Formation of gaseous protein ions from aqueous ammonium acetate droplets during native electrospray: insights from...

The transition of proteins from solution into the gas phase during native electrospray ionization (ESI) continues to be an enigmatic topic. Molecular dynamics (MD) simulations are an important avenue for gaining mechanistic insights, but most previous MD studies did not consider the role of proton transfer (PT) events. Here, we overcome this limitation by implementing a droplet mobile-proton MD (dMPMD) protocol for aqueous ammonium acetate solutions. The droplet radius (up to 8.5 nm) and protein size (up to 55 kDa) are the largest studied to date, bringing the simulations closer to experimental conditions than any earlier modeling studies. Consistent with experiments, our data show that PT among NH₄⁺, acetate (Ac^-), and protein sites produces NH₃ and acetic acid (HAc). NH₃ vaporizes rapidly, while HAc accumulation causes Ac^-/HAc buffering at pH 4.76 ± 1. Droplet evaporation near the Rayleigh limit produces gaseous proteins with a net charge close to that of protein-sized water droplets, in line with the charged residue model (CRM). The CRM/Rayleigh framework applies regardless of amino acid sequence and residue basicity. For proteins with very few basic sites, charging involves carbonyl-trapped NH₄⁺ adducts. Switching to gas-phase mobile-proton MD (gMPMD) simulations, we track proteins after their release from droplets, culminating in native-like conformers with numerous salt bridges. Our data provide unprecedented insights into the ESI process, and the maturation of nascent protein ions to metastable structures that are detectable in experiments.