Document Detail


Recovery of intact 2-aminobenzamide-labeled O-glycans released from glycoproteins by hydrazinolysis.
MedLine Citation:
PMID:  11969192     Owner:  NLM     Status:  MEDLINE    
Abstract/OtherAbstract:
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.
Authors:
Anthony H Merry; David C A Neville; Louise Royle; Brian Matthews; David J Harvey; Raymond A Dwek; Pauline M Rudd
Publication Detail:
Type:  Journal Article; Research Support, Non-U.S. Gov't    
Journal Detail:
Title:  Analytical biochemistry     Volume:  304     ISSN:  0003-2697     ISO Abbreviation:  Anal. Biochem.     Publication Date:  2002 May 
Date Detail:
Created Date:  2002-04-23     Completed Date:  2002-10-02     Revised Date:  2008-11-21    
Medline Journal Info:
Nlm Unique ID:  0370535     Medline TA:  Anal Biochem     Country:  United States    
Other Details:
Languages:  eng     Pagination:  91-9     Citation Subset:  IM    
Copyright Information:
(c)2002 Elsevier Science (USA).
Affiliation:
Oxford Glycobiology Institute, Department of Biochemistry, University of Oxford, South Parks Road, Oxford, OX1 3QU, United Kingdom.
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MeSH Terms
Descriptor/Qualifier:
Amino Acid Sequence
Animals
Anthranilic Acids
Carbohydrate Sequence
Cattle
Chromatography, High Pressure Liquid
Glycophorin / chemistry
Glycoproteins / chemistry*
Humans
Hydrazines
Molecular Sequence Data
Mucin-5B
Mucins / chemistry
Polysaccharides / chemistry,  isolation & purification*
Sequence Analysis / methods
Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization
alpha-Fetoproteins / chemistry
Chemical
Reg. No./Substance:
0/Anthranilic Acids; 0/Glycophorin; 0/Glycoproteins; 0/Hydrazines; 0/MUC5B protein, human; 0/Mucin-5B; 0/Mucins; 0/Polysaccharides; 0/alpha-Fetoproteins; 302-01-2/hydrazine; 88-68-6/anthranilamide

From MEDLINE®/PubMed®, a database of the U.S. National Library of Medicine


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