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.
Highlights
Ras proteins are small monomeric G-proteins that control key signalling pathways emanating from cell surface receptors
We have successfully used protein standard absolute quantitation (PSAQ) selected reaction monitoring (SRM) analysis to accurately quantify the endogenous abundance of Ras isoforms in cells
Whilst label free approaches provide an estimate of protein copy number, isotopic labelling strategies allow precise comparisons with a protein or peptide standard
Summary
Ras proteins are small monomeric G-proteins that control key signalling pathways emanating from cell surface receptors. Mutations at codons 12, 13 or 61 favour prolonged GTP binding and aberrant activation of the Ras signalling network. These mutations are present in approximately 30% of all tumours screened and oncogenic mutations and/or amplification of members of the Ras pathways are present in almost all cancers [1, 2]. Three Ras genes encode four isoforms (HRAS, KRAS4A, KRAS4B and NRAS) that are ubiquitously expressed and almost identical, but not functionally redundant [3]. Isoform-specific protein functions may mediate the particular oncogenic potency of KRAS [6, 7]. Understanding the absolute abundance of Ras isoforms and their mutated alleles is required for critical assessment of these models
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