Untitled

Abstract

Analysis of nucleotide sugar metabolism is essential in studying glycosylation in cells. Here we describe practical methods for both extraction of nucleotide sugars from cell lysates and for their analytical separation. Solid-phase extraction cartridges containing graphitized carbon can be used for the purification of nucleotide sugars by using triethylammonium acetate buffer as a ion-pairing reagent for decreasing retention. After that they are separated by high-performance liquid chromatography using a C18 reversed-phase column and the same ion-pairing reagent for increasing retention. These new sample preparation and analysis methods enable good separation of structurally similar sugar nucleotides, compatibility with rapid evaporative concentration, and possibility to automation. Monitoring the production of GDP-deoxyhexoses in genetically engineered yeast and native bacterial cells are described here as specific applications.

Functional Characterization of the Flagellar Glycosylation Locus in Campylobacter jejuni 81–176 Using a Focused Metabolomics Approach

“…However, nucleotide-activated sugars are highly polar molecules and, therefore, have limited affinity toward reverse-phase sorbents such as C18 and C8, which are typically found in SPE materials. Recently, Räbinä et al (22) reported impressive recoveries of sugar-nucleotides from yeast and bacterial cells using graphitized non-porous carbon SPE material along with the ion-pairing reagent, triethylammonium acetate, in the elution solvent. Subsequent ion-pairing LC experiments permitted separation of intracellular sugarnucleotides, and their identification was possible by matrix-assisted laser desorption ionization time-of-flight MS (22).…”

“…Recently, Räbinä et al (22) reported impressive recoveries of sugar-nucleotides from yeast and bacterial cells using graphitized non-porous carbon SPE material along with the ion-pairing reagent, triethylammonium acetate, in the elution solvent. Subsequent ion-pairing LC experiments permitted separation of intracellular sugarnucleotides, and their identification was possible by matrix-assisted laser desorption ionization time-of-flight MS (22). Using commercially available ENVI-Carb SPE cartridges, intracellular sugar-nucleotides were extracted from the cell lysates of C. jejuni 81-176 and the isogenic mutant pseC using an optimized SPE method based on that of Räbinä et al (22).…”

“…Subsequent ion-pairing LC experiments permitted separation of intracellular sugarnucleotides, and their identification was possible by matrix-assisted laser desorption ionization time-of-flight MS (22). Using commercially available ENVI-Carb SPE cartridges, intracellular sugar-nucleotides were extracted from the cell lysates of C. jejuni 81-176 and the isogenic mutant pseC using an optimized SPE method based on that of Räbinä et al (22). Briefly, the ENVI-Carb solid-phase extraction cartridges were activated using 6 ml of acetonitrile-trifluoroacetic acid (0.1%) (80 -20, v/v) followed by 4 ml of de-ionized water.…”

“…High performance anion-exchange chromatography and pulsed amperometric detection is an established technique for carbohydrate analysis. In addition, the use of ion-pair liquid chromatography with triethylammonium acetate as the ion-pairing reagent was recently shown to offer good selectivity toward sugarnucleotides (22). Although it is possible to use either of these techniques for the purification of the sugar-nucleotide precursors, pure standards for CMP-PseAm (I) and UDP-diacetamido-trideoxyhexose (II) would be necessary for the identification of these novel metabolites in a cell lysate extract.…”

Simultaneous determination of 19 intracellular nucleotides and nucleotide sugars in Chinese Hamster ovary cells by capillary electrophoresis

“…Ion-exchange chromatography and reversed phase liquid chromatography has been used for the determination of nucleotide sugars [20][21][22][23][24]. Ion-pairing chromatography [25,26] coupled with mass spectrometry and nuclear magnetic resonance [27] has likewise been applied to the determination of nucleotide sugars. CE has also been used for the separation and determination of nucleotide sugars in two reports [28,29] with promising results.…”