Molecular Dynamics Simulations of Native Protein Charging via Proton Transfer during Electrospray Ionization with...

Unraveling the mechanism by which native proteins are charged through electrospray ionization (ESI) has been the focus of considerable research because observable charge states can be correlated to biophysical characteristics, such as protein folding and, thus, solution conformation. Difficulties in characterizing electrosprayed droplets have catalyzed the use of molecular dynamics (MD) to provide insights into the mechanisms by which proteins are charged and transferred to the gas phase. However, prior MD studies have utilized metal ions, primarily Na⁺, as charge carriers, even though proteins are primarily detected as protonated ions in the mass spectra. Here, we propose a modified MD protocol for simulating discrete Grotthuss diffuse H₃O⁺ that is capable of dynamically altering amino-acid protonation states to model electrospray charging and gaseous ion formation of model proteins, ubiquitin, and myoglobin. Application of the protocol to the evaporation of acidic droplets enables a molecular perspective of H₃O⁺ coordination and proton transfer to/from proteins, which is unfeasible with the metal charge carriers used in previous MD studies of ESI. Our protocol recreates experimentally observed charge-state distributions and supports the charge residue model (CRM) as the dominant mechanism of native protein ionization during ESI. Additionally, our results suggest that protonation is highly specific to individual residues and is correlated to the formation of localized hydrated regions on the protein surface as droplets desolvate. Considering the use of discrete H₃O⁺ instead of Na⁺, the developed protocol is a necessary step toward developing a more comprehensive model of protein ionization during ESI.