Pushing the Boundaries of Native Mass Spectrometry: Structural Characterization of Ultra-High Molecular Weight Protein Complexes

Introduction: ADDomers are self-assembling dodecahedral virus-like particles (VLPs) derived from adenovirus, that represent a promising new synthetic antivenom platform to solve issues of snakebite envenomation (a neglected tropical disease1) due to their high stability, multivalency, and cost-efficiency . Their structural characterization remains challenging. This work introduces a native Mass Spectrometry (MS) and Ion Mobility (IM-MS) workflow to monitor the assembly kinetics and conformational stability of such 3.5 MDa complexes.
Methods: A modified Synapt G2-S (Waters) for high mass capabilities and native MS and Collision-Induced Unfolding (CIU) experiments was used on model proteins (Myoglobin, Insulin, and bovine serum albumin as BSA) in non-denaturing and denaturing conditions. Energy-resolved collision induced dissociation (or breakdown curves survival yield (SY) experiments) were acquired to determine the relative stability of the molecular assemblies (V50 values as voltage where 50% of SY of the assemblies).
Main results: Training and validation using Holo-Myoglobin [M+9H+]9+ demonstrated stable ion beam in non-denaturing MS conditions. CIU heatmaps showed three specific gas-phase population of conformation. Breakdown curves experiments and survival yield of insulin were successfully determined. Additionally, BSA native mass spectra in reduced and Oxidized forms identified overalkylation because of a carbamidomethylation patterns of +2 kDa (Corresponding to the 35 residues submitted to alkylation for 35 cysteines in BSA sequence). IM-MS and CIU will be applied to protein models of increasing mass before working with ADDomers for mapping their supramolecular assembly and avidity for toxins.