Synthesis Of D₀/d₃-MPPZ And Its Application In Amino Acids And Polypeptides

Nowadays,the research on proteomics is more and more in-depth,and the experimental methods and techniques are constantly improved.With the development of stable isotope labeling technology and liquid chromatography-mass spectrometry technology,it has become an important method for proteomics analysis.The method of chemical derivatization is used to introduce isotopes into the target substance.Qualitative and quantitative analysis of the peak intensity of the same target substance marked by the labeling reagent can make it easier to detect some substances without absorbing groups or poorly ionized.Thiol compounds are involved in a variety of complex biological processes in life and play a major role in disease detection.Therefore,they have a very important position in life sciences.However,their content is low and samples are more complex.By designing and synthesizing a derivative reagent and reacting it with the target substance,we have established a convenient method for the determination of amino-mercaptan compounds.The main research contents are as follows:1.The derivatization reagent 1-(2,4-dinitro5-fluorine)-4,4-dimethylpiperazine(MPPZ)and its deuterated reagent d₃-MPPZ were prepared by simple synthesis.The reagent can react with amino acids or amino groups in peptides to label the target.It has strong ultraviolet absorption,has a positive charge,and is easy to ionize,which can greatly enhance the detection sensitivity.In addition,the derivatization reagent has several advantages such as rapid and simple synthesis method,mild derivatization reaction conditions,low cost,and good stability.2.Explore the reaction of MPPZ with amino acids,peptides,amino acid side chain functional groups,and aminothiol compounds,and explore the best derivatization conditions and chromatographic separation conditions,and perform high performance liquid chromatography and liquid chromatography mass spectrometry on the products after the derivatization reaction Tandem detection and analysis.It was found that MPPZ cannot specifically bind to amino groups.In addition to amino groups,MPPZ also reacts with phenolic hydroxyl groups and sulfhydryl groups.However,compared with similar Sanger reagents,the advantage is that it does not react with the imidazole group and reduces the production of by-products.When there is only one reactive group of amino groups in amino acids and peptides,the retention time of the product peak increases with the number of labeled amino groups.When only one or two amino groups are labeled,the retention time of the product peak appears before the MPPZ peak;When there are other groups such as phenolic hydroxyl in the peptide that participate in the reaction in addition to the amino group,the product peak appears after the MPPZ peak;when the peptide is a phosphorylated peptide,the product retention time is longer than that of the non-phosphorylated peptide product,so MPPZ is more effective for phosphoric acid The identification and analysis of phosphopeptides and non-phosphorylated peptides play a certain role.When reacting with amino mercaptan compounds,as the distance between the amino group and the mercapto group is farther,the reaction activity becomes worse.3.Saliva was selected as the biological sample.After deproteinization,several aminothiol compounds and their oxides in the saliva were studied,and LC-MS/MS detection was performed,and glutathione and its oxidation were successfully detected in the saliva.The lowest detection limit of the determination is50 nM.