Project description:Sheep brain tissue was extracted using the Matyash protocol and analysed by HILIC-IMS-MS. A thin section was coated with DHB and analysed by MALDI-IMS-MS.
Project description:<p>Metabolic phenotyping of tissues uses metabolomics and lipidomics to measure the relative polar and non-polar (lipid) metabolite levels in biological samples. This approach aims to understand disease biochemistry and identify biochemical markers of disease. Sample preparation methods must be reproducible, sensitive (high metabolite and lipid yield), and ideally rapid. We evaluated three biphasic methods for polar and non-polar compound extraction (chloroform/methanol/water; dichloromethane/methanol/water; methyl tert-butyl ether [MTBE]/methanol/water), a monophasic method for polar compound extraction (acetonitrile/methanol/water) and a monophasic method for non-polar compound extraction (isopropanol/water). All methods were applied to sheep heart, kidney, and liver tissue. Polar extracts were analysed by hydrophilic interaction chromatography (HILIC) ultra-high-performance liquid chromatography-mass spectrometry (UHPLC-MS) and non-polar extracts by C18 reversed phase UHPLC-MS. Method reproducibility and yield was assessed using multiple annotated endogenous compounds (putatively and MS/MS annotated). Monophasic methods had the highest yield and high reproducibility for both polar (positive ion: median RSD<18%; negative ion: median RSD<28%) and non-polar (positive and negative ion: median RSD<15%) extractions for heart, kidney, and liver. The polar monophasic method extracted higher levels of lipid than biphasic polar extractions, and these lipids caused minimal detection suppression for other compounds during HILIC UHPLC-MS. The non-polar monophasic method had similar or greater detection responses of all detected lipid classes compared to biphasic methods (including increased phosphatidylinositol, phosphatidylserine and cardiolipin responses). Monophasic methods are quicker and simpler than biphasic methods and therefore most suited for future automation.</p>
Project description:Methyl tert-butyl ether (MTBE) has been shown to target developing vasculature in piscine and mammalian model systems. In the zebrafish, MTBE induces vascular lesions throughout development. These lesions result from exposure to MTBE at an early stage in development (6-somites to Prim-5 stages). During this time period, transcript levels of vegfa, vegfc, and vegfr1 were significantly decreased in embryos exposed to 5 mM MTBE. We performed global gene analysis as an unbiased approach to discover possible modes of action of MTBE vascular toxicity.
Project description:Comparative transcriptome analysis of Methylibium petroleiphilum PM1 exposed to the fuel-oxygenates methyl-tert-butyl ether and ethanol High-density whole genome cDNA microarrays were used to investigate substrate-dependent gene expression of Methylibium petroleiphilum PM1, one of the best-characterized aerobic methyl tert-butyl ether (MTBE)-degrading bacteria. Differential gene expression profiling was conducted with PM1 grown on MTBE and ethanol as sole carbon sources. Based on microarray high scores and protein similarity analysis, an MTBE regulon located on the megaplasmid was identified for further investigation. Putative functions for enzymes encoded in this regulon are described with relevance to the predicted MTBE degradation pathway. A new unique dioxygenase enzyme system that carries out the hydroxylation of TBA to 2-methyl-2-hydroxy-1-propanol in M. petroleiphilum PM1 was discovered. Based on the expression data, hypotheses regarding the acquisition and evolution of MTBE genes as well as the involvement of IS elements in these complex processes were formulated. The pathways for toluene, phenol, and alkane oxidation via toluene monooxygenase, phenol hydroxylase, alkane monooxygenase as well as propane monooxygenase, respectively, were upregulated in MTBE-grown cells compared to ethanol-grown cells. Four out of nine putative cyclohexanone monooxygenases were also upregulated in MTBE-grown cells. The global transcriptome response revealed the link between metabolism of MTBE and aromatic compounds (e.g. benzene, toluene) present in gasoline mixtures. The expression data aids our understanding of the regulation of metabolic processes that may occur in response to pollutant mixtures and perturbations in the environment. Keywords: bacterial metabolism
Project description:We present the lipidome of plasma collected from high-risk type 1 diabetes subjects. The methyl tert-butyl ether (MTBE) method was used for lipid extraction, followed by high performance liquid chromatography (HPLC) tandem mass spectrometry (LC-MS/MS) using a Q Exactive Orbitrap mass spectrometer and an Accela 600 HPLC. Lipid species were identified and quantified by analyzing the raw files in LipidSearch 4.2. Further analysis was conducted using Graphpad Prism and Ingenuity Pathway Analysis (IPA).