Native Electrospray Ionization of Multi-Domain Proteins via a Bead Ejection Mechanism

Native ion mobility mass spectrometry is potentially useful for the biophysical characterization of proteins, as the electrospray charge state distribution and the collision cross section distribution depend on their solution conformation. We examine here the charging and gas-phase conformation of multi-domain therapeutic proteins comprising globular domains tethered by disordered linkers. The charge and collision cross section distributions are multimodal, suggesting several conformations in solution, as confirmed by solution hydrogen/deuterium exchange. The most intriguing question is the ionization mechanism of these structures: a fraction of the population does not follow the charged residue mechanism but cannot ionize by pure chain ejection either. We deduce that a hybrid mechanism is possible, wherein globular domains are ejected one at a time from a parent droplet. The charge vs solvent accessible surface area correlations of denatured and intrinsically disordered proteins are also compatible with this “bead ejection mechanism”, which we propose as a general tenet of biomolecule electrospray.