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首页> 外文期刊>Chemistry: A European journal >One-pot solid-phase glycoblotting and probing by transoximization for high-throughput glycomics and glycoproteomics
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One-pot solid-phase glycoblotting and probing by transoximization for high-throughput glycomics and glycoproteomics

机译：一锅固相糖印迹法和通过转肟化进行的高通量糖组学和糖蛋白组学研究

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The development of rapid and efficient methods for high-throughput protein glycomics is of growing importance because the glycoform-focused reverse proteomics/genomics strategy will greatly contribute to the discovery of novel biomarkers closely related to cellular development, differentiation, growth, and aging as well as a variety of diseases such as cancers and vital infection. Recently, we communicated that rapid and efficient purification of carbohydrates can be achieved by employing sugar-specific chemical ligation with aminooxy-functionalized polymers, which we termed "glycoblotting" (see SA. Nishimura et al., Angew. Chem. 2005, 117, 93-98; Angew. Chem. Int. Ed. 2005, 44, 9196). The chemoselective blotting of oligosaccharides present in crude biological materials onto synthetic polymers relies on the unique oxime-bond formation between aminooxy group displayed on the supporting materials and aldehyde/ketone group at the reducing terminal of all oligosaccharides, thus enabling highly selective and rapid oligoosaccharide purification. Aiming to improve the detection sensitivity of the released oligosaccharides, we introduce here a novel strategy for one-pot solid-phase glycoblotting and probing by transoximization. We found that oligosaccharides captured by the polymer supports via the oxime bond can be released in the presence of excess O-substituted aminooxy derivatives in a weakly acidic condition. The released oligosaccharides could be recovered as newly formed oxime derivatives of the O-substituted aminooxy compound added, thus demonstrating the simultaneous releasing and probing. In addition, we synthesized a novel aminooxy-functionalized monomer, N-[2-[2-(2tert-butoxycarbonylaminooxyacetylami-no-ethoxy)ethoxy]ethyl]-2-methacrylamide, which allows for the large-scale preparation of a versatile polymer characterized by its high stability, high blotting capacity, and easy use. The one-pot protocol allowed to profile 23 kinds of N-glycan chains of human serum glycoproteins. This concept was further applied for the glycopeptides analysis in a crude mixture followed by galactose oxidase treatment to generate free aldehyde group at the non-reducing terminal of oligosaccharide moiety of glycopeptides. Our technique may be implemented in existing bio-chemistry and molecular diagnostics laboratories because enriched oligosaccharides and glycopeptides by solid-phase transoximization with high-sensitive labeling reagents are widely applicable in a variety of common analytical methods using two-dimensional HPLC, LC/MS, and capillary electrophoresis as well as modern mass spectrometry.
机译：快速高效的高通量蛋白质糖组学方法的开发变得越来越重要，因为以糖形式为中心的反向蛋白质组学/基因组学策略将极大地有助于发现与细胞发育，分化，生长和衰老密切相关的新型生物标志物作为多种疾病，例如癌症和重要感染。最近，我们传达出，通过将糖特异性的化学连接与氨基氧基官能化的聚合物（我们称为“糖印迹”），可以实现碳水化合物的快速有效纯化（参见SA。Nishimura等，Angew。Chem。2005，117， 93-98; Angew.Chem.Int.Ed.2005，44，9196）。粗生物材料中存在的寡糖对合成聚合物的化学选择性印迹依赖于支持物上显示的氨氧基与所有寡糖的还原末端处的醛/酮基之间的独特肟键形成，因此能够进行高度选择性和快速的寡糖纯化。为了提高释放的寡糖的检测灵敏度，我们在这里介绍一种新的策略，用于一锅固相糖印迹和通过转肟化进行探测。我们发现，在弱酸性条件下，在过量的O-取代的氨氧基衍生物的存在下，聚合物载体通过肟键捕获的寡糖可以被释放。释放的寡糖可以作为添加的O-取代的氨氧基化合物的新形成的肟衍生物回收，从而证明了同时释放和探测。此外，我们合成了一种新型的氨基氧基官能化单体，N- [2- [2-（2-叔丁氧基羰基氨基氧基乙酰氨基-无乙氧基）乙氧基]乙基] -2-甲基丙烯酰胺，可大规模制备通用聚合物具有高稳定性，高吸墨能力和易于使用的特点。一锅协议允许分析人血清糖蛋白的23种N-聚糖链。该概念进一步用于粗混合物中的糖肽分析，然后进行半乳糖氧化酶处理，以在糖肽的寡糖部分的非还原末端生成游离醛基。我们的技术可能会在现有的生物化学和分子诊断实验室中实施，因为通过高灵敏度标记试剂通过固相转肟化富集的寡糖和糖肽广泛适用于使用二维HPLC，LC / MS，毛细管电泳以及现代质谱。

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