Screening Of Biomarkers And Establishment Of Diagnostic Model For Tuberculosis Based On Plasma Proteomics

Background Tuberculosis(TB)is a contagious disease caused by tuberculosis,which still endangers human life and health to a great extent.The lack of accuracy and convenience of current tuberculosis diagnosis technology has greatly hindered the global control of tuberculosis morbidity and mortality.Discovering novel blood biological characteristics through proteomics is an important way to develop new diagnostic methods for tuberculosis,which is of great significance to the prevention and control of tuberculosis epidemics in all regions of the world.Objective We tried to explore the plasma proteins expression profile of tuberculosis patients and find the plasma protein biomarkers for the diagnosis of tuberculosis and establish diagnostic models.Methods(1)Proteomics technology based on tandem mass tagging(TMT)mass spectrometry was used to explore the protein expression differences in 18 plasma samples of each of group of bacterial positive tuberculosis,bacterial negative tuberculosis and healthy control.and the GO and KEGG of differentially expressed proteins in TMT mass spectrometry were analyzed by bioinformatics softwares.(2)The differences of 8candidate protein markers in plasma of 30 patients with tuberculosis and 15 healthy subjects were preliminarily verified by parallel reaction monitoring(PRM)targeted mass spectrometry.(3)Enzyme linked immunosorbent assay(ELSA)was used to verify the differences of 6 candidate protein markers in plasma of 80 patients with tuberculosis,76 patients with other non tuberculosis pulmonary respiratory disease(ORD)and 76 healthy subjects.The performance of plasma protein markers in diagnosis of tuberculosis was evaluated by using the subject’s working characteristics(ROC)curve and logic regression algorithm,and diagnostic models were established.Results(1)TMT mass spectrometry results showed that under the condition of fold change(FC)>1.2 or <0.833,P<0.05,compared with the healthy control group,there were43 up-regulated and 71 down-regulated proteins in the plasma of the bacterial positive tuberculosis group,in addition,42 up-regulated and 85 down-regulated plasma proteins existed in the bacterial negative tuberculosis group.The results of bioinformatics analysis showed that the differentially expressed proteins in plasma of TB patients were mainly distributed in extracellular space,involved in the regulation of enzyme activity,and involved in biological processes such as inflammatory response and fatty acid metabolism.Complement coagulation pathway and lipid metabolism are the signal pathways with high enrichment degree of differentially expressed proteins.(2)The results of PRM mass spectrometry showed that the expressions of CFH(P < 0.01),CFHR5(P < 0.01),FGB(P< 0.05),FGG(P < 0.01)and MBL2(P < 0.01)were up-regulated in the tuberculosis group,while the expressions of APOA4(P < 0.01)were down-regulated in the tuberculosis group.(3)ELISA results showed that compared with the healthy control group,the expressions of APOA4(P < 0.0001),CFH(P < 0.05),CFHR5(P < 0.05)and MBL2(P < 0.05)were up-regulated in the tuberculosis group,while the expressions of FGG(P < 0.0001)were down-regulated in the tuberculosis group.In addition,compared with the non-TB pulmonary respiratory disease group,CFH(P < 0.05)and CFHR5(P < 0.0001)were up-regulated in the TB group,while FGG(P < 0.0001)and MBL2(P < 0.001)were down-regulated in the TB group.There was no significant difference in FGB expression among the three groups.ROC curve analysis showed that,when differentiating TB patients from healthy subjects,the AUC of APOA4,CFH,CFHR5,FGG,and MBL2 were 0.73,0.61,0.60,0.73,and 0.57,respectively.when differentiating TB patients from ORD patients,The AUC of CFH,CFHR5,FGG and MBL2 were 0.77,0.61,0.87 and 0.66,respectively.The results of logical regression analysis showed that the diagnostic model consisting of(APOA4+CFH+CFHR5+FGG+MBL2)distinguishs TB patients from healthy subjects,with 71.4% sensitivity and 90.6% specificity in training set,and a sensitivity of62.5% and a specificity of 95.7% in test set.Secondly,the diagnostic model consisting of(CFH+CFHR5+ FGG+MBL2)identifies TB patients and ORD patients,with a sensitivity of 66.1% and a specificity of 98.1% in training set,and 87.5% sensitivity and 91.3%specificity in test set.Finally,the diagnostic model consisting of(APOA4+CFH+CFHR5+FGG)distinguishs between TB patients and non-TB patients,with a sensitivity of 89.3% and a specificity of 73.6% in the training set,and 91.7%sensitivity and 69.6% specificity in the test set.Conclusion(1)Through TMT mass spectroscopy,we found 173 abnormally expressed proteins in the plasma of tuberculosis patients.Complement and coagulation pathway is the signal pathway with the highest enrichment degree of differentially expressed proteins(2)By PRM and ELISA,we confirmed that the expression of CFH,CFHR5 and MBL2 in the plasma of TB patients was up-regulated,while the expression of APOA4 and FGG was down regulated.(3)We established three diagnostic models composed by multi biomarkers that may be helpful for the diagnosis of tuberculosis.The combination of APOA4,CFH,CFHR5,FGG and MBL2 was used to distinguish tuberculosis patients from healthy people.CFH,CFHR5,FGG and MBL2 were combined to distinguish tuberculosis patients from ORD patients.the combination of APOA4,CFH,CFHR5 and FGG was used to distinguish tuberculosis patients from non-tuberculous patients.