To investigate type II collagen (CII) as a joint‐specific target of the anti–citrullinated protein antibody (ACPA) response in rheumatoid arthritis (RA).

Potential citrullinated neoepitopes were identified by high‐resolution tandem mass spectrometry (MS/MS) of in vitro peptidylarginine deiminase 2 (PAD‐2)–treated CII, and the relationship between citrullination and CII conformation was investigated by circular dichroism and conformation‐dependent antibodies. Based on the MS analyses, synthetic peptides were designed and analyzed for serum IgG reactivity in the Epidemiological Investigation of RA (EIRA) case–control cohort of 1,949 RA patients and 278 healthy controls. Peptide‐specific antibodies were purified from RA patient serum and used to stain RA cartilage specimens.

We described the conformation‐dependent citrullination pattern of CII after PAD‐2 treatment at room temperature and 37°C and showed that CII could be citrullinated in its native triple‐helical conformation. Screening of Arg and Cit pairs of synthetic peptides revealed new citrullinated B cell epitopes on CII. Antibodies directed to 2 proximal epitopes close to the C‐terminus of the CII triple helix were recognized by autoantibodies in 21% and 17% of RA patients, respectively. Affinity‐purified antibodies from RA sera directed to these 2 epitopes, but not antibodies directed to citrullinated α‐enolase peptide 1, bound to RA cartilage.

These findings suggest that cartilage‐directed anticitrulline immunity contributes to the induction of joint inflammation in RA.