Carbohydrate release from picomole quantities of glycoprotein and characterisation of glycans by high-performance liquid chromatography and mass spectrometry.
Samples of 5 to 20 microg of human IgG were subjected to dithiothreitol treatment to reduce disulphide bridges, followed by tryptic digestion. Glycans released from the tryptic peptide mixture by PNGase F digestion were then derivatised with 2-aminoacridone. Labelled oligosaccharides were separated by normal-phase high-performance liquid chromatography and individual components were collected for matrix-assisted laser desorption ionization time-of-flight and electrospray mass spectrometric analysis.
Charlwood
J
J
SmithKline Beecham Pharmaceuticals, Harlow, Essex, UK.
Birrell
H
H
Camilleri
P
P
D000581:Amidohydrolases / Q000378:metabolism; D000609:Aminoacridines; D002851:Chromatography, High Pressure Liquid / Q000379:methods*; D004220:Disulfides / Q000378:metabolism; D004229:Dithiothreitol / Q000494:pharmacology; D006023:Glycoproteins / Q000378:metabolism*; D006801:Humans; D007074:Immunoglobulin G / Q000737:chemistry / Q000378:metabolism*; D008832:Microchemistry; D009844:Oligosaccharides / Q000032:analysis / Q000302:isolation & purification; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D011134:Polysaccharides / Q000737:chemistry / Q000302:isolation & purification* / Q000378:metabolism; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods*; D014357:Trypsin / Q000378:metabolism
D000609:Aminoacridines; D004220:Disulfides; D006023:Glycoproteins; D007074:Immunoglobulin G; D009844:Oligosaccharides; D011134:Polysaccharides; C072515:2-aminoacridone; D014357:Trypsin; D000581:Amidohydrolases; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase; D004229:Dithiothreitol