Analysis of N-linked Oligosaccharides Released from Glycoproteins Separated by Two-Dimensional Gel Electrophoresis

Protocols have been developed for the characterization of carbohydrate covalently attached (N-linked) to an asparagine residue in glycoproteins, after separation by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Mixtures of proteins (each at a level from 0.5 to 50 μg) were resolved i...

Full description

Saved in:
Bibliographic Details
Published inAnalytical biochemistry Vol. 284; no. 1; pp. 49 - 59
Main Authors Charlwood, Joanne, Skehel, J.Mark, Camilleri, Patrick
Format Journal Article
LanguageEnglish
Published United States Elsevier Inc 15.08.2000
Subjects
alpha-Fetoproteins - chemistry
Animals
Chromatography, High Pressure Liquid
Chromatography, Liquid
Electrophoresis, Gel, Two-Dimensional - methods
Electrophoresis, Polyacrylamide Gel
Glycoproteins - analysis
Glycoproteins - blood
Glycoproteins - metabolism
Hydrogen-Ion Concentration
Indicators and Reagents - pharmacology
Mice
Oligosaccharides - analysis
Oligosaccharides - blood
Polysaccharides - metabolism
Polyvinyls - pharmacology
Proflavine - analogs & derivatives
Proflavine - pharmacology
Rosaniline Dyes - pharmacology
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
Online AccessGet full text

Cover

More Information
Summary:Protocols have been developed for the characterization of carbohydrate covalently attached (N-linked) to an asparagine residue in glycoproteins, after separation by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE). Mixtures of proteins (each at a level from 0.5 to 50 μg) were resolved in the first dimension according to their isoelectric points (pI), followed by separation in the orthogonal axis on the basis of their molecular weights. Glycans were released directly from excised gel spots after digestion with PNGase F, with or without prior treatment with trypsin. In a third method, glycoproteins were electroblotted onto poly(vinylidene difluoride) before glycans were released by PNGase F. For all these procedures profiles of the neutral and sialic acid-containing oligosaccharide mixtures were obtained after derivatization with 3-acetamido-6-aminoacridine, and analysis by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry and/or high-performance liquid chromatography. Potential applications to proteomics are discussed.
Bibliography:ObjectType-Article-1
SourceType-Scholarly Journals-1
ObjectType-Feature-2
content type line 23
ISSN:0003-2697
1096-0309
DOI:10.1006/abio.2000.4687