Bioorthogonal Chemical Reporters for Analyzing Protein Sulfenylation in Cells

Abstract

Abstract Protein sulfenylation (RSOH), the redox‐based modification of cysteine thiol side chains by hydrogen peroxide (H 2 O 2 ), is an important mechanism in signal transduction. Likewise, dysregulated protein sulfenylation contributes to a range of human pathologies, including cancer. Efforts to elucidate the diverse roles of protein sulfenylation in physiology and disease have been hampered by the lack of techniques to probe these modifications in native environments. To address this problem, selective chemical reporters have been developed for the detection and identification of sulfenylated proteins directly in cells. In the approach described here, a cyclic β‐diketone warhead is functionalized with an azide or alkyne chemical handle. An orthogonally functionalized biotin or fluorescent reporter is then appended to the probe post‐homogenization via click chemistry for downstream analysis. These bi‐functional probes are exquisitely selective for protein sulfenyl modifications, non‐toxic, and do not perturb intracellular redox balance. These reagents have been utilized to investigate sulfenylation in vitro and to identify intracellular protein targets of H 2 O 2 during cell signaling. These methods provide a facile way to detect protein sulfenic acids and to study the biological role of cysteine oxidation with regard to physiological and pathological events. Curr. Protoc. Chem. Biol . 4:101‐122 © 2012 by John Wiley & Sons, Inc.