Understanding the charging mechanism of electrospray ionization (ESI) is central to explaining the relationship between protein structure and analyte charging in ESI mass spectrometry (MS). Today, a popular view of the ESI mechanism is that that low molecular weight species (&lt;6 kDa) are emitted by ion evaporation, that folded, globular proteins are released in accordance with the charged residue model (CRM), and that unfolded proteins and long, linear polymers are released by either a modified CRM (in which the droplet is distorted by the presence of the analyte) or by the chain ejection model (CEM, which envisions that unfolded proteins are gradually ejected from droplets as extended tails). Recently, investigators have suggested that a fourth mechanism applies to intrinsically disordered proteins (IDPs). The need for increasingly more models to explain analyte charge state distributions (CSDs) and gas phase structures could be evidence that existing models are deficient. We seek a single ESI model incorporating both solution and gas phase properties for analyte and solvent to explain protein charging. ESI can be viewed as a kinetically controlled process in which decomposing droplets dump their excess charge. Dispersed progeny droplets, solvent, analyte, and other solution components compete for this charge, with the ultimate allocation governed by the components’ changing basicity properties in the transition from solution to gas phase. This model successfully predicts many trends unaddressed by current paradigms, including CSD shifts to higher m/z for concentrated analytes and shifts to lower m/z for nanosprays employing smaller emitter diameters. From this view, a single mechanism can explain how reagents that promote analyte charging (“supercharging”), such as sulfolane, can increase analyte charge from “denaturing” and “native” solvent systems. Those additives alter how excess charge is allocated between solvent (H2O), buffer, and analyte, in part by disrupting hydrogen bonding between water molecules. Additives’ Brønsted basicities are inversely correlated to their ability to shift CSDs to lower m/z in positive ESI. This same mechanism explains how ESI from an aqueous solution can generate proteins so highly charged charge that they protonate argon.

Charging proteins by electrospray ionization

2019 Fall National Meeting

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