Lectin affinity columns

Glycoproteins generally bind to lectin affinity columns at pH values close to neutrality. Desorption may be achieved in some cases by alteration of the pH of the eluting buffer. The most common method of desorption, however, involves inclusion of free sugar molecules for which the lectin exhibits a high affinity in this elution buffer, i.e. the inclusion of a competing ligand. 

If only very small amounts of material are available (this is often the case with metabolically-labelled glycopeptides from cell cultures) and the sample weight cannot be determined accurately it is usually adequate to incubate the material overnight with 2.5 mg/ml pronase. Thereafter the procedure could follow that described above or, since only very small quantitites of protein are present in the sample, the pronase could be inactivated by heating at 100°C for 10 min and samples applied directly to lectin affinity columns. 

Membrane extracts from adult H. contortus were enriched 24-fold for cysteine protease activity by passage over a Thiol-Sepharose affinity column and the proteins obtained (abbreviated as TSBP) were clearly localized to the microvillar surface of the intestinal cells (Knox et al., 1995,1999). TSBP comprised a prominent 60 kDa protein and several minor bands between 35 and 45 kDa and 97 to 120 kDa (Fig. 13.2). Protease activity at 38, 52 and 70 kDa was attributable to cysteine proteases and at 70 and 88 kDa to serine/metalloproteases, as judged by inhibition analyses. Lectin-binding studies showed that most of the TSBPs were glycosylated. Expression library... 

Baues, R.J., and Cray, G.R. (1977) Lectin purification on affinity columns containing reductively ami-nated disaccharides./. Biol. Chem. 252, 57. 

Moroney, S.E., D Alarcao, L.J., Goldmacher, V.S., Lambert, H.M., and Blattlcr, W.A. (1987) Modification of the binding site(s) of lectins by an affinity column carrying an activated galactose-terminated ligand. Biochemistry 26, 8390. 

Many of the techniques used to enrich PTMs at the protein level are applicable at the peptide level. A popular method for enrichment of phosphopeptides is to use an immobilized metal affinity column. The molecular basis for the enrichment is the phosphate affinity to transition metal ions, such as copper, nickel, cobalt, iron, aluminum, gallium, and zinc (142). A comprehensive source for phosphopeptide enrichment strategies can be found in a recent review (143). Lectin-based chromatography is used to enrich for glycopep-tides (144). Lectins are proteins that have an affinity for carbohydrates. With multiple available enrichment options, choosing the right one for the experiment is important. For determining which proteins have a certain PTM, protein-level enrichment can be performed. However, if a type of PTM is of... 

Fig. 3a. Flow diagram for the separation of the components of a complex mixture of oligosaccharides by serial lectin affinity chromatography. Depending upon the lectin adsorbant, specific oligosaccharides are either unbound (not retarded by the adsorbant), retarded (and eluted without the need of a saccharide inhibitor), or are tightly bound and then require either lOmM methyl a-D-glucopyranoside or lOOmM methyl a-D-mannopyranoside for elution. Where appropriate, each eluted peak is concentrated and the saccharide inhibitor is removed prior to application to the second affinity column. The structures of the individual oligosaccharides are shown in Fig. 3b. (Adapted from ref 288.)...

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