title: Use of graphitised carbon negative ion LC-MS to analyse enzymatically digested glycosaminoglycans.
abstract: Capillary liquid chromatography-mass spectrometry using graphitised carbon stationary phase and ion trap mass spectrometry was shown to be a powerful technique for analysing glycosaminoglycans digested with endoglycosidases. Commonly found disaccharides from heparin/heparan sulphate digests at sub nanomole levels were found to be separated by mass and/or retention time and detected by negative ion electrospray mass spectrometry predominantly as [M-H]- ions using a standard electrospray interface and flow rate between 6-10 microL/min. Graphitised carbon liquid chromatography-fragmentation mass spectrometry provided sequence data of disaccharides and oligosaccharides. Sequence information was obtained from either collision of the [M-H]- ions (low sulphated disaccharides) or of the [M+Na-2H]- ions (highly sulphated disaccharides). This separation and identification method of endoglycosidase digestion and sample preparation using a combination of cation exchange and graphitised carbon, was used to successfully analyse digests of keratan sulphate (keratanase) and heparin (heparinase) standards, and hyaluronic acid (hyaluronidase) from synovial fluid samples.
authors.affiliation: Proteome Systems Ltd., Unit 1, 35-41 Waterloo Road, North Ryde, 2113 Sydney, NSW, Australia. Niclas.Karlsson@proteomesystems.com
authors.forename: Niclas G
authors.initials: NG
authors.lastname: Karlsson
authors.forename: Benjamin L
authors.initials: BL
authors.lastname: Schulz
authors.forename: Nicolle H
authors.initials: NH
authors.lastname: Packer
authors.forename: John M
authors.initials: JM
authors.lastname: Whitelock
mesh_terms: D002240:Carbohydrate Sequence; D002853:Chromatography, Liquid / Q000379:methods*; D006025:Glycosaminoglycans / Q000032:analysis* / Q000378:metabolism; D006026:Glycoside Hydrolases / Q000378:metabolism; D006108:Graphite; D006493:Heparin / Q000032:analysis / Q000378:metabolism; D019763:Heparin Lyase / Q000378:metabolism; D006820:Hyaluronic Acid / Q000032:analysis / Q000378:metabolism; D006821:Hyaluronoglucosaminidase / Q000378:metabolism; D007632:Keratan Sulfate / Q000032:analysis / Q000378:metabolism; D009844:Oligosaccharides / Q000032:analysis / Q000378:metabolism / Q000592:standards; D010084:Oxidation-Reduction; D012015:Reference Standards; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
chemical_list: D006025:Glycosaminoglycans; D009844:Oligosaccharides; D006108:Graphite; D006820:Hyaluronic Acid; D006493:Heparin; D007632:Keratan Sulfate; D006026:Glycoside Hydrolases; C026093:keratan-sulfate endo-1,4-beta-galactosidase; D006821:Hyaluronoglucosaminidase; D019763:Heparin Lyase