Shimaoka Hideyuki, Kuramoto Hiromitsu, Furukawa Jun-ichi, Miura Yoshiaki, Kurogochi Masaki, Kita Yoko, Hinou Hiroshi, Shinohara Yasuro, Nishimura Shin-Ichiro
Graduate School of Advanced Life Science, Frontier Research Center for the Post-Genome Science and Technology, Hokkaido University, N21, W11 Sapporo 001-0021, Japan.
Chemistry. 2007;13(6):1664-73. doi: 10.1002/chem.200601613.
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.
开发用于高通量蛋白质糖组学的快速高效方法变得越来越重要，因为以糖型为重点的反向蛋白质组学/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，我们报道了通过将糖特异性化学连接与氨氧基官能化聚合物结合使用，可以实现碳水化合物的快速高效纯化，我们将其称为“糖印迹”（见西村伸一等，《德国应用化学》，2005年，117卷，93 - 98页；《德国应用化学国际版》，2005年，44卷，91 - 96页）。将粗生物材料中存在的寡糖化学选择性印迹到合成聚合物上，依赖于支撑材料上显示的氨氧基与所有寡糖还原末端的醛/酮基之间独特的肟键形成，从而实现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，我们在此介绍一种通过转肟化进行一锅法固相糖印迹和探测的新策略。我们发现，通过肟键被聚合物载体捕获的寡糖在弱酸性条件下，在过量的O - 取代氨氧基衍生物存在下可以被释放。释放的寡糖可以作为添加的O - 取代氨氧基化合物新形成的肟衍生物被回收，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氧基官能化单体，N - [2 - [2 - (2 - 叔丁氧羰基氨氧基乙酰氨基 - 乙氧基)乙氧基]乙基] - 2 - 甲基丙烯酰胺，它允许大规模制备一种通用聚合物，其特点是具有高稳定性、高印迹容量且易于使用。一锅法方案能够分析人血清糖蛋白的23种N - 聚糖链。这个概念进一步应用于粗混合物中的糖肽分析，随后进行半乳糖氧化酶处理，以在糖肽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，因为通过与高灵敏度标记试剂进行固相转肟化富集的寡糖和糖肽广泛适用于使用二维高效液相色谱、液相色谱/质谱和毛细管电泳以及现代质谱的各种常见分析方法。
