Identification and quantification of protein S-nitrosation by nitrite in the mouse heart during ischemia

Nitrate (NO(3)(−)) and nitrite (NO(2)(−)) are known to be cardioprotective and to alter energy metabolism in vivo. NO(3)(−) action results from its conversion to NO(2)(−) by salivary bacteria, but the mechanism(s) by which NO(2)(−) affects metabolism remains obscure. NO(2)(−) may act by S-nitrosating protein thiols, thereby altering protein activity. But how this occurs, and the functional importance of S-nitrosation sites across the mammalian proteome, remain largely uncharacterized. Here we analyzed protein thiols within mouse hearts in vivo using quantitative proteomics to determine S-nitrosation site occupancy. We extended the thiol-redox proteomic technique, isotope-coded affinity tag labeling, to quantify the extent of NO(2)(−)-dependent S-nitrosation of proteins thiols in vivo. Using this approach, called SNOxICAT (S-nitrosothiol redox isotope-coded affinity tag), we found that exposure to NO(2)(−) under normoxic conditions or exposure to ischemia alone results in minimal S-nitrosation of protein thiols. However, exposure to NO(2)(−) in conjunction with ischemia led to extensive S-nitrosation of protein thiols across all cellular compartments. Several mitochondrial protein thiols exposed to the mitochondrial matrix were selectively S-nitrosated under these conditions, potentially contributing to the beneficial effects of NO(2)(−) on mitochondrial metabolism. The permeability of the mitochondrial inner membrane to HNO(2), but not to NO(2)(−), combined with the lack of S-nitrosation during anoxia alone or by NO(2)(−) during normoxia places constraints on how S-nitrosation occurs in vivo and on its mechanisms of cardioprotection and modulation of energy metabolism. Quantifying S-nitrosated protein thiols now allows determination of modified cysteines across the proteome and identification of those most likely responsible for the functional consequences of NO(2)(−) exposure.