Origin of the conformation dependence of protein charge‐state distributions in electrospray ionization mass spectrometry

Abstract

The influence of tertiary structure on the electrospray ionization mass spectra of proteins is a well known and broadly exploited phenomenon. However, the underlying mechanism is not well understood. This paper discusses the bases and the implications of the two current hypotheses (solvent accessibility and Coulombic repulsions), pointing out the remaining open questions. Evidence reported here supports a third hypothesis, i.e. that intramolecular interactions in folded proteins play a key role in determining the observed charge-state distributions. It is proposed that native protein structures stabilize to a large extent pre-existing charges of the opposite polarity to the net charge of the ion, preventing their neutralization during the electrospray process. Thus, the higher charge states of unfolded conformations, relative to the folded structure, would not derive from a more extensive ionization of the former, but rather from a higher content of neutralizing charges in the latter. This interpretation allows several other problematic observations to be explained, including the different shapes of the spectra of folded and unfolded proteins, the discrepancies between observed and predicted gas-phase reactivity of protein ions and the apparent inconsistency of positive- and negative-ion mode results.

A Computational Model for Protein Ionization by Electrospray Based on Gas-Phase Basicity

“…This finding is consistent with recent experimental evidence [6,37,38]. It also supports the hypothesis that a higher propensity for zwitterionic states of folded versus unfolded proteins can lower the net charge of the former, contributing to conformational effects in ESI-MS [39]. In contrast, there is no clear dependence of the number of salt bridges on the protein size ( Figure SI-4).…”

“…More generally, it is easy to see from a simple dimensional analysis that any stabilizing contribution (−ξS) that depends on the surface area (S) yields a ffiffiffiffi ffi M p dependence of the maximum charge possible. Indeed, the electrostatic potential is dimensionally proportional to charge 2 ×length -1 , whereas Comparison with the main charge state is made, instead of maximum or average charge state, because of its less ambiguous determination from literature data [39,51] the stabilizing contribution is proportional to length 2 . Thus, the square of maximum charge is proportional to a volume, and consequently the charge must be proportional to the square root of the mass.…”

Diffusion measurements by electrospray mass spectrometry for studying solution-phase noncovalent interactions

“…It is generally accepted that protein unfolding in solution leads to the formation of more highly charged protein ions in positive-ion ESI [77][78][79][80][81][82][83]. However, the physical basis of this phenomenon is still a matter of debate [23,84,85]. According to one hypothesis, a major determinant of the ESI charge state distribution is the "compactness" of a protein in solution [51, 83, 86 -88].…”

Co-populated Conformational Ensembles of β2-Microglobulin Uncovered Quantitatively by Electrospray Ionization Mass Spectrometry

“…Several models have been proposed to rationalize the observed differences in the charge state distributions of native and nonnative states. Differences in the solvent accessibility of ionizable groups (23,27), heightened Coulomb energies of folded compared with unfolded polyprotonated conformations (28,29), or the increased protection against charge neutralization offered by native protein conformations (30) have all been offered as plausible explanations. Whichever mechanism or combination of processes governs this phenomenon, the net result is that conformational species with more open structures carry, on average, more charges and produce broader ion envelopes centered on higher charge states than their more compact native counterparts (31).…”