Chemoseletive Ligation And Chemical Labeling Of Protein Methionine Sulfoxide

The reactive oxygen species,refered to a series of energetic oxygen clusters with strong capacity of oxidation,were produced in the process of aerobic cell metabolism which asisted by a arrays of redox modification.They mainly contained O2·-(superoxide anion),H2O2(hydrogen peroxide),·OH(hydroxyl radicals),etc.ROS could regulate various physiological functions through a succession of signal pathways by oxidating specific enzymes and reduced by other reductive substances.Excess of ROS may damaged the normal physiological metabolism,as a result,They were closely correlated with the occurrence of neurological and cardiovascular diseases.Methionine was a major amino acid with a specific methylthio group in biological systems.With its highly hydrophobicity,methionine could participated in initiation of protein translation and maintained the hydrophobicity of protein structure.Its’ sulfur atom was easily oxidatived by ROS to form R and S-type methionine sulfoxides(MetO)in organism,the MetO could also be overoxidated into sulfones and radicals.In addition.The methionine sulfoxides from the proteins could be reduced by the corresponding sulfoxide reductases(Msrs).As a result,this kind of redox could prevent oxdative stress and regulate signaling pathways by consuming ROS.In recent years,it has been found that the oxdiation and reduction of methionine were involved in the regulation of many oxidative stress-related diseases,such as neurodegenerative diseases,Parkinson’s disease,Alzheimer’s disease and cataracts,etc.Therefore,it is significant to study the sulfoxidation of protein methionine for understading the physiological process,detection and treatments of diseases.In this dissertation,a new chemical coupling reaction for labeling methionine sulfoxide was developed and the mechanism of this reaction was also discussed.Meanwhile,the corresponding probes were synthesized and successfully applied them in the detection of sulfoxide from simple protein or proteins in cells.The results provided a powerful tool for the study of methionine sulfoxide and its related physiological,pathological and signal transduction processes in life.The contents of this dissertation were as follows:1.Fluorescent and biotin labelling reagents were synthesized and labeled the methionine sulfoxides from OVA,β-lactoglobulin and CaM after oxidated by H2O2 and different oxidative times,by using gel electrophoresis and EASY-SprayTM mass spectrometry,we analyzed the number of sulfoxides,the markers and its’ product form to prove high efficiency of labeling methionine sulfoxides in recombiniant proteins.We also labeled methionine sulfoxides of β-lactoglobulin step by step by click chemistry and analyzed the labeling form and efficiency of each step.2.By using laser confocal microscopy and western blot,we qualitatively analysised the content and distributions of methionine sulfoxides from HeLa cells which treated with ROS(including H2O2 and ROS scavenger)and investigated the sites of sulfoxidation by using proteomics.3.By knocking down the transcriptive and translative RNA of MsrA and MsrB2 in HeLa cells,the content of Msrs were decreased which leaded to the increasement of methionine sulfoxides that react with labling reagents.The labeling was expanded the application scope of labeling method.At the same time,the efficiency of knockdown was verified by qPCR and western blot,and the optimal concentration of siRNA were also determined.4.Finally,we synthesized the single amino acid and dipeptides containing methionine sulfoxide,and combined the results of the protein EASY-SprayTM mass spectrometry,we investigated the principle of labeling reaction by changing the solvent,the structure and equivalence ratio of reagents,respectively.