Search

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. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

A new operational mode for an ion cyclotron mobility spectrometry instrument is explored as a possible means of performing high-resolution separations. The approach is based on oscillating fields that are applied to segmented regions of a circular drift tube. Ions with mobilities that are resonant with the frequency of field application are transmitted while nonresonant species are eliminated. An ion mobility spectrum is obtained by scanning the drift field application frequency. The approach is demonstrated by examining mixtures of ions produced by electrospraying the substance P peptide, as well as a mixture of tryptic peptides obtained by enzymatic digestion of cytochrome c. Drift field application frequency scans of substance P peptide ions show that it is possible to separate [[M+2H].sup.2+] ions, and compact and elongated forms of [[M+3H].sup.3+] ions. The resolution of different ions is related to the number of cycles for the analysis. At high cycle numbers (>50 3/4 or a drift length of 9242.03 cm) values of the resolving power can exceed 300 with a maximum resolving power of ~400. The ability to tune the resolving power of a mobility-based separation by varying the ion cycle number has substantial analytical utility. 10.1021/ac1017474

The ability to separate isotopes by high-resolution ion mobility spectrometry techniques is considered as a direct means for determining mass at ambient pressures. Calculations of peak shapes from the transport equation show that it should be possible to separate isotopes for low-mass ions (

Personalized medicine advocates have been frustrated by the issue of analyte component resolution in biomolecular profiling. Because complex human biological samples such as plasma, serum, or urine contain ~[10.sup.3]-[10.sup.7] unique [...]