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

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.