| Autor: |
Hood FE; Division of Cellular and Molecular Physiology, Institute of Translational Medicine, University of Liverpool, Liverpool, UK., Sahraoui YM; Division of Cellular and Molecular Physiology, Institute of Translational Medicine, University of Liverpool, Liverpool, UK., Jenkins RE; Centre for Drug Safety Science Bioanalytical Facility, Division of Molecular and Clinical Pharmacology, Institute of Translational Medicine, University of Liverpool, Liverpool, UK., Prior I; Division of Cellular and Molecular Physiology, Institute of Translational Medicine, University of Liverpool, Liverpool, UK. iprior@liv.ac.uk. |
| Jazyk: |
angličtina |
| Zdroj: |
Methods in molecular biology (Clifton, N.J.) [Methods Mol Biol] 2021; Vol. 2262, pp. 65-90. |
| DOI: |
10.1007/978-1-0716-1190-6_4 |
| Abstrakt: |
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. |
| Databáze: |
MEDLINE |
| Externí odkaz: |
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