title: Absolute Quantification of Endogenous Ras Isoform Abundance.
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.
authors.affiliation: Division of Cellular and Molecular Physiology, Institute of Translational Medicine, University of Liverpool, Liverpool, L69 3BX, United Kingdom.
authors.forename: Craig J
authors.initials: CJ
authors.lastname: Mageean
authors.affiliation: Cancer Research UK Manchester Institute, University of Manchester, Manchester, M20 4BX, United Kingdom.
authors.forename: John R
authors.initials: JR
authors.lastname: Griffiths
authors.affiliation: Cancer Research UK Manchester Institute, University of Manchester, Manchester, M20 4BX, United Kingdom.
authors.forename: Duncan L
authors.initials: DL
authors.lastname: Smith
authors.affiliation: Division of Cellular and Molecular Physiology, Institute of Translational Medicine, University of Liverpool, Liverpool, L69 3BX, United Kingdom.
authors.forename: Michael J
authors.initials: MJ
authors.lastname: Clague
authors.affiliation: Division of Cellular and Molecular Physiology, Institute of Translational Medicine, University of Liverpool, Liverpool, L69 3BX, United Kingdom.
authors.forename: Ian A
authors.initials: IA
authors.lastname: Prior
mesh_terms: D000596:Amino Acids / Q000737:chemistry; D002454:Cell Differentiation; D045744:Cell Line, Tumor; D049109:Cell Proliferation; D002471:Cell Transformation, Neoplastic / Q000235:genetics; D015179:Colorectal Neoplasms / Q000378:metabolism*; D020558:GTP Phosphohydrolases / Q000737:chemistry*; D015972:Gene Expression Regulation, Neoplastic; D011905:Genes, ras; D006801:Humans; D013058:Mass Spectrometry; D008565:Membrane Proteins / Q000737:chemistry*; D009154:Mutation; D020033:Protein Isoforms / Q000737:chemistry; D040901:Proteomics; D016283:Proto-Oncogene Proteins p21(ras) / Q000737:chemistry*; D015398:Signal Transduction; D053719:Tandem Mass Spectrometry
chemical_list: D000596:Amino Acids; C117307:KRAS protein, human; D008565:Membrane Proteins; D020033:Protein Isoforms; D020558:GTP Phosphohydrolases; C579846:NRAS protein, human; C501009:HRAS protein, human; D016283:Proto-Oncogene Proteins p21(ras)