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.

In-droplet hydrogen-deuterium exchange to examine protein/peptide solution conformer heterogeneity.

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 D2 O 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.

N6-methyladenosine (M6A) in fetal offspring modifies mitochondrial gene expression following gestational nano-TiO2 inhalation exposure.

N6-methyladenosine (m6A) is the most prominent epitranscriptomic modification to RNA in eukaryotes, but it's role in adaptive changes within the gestational environment are poorly understood. We propose that gestational exposure to nano titanium dioxide (TiO2) contributes to cardiac m6A methylation in fetal offspring and influences mitochondrial gene expression. 10-week-old pregnant female FVB/NJ wild-type mice underwent 6 nonconsecutive days of whole-body inhalation exposure beginning on gestational day (GD) 5. Mice were exposed to filtered room air or nano-TiO2 with a target aerosol mass concentration of 12 mg/m3. At GD 15 mice were humanely killed and cardiac RNA and mitochondrial proteins extracted. Immunoprecipitation with m6A antibodies was performed followed by sequencing of immunoprecipitant (m6A) and input (mRNA) on the Illumina NextSeq 2000. Protein extraction, preparation, and LC-MS/MS were used for mitochondrial protein quantification. There were no differences in maternal or fetal pup weights, number of pups, or pup heart weights between exposure and control groups. Transcriptomic sequencing revealed 3648 differentially expressed mRNA in nano-TiO2 exposed mice (Padj ≤ 0.05). Transcripts involved in mitochondrial bioenergetics were significantly downregulated (83 of 85 genes). 921 transcripts revealed significant m6A methylation sites (Padj ≤ 0.10). 311 of the 921 mRNA were identified to have both 1) significantly altered expression and 2) differentially methylated sites. Mitochondrial proteomics revealed decreased expression of ATP Synthase subunits in the exposed group (P ≤ 0.05). The lack of m6A modifications to mitochondrial transcripts suggests a mechanism for decreased transcript stability and reduced protein expression due to gestational nano-TiO2 inhalation exposure.

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

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 (qavg) increases for field-free cVSSI with increasing molecular weight similar to ESI. A clear difference is that the qavg 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.

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.

Physicochemical Property Correlations with Ionization Efficiency in Capillary Vibrating Sharp-Edge Spray Ionization (cVSSI).

The relative contributions to ionization efficiency by three molecular chemical properties have been examined for field-free and field-enabled capillary vibrating sharp-edge spray ionization (cVSSI) using mass spectrometry (MS) analysis. Ion intensities have been recorded for model compounds under each operational ionization mode as well as for aqueous and nonaqueous (methanol) solvent systems. Multiple regression analysis suggests that for field-free cVSSI, ion intensity is mostly associated with the log of the base dissociation constant (pKb) and proton affinity (PA) for both aqueous and methanol solutions. Comparatively, for field-enabled cVSSI using aqueous solutions, the dominant factor correlated with ion intensity is the log of the partition coefficient (log P). To a lesser degree, this is observed for methanol solutions as well. For ESI, pKb is the dominant factor associated with ion signal levels from methanol and aqueous solutions. These results are supported by studies conducted on two different mass spectrometers employing different cVSSI emitter tips. The relationship of ion intensity and pKb in ESI is supported by multiple studies; however, the shift to other chemical properties with the addition of cVSSI suggests the possibility that a different (or combinations of) ionization mechanism(s) may be operative for these ionization modes. These results are briefly considered in light of the different ESI mechanisms.

Investigating the interactions of the first 17 amino acid residues of Huntingtin with lipid vesicles using mass spectrometry and molecular dynamics.

The first 17 amino acid residues of Huntingtin protein (Nt17 of htt) are thought to play an important role in the protein's function; Nt17 is one of two membrane binding domains in htt. In this study the binding ability of Nt17 peptide with vesicles comprised of two subclasses of phospholipids is studied using electrospray ionization - mass spectrometry (ESI-MS) and molecular dynamics (MD) simulations. Overall, the peptide is shown to have a greater propensity to interact with vesicles of phosphatidylcholine (PC) rather than phosphatidylethanolamine (PE) lipids. Mass spectra show an increase in lipid-bound peptide adducts where the ordering of the number of such specie is 1,2-dioleoyl-sn-glycero-3-phosphocholine (DOPC) > 1-palmitoyl-2-oleoyl-glycero-3-phosphocholine (POPC) > 1-palmitoyl-2-oleoyl-sn-glycero-3 phosphoethanolamine (POPE). MD simulations suggest that the compactness of the bilayer plays a role in governing peptide interactions. The peptide shows greater disruption of the DOPC bilayer order at the surface and interacts with the hydrophobic tails of lipid molecules via hydrophobic residues. Conversely, the POPE vesicle remains ordered and lipids display transient interactions with the peptide through the formation of hydrogen bonds with hydrophilic residues. The POPC system displays intermediate behavior with regard to the degree of peptide-membrane interaction. Finally, the simulations suggest a helix stabilizing effect resulting from the interactions between hydrophobic residues and the lipid tails of the DOPC bilayer.