Kuno Atsushi, Uchiyama Noboru, Koseki-Kuno Shiori, Ebe Youji, Takashima Seigo, Yamada Masao, Hirabayashi Jun
Research Center for Glycoscience (RCG), National Institute of Advanced Industrial Science and Technology (AIST), AIST Tsukuba Central 2; Tsukuba, Ibaraki 305-8568, Japan.
Nat Methods. 2005 Nov;2(11):851-6. doi: 10.1038/nmeth803.
Glycans have important roles in living organisms with their structural diversity. Thus, glycomics, especially aspects involving the assignment of functional glycans in a high-throughput manner, has been an emerging field in the postproteomics era. To date, however, there has been no versatile method for glycan profiling. Here we describe a new microarray procedure based on an evanescent-field fluorescence-detection principle, which allows sensitive, real-time observation of multiple lectin-carbohydrate interactions under equilibrium conditions. The method allows quantitative detection of even weak lectin-carbohydrate interactions (dissociation constant, K(d) > 10(-6) M) as fluorescent signals for 39 immobilized lectins. We derived fully specific signal patterns for various Cy3-labeled glycoproteins, glycopeptides and tetramethylrhodamine (TMR)-labeled oligosaccharides. The obtained results were consistent with the previous reports of glycoprotein and lectin specificities. We investigated the latter aspects in detail by frontal affinity chromatography, another profiling method. Thus, the developed lectin microarray should contribute to creation of a new paradigm for glycomics.
聚糖凭借其结构多样性在生物体内发挥着重要作用。因此，糖组学，尤其是涉及以高通量方式对功能性聚糖进行鉴定的方面，已成为后蛋白质组学时代的一个新兴领域。然而，迄今为止，尚无通用的聚糖谱分析方法。在此，我们描述了一种基于倏逝场荧光检测原理的新型微阵列方法，该方法能够在平衡条件下灵敏、实时地观察多种凝集素 - 碳水化合物相互作用。该方法能够对39种固定化凝集素的微弱凝集素 - 碳水化合物相互作用（解离常数，K(d) > 10(-6) M）进行定量检测，并将其作为荧光信号。我们获得了各种Cy3标记的糖蛋白、糖肽和四甲基罗丹明（TMR）标记的寡糖的完全特异性信号模式。所得结果与先前关于糖蛋白和凝集素特异性的报道一致。我们通过另一种谱分析方法——前沿亲和色谱法，对后述方面进行了详细研究。因此，所开发的凝集素微阵列应为糖组学新范式的创建做出贡献。
