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; 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（由申请人提供）：高通量微阵列技术在蛋白质组学研究中变得极为有希望且重要。它允许对蛋白质相互作用的平行，大规模研究，从而使数千种化合物被研究。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。但是，该技术的许多局限性仍未解决，这阻止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本制备和数据分析等问题的限制，尤其是在固定和提供敏感检测方法期间捕获蛋白功能的保存中。近年来，在微阵列分析中，表面等离子体共振（SPR）成像已成为一种非常有吸引力的检测方法。它提供了很高的检测灵敏度，而无需标签，并启用各种测量功能，包括实时检测和动力学分析，以简单的仪器配置和操作过程进行。在这方面，该技术在微阵列技术中的任何现有方法无与伦比。然而，在微阵列分析中，获得高质量阵列和非特异性蛋白质相互作用的问题阻碍了SPR成像方法的广泛接受。脂质微阵列可能会提供一个独特的利基市场，用于推进高通量蛋白筛选技术。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以及在特定亚细胞位置的大分子信号传导复合物的形成来发挥其作为信号分子和第二个使者的作用。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，并允许鉴定功能上重要的脂质结合蛋白。与成熟的DNA微阵列法和快速改善蛋白质微阵列相比，脂质微阵列技术仍处于起步阶段。这大部分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，用于使用膜微阵列进行无标签的SPR检测，并对磷酸肌醇与PDZ（突触后密度蛋白，大量，大，Zonula cocludens）结构域进行高吞吐量分析。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。该提案中的具体目的如下：1）用Siox涂层SPR芯片设计和制造微阵列模板。 2）膜微阵列的形成和表征。 3）研究脂质 - 蛋白与脂质微阵列/SPR的相互作用。 PHS 398/2590（REV。09/04）页面延续格式公共卫生相关性：已知磷酸肌醇（PIPS）与稳定粘附结构，大信号传导复合物的靶向和组织有关，以及建立细胞极性。在癌症和神经生物学的背景下，正在研究它们的功能。对PIP蛋白相互作用的性质的理解还将促进新代谢酶的新抑制剂的发展。这些抑制剂可能可能被用作抗癌药。在设计PIP研究中，设计改进的技术技术需要改进的技术。提出的微阵列技术将显着增强在PIP研究中进行有效方法的能力，从而改善筛查的吞吐量。 PHS 398/2590（修订版09/04）页面延续格式页面