title: Comprehensive approach to structural and functional glycomics based on chemoselective glycoblotting and sequential tag conversion.
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.
authors.affiliation: Laboratory of Advanced Chemical Biology, Graduate School of Advanced Life Science, and Frontier Research Center for Post-Genome Science and Technology, Hokkaido University, Sapporo, Japan.
authors.forename: Jun-ichi
authors.initials: J
authors.lastname: Furukawa
authors.forename: Yasuro
authors.initials: Y
authors.lastname: Shinohara
authors.forename: Hiromitsu
authors.initials: H
authors.lastname: Kuramoto
authors.forename: Yoshiaki
authors.initials: Y
authors.lastname: Miura
authors.forename: Hideyuki
authors.initials: H
authors.lastname: Shimaoka
authors.forename: Masaki
authors.initials: M
authors.lastname: Kurogochi
authors.forename: Mika
authors.initials: M
authors.lastname: Nakano
authors.forename: Shin-Ichiro
authors.initials: S
authors.lastname: Nishimura
mesh_terms: D002240:Carbohydrate Sequence; D006020:Glycopeptides / Q000097:blood / Q000737:chemistry; D006023:Glycoproteins / Q000097:blood / Q000737:chemistry; D006801:Humans; D008969:Molecular Sequence Data; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase / Q000378:metabolism; D011134:Polysaccharides / Q000097:blood* / Q000737:chemistry*; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization; D013997:Time Factors
chemical_list: D006020:Glycopeptides; D006023:Glycoproteins; D011134:Polysaccharides; D043524:Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase