Glycobiology Advance Access published online on July 21, 2005
Glycobiology, doi:10.1093/glycob/cwj019
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1 CREST, Japan Science and Technology Agency, 4-1-8 Honcho Kawaguchi. Saitama 332-0012, Japan
* To whom correspondence should be addressed. Isolation of glycopeptides utilizing hydrogen bonding between glycopeptide glycans and a carbohydrate-gel matrix in the organic phase is useful for site-specific characterization of oligosaccharides of glycoproteins, when combined with mass spectrometry. In the present study, recovery of glycopeptides was improved by including divalent cations or increasing the organic solvent in the binding solution, without losing specificity, while it was still less effective for those with long peptide backbone exceeding 50 amino acid residues. The method was then applied to the analysis of glycan heterogeneities at seven N-glycosylation sites in each of the plasma and cellular fibronectins. There was a remarkable site-specific difference in fucosylation between these isoforms; Asn1244 selectively eacaped the global fucosylation of cellular fibronectin, while only Asn1007 and Asn2108 of the plasma isoform underwent modification. In addition, a new O-glycosylation site was identified at Thr279 in the connecting segment between the fibrin- and heparin-binding domain and the collagen-binding domain, and the glycopeptide was reactive to a peanut agglutinin lectin. Considering that another mucin-type O-glycosylation site lies within a different connecting segment, the O-glycosylation of fibronectin was suggested to play a significant role in segregating the neighboring domains and thus maintaining the topology of fibronectin and the domain functions. In addition, the method was applied to apolipoprotein B-100 whose N-glycan structures at 17 out of 19 potential sites have been reported, and characterized the remaining sites. The results also demonstrated that the enriched glycopeptide provides resources for site-specific analysis of oligosaccharides in glycoproteomics.
Received May 17, 2005
Revised July 16, 2005
Accepted July 18, 2005
Article
Differential analysis of site-specific glycans on plasma and cellular fibronectins: Application of a hydrophilic affinity method for glycopeptide enrichment
2 Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita 565-0871, Japan
3 Department of Molecular Medicine, Osaka Medical Center and Research Institute for Maternal and Child Health, 840 Murodo-cho Izumi, Osaka 594-1101, Japan; CREST, Japan Science and Technology Agency, 4-1-8 Honcho Kawaguchi. Saitama 332-0012, Japan; Graduate School of Medicine, Osaka University, 2-2 Yamadaoka, Suita 565-0871, Japan
Yoshinao Wada, E-mail: waday{at}mch.pref.osaka.jp
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