Chiral Imaging for High Throughput Proteome Screening

描述（由申请人提供）：拟定的研究描述了一种通过二次谐波发生（SHG）手性成像检测蛋白质与空间定位的表面固定化小分子（配体）和蛋白质微阵列缔合的创新方法。基于微阵列的蛋白质分析的使用具有显著增加活生物体内的全球蛋白质群体的分析的潜力。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。用于检测蛋白质-配体和蛋白质-蛋白质缔合或用于筛选蛋白质群体的大多数常规的基于微阵列的策略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。不能以受控的方式准确地进行该操作以获得细胞或生物体的完整蛋白质输出是目前提出的用于高通量蛋白质筛选的技术的主要缺点。该提案将解决蛋白质的内在手性可以用于在图案化微阵列上“成像”其关联的假设。这一假设最初将通过探索利用蛋白质的天然手性作为光谱探针，监测蛋白质与平面支持的脂质双层的外小叶上存在的配体的缔合的能力来进行测试。然后，基于蛋白质-配体的微阵列分析的SHG手性成像将被检查，使用微接触印刷方法产生有序的配体阵列用于蛋白质吸附研究。最后，将评估手性SHG成像测量图案化蛋白质微阵列上的蛋白质-蛋白质相互作用的能力。为了充分表征在这些研究中的界面蛋白质物种的空间排列，SHG显微镜将与荧光显微镜结合使用，以提供对所提出的手性SHG成像方法的精确评估。建议的研究代表了一种新的实验方法，应该提供答案的高通量蛋白质组筛选检测越来越多的问题。

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.