Glycoprotein Analysis by Capillary Zone Electrophoresis-Electrospray Mass Spectrometry
The study of glycoproteins as intact molecules or as smaller fragments generated by chemical or enzymatic digestion is well suited to the technique of capillary zone electrophoresis-electrospray mass spectrometry (CZE-ESMS). Separation of glycoform populations of intact glycoproteins or protein digests is possible using buffers compatible with mass spectrometric detection (1–8). CZE-ESMS analysis of intact proteins can provide semiquantitative information about the degree of heterogeneity of the glycoprotein (1,6,7). Enzymatic or chemical digestion of the glycoprotein, followed by CZE-ESMS, gives a direct measure of individual sites of heterogeneity (1–5). Combined CZE-collision induced dissociation (CID) mass spectrometric experiments (CZE-MS-MS) of digests enables the characterization of oligosaccharide structures (1–5). In particular, CID of glycopeptides is characterized by fragment ions corresponding to cleavage at each glycosidic bond. The occurrence of specific carbohydrate residues such as hexose (Man, Glc, Gal), N-acetylhexosamine (GlcNAc, GalNAc), or N-acetylneuraminic acid (NeuNAc) can be monitored by the observation of characteristic oxonium ions at m/z 163, 204, and 292, respectively. More recently, CZE-front-end CID-MS-MS has been shown to be a powerful tool for peptide sequencing of glycopeptides using subpicomole quantities of injected glycoprotein (3–5).
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