Targeted synovial fluid proteomics for biomarker discovery in rheumatoid arthritis

Objective: Rheumatoid arthritis (RA) is an autoimmune disease that targets the synovium. The autoantigens involved in the autoantibody responses in RA are unknown. A targeted proteomics approach was used to identify proteins in RA synovial fluid (SF) that are recognized by autoantibodies in RA sera. Methods: RA SF, depleted of abundant proteins, was fractionated by two-dimensional liquid chromatography (chromatofocusing followed by reverse phase HPLC). Protein arrays constructed from these fractions were probed with RA and normal control sera, and proteins within reactive fractions were identified by mass spectrometry. The reactivity of RA sera to an identified peptide was confirmed by ELISA. Results: RA sera specifically reacted to a SF fraction containing fibrin. Mass spectrometry analyses established the presence of a citrullinated arginine at position 271 of the fibrin fragment present in RA SF. A synthetic peptide corresponding to fibrin residues 259-287, containing the citrulline substitution at Arg 271, was recognized by 10 of 12 RA sera, but by two of 18 normal control sera and one of 10 systemic lupus erythematosus sera. Conclusion: Proteomics methodology can be used to directly characterize post-translational modifications in candidate autoantigens isolated from sites of disease activity. The finding that RA sera contain antibodies to the citrullinated fibrin 259-287 peptide may ultimately lead to improved diagnostic tests for RA and/or biomarkers for disease activity. © 2009 Humana Press.