title: An optimized approach for enrichment of glycoproteins from cell culture lysates using native multi-lectin affinity chromatography.
abstract: Lectins are capable of recognizing specific glycan structures and serve as invaluable tools for the separation of glycosylated proteins from nonglycosylated proteins in biological samples. We report on the optimization of native multi-lectin affinity chromatography, combining three lectins, namely, concanavalin A, jacalin, and wheat germ agglutinin for fractionation of cellular glycoproteins from MCF-7 breast cancer lysate. We evaluated several conditions for optimum recovery of total proteins and glycoproteins such as low pH and saccharide elution buffers, and the inclusion of detergents in binding and elution buffers. Optimum recovery was observed with overnight incubation of cell lysate with lectins at 4°C, and inclusion of detergent in binding and saccharide elution buffers. Total protein and bound recoveries were 80 and 9%, respectively. Importantly, we found that high saccharide strength elution buffers were not necessary to release bound glycoproteins. This study demonstrates that multi-lectin affinity chromatography can be extended to total cell lysate to investigate the cellular glycoproteome.
authors.affiliation: Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney, Australia.
authors.forename: Ling Y
authors.initials: LY
authors.lastname: Lee
authors.forename: Marina
authors.initials: M
authors.lastname: Hincapie
authors.forename: Nicolle
authors.initials: N
authors.lastname: Packer
authors.forename: Mark S
authors.initials: MS
authors.lastname: Baker
authors.forename: William S
authors.initials: WS
authors.lastname: Hancock
authors.forename: Susan
authors.initials: S
authors.lastname: Fanayan
mesh_terms: D001943:Breast Neoplasms / Q000737:chemistry* / Q000378:metabolism; D018929:Cell Culture Techniques; D002846:Chromatography, Affinity; D002853:Chromatography, Liquid; D005455:Fluorescent Antibody Technique; D006023:Glycoproteins / Q000032:analysis*; D006801:Humans; D006863:Hydrogen-Ion Concentration; D037102:Lectins / Q000737:chemistry*; D061986:MCF-7 Cells; D053719:Tandem Mass Spectrometry; D014407:Tumor Cells, Cultured
chemical_list: D006023:Glycoproteins; D037102:Lectins