APPLICATION OF MONOLITHIC AFFINITY HPLC COLUMN FOR RAPID DETERMINATION OF MALT GLYCOPROTEINS

Abstract

Of all known protein post-translational modifications, glycosylation is the most common and complex one, which plays an important role in many biological processes and shows great diversity. For identification and detailed analysis of glycoproteins, separation from complex samples is required. Lectin affinity chromatography has been frequently used for purification and enrichment of glycoproteins or glycopeptides. The most well characterized and widely used is concanavalin A (ConA), binding specifically to α-mannosyl and α-glucosyl residues. It is known that the protein as well as glycoprotein composition of barley malt is fundamental with regard to beer production. For this reason, water soluble glycoproteins were investigated. The aim of this study was to optimize separation and enrichment of individual modified proteins on a monolithic ConA affinity HPLC column. This chromatographic strategy demonstrates the potential of affinity chromatography on this type of column, providing higher reproducibility, efficiency, and faster performance in comparison with the previously used manually filled columns. Most importantly, the ConA column used is resistant to bleeding. Furthermore, ConA-bound proteins were separated on SDS-PAGE and then chymotryptic digested and identified using MALDI-TOF/TOF MS. Our proteomic analysis allowed successful determination of several putative glycoproteins present in barley malt.

Keywords:

• barley
• concanavalin A
• glycosylation
• MALDI
• malt
• proteomics

ACKNOWLEDGMENT

This work was supported by the Ministry of Education, Youth and Sports of the Czech Republic (Grant No.1M0570) and from the Institutional Research Plan AV0Z40310501.

Notes

*AA triplet, amino acid triplet.