Detection of Anti-RuvBL1/2 Autoantibodies by Targeted Liquid Chromatography–Tandem Mass Spectrometry

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BACKGROUND: Anti-RuvBL1/2 autoantibodies are found in patients with systemic sclerosis and systemic sclerosis-myositis overlap syndrome. Anti-RuvBL1/2 antibodies recognize conformational epitopes of the RuvBL1/2 complex, which complicates detection by solid phase assays. Here, we propose a method for detection of anti-RuvBL1/2 autoantibodies based on liquid phase immunoprecipitation combined with quantification of the precipitated RuvBL1/2 by LC-MS/MS. METHODS: Serum antibodies of patients (n = 13) and controls (n = 60) were captured by and cross-linked to protein A/G magnetic beads and incubated with HeLa nuclear cell extract. Captured proteins were eluted and digested with trypsin. The digested antigen peptides were analyzed by liquid chromatography linked to a triple quadrupole mass spectrometer operating in multiple reaction monitoring (MRM) acquisition mode. Detected signals (peak areas) were quantified. Synthetic proteotypic peptides were used for quality control and quantification purposes. RESULTS: The MRM analysis was based on 6 proteotypic peptides (3 for RuvBL1, 3 for RuvBL2) that are unique to each protein. The immunoprecipitation LC-MS/MS MRM approach allowed differentiation between anti-RuvBL1/2 positive and anti-RuvBL1/2 negative samples. CONCLUSIONS: We propose an immunoprecipitation-based LC-MS/MS MRM method for anti-RuvBL1/2 autoantibody testing. This technology is a promising detection method for autoantibodies targeting conformational epitopes.

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