When coupled with mass spectrometry, liquid chromatography forms a powerful technique with high sensitivity and selectivity. The technology is especially relevant to the specific detection and identification of chemicals in complex mixtures, which will be confirmed by the studies reviewed in this article.
“Lectin-affinity chromatography is a popular medium for the purification of oligosaccharides, glycopeptides, and glycoproteins,” noted Srinivasa Rao, Ph.D., technical manager at Dionex. Concanavalin A (Con A), a lectin derived from the jack bean, Canavalia ensiformis, is the lectin of choice for many purification protocols. At neutral and alkaline pH, it exists as a tetramer of four identical subunits weighing in at approximately 104 kDa.
Con A is noted as one of the best characterized and widely used lectins, given that it binds to α-mannose and to α-glucose, although with weaker affinity to the latter. “However, its utility is limited by the fact that it is ordinarily incorporated into manually operated agarose bead-based spin columns.”
As Dr. Rao explained, this limits the number of purification cycles for which the column can be used, with the result that costs are driven up and time is lost. With the growing interest in investigations in the realm of glycoproteomic studies, such as biomarker identification, there is an increasing need for a robust HPLC lectin column.
For this reason, Dr. Rao and his coworkers have focused on the development and function of new monolithic affinity columns. Their recently designed column is known as the ProSwift ConA-1S, a polymeric monolith prepared by in situ polymerization followed by functionalization with Con A.
The monolith is a cylindrical polymer rod containing uninterrupted, interconnected flow-through pores, with surface area greater than nonporous bead-based columns. The structure consists of small pores that contribute surface area and larger openings that allow reduced back pressure at elevated flow rates. This approach results in short mass-transfer distances that produce improved efficiency, even at elevated flow rates, Dr. Rao explained. The HPLC compatibility of this column allows automatic sample injection, high throughput, and excellent reproducibility, he remarked.
To give an example of successful application of their column technology, Dr. Rao described the purification of horseradish peroxidase, a glycoprotein rich in high-mannose type glycans. Bound to the Con A column, it was eluted with α-methyl-mannopyranoside. This high-capacity Con A column retained the high specificity toward alpha mannose residues as shown by binding and elution investigations of several standards.
Dionex’ new HPLC monolithic Con A affinity column, ProSwift™ ConA-1S, allows automatic injection and on-line elution monitoring, resulting in sharper peak shape and requiring smaller elution volume of the more enriched fractions, he explained. The column can also be used for glycan and glycoprotein analysis and purification. The ProSwift ConA-1S column can be regenerated easily by washing with conditioning buffer after sample binding and elution, said Dr. Rao. “Our durable column chemistry allows hundreds of run cycles with minimal capacity loss.”