细胞中的活性氧分子（ROS）在上皮间充质转化（EMT）和器官纤维化等生理过程中发挥着重要作用。ROS和细胞抗氧化体系相互作用导致蛋白质和代谢产物发生谷胱甘肽（GSH）化修饰。鉴定这些被GSH修饰的分子对理解ROS的作用机制具有重要意义。我们在前期工作中已建立了体外检测药物二相代谢产物和蛋白质谷胱甘肽化的方法，本课题拟发展一套定量检测体系来系统鉴定EMT和肝纤维化过程中谷胱甘肽化的内源性分子。具体的研究目标包括建立表达谷胱甘肽合成酶/转移酶突变体的稳转细胞系，通过代谢反应合成可发生点击化学反应的GSH探针分子；发展富集和检测方法鉴定EMT和肝纤维化相关过程中被GSH修饰的蛋白和内源性代谢产物；阐明谷胱甘肽化对细胞代谢通路和EMT过程的影响并开发表征肝纤维化相关氧化损伤的生物标志物。本项研究开发的检测体系可以改变修饰蛋白质组学重现性低的现状，并用于二相代谢过程以及重要蛋白谷胱甘肽化功能的研究。

Reactive oxygen species (ROS) are the most important biomolecules and play important roles in almost all biological and pathological processes including epithelial-mesenchymal transition and liver fibrosis. Glutathione as one of the most abundance molecules in cells is an essential component of the anti-oxidant system. Under oxidative stress, proteins and metabolites are glutathionylated that protects proteins from excessive and irreversible oxidation and detoxify reactive species. Our long term goal is to understand molecular mechanisms of the redox homeostasis network in ROS regulation and its roles in aging and aging-associated diseases. The specific aims of the present proposal include (1) establishing stable cell lines for expressing GSH synthase mutant, which can use azido-alanine for generating clickable GSH probes; and identifying EMT associated protein glutathionylation; (2) finding an GST mutant that can accommodate clickable GSH for reacting with cellular electrophiles and developing a new method to identify endogenous glutathionylated metabolites; and (3) identifying changes in glutathionylome in mouse models of live fabrosis for developing biomarkers of oxidative stress. The work proposed herein is to establish a reproducible techniques to investigate the functions of glutathionylation that play important roles in biological processes. The completion of the proposed work will establish the role of glutathionylation as a major regulator in EMT and fabrosis. The methods established in the present work for characterization of protein glutathionylation are useful tools for studying other biological systems.