Advances in Biopharmaceutical Analysis

Research tools that can decipher protein–protein interactions and binding interface contact points can aid in the successful development of biotherapeutics. Hydroxyl radical footprinting–mass spectrometry (HRF-MS) technologies are being developed as tools for deciphering protein–protein interactions. We have demonstrated the utility of using fast photochemical oxidation of proteins (FPOP) as an HRF-MS technology for biotherapeutic protein structural characterization and analysis of protein–protein interfaces; monoclonal antibody (mAb) epitope mapping has also been demonstrated using this technique. However, the postlabeling workflow that utilizes offline protein digestion and liquid chromatography–tandem mass spectrometry (LC–MS/MS) analysis is labor intensive and time consuming, and has significant sample consumption. In the present work, we demonstrate the potential of multidimensional online peptide mapping analysis (reduction, tryptic digestion, and separation) as a strategy for improving our postlabeling workflow for protein– protein interactions. The proof-of-concept studies were performed using a Fab antibody fragment and its antigen binding partner.