Oxidative Footprinting and Hydrogen/Deuterium Exchange Mass Spectrometry for Epitope Mapping of Cancer Therapeutic Immunotoxins

摘要

We first used an H/D exchange and mass spectrometry-based strategy for epitope mapping. The sequence coverage of the H/D exchange experiments was poor due to the use of MALDI-MS and due to differences in pepsin digestion between the unbound and bound forms of the immunotoxin. H/D exchange studies revealed two peptides that exhibited a decrease in solvent accessibility due to antibody binding. Oxidative footprinting studies were performed to generate complementary data and improve sequence coverage. Oxidative footprinting of the Ab-bound immunotoxin revealed three peptides with decreased solvent accessibility and two peptides with increased solvent accessibility. Changes in solvent accessibility detected using either H/D exchange or oxidative footprinting could be due to direct antibody:antigen interactions or could be due to conformational changes. However, we have significantly narrowed down the number of potential interaction regions.