PB0851 - Quantitative mass spectrometry-based monitoring of site-specific citrullination of fibrinogen

Background: Citrullination is an inflammation-related post-translational modification (PTM) comprising conversion of a positively charged arginine residue to neutral citrulline. Protein citrullination is associated with autoantibody formation, thereby contributing to autoimmune diseases. Early detection and quantification of protein citrullination will enable severity monitoring of systemic inflammation and has the potential to provide biological insight in thrombo-inflammation. However, current analytical methodologies for early recognition and quantification of protein citrullination are limited or lack analytical specificity.

Aims: Develop an analytical methodology for the quantification of the extent of citrullination of plasma-derived fibrinogen, a well-known citrullination target, as proof-of-concept.

Methods: Human plasma-derived fibrinogen was incubated with in-house recombinantly produced PAD4 in a time-course spanning 0-24 hours. The extent of citrullination was analyzed by a bottom-up proteomics approach including protein denaturation, tryptic digestion and peptide enrichment prior to liquid chromatography (LC) coupled to high resolution mass spectrometry (MS). Relative quantification of both citrullinated and non-citrullinated peptides was performed using parallel reaction monitoring combined with stable-isotope labelled synthetic peptides.

Results: Citrullinated and non-citrullinated amino acid residues were discriminated by their mass difference and LC retention time (Figure 1). PAD4 incubation resulted in time-dependent citrullination of fibrinogen (Figure 2). Several miscleaved peptides were observed upon citrullination, indicating impaired proteolytic processing by trypsin after citrullination of specific arginine residues. In vitro monitoring revealed complete citrullination of residue Arg591 which is part of the integrin-binding RGD-containing domain present within the fibrinogen alpha-chain. In addition, prominent time-dependent citrullination of Arg216, Arg271, Arg573 within the alpha chain and Arg84 within the beta chain was observed. Finally, native citrullination sites were detected in untreated plasma-derived fibrinogen.

Conclusion(s): The presented LC-MS strategy enables quantification of site-specific fibrinogen citrullination. Utilizing complementary unbiased and targeted proteomic strategies, this methodology can now be used to quantify a panel of citrullinated plasma proteins to monitor the relationship between citrullination and thrombo-inflammatory disease.