title: Extraction and Quantitation of Ketones and Aldehydes from Mammalian Cells Using Fluorous Tagging and Capillary LC-MS.
abstract: The extraction and quantitation of carbonyl metabolites from cell lysate was accomplished using a carbonyl-reactive fluorous tag and capillary liquid chromatography coupled to mass spectrometry (capLC-MS). Selective fluorous tagging for ketones and aldehydes provided a 30-fold increase in sensitivity using electrospray ionization MS. Separation of fluorous tagged carbonyl resulted in good separation of all components, and tandem MS was able to differentiate structural carbonyl isomers. The average limit of detection for carbonyl standards was 37 nM (range 1.5-250 nM), with linearity of R(2) > 0.99. Reproducibility for metabolites in cell lysate averaged 9% RSD. Human aortic endothelial cells (HAECs) were exposed to varying levels of glucose, and their carbonyl metabolite levels were quantified. Significant metabolite changes were seen in glycolysis and the propanoate pathway from a glucose challenge. Using an untargeted approach, 120 carbonyl metabolites were found to change in hyperglycemic HAECs. From this list of compounds, multiple metabolites from the pentose phosphate and tryptophan metabolic pathways were discovered. This system provides excellent sensitivity and quantitation of carbonyl metabolites without the need for isotope standards or labels.
authors.affiliation: †Institute for Bioscience and Biotechnology Research, University of Maryland, 9600 Gudelsky Drive, Rockville, Maryland 20850, United States.|‡Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742, United States.
authors.forename: Wei
authors.initials: W
authors.lastname: Yuan
authors.affiliation: †Institute for Bioscience and Biotechnology Research, University of Maryland, 9600 Gudelsky Drive, Rockville, Maryland 20850, United States.|‡Department of Chemistry and Biochemistry, University of Maryland, College Park, Maryland 20742, United States.
authors.forename: Shuwei
authors.initials: S
authors.lastname: Li
authors.affiliation: §Department of Chemistry, Saint Louis University, St. Louis, Missouri 63103, United States.
authors.forename: James L
authors.initials: JL
authors.lastname: Edwards
mesh_terms: D000447:Aldehydes / Q000032:analysis*; D000818:Animals; D002853:Chromatography, Liquid; D005461:Fluorine / Q000737:chemistry*; D006801:Humans; D007659:Ketones / Q000032:analysis*; D015203:Reproducibility of Results; D053719:Tandem Mass Spectrometry
chemical_list: D000447:Aldehydes; D007659:Ketones; D005461:Fluorine