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A new method for sequencing linear oligosaccharides on gels using charged, fluorescent conjugates.
K B Lee1, A al-Hakim, D Loganathan
1Division of Medicinal and Natural Products Chemistry, College of Pharmacy, University of Iowa, Iowa City 52242.
Carbohydrate Research
|July 18, 1991
Summary
A novel method uses fluorescently labeled oligosaccharides for sequencing on gels. This technique enables the determination of carbohydrate structures released from complex biological molecules.
Area of Science:
- Carbohydrate Chemistry
- Analytical Biochemistry
- Glycobiology
Background:
- Oligosaccharide sequencing is crucial for understanding biological processes.
- Existing methods for oligosaccharide analysis can be complex and time-consuming.
Purpose of the Study:
- To develop a new, efficient method for sequencing linear oligosaccharides.
- To enable the analysis of oligosaccharides derived from glycoproteins, glycolipids, and proteoglycans.
Main Methods:
- Conjugation of oligosaccharides with a charged fluorescent dye (monopotassium 7-amino-1,3-naphthalenedisulfonate) via reductive amination.
- Purification using preparative gradient polyacrylamide gel electrophoresis and semi-dry transfer to charged nylon membranes.
- Sequential treatment with exoglycosidases and endoglycosidases, followed by gel electrophoresis analysis.
Main Results:
- Successful conjugation and purification of mono-, di-, tri-, and tetra-saccharide-fluorescent conjugates.
- Structural elucidation of monosaccharide and trisaccharide conjugates using f.a.b.-m.s. and 2D n.m.r.
- Demonstrated utility of the method for determining oligosaccharide sequence through enzymatic degradation and gel electrophoresis.
Conclusions:
- The described method provides a robust approach for sequencing linear oligosaccharides.
- This technique offers a valuable tool for analyzing complex carbohydrate structures in glycobiology research.