General microarray technique for immobilization and screening of natural glycans
General microarray technique for immobilization and screening of natural glycans
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DOI：
10.1021/ac071187g
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发表时间：
2007-11-01
影响因子：
7.4
通讯作者：
Wuhrer, Manfred
中科院分区：
文献类型：
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作者：
de Boer, Arjen R.;Hokke, Cornelis H.;Wuhrer, Manfred
我们在这里提出了一个打印的共价聚糖微阵列的蛋白质结合研究，使用低飞摩尔量的聚糖。将聚糖（从来自天然来源的糖蛋白和糖脂释放的天然聚糖或合成聚糖）用常见的荧光标记物（例如，2-通过还原胺化反应制备2-氨基苯甲酰胺或2-氨基苯甲酸），并通过HPLC纯化。纯化的糖缀合物通过连接聚糖部分与荧光标签的仲胺基团共价固定在商业环氧活化的载玻片上。这种固定方法通常适用于具有不同荧光标记的还原胺化聚糖，并允许寡糖的空间排列。该微阵列包含来自各种生物来源的多种天然聚糖和合成聚糖，并为凝集素伴刀豆球蛋白A以及14种单克隆抗体提供了信息丰富的结合指纹。通过串联质谱法表征识别的聚糖，揭示结合基序。这种天然聚糖阵列允许表征来自稀疏生物来源的寡糖配体的碳水化合物结合蛋白的特异性。此外，它适用于通过使用已知的碳水化合物结合蛋白的微阵列聚糖的表征。
We here present a printed covalent glycan microarray for protein-binding studies, using low-femtomole quantities of glycans. Glycans, either natural glycans, which were released from glycoproteins and glycolipids from natural sources, or synthetic glycans, were labeled with common fluorescent labels (e.g., 2-aminobenzamide or 2-aminobenzoic acid) by reductive amination and purified by HPLC. The purified glycoconjugates were covalently immobilized on commercial epoxide-activated glass slides via the secondary amine group that links the glycan moiety with the fluorescent tag. This immobilization procedure is generally applicable to reductively aminated glycans with different established fluorescent labels and allows the spatial arrangement of oligosaccharides. The microarray comprised a variety of natural glycans from various biological sources and synthetic glycans and provided informative binding fingerprints for the lectin concanavalin A as well as 14 monoclonal antibodies. Recognized glycans were characterized by tandem mass spectrometry revealing binding motifs. This natural glycan array allowed the characterization of the specificity of carbohydrate-binding proteins for oligosaccharide ligands from sparse biological sources. Moreover, it was applied for the characterization of the microarray glycans by using known carbohydrate-binding proteins.