Ser) ATM/ATR Substrate(Ser/Thr) ATM/ATR Substrate(Ser) CDKs Substrate
CK2 Substrate
MAPK/CDK Substrate
PKA Substrate(Ser) PKC Substrate(Ser/Thr) PKD Substrate(Thr) PLK Binding Motif
Thr-Pro-Glu Motif
Thr-Pro Motif
Thr-X-Arg Motif

RXX(S/T)RXRXX(S/T)(L/M)XRXX(S/T)
SQ(S/T)QG, (S/T)Q(K/R)SPX(K/R)(S/T)DX(D/E)
PXSP and SPX(K/R)(K/R)(K/R)X(S/T)(K/R)XSX(K/R)
LXRXX(S/T)
STP
TPE, TPTP, TPP
TXR, TPR#9614#10001
#5759
#9607
#6966
#9477
#8738
#2325
#9624
#6967
#4381
#5243
#3004
#5757
#2351

#5561
#5563
#5564
#12267#12267–#12170
#4652
#5565
–
–
#5566
–
#5567
–

4
4
4
4
4
4
4
4
4
4
4
4
4
4
4

4
4
4
4
4
4
4
4
4
4
–
4
4
4
4

4
4
4
4
4
4
4
4
4
4
4
4
4
4
4

Tyrosine Rabbit (P-Tyr 1000)

Y

#8954

#14478/#8803

4

4

4

Service

Select PhosphoScan® Publications:Lee, M.E. et al. (2015) Endothelial Akt1 mediates angiogenesisby phosphorylating multiple angiogenic substrates. PNAS
111(35), 12865–12870.

Ren, H., et al. (2012) Identification of anaplastic lymphoma
kinase as a potential therapeutic target in ovarian cancer.
Cancer Res. 72(13) 3312–3323.

Rikova, K. et al. (2007) Global survey of phosphotyrosine signaling identifies oncogenic kinases in lung cancer. Cell 131(6),1190–1203.

Paardekooper Overman, J. et al. (2014) PZR Coordinates Shp2
Noonan and LEOPARD syndrome signaling in zebrafish andmice. Mol. Cell. Biol. 34(15), 2874–2889.

Carretero, J. et al. (2010) Integrative genomic and proteomic
analyses identify targets for Lkb1-deficient metastatic lungtumors. Cancer Cell 17(6), 547–559.

Siddoway, B. et al. (2013) Synaptic activity bidirectionallyregulates a novel sequence-specific S-Q phosphoproteome inneurons. Journal of Neurochemistry 128(6):841–851.

Moritz, A. et al. (2010) Akt-RSK-S6 kinase signaling networks
activated by oncogenic receptor tyrosine kinases. Sci. Signal
3(136), ra64.

Griswold, I.J., et al. (2006) Kinase domain mutants of Bcr-Abl
exhibit altered transformation potency, kinase activity, andsubstrate utilization, irrespective of sensitivity to imatinib. Mol
Cell Biol. 26(16) 6082–6093.
Rush, J. et al. (2005) Immunoaffinity profiling of tyrosine
phosphorylation in cancer cells. Nat. Biotechnol. 23(1), 94–101.
‡

Validated for use in other applications.

www.cellsignal.com/Proteomics 8

PTMScan® Discovery Products and Services (cont.)
MethylScan® (Methylation Proteomics)
Protein methylation is a common post-translational modification (PTM) that mostly occurs
on arginine and lysine residues. Arginine methylation regulates processes such as RNA
processing, gene transcription, DNA damage repair, protein translocation, and signaltransduction. Lysine methylation is best known to regulate histone function and is involvedin epigenetic regulation of gene transcription.

Features and Benefits• PTMScan® methyl antibodies are exceptionally specific, as demonstrated by peptide blocking experiments, to help ensure the mostaccurate results.

• P TMScan technology can be applied to many biological systems

MethylScan® proteomics uses proprietary methyl arginine (Me-R) or methyl lysine (Me-K)
antibodies to enrich methyl-containing peptides from trypsin digested samples prior to
LC-MS/MS analysis.

and species to encompass diverse research interests.

• Experienced CST scientists provide technical support throughout the
PTMScan workflow to facilitate research progress.

A

B

Brain
472

335

Embryo263

log2 ratio_Brain/Embryo

R-2Me(a) antibody

Blocking none
Peptidelog10 of total intensity
Quantitative analysis of arginine methylation in mouse brain and embryo: Venn diagrams of the number of monomethyl arginine(R-Me) and asymmetric dimethyl arginine (R-2Me(a)) sites identified in mouse brain and embryo (A). Quantitative comparison of arginine
monomethylation in the two tissues. Each dot in the scatter plot represents a unique arginine monomethylated peptide identified using
PTMScan® Mono-Methyl Arginine Motif [mme-RG] Kit #12235. The x-axis is the log10 value of the total intensity of the representative
peptide for a methylation site in mouse brain and embryo, and the y-axis shows the log2 ratio of intensity of the peptide in mouse brain vs.
embryo. A cutoff of 5-fold was set to indicate increased arginine monomethylation peptide abundance in either brain (green dots) or embryo(red dots). For the methyl peptides that uniquely existed in a specific tissue, arbitrary log2 ratios of 15 (brain specific) and -15 (embryo
specific) were assigned (B).*antigen R-2Me(s)Western blot analysis of lysates from
untreated (-) or AdOx-treated (+)
HCT116 cells using asymmetric
dimethyl arginine (R-2Me(a)) antibodies.
The specific signal was blocked byantigen R-2Me(a) peptides (middle
panel), but not by corresponding symmetric dimethyl arginine (R-2Me(s))
peptides (right panel).**This research was originally published in Molecular and Cellular Proteomics. Guo, A., et al. Immunoaffinity enrichment and mass spectrometry analysis of protein methylation. Mol. Cell. Biol. 2014; 13(1):372–387.
© the American Society for Biochemistry and Molecular Biology, and is licensed under CC BY 4.0 http://creativecommons.org/licenses/by/4.0/.

MethylScan® – Methylation Proteomics
TargetMotif

Mono-Methyl Arginine
Asymmetric Di-Methyl Arginine
Symmetric Di-Methyl Arginine
Pan-Methyl Lysine

K-Me/K-2Me/K-3Me

Antibody‡
No.

Kit

Service

Kit #9812

Service

R-Me

#8015

#122354–

4

R-2Me(a)R-2Me(s)#13522#13222#13474#13563

4
4

–
–

4
4

#14117 (2Me)#14680 (3Me)#148094–

4

Select MethylScan® Publications:
Guo, A. et al. (2014) Immunoaffinity enrichment and mass
spectrometry analysis of protein methylation. Mol. Cell.
Proteomics 13(1) 372–387.

‡

9

KinomeView®

PTMScan®

Sylvestersen, K. B. et al. (2014) Proteomic analysis ofarginine methylation sites in human cells reveals dynamic
regulation during transcriptional arrest. Mol. Cell. Proteomics
13(8):2072–2088.

Validated for use in other applications.

For Research Use Only. Not For Use in Diagnostic Procedures.

AcylScanTM (Acylation Proteomics)
O

O

HN

O

HN

O
O

HN

O

O
O

HN

O

HN

O

O

O

acetyl lysine

Features and BenefitsOsuccinyl lysine

Succinylation

B

NH

O

propionyl lysine

NH

O

NH

NH

AcylScanTM proteomics use proprietary acetyl- (Ac-K), glutaryl- (Glut-K),
malonyl- (Mal-K), propionyl- (Prop-K), or succinyl-lysine (Succ-K) antibodies
to enrich their respective acyl-containing peptides from trypsin digested
samples prior to LC-MS/MS analysis.

A

NH

Lysine residues are subject to a wide range of modifications. Acyl grouptransfer from the metabolic intermediates acetyl-, succinyl-, malonyl-,glutaryl-, butaryl-, and crotonyl- CoA all neutralize the positive charge oflysine and confer structural alterations affecting substrate protein function.

malonyl lysine

O

glutaryl lysine

Malonylation

2431

1048

• AcylScan methods allow quantitative profiling of thousands of acetyla-

663

tion, glutarylation, malonylation, propionylation, and succinylation
events by using antibodies with exquisite specificity and sensitivity.

2052

839

• PTMScan technology can be applied to many biological systems andspecies to encompass diverse research interests.

135

305

• Experienced CST scientists provide technical support throughout the

106

21

3

5
54

PTMScan workflow to facilitate research progress.

4

Acetylation

Glutarylation

Profiling lysine acylation in liver peptides from wild type and Sirt5 knockout mice:
Diagram of five acylation PTM types (A). The degree of overlap of sites identified using the four
specified acylation-specific antibodies (B).

Antibody‡
TargetKinomeView®

PTMScan®

Motif

No.

Kit

Service

Kit #9812

Service

Ac-K

#9814

#14499/#13416

4

–

4

–

#261014–

4–#93872

4–

4–#178484–

4–#137644–

4

AcetylScan® – Acetylation Proteomics
Acetyl-Lysine

GlutarylScanTM – Glutarylation Proteomics
Glutaryl-Lysine

Glut-K

MalonylScanTM – Malonylation Proteomics
Malonyl-Lysine

Mal-K

PropionylScanTM – Propionylation Proteomics
Propionyl-Lysine

Prop-K

SuccinylScanTM – Succinylation Proteomics
Succinyl-Lysine

Succ-K

Select AcylScanTM Publications:
Svinkina, T. et al. (2015) Deep, quantitative coverage of thelysine acetylome using novel anti-acetyl-lysine antibodies andan optimized proteomic workflow. Mol. Cell Proteomics 14(9),2429–2440.
Bouchut, A. et al. (2015) Proteome-wide lysine acetylation in cortical astrocytes and alterations that occur during Infection withbrain parasite toxoplasma gondii. PLoS ONE 10(3), e0117966.

Ryder, D.J. et al. (2015) Identification of the acetylation andubiquitin-modified proteome during the progression of skeletal
muscle atrophy. PLoS ONE 10(8), e0136247.

Mielcarek, M. et al. (2013) HDAC4 Does Not Act as a Protein
Deacetylase in the Postnatal Murine Brain In Vivo. PLoS One
8(11) e80849.

Rardin , M.J. et al. (2013) SIRT5 regulates the mitochondrial
lysine succinylome and metabolic networks. Cell Metab. 18(6),920–933.

Jeffers, V. et al (2012) Lysine acetylation is widespread onproteins of diverse function and localization in the protozoan
parasite Toxoplasma gondii. Eukaryot. Cell 11(6) 735–742.

‡

Validated for use in other applications.

www.cellsignal.com/Proteomics 10

PTMScan® Discovery Products and Services (cont.)
Caspase Cleavage Substrate Proteomics
The intrinsic and extrinsic apoptotic pathways involve a cascade of caspases. The humanproteome contains thousands of known or putative caspase cleavage sites. The majority of
caspase substrates are cleaved at an aspartic acid residue, generating fragments containing
a carboxy-terminal aspartate with a general DEXD motif.
PTMScan® technology for caspase cleavage substrate proteomics uses an antibody to the DEXD
motif to enrich even low abundance cleaved caspase substrate peptides from trypsin digested
samples prior to LC-MS/MS analysis.

Features and Benefits

Caspase Cleavage Substrate Proteomics: The motif logo was generated from a
PTMScan® LC-MS/MS experiment using 1044 nonredundant tryptic peptides with carboxyterminal aspartates derived from HeLa cells treated with Staurosporine #9953 (1 μM, 3 hr)
to induce apoptosis. Peptides were enriched using the PTMScan® Cleaved Caspase
Substrate Motif [DE(T/S/A)D] Kit #12810. The logo represents the relative frequency ofamino acids in each position leading up to the carboxy-terminal aspartate.

• The proprietary cleaved caspase substrate motif antibody is a unique and sensitive toolfor quantitative profiling of hundreds to thousands of caspase cleaved substrates.
• P TMScan technology can be applied to many biological systems and species toencompass diverse research interests.

• Experienced CST scientists provide technical support throughout the PTMScan
workflow to facilitate research progress.

Antibody‡

Caspase Cleavage Substrate Proteomics‡

KinomeView®

PTMScan®

Target

Motif

No.

Kit

Service

Kit #9812

Service

Caspase Cleavage Substrate[DE(T/S/A)D]#8698#128104–

4

Validated for use in other applications.

Select Caspase Cleavage Substrate Publications:
Anania, V.G. and Lill, J.R. (2015) Proteomic tools for thecharacterization of cell death mechanisms in drug discovery.
PROTEOMICS - Clinical Applications 9(7-8) 671–683.

Pham, V.C. et al. (2012) Complementary proteomic tools forthe dissection of apoptotic proteolysis events. J. Proteome Res.
11(5), 2947–2954.

PTMScan® Companion Products

AQUA™ peptides as identity
standards for post-translational modifications. Upper

To support your proteomics experiments, we offer the following fully validated companion reagents:

trace, extracted ion chromatogram for tryptic peptides fromthe activation loop of Met protein
kinase, corresponding to phosphorylation of Tyr-1234 (earlier
eluting peak) and Tyr-1235(later eluting peak). Lower trace,
extracted ion chromatogram for
the Tyr-1234 AQUA peptide.
Co-elution confirms the earlier
eluting peak is the peptide phosphorylated at Tyr-1234.

• Mouse Control Peptides for ensuring run-to-run performance of the LC-MS/MS system.
• Immunoaffinity Purification (IAP) Buffer for reconstitution of lyophilized peptides prior
to immunoaffinity purification.

• AQUA™ Peptides* for validating and quantifying protein markers and post-translational
modifications.

PTMScan® Companion Products
Product

No.

PTMScan Trypsin Digested Control Peptides I#12219PTMScan® Lys-C Digested Control Peptides I#12148PTMScan IAP Buffer (10X)

#9993

®

®

DMYDKE Y YSVHNK
1234

DMYD K E Y YSV H N K
1235*This method was developed by scientists at Harvard Medical School. Limited use of this method is permitted under a licensing arrangement with Harvard College.

For more information about AQUA peptides and other companion products visit: www.cellsignal.com/PTMScancompanion

11

For Research Use Only. Not For Use in Diagnostic Procedures.

PTMScan® Direct Services

Mass Spectrometry-based Antibody Arrays

PTMScan® Direct Services
CST offers six PTMScan® Direct services thatcan be used to investigate changes in PTMs of
specific or known protein targets in response
to a drug treatment or disease state.

Number of unique PTM sites*

Number of Proteins

PTMScan Direct Multi-Pathway

1,006

409

PTMScan® Direct PI3K / Akt Signaling

296

105

PTMScan Direct Apoptosis & Autophagy

175

100

PTMScan® Direct Cell Cycle & DNA Damage

263

168

PTMScan Direct Ser/Thr Kinases

385

130

PTMScan® Direct Tyrosine Kinases

671

120

®

®

®

* F or a full list of targets in each PTMScan Direct reagent visit: www.cellsignal.com/PTMScanDirect.

PTMScan® Direct Multi-Pathway

BCAP
Syk

PTEN

PP2A

Lyn

Gα
Gβγ GTPPHLPP1/2 CTMPNEDD1-4

TBK1

Membrane PDK1
Recruitment
and Activation

p70 S6K

PDK1

Membrane
Recruitment
and Activation

Neutrophil
Respiratory
Burst

p47phoxOrganizationof Nuclear
Proteins
NF-κB
PathwayLamin A
Tpl2

PDCD4

mTORC1

4EBP1

Inhibits
Autophagy

ATG13

eNOS

XIAP

Bim

Ataxin-1

Bad
14-3-3p53

Glycolysis
PIP5K AS160

PalladinGirdinApoptosis
CytosolicSequestration*The PI3 Kinase/Akt Signaling pathway is just one
pathway covered by the PTMScan Direct Multipathway reagent.

mTORC1GβL

RaptormTOR

DEPTOR

Skp2 SCF
Myt1
Wee1

Migration
Mig
rati
on

GS

p27 Kip1

ATP-Citrate
Lyase

p21
Waf1/Cip1
Cyclin D1

CDK2 Cyclin A

Aggregation
Neurodegeneration

PFKFB2

GSK-3

Bcl-2
Bax

Synaptic
Signaling

BlocksAggregation
Promotes
Neuroprotection

Huntingtin

MDM2

FoxO1

CardiovascularHomeostasis

GABAAR

Akt

PRAS40Inhibits Apoptosis

NOTE: Highlighted blue nodes
designate proteins within the
PI3K/Akt pathway with peptides
targeted by the PTMScan®
Direct Multi-Pathway Service.

IKKα

TSC2
TSC1

Other

PI3K

PI3K

mTORC2 PIP3

GPCR

mIg

science

TRAF6

Akt

PTEN

PIP3

S6

Jak1

DNA-PKα/β α/β

Neuro

ILK

DNA Damage

IRS-1

PI3K

Gab1

Gab2

PI3K

Paxillin

FAK

mIg

ATM/ATRAg

CD19

Integrin

Protein Synthesis

Adherens Junction Dynamics
AMPK Signaling
B Cell Receptor Signaling
Cell Cycle Control: G1/S Checkpoint
Cell Cycle Control: G2/M DNA Damage Checkpoint
ErbB/HER Signaling
G Protein-coupled Receptor Signaling to MAPK/Erk
Insulin Receptor Signaling
Jak/Stat Signaling: IL-6 Receptor Family
MAPK Signaling Cascades
MAPK/Erk in Growth and Differentiation
NF-κB Signaling
PI3 Kinase/Akt Signaling*
Regulation of Actin Dynamics
Regulation of Microtubule Dynamics
SAPK/JNK Signaling Cascades
Signaling Pathways Activating p38 MAPK
T cell Receptor Signaling
Toll-like Receptor Signaling

BCR

Cytokine
Receptor

RTK

PI3K

PTMScan® Direct Multi-Pathway Service quantitatively measuresa defined set of peptides spanning a large number of signaling
pathways, including:

Hexokinase II
GlucoseTransport

Fatty Acid
Synthesis
Glycogen
Synthesis

mTORC2
Sin1

Cell Cycle
Proliferation

PRR5

RictormTOR

GβL

DEPTOR

Select PTMScan® Direct Publications:
Stokes, M.P. et al. (2013) Quantitative profiling of DNA damage
and apoptotic pathways in UV damaged cells using PTMScan
Direct. Int. J. Mol. Sci. 14(1), 286–307.

Pease, B.N. etal. (2013) Global Analysis of Protein Expression and Phosphorylation of Three Stages of Plasmodium
falciparum Intraerythrocytic Development. J. Proteome Res.
2(9):4028–4045.

Stokes, M.P. et al. (2012) PTMScan Direct: identification andquantification of peptides from critical signaling proteins byimmunoaffinity enrichment coupled with LC-MS/MS. Mol. Cell
Proteomics 11(5), 187–201.

Post-PTMScan® Validation Studies
CST products to follow-up on candidates identified in PTMScan® Discovery or PTMScan® Direct include:• Modification state-specific and total protein antibodies produced and validated in-house for multiple applications• SignalSilence® siRNA for knockdown of specific human or mouse proteins• PathScan® Sandwich ELISA Kits and Antibody Pairs for analysis of a large selection of intracellular molecules• AQUATM peptides for multiple reaction monitoring (MRM) or immuno-MRM mass spectrometry assays

www.cellsignal.com/Proteomics 12

From Bench to Bedside
PTMScan® Technology in Translational Research
PTMScan® Discovery workflow for phospho-tyrosine modifications altered in NSCLCCST performed a global survey of tyrosine kinase activity in non-small cell lung cancer (NSCLC) to identify novel disease drivers (1). The PTMScan® PhosphoTyrosine Mouse mAb (P-Tyr-100) kit was used to enrich phosphorylated peptides from 41 NSCLC cell lines and 150 NSCLC tumors. This analysis identified
4,551 phospho-tyrosine residues on more than 2,700 proteins. The tyrosine kinase ALK (anaplastic lymphoma kinase) was among the top 10 candidates forfollow up analysis.

Cell or Tissue Samples

Kinase Family Targeting

Carcinoma-associated fusion proteins discovered as a result ofPTMScan® Technology
Further investigation revealed fusion of the N-terminus of EML4 (echinoderm
microtubule-associated protein-like 4) with the C-terminus of ALK in some
NSCLC cell lines and tumors. Additional studies showed that 3-7% of NSCLC
patients express the fusion protein in their tumors, suggesting that it is highly
oncogenic (1-4). Cancer cells expressing the EML4-ALK fusion protein are
sensitive to the small molecule ALK inhibitor crizotinib and in 2011 the FDA
approved crizotinib for the treatment of ALK positive NSCLC (5).

Immunoprecipitation
Using Motif Antibody

LC-MS/MS and
Bioinformatic Analysis

ALK fusion protein detection in human lung carcinoma
CST developed a highly specific and sensitive antibody, ALK (D5F3®) XP®
Rabbit mAb #3633, which detects full length ALK and the EML4-ALK fusion
protein. The FDA approved an immunohistochemistry (IHC) companion
diagnostic test that uses the ALK D5F3 clone licensed from CST (6). This
will help physicians determine which NSCLC patients are candidates for
crizotinib treatment.

ALK (D5F3®) XP® Rabbit mAb #3633: IHC analysis of paraffin-embedded human lungcarcinoma with high (left) and low levels (right) of ALK fusion protein expression using #3633.

References:
1. Rikova, K. et al (2007) Cell 131(6), 1190–203.
2. Soda, M., et al (2007) Nature 448(7153), 561–6.
3. Koivunen, J.P., et al (2008) Clin. Cancer Res.14(13), 4275–83.
4. Shaw, A.T., et al (2009) J. Clin. Oncol. 27(26), 4247–53.
5. Press Release: http://www.fda.gov/NewsEvents/Newsroom/PressAnnouncements/ucm269856.htm
6. Press Release: http://www.ventana.com/site/page?view=press-release-jan21-2015

13
9

For Research Use Only. Not For Use in Diagnostic Procedures.

www.cellsignal.com/SimplifyingProteomics
www.cellsignal.com/Proteomics

Support for Drug Discovery
Integrated Services and Tools
In addition to proteomic services and kits, CST offers products for a suite ofintegrated applications to support different stages of the drug discovery anddevelopment pipeline, and to enable diagnostic development.

CST Services and Tools

Proteomic Analysis» Post-Translational Modifications Analysis» Complete Service & Kit Portfolios

Immunofluorescence (ICC, IHC)

» Cellular/Tissue Biology Systems Analysis» Single/Multiplex Marker Analysis

Flow Cytometry» Cellular, Single/Multiplex Analysis

ELISA Assays» Low to High Throughput Screen

Antibody Arrays» Fast to Medium Throughput Profiling

ChIP Analysis/Sequencing» Gene Analysis

siRNA Portfolio» Gene Analysis

Product Customization» Development/Labeling/Formulation/Scale
Production

TargetIdentification
Validation

Hit/Lead
Discovery

Efficacy/Tox
Biomarkers

•

•

•

•

•

•

•

•

•

•

•

•

•

•

•

•

•

•

Technical Support
At CST, providing exceptionalcustomer service and technicalsupport are top priorities. Ourscientists work at the benchdaily to produce and validate
our antibodies, so they havehands-on experience and in-depth
knowledge of each antibody’s
performance. In the process, thesesame scientists generate valuablereference information that they
use to answer your questions andhelp troubleshoot your experimentby phone or email.
USA & Europe:www.cellsignal.com/support
China:
www.cst-c.com.cn/support
Japan:
www.cstj.co.jp/support© 2016 Cell Signaling Technology, Inc.
Cell Signaling Technology, CST, AcetylScan,
GlutarylScan, KinomeView, MalonoylScan,
MethylScan, MultiMab, PathScan, PhosphoScan,
PropionylScan PTMScan, SignalSilence,
SuccinylScan, UbiScan, XP, and D5F3 aretrademarks of Cell Signaling Technology, Inc.
All other trademarks are the property of theirrespective owners.

•
•

ist,
Troy, Product Sciente 2010.
has been with CST sinc•Printed on recycled paper (25% post-consumer
waste fiber) using vegetable inks and processed
chlorine free.

FPO
15
14

Cell Signaling Technology (CST) is a private, family-owned
company, founded by scientists and dedicated to providing highquality research tools to the biomedical research community.
Our employees operate worldwide from our U.S. headquartersin Massachusetts and our offices in the Netherlands, China,
and Japan.

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COVER IMAGE: Cellular Landscape: Vesicle Trafficking www.cellsignal.com/VesicleTrafficking.

16BROPTMS0135ENG

For Research Use Only. Not For Use in Diagnostic Procedures.