A Mass-Spectrometry-Based Modelling Workflow for Accurate Prediction of IgG Antibody Conformations in the Gas Phase

Abstract

Immunoglobulins are biomolecules involved in defence against foreign substances. Flexibility is key to their functional properties in relation to antigen binding and receptor interactions. We have developed an integrative strategy combining ion mobility mass spectrometry (IM-MS) with molecular modelling to study the conformational dynamics of human IgG antibodies. Predictive models of all four human IgG subclasses were assembled and their dynamics sampled in the transition from extended to collapsed state during IM-MS. Our data imply that this collapse of IgG antibodies is related to their intrinsic structural features, including Fab arm flexibility, collapse towards the Fc region, and the length of their hinge regions. The workflow presented here provides an accurate structural representation in good agreement with the observed collision cross section for these flexible IgG molecules. These results have implications for studying other nonglobular flexible proteins.

Keywords: conformation analysis; immunoglobulin; ion mobility; mass spectrometry; molecular dynamics.

Figure 1

Schematics and workflow for modelling antibody flexibility. a) Schematic representation of human IgG1–4 subclasses. b) Representative structure of IgG1, denoting hinge substructure and modes of Fab movement stemming from the upper hinge. c) Integrative workflow generating and comparing the calculated CCS values of initial, post‐sampling, and gas‐phase MD models with experimental CCS values.

Figure 2

Modelling the conformational flexibility of antibodies. a) Representative mobilogram and native mass spectrum (i) and CCS distributions for 21–23+ charge states of IgG2 (ii). b) Space occupied by IgG1–4 Fabs following upper‐hinge flexibility sampling. Each sphere represents one model for each IgG Fab heavy chain (teal and purple). Light chains are shown as blue. Initial models are shown as surface representations. c) Overlay of experimental CCS distribution with triplicate simulated collapse models. Experimental error is represented by the ±6 % dotted lines. Purple error bars represent the CCS range over the last 1 ns of gas‐phase simulation.

Figure 3

Summary of experimental and model CCS for IgG1–4. CCS was calculated for each stage of the modelling workflow. The reduction in CCS between modelling stages is shown by percentages. Error bars for the experimental data points (green) represent the standard deviation of measurement. Error bars for simulated collapse models (red) show CCS range over the last 1 ns of simulation.

Figure 4

Proposed collapse pathway of IgG during ESI. a) IgG molecules exhibit full flexibility in solution. b) Nanospray ESI produces charged droplets in which IgG molecules retain partial flexibility depending on droplet size. c) Gradual evaporation of droplets coerces flexible IgG molecules into more compact topologies. Solvent charges migrate to protein surfaces as they become exposed through desolvation (CRM). d) Dry protein ions are inflexible in vacuum and represent a distribution of compact conformations.