Quantitative Site-Specific Chemoproteomic Profiling of Protein Lipoylation

Abstract

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 <i>Escherichia coli</i> (<i>E. coli</i>) and human proteomes. The strategy enabled us to dissect the dependence of three evolutionarily related lipoylation sites in dihydrolipoamide acetyltransferase (ODP2) in <i>E. coli</i> 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.