Lectin Weak Affinity Chromatography (LWAC) Protocol

The lectin wheat germ agglutinin (WGA) has affinity for terminal N-acetylglucosamine (GlcNAc) and sialic acid residues. It has four binding sites, so can bind with high affinity to branched glycan structures with multiple terminal GlcNAc moieties. However, the affinity for a single GlcNAc residue, as encountered with the regulatory modification of O-GlcNAcylation, found on serines and threonines of nuclear and cytoplasmic proteins, is low. Hence, our Resource has developed and employs a protocol where WGA attached to POROS is used as a weak affinity column to enrich for O-GlcNAc-modified peptides.

Resin: WGA (Vector Laboratories) coupled to POROS-AL (Life Technologies)
Column: Stainless Steel, 250mm L x 2.0mm ID x 1/4in OD
LWAC Buffer: 100 mM Tris pH 7.5, 150 mM NaCl, 10 mM MgCl2, 10 mM CaCl2, 5% Acetonitrile
LWAC Elute Buffer: LWAC Buffer + 40mM GlcNAc
LWAC Storage Buffer: LWAC Buffer + 50 mM GlcNAc, 0.04% NaN3.

Making an LWAC Column

  1. Swell 257 mg POROS-AL with 25 mg WGA in 2.0 ml of 25 mM Bicine pH 7.5.
  2. Wash WGA vial with additional 1 ml 25 mM Bicine and add to POROS.
  3. Add 150 ul freshly made NaBH₃CN (100 mg/ml) to the POROS/WGA mixture and incubate at room temperature in end-over-end mixer for 4-5 hrs.
  4. Add 300 ul 2M Na2SO4 and continue to mix for 1 hour at room temperature.
  5. Move mixer to 4°C overnight.
  6. Pellet resin at 3000 x g for 5 min, remove and save supernatant to test for reaction efficiency.
  7. Quench reaction by adding 5 ml 100mM Tris + 300 ul NaBH₃CN (100 mg/ml) and mixing for 2 hr at room temperature.
  8. Wash beads 3 times with 10ml LWAC Buffer.
  9. Equilibrate necessary HPLC lines, a 10mm x 120mm Waters AP mini-column (used as a reservoir), and a 250 mm stainless steel column with a 2.0mm ID and 0.25” OD (with one frit at the bottom of the column) with LWAC buffer.
  10. Pack column at a flow rate of 0.600 ml/min.

LWAC Chromatography

  1. Roughly 2mg of peptides are resuspended in 50-200 ul LWAC buffer.
  2. LWAC buffer and the LWAC column are placed on ice for the enrichment runs.
  3. Peptides are injected and run over POROS-WGA column at 0.1 ml/min under isocratic conditions.
  4. After the flow through peak, 100 ul of LWAC Elution buffer is injected to elute any remaining peptides.
  5. Peptides are collected from roughly the last 10% of the flow through peak through the end of the LWAC elution peak.
  6. Enriched peptides are combined with peptides enriched in other primary LWAC runs, desalted, and resuspended in 50-200 ul LWAC buffer.
  7. Primary enriched LWAC peptides are further enriched 1 to 2 more times.
  8. Fractions corresponding to the same retention times are collected in these subsequent LWAC runs. A higher percentage of the flow through peak is collected with each additional enrichment step.
  9. The LWAC column is stored in LWAC buffer with 0.04% NaN3 and 50mM GlcNAc.

National Institute of General Medical Sciences Adelson Medical Research Foundation