Absolute Quantification of Endogenous Ras Isoform Abundance

作者：Mageean Craig J; Griffiths John R; Smith Duncan L; Clague Michael J; Prior Ian A

来源：PLos One, 2015, 10(11): e0142674.

DOI：10.1371/journal.pone.0142674

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.

• 出版日期2015-11-11