13075_2020_2252_MOESM2_ESM.pdf (195.25 kB)
Additional file 2 of Profiling of IgG antibodies targeting unmodified and corresponding citrullinated autoantigens in a multicenter national cohort of early arthritis in Germany
journal contribution
posted on 2020-07-07, 05:10 authored by Stefan Vordenbäumen, Ralph Brinks, Patrick Schriek, Angelika Lueking, Jutta G. Richter, Petra Budde, Peter Schulz-Knappe, Hans-Dieter Zucht, Johanna Callhoff, Matthias SchneiderAdditional file 2 : Supplemental Figure 1.A, Mass spectrometric analysis of in vitro citrullinated fibrinogen. Citrullination is a posttranslational modification of arginine resulting in a monoisotopic mass increase of + 0.984016 Da, which can be measured with mass spectrometry. Following in-vitro citrullination, proteins were digested by trypsin and resulting peptides were analysis by LC-ESI-MS using a Q Exactive Orbitrap mass spectrometer (ThermoFisherScientific, Bremen, Germany. Peptide spetra were investigated using MASCOT (Matrix Sciences, London, UK). The MASCOT search result of citrullinated fibrinogen are shown. Deamidated (citrullinated) peptides are indicated. B, Western Blot detection of in vitro citrullinated fibrinogen. Human fibrinogen (huFib, Sigma) was incubated with peptidylarginine deiminase for four hours. Afterwards proteins were separated by SDS-PAGE and subjected to Western blot analysis using a mouse monoclonal antibody that specifically detects citrullinated fibrinogen (anti-hFibC; Modiquest, AB Oss, Netherlands). Two different citrullinated antigens FibCit2 and FibCit4 (Modiquest, AB Oss, Netherlands) were included to confirm positive reactivity of the anti-huFIbC antibody. C, representative example of Reactivity of control sera with citrullinated und non-citrullinated antigen-coupled beads.