Kanski Jaroslaw, Behring Antje, Pelling Jill, Schöneich Christian
University of Kansas, Department of Pharmaceutical Chemistry, 2099 Constant Ave., Lawrence, KS 66047, USA.
Am J Physiol Heart Circ Physiol. 2005 Jan;288(1):H371-81. doi: 10.1152/ajpheart.01030.2003. Epub 2004 Sep 2.
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.
蛋白质组学技术被用于从Fisher 344/布朗挪威F1大鼠的全心匀浆和心脏线粒体中鉴定心脏蛋白质，这些大鼠因生物衰老而出现蛋白质硝化。通过一维和二维凝胶电泳分离年轻（5月龄）和年老（26月龄）动物的可溶性蛋白质。用抗硝基酪氨酸抗体进行的一维和二维蛋白质印迹显示，一些特定蛋白质的免疫反应随年龄增长而增加，这些蛋白质通过纳米电喷雾电离串联质谱（NSI-MS/MS）进行鉴定。用固定化抗硝基酪氨酸抗体免疫沉淀互补蛋白质，然后进行NSI-MS/MS分析。总共推定鉴定出48种蛋白质。鉴定出的蛋白质包括α-烯醇化酶、α-醛缩酶、结蛋白、乌头酸水合酶、甲基丙二酸半醛脱氢酶、3-酮酰基辅酶A硫解酶、乙酰辅酶A乙酰转移酶、甘油醛-3-磷酸脱氢酶、苹果酸脱氢酶、肌酸激酶、电子传递黄素蛋白、锰超氧化物歧化酶、F1-ATP酶和电压依赖性阴离子通道。一些污染性血液蛋白质，包括转铁蛋白和纤维蛋白原β链前体，其硝化水平也有所增加。MS/MS分析确定电子传递黄素蛋白的Y105位点发生了硝化。在鉴定出的蛋白质中，有负责能量产生和代谢的重要酶以及参与细胞结构完整性的蛋白质。我们的结果与年龄依赖性氧化应激增加以及蛋白质的自由基依赖性损伤一致。可能鉴定出的蛋白质的氧化修饰导致了心脏蛋白质的年龄依赖性退化和功能下降。
