title: Oligosaccharide mapping of heparinase I-treated heparins by hydrophilic interaction liquid chromatography separation and online fluorescence detection and electrospray ionization-mass spectrometry characterization.
abstract: Oligosaccharide mapping based on enzyme cleavage provides a useful molecular fingerprint of the heparin structure revealing detailed structural information regarding its sequence and the content of part of the ATIII-binding region. This approach is performed by strong-anion exchange (SAX)-HPLC separation which is incompatible with MS requiring purification of oligosaccharides for their conclusive identification. We report a novel oligosaccharide mapping strategy based on the HILIC separation of the main heparin disaccharides/oligosaccharides released by heparinase I, fluorotagged with 2-aminoacridone and on-line detected by a fluorescence detector and characterized by ESI-MS. The application of a polar solvent having a high pH with acetonitrile avoided desulfation enabling a simple and accurate structural oligosaccharide assignment. Oligosaccharide mapping, or merely complete disaccharide composition, may be performed on nanogram-scale by the fluorescence detector vs micrograms useful for classical SAX-HPLC. Additionally, only widely commercially available heparin lyase I is necessary, without the use of expensive heparinases II and III. Contrary to SAX-HPLC, this novel HILIC approach is able to separate and identify the saturated trisulfated disaccharide belonging to the non-reducing end of heparin chains. Finally, the content of 3-O-sulfo groups of the ATIII-binding region is determined.
authors.affiliation: Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy.
authors.forename: Fabio
authors.initials: F
authors.lastname: Galeotti
authors.affiliation: Department of Life Sciences, University of Modena and Reggio Emilia, Modena, Italy. Electronic address: volpi@unimo.it.
authors.forename: Nicola
authors.initials: N
authors.lastname: Volpi
mesh_terms: D000609:Aminoacridines / Q000737:chemistry; D002623:Chemistry Techniques, Analytical / Q000379:methods*; D002851:Chromatography, High Pressure Liquid; D004187:Disaccharides / Q000737:chemistry; D005453:Fluorescence; D006493:Heparin / Q000031:analogs & derivatives / Q000032:analysis / Q000737:chemistry*; D019763:Heparin Lyase / Q000378:metabolism; D057927:Hydrophobic and Hydrophilic Interactions; D009844:Oligosaccharides / Q000032:analysis* / Q000737:chemistry; D021241:Spectrometry, Mass, Electrospray Ionization
chemical_list: D000609:Aminoacridines; D004187:Disaccharides; D009844:Oligosaccharides; C464536:heparin disaccharide; C072515:2-aminoacridone; D006493:Heparin; D019763:Heparin Lyase
keywords: AMAC; Disaccharides; Glycosaminoglycans; HILIC; Heparan sulfate; Heparin; Oligosaccharide mapping