Label-Free Microarray Profiling of Phosphoinositide-PDZ Domain Interactions

磷酸肌醇-PDZ 结构域相互作用的无标记微阵列分析

• 批准号: 7660991
• 负责人: QUAN JASON CHENG
• 金额: $ 20.71万
• 依托单位: UNIVERSITY OF CALIFORNIA RIVERSIDE
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-04-01 至 2011-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7660991
• 关键词: Adhesions; Air; Antibodies; Antineoplastic Agents; Binding; Binding Proteins; Biosensing Techniques; Cell Polarity; Cell physiology; Cells; Complex; DNA; DNA Microarray Chip; Data Analyses; Detection; Development; Dimensions; Elements; Enzymes; Event; Film; Fluorescence; Fluorescence Microscopy; Genomics; Goals; Gold; Hand; Image; Imaging technology; Immobilization; Investigation; Kinetics; Label; Lead; Lipid Binding; Lipids; Location; Malignant Neoplasms; Measurement; Membrane; Membrane Protein Traffic; Methods; Microarray Analysis; Nature; Neurobiology; Non-Insulin-Dependent Diabetes Mellitus; Nuclear; Optics; Pattern; Performance; Phosphatidylinositols; Preparation; Principal Investigator; Printing; Procedures; Property; Protein Fingerprints; Protein Microarray Assay; Protein Microchips; Protein translocation; Proteins; Proteomics; Research; Research Personnel; Sampling; Screening procedure; Second Messenger Systems; Signal Transduction; Signaling Molecule; Silicates; Structure; Surface; Surface Plasmon Resonance; Surface Properties; Techniques; Technology; Testing; Tight Junctions; Time; Tissues; Work; base; cancer type; design; high throughput analysis; high throughput technology; imaging modality; improved; infancy; inhibitor/antagonist; instrument; novel; novel strategies; postsynaptic density protein; prevent; programs; protein complex; public health relevance; second messenger; tool

DESCRIPTION (provided by applicant): High-throughput microarray technology has become exceedingly promising and important in proteomics research. It allows parallel, large-scale investigation of protein interactions, enabling thousands of compounds to be studied en masse. Protein and antibody microarray technologies are two most propitious technologies for the screening of complex protein samples. However, many limitations of the technology are still unsolved, which have prevented protein microarray technology from reaching its full potential. Proteomics studies are limited by problems such as sample preparation and data analysis, especially in conservation of functionality of capture proteins during immobilization and provision of sensitive detection methods. In recent years, surface plasmon resonance (SPR) imaging has evolved to become a very attractive detection method in microarray analysis. It offers high detection sensitivity without the need of a label, and enables various measurement functions, including real time detection and kinetic analysis to be carried out with simple instrument configuration and operational procedure. In this respect, the technique is unsurpassed by any existing method in microarray technology. Nevertheless, problems in obtaining high quality arrays and non-specific protein interactions have impeded the wide acceptance of SPR imaging method in microarray analysis. Lipid microarray may offer a unique niche for advancing high throughput protein screening technique. Many cellular functions and signaling start with the binding events that take place between lipids and the proteins. For instance, phosphoinositides (PIP) exert their effect as signaling molecules and second messengers by directing protein translocation and the formation of macromolecular signaling complexes at specific subcellular locations. Lipid microarrays will allow researchers to obtain a comparable fingerprint of the proteins from a cell or tissue that bind to lipids, and enable the identification of functionally important lipid-binding proteins. Compared to the matured DNA microarray method and fast improving protein microarrays, lipid microarray technology is still only in its infancy. Much of this can be attributed to the great difficulty in array fabrication. The overall goal of this project is to develop new optical substrates for label-free SPR detection with membrane microarrays, and carry out a high throughput analysis to profile interactions of phosphoinositides with PDZ (postsynaptic density protein, disc large, zonula occludens) domains. Phosphoinositides are essential regulators of nuclear functions and membrane trafficking and are associated with cancers and type II diabetes. Specific aims in this proposal are as follows: 1) Design and fabrication of microarray templates with SiOx- coated SPR chips. 2) Formation and characterization of membrane microarrays. 3) Investigation of lipid-protein interactions with lipid microarray/SPR. PHS 398/2590 (Rev. 09/04) Page Continuation Format Page PUBLIC HEALTH RELEVANCE: Phosphoinositides (PIPs) are known to be associated to stabilization of adhesion structure, targeting and organization of large signaling complexes, and establishment of cell polarity. Their functions are being studied in the context of cancer and neurobiology. Understanding of the nature of PIP-protein interactions will also facilitate the development of new inhibitors of PIP-metabolizing enzymes. These inhibitors can potentially be used as anticancer drugs. There is a tremendous need in designing improved technology for high-throughput methods in PIP research. The proposed microarray technology will significantly enhance the capability to carry out effective approaches in PIP research and thus improve the throughput in screening. PHS 398/2590 (Rev. 09/04) Page Continuation Format Page

描述（申请人提供）：高通量微阵列技术在蛋白质组学研究中已经变得非常有前途和重要。它允许平行的，大规模的蛋白质相互作用的研究，使成千上万的化合物被研究在一起。蛋白质和抗体微阵列技术是筛选复杂蛋白质样品的两种最有利的技术。然而，该技术的许多限制仍未解决，这阻碍了蛋白质微阵列技术发挥其全部潜力。蛋白质组学研究受到样品制备和数据分析等问题的限制，特别是在固定过程中捕获蛋白质功能的保存和提供敏感的检测方法。近年来，表面等离子体共振（SPR）成像已经发展成为一种非常有吸引力的微阵列分析检测方法。它提供了高检测灵敏度，无需标签，并实现各种测量功能，包括实时检测和动力学分析，通过简单的仪器配置和操作程序进行。在这方面，该技术是微阵列技术中任何现有方法所无法超越的。然而，在获得高质量阵列和非特异性蛋白质相互作用方面的问题阻碍了SPR成像方法在微阵列分析中的广泛接受。脂质微阵列可能为推进高通量蛋白质筛选技术提供一个独特的利基。许多细胞功能和信号开始于脂质和蛋白质之间发生的结合事件。例如，磷酸肌苷（PIP）作为信号分子和第二信使发挥作用，通过在特定的亚细胞位置指导蛋白质易位和大分子信号复合物的形成。脂质微阵列将允许研究人员从与脂质结合的细胞或组织中获得可比较的蛋白质指纹，并使识别功能重要的脂质结合蛋白成为可能。与成熟的DNA微阵列方法和快速改进的蛋白质微阵列相比，脂质微阵列技术仍处于起步阶段。这在很大程度上可以归因于阵列制造的巨大困难。该项目的总体目标是开发新的光学底物，用于膜微阵列无标记SPR检测，并进行高通量分析，以描述磷酸肌苷与PDZ（突触后密度蛋白，大圆盘，闭带）结构域的相互作用。磷酸肌苷是核功能和膜运输的重要调节因子，与癌症和II型糖尿病有关。本课题的具体目标如下：1)用SiOx包覆的SPR芯片设计和制作微阵列模板。2)膜微阵列的形成与表征。3)脂质微阵列/SPR研究脂质-蛋白相互作用。公共卫生相关性：已知磷酸肌苷（PIPs）与粘附结构的稳定、大信号复合物的靶向和组织以及细胞极性的建立有关。它们的功能正在癌症和神经生物学的背景下进行研究。了解pip -蛋白相互作用的本质也将有助于开发新的pip代谢酶抑制剂。这些抑制剂有可能被用作抗癌药物。在PIP研究中，迫切需要为高通量方法设计改进的技术。所提出的微阵列技术将显著增强在PIP研究中开展有效方法的能力，从而提高筛选的吞吐量。小灵通398/2590 （Rev. 09/04）页延续格式页