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Link to original content: http://pubmed.ncbi.nlm.nih.gov/39052226/
Detection and Quantitation of Endogenous Membrane-Bound RAS Proteins and KRAS Mutants in Cancer Cell Lines Using 1D-SDS-PAGE LC-MS2 - PubMed Skip to main page content
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. 2024:2823:269-289.
doi: 10.1007/978-1-0716-3922-1_17.

Detection and Quantitation of Endogenous Membrane-Bound RAS Proteins and KRAS Mutants in Cancer Cell Lines Using 1D-SDS-PAGE LC-MS2

Jan A Kaczmarczyk  1   2 , Gordon R Whiteley  3 , Josip Blonder  4
Affiliations

Detection and Quantitation of Endogenous Membrane-Bound RAS Proteins and KRAS Mutants in Cancer Cell Lines Using 1D-SDS-PAGE LC-MS2

Jan A Kaczmarczyk et al. Methods Mol Biol. 2024.

Abstract

In healthy cells, membrane-anchored wild-type RAS proteins (i.e., HRAS, KRAS4A, KRAS4B, and NRAS) regulate critical cellular processes (e.g., proliferation, differentiation, survival). When mutated, RAS proteins are principal oncogenic drivers in approximately 30% of all human cancers. Among them, KRAS mutants are found in nearly 80% of all patients diagnosed with RAS-driven malignancies and are regarded as high-priority anti-cancer drug targets. Due to the lack of highly qualified/specific RAS isoform and mutant RAS monoclonal antibodies, there is a vital need for an effective antibody-free approach capable of identifying and quantifying membrane-bound RAS proteins in isoform- and mutation-specific manner. Here, we describe the development of a simple antibody-free protocol that relies on ultracentrifugation to isolate the membrane fraction coupled with single-dimensional (1D) sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to fractionate and enrich membrane-bound endogenous RAS isoforms. Next, bottom-up proteomics that utilizes in-gel digestion followed by reversed-phase high-performance liquid chromatography-tandem mass spectrometry (HPLC-MS2) is used for detection and relative quantitation of all wild-type RAS proteins (i.e., HRAS, KRAS4A, KRAS4B, and NRAS) and corresponding RAS mutants (e.g., G12D, G13D, G12S, G12V). Notably, this simple 1D-SDS-PAGE-HPLC-MS2-based protocol can be automated and widely applied to multiple cancer cell lines to investigate concentration changes in membrane-bound endogenous RAS proteins and corresponding mutants in the context of drug discovery.

Keywords: 1D-SDS-PAGE LC; Cancer cell lines; MS2; Membrane; Proteomics; Quantitation; RAS mutants; RAS proteins.

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