Exoglycosidase matrix-mediated sequencing of a complex glycan pool by capillary electrophoresis.
This paper discusses oligosaccharide sequencing by consecutive enzymatic digestion of carbohydrates using an exoglycosidase array, followed by capillary electrophoresis separation of the digests. Because of the high resolving power and good reproducibility of capillary electrophoresis, multistructure sequencing of a complex glycan pool can be performed in most instances requiring no prior isolation of the individual oligosaccharides. High sensitivity laser-induced fluorescence detection enables acquisition of complete sequence information from several picomoles of glycoproteins. Comparison of the migration times of the exoglycosidase digest fragments to the maltooligosaccharide ladder, enables calculation of migration shifts, due to cleavage based on the actual exoglycosidases used. The particular sequence of each oligosaccharide in a glycan pool can be proposed with high confidence based on the migration time shifts of the various oligosaccharide structures. However, possible combinations of various sequence fragments may have very similar charge to hydrodynamic volume ratios, resulting in electrophoretic co-migration when a mixture of different oligosaccharides is sequenced together. Then, capillary electrophoresis separations of the resulting fragments should be evaluated after each digestion step. In the instances of complex separation profiles when multiple peaks are present, the evaluation of peak shifts can get very complicated and solved only with the aid of a software program. Data about the monosaccharide composition of the glycan pool provides useful information in designing the digestion enzyme matrix.
Guttman
A
A
Genetic BioSystems, Inc., San Diego, CA 92121, USA.
Ulfelder
K W
KW
D002240:Carbohydrate Sequence; D019075:Electrophoresis, Capillary / Q000379:methods*; D006026:Glycoside Hydrolases / Q000378:metabolism*; D008969:Molecular Sequence Data; D011134:Polysaccharides / Q000737:chemistry* / Q000378:metabolism; D017421:Sequence Analysis / Q000379:methods*; D013050:Spectrometry, Fluorescence
D011134:Polysaccharides; D006026:Glycoside Hydrolases