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

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.

SUBMITTER: Adams LM

PROVIDER: S-EPMC8543976 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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

Adams Lauren M LM DeHart Caroline J CJ Kelleher Neil L NL

Methods in molecular biology (Clifton, N.J.) 20210101

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 immuno ...[more]

PMID: 33977470

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