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

Bio/Technology volume 14, pages 66–70 (1996)Cite this article

Abstract

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.

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Author notes

  1. Keith L. Williams: e-mail: keith.williams@mq.edu.au

Authors and Affiliations

  1. Macquarie University Centre for Analytical Biotechnology, Macquarie University, Sydney, NSW, 2109, Australia

    Nicolle H. Packer, Marc R. Wilkins, Margaret A. Lawson, Andrew A. Gooley & Keith L. Williams

  2. Central Clinical Chemistry Laboratory and Medical Computing Centre of the University of Geneva, CH 1211, Geneva, 14, Switzerland

    Olivier Golaz & Denis F. Hochstrasser

  3. Research School of Biological Sciences, Australian National University, Canberra, Australia

    John W. Redmond

Authors
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  6. Denis F. Hochstrasser
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Packer, N., Wilkins, M., Golaz, O. et al. Characterization of Human Plasma Glycoproteins Separated by Two-Dimensional Gel Electrophoresis. Nat Biotechnol 14, 66–70 (1996). https://doi.org/10.1038/nbt0196-66

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