The research team led by Prof. Chu Wang at the College of Chemistry and Molecular Engineering, Peking University and Peking-Tsinghua Center for Life Sciences has recently published a research article entitled "Quantitative Site-Specific Chemoproteomic Profiling of Protein Lipoylation" in the Journal of American Chemical Society (https://pubs.acs.org/doi/10.1021/jacs.2c01528). In this work, the authors 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, all known lipoylation sites in both Escherichia coli (E. coli) and human proteomes were successfully quantified. The strategy can dissect the dependence of the 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.
Lipoylation is a posttranslational modification that involves the covalent attachment of lipoic acid to a lysine residue via an amide bond in proteins. It is evolutionarily conserved from bacteria to mammals, which serves as the active-site cofactor and is located in the active pocket of several significant protein complexes in the core metabolic pathway. Lipoylation’s dysregulation is known to contribute to several human diseases, including metabolic disorders, cancers and Alzheimer’s disease. Therefore, advancing the understanding of the regulation of lipoylation will help elucidate the potential molecular mechanisms of these diseases.
The authors developed a chemical probe and incorporated it into a new quantitative chemoproteomic method to identify lipoylation sites in proteomes. Inspired by the thioacetal-based strategy that is commonly used in protecting carbonyl groups, they developed a butyraldehyde probe, which contains an aldehyde group that could react with the lipoylation and a bioorthogonal alkyne handle that could be conjugated with a cleavable biotin tag by the copper-catalyzed click chemistry reaction. Using this probe and an associated chemoproteomic pipeline with special enzyme digestion protocols, they successfully identified all the five lipoylation sites in the three known lipoylated proteins in E. coli and found that the regulation of the three lipoylation sites in the substrate protein ODP2 is independent. They also knocked out each of the three critical enzymes in the synthetic pathway of lipoylation in E. coli and found that the de novo pathway played a more important role than the salvage pathway in terms of lipoylation biosynthesis.
Finally, the authors established a quantitative chemoproteomic method for profiling lipoylation sites in human proteomes. Using an ionization-enhanced cleavable enrichment tag, all the six known lipoylation sites were successfully identified in HepG2 cells. Collectively, this quantitative and site-specific chemoproteomic method for lipoylation profiling will be a valuable tool to help decipher molecular mechanisms of lipoylation-related metabolisms and diseases.
Prof. Chu Wang is the corresponding author for this work. Dr. Shuchang Lai and Dr. Ying Chen are co-first authors of this paper. Other coworkers include Dr. Fan Yang, Dr. Weidi Xiao, and Dr. Yuan Liu. This research was funded in part by National Natural Science Foundation of China, Beijing National Laboratory for Molecular Science, Key Laboratory of Bioorganic Chemistry and Molecular Engineering of Ministry of Education.
Original link for the paper: https://pubs.acs.org/doi/10.1021/jacs.2c01528
