High throughput mass spectrometry and electrophoresis assay systems

描述（由申请人提供）：高通量化学分析已成为生物医学研究进展的关键。高通量用于药物发现、药物开发、组合合成反应的表征、诊断和新的生物技术，如蛋白质的定向进化。目前，高通量化学分析主要是在多孔板上进行荧光分析。这种方法有重要的局限性，包括假阳性以及将荧光变化构建为生化反应的成本和时间。质谱（MS）和电泳是非常有前途的高通量分析方法，可以通过直接检测参与反应的生物化学物质来进行很少或不需要标签的分析。它们还提供了通过在一次分析中分解多种化合物的多路复用的潜力。这些高通量分析方法的发展取得了一些进展；然而，目前的方法不能提供与荧光相当的吞吐量。我们建议耦合分段流，其中样品被划分为纳升液滴在不混溶的流体中，直接到质谱和电泳。这种方法将允许分析率高达

DESCRIPTION (provided by applicant): High-throughput chemical analysis has become critical to progress in biomedical research. High-throughput is used in drug discovery, drug development, characterizing combinatorial synthetic reactions, diagnostics, and new biotechnologies such as directed evolution of proteins. Presently high-throughput chemical analysis is dominated by fluorescence assays performed on multi-well plates. This approach has important limitations including false positives and the cost and time of building fluorescence changes into biochemical reactions. Mass spectrometry (MS) and electrophoresis are highly promising as high-throughput analysis methods that can be performed with minimal or no requirements for labels by directly detecting biochemicals involved in reactions. They also offer potential for multiplexing by resolving multiple compounds in one assay. Some progress has been made in developing these methods for high-throughput analysis; however, current approaches do not offer throughput comparable to fluorescence. We propose to couple segmented flow, where samples are compartmentalized as nanoliter droplets within an immiscible fluid, directly to MS and electrophoresis. This approach will allow analysis rates up to 10 Hz while consuming only nanoliters of sample. The latter is important because of the reduction in cost associated with screening. Because rapid analysis cannot be achieved without high-throughput sample preparation, we also propose to develop novel approaches to preparing samples for MS and electrophoresis including high-throughput extraction. These new methods will be applied to screening for modulators of novel protein targets that presently do not have high-throughput assays available. We will target Hsp70-protein complex formation, sirtuin 5 desuccinylase, sirtuin 6 deacetylase, and cytochrome P450 CYP2E1 activities. These proteins are implicated in Alzheimer's disease, type 2 diabetes, cancer, and alcohol liver disease, respectively. The significance of this work is that it provides powerful new technology for high-throughput chemical analysis on a highly miniaturized scale to increase the pace of drug development and reduce costs. It also will identify chemical leads for several novel protein targets. Important innovations include segmented flow sample introduction for MS and electrophoresis, high-throughput sample preparation, and development of assays for novel protein targets. PUBLIC HEALTH RELEVANCE: High-throughput chemical analysis has become increasingly important in biomedical research. High throughput is used in drug discovery, drug development, diagnostics, and biotechnology. This work will develop improved methods of high-throughput chemical analysis that may be used in such applications. The newly developed methods will be used in screens for modulators of proteins involved in type 2 diabetes, Alzheimer's disease, cancer, and alcohol liver disease. Lead compounds developed from these screens may prove useful in treatments for these diseases.