Proteomic identification of 3-nitrotyrosine-containing rat cardiac proteins: effects of biological aging.

Proteomic techniques were used to identify cardiac proteins from whole heart homogenate and heart mitochondria of Fisher 344/Brown Norway F1 rats, which suffer protein nitration as a consequence of biological aging. Soluble proteins from young (5 mo old) and old (26 mo old) animals were separated by one- and two-dimensional gel electrophoresis. One- and two-dimensional Western blots with an anti-nitrotyrosine antibody show an age-related increase in the immunoresponse of a few specific proteins, which were identified by nanoelectrospray ionization-tandem mass spectrometry (NSI-MS/MS). Complementary proteins were immunoprecipitated with an immobilized anti-nitrotyrosine antibody followed by NSI-MS/MS analysis. A total of 48 proteins were putatively identified. Among the identified proteins were alpha-enolase, alpha-aldolase, desmin, aconitate hydratase, methylmalonate semialdehyde dehydrogenase, 3-ketoacyl-CoA thiolase, acetyl-CoA acetyltransferase, GAPDH, malate dehydrogenase, creatine kinase, electron-transfer flavoprotein, manganese-superoxide dismutase, F1-ATPase, and the voltage-dependent anion channel. Some contaminating blood proteins including transferrin and fibrinogen beta-chain precursor showed increased levels of nitration as well. MS/MS analysis located nitration at Y105 of the electron-transfer flavoprotein. Among the identified proteins, there are important enzymes responsible for energy production and metabolism as well as proteins involved in the structural integrity of the cells. Our results are consistent with age-dependent increased oxidative stress and with free radical-dependent damage of proteins. Possibly the oxidative modifications of the identified proteins contribute to the age-dependent degeneration and functional decline of heart proteins.