Function search of glycoconjugates by selective laser desorption ionization mass spectronmeter using metal nanoparticle

本研究旨在从分子水平上了解糖偶联物的功能，即利用胶体金纳米粒和质谱仪寻找和鉴定与目标碳水化合物配对分子的新方法。制备显示碳水化合物的非特异性吸附抑制型水溶性金胶体纳米粒。由于数百个碳水化合物分子被展示在纳米颗粒的表面(碳水化合物团簇效应)，因此展示碳水化合物的纳米颗粒与配对分子有望产生强烈的相互作用。此外，通过与氨氧基和碳水化合物还原端的化学选择性反应，构建了能够显示任何碳水化合物的体系。利用该系统…构建了碳水化合物呈递纳米粒子文库更多的艾米。此外，纳米颗粒的表面还修饰了自杀底物类型的碳水化合物衍生物，这些衍生物可以通过共价键锚定目标蛋白质。利用激光裂解金属-硫醇键的MALDI-TOF MS表征了碳水化合物在纳米颗粒上的存在情况。为了降低蛋白质的非特异性粘附性，设计并合成了一种带有两性离子头基的硫醇化合物。本研究制备的所有显示碳水化合物的金属纳米颗粒均表现出较高的水溶性和稳定性。&lt；寻找和鉴定碳水化合物的伙伴分子的方法的建立&gt；高度水溶性的纳米颗粒捕获伙伴分子后可以直接进行各种生化分析和MALDI-TOF MS测量。将含有配对分子的纳米颗粒和样品溶液孵育，用缓冲液洗涤，然后将颗粒应用于SDS-PAGE。根据蛋白质组学的常规方法进行蛋白质鉴定是可能的。与传统的亲和层析等需要竞争碳水化合物洗脱的方法相比，这种方法通过减少样品损失实现了高灵敏度，并通过简化活性过程提高了效率。较少

this research was performed to understand the functions of glycoconjugates at the molecular level, that is to say, to develop a new methodology for hunting and identifying the partner molecules with target carbohydrates by using the gold colloidal nanoparticle and the mass spectrometer.<Preparation water -soluble and non-specific adsorption depression type gold colloidal nanoparticle displaying carbohydrates>in this study, the modification of nanoparticles was performed using specific affinity with a metallic surface and thiol group. Strong interaction of carbohydrate-displaying nanoparticle with a partner molecule is expected, because several hundred of carbohydrate molecules are displayed onto a surface of nanoparticle (carbohydrate cluster effect). In addition, the system that could display any carbohydrates was built by using chemoselective reaction with aminooxy1 group and a reducing end of carbohydrate. A library of carbohydrate presentation nano particle was made using this syst … More em. In addition, the surfaces of the nanoparticles were modified with suicide substrate type carbohydrate derivatives that could anchor the target proteins with covalent bonds. The presentation of carbohydrates on the nanoparticles was evaluated by MALDI-TOF MS using that the laser cleaved metal-thiol bond. In order to reduce the non-specific adhesion of proteins, a thiol compound with zwitterionic head group was designed and synthesized. All carbohydrate-displaying metal nanoparticles prepared in this study showed high water solubility and stability.<Establishment of the methods for hunting and identifying the partner molecules of carbohydrates>The highly water-soluble nanoparticles could be directly subjected to a various biochemical assays and MALDI-TOF MS measurement after the capture of partner molecules. Incubate nanoparticle and sample solution containing partner molecules, and wash the particle with buffer, then the particle was just applied to SDS-PAGE. The protein identification was possible according to the ordinary method of proteomics. In this way, a high sensitivity by reduced sample loss and a promotion of efficiency by simplification of activity process were achieved, compared with traditional approaches such as the affinity chromatography which needed elution by a competing carbohydrate. Less