Recovery of intact 2-aminobenzamide-labeled O-glycans released from glycoproteins by hydrazinolysis.
The initial step in quantitative analysis of O-linked glycans of glycoproteins is to release them in high yield, nonselectively, unmodified, and with a free reducing terminus. In contrast to other techniques, hydrazinolysis can meet these criteria. However, when analyzing pools of O-linked glycans as described in the accompanying article by L. Royle et al. (2002, Anal. Biochem. 304), some peeling of the glycans was observed. Critical steps in the sample preparation and glycan recovery were therefore evaluated by analyzing and identifying both intact O-glycans and degraded products. Synthetic O-glycopeptides were characterized by mass spectrometry. Released glycans were identical to those on the glycopeptide. O-Linked glycans from a range of glycoproteins of increasing complexity, namely, bovine serum fetuin, glycophorin A, and previously uncharacterized glycopeptides isolated from human salivary mucin Muc5B, were also analyzed. Quantitative analysis of the glycan profile confirmed that there was <2% peeling of O-glycans released by hydrazinolysis conditions of 60 degrees C for 6 h, and recovered using the optimised procedure now described. This demonstrated that O-glycans can be prepared by hydrazinolysis without degradation and, as part of an analytical strategy, makes the analysis of O-glycans attached to low-microgram levels of naturally occurring glycoproteins feasible.
Merry
Anthony H
AH
Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, United Kingdom.
Neville
David C A
DC
Royle
Louise
L
Matthews
Brian
B
Harvey
David J
DJ
Dwek
Raymond A
RA
Rudd
Pauline M
PM
D000595:Amino Acid Sequence; D000818:Animals; D002240:Carbohydrate Sequence; D002417:Cattle; D002851:Chromatography, High Pressure Liquid; D006021:Glycophorins / Q000737:chemistry; D006023:Glycoproteins / Q000737:chemistry*; D006801:Humans; D006834:Hydrazines; D008969:Molecular Sequence Data; D055257:Mucin-5B; D009077:Mucins / Q000737:chemistry; D011134:Polysaccharides / Q000737:chemistry / Q000302:isolation & purification*; D017421:Sequence Analysis / Q000379:methods; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D000509:alpha-Fetoproteins / Q000737:chemistry; D062367:ortho-Aminobenzoates
D006021:Glycophorins; D006023:Glycoproteins; D006834:Hydrazines; C524578:MUC5B protein, human; D055257:Mucin-5B; D009077:Mucins; D011134:Polysaccharides; D000509:alpha-Fetoproteins; D062367:ortho-Aminobenzoates; C029424:hydrazine; C000219:anthranilamide