Searching for citrullinated autoantigen biomarkers for interstitial lung disease associated with Rheumatoid Arthritis

Interstitial lung disease (ILD) associated with Rheumatoid arthritis (RA) is the cause of death in ~13% of RA patients. Biomarkers are therefore urgently needed to predict ILD development in RA patients, so that appropriate treatment can be instituted. Citrullination occurs in the lungs of RA-ILD patients. We hypothesize that, this post-translational modification leads to the formation of neo-epitopes, triggering autoreactivity & inflammation of the lungs. Because the formation of citrullinated autoantigens and associated autoantibodies is likely linked to ILD in RA patients, we are using RA & RA-ILD patient sera to differentially immunoprecipitate (IP) in-vitro citrullinated HeLa protein extracts containing potential citrullinated autoantigens. When using mass spectrometry (MS) for protein identification, IP results are confounded by an overwhelming amount of antibody-derived peptides. To solve this, we developed a secondary purification step using Biotin-CDM, a novel reversible protein capture reagent that tags all proteins in a cell lysate. The captured biotinylated IP proteins are then separated from non-biotinylated antibodies and released in a un-biotinylated state in the appropriate buffer for proteomic analysis, free of contaminating antibodies. With this modified approach, we have detected several differences in citrullinated autoantigen recognition profiles between RA & RA-ILD patient sera. We are currently identifying these differential IP proteins using MS, with the goal of using target antigens as “bait” in co-IP to isolate other interacting proteins from lung tissue extracts. Once validated, these results will help us further our understanding of the complex networks involved in RA-ILD pathogenesis.