ANALYZING GLYCOPROTEINS SEPARATED BY 2-DIMENSIONAL GEL-ELECTROPHORESIS

Two-dimensional (2-D) electrophoresis is the preferred method for sepa rating the glycoforms of proteins. The isoforms usually present as 'tr ains' of spots in the first dimension and may also differ in molecular weight. The primary goal for analyzing the carbohydrate content of gl ycoprotein spots is to understand the 'rules' which govern the migrati on of glycoproteins in 2-D electrophoresis. These rules can then be us ed to produce predictive vectors to interpret changes in glycosylation patterns. Techniques for the analysis of oligosaccharides released fr om glycoproteins which have been electroblotted to PVDF membrane after one-dimensional (1-D) and 2-D preparative gel electrophoresis are des cribed. The oligosaccharides are removed enzymatically (PNGase F of N- linked oligosaccharides) or chemically (beta-elimination of O-linked o ligosaccharides) and separated by high performance anion exchange chro matography (HPAEC-PAD) and identified by electrospray ionization mass spectrometry (ESI-MS) or analyzed directly by ESI-MS. After enzymic re moval of the N-linked oligosaccharides the protein spots can be furthe r analyzed by Edman sequence tagging for identification and quantitati on of the protein and by acid hydrolysis for monosaccharide analysis o f the O-linked oligosaccharides. These approaches have been proved on 1-D PAGE electroblotted bovine fetuin and human glycophorin A and then used to analyze two abundant proteins which separate as glycoforms on 2-D PAGE preparative narrow range (pH 4.5-5.5) blots of human plasma: alpha 2-HS glycoprotein (human fetuin) and alpha 1-antitrypsin (alpha 1-protease inhibitor). It is apparent that both the macroheterogeneit y (site occupation) and microheterogeneity (diversity of structures) o f the glycosylation contribute to the separation of protein isoforms i n 2-D PAGE.