F95 pan deimination antibody

For isolation of deiminated proteins from whale and orca sera, the F95 pan-deimination antibody (MABN328, Merck), which is developed against a deca-citrullinated peptide and specifically detects proteins modified by citrullination (Nicholas and Whitaker, 2002) , was used in conjunction with the Catch and Release immunoprecipitation kit (Merck, U.K.). According to the manufacturer's instructions (Merck), 50 μl of serum was used per sample for F95 enrichment and immunoprecipitation was carried out overnight on a rotating platform at 4 °C. The F95 bound proteins were eluted using denaturing elution buffer (according to the manufacturer's instructions, Merck) and the F95 enriched eluates were thereafter analysed both by Western blotting and by liquid chromatography mass spectrometry (LC-MS/MS) for identification of deiminated protein targets (Cambridge Proteomics, U.K.). For LC-MS/MS, peak files were submitted to in-house Mascot (Matrix Science, Cambridge Proteomics) using the following databases for identification of species-

Total deiminated proteins were isolated from lamprey plasma and plasma-EVs using the F95 pandeimination antibody (MABN328, Merck) and the Catch and Release®v2.0 immunoprecipitation kit (Merck, U.K.), according to previously described methods (Criscitiello et al., 2020a; Bowden et al, 2020c) . The F95-antibody is a pan-citrulline antibody which specifically detects proteins modified by citrullination/deimination (Nicholas and Whitaker, 2002) . Pools of plasma from five individual animals (5 x 20 μl) and pools of the corresponding plasma-EVs from 5 individuals (5x20 μl) were used for F95-enrichment, which was performed at 4 °C overnight, using a rotating platform. Elution of deiminated (F95-bound) proteins was performed with the 4x non-denaturing elution buffer according to the manufacturer's instructions (Merck), and the protein eluate was thereafter diluted 1:1 in 2 x Laemmli sample buffer (BioRad, UK), containing 5% beta-mercaptoethanol and boiled at 100°C for 5 min. Samples were thereafter kept frozen at -20 °C until further use for SDS-PAGE analysis, western blotting and in-gel digestion for LC-MS/MS analysis, as described below.