Background/Purpose: The protein/peptide citrullination is a post-translational modification and the citrullinated peptide epitopes are recognized by anti-citrullinated protein/peptide antibody (ACPA), which is a major diagnostic or prognostic marker for rheumatoid arthritis (RA). Together with ACPA, specific citrullinated endogenous protein/peptide autoantigens in the major inflammatory lesions of RA (e.g, synovial fluid) may provide valuable information for the development of early diagnosis and therapeutic intervention.

Methods: Synovial fluids obtained from RA (n=4) and osteoarthritis (OA) (n=4) patients were pooled, separately. Peptides in each pooled synovial fluid were extracted using centrifugal filtration and solid phase extraction, and then analyzed by a Q-Exactive mass spectrometer. ScaffoldPTM (v.2.1.3) was used to validate peptide identification and citrullination site mapping. Elevated levels of RA-specific citrullinated peptides are validated by targeted MS/MS analysis with synovial fluids from RA (n=30), OA (n=15), and ankylosing spondylitis (AS) (n=5).

Results: We have identified total 1094 endogenous peptides originated from 120 proteins using LC-MS peptide profiling experiment with pooled RA and osteoarthritis (OA) synovial fluids, separately. Among them 121 peptides derived from 9 proteins (FGA, FGB, SAA1, HMGN2, PTMA, SRGN, COL5A3, PRG4, and APOBR) were found to be citrullinated. The majority of citrullinated peptides were detected only in RA synovial fluid where some identified FGA citrullinated peptides are previously known citrullination sites of FGA, which validate our experiment. We also identified an RA unique citullinated peptide, which has not been reported in the past. To verify RA specific citrullinated peptides, targeted MS/MS analysis of selected citullinated peptides with synovial fluids from RA (n=30), OA (n=15), and ankylosing spondylitis (n=5) patients is being conducted.

Conclusion: We have identified unique citrullinated peptides present in RA synovial fluid. To validate representing citrullinated peptides in RA, we have undertaken targeted MS/MS analysis of selected citrullinated peptides using RA, OA, and AS synovial fluids. The eventual results from the independent cohort studies will confirm unique citullinated peptide markers present in RA synovial fluid, which may provide their diagnostic or therapeutic values for RA.

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ACR Meeting Abstracts - https://acrabstracts.org/abstract/targeted-quantitative-analysis-to-identify-citrullinated-peptides-in-the-synovial-fluid-of-patients-with-rheumatoid-arthritis/