LAUSR

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.