Biophysical and Proteomic Characterization Strategies for Cysteine Modifications in Ras GTPases
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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.
