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
- Swell 257 mg POROS-AL with 25 mg WGA in 2.0 ml of 25 mM Bicine pH 7.5.
- Wash WGA vial with additional 1 ml 25 mM Bicine and add to POROS.
- 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.
- Add 300 ul 2M Na2SO4 and continue to mix for 1 hour at room temperature.
- Move mixer to 4°C overnight.
- Pellet resin at 3000 x g for 5 min, remove and save supernatant to test for reaction efficiency.
- Quench reaction by adding 5 ml 100mM Tris + 300 ul NaBH₃CN (100 mg/ml) and mixing for 2 hr at room temperature.
- Wash beads 3 times with 10ml LWAC Buffer.
- 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.
- Pack column at a flow rate of 0.600 ml/min.
- Roughly 2mg of peptides are resuspended in 50-200 ul LWAC buffer.
- LWAC buffer and the LWAC column are placed on ice for the enrichment runs.
- Peptides are injected and run over POROS-WGA column at 0.1 ml/min under isocratic conditions.
- After the flow through peak, 100 ul of LWAC Elution buffer is injected to elute any remaining peptides.
- Peptides are collected from roughly the last 10% of the flow through peak through the end of the LWAC elution peak.
- Enriched peptides are combined with peptides enriched in other primary LWAC runs, desalted, and resuspended in 50-200 ul LWAC buffer.
- Primary enriched LWAC peptides are further enriched 1 to 2 more times.
- 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.
- The LWAC column is stored in LWAC buffer with 0.04% NaN3 and 50mM GlcNAc.