Quantitative Site-Specific Chemoproteomic Profiling of Protein Lipoylation

Protein lipoylation is an evolutionarily conserved post-translational modification from prokaryotes to eukaryotes. Lipoylation is implicated with several human diseases, including metabolic disorders, cancer, and Alzheimer's disease. While individual lipoylated proteins have been biochemically studied, a strategy for globally quantifying lipoylation with site-specific resolution in proteomes is still lacking. Herein, we developed a butyraldehyde-alkynyl probe to specifically label and enrich lipoylations in complexed biological samples. Combined with a chemoproteomic pipeline using customized tandem enzyme digestions and a biotin enrichment tag with enhanced ionization, we successfully quantified all known lipoylation sites in both Escherichia coli (E. coli) and human proteomes. The strategy enabled us to dissect the dependence of three evolutionarily related lipoylation sites in dihydrolipoamide acetyltransferase (ODP2) in E. coli and evaluated the functional connection between the de novo lipoylation synthetic pathway and the salvage pathway. Our chemoproteomic platform provides a useful tool to monitor the state of lipoylation in proteome samples, which will help decipher molecular mechanisms of lipoylation-related diseases.

Automated summary

Protein lipoylation is an important post-translational modification related to various human diseases. However, a comprehensive strategy for quantifying lipoylation sites is still lacking. In this study, we developed a novel approach to quantify lipoylation sites in complex biological samples and investigated their functional connection with the synthetic pathway. This research will contribute to the understanding of the molecular mechanisms underlying lipoylation-related diseases.