Study On The Mechanism Of Photodamage And Safflor Yellow Protection Of 661W Cells Based On Quantitative Proteomic Analysis Of TMT

Objective: To select different expressions of proteins which related to the protective effect of hyroxyaffor yellow A(HSYA)on 661 W cells,and in order to find a new way to study the mechanism of light damage and the protective effect of Hsya on 661 W cells.Methods: 1.In this experiment,661 W cells were used to make photoreceptor of cell light damage model,and HSYA was used to imitate safflor yellow to intervene the cells damaged by light.The cells were made into protein samples which had been put into illumination condition for 2 days.2.The protein was hydrolyzed to peptide level by trypsin,then labeled by TMT and graded by high p H reverse HPLC.3.The peptide was decomposed into small molecules by EASY NLC 1000.This data come from mass spectrometer after being ionized by Na spray ionization source.The data will be processed by quantitative proteomics software package of Maxquant.4.Finally,the data was processed by Gene ontology analysis,interpro domain database comparison,KEGG analysis,subcellular structure analysis,protein function enrichment,cluster analysis based on protein function enrichment,protein interaction network analysis and other technologies.Resluts :The protein quantitative method has good parallelism and accuracy,small differences,good repeatability and stable mass spectrographic analysis results.2.According to the bioinformatics analysis,55206.0 specific peptides were the results of of three groups of protein samples by mass spectrographic analysis.Among them,242 up-regulated proteins and 246 down regulated proteins were selected by 1.2-fold differential screening.57 up-regulated proteins and 21 down regulated proteins were found in the drug / light group.3.566 proteins seleted by 1.2-fold differential screening were analyzed by GO secondary annotation classification,subcellular structure localization,COG / KOG functional classification,GO enrichment analysis,KEGG pathway enrichment results,protein domain enrichment results,cluster analysis,protein interaction network and other technical analysis.The mechanism and cell composition of the significant differences in the expression of related photoproteins in this experiment were as follows: nucleosomes,Glutathione metabolism,ubiquinone,flavin adenine dinucleotide,cytochrome P450 on foreign body metabolism,histone,epidermal growth factor,metallothionein and other related proteins,and there are also 42 unknown functional proteins.4.Protein screening results: according to the principle of protein screening,the relationship between glutathione metabolism and P450 mechanism of drug metabolism is the most relevant;Cyp1b1 rose the most significantly under light conditions,and Gsta 4 rose obviously under Hsya and light conditions.Conclusion: 1.Mass spectrometry analysis can generally point out the differences among three groups of cell proteins,including nucleosome,glutathione metabolism,ubiquinone,flavin adenine dinucleotide,cytochrome P450,histone,epidermal growth factor and metallothionein,and the related proteins of cell structure are closely related to light damage and drug protection.2.Cytochrome P450 has the most significant correlation with the metabolisc mechanism of foreign body and glutathione.3.Cyp1b1 increased most obviously under light condition,and it decreased after adding medicine.Gsta1,gsta4,gstm2,gstm5,and gstp1 were increased under light and chemical conditions.The specific relationship between light damage and cyp1b1,HSYA and glutathione metabolism need to be further verified by Western blotting and other method.