S-nitrosylation (or S-nitrosation) has emerged as an important pathway of non-classical nitric oxide signaling. This post-translational modification involves the transfer of a nitroso group onto a cysteine residue and has been shown to regulate protein function. However, very little is known about the mechanism and structure-dependent factors of the modification. Understanding of S-nitrosothiol chemistry has lagged behind that for the classical nitric oxide signaling pathway due to challenges and limitations of current detection methods of S-nitrosothiols. The S-N bond is typically labile and indirect detection by traditional biotin switch method has low sensitivity and is prone to false positives. In this work, I have explored phosphine derivatization as a new direct approach to labeling protein S-nitrosothiols. Syntheses of aza-ylide derivatives of small organic S-nitrosothiols were successful and the termolecularity of the reaction was overcome by using a bisphosphine. Similarly, S-nitrosated cysteines of thioredoxin were successfully derivatized with the phosphine TCEP and identified by tandem mass spectrometry of the digested protein. Surprisingly, derivatization of S-nitrosoglutathione was found to be unsuccessful and ¹⁸O-labeling of the reaction indicated hydrolysis of the aza-ylide product. We hypothesize that solvent effects are the source of this discrepancy. In addition, x-ray crystallography studies were undertaken to investigate structural rearrangement of a thioredoxin helix to expose residue Cys 62 to S-nitrosation. A new structure of thioredoxin Q63A/C69S/C73S mutant was found to exhibit a highly dynamic N-terminal loop surrounding the pocket of Cys 62 which could have an effect on S-nitrosation of this residue.