title: Chemoselective detection and discrimination of carbonyl-containing compounds in metabolite mixtures by 1H-detected 15N nuclear magnetic resonance.
abstract: NMR spectra of mixtures of metabolites extracted from cells or tissues are extremely complex, reflecting the large number of compounds that are present over a wide range of concentrations. Although multidimensional NMR can greatly improve resolution as well as improve reliability of compound assignments, lower abundance metabolites often remain hidden. We have developed a carbonyl-selective aminooxy probe that specifically reacts with free keto and aldehyde functions, but not carboxylates. By incorporating (15)N in the aminooxy functional group, (15)N-edited NMR was used to select exclusively those metabolites that contain a free carbonyl function while all other metabolites are rejected. Here, we demonstrate that the chemical shifts of the aminooxy adducts of ketones and aldehydes are very different, which can be used to discriminate between aldoses and ketoses, for example. Utilizing the 2-bond or 3-bond (15)N-(1)H couplings, the (15)N-edited NMR analysis was optimized first with authentic standards and then applied to an extract of the lung adenocarcinoma cell line A549. More than 30 carbonyl-containing compounds at NMR-detectable levels, six of which we have assigned by reference to our database. As the aminooxy probe contains a permanently charged quaternary ammonium group, the adducts are also optimized for detection by mass spectrometry. Thus, this sample preparation technique provides a better link between the two structural determination tools, thereby paving the way to faster and more reliable identification of both known and unknown metabolites directly in crude biological extracts.
authors.affiliation: Center for Regulatory and Environmental Analytical Metabolomics, University of Louisville, Louisville, KY, USA; J.G. Brown Cancer Center, University of Louisville, Louisville, KY, USA.
authors.forename: Andrew N
authors.initials: AN
authors.lastname: Lane
authors.forename: Sengodagounder
authors.initials: S
authors.lastname: Arumugam
authors.forename: Pawel K
authors.initials: PK
authors.lastname: Lorkiewicz
authors.forename: Richard M
authors.initials: RM
authors.lastname: Higashi
authors.forename: Sébastien
authors.initials: S
authors.lastname: Laulhé
authors.forename: Michael H
authors.initials: MH
authors.lastname: Nantz
authors.forename: Hunter N B
authors.initials: HN
authors.lastname: Moseley
authors.forename: Teresa W-M
authors.initials: TW
authors.lastname: Fan
mesh_terms: D000447:Aldehydes / Q000737:chemistry / Q000378:metabolism*; D045744:Cell Line, Tumor; D006801:Humans; D007659:Ketones / Q000737:chemistry / Q000378:metabolism*; D008175:Lung Neoplasms / Q000737:chemistry / Q000378:metabolism*; D015336:Molecular Probe Techniques; D009587:Nitrogen Isotopes / Q000032:analysis / Q000737:chemistry; D066244:Proton Magnetic Resonance Spectroscopy / Q000379:methods*; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D000447:Aldehydes; D007659:Ketones; D009587:Nitrogen Isotopes
keywords: 15N-edited 1H NMR; aldehydes; aminooxy derivatives; chemoselection; ketones; metabolomics