Quantification of protein sulfenic acid modifications using isotope-coded dimedone and iododimedone.

Since its discovery almost 40 years ago, S-hydroxylation ( SOH) of cysteine thiol side chains at active and allosteric sites within proteins has emerged as a central post-translational modification. At present, more than 200 transcription factors, signaling proteins, metabolic enzymes, proteostasis regulators, and cytoskeletal components that undergo sulfenic acid modification have been identified. Like phosphorylation, S-hydroxylation can be a dynamic and reversible posttranslational modification whereby hydrogen peroxide (H2O2) and other reactive oxygen species (ROS) generated during the “oxidative burst” that accompanies many receptormediated signaling processes react with a thiolate anion to form sulfenic acid, and a family of enzymes reduces these modifications (or subsequent disulfides) in proteins (Scheme 1). Although protein sulfenic acids are often transient and labile, S-hydroxylation is significant in physiological and pathophysiological events. For example, it has been shown that this modification plays an essential role in eukaryotic H2O2 sensing, [5] T-cell activation, enzyme catalysis, as an intermediate in disulfide formation, and correlates with disease states. Given the rapidly expanding interest in this field, a number of strategies have been developed to detect protein Shydroxylation. Chemical probes based on the selective reaction between 5,5-dimethyl-1,3-cyclohexanedione (dimedone) and sulfenic acid are particularly well suited to monitor this modification in proteins under biological conditions (Scheme 2a). Using dimedone-based reagents, many Shydroxylated proteins have been observed by immunoblot and identified by mass spectrometry (MS); however, in vivo sulfenylation levels are virtually unknown. Knowledge regarding the degree or extent of S-hydroxylation within proteins is essential for understanding the function and