Gas Chromatography Coupled to Atmospheric Pressure Chemical Ionization High-Resolution Mass Spectrometry for Metabolite Fingerprinting of Grape (Vitis vinifera L) Berry.

This chapter describes the application of atmospheric pressure chemical ionization in conjunction with gas chromatography (APGC) coupled to high-resolution mass spectrometry for profiling metabolites in plant and fruit extracts. The APGC technique yields molecular ions and limited fragmentation of volatile or derivatized compounds. The data-independent acquisition mode, MSE, was used for measuring precursor and fragment ions with high resolution using a quadrupole ion mobility time-of-flight mass spectrometry system. We demonstrate the importance of acquiring accurate mass information in conjunction with accurate mass fragment ions for efficient database searching and compound assignments with high confidence. We demonstrate the application of APGC-MSE for obtaining metabolite data for grape berry extracts after derivatization.

Effects of Chronic Secondhand Smoke (SHS) Exposure on Cognitive Performance and Metabolic Pathways in the Hippocampus of Wild-Type and Human Tau Mice.

BACKGROUND: Exposure to secondhand smoke (SHS) is a risk factor for developing sporadic forms of sporadic dementia. A human tau (htau) mouse model is available that exhibits age-dependent tau dysregulation, neurofibrillary tangles, neuronal loss, neuroinflammation, and oxidative stress starting at an early age (3-4 months) and in which tau dysregulation and neuronal loss correlate with synaptic dysfunction and cognitive decline. OBJECTIVE: The goal of this study was to assess the effects of chronic SHS exposure (10 months' exposure to ∼30 mg/m3) on behavioral and cognitive function, metabolism, and neuropathology in mice. METHODS: Wild-type (WT) and htau female and male mice were exposed to SHS (90% side stream, 10% main stream) using the SCIREQ® inExpose™ system or air control for 168 min per day, for 312 d, 7 d per week. The exposures continued during the days of behavioral and cognitive testing. In addition to behavioral and cognitive performance and neuropathology, the lungs of mice were examined for pathology and alterations in gene expression. RESULTS: Mice exposed to chronic SHS exposure showed the following genotype-dependent responses: a) lower body weights in WT, but not htau, mice; b) less spontaneous alternation in WT, but not htau, mice in the Y maze; c) faster swim speeds of WT, but not htau, mice in the water maze; d) lower activity levels of WT and htau mice in the open field; e) lower expression of brain PHF1, TTCM1, IGF1ß, and HSP90 protein levels in WT male, but not female, mice; and f) more profound effects on hippocampal metabolic pathways in WT male than female mice and more profound effects in WT than htau mice. DISCUSSION: The brain of WT mice, in particular WT male mice, might be especially susceptible to the effects of chronic SHS exposure. In WT males, independent pathways involving ascorbate, flavin adenine dinucleotide, or palmitoleic acid might contribute to the hippocampal injury following chronic SHS exposure. https://doi.org/10.1289/EHP8428.

In-Vial Extraction Large Volume Gas Chromatography Mass Spectrometry for Analysis of Volatile PFASs on Papers and Textiles.

Volatile per- and polyfluorinated alkyl substances (PFASs) are found in consumer goods that contribute to human exposure to PFASs. Volatile PFAS precursors transform to perfluorinated carboxylates (PFCAs) and sulfonates (PFSAs) in both humans and the environment. Established methods for volatile PFASs in consumer goods exist, but higher sample throughput and greener sample preparation methods are needed to minimize analyte loss, while maintaining sensitivity. New analytical methodology was developed where a 1.5 × 1.5 cm piece of paper or textile is placed into an autosampler vial with solvent and mass-labeled internal standards, sonicated for 30 min, and directly injected without removal of material from the autosampler vial. Large volume injection (20 µL) gas chromatography mass spectrometry was applied for the quantification for 21 individual PFASs from five classes: fluorotelomer alcohols (FTOHs), fluorinated sulfonamides (N-MeFASA, N-EtFASA), and fluorinated sulfonamidoethanols (N-MeFASE, N-EtFASE). Nontargeted analysis revealed additional C2-C7 homologues of N-MeFASE and N-EtFASE, which accounted for 14-18% of the total volatile PFASs on three textiles. Overlooking short-chain (≤C7) N-MeFASE, N-EtFASE, and long-chained (10:2-14:2) FTOHs on older textiles from the 1980s leads to an underestimation of human and environmental exposure to volatile PFAS.

Using theoretical protein isotopic distributions to parse small-mass-difference post-translational modifications via mass spectrometry.

Small-mass-difference modifications to proteins are obscured in mass spectrometry by the natural abundance of stable isotopes such as (13)C that broaden the isotopic distribution of an intact protein. Using a ZipTip (Millipore, Billerica, MA, USA) to remove salt from proteins in preparation for high-resolution mass spectrometry, the theoretical isotopic distribution intensities calculated from the protein's empirical formula could be fit to experimentally acquired data and used to differentiate between multiple low-mass modifications to proteins. We could readily distinguish copper from zinc bound to a single-metal superoxide dismutase (SOD1) species; copper and zinc only differ by an average mass of 1.8 Da and have overlapping stable isotope patterns. In addition, proteins could be directly modified while bound to the ZipTip. For example, washing 11 mM S-methyl methanethiosulfonate over the ZipTip allowed the number of free cysteines on proteins to be detected as S-methyl adducts. Alternatively, washing with the sulfhydryl oxidant diamide could quickly reestablish disulfide bridges. Using these methods, we could resolve the relative contributions of copper and zinc binding, as well as disulfide reduction to intact SOD1 protein present from <100 µg of the lumbar spinal cord of a transgenic, SOD1 overexpressing mouse. Although techniques like ICP-MS can measure total metal in solution, this is the first method able to assess the metal-binding and sulfhydryl reduction of SOD1 at the individual subunit level and is applicable to many other proteins.

Cloning and functional characterization of a gene for capsanthin-capsorubin synthase from tiger lily (Lilium lancifolium Thunb. 'Splendens').

The orange color of tiger lily (Lolium lancifolium 'Splendens') flowers is due, primarily, to the accumulation of two κ-xanthophylls, capsanthin and capsorubin. An enzyme, known as capsanthin-capsorubin synthase (CCS), catalyzes the conversion of antheraxanthin and violaxanthin into capsanthin and capsorubin, respectively. We cloned the gene for capsanthin-capsorubin synthase (Llccs) from flower tepals of L. lancifolium by the rapid amplification of cDNA ends (RACE) with a heterologous non-degenerate primer that was based on the sequence of a gene for lycopene ß-cyclase (lcyB). The full-length cDNA of Llccs was 1,785 bp long and contained an open reading frame of 1,425 bp that encoded a polypeptide of 474 amino acids with a predicted N-terminal plastid-targeting sequence. Analysis by reverse transcription-PCR (RT-PCR) revealed that expression of Llccs was spatially and temporally regulated, with expression in flower buds and flowers of L. lancifolium but not in vegetative tissues. Stable overexpression of the Llccs gene in callus tissue of Iris germanica, which accumulates several xanthophylls including violaxanthin, the precursor of capsorubin, resulted in transgenic callus whose color had changed from its normal yellow to red-orange. This novel red-orange coloration was due to the accumulation of two non-native κ-xanthophylls, capsanthin and capsorubin, as confirmed by HPLC and ultraperformance liquid chromatography-tandem mass spectrometry (UPLC-MS/MS) analysis with authentic standards. Cloning of the Llccs gene should advance our understanding of the molecular and genetic mechanisms of the biosynthesis of κ-carotenoids in general and in the genus Lilium in particular, and will facilitate transgenic alterations of the colors of flowers and fruits of many plant species.

Metabolism of the anti-tuberculosis drug ethionamide by mouse and human FMO1, FMO2 and FMO3 and mouse and human lung microsomes.

Tuberculosis (TB) results from infection with Mycobacterium tuberculosis and remains endemic throughout the world with one-third of the world's population infected. The prevalence of multi-drug resistant strains necessitates the use of more toxic second-line drugs such as ethionamide (ETA), a pro-drug requiring bioactivation to exert toxicity. M. tuberculosis possesses a flavin monooxygenase (EtaA) that oxygenates ETA first to the sulfoxide and then to 2-ethyl-4-amidopyridine, presumably through a second oxygenation involving sulfinic acid. ETA is also a substrate for mammalian flavin-containing monooxygenases (FMOs). We examined activity of expressed human and mouse FMOs toward ETA, as well as liver and lung microsomes. All FMOs converted ETA to the S-oxide (ETASO), the first step in bioactivation. Compared to M. tuberculosis, the second S-oxygenation to the sulfinic acid is slow. Mouse liver and lung microsomes, as well as human lung microsomes from an individual expressing active FMO, oxygenated ETA in the same manner as expressed FMOs, confirming this reaction functions in the major target organs for therapeutics (lung) and toxicity (liver). Inhibition by thiourea, and lack of inhibition by SKF-525A, confirm ETASO formation is primarily via FMO, particularly in lung. ETASO production was attenuated in a concentration-dependent manner by glutathione. FMO3 in human liver may contribute to the toxicity and/or affect efficacy of ETA administration. Additionally, there may be therapeutic implications of efficacy and toxicity in human lung based on the FMO2 genetic polymorphism, though further studies are needed to confirm that suggestion.