Institute of Bioanalytical Chemistry, Center for Biotechnology and Biomedicine, Faculty of Chemistry and Mineralogy, Leipzig University, Deutscher Platz 5, 04103 Leipzig, Germany.
J Proteome Res. 2010 May 7;9(5):2516-26. doi: 10.1021/pr901182r.
Reactive oxidative species (ROS) play important roles in cellular signaling but can also modify and often functionally inactivate other biomolecules. Thus, cells have developed effective enzymatic and nonenzymatic strategies to scavenge ROS. However, under oxidative stress, ROS production is able to overwhelm the scavenging systems, increasing the levels of functionally impaired proteins. A major class of irreversible oxidative modifications is carbonylation, which refers to reactive carbonyl-groups. In this investigation, we have studied the production and clearance rates for skeletal muscle proteins in a rat model of acute oxidative stress over a time period of 24 h using a gel-based proteomics approach. Optimized ELISA and Western blots with 10-fold improved sensitivities showed that the carbonylation level was stable at 4 nmol per mg protein 3 h following ROS induction. The carbonylation level then increased 3-fold over 6 h and then remained stable. In total, the oxidative stress changed the steady state levels of 20 proteins and resulted in the carbonylation of 38 skeletal muscle proteins. Carbonylation of these proteins followed diverse kinetics with some proteins being highly carbonylated very quickly, whereas others peaked in the 9 h sample or continued to increase up to 24 h after oxidative stress was induced.
活性氧（ROS）在细胞信号转导中起着重要作用，但也可以修饰并经常使其他生物分子失活。因此，细胞已经开发出有效的酶和非酶策略来清除 ROS。然而，在氧化应激下，ROS 的产生能够超过清除系统，增加功能受损蛋白质的水平。不可逆氧化修饰的主要类型是羰基化，它是指反应性羰基基团。在这项研究中，我们使用基于凝胶的蛋白质组学方法在急性氧化应激的大鼠模型中研究了骨骼肌蛋白在 24 小时内的产生和清除率。经过优化的 ELISA 和 Western blot 具有 10 倍提高的灵敏度，结果表明，在 ROS 诱导后 3 小时，羰基化水平稳定在每毫克蛋白质 4 nmol。然后，羰基化水平在 6 小时内增加了 3 倍，然后保持稳定。总的来说，氧化应激改变了 20 种蛋白质的稳态水平，并导致 38 种骨骼肌蛋白发生羰基化。这些蛋白质的羰基化遵循不同的动力学，有些蛋白质很快就被高度羰基化，而其他蛋白质则在 9 小时的样本中达到峰值，或者在氧化应激诱导后继续增加到 24 小时。
