Quantitative Chemoproteomic Profiling Of Lipoic Acid Labels Cysteine Residues And Protein Lipoylation

Chemical proteomics,which combines chemical biology and mass spectrometry,and it is a powerful tool to study the interaction between small molecules-protein and protein-protein.In recent years,activity-based protein profiling(ABPP)has developed gradually.It can directly access proteome from a functional perspective by means of chemical probes,which is of great significance in target discovery,high-throughput drug screening and biomolecular recognition.lipoic acid(LA)is an antioxidant drug that helps treat cardiovascular disease,diabetes,liver and kidney diseases and many other chronic diseases.It is a cofactor related to mitochondrial energy production and catabolism.It can participate in acyl group transfer in substance metabolism in organisms,promote the catabolism of sugars,fats and other nutrients through active tricarboxylic acid cycle,and eliminate free radicals that accelerate aging and disease.The disulfide five-membered ring of lipoic acid can covalently bind with sulfhydryl groups on cysteine residues in protein,triggering apoptosis of human cancer cells,protecting enzymes from oxidative damage and achieving cell uptake.At present,the research on lipoylation protein is limited,which greatly limits the related physiological mechanism and clinical disease diagnosis,and the systematic research on lipoic acid binding protein is limited,which leads to the inaccurate and comprehensive analysis of lipoic acid drug target protein.Therefore,in this thesis,quantitative chemical proteomics method was used to conduct a comprehensive exploration of the target proteins and active sites of lipoic acid,through the design and synthesis of chemical probes with reporting groups,combined with high throughput identification of sulfhydryl proteins and active sites covalently interacting with lipoic acid in proteome,the lipoylation proteins were studied.The interpretation of the mode of action of key proteins and related signaling pathways is helpful for the development and clinical application of new drugs,and provides theoretical guidance for the prevention and treatment of related diseases.The work of this thesis mainly includes the following contents:1.We designed and synthesized functional probe LAN-yne and LAO-yne based on lipoic acid.The probe was used to identify the target protein and active site in He La cell lysate or live cells.Based on protein identification experiments,524 and 72 lipoic acid covalently binding proteins were identified.Most of them are lipoic acid-targeting proteins that have not been previously reported.Site identification experiments showed that the lipoic acid probe was highly selective,and 577 cysteine active sites were identified.Finally,bioinformatics analysis showed that lipoic acid target proteins are associated with a variety of diseases,which will help to further analyze therapeutic therapies for related diseases and fully explore the pharmacological efficacy of lipoic acid and its structurally similar small molecule drugs.2.Lipoylation is a post-translational modification in which the carboxylic acid of thioctyl carboxylate covalently modifies lysine residues via an amide bond.By using the reaction between phenyl-arsine oxide and five-membered thiocapic acid ring,we designed a probe of p-phenyl-arsine oxide to capture the thiocapsyl modified protein and use the azide group as the functionalized handle for subsequent fluorophore imaging or enrichment separation.This new method is a supplement to the existing methods for the identification of lipoylation proteins.It will contribute to the further understanding of lipoylation functional proteins and active sites,and ultimately provide important chemical and physiological information for the exploration of lipoylation related mechanisms and drug development.