Chapter title |
Biophysical and proteomic characterization strategies for cysteine modifications in ras GTPases.
|
---|---|
Chapter number | 6 |
Book title |
Ras Signaling
|
Published in |
Methods in molecular biology, January 2014
|
DOI | 10.1007/978-1-62703-791-4_6 |
Pubmed ID | |
Book ISBNs |
978-1-62703-790-7, 978-1-62703-791-4
|
Authors |
Hobbs GA, Gunawardena HP, Campbell SL, G. Aaron Hobbs, Harsha P. Gunawardena, Sharon L. Campbell, Hobbs, G. Aaron, Gunawardena, Harsha P., Campbell, Sharon L. |
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. |
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