Chiral Imaging for High Throughput Proteome Screening

描述（由申请人提供）：拟议的研究描述了一种创新的方法，通过二次谐波产生（SHG）的手性成像来检测蛋白质与空间定位的表面固定小分子（配体）和蛋白质微阵列的关联。使用微阵列为基础的蛋白质分析有可能大大增加活生物体内全球蛋白质种群的分析。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和蛋白质-蛋白质相互作用的研究产生巨大影响。大多数传统的基于微阵列的检测蛋白质配体、蛋白质-蛋白质结合或蛋白质群体筛选的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。目前提出的高通量蛋白质筛选技术的一个主要缺点是无法以受控的方式精确地对细胞或生物体的完整蛋白质输出执行该操作。这一建议将解决的假设，内在的手性的蛋白质可以用来“图像”他们的关联在图案微阵列。这一假设最初将通过探索监测蛋白质与平面支撑脂质双层外小叶上的配体结合的能力来验证，利用蛋白质的天然手性作为光谱探针。然后，将使用光刻和微接触印刷方法检查基于蛋白质配体微阵列分析的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成像在蛋白质微阵列上测量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物种的空间排列，SHG显微镜将与荧光显微镜结合使用，以提供所提出的手性SHG成像方法的精确评估。提出的研究代表了一种新的实验方法，应该为越来越多的高通量蛋白质组筛选检测问题提供答案。

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.