title: A high-performance liquid chromatography based strategy for rapid, sensitive sequencing of N-linked oligosaccharide modifications to proteins in sodium dodecyl sulphate polyacrylamide electrophoresis gel bands.
abstract: The majority of biologically active proteins are glycosylated, therefore any approach to proteomics which fails to address the analysis of oligosaccharides is necessarily incomplete. To appreciate the structure of a glycoprotein fully, to understand the roles for the attached oligosaccharides and to monitor disease associated changes it is necessary to visualise the sugars as well as the protein. To achieve this aim when biological samples are available at the low microgram level or less has involved increasing the sensitivity of the technology for glycan analysis. Since one protein may have many different oligosaccharides attached to it (glycoforms) this is a major technical challenge. CD59, for example, has over 100 different sugars at one N-linked glycosylation site. Applications of recently developed technology suggest that it is now becoming realistic to extend the proteomics analysis of glycoproteins to include details of glycosylation. This is achieved by releasing the N-glycans from the protein in a gel by optimised peptide-N-glycosidase F digestion. The released glycans are then tagged with the fluorophore, 2-amino benzamide. The labelled glycan pools (containing 50-100 femtomoles of glycans) are resolved by predictive normal phase high performance liquid chromatography (HPLC) on an amide based column or by reverse phase HPLC on a C18 column. Preliminary structural assignments are confirmed by exoglycosidase array digestions of the entire glycan pool. Complementary matrix-assisted laser desorption/ionization-mass spectrometry, which requires 10-20 times as much sugar for a single run, can be used where there is sufficient material. This provides a composition analysis but not linkage information.
authors.affiliation: Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford OX1 3QU, UK. pmr@oxglua.glycob.ox.ac.uk
authors.forename: P M
authors.initials: PM
authors.lastname: Rudd
authors.forename: C
authors.initials: C
authors.lastname: Colominas
authors.forename: L
authors.initials: L
authors.lastname: Royle
authors.forename: N
authors.initials: N
authors.lastname: Murphy
authors.forename: E
authors.initials: E
authors.lastname: Hart
authors.forename: A H
authors.initials: AH
authors.lastname: Merry
authors.forename: H F
authors.initials: HF
authors.lastname: Hebestreit
authors.forename: R A
authors.initials: RA
authors.lastname: Dwek
mesh_terms: D000581:Amidohydrolases; D002240:Carbohydrate Sequence; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D002852:Chromatography, Ion Exchange; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes; D006023:Glycoproteins / Q000737:chemistry*; D006031:Glycosylation; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry; D008958:Models, Molecular; D008969:Molecular Sequence Data; D009844:Oligosaccharides / Q000737:chemistry*; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011487:Protein Conformation; D020543:Proteome; D012967:Sodium Dodecyl Sulfate; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D062367:ortho-Aminobenzoates
chemical_list: D005456:Fluorescent Dyes; D006023:Glycoproteins; D007074:Immunoglobulin G; D009844:Oligosaccharides; D020543:Proteome; D062367:ortho-Aminobenzoates; D012967:Sodium Dodecyl Sulfate; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; C000219:anthranilamide