Formation of Gaseous Peptide Ions from Electrospray Droplets: Competition between the Ion Evaporation Mechanism and Charged Residue Mechanism.

The transfer of peptide ions from solution into the gas phase by electrospray ionization (ESI) is an integral component of mass spectrometry (MS)-based proteomics. The mechanisms whereby gaseous peptide ions are released from charged ESI nanodroplets remain unclear. This is in contrast to intact protein ESI, which has been the focus of detailed investigations using molecular dynamics (MD) simulations and other methods. Under acidic liquid chromatography/MS conditions, many peptides carry a solution charge of 3+ or 2+. Because of this pre-existing charge and their relatively small size, prevailing views suggest that peptides follow the ion evaporation mechanism (IEM). The IEM entails analyte ejection from ESI droplets, driven by electrostatic repulsion between the analyte and droplet. Surprisingly, recent peptide MD investigations reported a different behavior, that is, the release of peptide ions droplet evaporation to dryness which represents the hallmark of the charged residue mechanism (CRM). Here, we resolved this conundrum by performing MD simulations on a common model peptide (bradykinin) in Rayleigh-charged aqueous droplets. The primary focus was on pH 2 conditions (bradykinin solution charge = 3+), but we also verified that our MD strategy captured pH-dependent charge state shifts seen in ESI-MS experiments. In agreement with earlier simulations, we found that droplets with initial radii of 1.5-3 nm predominantly release peptide ions the CRM. In contrast, somewhat larger radii (4-5 nm) favor IEM behavior. It appears that these are the first MD data to unequivocally demonstrate the viability of peptide IEM events. Electrostatic arguments can account for the observed droplet size dependence. In summary, both CRM and IEM can be operative in peptide ESI-MS. The prevalence of one over the other mechanism depends on the droplet size distribution in the ESI plume.