title: Isolation and analysis of sugar nucleotides using solid phase extraction and fluorophore assisted carbohydrate electrophoresis.
abstract: The building blocks of simple and complex oligosaccharides, termed sugar nucleotides, are often overlooked for their role in metabolic diseases and may hold the key to the underlying disease pathogenesis. Multiple reasons may account for the lack of analysis and quantitation of these sugar nucleotides, including the difficulty in isolation and purification as well as the required expensive instrumentation such as a high performance liquid chromatography (HPLC), mass spectrometer, or capillary electrophoresis. We have established a simple yet effective way to purify and quantitate sugar nucleotides using solid phase extraction (SPE) chromatography combined with fluorophore assisted carbohydrate electrophoresis (FACE). The simplicity of use, combined with the ability to run multiple samples at one time, give this technique a distinct advantage over the established methods for isolation and analysis of sugar nucleotides from cell culture models. •Sugar nucleotides can be easily purified with solid phase extraction chromatography.•FACE can be used to analyze multiple nucleotide sugar extracts with a single run.•The proposed method is simple, affordable, and uses common everyday research labware.
authors.affiliation: Departments of Pathobiology, Cleveland Clinic, 9500 Euclid Ave, Cleveland, OH 44195, USA.
authors.forename: Jarrod
authors.initials: J
authors.lastname: Barnes
authors.affiliation: Departments of Pathobiology, Cleveland Clinic, 9500 Euclid Ave, Cleveland, OH 44195, USA.
authors.forename: Liping
authors.initials: L
authors.lastname: Tian
authors.affiliation: Departments of Pathobiology, Cleveland Clinic, 9500 Euclid Ave, Cleveland, OH 44195, USA.
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Loftis
authors.affiliation: Molecular Medicine, Cleveland Clinic, 9500 Euclid Ave, Cleveland, OH 44195, USA.
authors.forename: James
authors.initials: J
authors.lastname: Hiznay
authors.affiliation: Departments of Pathobiology, Cleveland Clinic, 9500 Euclid Ave, Cleveland, OH 44195, USA.
authors.forename: Suzy
authors.initials: S
authors.lastname: Comhair
authors.affiliation: Departments of Pathobiology, Cleveland Clinic, 9500 Euclid Ave, Cleveland, OH 44195, USA.
authors.forename: Mark
authors.initials: M
authors.lastname: Lauer
authors.affiliation: Departments of Pathobiology, Cleveland Clinic, 9500 Euclid Ave, Cleveland, OH 44195, USA; Pulmonary Critical Care Medicine, Respiratory Institute, Cleveland Clinic, 9500 Euclid Ave, Cleveland, OH 44195, USA.
authors.forename: Raed
authors.initials: R
authors.lastname: Dweik
keywords: AMAC, 2-aminoacridone; APS, ammonium persulfate; CMP, cytosine monophosphate; Carbohydrate; Electrophoresis; FACE, fluorophore assisted carbohydrate electrophoresis; Face; GDP, guanosine diphosphate; Gal, galactose; GalNAc, N-acetylgalactosamine; GlcNAc, N-acetylglucosamine; GlcUA, glucuronic acid; HPLC; HPLC, high performance liquid chromatography; Man, Mannose; NeuAc, sialic acid; SPE, solid phase extraction; Sugar nucleotide analysis by SPE and FACE; Sugar nucleotides; TEAA, triethylamine acetate; TEMED, N′,N′,N′N′-tetramethylenediamine; UDP, uridine diphosphate