Enhanced-fluidity, reversed-phase liquid chromatography was developed using custom instrumentation for separation and characterization of intact KRas proteins and tryptic peptides. The KRas, HRas and NRas function as GDP-GTP regulated binary switches in many signalling pathways, and mutations in Ras proteins are frequently found in human cancers and represent poor prognosis markers for patients. Mutations of the KRas isoform constitute some of the most common aberrations among all human cancers and intensive drug discovery efforts have been directed toward targeting the KRas protein. Separation and characterization of the KRas protein and tryptic peptides are helpful for exploring targeting, which has not been fully investigated using liquid chromatography-tandem mass spectrometry. EFLC-MS provided improved chromatographic performance compared to traditional HPLC-MS in terms of shorter analysis time, increased ion intensity and a shift to higher charge states for intact KRas proteins.
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