Comparative Proteomic Study Of Serum And Synovial Fluid In Patients With Rheumatoid Arthritis

Background and PurposeRheumatoid arthritis (RA) is a common chronic inflammatory autoimmune disease characterized by persistent inflammation of the synovium,leading to local destruction of bone and cartilage and a variety of systemic manifestations that could lead to disability.Although the pathogenesis of RA is not fully understood, autoimmune reactions are suggested to play pathological roles in chronic synovitis.So far,The pathological nature of citrullination of protein in joints tissue has been recognized as something that can help us understand fundamental etiologic and pathogenetic features of rheumatoid arthritis.Since then,several autoantibodies against citrullinated proteins have been identified in RA.The antibodies such as anti-keratin antibodies(AKA),anti-perinucIear factor(APF), anti-filaggrin antibodies(AFA), anti-cyclic citrullinated peptide(CCP) antibodies are very specific in RA.Antibodies against citrullinated proteins (ACPAs) are considered as very specific markers for the early diagnosis of RA and the reflection of destruction of bone and cartilage.Thecitrullinated protein and formation of IC were responsible for inducing a spectrum of pro-inflammatory cytokines by peripheral blood mononuclear cells and macrophages via Fc receptors. So far, a variety of candidate autoantigens such as collagen type Ⅱ,fibrin/fibrinogen,vimentin,cartilage intermediate layer protein, and Epstein-Barr virus nuclear antigen-1have been suggested to induce cellular and/or humoral autoimmune responses in RA,all of those proteins are citrullinated in RA synovial tissue.The citrullinated protein and immune complexes of rheumatoid arthritis patients serum and synovial fluid (SF) are involved in the activation of the complement cascade in RA synovial tissue,and induce the macrophages to secret cytokines, metalloproteases,etc, which caused the damage of joints and cartilage.However,the concrete pathogenic mechanism of citrullinated protein remains unclear. As RA is an serious inflammatory autoimmune disease,identify the antigens of RA is became very important. After identification of these antigens, a better understanding of the immunological process in the affected joints can be achieved.In our research,the protein were abstracted from serum by mulitiple affinity removal system(MARS) human-14,differential expression of protein in serum and synovial fluid between the control and RA group were researched through two-dimensional gel electrophoresis (2-DE) and mass spectrometry(MS).In addition,the differential expression of citrullinated protein in serum were also researched.The protein were abstracted from serum by affinity chromatography with immobilized anti-cyclic citrullinated peptide,then identified through proteomics.The protein profiles of differential expression between the control and experimental group were established and some differential proteins were found. The data obtained in this study might be helpful to illuminate the mechanism of citrullination in rheumatoid arthritis.MethodsThe patients,who were diagnosed according to the American Rheumatism Association criteria, obtained from clinic and hospitalization patients from Jinling hospital and Gulou hospital affiliated to medical college of Nanjing university, Serum samples from age-and gender-matched SLE and healthy donors were used as a control.The plasma samples was collected by centrifugalization and stored at-70℃.In order to research differential expression of protein in serum and synovial fluid,the mulitiple affinity removal system(MARS) human-14were used to removel high-abundance protein in serum and synovial fluid,then differential expression of protein between the control and RA group were researched through two-dimensional gel electrophoresis (2-DE) and mass spectrometry(MS).In addition,the differential expression of citrullinated protein in serum were also researched.Protein from serum of RA and healthy control were purified by affinity chromatography with immobolized anti-cyclic citrullinated peptide antibodies.The protein of serum and synovial fluid from RA patients and control were separated by two-dimensional gel electrophoresis(2-DE).After staining by silver,data analysing image by Image Master, several significantly different spots were excised and digested by tryspin, then identified by MALDI-TOF-MS.The functions of proteins in serum were analyzing according to NCBI human database.ResultsAfter remove high-abundance protein in serum and synovial fluid through mulitiple affinity removal system,the protein were separated under the same condition.Identification by MALDI-TOF-MS of differentially expressed protein in in serum and synovial fluid shows that1174spots in synovial fluid of RA can be detected while only1145spots in control,and1649spots can detectedin serum of RA,compare to1661spots in control.6of the34distinguishing proteins in serum were differentially3-fold more overexpressed in RA compared with controls. Meanwhile,12of92spots identified proteins in synovial fluid showed their expressions to be dereased in RA.Identification by MALDI-TOF-MS of differentially expressed protein in in serum and synovial fluid,according to the peptide mass fingerprinting,35proteins has been successfully identified.The anti-CCP antibodies were purified by affinity chromatography with immobilized protein G,then protein from serum of RA and healthy control were purified by affinity chromatography with immobolized anti-cyclic citrullinated peptide antibodies.After separated by two-dimensional gel electrophoresis(2-DE),791spots in serumof RA can be detected while only707spots in control.65of the167distinguishing proteins were differentially3-fold more overexpressed in RA compared with control. Meanwhile,101spots identified proteins showed their expressions to be decreased in RA.The51specifically protein spots in RA were selected for identification by MALDI-TOF-MS.Swiss-Prot and NCBI hunman database were used to analyze these diverse proteins in sera and synovial fluid from patients of rheumatoid arthritis and healthy controls.These diverse proteins may play an importment role in the pathogenesis of rheumatoid arthritis. ConclusionIn our reaserch,the protein of differentially expressed protein and citrullinated peptide from serum and synovial fluid of RA and healthy control were identified through comparative proteomics.The diverse proteins are involved into metabolism,cell signal protein,cell structure proteins,molecular chaperones and so on.Identification of differentially expressed protein and citrullinated peptide could greatly contribute to understanding the pathogenesis of RA,and provide important assistance for the early diagnosis,clinical observation and treatment for RA.Identification of key proteins could prompt us into further understand of RA pathophysiology and may reveal new treatment target.