Román-Meléndez GD, Monaco DR, Montagne JM, Quizon RS, Konig MF, Astatke M, Darrah E, Larman HB. *EBioMedicine* · September 2021 · DOI: [10.1016/j.ebiom.2021.103506](https://doi.org/10.1016/j.ebiom.2021.103506) How to cite ### AMA Román-Meléndez GD, Monaco DR, Montagne JM, et al. Citrullination of a phage-displayed human peptidome library reveals the fine specificities of rheumatoid arthritis-associated autoantibodies. *EBioMedicine*. 2021;71:103506. doi:10.1016/j.ebiom.2021.103506 ### APA ``` Román-Meléndez, G. D., Monaco, D. R., Montagne, J. M., Quizon, R. S., Konig, M. F., Astatke, M., Darrah, E., & Larman, H. B. (2021). Citrullination of a phage-displayed human peptidome library reveals the fine specificities of rheumatoid arthritis-associated autoantibodies. *EBioMedicine*, 71, 103506. https://doi.org/10.1016/j.ebiom.2021.103506 ``` ### BibTeX ``` @article{romanmelendez2021citrullination, author = {Rom{\'a}n-Mel{\'e}ndez, Gabriel D. and Monaco, Daniel R. and Montagne, Janelle M. and Quizon, Rachel S. and Konig, Maximilian F. and Astatke, Mekbib and Darrah, Erika and Larman, H. Benjamin}, title = {Citrullination of a phage-displayed human peptidome library reveals the fine specificities of rheumatoid arthritis-associated autoantibodies}, journal = {EBioMedicine}, volume = {71}, pages = {103506}, year = {2021}, doi = {10.1016/j.ebiom.2021.103506} } ``` Román-Meléndez and colleagues adapted Phage ImmunoPrecipitation Sequencing (PhIP-Seq) to the quantitative analysis of antibody reactivity against an enzymatically citrullinated human peptidome library, enabling proteome-scale, epitope-resolved characterization of anti-citrullinated protein antibody (ACPA) responses in rheumatoid arthritis. Comparing PAD2- and PAD4-modified versions of a ~250,000-peptide 90-mer library against unmodified counterparts, the assay distinguished citrulline-dependent autoreactivities and resolved PAD-isoform-specific ACPA fingerprints in individual patients — moving the RA serology biomarker from pan-CCP positivity to per-epitope, per-PAD-isoform resolution. [ Read publication at EBioMedicine ](https://doi.org/10.1016/j.ebiom.2021.103506) In this publication: - **~250,000-peptide 90-mer library** spanning the human proteome was enzymatically modified in vitro with recombinant human PAD2 and PAD4, then run alongside unmodified counterparts in a head-to-head PhIP-Seq workflow. - **90–95% sensitivity and 100% specificity** against the standard-of-care clinical anti-CCP assay in a cohort of 31 rheumatoid arthritis patients and 10 demographically matched healthy controls. - **20 of 21 anti-CCP+ RA patients reacted** to PAD2- and/or PAD4-dependent peptide epitopes, with PAD-isoform-specific ACPA fingerprints distinguished per patient. - **Generalizable framework for post-translational autoantigen profiling:** the enzymatic-modification + library-quality-control pipeline is portable to other PTMs (carbamylation, acetylation, phosphorylation), opening proteome-wide autoantibody profiling against modified self. 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. [← Back to all resources](/) ## Related resources [![](https://cdn.sanity.io/images/vbcbvda6/production/aa97363ccf2216d6365cdd27c5aaefcc489db819-1200x750.webp)Brochure ### HuSIGHT and EnviroSIGHT — InSIGHTs for Parkinson's Disease HuSIGHT + EnviroSIGHT for Parkinson's research: environmental triggers and autoimmune contributions in one assay. 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