Home Protocols N-Glycopeptide Binding Protein Typical Reaction Protocol (NEB #P8072)

N-Glycopeptide Binding Protein Typical Reaction Protocol (NEB #P8072)

The enrichment of N-glycopeptides by N-Glycopeptide Binding Protein typically follows protease digestion (most commonly, trypsin) and precedes LC-MS analysis. Please see typical protocols for trypsin digestion in the Notes section. Enrichment can be performed with either a filtration device or magnetic beads immobilized with N-Glycopeptide Binding Protein.

N-Glycopeptide Enrichment by Filtration

  1. Prepare cleaned-up, tryptic peptide sample in 100-300 μl in 100 mM NH4HCO3. A typical input amount of peptide is 100 μg.

  2. Load tryptic-peptide sample and 100 μl of N-Glycopeptide Binding Protein on a Microcon-30kDa Centrifugal Filter Unit with Ultracel-30 membrane (Millipore Sigma, P/N MRCF0R030). Incubate at 4°C for 1 h.

  3. Centrifuge at 14,000 x g for 12 min at 4°C to remove unbound peptides. Discard flowthrough.

  4. Add 300 μl of 100 mM NH4HCO3 to the filter unit and incubate at 4°C for 5 min to wash away non-glycopeptides. Centrifuge at 14,000 × g for 12 min at 4°C, discard flowthrough. Repeat twice.

  5. Add 200 μl 50% formic acid to the filter unit and incubate at room temperature for 5 min to dissociate bound glycopeptides. Centrifuge at 14,000 × g for 15 min, coll­­­­­ect the flowthrough containing the enriched N-glycopeptides. Repeat twice, combine all eluates, collect a total of 600 μl.

  6. Lyophilize the sample to dryness to remove formic acid and residual NH4HCO3.

  7. Reconstitute the enriched N-glycopeptide sample in 50 μl LC-MS grade water with 0.1% Formic Acid. If desired, measure the concentration using a Nanodrop Spectrophotometer or a UV-Vis spectrometer at A280 (with the sample type option set to 1 Abs = 1 mg/ml). Analyze using LC-MS or as desired.

N-Glycopeptide Enrichment by Magnetic Beads Immobilized with N-Glycopeptide Binding Protein

  1. Resuspend SNAP-Capture Magnetic bead slurry (NEB #S9145S) by vortexing or mixing.

  2. Immediately dispense 100 µl of the bead slurry to a 1.5 ml tube. Place the tube in a magnetic rack (NEB #S1506S) to pellet the beads. Remove and discard the supernatant.

  3. Add 150 µl of wash buffer to the bead pellet. Mix briefly to resuspend (wash buffer: 20 mM Tris-HCl, 50 mM NaCl, pH 8.0).

  4. Place the tube in a magnetic rack to pellet the beads. Remove and discard the supernatant. Repeat the wash step 3 times.

  5. Resuspend the beads in 100 µl wash buffer supplemented with 10 mM DTT (e.g., 100 µl wash buffer, 1 µl of 1M DTT). Note: DTT is required to efficiently conjugate N-Glycopeptide Binding Protein to SNAP-Capture Beads.

  6. Add 100 µl N-Glycopeptide Binding Protein (1 mg/ml) to the resuspended magnetic beads. Mix end-over-end overnight at 4°C to immobilize the protein to the beads.

  7. After overnight immobilization, place the tube in a magnetic rack to pellet the beads. Remove the supernatant and transfer to a fresh tube. Optional: to assess the immobilization reaction efficiency, analyze a sample of the supernatant by SDS-PAGE.

  8. Add 150 µl wash buffer to the bead pellet (now conjugated with N-Glycoprotein Binding Protein) and mix briefly to resuspend. Place the tube in a magnetic rack to pellet the beads. Remove and discard supernatant. Repeat twice.

  9. Add 150 µl Protein-Free Blocking Buffer (Pierce, P/N 37570) and mix end-over-end for 1 h at 4°C.

  10. Place the tube in a magnetic rack to pellet the beads. Remove and discard supernatant. The beads are now ready for glycopeptide or glycoprotein enrichment.

  11. Dilute or resuspend the peptide or protein sample to a concentration of 0.2 µg/µl in a total volume of 100 µl using wash buffer.

  12. Add the 100 µl peptide or protein sample to the beads and mix end-over-end at 4°C for 1 h.

  13. Place the tube in a magnetic rack to pellet the beads. Remove and discard supernatant.

  14. Add 150 µl wash buffer to the bead pellet and mix briefly to resuspend. Place the tube in a magnetic rack to pellet the beads. Remove and discard supernatant. Repeat twice. Add 50 µl of elution buffer (1:1 water: formic acid solution) to beads and mix the suspension end-over-end at 4°C for 5 minutes.

  15. Place the tube in a magnetic rack to pellet the beads, remove and keep the supernatant containing the eluted N-glycopeptides. Repeat twice and pool the eluates for a total of 150 µl.

  16. Lyophilize the pooled eluate to dryness.

  17. Reconstitute the enriched N-glycopeptide sample in 50 μl LC-MS grade water with 0.1% Formic Acid. If desired, measure the concentration using a Nanodrop Spectrophotometer or a UV-Vis spectrometer at A280 (with the sample type option set to 1 Abs = 1 mg/ml). Analyze using LC-MS or as desired.

Notes:

It is important to prepare your sample free of components that may reduce binding efficiency, such as denaturants and chaotropic agents used prior to proteolysis, as well as acid used to stop the trypsinization reaction.

Filtration Assisted Sample Preparation (FASP)

  1. Protein sample preparation, depending on the sample type, choose from the following protocols:

Serum

  1. Combine 5 μl serum (containing 300 - 400 μg protein) and 100 μl denaturing buffer (100 mM Tris-HCl, 100 mM DTT, 4% SDS, pH 7.5).

  2. Incubate at 95°C for 3 min.

  3. Transfer the reaction into a filter unit (Microcon-30kDa Centrifugal Filter Unit with Ultracel-30 membrane, Millipore Sigma, P/N MRCF0R030).

Cell lysate

  1. Lyse 50 µl of pelleted cells with 800 µl of lysis buffer (100 mM Tris-HCl, 1% SDS, pH 7.5).

  2. Clarify the cell lysate by centrifugation at 15,000 × g for 10 min to remove cell debris. Remove the supernatant and transfer to a fresh tube.

  3. Add 90 μl of fresh 100 mM DTT to the supernatant to a final concentration of 10 mM.

  4. Incubate at 95°C for 3 min.

  5. Transfer 400 µl of cell lysate (containing 300 – 400 µg protein) to a filter unit (Microcon-30kDa Centrifugal Filter Unit with Ultracel-30 membrane, Millipore Sigma, P/N MRCF0R030).

  6. Spin at 14,000 x g for ~5 min, or as needed, to concentrate to ~100 μg.

 

  1. Denaturation and alkylation
    1. Add 200 μl of fresh 8 M urea in 0.1 M Tris-HCl pH 8.5 to the filter unit. Centrifuge at 14,000 x g for 15 min. Discard flowthrough. Repeat once.

    2. Add 100 μl of iodoacetamide solution (fresh 0.05 M iodoacetamide and 8 M urea in 0.1 M Tris-HCl) to the filter and incubate for 20 min in the dark.

    3. Centrifuge at 14,000 x g for 10 min, discard flowthrough. Add 100 μl urea buffer, centrifuge at 14,000 x g for 10 min and discard flowthrough. Repeat twice.

    4. Add 50 μl of 50 mM NH4HCO3, centrifuge at 14,000 x g for 10 min. Discard flowthrough. Repeat 3 times.

 

  1. Trypsin Digestion
    1. Transfer the filter unit to a new collection tube. Add 100 μl Trypsin-Ultra (NEB #P8101S, 0.1 μg/μl in 50 mM NH4HCO3) to the filter. Incubate overnight at 37°.

    2. To collect tryptic peptides, centrifuge at 14,000 x g for 10 min, collect flowthrough. Add 100 mM NH4HCO3 buffer, centrifuge at 14,000 x g for 10 min, collect flowthrough. Repeat 3 times and combine all four flowthroughs (400 μl total volume).

    3. Add phenylmethylsulfonyl fluoride in ethanol (e.g., 4 μl of 0.1 M PMSF, Sigma-Aldrich, 93482) to the tryptic peptide solution to a final concentration of 1 mM to inactivate residual trypsin. Incubate at 4°C for ≥2 h.

    4. The concentration of tryptic peptides can be determined by a Nanodrop or UV-Vis spectrophotometer with A280 reading (sample type option set to 1 Abs = 1 mg/ml). Optional: Lyophilize the tryptic peptide sample. Resuspend in 100-300 μl 100mM NH4HCO3.

In-solution Trypsin digestion

Alternatively, a traditional in-solution digestion can be performed with a protocol of the user’s choice. Tryptic peptides will need to be cleaned up with a C18 spin column (e.g., The Nest Group Ultra microspin column, P/N: SUM SS18V.25) according to manufacturer’s instructions. A typical protocol using a microspin C18 columns is as follows1:

  1. Wash the column by adding 200 μl 100% acetonitrile. Centrifuge and discard flowthrough. Repeat twice.

  2. Wash the column by adding 200 μl 0.1% Trifluoroacetic acid (TFA). Centrifuge and discard flowthrough. Repeat twice.

  3. To the tryptic-digest sample, add 10% TFA to bring the final concentration to 0.1% TFA.

  4. Load the tryptic-digest sample onto the column and centrifuge. The flowthrough may be collected and reapplied to the column or cartridge if desired.

  5. Wash the column by adding 200 μl 0.1% TFA. Centrifuge and discard flowthrough. Repeat 5 times.

  6. Wash the column by adding 200 μl water to remove TFA. Centrifuge and discard flowthrough. Repeat twice.

  7. Place microspin column in a new collection tube, and elute peptides from the column by adding 30 μl of elution buffer (50% acetonitrile, 0.1% formic acid, 49.9% H2O). Collect the flowthrough.

  8. The concentration of tryptic peptides can be determined by a Nanodrop spectrophotometer at 280 nm with the sample type option set to 1 Abs = 1 mg/mL. Optional: Lyophilize the tryptic-peptide sample to dryness. Resuspend in 100-300 μl 100mM NH4HCO3.

Reference:

  1. Gundry, R. L. et al, (2010). Curr. Protoc. Mol. Biol. 1 April 2010