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

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


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.

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

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, Non-P.H.S.

MeSH terms

  • Ascorbic Acid / chemistry
  • Ascorbic Acid / metabolism
  • Cyclohexanones / chemistry
  • Cyclohexanones / metabolism
  • Cysteine / chemistry*
  • Cysteine / metabolism
  • Dithiothreitol / chemistry*
  • Dithiothreitol / metabolism
  • Ethylmaleimide / chemistry
  • Ethylmaleimide / metabolism
  • Iodoacetamide / chemistry
  • Iodoacetamide / metabolism
  • Methyl Methanesulfonate / analogs & derivatives
  • Methyl Methanesulfonate / chemistry
  • Methyl Methanesulfonate / metabolism
  • Oxidation-Reduction
  • Proteins / chemistry*
  • Proteins / metabolism
  • Reactive Oxygen Species / metabolism
  • Spectrometry, Mass, Electrospray Ionization
  • Sulfenic Acids / chemistry*
  • Sulfenic Acids / metabolism
  • Sulfhydryl Compounds / chemistry*
  • Sulfhydryl Compounds / metabolism


  • Cyclohexanones
  • Proteins
  • Reactive Oxygen Species
  • Sulfenic Acids
  • Sulfhydryl Compounds
  • methyl methanethiosulfonate
  • Methyl Methanesulfonate
  • dimedone
  • Cysteine
  • Ethylmaleimide
  • Ascorbic Acid
  • Dithiothreitol
  • Iodoacetamide