Citrullination of a phage-displayed human peptidome library reveals the fine specificities of rheumatoid arthritis-associated autoantibodies

Rheumatoid arthritis (RA) is associated with autoantibodies that recognize citrullinated proteins — proteins in which the amino acid arginine has been enzymatically converted to citrulline by peptidylarginine deiminase (PAD) enzymes. The clinical anti-cyclic citrullinated peptide (CCP) test is highly informative for RA diagnosis but reports a single global readout: it doesn’t tell clinicians which citrullinated targets a given patient’s antibodies actually recognize, nor whether those antibodies prefer PAD2- or PAD4-citrullinated substrates.

The authors adapted Phage ImmunoPrecipitation Sequencing (PhIP-Seq) — a programmable phage-display platform that displays ~250,000 overlapping 90-amino-acid peptide tiles spanning the human proteome — to this problem. They enzymatically citrullinated the library in vitro using recombinant human PAD2 and PAD4 enzymes, established quality-control criteria for the modification, and then mixed each modified library, alongside its unmodified counterpart, with patient serum. Antibody-bound phage clones are immunoprecipitated, PCR-amplified, and quantified by deep DNA sequencing. By comparing read counts between modified and unmodified libraries, peptides whose binding depended on citrullination — the citrulline-specific ACPA targets — could be identified epitope-by-epitope, across the proteome, in an unbiased way.

Applied to a cohort of 31 RA patients and 10 demographically matched healthy controls, the assay was highly concordant with clinical anti-CCP serostatus and resolved PAD-isoform-specific ACPA reactivity profiles among individual patients. The authors propose PAD PhIP-Seq as a powerful, quantitative tool for proteome-scale antibody-binding analyses in RA and, by extension, as a generalizable framework for studying autoantibodies against any post-translationally modified epitope — carbamylation, acetylation, phosphorylation, and others — opening a path to longitudinal monitoring of pre-clinical RA, response to PAD-targeted therapeutics, and novel PTM-associated autoantigen discovery.