Identification of neutral and sialyl N-linked oligosaccharide structures from human serum glycoproteins using three kinds of high-performance liquid chromatography.
The distribution of neutral and sialyl N-linked oligosaccharides from human serum glycoproteins has been studied using three kinds of HPLC columns as described below. N-linked oligosaccharides were released from chymotrypsin- and trypsin-digested glycopeptides of human serum by means of glycoamidase (from almond) digestion. The reducing ends of the oligosaccharides were derivatized with the fluorescent reagent 2-aminopyridine (PA). The mixture of PA-oligosaccharides was separated by high-performance liquid chromatography on a diethylaminoethyl column according to the sialic acid content. Each fraction separated was then applied on an octadecylsilyl (ODS) column, and the elution volume of each peak was recorded as a glucose unit on the X-axis. Then, each of the separated oligosaccharides was applied to the amide column. Each peak's elution volume was recorded as a glucose unit on the Y-axis. The elution volumes from these two columns (ODS and amide) provide a unique set of coordinates. The structure of each sialyl oligosaccharide fraction was analyzed by the same two-dimensional sugar-mapping technique as previously developed for neutral oligosaccharides. This was combined with exoglycosidase digestion and high-resolution proton NMR measurement. Fourteen neutral, eight mon-sialyl, four di-sialyl, and five tri-sialyl oligosaccharides were isolated from human serum and their structures were characterized.
Nakagawa
H
H
GlycoLab, Nakano Vinegar Company Ltd., Handa-City, Japan.
Kawamura
Y
Y
Kato
K
K
Shimada
I
I
Arata
Y
Y
Takahashi
N
N
D000581:Amidohydrolases / Q000378:metabolism; D000631:Aminopyridines / Q000737:chemistry; D001798:Blood Proteins / Q000737:chemistry*; D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002918:Chymotrypsin / Q000378:metabolism; D006023:Glycoproteins / Q000737:chemistry*; D006026:Glycoside Hydrolases / Q000378:metabolism; D006801:Humans; D009682:Magnetic Resonance Spectroscopy; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000032:analysis* / Q000737:chemistry; D014357:Trypsin / Q000378:metabolism
D000631:Aminopyridines; D001798:Blood Proteins; D006023:Glycoproteins; D009844:Oligosaccharides; C016735:sialooligosaccharides; D006026:Glycoside Hydrolases; D002918:Chymotrypsin; D014357:Trypsin; D000581:Amidohydrolases; C032439:alpha-aminopyridine