Absolute Quantitation of GTPase Protein Abundance

Hood, Fiona E ORCID: 0009-0009-7639-4695, Sahraoui, Yasmina M, Jenkins, Rosalind E and Prior, Ian ORCID: 0000-0002-4055-5161 (2021) Absolute Quantitation of GTPase Protein Abundance RAS ACTIVITY AND SIGNALING, 2262. pp. 65-90. ISSN 1064-3745, 1940-6029

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Abstract

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:	GTPase, Protein abundance, Proteomics, PSAQ, RAS
Divisions:	Faculty of Health & Life Sciences Faculty of Health & Life Sciences > Inst. Systems, Molec & Integrative Biology > Inst. Systems, Molec & Integrative Biology
Depositing User:	Symplectic Admin
Date Deposited:	21 May 2021 10:21
Last Modified:	23 May 2026 05:53
DOI:	10.1007/978-1-0716-1190-6_4
Related Websites:	Author Publisher
URI:	https://livrepository.liverpool.ac.uk/id/eprint/3123542
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