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

Authors

  • Julie A. Reisz,

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

    1. Department of Chemistry, Wake Forest University, Winston-Salem, NC, USA
    Current affiliation:
    1. Office for Technology Licensing & Industry Collaboration, Tufts University, Boston, MA, USA
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  • S. Bruce King,

    1. Department of Chemistry, Wake Forest University, Winston-Salem, NC, USA
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  • Leslie B. Poole,

    1. Department of Biochemistry, Wake Forest School of Medicine, Winston-Salem, NC, USA
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  • Cristina M. Furdui

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

      C. M. Furdui, Section on Molecular Medicine, Department of Internal Medicine, Wake Forest School of Medicine, Winston-Salem, NC 27157, USA

      Fax: +1 336 716 1214

      Tel: +1 336 716 2697

      E-mail: cfurdui@wakehealth.edu;

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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.

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