TY - JOUR
T1 - Simultaneous determination of nucleotide sugars with ion-pair reversed-phase HPLC
AU - Nakajima, Kazuki
AU - Kitazume, Shinobu
AU - Angata, Takashi
AU - Fujinawa, Reiko
AU - Ohtsubo, Kazuaki
AU - Miyoshi, Eiji
AU - Taniguchi, Naoyuki
N1 - Funding Information:
KN was a special postdoctoral fellow at RIKEN (2006–2009). This work was partly supported by Grant-in-Aid for Scientific Research (A), 20249018, and by the global COE program from the Ministry of Education, Culture, Sports, Science and Technology of Japan.
PY - 2010/7
Y1 - 2010/7
N2 - 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 1×106 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.
AB - 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 1×106 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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U2 - 10.1093/glycob/cwq044
DO - 10.1093/glycob/cwq044
M3 - Article
C2 - 20371511
AN - SCOPUS:77956838656
SN - 0959-6658
VL - 20
SP - 865
EP - 871
JO - Glycobiology
JF - Glycobiology
IS - 7
ER -
