Identification and quantification of protein
| Autor: | Edward T, Chouchani, Andrew M, James, Carmen, Methner, Victoria R, Pell, Tracy A, Prime, Brian K, Erickson, Marleen, Forkink, Gigi Y, Lau, Thomas P, Bright, Katja E, Menger, Ian M, Fearnley, Thomas, Krieg, Michael P, Murphy |
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| Rok vydání: | 2017 |
| Předmět: |
inorganic chemicals
Proteomics Cardiotonic Agents Cell Membrane Permeability Potassium Compounds Nitrosation Myocardial Ischemia Mitochondria Liver heart ischemia Bioenergetics complex mixtures Mitochondria Heart redox regulation Mice Animals Cysteine Rats Wistar nitrite Nitrites Nitrates Myocardium Affinity Labels respiratory system S-nitrosation Up-Regulation Mice Inbred C57BL mitochondria Disease Models Animal redox Female Mitochondrial Swelling OxICAT Protein Processing Post-Translational |
| Zdroj: | The Journal of Biological Chemistry |
| ISSN: | 1083-351X |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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