Biophysical and Proteomic Characterization Strategies for Cysteine Modifications in Ras GTPases

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.