Key Laboratory of Bioorganic Synthesis of Zhejiang Province, College of Biotechnology and Bioengineering, Zhejiang University of Technology, 18 Chaowang Road, Hangzhou, 310014, China.
Institute of Drug Metabolism and Pharmaceutical Analysis, College of Pharmaceutical Sciences, Zhejiang University, 866 Yuhangtang Road, Hangzhou, 310058, China.
Chembiochem. 2019 May 2;20(9):1155-1160. doi: 10.1002/cbic.201800735. Epub 2019 Mar 26.
The mitochondrion is one of the most important organelles in the eukaryotic cell. Characterization of the mitochondrial proteome is a prerequisite for understanding its cellular functions at the molecular level. Here we report a proteomics method based on mitochondrion-targeting groups and click chemistry. In our strategy, three different mitochondrion-targeting moieties were each augmented with a clickable handle and a cysteine-reactive group. Fluorescence-based bioimaging and fractionation experiments clearly showed that most signals arising from the labels were localized in the mitochondria of cells, as a result of covalent attachment between probe and target proteins. The three probes had distinct profiling characteristics. Furthermore, we successfully identified more than two hundred mitochondrial proteins. The results showed that different mitochondrion-targeting groups targeted distinct proteins with partial overlap. Most of the labeled proteins were localized in the mitochondrial matrix and inner mitochondrial membrane. Our results provide a tool for chemoproteomic analysis of mitochondrion-related proteins.
线粒体是真核细胞中最重要的细胞器之一。对线粒体蛋白质组进行特征分析是在分子水平上理解其细胞功能的前提。在这里，我们报告了一种基于线粒体靶向基团和点击化学的蛋白质组学方法。在我们的策略中，三个不同的线粒体靶向部分分别与一个可点击的接头和一个半胱氨酸反应基团相连。基于荧光的生物成像和分级实验清楚地表明，由于探针与靶蛋白之间的共价连接，标记物产生的大多数信号都定位于细胞的线粒体中。这三种探针具有不同的分析特性。此外，我们成功地鉴定了两百多种线粒体蛋白质。结果表明，不同的线粒体靶向基团与部分重叠的不同蛋白质靶向。大多数标记的蛋白质定位于线粒体基质和线粒体内膜。我们的结果为线粒体相关蛋白质的化学蛋白质组学分析提供了一种工具。
