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

基本信息

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
项目类别：
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

项目摘要

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（由申请人提供）：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景和重要。它允许对蛋白质相互作用进行并行、大规模的研究，从而能够对数千种化合物进行集体研究。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，该技术的许多局限性仍未解决，这阻碍了蛋白质微阵列技术充分发挥其潜力。蛋白质组学研究受到样品制备和数据分析等问题的限制，特别是在固定化过程中捕获蛋白功能的保护和提供灵敏的检测方法方面。近年来，表面等离子共振（SPR）成像已发展成为微阵列分析中非常有吸引力的检测方法。它无需标签即可提供高检测灵敏度，并通过简单的仪器配置和操作程序实现实时检测和动力学分析等各种测量功能。在这方面，该技术是微阵列技术中任何现有方法都无法超越的。然而，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独特的利基。许多细胞功能和信号传导始于脂质和蛋白质之间发生的结合事件。例如，磷酸肌醇（PIP）通过指导蛋白质易位和在特定亚细胞位置形成大分子信号复合物来发挥其作为信号分子和第二信使的作用。脂质微阵列将使研究人员能够从与脂质结合的细胞或组织中获得可比较的蛋白质指纹，并能够识别功能上重要的脂质结合蛋白。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。这很大程度上可以归因于阵列制造的巨大难度。该项目的总体目标是开发用于膜微阵列无标记 SPR 检测的新型光学底物，并进行高通量分析以分析磷酸肌醇与 PDZ（突触后密度蛋白、椎间盘大、闭锁小带）结构域的相互作用。磷酸肌醇是核功能和膜运输的重要调节剂，与癌症和 II 型糖尿病有关。本提案的具体目标如下： 1）设计和制造带有 SiOx 涂层 SPR 芯片的微阵列模板。 2）膜微阵列的形成和表征。 3) 用脂质微阵列/SPR 研究脂质-蛋白质相互作用。 PHS 398/2590 (Rev. 09/04) 页继续格式页公共卫生相关性：众所周知，磷酸肌醇 (PIP) 与粘附结构的稳定、大型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-蛋白质相互作用的性质也将有助于开发新的 PIP 代谢酶抑制剂。这些抑制剂有可能用作抗癌药物。 PIP 研究中迫切需要设计改进的高通量方法技术。所提出的微阵列技术将显着增强在 PIP 研究中实施有效方法的能力，从而提高筛选的通量。 PHS 398/2590 (Rev. 09/04) 页面延续格式页面