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Simultaneous determination of nucleotide sugars with ion-pair reversed-phase HPLC.

Authors
  • Nakajima, Kazuki
  • Kitazume, Shinobu
  • Angata, Takashi
  • Fujinawa, Reiko
  • Ohtsubo, Kazuaki
  • Miyoshi, Eiji
  • Taniguchi, Naoyuki
Type
Published Article
Journal
Glycobiology
Publisher
Oxford University Press
Publication Date
Jul 01, 2010
Volume
20
Issue
7
Pages
865–871
Identifiers
DOI: 10.1093/glycob/cwq044
PMID: 20371511
Source
Medline
License
Unknown

Abstract

Nucleotide sugars are important in determining cell surface glycoprotein glycosylation, which can modulate cellular properties such as growth and arrest. We have developed a conventional HPLC method for simultaneous determination of nucleotide sugars. A mixture of nucleotide sugars (CMP-NeuAc, UDP-Gal, UDP-Glc, UDP-GalNAc, UDP-GlcNAc, GDP-Man, GDP-Fuc and UDP-GlcUA) and relevant nucleotides were perfectly separated in an optimized ion-pair reversed-phase mode using Inertsil ODS-4 and ODS-3 columns. The newly developed method enabled us to determine the nucleotide sugars in cellular extracts from 1 x 10(6) cells in a single run. We applied this method to characterize nucleotide sugar levels in breast and pancreatic cancer cell lines and revealed that the abundance of UDP-GlcNAc, UDP-GalNAc, UDP-GlcUA and GDP-Fuc were a cell-type-specific feature. To determine the physiological significance of changes in nucleotide sugar levels, we analyzed their changes by glucose deprivation and found that the determination of nucleotide sugar levels provided us with valuable information with respect to studying the overview of cellular glycosylation status.

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