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

  1. Author:
    D'Ippolito,Robert
    Scheidemantle,Grace
    Smith,Brian
    Powell,Katie
    Eury,Scott
    Neish, Abigail
    Mehalko,Jennifer
    Beaumont,Lauren
    Fer,Nicole
    Wall,Vanessa
    Burgan,William
    Maciag,Anna
    Esposito,Dom
    Dehart,Caroline

  2. Author Address

    NCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, MD, USA., NCI RAS Initiative, Cancer Research Technology Program, Frederick National Laboratory for Cancer Research, Frederick, MD, USA. caroline.dehart@nih.gov.,
    1. Year: 2024
  2. Journal: Methods in Molecular Biology (Clifton, N.J.)
    1. 2797
    2. Pages: 299-322
  4. Type of Article: Article
  1. Abstract:

    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. © 2024. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.

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External Sources

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  2. DOI: 10.1007/978-1-0716-3822-4_22
  3. PMID: 38570469

Library Notes

  1. Fiscal Year: FY2023-2024
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