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Mass spectrometry-based identification of new anti-Ly and known antisynthetase autoantibodies
  1. Jean-Baptiste Vulsteke1,2,
  2. Rita Derua3,4,
  3. Sylvain Dubucquoi5,
  4. Frédéric Coutant6,7,
  5. Sebastien Sanges8,9,
  6. David Goncalves7,
  7. Greet Wuyts10,
  8. Petra De Haes11,12,
  9. Daniel Blockmans13,14,
  10. Wim A Wuyts15,16,
  11. Kristl G Claeys17,18,
  12. Ellen De Langhe19,20,
  13. Nicole Fabien7,
  14. Xavier Bossuyt10,21
  1. 1Development and Regeneration, Skeletal Biology Engineering and Research Center, KU Leuven, Leuven, Belgium
  2. 2Rheumatology, KU Leuven University Hospitals Leuven, Leuven, Belgium
  3. 3Molecular and Cellular Medicine: Laboratory of Protein Phosphorylation and Proteomics, KU Leuven, Leuven, Belgium
  4. 4SyBioMa, KU Leuven, Leuven, Belgium
  5. 5Inserm, CHU Lille, U1286 - INFINITE - Institute for Translational Research In Inflammation, University of Lille, Lille, France
  6. 6Eduard Herriot Hospital, Immunogenomics and Inflammation Research Team, University of Lyon, Lyon, France
  7. 7Immunology, Lyon-Sud Hospital, Hospices Civils de Lyon, Pierre-Bénite, France
  8. 8Inserm, CHU Lille, U1286 - INFINITE - Institute for Translational Research in Inflammation, University of Lille, Lille, France
  9. 9Service de Médecine Interne et Immunologie Clinique, Centre de référence des maladies autoimmunes systémiques rares du Nord et Nord-Ouest de France (CeRAINO), CHU Lille, Lille, France
  10. 10Microbiology, Immunology and Transplantation, Clinical and Diagnostic Immunology, KU Leuven, Leuven, Belgium
  11. 11Microbiology, Immunology and Transplantation, KU Leuven University Hospitals Leuven, Leuven, Belgium
  12. 12Dermatology, KU Leuven University Hospitals Leuven, Leuven, Belgium
  13. 13Microbiology, Immunology and Transplantation, Laboratory for Clinical Infectious and Inflammatory Disorders, KU Leuven, Leuven, Belgium
  14. 14General Internal Medicine, KU Leuven University Hospitals Leuven, Leuven, Belgium
  15. 15Chronic Diseases and Metabolism, Laboratory of Respiratory Diseases and Thoracic Surgery, KU Leuven, Leuven, Belgium
  16. 16Respiratory Diseases, KU Leuven University Hospitals Leuven, Leuven, Belgium
  17. 17Neurosciences, Laboratory for Muscle Diseases and Neuropathies, KU Leuven, Leuven, Belgium
  18. 18Neurology, European Reference Network on Rare Neuromuscular Diseases (ERN EURO-NMD), KU Leuven University Hospitals Leuven, Leuven, Belgium
  19. 19Development and Regeneration, Skeletal Biology and Engineering Research Center, KU Leuven, Leuven, Belgium
  20. 20Rheumatology, European Reference Network on Rare and Complex Connective Tissue and Musculoskeletal Diseases (ReCONNET), KU Leuven University Hospitals Leuven, Leuven, Belgium
  21. 21Laboratory Medicine, KU Leuven University Hospitals Leuven, Leuven, Belgium
  1. Correspondence to Xavier Bossuyt, Microbiology, Immunology and Transplantation, Clinical and Diagnostic Immunology, KU Leuven, 3000 Leuven, Belgium; xavier.bossuyt{at}uzleuven.be

Abstract

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.

  • Autoantibodies
  • Polymyositis
  • Autoimmune Diseases
  • Autoimmunity

Data availability statement

Data are available on reasonable request.

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Data availability statement

Data are available on reasonable request.

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Footnotes

  • EDL, NF and XB are joint senior authors.

  • Handling editor Josef S Smolen

  • Twitter @JBVulsteke

  • 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.

  • Supplemental material This content has been supplied by the author(s). It has not been vetted by BMJ Publishing Group Limited (BMJ) and may not have been peer-reviewed. Any opinions or recommendations discussed are solely those of the author(s) and are not endorsed by BMJ. BMJ disclaims all liability and responsibility arising from any reliance placed on the content. Where the content includes any translated material, BMJ does not warrant the accuracy and reliability of the translations (including but not limited to local regulations, clinical guidelines, terminology, drug names and drug dosages), and is not responsible for any error and/or omissions arising from translation and adaptation or otherwise.