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Anal Chem. 2008 Feb 15;80(4):1094-101. doi: 10.1021/ac702124d. Epub 2008 Jan 19.

Comprehensive approach to structural and functional glycomics based on chemoselective glycoblotting and sequential tag conversion.

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  • 1Laboratory of Advanced Chemical Biology, Graduate School of Advanced Life Science, and Frontier Research Center for Post-Genome Science and Technology, Hokkaido University, Sapporo, Japan.

Abstract

Changes in protein glycosylation profoundly affect protein function. To understand these effects of altered protein glycosylation, we urgently need high-throughput technologies to analyze glycan expression and glycan-protein interactions. Methods are not available for amplification of glycans; therefore, highly efficient sample preparation is a major issue. Here we present a novel strategy that allows flexible and sequential incorporation of various functional tags into oligosaccharides derived from biological samples in a practical manner. When combined with a chemoselective glycoblotting platform, our analysis enables us to complete sample preparation (from serum to released, purified, methyl-esterified, and labeled glycans) in 8 h from multiple serum samples (up to 96 samples) using a 96-well microplate format and a standard de-N-glycosylation protocol that requires reductive alkylation and tryptic digestion prior to PNGase F digestion to ensure maximal de-N-glycosylation efficiency. Using this technique, we quantitatively detected more than 120 glycans on human carcinoembryonic antigens for the first time. This approach was further developed to include a streamlined method of purification, chromatographic fractionation, and immobilization onto a solid support for interaction analysis. Since our approach enables rapid, flexible, and highly efficient tag conversion, it will contribute greatly to a variety of glycomic studies.

[PubMed - indexed for MEDLINE]
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