Chiral Imaging for High Throughput Proteome Screening

用于高通量蛋白质组筛选的手性成像

• 批准号: 6891422
• 负责人: JOHN C CONBOY
• 金额: $ 19.73万
• 依托单位: UNIVERSITY OF UTAH
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-05-01 至 2008-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6891422
• 关键词: adsorption; antigen antibody reaction; biotechnology; chemical kinetics; confocal scanning microscopy; experimental designs; fluorescence microscopy; high throughput technology; imaging /visualization /scanning; immunoglobulin structure; ligands; lipid bilayer membrane; mathematics; microarray technology; optics; protein protein interaction; protein structure; proteomics; ricin; spectrometry; stereoisomer; thermodynamics

DESCRIPTION (provided by applicant): The proposed studies describe an innovative approach for detecting protein association to spatially localized surface-immobilized small molecule (ligand) and protein microarrays via chiral imaging with second harmonic generation (SHG). The use of microarray-based protein assays has the potential to dramatically increase the analysis of global protein populations within living organisms. The practical implementation of these technologies will have a dramatic impact in proteomics, pharmacological screening of drug candidates, and the investigation of protein-protein interactions. Most conventional microarray-based strategies for detecting protein-ligand, and protein-protein association or for the screening of protein populations are based on fluorescence methods to visualize the interaction of interest. Such methodologies require the fluorescent labeling of a large population of proteins with fluorophores. The inability to accurately perform this operation in a controlled manner for the complete protein output of a cell or organism is a major drawback to currently proposed techniques for high throughput protein screening. This proposal will address the hypothesis that the intrinsic chirality of proteins can be used to "image" their association on patterned microarrays. This hypothesis will initially be tested by exploring the ability to monitor protein association to ligands presented on the outer leaflet of planar supported lipid bilayers, utilizing the proteins' native chirality as a spectroscopic probe. Then, SHG chiral imaging for protein-ligand based microarray assay will be examined using photolithographic and micro-contact printing methods to produce ordered arrays of ligands for protein adsorption studies. Finally, the ability of chiral SHG imaging to measure protein-protein interactions on patterned protein microarrays will be assessed. In order to fully characterize the spatial arrangement of interfacial protein species in these studies, SHG microscopy will be used in combination with fluorescence microscopy to provide a precise evaluation of the proposed chiral SHG imaging methodology. The proposed studies represent a novel experimental approach that should provide answers to a growing number of questions concerning detection for high throughput proteome screening.

描述（由申请人提供）：拟议的研究描述了一种通过二次谐波产生（SHG）手性成像检测蛋白质与空间局部表面固定小分子（配体）和蛋白质微阵列的关联的创新方法。基于微阵列的蛋白质检测的使用有可能显着增加对生物体内整体蛋白质群体的分析。这些技术的实际应用将对蛋白质组学、候选药物的药理学筛选以及蛋白质-蛋白质相互作用的研究产生巨大影响。大多数用于检测蛋白质-配体、蛋白质-蛋白质关联或筛选蛋白质群体的传统微阵列策略都是基于荧光方法来可视化感兴趣的相互作用。这种方法需要用荧光团对大量蛋白质进行荧光标记。无法以受控方式准确地执行该操作以获得细胞或生物体的完整蛋白质输出是当前提出的高通量蛋白质筛选技术的主要缺点。该提案将解决这样的假设：蛋白质的内在手性可用于在图案化微阵列上“成像”它们的关联。该假设最初将通过利用蛋白质的天然手性作为光谱探针，探索监测蛋白质与平面支持的脂质双层外叶上的配体关联的能力来进行测试。然后，将使用光刻和微接触印刷方法检查用于基于蛋白质配体的微阵列测定的SHG手性成像，以产生用于蛋白质吸附研究的有序配体阵列。最后，将评估手性 SHG 成像测量图案化蛋白质微阵列上蛋白质-蛋白质相互作用的能力。为了在这些研究中充分表征界面蛋白质种类的空间排列，SHG 显微镜将与荧光显微镜结合使用，以对所提出的手性 SHG 成像方法进行精确评估。拟议的研究代表了一种新颖的实验方法，应该为越来越多的有关高通量蛋白质组筛选检测的问题提供答案。