Material Analyzed and Expected Results

The sample preparation necessary to analyze glycoproteins depends on the analytical method selected; however, the glycoprotein usually needs to be affinity purified. Once a purified glycoprotein has been obtained, many of the analytical techniques are sensitive to salts. A desalting step is usually conducted prior to analysis. The whole or intact glycoprotein can be analyzed directly, or digested and the individual components analyzed. Figure 6 shows the breakdown of an intact glycoprotein for analysis into a glycopeptide mixture, individual glycopeptides, oligosaccharides, and monosaccharides. Each pool shown in Fig. 6 can be analyzed by a variety of methods. Glycopeptides are obtained by protease digestion of the glycoprotein. Trypsin is a commonly used protease (120). The glycopeptide mixture is separated into glycopeptide pools with the same peptide backbone. The individual glycopep-tides can be analyzed for glycosylation or the oligosaccharides and monosaccharides can be analyzed after removal from the peptide. Oligosaccharides can be enzymati-cally or chemically cleaved from the glycoprotein or separated glycopeptides. Mono-saccharides can be obtained directly from the glycoprotein or glycopeptide via acid

Protease Digestion

Glycan Release

Monosaccharides

Glycan Release

Protease Digestion

Monosaccharides

Glycopeptide Mixture

Separation

Glycopeptide Mixture

Separation

Monosaccharide

Monosaccharide

Giycan Release

Glycopeptide

Oligosaccharides

Figure 6 Fractionation methods to characterize glycoproteins. [Adapted from Refs. (20,54,121-132).] (See color insert p. 3.)

hydrolysis. Monosaccharides can also be obtained via acid hydrolysis of the oligosaccharides (120).

In this review, the type of information that can be obtained from analyzing the intact glycoprotein, glycopeptides, oligosaccharides, and monosaccharides will be discussed independently of the analytical techniques. Then, the analytical techniques will be discussed individually. Since no single technique can routinely provide complete characterization, how the various techniques are used together to obtain the glycosylation information for a glycoprotein will be discussed. Table 1 lists the analytical techniques commonly used to characterize the glycosylation of a glycoprotein. Details are presented regarding starting material, information obtained, sample size, and analysis difficulty.

Glycoprotein Analysis

Whole or intact glycoproteins can be analyzed based on size (molecular weight), charge, affinity, and oligosaccharide content. The presence of different oligosacchar-ides can change the molecular weight of the glycoprotein. The glycoprotein glyco-forms can be separated from each other by size. Since sialic acid is a negatively charged sugar, and under some conditions other sugars will take a charge, the presence of different oligosaccharides can be used to separate the glycoforms by charge. The oligosaccharides attached to the glycoprotein also alter the shape of the glyco-protein. The glycoforms can be analyzed for affinity with lectins or antibodies against oligosaccharide groups. The level of resolution for the glycosylation analysis of a glycoprotein is relatively low. However, the analytical techniques used are also relatively easy, fast, and inexpensive. Analysis techniques used on the intact

Table 1 Analysis Techniques to Characterize the Glycosylation of a Glycoprotein

Technique

Sample Analysis

Glycoprotein Glycopeptide Oligosaccharide Monosaccharide size difficulty

Comments (Refs.)

Electrophoresis SDS-PAGE

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