Precise Characterization of KRAS4B Proteoforms by Combining Immunoprecipitation with Top-Down Mass Spectrometry
| Autor: | Lauren M. Adams, Neil L. Kelleher, Caroline J. DeHart |
|---|---|
| Rok vydání: | 2021 |
| Předmět: |
Gene isoform
Proteome Immunoprecipitation Peptide Mass spectrometry 01 natural sciences Article Proto-Oncogene Proteins p21(ras) 03 medical and health sciences Tandem Mass Spectrometry Neoplasms Tumor Cells Cultured Humans Protein Isoforms 030304 developmental biology chemistry.chemical_classification 0303 health sciences biology Chemistry 010401 analytical chemistry Intact protein 0104 chemical sciences Characterization (materials science) Biochemistry biology.protein Antibody Cancer cell lines Protein Processing Post-Translational Chromatography Liquid |
| Zdroj: | Methods in Molecular Biology ISBN: 9781071611890 Methods Mol Biol |
| DOI: | 10.1007/978-1-0716-1190-6_3 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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