Home Protocols Protocol for Enrichment of O-glycopeptides or N-glycans with terminal GlcNAcs with Boletopsis grisea Lectin (BGL) (NEB #P0867)

Protocol for Enrichment of O-glycopeptides or N-glycans with terminal GlcNAcs with Boletopsis grisea Lectin (BGL) (NEB #P0867)

Introduction:

Highly specific lectins can be used to reduce sample complexity by permitting subtraction of structures that bear a specific sugar epitope using a filter-assisted sample preparation technique. We defined this method for UPLC-HILIC-FLR analyses and have given it the acronym epitope-directed glycan enrichment (EDGE) UPLC-HILIC-FLR profiling (1,2).

Boletopsis grisea Lectin (BGL) is one such specific lectin that can be used to enrich GlcNAc-capped N-glycans or mucin type O-glycopeptides from complex samples in glycomics and glycoproteomics analytical workflows.

Reaction Setup:

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. Pass sample through a 10 kDa MWCO filter (e.g., Nanosep® Centrifugal Device with Omega™ Membrane 10K (PALL# OD10C34)).

  3. Wash the filter 2 times by centrifugation (10 minutes at 13,000 x g) with 20 mM Tris-HCl pH 7.5. Combine the washes, dry by vacuum evaporation and save for analysis (flow-through).

Elution Protocols

For O-glycopeptides:

  1. Add 120 µl elution buffer (0.2% formic acid, 50% acetonitrile)

  2. Incubate for 30 minutes at room temperature.

  3. Collect the eluate by centrifugation (10 minutes at 13,000 x g), wash as above and dry it by vacuum evaporation.

  4. Analyze the flow-through (from Step 3) and eluate by LC-MS or UPLC-HILIC-FLR.

For N-glycans:

  1. Add 120 µl elution buffer (6U Proteinase K in 20 mM Tris-HCl pH 7.5 and lacking glycerol (see Footnote).

  2. Incubate the reaction at 37 °C overnight.

  3. Collect the eluate by centrifugation (10 minutes at 13,000 x g), wash as above and dry it by vacuum evaporation.

  4. Analyze the flow-through and eluate 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 20mM 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.

References:

  1. Ganatra, M. B. et al. A bi‑specific lectin from the mushroom Boletopsis grisea and its application in glycoanalytical workflows. Sci. Rep. 11, 160.
  2. Vainauskas, S. et al. Profiling of core fucosylated N-glycans using a novel bacterial lectin that specifically recognizes α1,6 fucosylated chitobiose. Sci. Rep. 6, 34195. https://doi.org/10.1038/srep3 4195 (2016).