OC 37.3 - A novel mass-spectrometry-based approach enabling site-specific detection of plasma protein citrullination in inflammation-induced thrombosis

Background: Citrullination of arginine residues by peptidyl arginine deiminase (PAD) enzymes drives autoimmune diseases. This post-translational modification can inactivate ADAMTS13 and SERPINs which suggests involvement of citrullination in inflammation-related thrombosis. However, citrullination is notoriously difficult to detect by conventional methods, especially in complex proteomes.

Aims: Here, we developed a novel citrulline probe which enables enrichment of citrullinated peptides prior to analysis via mass spectrometry (MS). We utilized this method to analyze citrullination of fibrinogen and ADAMTS13 by PAD4 and show the utility of this probe for assessment of plasma protein citrullination.

Methods: The citrulline probe was synthesized and its specificity was assessed by NMR. PAD4 was used for in vitro citrullination of fibrinogen and ADAMTS13. Following tryptic digestion citrullinated peptides were labelled, enriched and analyzed by MS. Probe-specific peptide mass shift was used for reliable identification of citrullination sites using MaxQuant.

Results: The newly developed probe selectively modifies citrulline. Using a time-course of PAD4-incubated fibrinogen we measured an increasing intensity and number of citrullinated fibrinogen residues with a maximum of 52 sites. Clustered fibrinogen alpha-chain residues Arg218, Arg271, Arg458 and Arg573 are preferentially citrullinated (Figure 1). Analysis of in vitro citrullinated ADAMTS13 showed primarily citrullination of spacer-domain residue Arg636. Applicability of our method for complex proteomes was showcased by measuring a dose-response curve of spiked citrullinated fibrinogen in human plasma.

Conclusion(s): We applied a novel chemical probe for site-specific enrichment and analysis of citrullinated peptides. Our findings indicate that PAD4 preferentially modifies specific substrate sites. For fibrinogen, preferred citrullination sites overlap with known targets of anti-citrullinated protein antibodies (ACPA) present during rheumatoid arthritis. We thus propose that preferred citrullination sites drive autoimmune responses. Ongoing studies aim at determining the citrullinome in patients with cancer, venous thrombosis, sepsis and immune thrombotic thrombocytopenic purpura.