Objectives To discover new and detect known antisynthetase autoantibodies (ASAs) through protein immunoprecipitation combined with gel-free liquid chromatography-tandem mass spectrometry (IP-MS).
Methods IP-MS was performed using sera of individuals showing features of antisynthetase syndrome (ASyS) without (n=5) and with (n=12) previously detected ASAs, and healthy controls (n=4). New candidate aminoacyl-tRNA-synthetase (ARS) autoantigens identified through unbiased IP-MS were confirmed by IP-western blot. A targeted IP-MS assay for various ASA specificities was developed and validated with sera of patients with known ASAs (n=16), disease controls (n=20) and healthy controls (n=25). The targeted IP-MS assay was applied in an additional cohort of patients with multiple ASyS features or isolated myositis without previously detected ASAs (n=26).
Results Autoantibodies to cytoplasmic cysteinyl-tRNA-synthetase (CARS1) were identified by IP-MS and confirmed by western blot as a new ASA specificity, named anti-Ly, in the serum of a patient with ASyS features. Rare ASAs, such as anti-OJ, anti-Zo and anti-KS, and common ASAs could also be identified by IP-MS. A targeted IP-MS approach for ASA detection was developed and validated. Application of this method in an additional cohort identified an additional patient with anti-OJ autoantibodies that were missed by line and dot immunoassays.
Discussion CARS1 is the dominant cognate ARS autoantigen of the newly discovered anti-Ly ASA specificity. Rare and common ASA specificities could be detected by both unbiased and targeted IP-MS. Unbiased and targeted IP-MS are promising methods for discovery and detection of autoantibodies, especially autoantibodies that target complex autoantigens.
- Autoimmune Diseases
Data availability statement
Data are available on reasonable request.
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EDL, NF and XB are joint senior authors.
Handling editor Josef S Smolen
Contributors J-BV, RD and XB designed the study, analysed the data and drafted the manuscript. J-BV, RD and GW performed the experiments. SD, FC, SS, DG, PDH, DB, WAW, EDL and NF are involved in clinical patient management or clinical laboratory evaluation of autoantibodies. All authors critically revised the manuscript. J-BV and XB act as guarantor for the overall content.
Funding J-BV holds a Research Foundation – Flanders (FWO) SB Fellowship (1S62419N). This study was supported by the Research Fund KU Leuven (C3/20/042).
Competing interests SS received consulting fees from Novartis and Biotest, and support for attending meetings from Novartis, Sobi, Shire Takeda and Sanofi Genzyme. WAW has received research grants and/or consultancy fee’s from Roche, Boehringer-Ingelheim and Galapagos. XB was part of a scientific advisory committee for Werfen and Thermo Fisher Scientific, and received speaker’s fees from Werfen and Thermo Fisher Scientific. All abovementioned interests were outside the scope of the current work. J-BV, RD, SD, FC, DG, GW, PDH, DB, EDL and NF do not declare any competing interests.
Patient and public involvement Patients and/or the public were not involved in the design, or conduct, or reporting, or dissemination plans of this research.
Provenance and peer review Not commissioned; externally peer reviewed.
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