Precise Characterization of KRAS4B Proteoforms by Combining Immunoprecipitation with Top-Down Mass Spectrometry

Lauren M. Adams, Caroline Jane DeHart, Neil L. Kelleher*

*Corresponding author for this work

Research output: Chapter in Book/Report/Conference proceedingChapter

Abstract

The characterization of biologically relevant post-translational modifications (PTMs) on KRAS4B has historically been carried out through methodologies such as immunoblotting with PTM-specific antibodies or peptide-based proteomic methods. While these methods have the potential to identify a given PTM on KRAS4B, they are incapable of characterizing or distinguishing the different molecular forms or proteoforms of KRAS4B from those of related RAS isoforms. We present a method that combines immunoprecipitation of KRAS4B with top-down mass spectrometry (IP-TDMS), thus enabling the precise characterization of intact KRAS4B proteoforms. We provide detailed protocols for the IP, LC-MS/MS, and data analysis comprising a successful IP-TDMS assay in the contexts of cancer cell lines and tissue samples.

Original languageEnglish (US)
Title of host publicationMethods in Molecular Biology
PublisherHumana Press Inc.
Pages47-64
Number of pages18
DOIs
StatePublished - 2021

Publication series

NameMethods in Molecular Biology
Volume2262
ISSN (Print)1064-3745
ISSN (Electronic)1940-6029

Keywords

  • Immunoprecipitation
  • Intact protein
  • Isoform
  • Proteoform
  • RAS
  • Top-down mass spectrometry

ASJC Scopus subject areas

  • Molecular Biology
  • Genetics

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