Snap capture beads conjugated to snap tag purified protein

Example 6

When PNGase F is used to trim N-glycans away from the peptides or proteins, the terminal GlcNAc residue becomes available for modification. A common modification is fluorophore labeling using 2-AB dye. The primary amino group of the dye performs a nucleophilic attack on the carbonyl carbon of the acyclic reducing terminal residue to form a partially stable Schiff's base. The Schiff's base imine group is chemically reduced to give a stable labeled glycan. The conserved terminal GlcNAc residue is thus referred to as the “reducing end” of a glycan species. Upon 2-AB labeling, the carbohydrate ring structure is no longer capable of forming. Fbs binding to 2-AB labeled M3N2 was tested and the results are shown in FIG. 4. Fifty picomoles of 2-AB labeled M3N2 in low salt buffer were incubated with SNAP-Fbs beads or SNAP control beads (SNAP Capture beads conjugated to SNAP-tag Purified Protein, New England Biolabs, Ipswich, Mass.). The majority of the 2-AB labeled fluorophore was found in the flow-through and low salt buffer wash fractions in both samples indicating that 2-AB labeling disrupts the interaction between glycan and Fbs. This consequence does not affect the application of Fbs for N-glycan linked glycomolecule enrichment as the 2-AB labeling step is an optional step downstream from the enrichment step.

Example 6

When PNGase F is used to trim N-glycans away from the peptides or proteins, the terminal GlcNAc residue becomes available for modification. A common modification is fluorophore labeling using 2-AB dye. The primary amino group of the dye performs a nucleophilic attack on the carbonyl carbon of the acyclic reducing terminal residue to form a partially stable Schiff's base. The Schiff's base imine group is chemically reduced to give a stable labeled glycan. The conserved terminal GlcNAc residue is thus referred to as the “reducing end” of a glycan species. Upon 2-AB labeling, the carbohydrate ring structure is no longer capable of forming. Fbs binding to 2-AB labeled M3N2 was tested and the results are shown in FIG. 4. Fifty picomoles of 2-AB labeled M3N2 in low salt buffer were incubated with SNAP-Fbs beads or SNAP control beads (SNAP Capture beads conjugated to SNAP-tag Purified Protein, New England Biolabs, Ipswich, Mass.). The majority of the 2-AB labeled fluorophore was found in the flow-through and low salt buffer wash fractions in both samples indicating that 2-AB labeling disrupts the interaction between glycan and Fbs. This consequence does not affect the application of Fbs for N-glycan linked glycomolecule enrichment as the 2-AB labeling step is an optional step downstream from the enrichment step.