Murray James, Oquendo C Elisa, Willis John H, Marusich Michael F, Capaldi Roderick A
Mitosciences Inc, Eugene OR 97403-2095, USA.
Adv Drug Deliv Rev. 2008 Oct-Nov;60(13-14):1497-503. doi: 10.1016/j.addr.2008.05.003. Epub 2008 Jul 4.
High levels of free radicals produced by the mitochondrial respiratory chain, with subsequent damage to mitochondria have been implicated in a large and growing number of diseases. The underlying pathology of these diseases is oxidative damage to mitochondrial DNA, lipids and proteins which accumulate over time to produce a metabolic deficiency. We are developing an antibody based immunocapture array for many important mitochondrial proteins involved in free radical production, detoxification and mitochondrial energy production. Our array is capable of a multi-parameter measurement including enzyme activity, quantity, and oxidative protein modifications. Here we demonstrate the use of this array by analyzing the proteomic differences in OXPHOS (oxidative phosphorylation) enzymes between human heart and liver tissues, cells grown in media promoting aerobic versus anaerobic metabolism, and the catalytic/proteomic effects of mitochondria exposed to oxidative stress. Protein oxidation is identified as carbonyl formation arising from reactive oxygen species and 3-nitrotyrosine as a marker of reactive nitrogen species. Several identified modifications are confirmed by electrophoresis and mass spectrometry of immunocaptured material. We continue to expand this array as antibodies for enzyme isolation and detection become available.
线粒体呼吸链产生的高水平自由基以及随后对线粒体的损伤，已被认为与大量且日益增多的疾病有关。这些疾病的潜在病理是线粒体DNA、脂质和蛋白质的氧化损伤，随着时间的推移会累积导致代谢缺陷。我们正在开发一种基于抗体的免疫捕获阵列，用于检测许多参与自由基产生、解毒和线粒体能量产生的重要线粒体蛋白。我们的阵列能够进行多参数测量，包括酶活性、数量以及蛋白质的氧化修饰。在这里，我们通过分析人类心脏和肝脏组织中氧化磷酸化（OXPHOS）酶的蛋白质组差异、在促进有氧与无氧代谢的培养基中生长的细胞以及暴露于氧化应激的线粒体的催化/蛋白质组效应，来展示该阵列的用途。蛋白质氧化被确定为活性氧产生的羰基形成，而3-硝基酪氨酸作为活性氮物种的标志物。通过对免疫捕获物质进行电泳和质谱分析，证实了几种已鉴定的修饰。随着用于酶分离和检测的抗体不断出现，我们继续扩展这个阵列。
