权益分类	功能权益	普通用户	{{item.name}}会员
{{category.name}}	{{benefitItem.name}}

High throughput mass spectrometry and electrophoresis assay systems

高通量质谱和电泳分析系统

基本信息

批准号：
8340559
负责人：
ROBERT T KENNEDY
金额：
$ 27.55万
依托单位：
UNIVERSITY OF MICHIGAN AT ANN ARBOR
依托单位国家：
美国
项目类别：
财政年份：
2012
资助国家：
美国
起止时间：
2012-09-30 至 2016-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8340559
关键词：
Air Alcohols Alzheimer&apos s Disease Biochemical Biochemical Reaction Biological Assay Biomedical Research Biotechnology CYP2E1 gene Chemicals Clinical Complement Complex Complex Mixtures Consumption Coupling Cytochrome P450 Deacetylase Detection Diagnostic Disease Electrophoresis Electrospray Ionization Enzymes Fluorescence Goals Health Label Lead Liquid substance Liver diseases Malignant Neoplasms Mass Spectrum Analysis Methods Microchip Electrophoresis Microfabrication Non-Insulin-Dependent Diabetes Mellitus Oils Optics Performance Pharmaceutical Preparations Phase Preparation Procedures Proteins Protocols documentation Pump Reaction Reader Reagent Sampling Scanning Screening procedure Solid Source Spectrometry, Mass, Electrospray Ionization Speed System Technology Time Tube Work assay development base combinatorial cost density directed evolution drug development drug discovery drug metabolism high throughput analysis high throughput screening improved innovation instrument interest mass spectrometer metabolic abnormality assessment miniaturize nanolitre nanolitre scale new technology novel novel diagnostics novel strategies protein complex protein phosphatase inhibitor-2 tool

项目摘要

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.

描述(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。目前高通量的化学分析主要是在多孔板上进行的荧光分析。这种方法有重要的局限性，包括假阳性和在生化反应中建立荧光变化的成本和时间。质谱仪(MS)和电泳法作为高通量分析方法非常有前景，通过直接检测参与反应的生物化学物质，可以进行最小甚至不需要标记的分析。它们还提供了在一次检测中分解多个化合物的多重潜力。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可与荧光相媲美的吞吐量。我们建议将分段流动直接耦合到MS和电泳，其中样品被分隔为不混溶流体中的纳升液滴。这种方法将允许分析率高达 10赫兹，同时只消耗纳升的样品。后者很重要，因为与筛查相关的成本降低了。由于没有高通量的样品制备就无法实现快速分析，我们还建议开发新的方法来为MS和电泳法制备样品，包括高通量提取。这些新方法将被应用于筛选目前尚无高通量分析方法的新蛋白质靶标的调节子。我们的目标是Hsp70-蛋白质复合体的形成，sirtuin 5脱琥珀酸酶，sirtuin 6脱乙酰酶和细胞色素P450细胞色素P450细胞色素P2E1的活性。这些蛋白质分别与阿尔茨海默氏症、2型糖尿病、癌症和酒精性肝病有关。这项工作的意义在于，它为高通量、高度小型化的化学分析提供了强大的新技术，加快了药物开发的步伐，降低了成本。它还将为几个新的蛋白质靶标确定化学线索。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针对新蛋白质靶标的分析方法的开发。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。高通量被用于药物发现、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的调节器。从这些筛查中开发出的先导化合物可能被证明在治疗这些疾病方面是有用的。