Chouchani, ET; James, AM; Methner, C; Pell, VR; Prime, TA; Erikson, BK; Forkink, M; ... Murphy, MP; + view all Chouchani, ET; James, AM; Methner, C; Pell, VR; Prime, TA; Erikson, BK; Forkink, M; Lau, GY; Bright, TP; Menger, KE; Fearnley, IM; Krieg, T; Murphy, MP; - view fewer (2017) Identification and quantification of protein S-nitrosation by nitrite in the mouse heart during ischemia. Journal of Biological Chemistry , 292 (35) pp. 14486-14495. 10.1074/jbc.M117.798744.

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Abstract

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.

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