Extended Protein Ions Are Formed by the Chain Ejection Model in Chemical Supercharging Electrospray Ionization

Abstract

Supercharging electrospray ionization can be a powerful tool for increasing charge states in mass spectra and generating unfolded ion structures, yet key details of its mechanism remain unclear. The structures of highly extended protein ions and the mechanism of supercharging were investigated using ion mobility-mass spectrometry. Head-to-tail-linked polyubiquitins (Ubq) were used to determine size and charge state scaling laws for unfolded protein ions formed by supercharging while eliminating amino acid composition as a potential confounding factor. Collisional cross section was found to scale linearly with mass for these ions and several other monomeric proteins, and the maximum observed charge state for each analyte scales with mass in agreement with an analytical charge state scaling law for protein ions with highly extended structures that is supported by experimental gas-phase basicities. These results indicate that these highly unfolded ions can be considered quasi-one-dimensional, and collisional cross sections modeled with the Trajectory Method in Collidoscope show that these ions are significantly more extended than linear α-helices but less extended than straight chains. The effect of internal disulfide bonds on the extent of supercharging was probed using bovine serum albumin, β-lactoglobulin, and lysozyme, each of which contains multiple internal disulfide bonds. Reduction of the disulfide bonds led to a marked increase in charge state upon supercharging without significantly altering folding in solution. This evidence supports a supercharging mechanism in which these proteins unfold before or during evaporation of the electrospray droplet and ionization occurs by the Chain Ejection Model.

The use of chromium(III) complexes to enhance peptide protonation by electrospray ionization mass spectrometry

“…Numerous studies have found that more highly charged ions are generated by the addition of small organic molecules to the biomolecule solutions undergoing ESI. This process is known as “supercharging.” Some studies suggest that supercharging is the result of denaturation; the organic reagents induce the unfolding of a molecule (ie, conformational change) and changes in droplet surface tension . Other studies suggest that supercharging is due to a direct interaction between biomolecules and supercharging reagents .…”

“…[32][33][34][35][36][37][38][39][40][41][42][43][44][45] Some studies suggest that supercharging is the result of denaturation; the organic reagents induce the unfolding of a molecule (ie, conformational change) and changes in droplet surface tension. [46][47][48][49][50][51] Other studies suggest that supercharging is due to a direct interaction between biomolecules and supercharging reagents. [52][53][54] Loo and coworkers proposed an ESI mechanism to predict and explain how supercharging reagents can increase analyte charge.…”

Conformational changes and the self-assembly of alpha-synuclein

“…This leads to protons attaching to the exposed portion of the ion, followed by further extrusion and ultimate ejection of the extended chain (Konermann et al, 2013). Much of the current evidence suggests that folded proteins formed by ESI from buffered aqueous solution ionize by the CRM, small ions by the IEM, and unfolded, disordered proteins by the CEM (Donor et al, 2017). A schematic illustrating these three models of ion formation by ESI is shown in Figure 1.7.…”

Aggregation and conformation of alpha-synuclein: effects of ligand binding and phosphomimetics

“…This model of ionisation preserves non-covalent complexes, as it requires less energy, as the ions are not expelled from within the droplet, but the droplet is removed from around them, and is therefore less damaging. It is generally accepted that compact proteins are ionised via the charge residue model (Donor et al, 2017).…”

“…The unfolded protein chain is partially ejected from the solvent droplet, leading protons to attach to the exposed portion of the ion. The ion is then further extruded from the droplet, gaining more charges, until it is fully ejected from the solvent (Konermann et al, 2013;Donor et al, 2017).…”