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

Abstract

Background: Post-translational modifications (PTMs) on proteins can be targeted by antibodies associated with autoimmunity. Despite a growing appreciation for their intrinsic role in disease, there is a lack of highly multiplexed serological assays to characterize the fine specificities of PTM-directed autoantibodies. Methods: In this study, we used the programmable phage display technology, Phage ImmunoPrecipitation Sequencing (PhIP-Seq), to profile rheumatoid arthritis (RA) associated anti-citrullinated protein antibody (ACPA) reactivities. Findings: Using both unmodified and peptidylarginine deiminase (PAD)-modified phage display libraries consisting of~250,000 overlapping 90 amino acid peptide tiles spanning the human proteome, PTM PhIP-Seq robustly identified antibodies to citrulline-dependent epitopes. Interpretation: PTM PhIP-Seq was used to quantify key differences among RA patients, including PAD isoform specific ACPA profiles, and thus represents a powerful tool for proteome-scale antibody-binding analyses.

Autoantibody discovery across monogenic, acquired, and COVID-19-associated autoimmunity with scalable PhIP-seq

“…PhIP-seq is a powerful tool for antigen discovery due to its throughput and scalability. Continually declining costs in sequencing paired with scaled protocols such as the ones presented here result in a low per-sample cost and experimental time, and declining costs of custom oligonucleotide based-libraries allow for extensive adaptation ( Román-Meléndez et al, 2021 ; Vogl et al, 2021 ). Yet, as the technology is relatively new, increased discussion around best practices in experimental design, methodology, and data interpretation would be beneficial.…”

“…Similarly, the increased sensitivity of whole protein validation relative to PhIP-seq for BTNL8 autoantibodies highlights the need for rapid and sensitive secondary assays to confirm or even increase the importance of a given candidate antigen. These principles may be extended to other related PhIP-seq modalities ( Mina et al, 2019 ; Román-Meléndez et al, 2021 ; Vogl et al, 2021 ).…”

Phage ImmunoPrecipitation Sequencing (PhIP-Seq): The Promise of High Throughput Serology

“…Similarly, as phage displayed peptide tiles are products of bacteria, PhIP-Seq inherently lacks eukaryotic post-translational modifications (PTMs) of proteins and therefore can pose a challenge in cases where PTMs on pathogens are targeted by the humoral immune response. The PTM problem is partially addressed in a recent work where the investigators used a phage library modified by a PTM enzyme, calcium-dependent peptidylarginine deaminase (PAD), to citrullinate relevant peptide tiles [ 40 ]. While this proof-of-concept study was successful in improving the detection of citrullinated proteins in patients with rheumatoid arthritis, more work must be performed to cover the numerous other PTM processes in eukaryotic cells.…”

New technologies laying a foundation for next generation clinical serology

“…This has previously also been demonstrated using the VirScan platform for screening of antibody responses to common viruses [4]. In Rom an-Mel endez et al [2], this concept is further advanced by developing a T7 proteomic platform for mapping autoantibody responses to PTM antigens targeted by ACPA in RA.…”

“…In the current study by Rom an-Mel endez et al [2], the scope has been to use phage display technology to help define the breadth of anti-citrullinated peptide autoantibodies (ACPA) in rheumatoid arthritis (RA). Within the field of clinical immunology, a positive serological antibody test is often part of the clinical diagnostic criteria, and in many disease settings the precise target(s) of autoantibodies are well understood.…”