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

Abstract

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.

Chemical methods for mapping cysteine oxidation

“…77 Recently, Murphy and co-workers devised an improved strategy to detect nitrosylated cysteines coined S-nitrosothiol redox isotope-coded affinity tag (SNOxICAT). 78 This technique is based on the isotope-coded affinity tag labelling technique (OxICAT) commonly applied in the analysis of reversibly oxidised protein thiols. Samples were stabilised by solubilisation in 20% TCA in the presence of 0.5% sulfanilamide to both preserve endogenous S-nitrosothiols and prevent artefactual nitrosylation during work-up.…”

Oral nitrite restores age-dependent phenotypes in eNOS-null mice

“…The use of nitrite in humans is grounded on the known ability of nitrite to dilate isolated blood vessels and original studies testing the efficacy of nitrite to induce vasodilation in humans (22)(23)(24). We now appreciate that the acidity of the gastric lumen as well as metalloenzymes produce an array of secondary reactive NO metabolites that elicit production of cGMP as well as formation of protein S-nitrosocysteine after oral administration of nitrite (25)(26)(27). Currently, several ongoing clinical trials are testing the efficacy of nitrite as a replenishing NO-based therapy in both acute and chronic cardiovascular disorders.…”

Detection and quantification of nitric oxide–derived oxidants in biological systems

“…Both methods are based on using a light and a heavy isotope-containing tag. In ICAT, samples to compare are processed in parallel and tagged with biotin derivatives that include either light or heavy isotope linkers, and then mixed and further processed (135). SILAC involves adding either light or heavy isotope-containing arginine and lysine to control or stimulated cell cultures (136).…”