title: Screening citrullinated proteins in synovial tissues of rheumatoid arthritis using 2-dimensional western blotting.
abstract: OBJECTIVE
  Citrullination, a reaction converting arginine residue into citrulline residue, is essential for autoimmunity of rheumatoid arthritis (RA). We conducted 2-dimensional Western blot analyses (2-D WB) to screen for novel citrullinated proteins in synovial tissues from patients with RA.
  
  
  METHODS
  Total proteins were extracted from the synovial membranes of patients with RA (n = 10) and pooled. Four identical 2-D electrophoresis (2-DE) gels were prepared, and 2 gels were transblotted to polyvinylidene fluoride membranes that were separately probed with sera from patients with RA (n = 10) or an anticitrulline antibody. The protein profiles of the 2-DE gels were compared with the hybridization results on a global level. The immunoreactive protein spots were collected from the 2-DE gels and identified using mass spectrometry. Proteins that were detected by both RA sera and anticitrulline antibody were considered citrullinated proteins. The result was confirmed through routine WB, immunoprecipitation, and ELISA. Autoantibodies against these potential antigens were also examined in the blood of patients with RA by ELISA.
  
  
  RESULTS
  RA sera and the anticitrulline antibody on 2-D WB detected α-1-antitrypsin (A1AT), dynein heavy-chain 3, fibrinogen β chain, keratin type II cuticular Hb4 (KRT84), lumican, tubulin β-chain (TUBB), and vimentin. A1AT, KRT84, and TUBB had high expression in the synovial membranes (n = 5) of patients with RA and A1AT and KRT84 had high expression in RA synovial fluids (n = 40). A1AT, KRT84, and TUBB immunoprecipitated from synovial tissues showed citrullination. A high level of autoantibodies against KRT84 was detected in the blood of patients with RA (n = 92) compared to that of healthy controls (n = 92).
  
  
  CONCLUSIONS
  Our study identified some new citrullinated proteins in RA synovial tissues using 2-D WB.
authors.affiliation: Medical Research Center and the Orthopedic Surgery Center of Shandong Provincial Qianfoshan Hospital, Shandong University, Shandong, China. changxt@126.cn
authors.forename: Xiaotian
authors.initials: X
authors.lastname: Chang
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhao
authors.forename: Yuejian
authors.initials: Y
authors.lastname: Wang
authors.forename: Yu
authors.initials: Y
authors.lastname: Chen
authors.forename: Xinfeng
authors.initials: X
authors.lastname: Yan
mesh_terms: D000328:Adult; D000368:Aged; D001172:Arthritis, Rheumatoid / Q000276:immunology / Q000378:metabolism*; D001323:Autoantibodies / Q000276:immunology / Q000378:metabolism; D015551:Autoimmunity / Q000276:immunology*; D002956:Citrulline / Q000276:immunology / Q000378:metabolism*; D005260:Female; D006801:Humans; D007719:Knee Joint / Q000276:immunology / Q000378:metabolism; D008297:Male; D008875:Middle Aged; D010003:Osteoarthritis / Q000276:immunology / Q000378:metabolism; D040901:Proteomics; D013582:Synovial Fluid / Q000276:immunology / Q000378:metabolism*; D013583:Synovial Membrane / Q000276:immunology / Q000378:metabolism*
chemical_list: D001323:Autoantibodies; D002956:Citrulline