Figure 1.

Workflow for experimentally assessing the criticality of modifications by (a) stressing antibody, (b) mixing with target protein, (c) separating by SEC on bound antibody-target complex and unbound antibody, (d) collecting fractions, (e) digesting by trypsin or other proteases, (f) LC-MS/MS peptide mapping identification and relative quantitation of modifications in bound and unbound antibody. Statistical analysis performed and presented as (g) relative abundance plot and (h) volcano plot, where every dot is defined by the fold change of a chemical modification level in unbound versus bond fraction as X-axis and confidence as Y-axis. The round dots in the gray area of the volcano plot represent modifications that are not affecting binding and the artificial modifications caused by the sample preparation. The modifications affecting binding appeared in the top right corner of the volcano plot and were mapped on the crystal structure (i) and represented by stars