Characterization of human plasma glycoproteins separated by two-dimensional gel electrophoresis

Purification of protein isoforms for the characterization of post-translational modifications, such as glycosylation, can be laborious and demanding, We report a means of determining monssaccharide composition and the identity of glycoproteins from a single spot on a two-dimensional (2-D) gel, The sensitivity of the method depends on the degree of glycosylation of the protein, We show that bovine fetuin can be analyzed and identified at the level of 100 pmol, 2-D reference maps enable quick identification of glycoprotein isoforms, and the nature of glycosylation differences, Human sera glycoforms were isolated by micropreparative 2-D PAGE using a narrow-range immobilized PH gradient, Single spots excised from one polyvinylidene difluoride blot of a 2-D gel were used sequentially for sialic acid analysis, neutral and amino sugar analysis, and finally amino acid analysis, The glycosylation variations in isoforms of human fetuin and alpha-1-antitrypsin were determined, The amino acid composition, in conjunction,vith protein pi and MW, successfully identified the glycoproteins.