Versatile High Resolution Oligosaccharide Microarrays for Plant Glycobiology and Cell Wall Research
Versatile High Resolution Oligosaccharide Microarrays for Plant Glycobiology and Cell Wall Research
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DOI：
10.1074/jbc.m112.396598
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发表时间：
2012-11-16
影响因子：
4.8
通讯作者：
Willats, William G. T.
中科院分区：
文献类型：
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作者：
Pedersen, Henriette L.;Fangel, Jonatan U.;Willats, William G. T.
微阵列是高通量分析的强大工具，使用非常少量的分析物可以同时评估数百或数千个分子相互作用。核苷酸微阵列在植物研究中建立得很好，但碳水化合物微阵列的建立要少得多，其中一个原因是缺乏合适的聚糖来填充阵列。多糖微阵列是相对容易生产的，因为易于将大聚合物非共价固定在各种微阵列表面上，但它们缺乏分析分辨率，因为多糖通常含有多个不同的碳水化合物亚结构。微阵列确定的寡糖有可能克服这个问题，但更难生产，因为寡糖通常需要在固定之前偶联。我们已经组装了一个具有良好特征的植物低聚糖库，这些低聚糖要么是由多糖部分水解产生的，要么是通过重新化学合成产生的。一旦与蛋白质偶联，这些新糖缀合物是多功能试剂，可以作为微阵列打印到各种类型的载玻片和膜上。我们表明，这些微阵列适用于单克隆抗体、碳水化合物结合模块和其他具有生物学意义的低聚糖结合蛋白的识别能力的高通量表征，并且它们也具有表征碳水化合物活性酶的潜力。
Microarrays are powerful tools for high throughput analysis, and hundreds or thousands of molecular interactions can be assessed simultaneously using very small amounts of analytes. Nucleotide microarrays are well established in plant research, but carbohydrate microarrays are much less established, and one reason for this is a lack of suitable glycans with which to populate arrays. Polysaccharide microarrays are relatively easy to produce because of the ease of immobilizing large polymers noncovalently onto a variety of microarray surfaces, but they lack analytical resolution because polysaccharides often contain multiple distinct carbohydrate substructures. Microarrays of defined oligosaccharides potentially overcome this problem but are harder to produce because oligosaccharides usually require coupling prior to immobilization. We have assembled a library of well characterized plant oligosaccharides produced either by partial hydrolysis from polysaccharides or by de novo chemical synthesis. Once coupled to protein, these neoglycoconjugates are versatile reagents that can be printed as microarrays onto a variety of slide types and membranes. We show that these microarrays are suitable for the high throughput characterization of the recognition capabilities of monoclonal antibodies, carbohydrate-binding modules, and other oligosaccharide-binding proteins of biological significance and also that they have potential for the characterization of carbohydrate-active enzymes.