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 alpha1-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.
Kannicht
Christoph
C
Molecular Biochemistry Department, Octapharma Research and Development, Berlin, Germany.
Grunow
Detlef
D
Lucka
Lothar
L
D002236:Carbohydrate Conformation; D006026:Glycoside Hydrolases / Q000378:metabolism*; D016256:Lewis X Antigen / Q000737:chemistry*; D011134:Polysaccharides / Q000737:chemistry* / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*
D016256:Lewis X Antigen; D011134:Polysaccharides; D006026:Glycoside Hydrolases