Peptides were synthesized and purified by the manufacturer (Sigma). Putative citrullinated epitopes were predicted based on published binding data for anchor residues at pockets 1, 4, 6, 7, and 9 and our data about accommodation of citrulline versus arginine (17 (link)). As summarized in Supplemental Table 1 , DR0401 motifs were defined by calculating the product of the coefficients for each possible combination of residues that included a citrulline and scores of 0.1 or higher were selected for synthesis.
For peptide binding assays, increasing concentrations of each non-biotinylated test peptide were incubated in competition with 0.01 µM biotinylated HA306–318 peptide in wells coated with HLA-DR0401 protein as previously described (18 (link)). Europium-conjugated streptavidin (PerkinElmer) was used to label biotinylated peptide bound to the HLA-DR protein and was quantified using a Victor2 multilabel time resolved-fluorometer (PerkinElmer). Binding curves were fitted by non-linear regression with a sigmoidal dose response curve model using Prism software (version5.0, GraphPad Software Inc.). Peptides selected for further study based on positive binding results were re-synthesized at a higher purity by another manufacturer (Genscript).
For peptide binding assays, increasing concentrations of each non-biotinylated test peptide were incubated in competition with 0.01 µM biotinylated HA306–318 peptide in wells coated with HLA-DR0401 protein as previously described (18 (link)). Europium-conjugated streptavidin (PerkinElmer) was used to label biotinylated peptide bound to the HLA-DR protein and was quantified using a Victor2 multilabel time resolved-fluorometer (PerkinElmer). Binding curves were fitted by non-linear regression with a sigmoidal dose response curve model using Prism software (version5.0, GraphPad Software Inc.). Peptides selected for further study based on positive binding results were re-synthesized at a higher purity by another manufacturer (Genscript).
