Accessing Different Protein Conformer Ensembles with Tunable Capillary Vibrating Sharp-Edge Spray Ionization

Capillary vibrating sharp-edge spray ionization (cVSSI) has been used to control the droplet charging of nebulized microdroplets and monitor effects on protein ion conformation makeup as determined by mass spectrometry (MS). Here it is observed that the application of voltage results in noticeable differences to the charge state distributions (CSDs) of ubiquitin ions. The data can be described most generally in three distinct voltage regions: Under low-voltage conditions (<+200 V, LV regime), low charge states (2+ to 4+ ions) dominate the mass spectra. For midvoltage conditions (+200 to +600 V, MV regime), higher charge states (7+ to 12+ ions) are observed. For high-voltage conditions (>+600 V, HV regime), the "nano-electrospray ionization (nESI)-type distribution" is achieved in which the 6+ and 5+ species are observed as the dominant ions. Analysis of these results suggests that different pathways to progeny nanodroplet production result in the observed ions. For the LV regime, aerodynamic breakup leads to low charge progeny droplets that are selective for the native solution conformation ensemble of ubiquitin (minus multimeric species). In the MV regime, the large droplets persist for longer periods of time, leading to droplet heating and a shift in the conformation ensemble to partially unfolded species. In the HV regime, droplets access progeny nanodroplets faster, leading to native conformation ensemble sampling as indicated by the observed nESI-type CSD. The notable observation of limited multimer formation and adduct ion formation in the LV regime is hypothesized to result from droplet aero breakup resulting in protein and charge carrier partitioning in sampled progeny droplets. The tunable droplet charging afforded by cVSSI presents opportunities to study the effects of the droplet charge, droplet size, and mass spectrometer inlet temperature on the conformer ensemble sampled by the mass spectrometer. Additionally, the approach may provide a tool for rapid comparison of protein stabilities.