Fig. 3: Covalent RaPID selection against PADI4 and peptide characterisation.
A DNA recovery from qPCR after each round of selection from biotinylated beads (negative) and PADI4 beads (positive), compared to the input DNA from each round. B Enrichment in warhead-containing peptides. Percentage of sequences from each round of selection which do not contain an internal methionine residue, which encodes the FAO warhead. C Enrichment of 6 key sequences over the five rounds of selection. D Inhibition COLDER assays with PADI4 and 6 peptides identified in the selection or PADI4_3_H4(2). COLDER assays were performed at different peptide concentration (50–0.003 µM) in the presence of 10 mM CaCl2 either with (1 h) or without (0 h) preincubation of peptide and PADI4. Data is normalised to activity of PADI4 in the presence of 0.1% DMSO. Data shows mean ± SEM of two independent replicates. Each replicate was done in triplicate. E COLDER assays to determine KI and kinact. Apparent IC50 values were determined at 15-minute intervals from three independent replicates and the Krippendorff equation was fitted. F COLDER assays to determine selectivity of cP4_15 for PADI4 over PADIs 1-3. Apparent IC50 curves were determined at 15-minute intervals with each of PADIs 1-4. Data for the 60-minute time point is shown. Other time points for PADI3 are shown in Fig. S11A. COLDER assays were performed at different peptide concentration (50–0.08 µM) in the presence of 10 mM CaCl2 without preincubation with PADI protein. Data is normalised to activity of each PADI in the presence of 0.1% DMSO. Data shows mean ± SEM of at least two independent replicates.
