Multistructure sequencing of N-linked fetuin glycans by capillary gel electrophoresis and enzyme matrix digestion.
Oligosaccharide sequencing by specific enzymatic digestion of N-linked fetuin glycans using a carefully designed exoglycosidase matrix in conjunction with separation of the combined digests by high performance capillary gel electrophoresis is described. Due to the extremely high separation power and excellent reproducibility of capillary gel electrophoresis, in most instances no isolation of the individual oligosaccharides is necessary, enabling multistructure sequencing from a released glycan pool. By comparing the positions of the separated exoglycosidase digest fragments to maltooligosaccharides of known size, relative migration times and migration shifts are easily calculated. Hence, the particular sequence of each oligosaccharide in a released glycan pool can be proposed with high confidence. Additionally, with the use of high sensitivity laser-induced fluorescence detection, complete sequence information can be attained from picomolar amounts of purified glycoproteins.
Guttman
A
A
Beckman Instruments, Inc., Fullerton, CA, USA. aguttman@genbiosys.com
D000818:Animals; D002240:Carbohydrate Sequence; D002417:Cattle; D019075:Electrophoresis, Capillary / Q000379:methods*; D005456:Fluorescent Dyes; D006026:Glycoside Hydrolases / Q000378:metabolism; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000737:chemistry*; D011721:Pyrenes; D017421:Sequence Analysis / Q000379:methods*; D000509:alpha-Fetoproteins / Q000737:chemistry*
C103430:8-aminopyrene-1,3,6-trisulfonic acid; D005456:Fluorescent Dyes; D011134:Polysaccharides; D011721:Pyrenes; D000509:alpha-Fetoproteins; D006026:Glycoside Hydrolases