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Separation of 1‐aminopyrene‐3,6,8‐trisulfonate‐labeled asparagine‐linked fetuin glycans by capillary gel electrophoresis
Author(s) -
Guttman András,
Chen FuTai A.,
Evangelista Ramon A.
Publication year - 1996
Publication title -
electrophoresis
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.666
H-Index - 158
eISSN - 1522-2683
pISSN - 0173-0835
DOI - 10.1002/elps.1150170221
Subject(s) - capillary electrophoresis , chromatography , glycan , fetuin , chemistry , asparagine , electrophoresis , biochemistry , glycoprotein , amino acid
Abstract Asparagine‐linked glycans of bovine fetuin were separated by capillary gel electrophoresis after enzymatic release (peptide‐ N ‐glycosidase F) and labeling via reductive amination by a fluorescent dye, 1‐aminopyrene‐3,6‐8‐trisulfonate (APTS). At low separation pH (2.5) only two dominant peaks were observed. Increasing the separation buffer pH to 4.75 resulted in complete separation of two primary doublets and several minor peaks from the fetuin N ‐linked glycan pool. Two of the four major peaks were spiked with purified individual standards and were identified as trisialylated triantennary structures with different sialylation linkages. The other two larger peaks were postulated to be tetrasialylated triantennary structures, based on calculations considering their corresponding glucose unit (GU) values. Effects of the electrophoretic separation parameters, such as gel concentration, electric field strength and temperature on the migration behavior of the two major doublets of the fetuin glycan pool were also thoroughly examined. Our data suggest that the capillary gel electrophoresis separation of the multisialylated branched oligosaccharides with different linkage isomers, released from bovine fetuin, is fundamentally based on their degree of sialylation and hydrodynamic volumes.