Determination of Binding Sites on Trastuzumab and Pertuzumab to Selective Affimers Using Hydrogen-Deuterium Exchange Mass Spectrometry

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a method to probe the solvent accessibility and conformational dynamics of a protein or a protein-ligand complex with respect to exchangeable amide hydrogens. Here, we present the application of HDX-MS to determine the binding sites of Affimer reagents to the monoclonal antibodies trastuzumab and pertuzumab, respectively. Intact and subunit level HDX-MS analysis of antibody-affimer complexes showed significant protection from HDX in the antibody Fab region upon affimer binding. Bottom-up HDX-MS experiments including online pepsin digestion revealed that the binding sites of the affimer reagents were mainly located in the complementarity-determining region (CDR) 2 of the heavy chain of the respective antibodies. Three-dimensional models of the binding interaction between the affimer reagents and the antibodies were built by homology modeling and molecular docking based on the HDX data.

Automated summary

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a method to study the solvent accessibility and conformational dynamics of proteins. In this study, HDX-MS was used to determine the binding sites of Affimer reagents on monoclonal antibodies trastuzumab and pertuzumab. The results showed that the affimer binding led to significant protection in the Fab region of the antibodies. The binding sites of the affimer reagents were mainly located in the CDR2 of the heavy chain of the antibodies, as revealed by bottom-up HDX-MS experiments.