Absolute Quantitation of GTPase Protein Abundance.

Hood, Fiona E, Sahraoui, Yasmina M, Jenkins, Rosalind E ORCID: 0000-0002-3730-1136 and Prior, Ian ORCID: 0000-0002-4055-5161
(2021) Absolute Quantitation of GTPase Protein Abundance. Methods in molecular biology (Clifton, N.J.), 2262. 65 - 90.

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Ras proteins and other small molecular weight GTPases are molecular switches controlling a wide range of cellular functions. High homology and functional redundancy between closely related family members are commonly observed. Antibody-based methods are commonly used to characterize their protein expression. However, these approaches are typically semi-quantitative, and the requirement to use different antibodies means that this strategy is not suited for comparative analysis of the relative expression of proteins expressed by different genes. We present a mass spectrometry-based method that precisely quantifies the protein copy number per cell of a protein of interest. We provide detailed protocols for the generation of isotopically labeled protein standards, cell/tissue processing, mass-spectrometry optimization, and subsequent utilization for the absolute quantitation of the abundance of a protein of interest. As examples, we provide instructions for the quantification of HRAS, KRAS4A, KRAS4B, NRAS, RALA, and RALB in cell line and tissue-derived samples.

Item Type: Article
Uncontrolled Keywords: Tumor Cells, Cultured, Humans, Neoplasms, ras Proteins, Chromatography, Affinity, Isotope Labeling, Protein Processing, Post-Translational, Mass Spectrometry
Divisions: Faculty of Health and Life Sciences
Faculty of Health and Life Sciences > Institute of Systems, Molecular and Integrative Biology
Depositing User: Symplectic Admin
Date Deposited: 21 May 2021 10:21
Last Modified: 25 Jan 2022 18:30
DOI: 10.1007/978-1-0716-1190-6_4
URI: https://livrepository.liverpool.ac.uk/id/eprint/3123542