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。高通量被用于药物发现、药物开发、表征组合合成反应、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的荧光测定。这种方法具有重要的局限性，包括假阳性以及将荧光变化构建到生化反应中的成本和时间。质谱（MS）和电泳作为高通量分析方法是非常有前途的，其可以通过直接检测反应中涉及的生化物质而在对标记的要求最低或没有要求的情况下进行。它们还通过在一次测定中解析多种化合物提供了多重检测的潜力。在开发这些用于高通量分析的方法方面已经取得了一些进展;然而，目前的方法不能提供与荧光相媲美的通量。我们建议耦合分段流，其中样品被划分为纳升液滴内的不混溶流体，直接MS和电泳。这种方法将允许分析率高达 10 Hz，同时仅消耗纳升样品。后者很重要，因为与筛查相关的成本降低。由于快速分析不能实现没有高通量的样品制备，我们还建议开发新的方法来制备样品的MS和电泳，包括高通量提取。这些新方法将被应用于筛选目前没有高通量检测的新型蛋白质靶点的调节剂。我们将靶向Hsp 70蛋白复合物的形成，沉默调节蛋白5脱琥珀酰酶，沉默调节蛋白6脱乙酰酶和细胞色素P450 CYP 2 E1活性。这些蛋白质分别与阿尔茨海默病、2型糖尿病、癌症和酒精性肝病有关。这项工作的意义在于，它为高度小型化的高通量化学分析提供了强大的新技术，以提高药物开发的速度并降低成本。它还将确定几种新型蛋白质靶点的化学先导物。重要的创新包括用于MS和电泳的分段流动样品引入、高通量样品制备以及新型蛋白质靶点测定的开发。 公共卫生相关性：高通量化学分析在生物医学研究中变得越来越重要。高通量用于药物发现、药物开发、诊断和生物技术。这项工作将开发可用于此类应用的高通量化学分析的改进方法。新开发的方法将用于筛选与2型糖尿病、阿尔茨海默病、癌症和酒精性肝病有关的蛋白质调节剂。从这些筛选中开发的先导化合物可能被证明在治疗这些疾病中是有用的。

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.