Protein Protocols Protein Protocols

Glycoprofiling and SPR 885
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Glycoprofiling Purified Glycoproteins Using
Surface Plasmon Resonance
Angeliki Fotinopoulou and Graham A. Turner
1. Introduction
1.1. Glycosylation and Its Investigation
The carbohydrate part of glycoproteins can define several of their biological properties,
including the clearance rate, immunogenicity, thermal stability, solubility, the specific
activity, and conformation (1). Often, small differences in the composition of the
sugar side chains of a glycoprotein can affect the biological properties (2). Characterization
of oligosaccharide structures on glycoproteins is essential for glycoprotein
therapeutic products, because inflammatory and systemic responses may arise if
glycosylation of the product is different from that of the native substance.
Determination of the precise oligosaccharide profile of a glycoprotein, however,
cannot yet be considered as a routine laboratory task. Methods for glycosylation analysis
can be generally divided in two major categories, direct methods and methods using
lectins. This chapter describes a recently developed method that measures the binding
of lectins to glycoproteins using surface plasmon resonance (SPR). Lectins are a class
of proteins that bind to carbohydrates in a noncovalent reversible way that does not
chemically modify the sugar molecule (3). The lectin specificity is not absolute, but
usually there is one carbohydrate grouping to which a lectin binds with much higher
affinity than to other carbohydrate structures. This property makes them ideal for recognizing
oligosaccharide structures. Although lectin methods are indirect, they can be
used when comparisons are needed. They provide many advantages compared to other
methods for investigating carbohydrate structures. There are a large number of lectins
available with different and distinct specificities. Lectin methods are also quick and
simple (4).
1.2. Surface Plasmon Resonance and Lectins
SPR is an optical sensing phenomenon that allows one to monitor biomolecule interactions
in real time (Fig. 1). The sensor device is composed of a sensor chip consisting
of three layers (glass, a thin gold film, and a carboxymethylated dextran matrix); a prism
placed on the glass surface of the chip; and a microfluidic cartridge, which controls the
delivery of liquid to the sensor chip surface (5). When light illuminates the thin gold
From: The Protein Protocols Handbook, 2nd Edition
Edited by: J. M. Walker © Humana Press Inc., Totowa, NJ
885