Comprehensive approach to structural and functional glycomics based on chemoselective glycoblotting and sequential tag conversion
Comprehensive approach to structural and functional glycomics based on chemoselective glycoblotting and sequential tag conversion
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DOI：
10.1021/ac702124d
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发表时间：
2008-02-15
影响因子：
7.4
通讯作者：
Nishimura, Shin-Ichiro
中科院分区：
文献类型：
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作者：
Furukawa, Jun-ichi;Shinohara, Yasuro;Nishimura, Shin-Ichiro
蛋白质糖基化的变化深刻地影响蛋白质功能。为了了解蛋白质糖基化改变的这些影响，我们迫切需要高通量技术来分析聚糖表达和聚糖-蛋白质相互作用。没有方法可用于聚糖的扩增;因此，高效的样品制备是一个主要问题。在这里，我们提出了一种新的策略，允许灵活和顺序掺入各种功能标签到寡糖来源于生物样品中的一个实用的方式。当与化学选择性糖印迹平台相结合时，我们的分析使我们能够完成样品制备（从血清到释放的、纯化的、甲基酯化的，和标记的聚糖），使用96孔微孔板格式和标准去N-糖基化方案（需要在PNGase F消化前进行还原性烷基化和胰蛋白酶消化，以确保最大程度的去N-糖基化），糖基化效率利用该技术，我们首次定量检测了人癌胚抗原上的120多个聚糖。进一步开发了该方法，以包括纯化、色谱分离和固定到固体支持物上用于相互作用分析的简化方法。由于我们的方法能够快速，灵活，高效的标签转换，它将大大有助于各种糖组学研究。
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.