Carbohy drate Research, 214 (1991) 155-168 Elsevier Science Publishers B.V., Amsterdam 155 A new method for sequencing linear oligosaccharides on gels using charged, fluorescent conjugates* Kyung-Bok. Lee, Ali Al-Hakim, Duraikkannu Loganathan, and Robert J. Linhardt+ Division of Medicinal and Naturul Products Chemistry, College of Phurmuq. Uninersii): qf Iowa, Iowa City, IA 52242 (U.S.A.) (Received September 13th, 1990; accepted for pubhcdtion, in revised form, December 5th, 1990) ABSTRACT A new method is described for sequencing linear oligosaccharides on gels using charged, fluorescent conjugates. The reducing ends of various mono-, di-, tri-. and tetra-saccharides were conjugated with monopotassium 7-amino-1,3-naphthalenedisulfonatc (a fluorescent and negatively charged compound) by reductive amination using sodium cyanoborohydride. The sugar conjugates were purified by preparative gradient polyacrylamide gel electrophoresis followed by a newly developed technique involving their semi-dry transfer to positively charged nylon membranes and elution with sodium chloride. The structures ofa monosaccharide- and trisaccharide-conjugate were established by f.a.b.-m.s. and 2D n.m.r. Seven linear oligosaccharideefluorescent conjugates were treated sequentially with exoglycosidases and with endoglyco- sidases. Analysis of the products by gel electrophoresis provided sequence information. These methods may be useful for sequencing oligosaccharides that are chemically or enzymically (endoglycosidase) released from glycoproteins, glycolipids, and proteoglycans. INTRODUCTION Many biological roles of carbohydrate units in glycoproteins have been reported’. These include: protection of the polypeptide component against uncontrolled proteo- lytic attack’,‘, facilitation of the secretion of certain proteins or their mobilization to the cell surface4, maintenance of glycoprotein conformation in a biologically active form’, clearance of glycoproteins from plasma’, direction of the immune response by acting as immune decoys7,x, and their importance as antigenic determinants in differentiation and development’. Therefore, information about glycoprotein sugar composition, and more importantly their sequence is required to establish structureefunction relation- ships. However, glycoproteins are usually available in only limited quantities (typically l&100 pg), making it difficult to determine the sequence, position, and anomeric configurations of glycosidic linkages in their carbohydrate chains’. Highly sensitive detection methods have been reported for oligosaccharides, such as tritium labeling at the reducing end of sugars by sodium [‘Hlborohydride reduc- tion” I2 and fluorescent labeling by reductive amination. Fluorophores, including 2-aminopyridine’3m’5, 7-amino-4-methylcoumarin”, monodansylethylenediamine17, dan- * Presented at the 15th International Carbohydrate Symposium. Yokohama, Japan. August 12- 17, 1990. ’ To whom correspondence should be addressed. OOOg-6215/91/$03.50 @ 1991 ~ Elsevier Science Publishers B.V.