title: Glycan analysis via derivatization with a fluorogenic pyrylium dye.
abstract: The expansion of glycomics analysis is reliant upon the development of robust, routine methods for carbohydrate characterization. Simple protocols to derivatize sugars with functionality that facilitate analysis-chromophores, fluorophores, charges, ionizable groups-are therefore necessary. Here we describe a method for the labeling of oligosaccharide mixtures with a fluorogenic pyrylium dye to enable analysis by capillary electrophoresis (CE) and matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF-MS). The unreacted free dye, Py-1, is effectively non-fluorescent but when conjugated to the analyte it displays strong fluorescence at 600-640 nm. Removal of excess dye following labeling is not required prior to analysis unlike for many traditional oligosaccharide labels. Labeling is achieved in two steps; the oligosaccharide mixtures are first functionalized with an ethylenediamine moiety via reductive amination at the reducing-end sugar, then the remaining free primary amine is reacted with the pyrylium dye (Py-1) under basic conditions to form a pyridinium ion. We have labeled mixtures of maltooligosaccharides and observed good peak separation in CE analysis using a SDS/borate pH 9.3 running buffer. Excellent sensitivity in MALDI-ToF-MS analysis enabled detection of oligosaccharides with up to 58 glucose units.
authors.affiliation: Carlsberg Laboratory, Gamle Carlsberg Vej 10, Copenhagen V DK-1799, Denmark.
authors.forename: Sine A
authors.initials: SA
authors.lastname: Johannesen
authors.forename: Sophie R
authors.initials: SR
authors.lastname: Beeren
authors.forename: Dennis
authors.initials: D
authors.lastname: Blank
authors.forename: Byung Y
authors.initials: BY
authors.lastname: Yang
authors.forename: Rudolf
authors.initials: R
authors.lastname: Geyer
authors.forename: Ole
authors.initials: O
authors.lastname: Hindsgaul
mesh_terms: D000586:Amination; D000588:Amines / Q000737:chemistry; D019075:Electrophoresis, Capillary; D005456:Fluorescent Dyes / Q000737:chemistry*; D006575:Heterocyclic Compounds, 3-Ring / Q000737:chemistry*; D009844:Oligosaccharides / Q000032:analysis* / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
chemical_list: D000588:Amines; D005456:Fluorescent Dyes; D006575:Heterocyclic Compounds, 3-Ring; D009844:Oligosaccharides; C505939:pyrylium 1