Capture of Endogenous S-Nitrosothiols with Triarylphosphine Probes

Protein S-nitrosation, the modification of thiol (SH) group to nitrosothiol (SNO), is an important posttranslational modification that regulates central components in virtually every known signaling pathway. Understanding the behavior of SNOs in vivo is therefore important. This task, however, is far from trivial due to chemical instability of the species and the limitations in current SNO detection methods, i.e., (1) blocking free thiols, (2) reducing the SNO to SH, and then 3. derivatizing the nascent thiol. More importantly, these indirect methods limit their overall specificity and reliability due to incomplete blocking and difficulty in distinguishing unblocked from S-nitrosated thiols. To circumvent these limitations, we recently described a direct detection strategy based on phosphine ligation coupled to LC-MS/MS for SNO analysis in biological matrices. While retaining the elements of the SNO functional group the protocol allows unequivocal identification of endogenous low molecular weight S-nitrosated peptides, particularly GSNO, and also permits precise quantiftation using isotopically labeled-SNOs as internal standards. These peptides, as well as proteins such as thioredoxin, are key to some of the major mechanisms of transnitrosation, and we have shown in cells in culture that they represent an index of NO production from the endogenous NO synthases in the cells themselves. Ongoing studies using phosphine capturing technology for the quantitation of GSNO in blood, RSNO-related oxidative damage in malaria parasite infected RBCs, and discovery of novel S-nitrosated thiols in prokaryotic bacteria are currently in progress and will expand our knowledge on the complex physiological roles of S-nitrosation.