Proteomic and mass spectroscopic quantitation of protein s-nitrosation differentiates NO-donors

Protein S-nitrosation has been argued to be the most important signaling pathway mediating the bioactivity of NO. This post-translational modification of protein thiols is the result of chemical nitrosation of cysteine residues. The term NO-donors covers very different chemical classes, from clinical therapeutics to probes of routine use in chemical biology; their different chemistry is predicted to result in distinctive biology regulated by protein S-nitrosation. To measure the extent of protein S-nitrosation by NO-donors, a proteomic mass spectrometry method was developed, which quantitates free thiol versus nitrosothiol for each modified cysteine residue, coined d-Switch. This method is adapted from the biotin switch (BST) method, used extensively to identify S-nitrosated proteins in complex biological mixtures; however, BST does not quantitate free thiol. Since glutathione-S-transferase P1-1 (GST-P1) has been proposed to be a biological "NO-carrier", GST-P1 was used as a reporter protein. The 5 different chemical classes of NO-donors compared by d-Switch demonstrated very different profiles of protein S-nitrosation and response to O ₂ and cysteine, although all NO-donors were oxidants toward GST-P1. The low limits of detection and the ability to use established MS database searching allowed facile generalization of the d-Switch method. Therefore after incubation of neuronal cell cultures with nitrosothiol, it was possible to quantitate not only S-nitrosation of GST-P1 but also many other proteins, including novel targets such as ubiquitin carboxyl-terminal esterase L1 (UCHL1). Moreover, d-Switch also allowed identification of non-nitrosated proteins and quantitation of degree of nitrosation for individual protein thiols.