Protocol

Glycobiology Protocols

Volume 347 of the series Methods in Molecular Biology pp 125-143

Detailed Structural Analysis of N-Glycans Released From Glycoproteins in SDS-PAGE Gel Bands Using HPLC Combined With Exoglycosidase Array Digestions

  • Louise RoyleAffiliated withDepartment of Biochemistry, Glycobiology Institute, University of Oxford
  • , Catherine M. RadcliffeAffiliated withDepartment of Biochemistry, Glycobiology Institute, University of Oxford
  • , Raymond A. DwekAffiliated withDepartment of Biochemistry, Glycobiology Institute, University of Oxford
  • , Pauline M. RuddAffiliated withDepartment of Biochemistry, Glycobiology Institute, University of Oxford

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Abstract

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.