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:

Component Volume Final Concentration
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
  1. Incubate the reaction at 25°C for 90 minutes.

  2. 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.

  3. Transfer the reaction mixture from Step. 1 to the Nanosep filter. Centrifuge for 10 minutes at 13,000 x g.

  4. 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.

  5. Combine the washes to a fresh tube, dry by vacuum evaporation, and save for analysis (Flow Through sample).

  6. (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).

  7. Analyze the flow-through and elution samples by LC-MS or UPLC-HILIC-FLR.

 

Footnote:

Removal of Glycerol from Proteinase K (NEB# P8107S)

  1. Dilute 50 μl of 800 units/ml Proteinase K, Molecular Biology Grade (NEB #P8107S) with 450 μl 20mM Tris HCl pH 7.5.
  2. 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.
  3. 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.
  4. 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.