Glycan analysis via derivatization with a fluorogenic pyrylium dye Sine A. Johannesen a , Sophie R. Beeren a , Dennis Blank b , Byung Y. Yang c , Rudolf Geyer b , Ole Hindsgaul a,⇑ a Carlsberg Laboratory, Gamle Carlsberg Vej 10, Copenhagen V DK-1799, Denmark b Institute of Biochemistry, Faculty of Medicine, Justus-Liebig-University of Giessen, Friedrichstrasse 24, 35392 Giessen, Germany c Grain Processing Corporation, 1600 Oregon St. Muscatine, IA 52761, USA article info Article history: Received 30 November 2011 Received in revised form 13 February 2012 Accepted 15 February 2012 Available online 24 February 2012 Dedicated to Professor Hans Paulsen on the occasion of his 90th birthday. Keywords: Carbohydrate analysis Fluorogenic dye Capillary electrophoresis MALDI-ToF-MS abstract The expansion of glycomics analysis is reliant upon the development of robust, routine methods for car- bohydrate characterization. Simple protocols to derivatize sugars with functionality that facilitate anal- ysis—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 spec- trometry (MALDI-ToF-MS). The unreacted free dye, Py-1, is effectively non-fluorescent but when conju- gated 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 oligosaccha- rides with up to 58 glucose units. Ó 2012 Elsevier Ltd. All rights reserved. 1. Introduction Research in glycobiology is dependent upon the availability of efficient and reliable analytical techniques to quantify and charac- terize carbohydrates. 1–3 Since the carbohydrates themselves lack chromophores or fluorophores, and have poor ionization efficiency, derivatization is generally required for analysis using HPLC or capillary electrophoresis (CE), and for detection by mass spectrom- etry. 4–8 Derivatization at the reducing-end aldehyde (hemi-acetal) ensures that each oligosaccharide receives precisely one label, enabling direct quantification of glycan mixtures. Here we describe a two step method for the functionalization of oligosaccharides with a fluorogenic pyrylium dye (Py-1) that generates a highly fluorescent sugar conjugate and incorporates a permanent positive charge, thus allowing sensitive detection by CE and matrix assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-ToF-MS). While there are many different fluorescent labels that have been developed for oligosaccharide labeling using, for example, reductive amination or hydrazone formation at the reducing-end aldehyde as a means of conjugation, 5,6 a common limitation is the need to remove the large quantities of excess dye required for the derivatization before analyses can be made. Fluorogenic dyes, themselves, are non-fluorescent but become fluorescent only after reaction with the analyte. 9–11 Purification of fluorogenically- labeled samples, therefore, or careful washing of a fluorogenical- ly-stained gel to minimize background fluorescence, is not required before analysis. Pyrylium salts selectively react with primary amines to form pyridinium salts and have been used to functionalize proteins with various labels via reaction with the e-amino group of lysine. 11–13 Py-1, recently developed by Wolfbeis as a fluorogenic protein stain for gel electrophoresis, is blue and virtually non-fluorescent. 11 Upon reaction with a primary amine it forms a red pyridinium con- jugate (k abs. max.  500 nm) with strong fluorescence emission at 600–640 nm (Fig. 1). Commercially sold as Chromeo P503, Py-1 has been effectively utilized as a stain for SDS–PAGE separation of proteins, 11,14 to label proteins for highly sensitive quantitative analysis using CE, 15–19 as well as to investigate protein–aptamer complexation by means of kinetic capillary electrophoresis (KCE). 20 In this article we explore, for the first time, the use of Py-1 to label, characterize, and profile oligosaccharide mixtures. In an easy, two-step process, the oligosaccharides are first equipped with a primary amino group, by reaction with one end of ethylenedia- mine, leaving a free primary amine, which is subsequently reacted with Py-1 (Scheme 1). Though not made use of in the present study, the remaining secondary amine is available for introduction of additional functionalities such as stable isotope tags for mass 0008-6215/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved. doi:10.1016/j.carres.2012.02.016 ⇑ Corresponding author. Tel.: +45 33275382; fax: +45 33274708. E-mail address: Ole.Hindsgaul@carlsberglab.dk (O. Hindsgaul). Carbohydrate Research 352 (2012) 94–100 Contents lists available at SciVerse ScienceDirect Carbohydrate Research journal homepage: www.elsevier.com/locate/carres