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

J Biol Chem. 2017 Sep 1;292(35):14486-14495. doi: 10.1074/jbc.M117.798744. Epub 2017 Jul 14.


Nitrate (NO3-) and nitrite (NO2-) are known to be cardioprotective and to alter energy metabolism in vivo NO3- action results from its conversion to NO2- by salivary bacteria, but the mechanism(s) by which NO2- affects metabolism remains obscure. NO2- 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 NO2--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 NO2- under normoxic conditions or exposure to ischemia alone results in minimal S-nitrosation of protein thiols. However, exposure to NO2- 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 NO2- on mitochondrial metabolism. The permeability of the mitochondrial inner membrane to HNO2, but not to NO2-, combined with the lack of S-nitrosation during anoxia alone or by NO2- 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 NO2- exposure.

Keywords: OxICAT; S-nitrosation; heart; ischemia; mitochondria; nitrite; proteomics; redox; redox regulation.

Publication types

  • Comparative Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Affinity Labels / metabolism
  • Animals
  • Cardiotonic Agents / pharmacology
  • Cell Membrane Permeability / drug effects
  • Cysteine / metabolism
  • Disease Models, Animal*
  • Female
  • Heart / drug effects
  • Mice
  • Mice, Inbred C57BL
  • Mitochondria, Heart / drug effects
  • Mitochondria, Heart / metabolism*
  • Mitochondria, Liver / drug effects
  • Mitochondria, Liver / metabolism
  • Mitochondrial Swelling / drug effects
  • Myocardial Ischemia / drug therapy
  • Myocardial Ischemia / metabolism*
  • Myocardium / metabolism*
  • Nitrates / pharmacology
  • Nitrites / metabolism*
  • Nitrites / pharmacology
  • Nitrosation / drug effects
  • Potassium Compounds / pharmacology
  • Protein Processing, Post-Translational*
  • Proteomics / methods
  • Rats, Wistar
  • Up-Regulation* / drug effects


  • Affinity Labels
  • Cardiotonic Agents
  • Nitrates
  • Nitrites
  • Potassium Compounds
  • potassium nitrite
  • Cysteine
  • potassium nitrate