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.
Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill, Chapel Hill, NC, USA.
G Aaron
GA
Hobbs
Harsha P
HP
Gunawardena
Sharon L
SL
Campbell
D001583:Benzoxazoles / Q000378:metabolism; D001703:Biophysics / Q000379:methods*; D003545:Cysteine / Q000378:metabolism*; D013058:Mass Spectrometry; D015538:Nitrosation; D040901:Proteomics / Q000379:methods*; D026361:Reactive Nitrogen Species / Q000378:metabolism; D017382:Reactive Oxygen Species / Q000378:metabolism; D018631:ras Proteins / Q000737:chemistry* / Q000378:metabolism*
D001583:Benzoxazoles; D026361:Reactive Nitrogen Species; D017382:Reactive Oxygen Species; C020160:benzofurazan; D018631:ras Proteins; D003545:Cysteine