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.