ac400653z_si_001.pdf (224.13 kB)
Serial Affinity Chromatography as a Selection Tool in Glycoproteomics
journal contribution
posted on 2013-08-06, 00:00 authored by Kwanyoung Jung, Wonryeon ChoGlycan-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-LexAb → anti-sLexAb series and the anti-sLexAb → anti-LexAb → 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.