Enhancing Protein Electrospray Charge States by Multivalent Metal Ions: Mechanistic Insights from MD Simulations and Mass Spectrometry Experiments.

Authors

Leanne M Martin
Lars Konermann

Document Type

Article

Publication Date

1-2-2020

Journal

Journal of the American Society for Mass Spectrometry

Volume

31

Issue

1

First Page

25

Last Page

33

Abstract

The structure and reactivity of electrosprayed protein ions is governed by their net charge. Native proteins in non-denaturing aqueous solutions produce low charge states. More highly charged ions are formed when electrospraying proteins that are unfolded and/or exposed to organic supercharging agents. Numerous studies have explored the electrospray process under these various conditions. One phenomenon that has received surprisingly little attention is the charge enhancement caused by multivalent metal ions such as La³⁺ when electrospraying proteins out of non-denaturing solutions. Here, we conducted mass spectrometry and ion mobility spectrometry experiments, in combination with molecular dynamics (MD) simulations, to uncover the mechanistic basis of this charge enhancement. MD simulations of aqueous ESI droplets reproduced the experimental observation that La³⁺ boosts protein charge states relative to monovalent metals (e.g., Na⁺). The simulations showed that gaseous proteins were released by solvent evaporation to dryness, consistent with the charged residue model. Metal ion ejection kept the shrinking droplets close to the Rayleigh limit until ∼99% of the solvent had left. For droplets charged with Na⁺, metal adduction during the final stage of solvent evaporation produced low protein charge states. Droplets containing La³⁺ showed a very different behavior. The trivalent nature of La³⁺ favored adduction to the protein at a very early stage, when most of the solvent had not evaporated yet. This irreversible binding via multidentate contacts suppressed La³⁺ ejection from the vanishing droplets, such that the resulting gaseous proteins carried significantly more charge. Our results illustrate that MD simulations are suitable for uncovering intricate aspects of electrospray mechanisms, paving the way toward an atomistic understanding of mass spectrometry based analytical workflows.