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

[Image: see text] 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.