Combined charged residue-field emission model of macromolecular electrospray ionization

The mechanism of the multiple charging of macromolecules in electrospray ionization (ESI) continues to inspire debate and controversy. Recently, we proposed that the number of charges on a macromolecule is determined by the emission of small charge carriers from macromolecule-containing nanodroplets and that, after solvent evaporation, the remaining charge is transferred to the macromolecule. In this study, we tested the applicability of this new theory for macromolecular, positive-ion ESI mass spectrometry by measuring the mean charge states and charge distributions of globular proteins under non-denaturing and denaturing conditions. Predictions of protein mean charge states for native state proteins are in excellent agreement with mass spectrometric measurements, giving strong credence to the proposed theory. Theoretical predictions are also in good agreement with mean charge states measured for proteins in basic solutions (pH = 11.5). For some proteins in acidic solutions (pH = 2.1), the measured mean charge states are anomalously higher than the Rayleigh limit of a water droplet with a volume equivalent to that of the protein. We propose that some macromolecules that are highly charged in solution may desorb from charged droplets of the same polarity in a similar manner to that whereby charge carriers emit from nanodroplets, leading to "supercharged" macromolecular ions. Examination of rate expressions for solvent evaporation and charge-carrier emission demonstrates that the newly proposed model for ESI is consistent with previously reported ion-emission kinetics. Overall, we obtained additional experimental evidence for the charge carrier emission model for macromolecular charging, suggesting opportunities for understanding and applying ESI-MS.