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- Title
Thiol-blocking electrophiles interfere with labeling and detection of protein sulfenic acids.
- Authors
Reisz, Julie A; Bechtold, Erika; King, S Bruce; Poole, Leslie B; Furdui, Cristina M
- Abstract
Cellular exposure to reactive oxygen species induces rapid oxidation of DNA, proteins, lipids and other biomolecules. At the proteome level, cysteine thiol oxidation is a prominent post-translational process that is implicated in normal physiology and numerous pathologies. Methods for investigating protein oxidation include direct labeling with selective chemical probes and indirect tag-switch techniques. Common to both approaches is chemical blocking of free thiols using reactive electrophiles to prevent post-lysis oxidation or other thiol-mediated cross-reactions. These reagents are used in large excess, and their reactivity with cysteine sulfenic acid, a critical oxoform in numerous proteins, has not been investigated. Here we report the reactivity of three thiol-blocking electrophiles, iodoacetamide, N-ethylmaleimide and methyl methanethiosulfonate, with protein sulfenic acid and dimedone, the structural core of many sulfenic acid probes. We demonstrate that covalent cysteine -SOR (product) species are partially or fully susceptible to reduction by dithiothreitol, tris(2-carboxyethyl)phosphine and ascorbate, regenerating protein thiols, or, in the case of ascorbate, more highly oxidized species. The implications of this reactivity on detection methods for protein sulfenic acids and S-nitrosothiols are discussed.
- Publication
The FEBS journal, 2013, Vol 280, Issue 23, p6150
- ISSN
1742-4658
- Publication type
Journal Article
- DOI
10.1111/febs.12535