Proteomic Analys Is Of Immune Complexes Deposited In Synovial Tissues From RA Patients Using LC-MS

BackgroundRheumatoid arthritis(RA)is a chronic autoimmune disease which is characterized with persistent synovial inflammation,cartilage or bone destruction and systemic complications,including cardiovascular,pulmonary and skeletal disorders.However,the pathogenesis of rheumatoid arthritis is still unknown.Many immune complexes(ICs)were found in blood and synovial tissues(fluid)from RA patients,which were formed by the interaction between autoantibodies and antigens.ICs are likely to contribute to cartilage or bone destruction through the activation of complement cascade and production of pro inflammatory cytokines.Recent research shows that ICs in synovial tissues play a great role in the pathogenesis of RA.Although a variety of autoantigens have been found in the blood and synovial tissues(fluid)of RA patients,including vimentin fibrinogen,collagen type II,fibronectin and so on,the specific antigens that lead to joint destruction were not identified.Antigen profile study on ICs from RA synovial tissues is still limited.Our study was conducted with an enrichment strategy based on immunoprecipitation,in-gel digestion and a liquid chromatography-tandem mass spectrometry(LC-MS/MS)analysis,aiming to identify the protein profile of ICs deposited in synovial tissues from RA patients.By comparation of protein profile between RA and OA synovial ICs,on the one hand,we verified autoantigens that have been identified in previous studies,provided a basis for novel biomarkers for RA early diagnosis.ObjectThe object of the study is to scan protein expression profile of ICs and find specific autoantigens derived from the synovial tissue of RA patients based on LC-MS.To establish an indirect ELISA for the detection of serum anti H2A antibody and preliminary apply to the detection of clinical samples.MethodsSamples of SFs were obtained from knee joints of RA and OA patients during knee therapeutic arthrocentesis at the Department of Osteology,Jinling Hospital,School of Medicine,Nanjing University.The protein expression profile of ICs was identified by an enrichment strategy based on immunoprecipitation and a liquid chromatography-tandem mass spectrometry(LC-MS/MS)analysis.The fraction of total(FOT)was used to estimate protein abundance and screen up and down regulated proteins.The differential proteins were subjected to enrichment analysis,interaction network and signal pathway analysis in David and String.The original data was generated from the RefSeq(NCBI Reference Sequence Database)by Mascot.Further verification experiment was conducted with histone H2A which only appeared in RA-ICs.Histone H2A was got by prokaryotic expression,protein extraction and purification techniques.Indirect ELISA was established using purified H2Aas coating antigen to detect the anti H2A antibody level in serum.ResultsA total of 511 and 526 protein spots were identified in ICs of RA and OA patients respectively.Among them,170 proteins exist only in RA group,340 proteins exist only in RA group,45 and 85 proteins in RA group were statistically up and down expressed compared with controls.Histone H2A,Thioredoxin,vitronectin and HLA-G appeared only in RA-IC while HSP90AA1 was up expressed in RA-IC compared with controls.Recombinant plasmid H2A/pET28a was successfully constructed and purified H2A was got through the progress of prokaryotic expression,protein extraction and purification.Indirect ELISA was successfully established using purified H2A as coating antigen.In the indirect ELISA,blocking rate was 66.4%in the antibody blocking experiment,intraassay CV of the repeated tests was 5.1%,7.5%and 6.6%while interassay CV was 7.7%,9.5%and 8.4%in high,medium and low levels respectively.The sensitivity and specificity of this method was 84%and 80%respectively,the cutoff value was 0.494.The positive rate of anti H2A antibody in RA group was 84%,significantly higher than 20%in healthy control group(p<0.01).ConclusionHSP90AA1,HSP70,HLA-G,Thioredoxin,Annexin A2,vitronectin and histone H2A were involved in the pathogenesis of RA through different paths and may be promising diagnostic indicators or new therapeutic targets for RA.The anti H2A antibody level was significantly higher in RA patients serum than that of healthy controls,serum anti H2A antibody may be an useful index for the auxiliary diagnosis of RA.