Fig. 7: Identification of pyrK-containing peptides recognized by VH domain Abs.
From: Molecular and structural basis of anti-DNA antibody specificity for pyrrolated proteins
a Fractionation of digested peptides from pyrBSA by HPLC and competitive ELISA. Bars indicate competition rates (B/B0) for competitive ELISA using VH DO2 as shown by mean of triplicate samples (representative of three independent experiments). Eluate was monitored by absorbance at 220 nm and collected every 5 min for 0–60 min. The fraction with retention times of 45–50 min is indicated by a red arrow. b, c MS spectra of fractions containing pyrK-containing peptides. The fraction with retention time of 45–50 min for pyrBSA-digested peptides was further separated into ten fractions and the MS spectra of fractions 1 and 4 (with retention time 45.0–45.5 and 46.5–47.0, respectively) are shown. d Amino acid sequence of identified pyrK-containing peptides. PyrK is shown as K* highlighted in red. e, f Competitive ELISA with native or pyrrolated recombinant peptides as competitors. Binding of VH DO1 (left panel) and VH DO2 (right panel) to pyrBSA was evaluated in the presence of BSA504-519, pyrBSA504-519, BSA300-320, or pyrBSA300-320 (e), and BSA310-320, pyrBSA310-320, and pyrBSA310-320 (D > N; E > Q) (f). Data are mean ± S.D. of triplicate samples (representative of three independent experiments).
