Two-dimensional poly(acrylamide) electrophoresis of fluoresceinated glycopeptides. Resolution and structural characterization of ovalbumin glycans.

The microheterogeneous mixture of fluoresceinated glycopeptides (FGPs) obtained from the single site of glycosylation of chicken ovalbumin was resolved by a combination of discontinuous electrophoresis in a high-density poly(acrylamide) gel (PAGE) for sizing, in conjunction with borate-PAGE. Two FGPs of similar size but with different mobilities in borate-PAGE were purified and characterized by sequential exoglycosidase digestion and sizing on the discontinuous PAGE system, as well as by methylation analysis. The two FGPs of identical size are distinct and have structures beta-D-Glc pNAc-(1-->2)-alpha-D-Man p-(1-->3)-[beta-D-Glc pNAc-(1-->4)]-[beta-D-Glc pNAc-(1-->2)-alpha-D- Man p-(1-->6)]-beta-D-Man-p-(1-->4)-beta-D-Glc pNAc-(1-->4)-beta-D-Glc pNAc-1-->R and alpha-D-Man p-(1-->2)-alpha-D-Man p-(1-->3 or 6)-[alpha-D-Man p-(1-->3)-[alpha-D-Man p-(1-->6)]-alpha-D-Man p-(1-->6 or 3)]-beta-D-Man p-(1-->4)-beta-D-Glc pNAc-(1-->4)-beta-D-Glc pNAc-1-->R (R = Asn-(amino acids)-fluorescein). The results demonstrate that two-dimensional PAGE is applicable to the separation and characterization of complex mixtures of FGPs. The procedure is rapid, sensitive, and convenient for glycopeptide mapping, and for the purification and structural characterization of glycans. Furthermore, the FGPs can be characterized with affinity matrices, such as lectins, and by methylation analysis.