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 microg) 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.
SmithKline Beecham Pharmaceuticals, New Frontiers Science Park, Third Avenue, Harlow, Essex CM19 5AW, United Kingdom.
J
J
Charlwood
J M
JM
Skehel
P
P
Camilleri
D000818:Animals; D002851:Chromatography, High Pressure Liquid; D002853:Chromatography, Liquid; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods*; D004591:Electrophoresis, Polyacrylamide Gel; D006023:Glycoproteins / Q000032:analysis / Q000097:blood / Q000378:metabolism*; D006863:Hydrogen-Ion Concentration; D007202:Indicators and Reagents / Q000494:pharmacology; D051379:Mice; D009844:Oligosaccharides / Q000032:analysis* / Q000097:blood; D011134:Polysaccharides / Q000378:metabolism; D011145:Polyvinyls / Q000494:pharmacology; D011370:Proflavine / Q000031:analogs & derivatives / Q000494:pharmacology; D012394:Rosaniline Dyes / Q000494:pharmacology; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D000509:alpha-Fetoproteins / Q000737:chemistry; D005465:Fluorocarbon Polymers
D006023:Glycoproteins; D007202:Indicators and Reagents; D009844:Oligosaccharides; D011134:Polysaccharides; D011145:Polyvinyls; D011370:Proflavine; D012394:Rosaniline Dyes; D000509:alpha-Fetoproteins; C059684:3-acetamido-5-iodoproflavine; C024865:polyvinylidene fluoride; D005465:Fluorocarbon Polymers; C048139:Coomassie blue