Analysis of nucleotide sugars from cell lysates by ion-pair solid-phase extraction and reversed-phase high-performance liquid chromatography.
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.
Räbinä
J
J
Biomedicum Helsinki, Rational Drug Development and Department of Bacteriology and Immunology, P.O. Box 63, FIN-00014 University of Helsinki, Finland. jarkko.rabina@bts.redcross.fi
Mäki
M
M
Savilahti
E M
EM
Järvinen
N
N
Penttilä
L
L
Renkonen
R
R
D000327:Adsorption; D002244:Carbon; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D002852:Chromatography, Ion Exchange / Q000379:methods; D003837:Deoxy Sugars / Q000032:analysis* / Q000096:biosynthesis; D009711:Nucleotides / Q000032:analysis*; D011550:Pseudomonas aeruginosa / Q000737:chemistry / Q000235:genetics; D012441:Saccharomyces cerevisiae / Q000737:chemistry / Q000235:genetics; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods
D003837:Deoxy Sugars; D009711:Nucleotides; D002244:Carbon