title: A platform for the structural characterization of glycans enzymatically released from glycosphingolipids extracted from tissue and cells.
abstract: BACKGROUND
  Glycosphingolipids (GSLs) constitute a highly diverse class of glyco-conjugates which are involved in many aspects of cell membrane function and disease. The isolation, detection and structural characterization of the carbohydrate (glycan) component of GSLs are particularly challenging given their structural heterogeneity and thus rely on the development of sensitive, analytical technologies.
  
  
  METHODS
  Neutral and acidic GSL standards were immobilized onto polyvinylidene difluoride (PVDF) membranes and glycans were enzymatically released using endoglycoceramidase II (EGCase II), separated by porous graphitized carbon (PGC) liquid chromatography and structurally characterized by negative ion mode electrospray ionization tandem mass spectrometry (PGC-LC/ESI-MS/MS). This approach was then employed for GSLs isolated from 100 mg of serous and endometrioid cancer tissue and from cell line (10(7) cells) samples.
  
  
  RESULTS
  Glycans were released from GSL standards comprising of ganglio-, asialo-ganglio- and the relatively resistant globo-series glycans, using as little as 1 mU of enzyme and 2 µg of GSL. The platform of analysis was then applied to GSLs isolated from tissue and cell line samples and the released isomeric and isobaric glycan structures were chromatographically resolved on PGC and characterized by comparison with the MS(2) fragment ion spectra of the glycan standards and by application of known structural MS(2) fragment ions. This approach identified several (neo-)lacto-, globo- and ganglio-series glycans and facilitated the discrimination of isomeric structures containing Lewis A, H type 1 and type 2 blood group antigens and sialyl-tetraosylceramides.
  
  
  CONCLUSIONS
  We describe a relatively simple, detergent-free, enzymatic release of glycans from PVDF-immobilized GSLs, followed by the detailed structural analysis afforded by PGC-LC-ESI-MS/MS, to offer a versatile method for the analysis of tumour and cell-derived GSL-glycans. The method uses the potential of MS(2) fragmentation in negative ion ESI mode to characterize, in detail, the biologically relevant glycan structures derived from GSLs.
authors.affiliation: Department of Chemistry and Biomolecular Sciences, Biomolecular Frontiers Research Centre, Faculty of Science, Macquarie University, Sydney, 2109, Australia.
authors.forename: Merrina
authors.initials: M
authors.lastname: Anugraham
authors.affiliation: Department of Chemistry and Biomolecular Sciences, Biomolecular Frontiers Research Centre, Faculty of Science, Macquarie University, Sydney, 2109, Australia.
authors.forename: Arun Vijay
authors.initials: AV
authors.lastname: Everest-Dass
authors.affiliation: Gynecological Research Group, Department of Biomedicine, University Hospital Basel, University of Basel, Basel, 4031, Switzerland.
authors.forename: Francis
authors.initials: F
authors.lastname: Jacob
authors.affiliation: Department of Chemistry and Biomolecular Sciences, Biomolecular Frontiers Research Centre, Faculty of Science, Macquarie University, Sydney, 2109, Australia.
authors.forename: Nicolle H
authors.initials: NH
authors.lastname: Packer
mesh_terms: D001426:Bacterial Proteins / Q000378:metabolism; D045744:Cell Line, Tumor; D002853:Chromatography, Liquid / Q000379:methods; D005260:Female; D006026:Glycoside Hydrolases / Q000378:metabolism; D006028:Glycosphingolipids / Q000032:analysis* / Q000737:chemistry* / Q000378:metabolism; D006801:Humans; D010051:Ovarian Neoplasms / Q000737:chemistry*; D011134:Polysaccharides / Q000032:analysis* / Q000737:chemistry* / Q000378:metabolism; D011145:Polyvinyls; D011994:Recombinant Proteins / Q000378:metabolism; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods; D053719:Tandem Mass Spectrometry / Q000379:methods; D005465:Fluorocarbon Polymers
chemical_list: D001426:Bacterial Proteins; D006026:Glycoside Hydrolases; D006028:Glycosphingolipids; D011134:Polysaccharides; D011145:Polyvinyls; D011994:Recombinant Proteins; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers; C050427:endoglycoceramidase