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Quantitative Assays for RAS Pathway Proteins and Phosphorylation States

December 1, 2014, by Jim Hartley and Gordon Whiteley

In cooperation with the RAS Initiative, the NCI's Clinical Proteomic Tumor Analysis Consortium (CPTAC) has launched a project to develop quantitative assays for proteins and phosphopeptides involved in RAS signaling. Within the next 1-2 years these assays should allow the amounts and phosphorylation states of tens of RAS and RAS-related proteins to be determined in tumor samples, cell lines, or cancer models in a single run.

The initial target list for the CPTAC - RAS project is shown below.  We invite your input on the best targets to improve our understanding of how the RAS pathway operates. Please submit a comment or send an email to SolveRAS@nih.gov.

Quantitative Mass Spectrometry of Peptides using Immuno-MRM.

Quantitative Mass Spectrometry of Peptides using Immuno-MRM.

The capability to use mass spectrometry to determine the absolute amounts of peptides (and thus, proteins) is the culmination of years of developing concepts and technologies. First, pre-selected peptides derived from protein targets by trypsin digestion can be detected within a complex mixture using a targeted mass spectrometric technique called multiple reaction monitoring (MRM)1. Second, the quantities of these peptides in the mixture can be determined by spiking the samples with synthetic peptides of exactly the same amino acid sequences (and post-translational modifications, if appropriate) made distinguishable by the incorporation of heavy isotopes2.  MRM assays can be developed to establish linear response curves, repeatability, lower limit of quantitation, etc. Third, in cases where protein targets are of low abundance, antibodies raised against their tryptic peptides can enrich for the target peptides and their spiked synthetic counterparts prior to MRM measurement, a process called immuno-MRM3. Fourth, both MRM and immuno-MRM assays can be highly multiplexed so that many peptide and protein targets can be determined from a single analytical run4.  Finally, an extensive program of interlaboratory testing has established good reproducibility of these new techniques5.

The CPTAC program recently awarded contracts to the Fred Hutchinson Cancer Research Center (Dr. Amanda Paulovich), the Moffitt Cancer Center (Dr. John Koomen), and the Broad Institute (Dr. Steven Carr) to develop assays for the RAS pathway targets. The target list is being refined by comparing desired target peptides to databases of peptides that have already been detected in global proteomic screens. Look for updated information on progress here at www.cancer.gov/rascentral.

Top 100 RAS Pathway Targets for Immuno-MRM
Target Protein Specific Target Phosporylation Site Status
Akt1 Total   Downstream
Akt1 P S473 Downstream
Akt1 P T308 Downstream
Akt2 Total   Downstream
Akt3 Total   Downstream
Akt3 P S485 Downstream
E-Cadherin Total   Downstream
N-Cadherin Total   Downstream
b-catenin P T41/S45 Downstream
b-catenin P S552 Downstream
b-catenin non-P (S33/S37/T41) Downstream
PTEN Total   Downstream
PTEN P S380 Downstream
ARAF Total   Effector
ARAF P S299 Effector
BRAF Total   Effector
BRAF P T401 Effector
BRAF P S467 Effector
BRAF P S465 Effector
BRAF P S445 Effector
CRAF Total   Effector
CRAF P S259 Effector
CRAF P S359 Effector
CRAF P S338 Effector
MEK1 Total   Downstream
MEK1 P S298 Downstream
MEK1 P T386 Downstream
MEK2 Total   Downstream
MEK2 P S221 Downstream
mTOR Total   Downstream
mTOR P S2448 Downstream
mTOR P S2481 Downstream
GSK-b Total   Downstream
GSK-b P S9 Downstream
ERK1/2 Total   Downstream
ERK1/2 P T202/Y204 Downstream
c-Fos Total   Downstream
EGFR Total   Upstream
EGFR P S1046/1047 Upstream
EGFR P Y1045 Upstream
EGFR P Y1068 Upstream
EGFR P Y1086 Upstream
EGFR P Y1148 Upstream
EGFR P Y1173 Upstream
EGFR P Y845 Upstream
EGFR P Y992 Upstream
ErbB2 Total   Upstream
ErbB2 P Y1248 Upstream
ErbB2 P Y877 Upstream
ErbB3 Total   Upstream
ErbB3 P Y1289 Upstream
ErbB4 Total   Upstream
ErbB4 P Y984 Upstream
ErbB4 P Y1284 Upstream
Cyclin D1 Total   Downstream
Cyclin D1 P T286 Downstream
Raptor Total   Downstream
Raptor P S792 Downstream
RasGRF1 Total   Regulator
RasGRF1 P S916 Regulator
Rictor Total   Downstream
IRS-1 Total   Upstream
IRS-1 P S612 Upstream
SPRED1 Total   Regulator
SPRED2 Total   Regulator
SPRED3 Total   Regulator
Sprouty1 Total   Regulator
Sprouty2 Total   Regulator
Sprouty3 Total   Regulator
Sprouty4 Total   Regulator
NF1 Total   Regulator
SYNGAP Total   Regulator
p120GAP Total   Regulator
RASGRP Total   Regulator
RASGRF2 Total   Regulator
Sos1 Total   Regulator
H-Ras Total   RAS
K-Ras 4A Total   RAS
K-Ras 4B Total   RAS
N-Ras Total   RAS
K-Ras 4B G12A Total   RAS
K-Ras 4B G12C Total   RAS
K-Ras 4B G12D Total   RAS
K-Ras 4B G12V Total   RAS
K-Ras 4B Q61L Total   RAS
K-Ras 4B Q61R Total   RAS
K-Ras 4B P S181 RAS
Calmodulin Total   Effector
CaM kinase Total   Downstream
CaM kinase P S286 Downstream
KSR1 Total   Downstream
KSR2 Total   Downstream
IQGAP Total   Regulator
DUSP1 Total   Downstream
DUSP2 Total   Downstream
DUSP4 Total   Downstream
DUSP6 Total   Downstream
DUSP7 Total   Downstream
The RAS pathway target list comprises nearly 100 target peptides from 52 different proteins. Column A contains the names of the target proteins. Column B describes the kind of assay, either "Total" (measures the total amount of protein, using any convenient peptide), "P" (indicating that a specific phosphopeptide will be determined), or "non-P" (specifying a beta-catenin peptide important for regulating degradation). Column C gives the specific amino acid that is phosphorylated for the "P" targets in column B (for example, S473 for AKT1). Column D describes the class of each target, whether "upstream", "downstream", "effector", "regulator", or "RAS" itself. For some proteins several different assays for specific phosphopeptides will be developed (for example, assays for total BRAF protein and and BRAF phosphorylated at T401, S445, S465, and S467).The target list includes 11 RAS proteins: HRAS, NRAS, KRAS-4a, KRAS-4b, and six mutants of KRAS-4b.

Selected References

  1. Boja, ES, Rodriguez, H. Mass spectrometry-based targeted quantitative proteomics: achieving sensitive and reproducible detection of proteins. Proteomics. 2012; 12(8): 1093-110.
  2. Hoofnagle, AN, et al. Multiple-reaction monitoring-mass spectrometric assays can accurately measure the relative protein abundance in complex mixtures. Clin Chem. 2012; 58(4): 777-781.
  3. Kuhn, E, et al. Interlaboratory evaluation of automated, multiplexed peptide immunoaffinity enrichment coupled to multiple reaction monitoring mass spectrometry for quantifying proteins in plasma. Mol Cell Proteomics. 2012; 11(6): M111.013854.
  4. Whiteaker JR, et al. Sequential multiplexed analyte quantification using peptide immunoaffinity enrichment coupled to mass spectrometry. Mol Cell Proteomics. 2012; 11(6): M111.015347.
  5. Kennedy, JJ, et al., Nat Methods 11, 149, 2014, doi: 10.1038/nmeth.2763.
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