]. Briefly, the analytical platform employs a Vanquish ultra-high performance liquid chromatography system coupled online to a Q Exactive mass spectrometer . Polar extracts were resolved over a Kinetex C18 column, 2.1 × 150 mm, 1.7-µm particle size equipped with a guard column using an aqueous phase of water and 0.1% formic acid and a mobile phase of acetonitrile and 0.
Metabolite molar concentrations were determined by multiplying the ratio of the monoisotope to the respective spiked in isotopologue standard by the standard known concentration, with a subsequent correction for the dilution factor during extraction as reported [ ]. Lipidomics data were analyzed using LipidSearch 5.0 , which provides lipid identification on the basis of accurate intact mass, isotopic pattern, and fragmentation pattern to determine lipid class and acyl chain composition.
Graphs, heat maps, and statistical analyses , metabolic pathway analysis, partial least squares-discriminant analysis, and hierarchical clustering were performed using the MetaboAnalyst 4.0 package []. XY graphs were plotted through GraphPad Prism 8 . Pathway graphs were prepared on BioRender.com. Fuzzy c-means clustering was performed using the R package ‘Mfuzz’ using four centers, and an m value of 1.5 and an min.acore of 0.7.
Metabolite pathway enrichment analysis was performed using the Peaks To Pathways of MetaboAnalyst 4.0. Feature regions identified with the longitudinal patterns selected from c-means clustering were subjected to pathway analysis and enrichments were plotted on pie charts demonstrating pathway enrichment as a function of observed versus total pathway hits.
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