title: Serial affinity chromatography as a selection tool in glycoproteomics.
abstract: Glycan-targeting affinity chromatography systems are becoming increasingly important as tools in the purification, enrichment, and identification of glycoproteins. The great advantage of this strategy is that immobilized lectin and antibody selectors allow specific glycan structures to be matched with a particular protein. It is also possible to show that a glycan seen at one site in a glycoprotein may not be present at another glycosylation site in the same glycoprotein. A problem with single-column affinity chromatography is how to obtain information on glycan diversity within the oligosaccharide portions of captured glycoproteins. Although all the glycoprotein species bearing a particular glycan feature will be captured by an affinity column, there is no way of knowing whether the ligand being targeted appears alone or coresides with a series of other glycan features in the same oligosaccharide conjugate. The work being described here examines the utility of serial affinity columns in determining whether individual glycan structures appear alone or together with other glycans in specific proteins. Four different types of affinity columns were examined in two series; the LEL → HPA → anti-Le(x)Ab → anti-sLe(x)Ab series and the anti-sLe(x)Ab → anti-Le(x)Ab → HPA → LEL series. Patterns in protein capture from these two series were very different. Thus, serial affinity chromatography (SAC) can be a valuable tool in recognizing diversity in protein glycosylation, especially when the order of columns in the SAC series is varied. Two clear types of diversity were recognized. One is the independent occurrence of different affinity-targetable glycan features in the same glycoprotein. The other is that multiple targetable glycan features were coresident in the same glycoprotein. The great advantage of this method is that it couples easily with current methods used in glycoproteomics.
authors.affiliation: Department of Chemistry, Seoul Science High School, Jongno-gu, Seoul, Republic of Korea.
authors.forename: Kwanyoung
authors.initials: K
authors.lastname: Jung
authors.forename: Wonryeon
authors.initials: W
authors.lastname: Cho
mesh_terms: D002846:Chromatography, Affinity / Q000379:methods*; D006023:Glycoproteins / Q000097:blood / Q000302:isolation & purification* / Q000378:metabolism; D006801:Humans; D059748:Proteolysis; D040901:Proteomics / Q000379:methods*
chemical_list: D006023:Glycoproteins