Chemoseletive Ligation And Chemical Labeling Of Protein Sulfinic Acid With Hydroxylamine-o-sulfonic Acid Derivatives

Reactive oxygen species(ROS)are generated by the incomplete reduction of oxygen during normal metabolism processes.ROS could react with a range of biomolecules(such as proteins,nucleic acids,liposomes,etc.)triggering oxidative stress or being involved in various signal transduction processes.And thus they were closely related to many physiological functions(such as cell aging,apoptosis,etc.)and pathological processes(such as cardiovascular,cerebrovascular diseases,cancer,Alzheimer's disease,etc.).As the most redox sensitive side chain,the protein cysteine residues can be easily modified by ROS to form sulfenic acids which change the structure and function of proteins.The proteins sulfenylation is one of the multitudinous key steps in reactive oxygen signal transduction which affects many signaling pathways such as PI3K,MAPK,and NF-?B.However,sulfenic acids can be further oxidized to sulfinic acids in the presence of excessive intracellular ROS.As a new post-translational modification,S-sulfinylation plays an important role in the redox biology,and also exerts various important physiological and pathological functions.As an extensive and stable product of oxidative modification,protein sulfinic acids are one of the key biomarkers of oxidative stress.It has also been reported that the S-sulfinylation was closely related to a range of diseases,cellular activities and processes.In addition,due to the discovery of protein sulfinic acid reductase(sulfiredoxin),the reversible S-sulfinylation is suggested to play an important role in regulating cell redox status and signal transduction.In this dissertation,the investigation of ligation between protein sulfrnic acids and hydroxylamine-O-sulfonic acid derivatives was illustrated.Based on this reaction,highly efficient and selective labeling reagents for protein sulfinic acids were designed and synthesized,which have also been applied successfully in biological system.Besides,with biotin-conjugated labeling reagent,we also performed proteomic analysis of the cell lysate and then achieved many ROS-sensitive sulfinated proteins intracellular by enrichment,which is of great significance for studying physiological activities,pathological processes and signal transduction.The main innovation of this paper is listed as follows:1.Based on the difference between the stability of sulfonamide and sulfenamide,a series of compounds were designed and synthesized.They selectively react with sulfinic acids to form sulfonamides.The reaction worked well with the hydroxylamine-O-sulfonic acid derivatives and sulfinic acids in different buffer solution systems with broad range of pH values.2.Based on the optimal model compound,the protein sulfinic acids labeling reagents were designed and synthesized.3.The reaction sites of the model compound with ?-lactoglobulin protein sulfinic acids were characterized by EASY-SprayTM protein mass spectrometry,which indicated that hydroxylamine-O-sulfonic acid derivatives have a good selectivity toward protein sulfinic acid.4.The performance of fluorescent-and biotin-conjugated labeling reagents was verified by gel electrophoresis experiments using ?-lactoglobulin proteins.5.The successful labeling of intracellular protein sulfinic acids indicated that the above-mentioned labeling reagents can also be used into complex biological system.The effects of ROS and sulfiredoxin on proteins S-sulfinylation were investigated by laser confocal imaging and western blotting.The distribution of intracellular protein sulfinic acids was also studied,and the results showed that the protein sulfinic acids were mainly distributed in the cytoplasm.6.The effects of ROS on S-sulfinylation were studied by the enrichments of intracellular S-sulfinylation proteins.The results indicated that 145 proteins(involving in cell metabolism,signal transduction processes,endocrine and metabolic diseases,neurodegenerative diseases,cancer and infectious diseases)were finally sulfinated by H2O2.Meanwhile,the sulfinations of 197 proteins were not sensitive to H2O2,but were also regulated by redox environment.These proteins are mainly involved in cell translation,redox,metabolism and phosphorylation,as well as related to signal transduction and many diseases.