Comprehensive Approach to Structural and Functional Glycomics Based on Chemoselective Glycoblotting and Sequential Tag Conversion
| Autor: | Shin-Ichiro Nishimura, Mika Nakano, Yoshiaki Miura, Masaki Kurogochi, Yasuro Shinohara, Hiromitsu Kuramoto, Jun-ichi Furukawa, Hideyuki Shimaoka |
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| Rok vydání: | 2008 |
| Předmět: |
PNGase F
chemistry.chemical_classification Glycan Time Factors Glycosylation biology Molecular Sequence Data Glycopeptides Oligosaccharide Serum samples Analytical Chemistry carbohydrates (lipids) Glycomics chemistry.chemical_compound Matrix-assisted laser desorption/ionization Carbohydrate Sequence Biochemistry chemistry Polysaccharides Spectrometry Mass Matrix-Assisted Laser Desorption-Ionization biology.protein Humans Peptide-N4-(N-acetyl-beta-glucosaminyl) Asparagine Amidase Sample preparation Glycoproteins |
| Zdroj: | Analytical Chemistry. 80:1094-1101 |
| ISSN: | 1520-6882 0003-2700 |
| Popis: | 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. |
| Databáze: | OpenAIRE |
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