Separation of 1‐aminopyrene‐3,6,8‐trisulfonate‐labeled asparagine‐linked fetuin glycans by capillary gel electrophoresis

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.

Neutral polar methacrylate‐based monoliths for normal phase nano‐LC and CEC of polar species including N‐glycans

“…The PNGase F was employed to catalyze the hydrolysis of asparagine-linked high-mannose, hybrid, and complex type N-glycans from standard glycoproteins [27,28]. Glycoprotein (2 mg) was added to an Eppendorf tube, to which 350 lL of deionized water was added to dissolve all of the protein.…”

Comparison of separation techniques for the elucidation of IgG N-glycans pooled from healthy mammalian species

“…CE-LIF has proven to be a sensitive and high resolution separation method that is already broadly utilized in the analysis of complex carbohydrates, both by the biomedical as well as the biotechnology 90 industries. [24][25][26] CE separation is driven by an electric field mediated migration and is therefore based on charge to hydrodynamic volume ratio. Highly charged species migrate first.…”

“…The most commonly used fluorophore which meets these criteria is 8-aminopyrene-1,3,6-trisulfonic acid (APTS). 24,28 For each of the selected separation techniques peak retention/ migration times are directly correlated to a standard oligosaccharide ladder used to allow for initial structural assignment of the glycans of interest. The choice of the ladder depends on the matrix by which the separation occurs.…”

Monolithic silica capillary columns having immobilized lectins and surface bound polar functionalities for lectin affinity and normal phase nano‐LC and CEC of glycoconjugates, respectively

“…The PNGase F was employed to catalyze the hydrolysis of asparagine-linked high-mannose (Man), hybrid, and complex types oligosaccharides from glycoproteins [50,51]. Glycoprotein of 2 mg was added to an Eppendorf tube, to which 350 lL of deionized water was added to dissolve all of the proteins.…”