4.2. S-Nitrosylation (SNO)

SNO is another important, but highly unstable redox PTM. Phenylmercury-based reagents, including organomercury-conjugated resin (MRC) and phenylmercury-polyethyleneglycol-biotin (mPEGb) (Fig. 3B) have been developed based on the reactivity of phenylmercury to SNO, which forms a covalent thiol-mercury bond (S-Hg) [86]. The direct covalent capture of SNO alleviates some of the inherent limitations of BST and other selective reduction and tagging approaches, consequently providing more SNO specificity and detection by MS [87]. Using MRC, Raju et al. observed decreased S-nitrosylation of proteins involved in glutamate metabolism and uptake in mice lacking nNOS (neuronal isoform of nitric oxide synthase) [88]. Wang et al. reported another tagging concept to generate stable sulfenamide analogs with phosphine esters [89]. The reaction of triphenylphosphine esters with protein-SNO is selective without detectable interference from protein disulfides (e.g. SSG, S-S) or SOH [90]. Since then, several phosphine-based chemical probes linked to fluorophores, biotin, or clickable alkyne have emerged for detecting SNOs in living cells or via gel- and MS-based proteomics approaches [91–94]. In the SNOTRAP method, the phosphine probe is triphenylphosphine thioester (Fig. 3B) linked to biotin through a polyethyleneglycol (PEG) group [92]. This probe enables direct tagging of endogenous SNO leading to identification of ~300 endogenous SNO sites in mouse brain [92]. More recently, Clements et al. reported a clickable alkyne probe called “PBZyn”, which contains a o-phosphino-benzoyl group warhead which can conjugate to both small molecule SNOs (e.g. GSNO, SNAP) and protein-SNO to form a disulfide linker [94]. The PBZyn probe has a potential versatile utility in imaging, protein blotting, and proteomics; however, its utility in SNO profiling by MS has not been demonstrated [94].