Capillary Vibrating Sharp-Edge Spray Ionization Augments Field-Free Ionization Techniques to Promote Conformer Preservation in the Gas-Phase for Intractable Biomolecular Ions

Abstract

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.

In‐droplet hydrogen–deuterium exchange to examine protein/peptide solution conformer heterogeneity

“…Recently it has been shown that this allows improved preservation of the structure of fragile proteins relative to nESI. 48,49 These unique attributes (softness, ionization efficiency, and simplicity/accessibility) of cVSSI suggest that it may be possible and indeed useful to couple native MS with in-droplet HDX for examining challenging biomolecular solution structures including coexisting conformers and structural transformations. In seeking to apply new techniques to the study of the biomolecular structure, this work builds upon the seminal experiments of Derrick and Williams and their coworkers who studied the nature of in-droplet processes for biomolecules using theta capillaries.…”

“…This was accomplished while preserving biomolecular structures such as that demonstrated by the mass spectra for G‐quadruplex DNA ions, which were shown to be similar to those acquired using ESI under native conditions. The second advantage of cVSSI has been demonstrated recently, where the new ionization approach is shown to be a remarkably gentle technique allowing the preservation of solution structures for fragile proteins that may unravel during the ESI process 48 . A third attribute of cVSSI is its relative simplicity in that a droplet plume is generated in a straightforward fashion without the need for an auxiliary nebulization gas that would not allow facile in‐droplet HDX measurements.…”

“…In addition, the technique decouples droplet production from a coulombically driven process such as encountered with nESI. Recently it has been shown that this allows improved preservation of the structure of fragile proteins relative to nESI 48,49 . These unique attributes (softness, ionization efficiency, and simplicity/accessibility) of cVSSI suggest that it may be possible and indeed useful to couple native MS with in‐droplet HDX for examining challenging biomolecular solution structures including coexisting conformers and structural transformations.…”

Integration of capillary vibrating sharp-edge spray ionization as a nebulization device for ICP-MS

“…Since the development of cVSSI in 2019, several applications for mass spectrometry have been explored, including uses in droplet hydrogen/deuterium exchange, liquid chromatography, solid phase microextraction, and native biomolecule analysis. [28][29][30][31][32] Here, we present the rst application of cVSSI as a nebulization device for ICP-MS, and also discuss the fused deposition modeling (3D printing) of a sample introduction chamber to interface the cVSSI nebulization device with the ICP. Previous studies have shown the feasibility of using fused deposition modeling to create spray chambers for ICP-MS. Thompson demonstrated the ability to 3D model, print, and use cyclonic spray chambers for ICP optical emissions spectroscopy.…”

“…Since the development of cVSSI in 2019, several applications for mass spectrometry have been explored, including uses in droplet hydrogen/deuterium exchange, liquid chromatography, solid phase microextraction, and native biomolecule analysis. 28–32…”