Journal of Chromatography A, 1004 (2003) 121–129 www.elsevier.com / locate / chroma Improved capillary electrophoretic separation and mass spectrometric detection of oligosaccharides * Hyun Joo An, Andreas H. Franz,, Carlito B. Lebrilla Department of Chemistry, University of California, Davis, CA 95616, USA Abstract We have developed a CE method for the separation of structural isomers of oligosaccharides labeled with N-quaternized benzylamine. Oligosaccharides with reducing ends were derivatized with benzylamine by reductive amination followed by quaternization to yield a fixed cation label. The benzylamine-derivatized oligosaccharides were analyzed by CE–UV in ammonium acetate buffer and off-line matrix-assisted laser desorption ionization (MALDI) MS. The method was applied to a 1 nmol sample of a model oligosaccharide (LNDFH 1). From this sample a 38 fmol diluted standard was detected. The quaternization of benzylamine-labeled products significantly improved CE separation of neutral oligosaccharides along with several structural isomers. Two hexasaccharide isomers (LNDFH I and LNDFH II) were baseline resolved using an ammonium acetate buffer. This method was also applied successfully to the profiling of oligosaccharides released from the glycoprotein RNase B. The release of 6 pmol of glycans followed by workup showed the detection of all components, with one component corresponding to 100 fmol. Micropreparative collection of CE enabled successful off-line CE–MALDI-MS without additional sample clean up. This report provides a simple and rapid method to separate and analyze oligosaccharides.  2003 Elsevier B.V. All rights reserved. Keywords: Derivatization, electrophoresis; Oligosaccharides; Glycoproteins; Proteins 1. Introduction tures because of its inherent high resolving power and sensitivity [4–7]. The small volume and sample The complete characterization of the glycan is amounts necessary for analyses are ideally suited to essential for understanding the biological functions the sample limitations common to oligosaccharides of glycoproteins [1–3]. However, the separation of analyses. However, the CE analysis of oligosac- oligosaccharide mixtures released from glycoproteins charides remains far from routine. One major limita- is often challenging due to their heterogeneity. tion is the oligosaccharides’ lack of chromophoric Structural isomers are of particular concern as they activity in the operational range of existing detectors. frequently suffer from poor chromatographic sepa- To overcome this problem, oligosaccharides are ration. often labeled with UV active and/or fluorescent Capillary electrophoresis (CE) can play an im- labels for increased sensitivity [8,9]. The most portant role in the analysis of oligosaccharide mix- common method for labeling oligosaccharides is via reductive amination [10]. 2-Aminopyridine was the first introduced as an oligosaccharide label, however, *Corresponding author. Tel.: 11-530-752-6364; fax: 11-530- since then many more have been used successfully. 752-8995. E-mail address: cblebrilla@ucdavis.edu (C.B. Lebrilla). In addition to their chromophoric activities, other 0021-9673 / 03 / $ – see front matter  2003 Elsevier B.V. All rights reserved. doi:10.1016 / S0021-9673(03)00718-0