By Jantima Tanboon, MD


doi: 10.1136/ard-2022-222686. Online ahead of print.

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

Vulsteke JB, Derua R, Dubucquoi S, et al.


Antisynthetase syndrome (ASS) is a subtype of idiopathic inflammatory myopathy characterised by the presence of antisynthetase antibodies (ASA, i.e., autoantibodies against cytoplasmic aminoacyl-tRNA-synthetases, ARS) together with various combinations of the following clinical features: myositis, interstitial lung disease, arthritis, Raynaud’s phenomenon, and mechanic hands. Identification of ASA and the diagnosis of ASS in patients with compatible clinical features could be challenging because of the low sensitivity and the availability of the commonly used solid phase assays against some rare ASA (e.g. anti-OJ). In some patients with compatible clinical features the diagnosis of ASS could not be rendered because no known ASAs could be identified.

In a recent study, Vulsteke JB., et al (Ann Rheum Dis. 2022 Dec 26, online ahead of print) used protein immunoprecipitation combined with gel-free liquid chromatography-tandem mass spectrometry (IP-MS) to discover the new ASA, anti-Ly, and effectively detect the known ASA. For unbiased IP-MS and development of targeted IP-MS, sera from five patients clinically suggestive of ASS by Connors criteria with a cytoplasmic pattern on the HEp-2 indirect immunofluorescence (Hep-2 IIF) assay but negative for antibodies by dot immunoassay (ASA-neg) were tested against serum from 12 patients positive for ASA (ASA-pos: anti-Jo1, anti-PL7, anti-PL12, and anti-EJ) by dot immunoassay and four healthy controls.

Cytoplasmic cysteinyl-tRNA synthetase (CARS1) and valyl-tRNA synthetase (VARS1) were precipitated by one serum from ASA-neg patient, with a total spectral count of 51 and 10 respectively. This reactivity was named as anti-Ly (because the serum was from CHU Lyon, France). CARS1 but not VARS1 was considered as the dominant cognate ARS autoantigen of anti-Ly autoantibodies because (1) CARS1 was more abundant in immunoprecipitate; (2) showed stronger precipitation by anti-Ly in western blot (WB); (3) the Ly serum showed strong reactivity with recombinant CARS1 on WB; and (4) did not clearly coimmunoprecipitate with VARS1. Proteins associated with known ASA were detected by IP-MS in the other four ASA-neg patients shifting the diagnosis to two anti-OJ, one anti-Zo, and one anti-KS ASS. In one of three patients with anti-EJ on dot immunoassay, RuvB-like 1/2 dimer was identified by IP-MS instead of protein associated with anti-EJ; the patient was confirmed to have anti-RuvBL1/2 antibodies by IP-WB.

To develop targeted IP-MS, proteotypic peptides specific for the precipitated proteins associated with each ASA subtype were identified and selected. The summation of the five most intense spectral fragment peak areas (i.e., sum peak area) of the proteotypic peptide normalised by the maximal peptide intensity observed across all samples was sufficient to discriminate the different ASA subtype in the discovery cohort. The targeted IP-MS was validated against 16 sera from patients positive for ASA by line immunoassay, 20 disease controls and 25 healthy controls. Fourteen of 16 sera from patients with known ASA by line immunoassay showed concordance results with IP-MS. One serum positive for concurrent anti-PL-7 and anti-OJ by line immunoassay was concordant only for anti-PL-7 by IP-MS. Another serum identified as anti-PL-7 by line immunoassay was negative by IP-MS and dot immunoassay.

The IP-MS was also tested against the addition cohort of individuals with multiple ASS features (≥ two clinical features from Connors’ criteria or one clinical feature in combination with a cytoplasmic HEp-2 IIF pattern≥1/80, n=17) or biopsy-proven myositis without other ASyS features (n=9, all HEp-2 IIF negative) and without known ASAs or other relevant autoantibodies was evaluated. In this additional cohort, anti-OJ was identified by IP-MS among 17 patients with ASS clinical features. No other ASS patient was identified in the additional cohort.

In summary, unbiased and target IP-MS detected proteins associated with known ASA and discovered CARS1 as the dominant cognate ARS antigen of the new anti-Ly ASA, in a clinically compatible ASS patient. The methods could detect heterogenous compositions such as multisynthetase complex, the target of anti-OJ. Thus, unbiased and target IP-MS are promising methods for discovery and detection of antibodies including those target complex antigens.

About the author:

​Jean-Baptiste Vulsteke is a rheumatology fellow and PhD student in the University Hospitals Leuven-KU Leuven (Belgium). His research focuses on new and rare autoantibodies in systemic autoimmune diseases, including myositis-spectrum disorders.​


Reviewer: Jantima Tanboon, MD

A photo of Jantima Tanboon. Jantima is smiling at the camera.

This article is presented by the

Publication Highlights Committee.

Published on 15 November 2023.


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