Field-free capillary vibrating sharp-edge spray ionization (cVSSI) is evaluated for its ability to conduct native mass spectrometry (MS) experiments. The charge state distributions for nine globular proteins are compared using field-free cVSSI, field-enabled cVSSI, and electrospray ionization (ESI). In general, for both positive and negative ion mode, the average charge state (q avg) increases for field-free cVSSI with increasing molecular weight similar to ESI. A clear difference is that the q avg is significantly lower for field-free conditions in both analyses. Two proteins, leptin and thioredoxin, exhibit bimodal charge state distributions (CSDs) upon the application of voltage in positive ion mode; only a monomodal distribution is observed for field-free conditions. In negative ion mode, thioredoxin exhibits a multimodal CSD upon the addition of voltage to cVSSI. Extensive molecular dynamics (MD) simulations of myoglobin and leptin in nanodroplets suggest that the multimodal CSD for leptin may originate from increased conformational “breathing” (decreased packing) and association with the droplet surface. These properties along with increased droplet charge appear to play critical roles in shifting ionization processes for some proteins. Further exploration and development of field-free cVSSI as a new ionization source for native MS especially as applied to more flexible biomolecular species is warranted.

Rationale Many different structure analysis techniques are not capable of probing the heterogeneity of solution conformations. Here, we examine the ability of in‐droplet hydrogen–deuterium exchange (HDX) to directly probe solution conformer heterogeneity of a protein with mass spectrometry (MS) detection. Methods Two vibrating capillary vibrating sharp‐edge spray ionization (cVSSI) devices have been arranged such that they generate microdroplet plumes of the analyte and D2O reagent, which coalesce to form reaction droplets where HDX takes place in the solution environment. The native HDX–MS setup has been first explored for two model peptides that have distinct structural compositions in solution. The effectiveness of the multidevice cVSSI–HDX in illustrating structural details has been further exploited to investigate coexisting solution‐phase conformations of the protein ubiquitin. Results In‐droplet HDX reveals decreased backbone exchange for a model peptide that has a greater helix‐forming propensity. Differences in intrinsic rates of the alanine and serine residues may account for much of the observed protection. The data allow the first estimates of backbone exchange rates for peptides undergoing in‐droplet HDX. That said, the approach may hold greater potential for investigating the tertiary structure and structural transitions of proteins. For ubiquitin protein, HDX reactivity differences suggest that multiple conformers are present in native solutions. The addition of methanol to buffered aqueous solutions of ubiquitin results in increased populations of solution conformers of higher reactivity. Data analysis suggests that partially folded conformers such as the A‐state of ubiquitin increase with methanol content; the native state may be preserved to a limited degree even under stronger denaturation conditions. Conclusion The deuterium uptake after in‐droplet HDX has been observed to correspond to some degree with peptide backbone hydrogen protection based on differences in intrinsic rates of exchange. The presence of coexisting protein solution structures under native and denaturing solution conditions has been distinguished by the isotopic distributions of deuterated ubiquitin ions.