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

使用金属纳米颗粒的选择性激光解吸电离质谱仪进行糖复合物的功能搜索

基本信息

批准号：
17205015
负责人：
NISIMURA Shin-ichitro
金额：
$ 31.28万
依托单位：
Hokkaido University
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (A)
财政年份：
2005
资助国家：
日本
起止时间：
2005 至 2007
项目状态：
已结题

项目摘要

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

本研究旨在从分子水平上了解糖缀合物的功能，即开发一种利用胶体金纳米颗粒和质谱仪来寻找和鉴定与目标糖缀合物的伴侣分子的新方法。在本研究中，使用与金属表面和硫醇基团的特异性亲和力进行纳米颗粒的改性。预期碳水化合物展示纳米颗粒与配偶体分子的强相互作用，因为数百个碳水化合物分子展示在纳米颗粒的表面上（碳水化合物簇效应）。此外，利用氨基与糖的还原性末端的化学选择性反应，构建了一个可以显示任意糖的体系。利用该系统制备了一个糖展示纳米粒子库， ...更多信息 em.此外，纳米粒子的表面修饰的自杀底物类型的碳水化合物衍生物，可以锚与共价键的靶蛋白。通过MALDI-TOF MS使用激光切割的金属-硫醇键来评估碳水化合物在纳米颗粒上的呈现。为了降低蛋白质的非特异性粘附，设计并合成了一种带有两性离子头基的巯基化合物。本研究中制备的所有碳水化合物展示金属纳米颗粒均显示出高的水溶性和稳定性。<Establishment of the methods for hunting and identifying the partner molecules of carbohydrates>高水溶性纳米颗粒可以直接进行各种生化分析和MALDI-TOF MS测量后，捕获的伴侣分子。将纳米颗粒与含有配偶体分子的样品溶液孵育，并用缓冲液洗涤颗粒，然后将颗粒直接应用于SDS-PAGE。根据蛋白质组学的常规方法进行蛋白质鉴定是可行的。通过这种方式，与传统的方法如需要通过竞争性碳水化合物洗脱的亲和色谱相比，通过减少样品损失实现了高灵敏度，并且通过简化活性过程实现了效率的提高。少