A pyridinium-based strategy for lysine-selective protein modification and chemoproteomic profiling in live cells
Chuan Wan, Dongyan Yang, Chunli Song, Mingchan Liang, Yuhao An, Chenshan Lian, Chuan Dai, Yuxin Ye, Feng Yin, Rui Wang, Zigang Li
Abstract
Protein active states are dynamically regulated by various modifications; thus, endogenous protein modification is an important tool for understanding protein functions and networks in complicated biological systems. Here we developed a new pyridinium-based approach to label lysine residues under physiological conditions that is low-toxicity, efficient, and lysine-selective. Furthermore, we performed a large-scale analysis of the ∼70% lysine-selective proteome in MCF-7 cells using activity-based protein profiling (ABPP). We quantifically assessed 1216 lysine-labeled peptides in cell lysates and identified 386 modified lysine sites including 43 mitochondrial-localized proteins in live MCF-7 cells. Labeled proteins significantly preferred the mitochondria. This pyridinium-based methodology demonstrates the importance of analyzing endogenous proteins under native conditions and provides a robust chemical strategy utilizing either lysine-selective protein labeling or spatiotemporal profiling in a living system.