ARTICLES Targeted Use of Exoglycosidase Digestion for the Structural Elucidation of Neutral O-Linked Oligosaccharides Yongming Xie, Ken Tseng, Carlito B. Lebrilla Department of Chemistry, University of California, Davis, California, USA Jerry L. Hedrick Section of Molecular and Cellular Biology, University of California, Davis, California, USA Exoglycosidase digestion in combination with the catalog-library approach (CLA) is used with matrix-assisted laser desorption/ionization Fourier transform mass spectrometry (MALDI- FTMS) to obtain the complete structure of oligosaccharides. The CLA is a collision-induced dissociation (CID)-based method used to determine the structure of O-linked neutral oligo- saccharides. It provides both linkage and stereochemical information. Exoglycosidases are used to confirm independently the validity of the CLA. In some cases, the CLA provides structural information on all but a single residue. Exoglycosidase is used to refine these structures. In this way, exoglycosidase use is targeted employing only a small number of enzymes. Exoglycosidase arrays, which have been used with N-linked oligosaccharides, is avoided despite the larger variations in structures of O-linked species. (J Am Soc Mass Spectrom 2001, 12, 877– 884) © 2001 American Society for Mass Spectrometry O ligosaccharides, as glycoconjugates, are widely distributed in nature and play key roles in several fundamental cellular processes [1]. Their relative importance in cellular recognition has spurred the development of a large number of methods for structural elucidation. Despite the significant efforts in their developments however, no single method has emerged. The problem lies in the complexity of oligo- saccharides. The variations in connectivity, the number of different linkage combinations, the possibility of branching, and the stereochemistry of each monosac- charide unit all conspire to produce major challenges in structural elucidation. Moreover, the large number of oligosaccharides and the variations in their structures from even a single source produce an often intractable problem that has hindered the study of structure– function relationships in oligosaccharides. Mass spectrometry offers an attractive method for structural elucidation as it is unsurpassed in both speed and sensitivity. Mass spectrometry has long been used to obtain the composition and the connectivity of un- known oligosaccharides. Several studies have been published where mass spectrometry, and specifically CID was employed to obtain linkage information [2–7]. There have even been efforts towards obtaining stereo- chemistry with CID [8]. These latter studies have fo- cused on monosaccharides but they may be extended to larger oligomers. Until recently, the use of mass spectrometry to determine the complete structure was still not possible. In a preceding publication, we described a mass spec- trometry (MS) based method to obtain the structure of oligosaccharides complete with linkage, stereochemis- try, and anomericity [9]. The method was used to profile most of the O-linked neutral oligosaccharide components in a library released from the jelly coat of the Xenopus laevis egg [10]. This frog is a common biological model for the study of reproduction [11–13]. Oligosaccharides released from this source were found to have several substructural motifs in common (Chart 1). Even the small number of motifs combine to produce large variation in structures. The motifs also yield distinct fragmentation patterns under CID that are preserved from one compound to the next. These pat- terns are sensitive to the linkage and even the stereo- chemistry of the substructural unit. The determination of new structures requires the identification of various patterns corresponding to substructures in the spectra. Unknown structures can be deduced by recombining various motifs found in the CID spectra (Scheme 1). The key to this method is to identify the substructural motifs and their corresponding CID spectra to develop a catalog for the library [9]. For this reason the method was named the catalog-library approach (CLA). CLA Published online May 16, 2001 Address reprint requests to Dr. C. B. Lebrilla, Department of Chemistry, University of California, Davis, CA 95616. E-mail: cblebrilla@ucdavis.edu © 2001 American Society for Mass Spectrometry. Published by Elsevier Science Inc. Received December 18, 2000 1044-0305/01/$20.00 Revised March 19, 2001 PII S1044-0305(01)00267-7 Accepted March 19, 2001