Characterization of Human Plasma Glycoproteins Separated by Two-Dimensional Gel Electrophoresis

• Published in: Bio/Technology 1996-01, Vol.14 (1), p.66-70
• Main Authors: Packer, Nicolle H, Wilkins, Marc R, Golaz, Olivier, Lawson, Margaret A, Gooley, Andrew A, Hochstrasser, Denis F, Redmond, John W, Williams, Keith L
• Format: Article
• Language: English
• Subjects: Agriculture; alpha 1-Antitrypsin - analysis; alpha-Fetoproteins - analysis; Analytical, structural and metabolic biochemistry; Animals; Bioinformatics; Biological and medical sciences; Biomedical and Life Sciences; Biomedical Engineering/Biotechnology; Biomedicine; Biotechnology; Cattle; Electrophoresis, Gel, Two-Dimensional; Fundamental and applied biological sciences. Psychology; Glycoproteins; Glycoproteins - blood; Glycoproteins - isolation & purification; Glycosylation; Humans; Life Sciences; Monosaccharides - analysis; Oligosaccharides - analysis; Proteins
• ISSN: 0733-222X; 1087-0156; 2331-3684; 1546-1696
• DOI: 10.1038/nbt0196-66

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 monosaccharide 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 α-1-antitrypsin were determined. The amino acid composition, in conjunction with protein pI and MW, successfully identified the glycoproteins.