One-pot solid-phase glycoblotting and probing by transoximization for high-throughput glycomics and glycoproteomics.
One-pot solid-phase glycoblotting and probing by transoximization for high-throughput glycomics and glycoproteomics.
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DOI：
10.1002/chem.200601613
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发表时间：
2007-02
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通讯作者：
Hideyuki Shimaoka;Hiromitsu Kuramoto;J. Furukawa;Y. Miura;Masaki Kurogochi;Y. Kita;H. Hinou;Y. Shinohara;S. Nishimura
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文献类型：
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作者：
Hideyuki Shimaoka;Hiromitsu Kuramoto;J. Furukawa;Y. Miura;Masaki Kurogochi;Y. Kita;H. Hinou;Y. Shinohara;S. Nishimura
发展快速有效的高通量蛋白质糖组学方法越来越重要，因为以糖型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、生长和衰老以及各种疾病（如癌症和病毒感染）密切相关的新型生物标志物。最近，我们传达了通过使用糖特异性化学连接与氨氧基官能化聚合物可以实现碳水化合物的快速和有效纯化，我们称之为“糖印迹”（参见S. I. Nishimura等人，Angew. 2005，117，93-98; Angew. Chem.Int.Ed.2005，44，91-96）。存在于粗生物材料中的寡糖到合成聚合物上的化学选择性印迹依赖于在支撑材料上展示的氨基氧基与所有寡糖的还原末端处的醛/酮基团之间的独特肟键形成，因此能够实现高选择性和快速的寡糖纯化。为了提高释放的低聚糖的检测灵敏度，我们在这里介绍了一种新的一锅固相糖印迹和通过转胺肟化进行探测的策略。我们发现，低聚糖捕获的聚合物支持通过肟键可以释放过量的O-取代的氨基氧基衍生物的存在下，在弱酸性条件下。释放的低聚糖可以作为添加的O-取代的氨基氧基化合物的新形成的肟衍生物回收，从而证明同时释放和探测。此外，我们合成了一种新的氨氧基官能化单体，N-[2-[2-（2-叔丁氧基羰基氨氧基乙酰基氨基-乙氧基）乙氧基]乙基]-2-甲基丙烯酰胺，其允许大规模制备多功能聚合物，其特征在于其高稳定性，高印迹能力和易于使用。该一锅法可对人血清糖蛋白的23种N-聚糖链进行谱分析。这一概念进一步应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产生游离醛基。我们的技术可以在现有的生物化学和分子诊断实验室中实施，因为通过固相转肟化与高灵敏度标记试剂富集的寡糖和糖肽广泛适用于各种常用的分析方法，使用二维HPLC，LC/MS，毛细管电泳以及现代质谱。
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 viral 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 S.-I. Nishimura et al., Angew. Chem. 2005, 117, 93-98; Angew. Chem. Int. Ed. 2005, 44, 91-96). 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 oligosaccharide 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-(2-tert-butoxycarbonylaminooxyacetylamino-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 biochemistry 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.