A Typical Epitope Directed Glycan Enrichment (EDGE) Profiling Protocol using Boletopsis grisea Lectin (BGL) (NEB #P0867)
We recommend a starting amount of 100 μg BGL for EDGE enrichment (A lower starting amount could be used for O-glycopeptides. This amount should be empirically determined).
Combine the following components in a microcentrifuge tube:
|O-glycopeptides or N-glycans released from glycoproteins||Variable||up to 200 pmol|
|BGL (1 mg/ml)||Variable||100 µg (100 µl)|
|Tris-HCl pH 7.5 (20 mM)||up to 120 µl|
- Incubate the reaction at 25°C for 90 minutes.
- Wash a Nanosep® Centrifugal Device with Omega™ Membrane 10K (PALL# OD10C34) with 500 μl H2O. Centrifuge for 10 minutes at 13,000 x g. Discard the filtrate.
- Transfer the reaction mixture from Step. 1 to the Nanosep filter. Centrifuge for 10 minutes at 13,000 x g.
- Wash the filter 2 times with 400 μl of 20 mM Tris-HCl pH 7.5. Centrifuge for 10 minutes at 13,000 x g after each wash.
- Combine the washes to a fresh tube, dry by vacuum evaporation, and save for analysis (Flow Through sample).
- (A) Elution Protocol For O-glycopeptides:
Add 120 μl elution buffer (0.2% formic acid, 50% acetonitrile) and incubate for 30 minutes at room temperature. Collect the eluate by centrifugation (10 minutes at 13,000 x g), wash as above, and dry it by vacuum evaporation (Elution sample).
(B) Elution Protocol For N-glycans:
Add 120 μl elution buffer (6U Proteinase K in 20 mM Tris-HCl pH 7.5 and lacking glycerol (see Footnote). Incubate the reaction at 37°C overnight.
Collect the eluate by centrifugation (10 minutes at 13,000 x g), wash as above, and dry it by vacuum evaporation (Elution sample).
- Analyze the flow-through and elution samples by LC-MS or UPLC-HILIC-FLR.
Removal of Glycerol from Proteinase K (NEB# P8107S)
- Dilute 50 μl of 800 units/ml Proteinase K, Molecular Biology Grade (NEB #P8107S) with 450 μl 20mM Tris HCl pH 7.5.
- Apply to a 0.5 ml 3K Millipore Amicon Ultra Filter Unit (cat. # UFC500324) and spin in a microcentrifuge for 30 minutes at 12,000 rpm.
- Discard flow-through and add an additional 450 μl of 20 mM Tris HCl pH 7.5 to the sample. Spin in a microcentrifuge for 30 minutes at 12,000 rpm.
- Place the Amicon filter device upside-down in a clean microcentrifuge tube and spin for 2 minutes at 1,000 rpm to transfer glycerol-free Proteinase K to the tube.