Examining DNA Structures with In-droplet Hydrogen/Deuterium Exchange Mass Spectrometry.

Capillary vibrating sharp-edge spray ionization (cVSSI) combined with hydrogen/deuterium exchange-mass spectrometry (HDX-MS) has been utilized to characterize different solution-phase DNA conformers including DNA G-quadruplex topologies as well as triplex DNA and duplex DNA. In general, G-quadruplex DNA shows a wide range of protection of hydrogens extending from ~12% to ~21% deuterium incorporation. Additionally, the DNA sequences selected to represent parallel, antiparallel, and hybrid G-quadruplex topologies exhibit slight differences in deuterium uptake levels which appear to loosely relate to overall conformer stability. Notably, the exchange level for one of the hybrid sequence sub topologies of G-quadruplex DNA (24 TTG) is significantly different (compared with the others studied here) despite the DNA sequences being highly comparable. For the quadruplex-forming sequences, correlation analysis suggests protection of base hydrogens involved in tetrad hydrogen bonding. For duplex DNA ~19% deuterium incorporation is observed while only ~16% is observed for triplex DNA. This increased protection of hydrogens may be due to the added backbone scaffolding and Hoogsteen base pairing of the latter species. These experiments lay the groundwork for future studies aimed at determining the structural source of this protection as well as the applicability of the approach for ascertaining different oligonucleotide folds, co-existing conformations, and/or overall conformer flexibility.

Development of cVSSI-APCI for the Improvement of Ion Suppression and Matrix Effects in Complex Mixtures.

The new ionization technique termed vibrating sharp-edge spray ionization (cVSSI) has been coupled with corona discharge to investigate atmospheric pressure chemical ionization (APCI) capabilities. The optimized source was evaluated for its ability to enhance ion signal intensity, overcome matrix effects, and limit ion suppression. The results have been compared with state-of-the-art ESI source performance as well as a new APCI-like source. In methanol, the ion signal intensity increased 10-fold and >10-fold for cocaine and the suppressed analytes, respectively. The ability to overcome ion suppression was improved from 2-fold to 16-fold for theophylline and vitamin D2, respectively. For aqueous samples, ion signal levels increased by two orders of magnitude for all analytes. In both solvent systems, the signal-to-noise ratios also increased for all suppressed analytes. One example of the characterization of low-ionizing (by ESI or cVSSI alone) species in the presence of high-ionizing species by direct analysis from a cotton swab is presented. The work is discussed with respect to the advantages of cVSSI-APCI for direct, in situ, and field analyses.

Characterizing Multidevice Capillary Vibrating Sharp-Edge Spray Ionization for In-Droplet Hydrogen/Deuterium Exchange to Enhance Compound Identification.

Multidevice capillary vibrating sharp-edge spray ionization (cVSSI) source parameters have been examined to determine their effects on conducting in-droplet hydrogen/deuterium exchange (HDX) experiments. Control experiments using select compounds indicate that the observed differences in mass spectral isotopic distributions obtained upon initiation of HDX result primarily from solution-phase reactions as opposed to gas-phase exchange. Preliminary studies have determined that robust HDX can only be achieved with the application of same-polarity voltage to both the analyte and the deuterium oxide reagent (D2O) cVSSI devices. Additionally, a similar HDX reactivity dependence on the voltage applied to the D2O device for various analytes is observed. Analyte and reagent flow experiments show that, for the multidevice cVSSI setup employed, there is a nonlinear dependence on the D2O reagent flow rate; increasing the D2O reagent flow by 100% results in only an ∼10-20% increase in deuterium incorporation for this setup. Instantaneous (subsecond) response times have been demonstrated in the initiation or termination of HDX, which is achieved by turning on or off the reagent cVSSI device piezoelectric transducer. The ability to distinguish isomeric species by in-droplet HDX is presented. Finally, a demonstration of a three-component cVSSI device setup to perform multiple (successive or in combination) in-droplet chemistries to enhance compound ionization and identification is presented and a hypothetical metabolomics workflow consisting of successive multidevice activation is briefly discussed.

Combining Field-Enabled Capillary Vibrating Sharp-Edge Spray Ionization with Microflow Liquid Chromatography and Mass Spectrometry to Enhance 'Omics Analyses.

Field-enabled capillary vibrating sharp-edge spray ionization (cVSSI) has been combined with high-flow liquid chromatography (LC) and mass spectrometry (MS) to establish current ionization capabilities for metabolomics and proteomics investigations. Comparisons are made between experiments employing cVSSI and a heated electrospray ionization probe representing the state-of-the-art in microflow LC-MS methods for 'omics studies. For metabolomics standards, cVSSI is shown to provide an ionization enhancement by factors of 4 ± 2 for both negative and positive ion mode analyses. For chymotryptic peptides, cVSSI is shown to provide an ionization enhancement by factors of 5 ± 2 and 2 ± 1 for negative and positive ion mode analyses, respectively. Slightly broader high-performance liquid chromatography peaks are observed in the cVSSI datasets, and several studies suggest that this results from a slightly decreased post-split flow rate. This may result from partial obstruction of the pulled-tip emitter over time. Such a challenge can be remedied with the use of LC pumps that operate in the 10 to 100 µL·min-1 flow regime. At this early stage, the proof-of-principle studies already show ion signal advantages over state-of-the-art electrospray ionization (ESI) for a wide variety of analytes in both positive and negative ion mode. Overall, this represents a ∼20-50-fold improvement over the first demonstration of LC-MS analyses by voltage-free cVSSI. Separate comparisons of the ion abundances of compounds eluting under identical solvent conditions reveal ionization efficiency differences between cVSSI and ESI and may suggest varied contributions to ionization from different physicochemical properties of the compounds. Future investigations of parameters that could further increase ionization gains in negative and positive ion mode analyses with the use of cVSSI are briefly presented.

Rapid Solution-Phase Hydrogen/Deuterium Exchange for Metabolite Compound Identification.

Rapid, solution-phase hydrogen/deuterium exchange (HDX) coupled with mass spectrometry (MS) is demonstrated as a means for distinguishing small-molecule metabolites. HDX is achieved using capillary vibrating sharp-edge spray ionization (cVSSI) to allow sufficient time for reagent mixing and exchange in micrometer-sized droplets. Different compounds are observed to incorporate deuterium with varying efficiencies resulting in unique isotopic patterns as revealed in the MS spectra. Using linear regression techniques, parameters representing contribution to exchange by different hydrogen types can be computed. In this proof-of-concept study, the exchange parameters are shown to be useful in the retrodiction of the amount of deuterium incorporated within different compounds. On average, the exchange parameters retrodict the exchange level with ~ 2.2-fold greater accuracy than treating all exchangeable hydrogens equally. The parameters can be used to produce hypothetical isotopic distributions that agree (± 16% RMSD) with experimental measurements. These initial studies are discussed in light of their potential value for identifying challenging metabolite species.

Comparison of Peptide Ion Conformers Arising from Non-Helical and Helical Peptides Using Ion Mobility Spectrometry and Gas-Phase Hydrogen/Deuterium Exchange.

The dominant gas-phase conformer of [M+3H]3+ ions of the model peptide acetyl-PSSSSKSSSSKSSSSKSSSSK has been examined with ion mobility spectrometry (IMS), gas-phase hydrogen deuterium exchange (HDX), and mass spectrometry (MS) techniques. The [M+3H]3+ peptide ions are observed predominantly as a relatively compact conformer type. Upon subjecting these ions to electron transfer dissociation (ETD), the level of protection for each amino acid residue in the peptide sequence is assessed. The overall per-residue deuterium uptake is observed to be relatively more efficient for the neutral residues than for the model peptide acetyl-PAAAAKAAAAKAAAAKAAAAK. In comparison, the N-terminal and C-terminal regions of the serine peptide show greater relative protection compared with interior residues. Molecular dynamics (MD) simulations have been used to generate candidate structures for collision cross section and HDX reactivity matching. Hydrogen accessibility scoring (HAS) for select structural candidates from MD simulations has been used to suggest conformer types that could contribute to the observed HDX patterns. The results are discussed with respect to recent studies employing extensive MD simulations of gas-phase structure establishment of a peptide system. Graphical Abstract ᅟ.

Ion Mobility Spectrometry-Mass Spectrometry Coupled with Gas-Phase Hydrogen/Deuterium Exchange for Metabolomics Analyses.

Ion mobility spectrometry-mass spectrometry (IMS-MS) in combination with gas-phase hydrogen/deuterium exchange (HDX) and collision-induced dissociation (CID) is evaluated as an analytical method for small-molecule standard and mixture characterization. Experiments show that compound ions exhibit unique HDX reactivities that can be used to distinguish different species. Additionally, it is shown that gas-phase HDX kinetics can be exploited to provide even further distinguishing capabilities by using different partial pressures of reagent gas. The relative HDX reactivity of a wide variety of molecules is discussed in light of the various molecular structures. Additionally, hydrogen accessibility scoring (HAS) and HDX kinetics modeling of candidate (in silico) ion structures is utilized to estimate the relative ion conformer populations giving rise to specific HDX behavior. These data interpretation methods are discussed with a focus on developing predictive tools for HDX behavior. Finally, an example is provided in which ion mobility information is supplemented with HDX reactivity data to aid identification efforts of compounds in a metabolite extract. Graphical Abstract ᅟ.

Magnifying ion mobility spectrometry-mass spectrometry measurements for biomolecular structure studies.

Ion mobility spectrometry-mass spectrometry (IMS-MS) provides information about the structures of gas-phase ions in the form of a collision cross section (CCS) with a neutral buffer gas. Indicating relative ion size, a CCS value alone is of limited utility. Although such information can be used to propose different conformer types, finer details of structure are not captured. The increased accessibility of IMS-MS measurements with commercial instrumentation in recent years has ballooned its usage in combination with separate measurements to provide enhanced data from which greater structural inferences can be drawn. This short review presents recent outstanding developments in scientific research that employs complementary measurements that when combined with IMS-MS data are used to characterize the structures of a wide range of compounds.