CST Guide:

Section III: Workflow Tools
chapter 19: Discovery
How do I select the most relevant PTM to study
The first step to discovery of novel PTM alterations in response to disease states or drug treatments
is selecting the appropriate class of modifications for enrichment. If you are not certain which specific
classes of modifications are altered in your experimental system, you can first perform a series of
western blot analyses employing a panel of motif antibodies that detect a range of different PTMs
(KinomeView ® ).
What type of data can I expect
from PTMScan ® kits and services
The data set generated by a PTMScan service experiment includes not only quantification of PTM
changes, but also the identity of each protein and specific location of each modification site. For ease
of interpretation, the experimental results can be viewed at a global level as a scatter plot, as a detailed
table view (below), or as a domain map for a selected protein. Using the appropriate LC-MS/MS platforms
and informatics processing, the PTMScan kits can be used to generate comparable results.
Identify Key Targets from Thousands of Peptides
The process of data refinement can be illustrated by a simple scenario. A typical PTMScan proteomic
analysis focused on a single class of PTM might identify 1,000–3,000 unique modified peptides. Of
these, perhaps 50–100 might exhibit a clear change in modification that correlates with tissue type or
with the expected biology and pathway associated with the cell treatment employed in the study. With
further WB and/or ELISA experimentation, you can shorten this list of candidate PTM sites to a limited
subset of actionable targets (1,4,6).
Identify 1,000-3,000 unique PTM-containing peptides
Adjust signal:noise cutoff
to optimize results
Representative PTMScan ® data, revealing the protein identity,
site location, and relative abundance for each PTM detected.
Normalized Fold Change
SU11274 vs.
DMSO Control
Staurosporine
vs. DMSO Control
Protein
Name Site -7/+ 7 Peptide Upstream Kinase
-5.0 -4.6 EphA2 897 RVSIRLPsTSGSEGV LPS*T*SGSEGVPFR Akt1
-13.6 -2.1 FOXO1A 319 TFRPRTSsNASTISG TSS*NASTISGR Akt1
-158.0 -7.2 FOXO4 32 QSRPRSCtWPLPRPE SCT*WPLPRPEIANQPSEPPEVEPDLGEK Akt1
-3.4 1.8 QIK 358 DGRQRRPsTIAEQTV RPS*TIAEQTVAK Akt1, Akt2
-13.3 -29.4 S6 235,
236,
240
-7.0 -24.5 S6 236,
240
IAKRRRLsSLRASTS RLS*S*LRAS*TSK Akt1, Akt2,
P70S6KB, PKACA,
PKCA, PKCD
AKRRRLSsLRASTSK RLSS*LRAS*TSK Akt1, Akt2,
P70S6KB, PKACA,
PKCA, PKCD
2.6 1.1 BRAF 365 GQRDRSSsAPNVHIN SSS*APNVHINTIEPVNIDDLIR Akt1, Akt3
-7.0 -9.4 GSK3B 9 SGRPRTTsFAESCKP TTS*FAESCKPVQQPSAFGSMK Akt1, AurA,
CAMK2B, GSK3B,
KHS1, PKACA,
PKCA
-5.3 N.D. GSK3B 9, 21 SGRPRTTsFAESCKP TTS*FAESCKPVQQPS*AFGSMK Akt1, AurA,
CAMK2B, GSK3B,
KHS1, PKACA,
PKCA
-21.3 -3.0 PEA-15 116 KDIIRQPsEEEIIKL DIIRQPS*EEEIIK Akt1, CAMK2A,
CK2A1
-2.1 -2.9 GSK3A 21 SGRARTSsFAEPGGG TSS*FAEPGGGGGGGGGGPGGSASGPGGTGGGK Akt1, CAMK2B,
PKACA, PKCA,
PKCB
-10.3 -1.8 RANBP3 126 VKRERTSsLTQFPPS TSS*LTQFPPSQSEER Akt1, ERK1, RSK2,
p90RSK
2.7 2.5 eIF4B 422 RERSRTGsESSQTGT TGS*ESSQTGTSTTSSR Akt1, p70S6K,
p90RSK
4.8 2.5 eIF4B 422,
425
RERSRTGsESSQTGT TGS*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 the basophilic Akt substrate motif RXRXX(s/t) or RXX(s/t). Relative abundance changes of 2.5-fold or greater
(treated versus control) for phosphorylated peptides are indicated by green (increase) or light red (reduction) highlighting. Incrementally
darker highlighting in this image indicates abundance changes in the ranges of 2.5- to 24.9-fold and of greater than 25-fold, respectively.
Analyze in context of known biology
of treatment or system
Map results using protein
interaction database service
to reveal novel pathways
WB and/or
ELISA validation
Potential
key nodes
(