The NADPH oxidase (NOX) family of proteins is involved in regulating many diverse cellular processes, which is largely mediated by NOX-mediated reversible oxidation of target proteins in a process known as redox signaling. Protein cysteine residues are the most prominent targets in redox signaling, and to understand the mechanisms by which NOX affect cellular pathways, specific methodology is required to detect specific oxidative cysteine modifications and to identify targeted proteins. Among the many potential redox modifications involving cysteine residues, reversible modifications most relevant to NOX are sulfenylation (P-SOH) and S-glutathionylation (P-SSG), as both can induce structural or functional alterations. Various experimental approaches have been developed to detect these specific modifications, and this chapter will detail state-of-the-art methodology to selectively evaluate these modifications in specific target proteins in relation to NOX activation. We also discuss some of the limitations of these procedures and potential complementary approaches.
Keywords: DUOX; Dimedone; H2O2; NADPH oxidases; Redox signaling; S-glutathionylation; Sulfenylation.