Mass Spectrometric Identification Of Tissue Synovial Tissue Antigen And Citrullinated Modified Epitope Verification Based On RA Serum Antibody Binding Reaction

Objective Rheumatoid arthritis(RA)is a highly heterogeneous multisystem autoimmune disease.This study investigated the differentially expressed proteins,citrullinated proteins and citrulline modification sites in RA synovial tissue,and analyzed the biological functions of up-regulated proteins and citrulline short peptides,looking for short peptides with high correlation with RA,and studied the binding activity of citrulline-modified short peptide to serum ACPA and its diagnostic value for RA.Methods The antigen,citrullinated protein and its modification sites of RA serum antibody binding reaction synovial tissue were identified by Thermo Q Exactive HF mass spectrometry.Bioinformation analysis was performed by online software STRING,Cytoscape software and online software SYFPEITHI.Principle,two citrulline modified short peptides(COL6A3_C: QDVVNAV-(Cit)-QLTLLGG;KRT10-C: RLAADDF-(Cit)-LKYENEV)of COL6A3 and KRT10 were synthesized as target antigens,and HRP-labeled anti-human IgA was used respectively.The IgG and IgM antibodies are secondary antibodies,and the binding activity of serum ACPA to two anti-citrullin short peptide antibodies is tested,and the effects of two short peptides on RA diagnosis are evaluated.Result(1)A total of 594 proteins were identified by mass spectrometry.Among them,125 proteins were up-regulated in RA anti-CCP antibody(+)group and 87 proteins were up-regulated in RA(-)anti-CCP antibody group.57 kinds of melon ammonia were identified.Acid modified protein and 97 modification sites;(2)RA anti-CCP antibody(+)group up-regulated protein is mainly involved in leukocyte activation,regulation of exocytosis,complement activation,regulation of coagulation process and acute inflammatory reaction;RA anti-CCP antibody(-)group up-regulated protein mainly involved in tissue development,epithelium Cytokeratinization,protein folding,and material catabolism.The short peptide sequence of COL6A3,QDVVNAVRQLTLLGG,has a higher binding capacity to MHC class II molecules;(3)ELISA detection value of anti-COL6A3_C short peptide antibody(with anti-human IgM as secondary antibody)sensitivity for RA diagnosis is 65.52%,specificity is 78.95%,positive predictive value is 82.61%,negative predictive value is 60%,ROC The area under the curve was 0.724,but the area of this antibody was 0.956 under the ROC curve for patients diagnosed with anti-CCP antibody(-)RA.The ELISA value of anti-KRT10_C short peptide antibody(with anti-human IgG as secondary antibody)was 58.62%,the specificity was 52.17%,the positive predictive value was 60.71%,the negative predictive value was 52.17%,and the area under the ROC curve.At 0.686,the area under the ROC curve for patients diagnosed with anti-CCP antibody(+)RA was only 0.895.Conclusion:(1)Identification of citrulline-modified antigen from RA synovial tissue provides direct evidence for the presence of citrulline modification in RA.(2)The up-regulated protein of RA anti-CCP antibody(+)group is mainly involved in the immune reaction process,and the up-regulated protein of RA anti-CCP antibody(-)group is mainly involved in cell growth and development.COL6A3 is highly correlated with RA and may be the target antigen for RA autoantibodies.(3)Anti-COL6A3_C short peptide antibody is only used for the anti-CCP antibody(-)RA patients with high diagnostic value,anti-KRT10_C short peptide antibody is only used for anti-CCP antibody(+)RA patients with better diagnostic effect,will be two The addition of anti-short peptide antibodies to the RA laboratory diagnostic system can improve the diagnostic rate of RA.