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.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
Similar content being viewed by others
References
Stephen AG, Esposito D, Bagni RK, McCormick F (2014) Dragging Ras back in the ring. Cancer Cell 25(3):272–281. https://doi.org/10.1016/j.ccr.2014.02.017
Mo SP, Coulson JM, Prior IA (2018) RAS variant signalling. Biochem Soc Trans 46(5):1325–1332. https://doi.org/10.1042/BST20180173
Gentry LR, Martin TD, Reiner DJ, Der CJ (2014) Ral small GTPase signaling and oncogenesis: more than just 15minutes of fame. Biochim Biophys Acta 1843(12):2976–2988. https://doi.org/10.1016/j.bbamcr.2014.09.004
Prior IA, Lewis PD, Mattos C (2012) A comprehensive survey of Ras mutations in cancer. Cancer Res 72(10):2457–2467
Li S, Balmain A, Counter CM (2018) A model for RAS mutation patterns in cancers: finding the sweet spot. Nat Rev Cancer 18(12):767–777. https://doi.org/10.1038/s41568-018-0076-6
Pershing NL, Lampson BL, Belsky JA, Kaltenbrun E, MacAlpine DM, Counter CM (2015) Rare codons capacitate Kras-driven de novo tumorigenesis. J Clin Invest 125(1):222–233. https://doi.org/10.1172/JCI77627
Brun V, Masselon C, Garin J, Dupuis A (2009) Isotope dilution strategies for absolute quantitative proteomics. J Proteome 72(5):740–749. https://doi.org/10.1016/j.jprot.2009.03.007
Ankney JA, Muneer A, Chen X (2018) Relative and absolute quantitation in mass spectrometry-based proteomics. Annu Rev Anal Chem (Palo Alto, Calif) 11(1):49–77. https://doi.org/10.1146/annurev-anchem-061516-045357
Mageean CJ, Griffiths JR, Smith DL, Clague MJ, Prior IA (2015) Absolute quantification of endogenous Ras isoform abundance. PLoS One 10(11):e0142674. https://doi.org/10.1371/journal.pone.0142674
Lebert D, Dupuis A, Garin J, Bruley C, Brun V (2011) Production and use of stable isotope-labeled proteins for absolute quantitative proteomics. Methods Mol Biol 753:93–115. https://doi.org/10.1007/978-1-61779-148-2_7
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2021 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
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
Download citation
DOI: https://doi.org/10.1007/978-1-0716-1190-6_4
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-1189-0
Online ISBN: 978-1-0716-1190-6
eBook Packages: Springer Protocols