Screening THBS4 Based On TMT Quantitative Proteomics And Its Expression And Diagnostic Value In Myasthenia Gravis

ObjectiveMyasthenia gravis(MG)is a kind of acquired autoimmune disease,which is mainly mediated by autoantibodies and characterized by neuromuscular junction(NMJ)excitatory transfer disorder.The destruction of NMJ structure mediated by autoantibody is the main cause of signal transmission block.Under the action of autoantibody and its induced immune response,the fold structure of NMJ postsynaptic membrane will be simplified and lose its normal shape.The internalization and degradation of ACh R on the surface of sarcolemma will be accelerated,and some ACh R-Ab will directly block the normal function of ACh R.The clinical diagnosis of MG was confirmed by typical symptoms,muscle electrophysiological test,neuromuscular junction dysfunction and positive specific antibody test.About 90% of MG patients are positive for ACh R-Ab,Mu Sk-Ab and LRP4-Ab,but about 10% of MG patients have not detected specific autoantibodies.The diagnosis of MG patients with negative serum antibodies may not be clear.Therefore,it is necessary to explore new biomarkers with high sensitivity and specificity to improve the clinical diagnostic value of MG.Proteomic analysis based on tandem mass tag(TMT)has been one of the most popular methods in the past few years for its high throughput,high sensitivity and wide application.In previous studies of proteomics of myasthenia gravis,most of them focused on two-dimensional gel electrophoresis and mass spectrometry to analyze the differential proteins in the muscle tissues of patients with serum and disease models.There are no studies using this technology to analyze the differential proteins in muscle tissue of MG.Therefore,this study aims to screen potential mg markers by TMT quantitative proteomics.The EAMG mouse model was constructed to detect the gene and protein expression levels of the marker in different muscle tissues.At the same time,the expression level of this marker in the population was detected,and the optimal cut-off value was selected to evaluate its diagnostic value for MG,so as to provide technical methods and theoretical basis for finding novel,high sensitivity and high specificity MG marker.Methods1.TMT quantitative proteomics screening potential MG markerBased on TMT quantitative proteomics,differential proteins were screened and identified in intercostal muscles of 5 MG patients and 5 esophageal cancer patients.THBS4 was selected as a potential MG marker by nonparametric test(MG expression level > control grup expression level,P < 0.05),Fold Change(MG average expression level / control average expression level,FC > 1.5 or FC < 0.7),the number of specific peptides identified by mass spectrometry(specific peptides ≥ 5)and GO enrichment analysis.2.Construct EAMG mouse model to verify the expression level of THBS4 in different muscle tissues(1)Based on q PCR experiment,the m RNA expression level of THBS4 in different muscle tissues was detected.(2)In order to explore the protein expression level of THBS4 in different muscle tissues and verify whether the marker is related to endoplasmic reticulum stress.The expression levels of THBS4,GRP78 and calreticulin in different muscle tissues were detected by Western blot.3.The expression level of THBS4 in human serum and its diagnostic value in MG(1)The Double antibody sandwich enzyme linked immunosorbent assay(ELISA)was used to detect the expression level of THBS4 in serum samples of 87 MG patients,37 MP patients and 49 NCs.(2)The maximum Youden index method,95 th percentile method and mean plus standard deviation method were used to screen the best cut-off value of THBS4 in the diagnosis of MG.The diagnostic value of THBS4 with different cut-off values for MG was evaluated by screening experiment evaluation system,and the optimal cut-off value was determined,and its diagnostic value for MG subgroups was evaluated.(3)To evaluate the diagnostic value of combined detection of thbs4 and ACh RAb in MG.Result1.The differentially expressed proteins in intercostal muscles of MG were screened based on TMT quantitative proteomics,and THBS4 was selected as a potential biomarker of MG through a variety of screening conditions.2.The relative expression levels of THBS4 m RNA in forelimb muscle,gastrocnemius muscle,intercostal muscle and myocardium were detected by q PCR experiment.The results showed that the expression level of THBS4 in intercostal muscle was significantly higher than that in other muscle tissues(P < 0.05).However,there was no significant difference in the expression level of THBS4 in the same muscle tissue among the three groups(P > 0.05).The relative protein expression levels of THBS4,GRP78 and calreticulin were detected by Western blot.By comparing the expression levels of THBS4 protein in the above muscle tissues,the results showed that the protein expression profiles of THBS4 protein in NC and vehicle groups were similar.Compared with gastrocnemius,the expression of THBS4 was higher in myocardium and forelimb muscle,and lower in intercostal muscle.In EAMG,the expression level of THBS4 in intercostal muscle was the highest,but there was no significant difference compared with other tissues(P >0.05).The results showed that the expression levels of the three proteins in gastrocnemius of EAMG group were significantly higher than those in NC group and vehicle group(P < 0.05).3.The Double antibody sandwich ELISA was used to detect the expression level of THBS4 in 173 serum samples(87 MG patients,37 MP patients and 49 NCs).In strict accordance with the experimental quality control requirements,99 samples(46MG patients,29 NCs,24 MP patients)were included for follow-up analysis.By ROC analysis,THBS4 may be a potential diagnostic marker of MG,and its AUC was 0.620.By comparing the diagnostic value of different cut-off values for MG,it was determined that when the cut-off value 4.47 ng/m L,THBS4 had the highest diagnostic value for MG,and its diagnostic sensitivity,specificity and accuracy were 56.5%,72.4%and 62.57% respectively.The detection of THBS4 and ACh R-Ab could significantly improve the positive of MG(P < 0.05).Conclusion1.Based on TMT quantitative proteomics,THBS4 was selected as MG potential biomarker.2.THBS4,GRP78 and calreticulin were significantly up regulated in gastrocnemius in EAMG.3.When the optimal cut-off value is the cut-off value corresponding to the maximum Youden index(4.47 ng / ml),THBS4 has the highest diagnostic value for MG.4.Detection of THBS4 and ACh R-Ab can significantly improve the positive of MG.