Probing Antibody Binding to Canine Parvovirus with Charge Detection Mass Spectrometry

Abstract

There are many techniques for monitoring and measuring the interactions between proteins and ligands. Most of these techniques are ensemble methods that can provide association constants and in some cases stoichiometry. Here we use charge detection mass spectrometry (CDMS), a single particle technique, to probe the interactions of antigen binding fragments (Fabs) from a series of antibodies with the canine parvovirus (CPV) capsid. In addition to providing the average number of bound Fabs as a function of Fab concentration (i.e., the binding curve), CDMS measurements provide information about the distribution of bound Fabs. We show that the distribution of bound ligands is much better at distinguishing between different binding models than the binding curve. The binding of Fab E to CPV is a textbook example. A maximum of 60 Fabs bind and the results are consistent with a model where all sites have the same binding affinity. However, for Fabs B, F, and 14, the distributions can only be fit by a model where there are distinct virus subpopulations with different binding affinities. This behavior can be distinguished from a situation where all CPV particles are identical, and each particle has the same distribution of sites with different binding affinities. The different responses to viral heterogeneity can be traced to the Fab binding sites. A comparison of Fab binding to new and aged CPV capsids reveals that a post-translational modification at the binding site for Fab E (M569) probably reduces the binding affinity.

Mechanisms of antibody binding revealed by asymmetric Fab-virus complexes

“…The binding and occupancy of Fabs on parvovirus have also been determined previously using charge detection mass spectrometry (CDMS), which revealed that some of the tested monoclonal antibody-derived Fabs, including Fab 14, could fully occupy all 60 epitopes of the capsid, but with some differences in the kinetics of attachment (20). Incubating with excess Fab molecules to occupy all icosahedrally-equivalent sites on capsids has long been the preferred cryo-EM structural approach since this allows icosahedral symmetry averaging to be imposed during the reconstruction process for maximizing resolution (19,21,22).…”

High-resolution asymmetric structure of a Fab–virus complex reveals overlap with the receptor binding site

“…The binding and occupancy of Fabs on the CPV capsid have also been determined previously using charge detection mass spectrometry, which revealed that some of the tested monoclonal antibody–derived Fabs, including Fab 14, could fully occupy all 60 epitopes of the capsid but with some differences in the kinetics of attachment ( 24 ). Incubating with excess Fab molecules to occupy all icosahedrally equivalent sites on capsids has long been the preferred cryo-EM structural approach since this allows icosahedral symmetry averaging to be imposed during the reconstruction process for maximizing resolution ( 18 , 25 , 26 ).…”

Carbon Nanodot–Based Fluorescent Method for Virus DNA Analysis with Isothermal Strand Displacement Amplification

“…Up to now, traditional virus detection techniques include serological assay, mass spectrometry, and surface plasmon resonance (SPR) . However, those traditional methods are time‐consuming, laborious, less sensitive, which limit their clinical applications.…”