Hood, Fiona E, 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.
Text
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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 |
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Uncontrolled Keywords: | GTPase, Protein abundance, Proteomics, PSAQ, RAS |
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: | 07 Mar 2023 15:38 |
DOI: | 10.1007/978-1-0716-1190-6_4 |
Related URLs: | |
URI: | https://livrepository.liverpool.ac.uk/id/eprint/3123542 |