Analysis Of Differential Expression Proteins And The Citrullinated Modification In Serum Immune Complexes And Synovial Tissue Of Patients With Rheumatoid Arthritis By Mass Spectrometry

Objective Rheumatoid arthritis(RA)is a systemic autoimmune disease characterized by chronic inflammatory hyperplasia of the synovial membrane,progressive destruction of the bone and cartilage tissue,and with the production of various autoantibodies.Anti-cyclic citrullinated peptide antibodies.ACPA(Anti-cyclic citrullinated polypeptide antibodies)are now widely used in the clinical diagnosis and prediction of RA,but ACPA were still negative in some RA patients.In this study,we try to explore the autoantigen profile of IC and synovial tissue in RA,in order to provide a theoretical data for finding new biomarkers from the perspective of immunology.Methods IC and Ig G globulin were extracted from 4 groups of mixed serum(41ACPA(+)RA patients,14 ACPA(-)RA,41 systemic lupus erythematosus(SLE)and41 healthy as controls)by polyethylene glycol(PEG)precipitation.The mixed serum Ig G of ACPA(+)RA was reacted with whole protein solution extracted from ACPA(+)RA,ACPA(-)RA,osteoarthritis,traumatic arthritis(TA)synovial tissue,respectively.The serum IC and the antigens extracted from synovial were separated by immuneprecipitation,digested with trypsin in gel and then subjected to mass spectrometry for identifying.Differential comparisons between groups were performed using Vennny 2.1.0.The up-regulated protein was determined when the fraction of total(FOT)value of the RA group was more than 2 times of the control groups,when down-regulated protein was judged when the FOT value was less than0.5 times of the control groups.STRING software was used to perform further functional analysis of the differential proteins in RA.Result 1 Compared with SLE and healthy groups,129 proteins were found only in IC of RA patients,among which there were 38 proteins in ACPA(+)RA,109 proteins in ACPA(-)RA,and 18 proteins were found both in the two subgroups above.Among the proteins in both RA and controls,2 proteins were up-regulated in ACPA(+)RA group when 11 proteins were up-regulated in ACPA(-)RA group.In addition,17proteins were down-regulated in ACPA(+)RA group when 11 proteins were up-regulated in ACPA(-)RA group.2 In the IC of ACPA(+)RA patients,28 kinds of citrullinated peptides and 16 kinds of citrullinated protein were found,including keratin family,desmoglein,histone and complement components;In the IC of ACPA(-)RA,62 kinds of citrullinated peptide,30 kinds of citrullinated protein were found,including keratin family,histone,apolipoprotein,fibronectin,desmoglein and complement components.3 77 proteins were found only in the synovial tissue of ACPA(+)RA with 90 proteins only in ACPA(-)RA synovial tissue.There were 192 proteins in both RA and the control groups,of which 9 proteins were down-regulated in RA group.41 proteins were overexpression in ACPA(+)RA group and 34 were up-regulated in ACPA(-)RA group.4 A total of 32 citrullinated peptides and 16 citrullinated proteins were identified in synovial tissue of RA patients.Among them,26 kinds of citrullinated peptides and 12 kinds of citrullinated protein were found in ACPA(+)RA group,including keratin family,caspase,collagen,fibronectin and so on.A total of 18 citrullinated peptides and 11 kinds of citrullinated proteins were found in the ACPA(-)RA group,including keratin family,histone,fibronectin,collagen and so on.There were 3 kinds of citrulline proteins that appeared only in ACPA(+)RA group.There were 4 kinds of citrulline proteins that appeared only in ACPA(-)RA group.Conclusion: 1 141 potential autoantibody target antigens were identified in the serum circulating immune complexes of RA,of which only 129 specific appeared in RA.The numbers and species of differentially expressed proteins in the IC of ACPA(-)RA were more than ACPA(+)RA.The RA-related proteins,like fibrinogen,myosin,heat shock protein-90,were up-expression in some ACPA(-)RA patients,which provided a good evidence to support the phenomenon that some people were finally diagnosis for RA with the negative seralogical result.2 A total of 207 potential autoantibody target antigens were identified in RA synovial tissue.145 kinds of antigens were expressed only in RA synovium,and the antigens spectrum derived from ACPA(-)RA were more comprehensive.Phosphotreptonase,sulfur redox protein 5,peroxiredoxin and other proteins were up-regulated in the ACPA(-)RA synovial membrane,which will provide a new interpretation to explore RA-related potential autoantibody target antigen in ACPA(-)RA.3 A total of 72 citrullinated peptides and 33 citrullinated proteins were identified in the serum IC of RA patients.The numbers and species of citrullinated proteins in IC of ACPA(-)RA were significantly higher than ACPA(+)RA.At the same time,we found that ACPA(-)RA synovial membrane also has a variety of citrullinated protein deposition,including a variety of subtypes of keratin,collagen fibers,histones,and so on.Although the number of samples is limited but the result still has a more important hint meaning: ACPA-negative patients also have citrullinated modification,but they have not been found because of the limitation of the existing techniques.That means new serological markers are still needed with a great value in improving the detection of ACPA-negative patients.