GeLC-MRM quantitation of mutant KRAS oncoprotein in complex biological samples.
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.
Department of Biochemistry, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-6350, USA.
Patrick J
PJ
Halvey
Cristina R
CR
Ferrone
Daniel C
DC
Liebler
D045744:Cell Line, Tumor; D004591:Electrophoresis, Polyacrylamide Gel; D006801:Humans; D050505:Mutant Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D010181:Pancreatic Cyst / Q000378:metabolism*; D010446:Peptide Fragments / Q000737:chemistry; D059748:Proteolysis; D011518:Proto-Oncogene Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*; D016283:Proto-Oncogene Proteins p21(ras); D012015:Reference Standards; D053719:Tandem Mass Spectrometry / Q000592:standards; D018631:ras Proteins / Q000737:chemistry / Q000235:genetics / Q000302:isolation & purification / Q000378:metabolism*
C117307:KRAS protein, human; D050505:Mutant Proteins; D010446:Peptide Fragments; D011518:Proto-Oncogene Proteins; D016283:Proto-Oncogene Proteins p21(ras); D018631:ras Proteins