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

Prior 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 end...

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Published inMethods in molecular biology (Clifton, N.J.) Vol. 2797; p. 299
Main Authors D'Ippolito, Robert A, Scheidemantle, Grace M, Smith, Brian P, Powell, Katie, Eury, Scott, Neish, Abigail, Mehalko, Jennifer, Beaumont, Lauren, Fer, Nicole, Wall, Vanessa, Burgan, William, Maciag, Anna E, Esposito, Dominic, DeHart, Caroline J
Format Journal Article
LanguageEnglish
Published United States 2024
Subjects
Chromatography, Liquid
Liquid Chromatography-Mass Spectrometry
Protein Processing, Post-Translational
Proteomics - methods
Tandem Mass Spectrometry - methods
Top-down mass spectrometry
Proteoform
Immunoprecipitation
Posttranslational modifications
Farnesylation
FLAG tag
KRAS4B
Geranylgeranylation
Hydroxyfarnesylation
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Summary:Prior 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.
ISSN:1940-6029
DOI:10.1007/978-1-0716-3822-4_22