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 agarose is used as a weak affinity column to enrich for O-GlcNAc-modified peptides.
|Resin:||WGA-Agarose (Vector Laboratories)|
|Tubing:||Teflon PFA 1/16” (1.6mm) OD, 1/25” (1mm) ID|
|LWAC Buffer:||25 mM Tris pH 7.8, 300 mM NaCl, 5 mM CaCl, 1 mM MgCl2|
|LWAC Elute Buffer:||LWAC Buffer + 20mM GlcNAc|
Making an LWAC Column
We use a large empty column (10mm x 120mm, Waters AP mini-column) as a reservoir to pack our LWAC column. We use as a column the largest inner diameter Teflon tubing with a 1/16” OD, and use a 0.5µm frit. Use an HPLC system to pack the column using the LWAC Buffer as solvent. We recommend packing a column of at least three meters in length (we have used columns up to 10 meters in length). To pack the tubing requires 0.8 ml of resin per meter of column. An issue with these columns is that the agarose can compress. Hence, we recommend keeping the column pressure below 2 MPa (20 mbar). This means operating at a flow rate between 50-150 µl / min (longer columns will require lower flow rates). If the column seems to stop packing and the pressure increases, then reverse the direction of the column (move the frit to the other end). Packing these columns is slow — expect it to take all day to make a column.
The chromatography is isocratic, so very straightforward. Make sure you do not use a too high flow rate (keep the pressure below 2 MPa (20 mbar)). Load sample in LWAC buffer (50-200µl) and wait for the main peak to elute. Depending on the length of your column, the O-GlcNAc-modified peptides will elute in the tail of the main peak or a little afterwards. Do not worry if you do not see a distinct peak for modified peptides, and collect fractions even beyond the point at which there is any obvious UV absorbance (there will probably still be peptides there). Inject LWAC Elute Buffer to elute any complex glycans that may be bound to the column. The GlcNAc in this buffer absorbs at 214nm, so do not be concerned if you observe a large UV peak.
We recommend storing the column in the LWAC Elute Buffer at 4°C.
Publications using this protocol
Vosseller, K., Trinidad, J. C., Chalkley, R. J., Specht, C. G., Thalhammer, A., Lynn, A. J., Snedecor, J. H., Guan, S., Medzihradszky, K. F., Maltby, D. A., Schoepfer, R., Burlingame, A. L. O-linked N-acetylglucosamine proteomics of postsynaptic density preparations using lectin weak affinity chromatography and mass spectrometry, Mol Cell Proteomics (2006) 5(5): p.923-924. [Pubmed]
Chalkley, R.J., Thalhammer, A., Schoepfer, R., and Burlingame, A.L., Identification of protein O-GlcNAcylation sites using electron transfer dissociation mass spectrometry on native peptides, Proc Natl Acad Sci U S A (2009) 106(22) p.8894-8899. [Pubmed]