Enzymatic Sequence Analysis of N-Glycans by Exoglycosidase Cleavage and Mass Spectrometry – detection of Lewis X Structures
Enzymatic sequencing of oligosaccharides gives structural information on sequence of monosaccharides and type of linkage within the oligosaccharide chain. This data can be obtained by stepwise enzymatic digestion of a single, isolated oligosaccharide using individual or mixtures of specific exoglycosidases. N-glycans have to be fractionated from mixtures prior to sequence analysis to assign this type of structural information to a specific glycan. Enzymatic sequencing can be applied to oligosaccharide mixtures as well to evaluate the occurrence of distinct oligosac-charide motives of functional and/or structural interest.
Here we describe the application of enzymatic sequence analysis to a mixture of N-glycans released from α1-acid glycoprotein. The experimental conditions are optimized for detection of possible Lewis X structures after stepwise exoglycosi-dase digestion by MALDI-TOF mass spectrometry. However, the described method is generally applicable to analyze other structural properties of N-glycans by use of the respective specific exoglycosidases.
Key WordsEnzymatic sequencing N-glycan exoglycosidase MALDI-TOF Lewis x oligosaccharide structure
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