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 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 alpha-1-antitrypsin were determined. The amino acid composition, in conjunction with protein pI and MW, successfully identified the glycoproteins.
Packer
N H
NH
Macquarie University Centre for Analytical Biotechnology, Macquarie University, Sydney, NSW, Australia.
WIlkins
M R
MR
Golaz
O
O
Lawson
M A
MA
Gooley
A A
AA
Hochstrasser
D F
DF
Redmond
J W
JW
Williams
K L
KL
D000818:Animals; D002417:Cattle; D015180:Electrophoresis, Gel, Two-Dimensional; D006023:Glycoproteins / Q000097:blood* / Q000302:isolation & purification; D006031:Glycosylation; D006801:Humans; D009005:Monosaccharides / Q000032:analysis*; D009844:Oligosaccharides / Q000032:analysis*; D000515:alpha 1-Antitrypsin / Q000032:analysis; D000509:alpha-Fetoproteins / Q000032:analysis
D006023:Glycoproteins; D009005:Monosaccharides; D009844:Oligosaccharides; D000515:alpha 1-Antitrypsin; D000509:alpha-Fetoproteins