Screening Differentially Expressed Plasma Proteins In Rat Plasma With Whole Body Radiation Exposure By TMT Quantitative Proteomics

ObjectiveIt is very important to adopt accurate and rapid biological analysis technology to estimate the exposure dose of exposed population in nuclear accident and radiation accident.The"gold standard"chromosome aberration analysis of radiation dose estimation has the shortcomings,such as long experimental period and high technical requirements,which could not meet the above requirements.In recent years,proteomics technology,which can achieve rapid and high throughput screening,has been applied in radiation research,and provided an effective method for the discovery of radiation biomarkers.Among them,the wide application of quantitative proteomics method is in vitro labeling quantitative technology-tandem mass spectrometry(TMT)and isotope labeling relative and absolute quantitative technology.This method can analyze different proteins in plasma of rats following exposure to different doses of radiation.However,in previous researchs of differentially expressed proteins in irradiated rats using proteomic methods,the selected radiation dose points are few,the observation time is short,and there is no validation experiment.Therefore,it is difficult to obtain the same candidate proteins for radiation sensitivity in different experiments.Therefore,the purpose of this study is to study the changes of plasma proteomics in rats at 1,3,5 and 7 days after different doses of radiation with TMT quantitative proteomics method on the basis of existing studies,and to conduct bioinformatics analysis.At the same time,to verify the differentially expressed proteins in plasma with good radiation dose-response relationship,so as to provide experimental basis for further development and application.Methods1.Laboratory animals,grouping and radiation:SPF grade SD rats,male,8 weeks,weighing(200 + 10)g,were fed with standard intake and drinking water for one week before the experiment.The growth status of rats was observed and healthy rats were incorporated in the study.Eighty healthy rats were weighed and randomly divided into four groups,20 rats in each group.Rats were irradiated at a dose rate of 1 Gy/min,doses were 0 Gy,1 Gy,3 Gy and 5 Gy.2.Sample preparation:After 1 d,3 d,5 d and 7 d irradiation,peripheral blood was collected,blood routine examination and then centrifuged to obtain plasma stored in a refrigerator at-80?.The rats after blood collection were anesthetized,sacrificed;the thymus and spleen were removed,weighed,frozen at-80 ?3.Mass spectrometry analysis:The IgG in plasma was removed by the high abundance protein removal kit and quantified by BCA according to the instructions.SDS-PAGE electrophoresis was used to detect the effect of removing high abundance proteins from plasma samples.Peptide segment preparation,proteolysis,TMT labeled peptide segment labeling,each group of labeled peptide segments were mixed into a sample,after desalination,high pH reverse chromatography was prepared for pre-separation,and then into tandem mass spectrometry detection and protein identification,a total of 503 proteins were identified..4.Bioinformatics analysis:GO annotation analysis,KEGG pathway and STING protein interaction analysis were performed for differentially expressed proteins.5.ELISA verification:The expression levels of Flt-3L,SAA,GDF-15,A2m,CHGA,GPX3,Cp and Clu proteins in plasma were detected by ELISA kit.6.Data analysis:One-way analysis of variance(ANOVA)was used to test the normality and homogeneity of variance of multiple samples.LSD-t test was used to further analyze the differences between groups if the analysis of variance had statistical significance.The dose-effect equation was established by linear regression method.P<0.05 was statistically significant.Results1.The Results of Animal ExperimentCompared with the control group,the weight of rats in the experimental group decreased significantly,while the WBC,ALC and ANC decreased.In addition,the thymus index and spleen index also decreased with the increase of irradiation dose.2.The Results of Mass Spectrometry Screening and Bioinformatics Analysis(1)A total of 503 plasma proteins were identified by mass spectrometry.249 proteins were up-regulated and 328 proteins were down-regulated,of which 74 proteins were up-regulated and down-regulated at different time or dose points.A total of 74 proteins were screened out,including A2m,CHGA,GPX3,Cp and Clu proteins,which showed a better dose-response trend after irradiation.(2)Based on the information of Go categories,these differently expressed proteins were located in the extracellular exosome;the main function was antigen binding;mainly to participate in immune.KEGG pathway analysis showed that differently expressed proteins are mainly involved in complement and coagulation cascades,metabolic process and signaling pathways.STRING protein-protein interaction analysis showed that the interactions of 189 proteins,which were up-regulated proteins.3.The Results of Verification Protein(1)At 1 day after irradiation,the expression of GPX3 and GDF-15 protein increased with the increase of dose.The dose-effect relationship of GDF-15 protein was the best.The dose-effect curve was y=432.887x-465.154 and R2=0.936(y:pg/ml,x:Gy).(2)At 3 days after irradiation,the expression of A2m,CHGA,GDF-15,GPX 3 and Flt-3L proteins tended to increase with the increase of dose.Among them,A2m,CHGA and GDF-15 proteins were fitted to obtain better dose-response curves.The dose-response curves of A2m protein were y=3.931x+36.083,R2=0.960(y:ug/ml,x:Gy);the dose-response curves of CHGA protein were y=6.257x+41.429,R2=0.951(y:ng/ml,x:Gy);the dose-response curves of GDF-15 protein were y=64.280x-21.370,R2=0.913(y:pg/ml,x:Gy).(3)At 5 days after irradiation,the expression of A2m,CHGA,GDF-15 and Flt-3L protein increased with the increase of dose.The dose-effect relationship of Flt-3L protein was the best.The dose-effect curve was y=27.265x+65.764 and R2=0.965(y:pg/ml,x:Gy)Conclusion(1)74 proteins were screened out from 503 differentially expressed proteins by TMT quantitative proteomics.The expression of 74 proteins increased with the increase of dose in plasma after irradiation.Compared with traditional methods,proteomics can screen more proteins with dose-response trend in one study,and find proteins that have not been explored in other studies.(2)Bioinformatics analysis showed that 503 differentially expressed proteins were mainly involved in immune response,resisting radiation-induced damage and maintaining homeostasis.(3)The dose-effect relationship of GDF-15 protein was the best one day after irradiation;the dose-effect relationship of A2m,CHGA and GDF-15 protein was the best three days after irradiation;and the dose-effect relationship of Flt-3L protein was the best five days after irradiation.Among the eight proteins quantitatively analyzed,A2m,CHGA,GPX3,Flt-3L and GDF-15 proteins are expected to be used as radiation biomarkers.