The use of polyacrylamide gel electrophoresis for the analysis of acidic glycans labeled with the fluorophore 2-aminoacridone.
A method is described for the electrophoretic analysis of fluorophore-labeled acidic glycans. Various glycoproteins were treated with the enzyme, peptide N-glycosidase F, to release mixtures of asparagine-linked glycans that were then labeled with the fluorophore 2-aminoacridone, and the resulting derivatives electrophoresed in high-density polyacrylamide gels. The acidic glycans were separated with high resolution and distinct, well-resolved fluorescent band patterns were produced. Neutral saccharides did not electrophorese in the system. Information on the structure of the glycans was revealed by the changes in the band patterns observed after the mixtures of the glycan fluorophore derivatives were treated with either of the enzymes neuraminidase or beta-galactosidase. Quantities of glycoprotein as little as 10 micrograms were analysed without difficulty. The electrofluorograms were viewed using a digital imaging system based on a cooled charge-coupled device. The method was also demonstrated using purified acidic glycans of known structure. The method was easy to use, enabled the sensitive analysis of multiple samples in parallel and should be a useful addition to the range of glycan analytical techniques.
Jackson
P
P
Millipore Corporation, Bedford, MA.
Pluskal
M G
MG
Skea
W
W
D000143:Acids / Q000032:analysis*; D000581:Amidohydrolases; D000609:Aminoacridines; D002240:Carbohydrate Sequence; D004591:Electrophoresis, Polyacrylamide Gel; D005456:Fluorescent Dyes; D008969:Molecular Sequence Data; D009439:Neuraminidase; D009844:Oligosaccharides / Q000032:analysis; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011134:Polysaccharides / Q000032:analysis*; D001616:beta-Galactosidase
D000143:Acids; D000609:Aminoacridines; D005456:Fluorescent Dyes; D009844:Oligosaccharides; D011134:Polysaccharides; C072515:2-aminoacridone; D009439:Neuraminidase; D001616:beta-Galactosidase; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase