On the formation of highly charged gaseous ions from unfolded proteins by electrospray ionization

Electrospray ionization (ESI) of native proteins results in a narrow distribution of low protonation states. ESI for these folded species proceeds via the charged residue mechanism. In contrast, ESI of unfolded proteins yields a wide distribution of much higher charge states. The current work develops a model that can account for this effect. Recent molecular dynamics simulations revealed that ESI for unfolded polypeptide chains involves protein ejection from nanodroplets, representing a type of ion evaporation mechanism (IEM). We point out the analogies between this IEM, and the dissociation of gaseous protein complexes after collisional activation. The latter process commences with unraveling of a single subunit, in concert with Coulombically driven proton transfer. The subunit then separates from the residual complex as a highly charged ion. We propose that similar charge equilibration events accompany the IEM of unfolded proteins, thereby causing the formation of high ESI charge states. A bead chain model is used for examining how charge is partitioned as protein and droplet separate. It is shown that protein ejection from differently sized ESI droplets generates a range of protonation states. The predicted behavior agrees well with experimental data.