Mass spectrometry-based identification of new anti-Ly and known antisynthetase autoantibodies

ObjectivesTo discover new and detect known antisynthetase autoantibodies (ASAs) through protein immunoprecipitation combined with gel-free liquid chromatography-tandem mass spectrometry (IP-MS). MethodsIP-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). ResultsAutoantibodies 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. DiscussionCARS1 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.

Automated summary

New and known antisynthetase autoantibodies (ASAs) were discovered and detected through protein immunoprecipitation combined with gel-free liquid chromatography-tandem mass spectrometry (IP-MS). The dominant cognate ARS autoantigen of the newly discovered anti-Ly ASA specificity was identified. Unbiased and targeted IP-MS are promising methods for discovery and detection of autoantibodies.