Many effects of oxidative stress are thought to be mediated by oxidation and alkylation of protein cysteinyl thiols.  Analysis of labile and low abundant modifications to protein thiols remains a challenging analytical problem.  We have employed chemoselective probes, Click chemistry and mass spectrometry to identify protein targets and sequences for cysteinyl modifications and to quantify changes produced by lipid electrophiles and oxidants.  A key element of our approach is a photocleavable azidobiotin reagent, which enables efficient photorelease of Click-labeled electrophile adducts.  Application of this strategy identified over 1,600 proteins alkylated by the lipid electrophile probes alkynyl-4-hydroxynonenal (aHNE) and alkynyl-4-oxononenal (aONE) in electrophile-exposed human cells.  We adapted this approach to analyze S-sulfenyl (S-OH) peptides in intact cells.   Selective labeling of S-sulfenylcysteine residues with DYn-2, an alkynyl beta-diketone was followed by digestion, biotinylation of labeled peptides and streptavidin capture and photorelease.  High resolution tandem mass spectrometry (MS/MS) identified 1105 localized S-sulfenylcysteine sites on 778 proteins in RKO cells.  Tag-directed fragmentation of the DYn-2 modified peptides yielded diagnostic fragment ions, which enabled confident identifications.   Use of isotopically-labeled DYn-2 afforded quantitative analyses of site-specific S-sulfenyl changes induced by hydrogen peroxide exposure.  The results revealed that multiple S-sulfenylated sites on the same protein could sense exogenous hydrogen peroxide in a markedly different manner.  We have also synthesized a 13C-labeled azidobiotin reagent, which enables quantitative comparisons without the use of isotope-labeled electrophile or thiol redox probes.  This collection of reagents and methods makes possible systematic, quantitative evaluation of global changes in thiol alkylation and redox status.  (Supported by NIH grants CA159988, GM102187 and ES013125.)

Quantitative analysis of thiol electrophile and redox modifications by LC-MS/MS

248th National Meeting (2014)

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