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FEBS J. 2013 Dec;280(23):6150-61. doi: 10.1111/febs.12535. Epub 2013 Oct 16.

Thiol-blocking electrophiles interfere with labeling and detection of protein sulfenic acids.

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  • 1Section on Molecular Medicine, Department of Internal Medicine, Wake Forest School of Medicine, Winston-Salem, NC, USA.

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.

© 2013 FEBS.

KEYWORDS:

S-nitrosation; dimedone; mass spectrometry; protein sulfenic acid; thiol blocking

PMID:
24103186
[PubMed - indexed for MEDLINE]
PMCID:
PMC3928805
Free PMC Article
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