GeLC-MRM quantitation of mutant KRAS oncoprotein in complex biological samples

J Proteome Res. 2012 Jul 6;11(7):3908-13. doi: 10.1021/pr300161j. Epub 2012 Jun 26.


Tumor-derived mutant KRAS (v-Ki-ras-2 Kirsten rat sarcoma viral oncogene) oncoprotein is a critical driver of cancer phenotypes and a potential biomarker for many epithelial cancers. Targeted mass spectrometry analysis by multiple reaction monitoring (MRM) enables selective detection and quantitation of wild-type and mutant KRAS proteins in complex biological samples. A recently described immunoprecipitation approach (Proc. Nat. Acad. Sci.2011, 108, 2444-2449) can be used to enrich KRAS for MRM analysis, but requires large protein inputs (2-4 mg). Here, we describe sodium dodecyl sulfate-polyacrylamide gel electrophoresis-based enrichment of KRAS in a low molecular weight (20-25 kDa) protein fraction prior to MRM analysis (GeLC-MRM). This approach reduces background proteome complexity, thus, allowing mutant KRAS to be reliably quantified in low protein inputs (5-50 μg). GeLC-MRM detected KRAS mutant variants (G12D, G13D, G12V, G12S) in a panel of cancer cell lines. GeLC-MRM analysis of wild-type and mutant was linear with respect to protein input and showed low variability across process replicates (CV = 14%). Concomitant analysis of a peptide from the highly similar HRAS and NRAS proteins enabled correction of KRAS-targeted measurements for contributions from these other proteins. KRAS peptides were also quantified in fluid from benign pancreatic cysts and pancreatic cancers at concentrations from 0.08 to 1.1 fmol/μg protein. GeLC-MRM provides a robust, sensitive approach to quantitation of mutant proteins in complex biological samples.

Publication types

  • Research Support, N.I.H., Extramural

MeSH terms

  • Cell Line, Tumor
  • Electrophoresis, Polyacrylamide Gel
  • Humans
  • Mutant Proteins / chemistry
  • Mutant Proteins / genetics
  • Mutant Proteins / isolation & purification
  • Mutant Proteins / metabolism*
  • Pancreatic Cyst / metabolism*
  • Peptide Fragments / chemistry
  • Proteolysis
  • Proto-Oncogene Proteins / chemistry
  • Proto-Oncogene Proteins / genetics
  • Proto-Oncogene Proteins / isolation & purification
  • Proto-Oncogene Proteins / metabolism*
  • Proto-Oncogene Proteins p21(ras)
  • Reference Standards
  • Tandem Mass Spectrometry / standards
  • ras Proteins / chemistry
  • ras Proteins / genetics
  • ras Proteins / isolation & purification
  • ras Proteins / metabolism*


  • KRAS protein, human
  • Mutant Proteins
  • Peptide Fragments
  • Proto-Oncogene Proteins
  • Proto-Oncogene Proteins p21(ras)
  • ras Proteins