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