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Ras Signaling pp 75–96Cite as

Biophysical and Proteomic Characterization Strategies for Cysteine Modifications in Ras GTPases

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Part of the book series: Methods in Molecular Biology ((MIMB,volume 1120))

Abstract

Cysteine is one of the most reactive amino acids and is modified by a number of oxidants. The reactivity of cysteines is dependent on the thiol pK a; however, measuring cysteine pK a values is nontrivial. Ras family GTPases have been shown to contain a free cysteine that is sensitive to oxidation, and free radical-mediated oxidation of this cysteine has been shown to be activating. Here, we present a new technique that allows for measuring cysteine pK a values using a fluorescent detection system with the molecule 4-fluoro-7-aminosulfonylbenzofurazan (ABD-F). In addition, we also describe how to generate several oxidants. Lastly, we describe several mass spectrometry-based experiments and the necessary adjustments to the experiments to detect cysteine oxidation.

An erratum to this chapter is available at http://dx.doi.org/10.1007/978-1-62703-791-4_26

An erratum to this chapter can be found at http://dx.doi.org/10.1007/978-1-62703-791-4_26

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Acknowledgements

We would like to thank Dan Isom for his technical insights. The research efforts described herein were supported by NIH RO1GM75431 and RO1CA089614 to SLC, and GAH was partially funded by the Program in Molecular and Cellular Biophysics (NIH T32GM008570).

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Authors and Affiliations

  1. Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    G. Aaron Hobbs & Sharon L. Campbell

  2. Program in Molecular Biology and Biotechnology, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Harsha P. Gunawardena

  3. Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA

    Sharon L. Campbell

Authors
  1. G. Aaron Hobbs
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  2. Harsha P. Gunawardena
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  3. Sharon L. Campbell
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Editors and Affiliations

  1. University of Siena, Italy

    Lorenza Trabalzini

  2. University of Torino, Italy

    Saverio Francesco Retta

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Hobbs, G.A., Gunawardena, H.P., Campbell, S.L. (2014). Biophysical and Proteomic Characterization Strategies for Cysteine Modifications in Ras GTPases. In: Trabalzini, L., Retta, S. (eds) Ras Signaling. Methods in Molecular Biology, vol 1120. Humana Press, Totowa, NJ. https://doi.org/10.1007/978-1-62703-791-4_6

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  • DOI: https://doi.org/10.1007/978-1-62703-791-4_6

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