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.
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Hood, F.E., Sahraoui, Y.M., Jenkins, R.E., Prior, I. (2021). Absolute Quantitation of GTPase Protein Abundance. In: Rubio, I., Prior, I. (eds) Ras Activity and Signaling. Methods in Molecular Biology, vol 2262. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1190-6_4
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DOI: https://doi.org/10.1007/978-1-0716-1190-6_4
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Publisher Name: Humana, New York, NY
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Online ISBN: 978-1-0716-1190-6
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