Detailed structural analysis of N-glycans released from glycoproteins in SDS-PAGE gel bands using HPLC combined with exoglycosidase array digestions.
In contrast to the linear sequences of protein and DNA, oligosaccharides are branched structures. In addition, almost all glycoproteins consist of a heterogeneous collection of differently glycosylated variants. Glycan analysis therefore requires high-resolution separation techniques that can provide detailed structural analysis, including both monosaccharide sequence and linkage information. This chapter describes how a combination of high-performance liquid chromatography (HPLC) and exoglycosidase enzyme array digestions can deliver quantitative glycan analysis of sugars released from glycoproteins in sodium dodecyl sulfate-polyacrylamide gel electrophoresis gel bands by matching HPLC elution positions with a database of standard glycans.
Royle
Louise
L
Department of Biochemistry, Glycobiology Institute, University of Oxford, Oxford, England.
Radcliffe
Catherine M
CM
Dwek
Raymond A
RA
Rudd
Pauline M
PM
D002236:Carbohydrate Conformation; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005456:Fluorescent Dyes / Q000737:chemistry; D006023:Glycoproteins / Q000737:chemistry; D006026:Glycoside Hydrolases / Q000737:chemistry / Q000378:metabolism*; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000032:analysis* / Q000737:chemistry* / Q000378:metabolism
D005456:Fluorescent Dyes; D006023:Glycoproteins; D011134:Polysaccharides; D006026:Glycoside Hydrolases