| Abstract: | Surface plasmon resonance was used to characterize the carbohydrate moieties of bovine fetuin. This technique, requiring no sample derivitization or labeling, identified the presence and composition of both N- and O-linked oligosaccharides, using a combination of lectin probes and in situ glycosidase digestion. A complete analysis was achieved using 1.4 μg of pure bovine fetuin, and was fully automated using Pharmacia′s BIAcore. The presence or absence of specific oligosaccharide structures was determined by the binding of a panel of unlabeled lectins. Monosaccharide order and linkages were determined by sequential digestion using a range of specific exoglycosidases. This novel method implementing in situ digestion was achieved using a modification to the BIAcore software, allowing the flow of reagent over the sensor chip to be stopped for variable lengths of time, thereby permitting enzymatic digestion to occur. This technique can be applied to other commercial SPR biosensors currently available. Glycoanalysis by SPR uses approximately 100- to 1000-fold less protein than comparable analyses using alternative techniques such as gel permeation chromatography of released oligosaccharides, labeled lectin binding, or mass spectrometry. |
