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Rapid and Sensitive Photoaffinity Labeling of Drug Receptors with Immobilized Probes

用固定探针快速灵敏地光亲和标记药物受体

基本信息

批准号：
12470504
负责人：
HATANAKA Yasumaru
金额：
$ 8.64万
依托单位：
TOYAMA MEDICAL AND PHARMACEUTICAL UNIVERSITY
依托单位国家：
日本
项目类别：
Grant-in-Aid for Scientific Research (B)
财政年份：
2000
资助国家：
日本
起止时间：
2000 至 2003
项目状态：
已结题

项目摘要

Since there is no strict linear relationship between genome and proteome, proteomics is desirable to address protein networks. Until recently, proteomics was almost synonymous with the mapping of denatured proteins by two-dimensional gel electrophoresis. However, the major question about protein functions is how they interact with their partners. Thus, affinity-based methods have become increasingly appreciated as the approach of chemical genomics for sorting proteins based on their functions.Photoaffinity methods introduces a cross-link between a ligand and its specific receptor for probing their affinity correlation through a covalent bond, which enables the direct identification of target proteins as well as ligand binding domains. ^<1)>Because the proteomics of is the extensive study of proteins, it is clear that conventional photoaffinity methods are required to increase in the throughput throughout the analytical steps. We firstly developed efficient methods for tethering photoreactive diazirine to various biological ligands. The method significantly increases the synthetic routine in the preparation of various photoreactive derivatives of important biological ligands such as DNA,RNA,carbohydretes, peptides, and proteins. Analogous to DNA technologies, protein chips and protein arrays serve an efficient device for the Screening of samples in a massively parallel fashion. Thus, we secondly developed a rapid and sensitive photoaffinity device for capturing ligand-receptor pair on a solid matrix. The technique based on surface plasmon resonance spectroscopy was developed for the real-time analysis of ligand-receptor interaction. In contrast to this, the photoaffinity device provides a rapid method for the isolation of target proteins, which enables the high-throughput mass spectrometric analysis for the identification of targets as well as their functional sites.

由于基因组和蛋白质组之间没有严格的线性关系，蛋白质组学是解决蛋白质网络的理想方法。直到最近，蛋白质组学几乎是通过二维凝胶电泳绘制变性蛋白质图谱的同义词。然而，关于蛋白质功能的主要问题是它们如何与伴侣相互作用。因此，基于亲和的方法越来越受到重视，作为化学基因组学的方法，根据蛋白质的功能对其进行分类。光亲和方法引入了配体与其特定受体之间的交联，通过共价键探测它们的亲和关系，从而可以直接识别靶蛋白以及配体结合域。由于蛋白质组学是对蛋白质的广泛研究，很明显，传统的光亲和方法需要在整个分析步骤中增加通量。我们首先开发了将光反应性重氮嘧啶与各种生物配体拴住的有效方法。该方法显著增加了制备重要生物配体（如DNA、RNA、碳水化合物、肽和蛋白质）的各种光反应衍生物的合成程序。与DNA技术类似，蛋白质芯片和蛋白质阵列是一种有效的设备，用于大规模并行筛选样品。因此，我们开发了一种快速灵敏的光亲和装置，用于捕获固体基质上的配体-受体对。建立了基于表面等离子体共振光谱的配体-受体相互作用实时分析技术。与此相反，光亲和装置提供了一种快速分离靶蛋白的方法，使高通量质谱分析能够识别靶蛋白及其功能位点。