Absolute Quantification of Endogenous Ras Isoform Abundance
Craig J Mageean1, John R Griffiths2, Duncan L Smith2
1Division of Cellular and Molecular Physiology, Institute of Translational Medicine, University of Liverpool, Liverpool, L69 3BX, United Kingdom.
Abstract:
Ras proteins are important signalling hubs situated near the top of networks controlling cell proliferation, differentiation and survival. Three almost identical isoforms, HRAS, KRAS and NRAS, are ubiquitously expressed yet have differing biological and oncogenic properties. In order to help understand the relative biological contributions of each isoform we have optimised a quantitative proteomics method for accurately measuring Ras isoform protein copy number per cell. The use of isotopic protein standards together with selected reaction monitoring for diagnostic peptides is sensitive, robust and suitable for application to sub-milligram quantities of lysates. We find that in a panel of isogenic SW48 colorectal cancer cells, endogenous Ras proteins are highly abundant with ≥260,000 total Ras protein copies per cell and the rank order of isoform abundance is KRAS>NRAS≥HRAS. A subset of oncogenic KRAS mutants exhibit increased total cellular Ras abundance and altered the ratio of mutant versus wild type KRAS protein. These data and methodology are significant because Ras protein copy number is required to parameterise models of signalling networks and informs interpretation of isoform-specific Ras functional data.
Insights
Researchers developed a quantitative proteomics method to measure Ras protein isoforms in cells. They found KRAS is the most abundant isoform in colorectal cancer cells, with oncogenic mutants altering Ras protein levels.
Area of Science:
- Molecular Biology
- Cellular Signaling
- Proteomics
Background:
- Ras proteins are key regulators of cell proliferation, differentiation, and survival.
- Three highly similar isoforms (HRAS, KRAS, NRAS) exist with distinct biological and oncogenic roles.
Purpose of the Study:
- To optimize a quantitative proteomics method for precise measurement of Ras isoform protein copy number per cell.
- To understand the relative biological contributions of each Ras isoform.
Main Methods:
- Utilized isotopic protein standards and selected reaction monitoring (SRM) for diagnostic peptides.
- Applied the method to sub-milligram quantities of cell lysates.
- Analyzed Ras isoform abundance in isogenic SW48 colorectal cancer cells.
Main Results:
- Endogenous Ras proteins are highly abundant (≥260,000 copies/cell) in SW48 cells.
- The rank order of isoform abundance is KRAS > NRAS ≥ HRAS.
- Oncogenic KRAS mutants showed increased total Ras abundance and altered mutant/wild-type ratios.
Conclusions:
- The developed proteomics method is sensitive, robust, and suitable for quantifying Ras isoforms.
- Ras protein copy number is crucial for modeling signaling networks and interpreting functional data.
- Findings provide essential quantitative data for understanding Ras isoform-specific functions in cancer.
More Related Videos
08:24Nitrogen Cavitation and Differential Centrifugation Allows for Monitoring the Distribution of Peripheral Membrane Proteins in Cultured Cells
Published on: August 18, 2017
09:04Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification
Published on: August 17, 2015
