Chiral Imaging for High Throughput Proteome Screening

描述(申请人提供)：建议的研究描述了一种创新的方法，通过二次谐波产生(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)和蛋白质微阵列的结合。基于微阵列的蛋白质分析的使用有可能极大地增加对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重大影响。大多数传统的基于微阵列的检测蛋白质-配体、蛋白质-蛋白质结合或筛选蛋白质种群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。对于目前提出的高通量蛋白质筛选技术来说，不能以受控方式准确地对细胞或生物体的完整蛋白质输出执行这一操作是一个主要缺点。这项建议将解决这样一种假设，即蛋白质的内在手性可以用来在图案化的微阵列上“成像”它们的关联。这一假说最初将通过利用蛋白质的天然手性作为光谱探针来探索监测蛋白质与平面支撑的脂双层外层小叶上的配体的联系的能力来检验。然后，将使用光刻和微接触打印方法来检测基于蛋白质配体的微阵列分析的倍频手性成像，以产生用于蛋白质吸附研究的有序配体阵列。最后，将评估手性倍频成像在图案化蛋白质微阵列上测量蛋白质-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物种的空间排列，倍频显微镜将与荧光显微镜结合使用，以提供对所提出的手性倍频成像方法的准确评估。建议的研究代表了一种新的实验方法，它应该为高通量蛋白质组筛选的检测提供越来越多的问题的答案。

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.