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Updated: May 21, 2026
Fully Processed Recombinant KRAS4b: Isolating and Characterizing the Farnesylated and Methylated Protein
Published on: January 16, 2020
GeLC-MRM quantitation of mutant KRAS oncoprotein in complex biological samples
Patrick J Halvey1, Cristina R Ferrone, Daniel C Liebler
1Department of Biochemistry, Vanderbilt-Ingram Cancer Center, Vanderbilt University School of Medicine, Nashville, Tennessee 37232-6350, USA.
Abstract:
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.
Insights
A new GeLC-MRM method accurately quantifies mutant KRAS proteins in cancer samples using low protein input. This sensitive approach aids in detecting cancer biomarkers and understanding tumor drivers.
Area of Science:
- Biochemistry
- Proteomics
- Cancer Biology
Background:
- Tumor-derived mutant KRAS oncoprotein drives cancer phenotypes and serves as a biomarker for epithelial cancers.
- Targeted mass spectrometry (MS) using multiple reaction monitoring (MRM) can detect and quantify wild-type and mutant KRAS.
- Existing KRAS enrichment methods require substantial protein input (2-4 mg), limiting their application.
Purpose of the Study:
- To develop a more sensitive method for KRAS protein quantification using lower protein inputs.
- To enable reliable detection of mutant KRAS variants in complex biological samples.
- To improve the accuracy of KRAS measurements by accounting for homologous proteins.
Main Methods:
- Developed GeLC-MRM, a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) based enrichment strategy for KRAS in a low molecular weight fraction.
- Applied GeLC-MRM to analyze protein inputs ranging from 5-50 μg, significantly reducing the required sample amount.
- Incorporated concomitant analysis of HRAS and NRAS peptides to correct KRAS-targeted measurements.
Main Results:
- GeLC-MRM successfully detected KRAS mutant variants (G12D, G13D, G12V, G12S) in cancer cell lines.
- Quantification of wild-type and mutant KRAS showed linearity with protein input and low variability (CV = 14%).
- KRAS peptides were quantified in pancreatic cyst and cancer fluid, with concentrations ranging from 0.08 to 1.1 fmol/μg protein.
Conclusions:
- GeLC-MRM offers a robust and sensitive approach for quantifying mutant KRAS proteins in complex biological samples.
- This method significantly reduces protein input requirements compared to previous techniques.
- GeLC-MRM facilitates the detection of KRAS biomarkers in various cancer types and clinical samples.
