Glycan analysis via derivatization with a fluorogenic pyrylium dye

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• 作者：Sine A. Johannesen^a ; Sophie R. Beeren^a ; Dennis Blank^b ; Byung Y. Yang^c ; Rudolf Geyer^b ; Ole Hindsgaul^a ; ^{Ole.Hindsgaul@carlsberglab.dk}
• 关键词：Carbohydrate analysis ; Fluorogenic dye ; Capillary electrophoresis ; MALDI-ToF-MS
• 刊名：Carbohydrate Research
• 出版年：2012
• 出版时间：1 May, 2012
• 年：2012
• 卷：352
• 期：Complete
• 页码：94-100
• 全文大小：558 K

文摘

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¡ªchromophores, fluorophores, charges, ionizable groups¡ªare 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.