Evanescent-field fluorescence-assisted lectin microarray: a new strategy for glycan profiling
Evanescent-field fluorescence-assisted lectin microarray: a new strategy for glycan profiling
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DOI：
10.1038/nmeth803
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发表时间：
2005-11-01
期刊：
影响因子：
48
通讯作者：
Hirabayashi, J
中科院分区：
文献类型：
--
作者：
Kuno, A;Uchiyama, N;Hirabayashi, J
聚糖以其结构多样性在生物体中具有重要作用。因此，糖组学，特别是涉及以高通量方式分配功能性聚糖的方面，已成为后蛋白质组学时代的新兴领域。然而，到目前为止，还没有通用的聚糖盈利方法。在这里，我们描述了一个新的微阵列程序的基础上渐逝场！荧光检测原理，其允许在平衡条件下灵敏地实时观察多种凝集素-碳水化合物相互作用。该方法允许定量检测甚至微弱的凝集素-碳水化合物相互作用（解离常数，Kd> 10（-6）M）作为39种固定凝集素的荧光信号。我们推导出各种Cy 3标记的糖蛋白，糖肽和四甲基罗丹明（TMR）标记的寡糖完全特定的信号模式。所得结果与以前报道的糖蛋白和凝集素特异性一致。我们通过另一种有益的方法--前沿亲和层析法对后者进行了详细的研究。因此，开发的凝集素微阵列应有助于创建一个新的模式，糖组学。
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 profiting. 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 tetra methylrhodamine (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 profiting method. Thus, the developed Lectin microarray should contribute to creation of a new paradigm for glycomics.