Molecular Dynamics Simulations of Native Protein Charging in Electrosprayed Droplets with Experimentally Relevant Compositions

Electrospray ionization-mass spectrometry (ESI-MS) has been widely used to study proteins given its preservation of native-protein structure when transitioning to the gas-phase. Understanding the influence of experimental factors on ESI can provide insight into the resulting charge states, as well as the degree to which “native” structure is maintained. Experimentally, it is challenging to characterize nanometer-scale electrosprayed droplets; however, molecular dynamics (MD) simulations pose an attractive solution by providing a molecular perspective of protein-ion formation. By resolving many approximations used in past MD simulations of ESI, we demonstrate the capability of simulating electrosprayed droplets with experimentally relevant droplet compositions and behavior. This is accomplished by modeling proton transfers between all titratable molecules in simulated droplets under atmospheric conditions; thus, enabling simulated droplets containing ammonium acetate that form experimentally observed (de)protonated protein ions. Application of the proposed protocol to several native proteins in positive- and negative-ion mode ESI produced simulated weighted averages of charge-state distributions that differed by 0.14 compared to experimental values. Our simulations suggest that changes in residue basicity during the transition to the gas-phase play a significant role in moderating protein charging during native-ESI and can explain many experimentally observed trends. Ionic protein structures produced via the simulated model maintained, on average, 73% of their native contacts into the gas phase relative to solution-phase structures. While applied towards native proteins here, novel insights into effects of the transition to gas-phase enable a deeper understanding of the ESI process itself and thus, are informative regardless of analyte.