Isolation and Purification of Glycoproteins
Many glycoproteins are present in low concentrations in complex biological matrices. Concentrations in human plasma, for example, are estimated to span 10 orders of magnitude. Thus, for the less abundant glycoproteins, the first stage of analysis is to extract them in a sufficient state ofpurity. Immunodepletion of the major proteins such as albumin, a1-acid glycoprotein, a1-antitrypsin, apolipoprotein A, fibrinogen, haptoglobin, IgA, IgG, IgM, and transferrin is frequently used, and kits for this process are available commercially. A large number of specific methods have been developed and space only permits a brief summary of the more important ones.
Lectin Affinity Chromatography
Lectins are proteins that have affinity for glycans and have been used extensively for extractions and for profiling glycans. Over 160 lectins have been described with some 60 being commercially available [33, 34]. The affinity of these proteins tends to be specific for certain features of the glycans; for example, concanavalin A (ConA) mainly recognizes a-mannose and glucose, Lens culinaris agglutinin (LCA) recognizes hybrid or bi- and triantennary complex structures that are core fucosylated, peanut agglutinin (PNA) recognizes Galpi-3GalNAc and p-linked Gal, whereas wheat germ agglutinin (WGA) recognizes GlcNAc and sialic acid. The lectins are usually immobilized on silica- based materials or agarose but magnetic nanoparticles have also been used [35-37].
Although these lectins individually are not suitable for total glycoprotein extractions, they are invaluable when certain sets of glycan, such as high- mannose glycans, are required. For use with a wider range of glycoproteins, multilectin [38] or sequential lectin columns [39] can be used. Madera et al. [40] have compared the two approaches for extraction of glycoproteins from human blood serum. Four silica-bound lectins (ConA, Sambucus nigra agglutinin (SNA-I), Ulex europaeus agglutinin I (UEA-I), and Phaseolus vulgaris lectin (PHA-L)) were used individually or mixed. 108 glycoproteins were found with the individual columns, whereas only 67 were recovered from the mixed column. This situation may not, of course, apply to all glycoprotein mixtures.
A list of suitable lectins is available in the review by Alley et al. [3]. It has been pointed out that although the affinity of any particular lectin for specific carbohydrates may be known for the carbohydrate itself, this may not apply to the glycan when it is bound to the protein.
