Yuting Huang

Ion mobility studies of carbohydrates as group i adducts: Isomer specific collisional cross section dependence on metal ion radius

Carbohydrates play numerous critical roles in biological systems. Characterization of oligosaccharide structures is essential to a complete understanding of their functions in biological processes; nevertheless, their structural determination remains challenging in part due to isomerism. Ion mobility spectrometry provides the means to resolve gas phase ions on the basis of their shape-to-charge ratios, thus providing significant potential for separation and differentiation of carbohydrate isomers. Here, we report on the determination of collisional cross sections for four groups of isomeric carbohydrates (including five isomeric disaccharides, four isomeric trisaccharides, two isomeric pentasaccharides, and two isomeric hexasaccharides) as their group I metal ion adducts (i.e., [M + Li](+), [M + Na](+), [M + K](+), [M + Rb](+), and [M + Cs](+)). In all, 65 collisional cross sections were measured, the great majority of which have not been previously reported. As anticipated, the collisional cross sections of the carbohydrate metal ion adducts generally increase with increasing metal ion radius; however, the collisional cross sections were found to scale with the group I cation size in isomer specific manners. Such measurements are of substantial analytical value, as they illustrate how the selection of charge carrier influences carbohydrate ion mobility determinations. For example, certain pairs of isomeric carbohydrates assume unique collisional cross sections upon binding one metal ion, but not another. On the whole, these data suggest a role for the charge carrier as a probe of carbohydrate structure and thus have significant implications for the continued development and application of ion mobility spectrometry for the distinction and resolution of isomeric carbohydrates.

Discrimination of Isomeric Carbohydrates as the Electron Transfer Products of Group II Cation Adducts by Ion Mobility Spectrometry and Tandem Mass Spectrometry

The rapid and unambiguous distinction of isomeric carbohydrate structures persists as a tremendous analytical challenge. This paper reports the first exploitation of carbohydrate/metal ion interactions in concert with gas-phase ion chemistry to improve discrimination of oligosaccharide isomers by both ion mobility spectrometry and tandem mass spectrometry. This is demonstrated for two isomeric pentasaccharides and two isomeric hexasaccharides, each studied in an underivatized form as their calcium ion adducts, barium ion adducts, and gas-phase electron transfer products thereof. With appropriate selection of the charge carrier, transfer of a single electron to the carbohydrate metal ion adducts resulted in isomer-distinguishing shifts in their ion/neutral collision cross sections and the appearance of unique features in their vibrational activation/dissociation spectra. These findings suggest novel and elegant gas-phase strategies for rapid differentiation of isomeric oligosaccharides.

Alterations in energy/redox metabolism induced by mitochondrial and environmental toxins: a specific role for glucose-6-phosphate-dehydrogenase and the pentose phosphate pathway in paraquat toxicity.

Parkinson’s disease (PD) is a multifactorial disorder with a complex etiology including genetic risk factors, environmental exposures, and aging. While energy failure and oxidative stress have largely been associated with the loss of dopaminergic cells in PD and the toxicity induced by mitochondrial/environmental toxins, very little is known regarding the alterations in energy metabolism associated with mitochondrial dysfunction and their causative role in cell death progression. In this study, we investigated the alterations in the energy/redox-metabolome in dopaminergic cells exposed to environmental/mitochondrial toxins (paraquat, rotenone, 1-methyl-4-phenylpyridinium [MPP+], and 6-hydroxydopamine [6-OHDA]) in order to identify common and/or different mechanisms of toxicity. A combined metabolomics approach using nuclear magnetic resonance (NMR) and direct-infusion electrospray ionization mass spectrometry (DI-ESI-MS) was used to identify unique metabolic profile changes in response to these neurotoxins. Paraquat exposure induced the most profound alterations in the pentose phosphate pathway (PPP) metabolome. 13C-glucose flux analysis corroborated that PPP metabolites such as glucose-6-phosphate, fructose-6-phosphate, glucono-1,5-lactone, and erythrose-4-phosphate were increased by paraquat treatment, which was paralleled by inhibition of glycolysis and the TCA cycle. Proteomic analysis also found an increase in the expression of glucose-6-phosphate dehydrogenase (G6PD), which supplies reducing equivalents by regenerating nicotinamide adenine dinucleotide phosphate (NADPH) levels. Overexpression of G6PD selectively increased paraquat toxicity, while its inhibition with 6-aminonicotinamide inhibited paraquat-induced oxidative stress and cell death. These results suggest that paraquat “hijacks” the PPP to increase NADPH reducing equivalents and stimulate paraquat redox cycling, oxidative stress, and cell death. Our study clearly demonstrates that alterations in energy metabolism, which are specific for distinct mitochondiral/environmental toxins, are not bystanders to energy failure but also contribute significant to cell death progression.

A Study of Calibrant Selection in Measurement of Carbohydrate and Peptide Ion-Neutral Collision Cross Sections by Traveling Wave Ion Mobility Spectrometry

While ion-neutral collision cross sections (CCSs) can be directly calculated from drift tube ion mobility spectrometry (DTIMS) data, measurements made using the more recently introduced traveling wave ion mobility spectrometry (TWIMS) technique are usually calibrated using standards with known CCS. Presently, there remains some question regarding how selection of calibrants influences TWIMS CCS measurements. This is of particular concern when calibrants of the same molecular class (e.g., carbohydrate versus peptide) or charge state as the unknowns are unavailable. This report presents a study of calibrant ion influence on CCS determination via TWIMS. Drift times from TWIMS were calibrated to CCSs using either carbohydrates or peptides as standards. These calibrations were then applied to other carbohydrates and peptides with known CCSs, and the errors of the measurements were assessed. In addition, calibrations with standards having charge states either matched or mismatched with those of the target analytes were applied and evaluated for accuracy. The use of carbohydrates to calibrate peptide CCSs and vice versa was found to introduce errors only modestly larger than the inherent uncertainties of the measurements (on average, 1.0%). Charge state mismatching while the same molecular class of calibrant and analyte was maintained yielded larger errors (on average, 3.5%). Mismatching of both calibrant molecular class and charge state resulted in the largest errors (on average, 4.7%). These results suggest that matching of both molecular class and charge state is recommended when possible, while matching at least the charge state is strongly advisable.

Combining DI-ESI–MS and NMR datasets for metabolic profiling

Metabolomics datasets are commonly acquired by either mass spectrometry (MS) or nuclear magnetic resonance spectroscopy (NMR), despite their fundamental complementarity. In fact, combining MS and NMR datasets greatly improves the coverage of the metabolome and enhances the accuracy of metabolite identification, providing a detailed and high-throughput analysis of metabolic changes due to disease, drug treatment, or a variety of other environmental stimuli. Ideally, a single metabolomics sample would be simultaneously used for both MS and NMR analyses, minimizing the potential for variability between the two datasets. This necessitates the optimization of sample preparation, data collection and data handling protocols to effectively integrate direct-infusion MS data with one-dimensional (1D) 1H NMR spectra. To achieve this goal, we report for the first time the optimization of (i) metabolomics sample preparation for dual analysis by NMR and MS, (ii) high throughput, positive-ion direct infusion electrospray ionization mass spectrometry (DI-ESI–MS) for the analysis of complex metabolite mixtures, and (iii) data handling protocols to simultaneously analyze DI-ESI–MS and 1D 1H NMR spectral data using multiblock bilinear factorizations, namely multiblock principal component analysis (MB-PCA) and multiblock partial least squares (MB-PLS). Finally, we demonstrate the combined use of backscaled loadings, accurate mass measurements and tandem MS experiments to identify metabolites significantly contributing to class separation in MB-PLS-DA scores. We show that integration of NMR and DI-ESI–MS datasets yields a substantial improvement in the analysis of metabolome alterations induced by neurotoxin treatment.Graphical abstract

Metabolic Investigations of the Molecular Mechanisms Associated with Parkinson’s Disease

Parkinson’s disease (PD) is a neurodegenerative disorder characterized by fibrillar cytoplasmic aggregates of α-synuclein (i.e., Lewy bodies) and the associated loss of dopaminergic cells in the substantia nigra. Mutations in genes such as α-synuclein (SNCA) account for only 10% of PD occurrences. Exposure to environmental toxicants including pesticides and metals (e.g., paraquat (PQ) and manganese (Mn)) is also recognized as an important PD risk factor. Thus, aging, genetic alterations, and environmental factors all contribute to the etiology of PD. In fact, both genetic and environmental factors are thought to interact in the promotion of idiopathic PD, but the mechanisms involved are still unclear. In this study, we summarize our findings to date regarding the toxic synergistic effect between α-synuclein and paraquat treatment. We identified an essential role for central carbon (glucose) metabolism in dopaminergic cell death induced by paraquat treatment that is enhanced by the overexpression of α-synuclein. PQ “hijacks” the pentose phosphate pathway (PPP) to increase NADPH reducing equivalents and stimulate paraquat redox cycling, oxidative stress, and cell death. PQ also stimulated an increase in glucose uptake, the translocation of glucose transporters to the plasma membrane, and AMP-activated protein kinase (AMPK) activation. The overexpression of α-synuclein further stimulated an increase in glucose uptake and AMPK activity, but impaired glucose metabolism, likely directing additional carbon to the PPP to supply paraquat redox cycling.