FLAG-KRAS4B as a Model System for KRAS4B Proteoform and PTM Evaluation by Mass Spectrometry

Methods Mol Biol. 2024;2797:299-322. doi: 10.1007/978-1-0716-3822-4_22.ABSTRACTPrior analysis of intact and modified protein forms (proteoforms) of KRAS4B isolated from cell lines and tumor samples by top-down mass spectrometry revealed the presence of novel posttranslational modifications (PTMs) and potential evidence of context-specific KRAS4B modifications. However, low endogenous proteoform signal resulted in ineffective characterization, making it difficult to visualize less abundant PTMs or perform follow-up PTM validation using standard proteomic workflows. The NCI RAS Initiative has developed a model system, whereby KRAS4B bearing an N-terminal FLAG tag can be stably expressed within a panel of cancer cell lines. Herein, we present a method for combining immunoprecipitation with complementary proteomic methods to directly analyze N-terminally FLAG-tagged KRAS4B proteoforms and PTMs. We provide detailed protocols for FLAG-KRAS4B purification, proteoform analysis by targeted top-down LC-MS/MS, and validation of abundant PTMs by bottom-up LC-MS/MS with example results.PMID:38570469 | DOI:10.1007/978-1-0716-3822-4_22
Source: Mol Biol Cell - Category: Molecular Biology Authors: Source Type: research