Screening Of Biomarkers Of 2,6-dichlorobenzoquinone Based On Chemical Labeling Method

Water is the source of life,carrying human life activities,and drinking water safety is closely related to people’s livelihood and health;drinking water disinfection can effectively eliminate pathogens,control and prevent the transmission of epidemics,and is one of the greatest public health safety measures since the 20th century.However,during the disinfection process,a variety of chemical reactions occur between commonly used disinfectants and some substances in water,such as amino acids,humus and other substances,resulting in a variety of disinfection by-products.2,6-dichloro-1,4-benzoquinone is a typical halogenated quinone disinfection by-product with the highest detection rate and high toxicity in drinking water.Many studies at home and abroad have found that it has cytotoxicity,developmental toxicity and organ toxicity.However,there are no suitable biomarkers for evaluating the organ toxicity and health risks of 2,6-DCBQ.Screening characteristic phosphorylated proteins is one of the effective ways to identify biomarkers.Proteins are the main executors of life activities,and many protein activities are activated by post-translational modification,among which phosphorylation modification is one of the main forms of post-translational modification and the largest number.Both normal life activities and individual diseases are often regulated by phosphorylated proteins.Therefore,through in-depth study of the process of post-translational modification and quantitative analysis of phosphorylated protein changes,we can reveal the mechanism of normal life activities,screen disease and environmental biomarkers,and identify drug targets.At present,techniques for high-throughput analysis of phosphorylated proteins is mainly based on"bottom-up"proteomics approaches.However,the existing quantitative methods have more or less some defects.In response to these problems,this project plans to establish a new method for the relative quantification of phosphorylated proteins,which is widely applicable,can reduce the loss of phosphoryl neutral,low cost,and improve the ionization efficiency in positive ion mode,and then use this method to screen and identify the organ toxicity markers of 2,6-DCBQ,which is expected to be used for environmental risk assessment and potential drug target screening.Main research contents and conclusions:1.Phosphorylated proteins were digested with the mixture enzyme of lyacyl endonuclease and Trypsin and then derived from dimethylamine.The labeling method based on carboxyl amidation has high specificity and usually only occurs on the carboxyl group but have no significant effect on phosphoric acid group.The qualitative and relative quantitative analysis of phosphorylated peptides was achieved by labeling the peptides with dimethylamine and deuterated dimethylamine,respectively.This method not only has good reliability and repeatability,but also enhances the identification efficiency of phosphorylated peptides to a certain extent.2.Phosphorylated protein digested by the mixture enzyme of Lys-C and glutamyl endopeptidase derived from dimethylamine.Compared with Lys-C+Trypsin,Glu-C digestion under the acid buffer system could reduce the generation of lysine-terminating polypeptides and greatly reduce the proportion of self-condensation of peptides.Reduce the occurrence of deamidation under alkaline environment,the ratio of deamidation is less than2%.At the same time,the length of peptide and the number of phosphorylation sites in peptide were decreased.After carboxyl amidation,the specific enrichment of peptide and the mass spectrum response signal were significantly enhanced.Secondary fragmentation produces more characteristic fragment ions,more abundant ion species and less neutral loss,which is helpful for the determination and quantification of polypeptide sequence.3.Phosphopeptides were determined by MicroLC-MS after double derivatization.After the carboxyl group is aminated with methylamine,dimethylamine is further used to replace the phosphoacyl group under alkaline conditions.Under the action of double derivatization,on the one hand,the neutralized carboxyl group enhances the adsorption specificity of the enrichment material for phosphorylated peptides.On the other hand,the negative carboxyl group and phosphorylacyl group are simultaneously derivated,which greatly enhances the mass spectrum response of the peptide and ultimately improves the identification efficiency of phosphorylated peptides,especially polyphosphorylated peptides.4.Effects of low concentration 2,6-DCBQ exposure on phosphorylated proteins of intestinal tissue in fish.Phosphorylated proteins were extracted from the intestinal tissues of goldfish in the control group and the experimental group.After digested by Lys-C+Glu-C mixed enzyme and methylamidation of carboxyl group,phosphoacyl group was replaced by d₀-DMA and d₆-DMA under alkaline conditions,respectively.Then,based on the proteomics method,the differentially expressed proteins before and after 2,6-DCBQ exposure were screened,and the characteristic phosphorylated proteins were further selected as potential biomarkers.