Figure 4
Identification of binding interfaces between nIgG and H-ficolin by HDMS.
(a) and (c) HDMS to identify interaction sites between (a) nIgG and (c) H-ficolin. Deuterium incorporation in proteins over time is annotated as follows: solid lines, presence of both nIgG and H-ficolin; dashed lines (control), presence of either nIgG or H-ficolin; black lines, normal condition; and orange lines, infection-inflammation condition. The amino acid sequence of the peptides is indicated in each panel. 18 and 16 peptides, respectively, of nIgG (70% coverage) and H-ficolin (85% coverage) were selected for plotting the graphs based on the mass spectrometric peak quality. Binding peptides of nIgG and H-ficolin showing decreased deuterium incorporation in the presence of both the proteins as compared to individual protein under both conditions, in particular under infection-inflammation condition are indicated by downward orange arrow. H-ficolin non-binding peptides showing increased deuterium incorporation in the presence of both proteins as compared to H-ficolin alone (under infection-inflammation condition) are indicated by upward orange arrow. Representative non-binding peptides showing no difference in deuterium incorporation in the presence of individual proteins or both proteins serve as negative controls. The structure diagrams indicate (b) nIgG Fc and (d) H-ficolin FBG, highlighting the interacting peptides in orange. The additional H-ficolin peptide (YDADHDSSNSNC234β245) interacting with nIgG only under the infection-inflammation condition is highlighted in pink. Results are mean Β± S.D. from 3 independent experiments.
