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：
8545872
负责人：
ROBERT T KENNEDY
金额：
$ 27.2万
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/8545872
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 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 screening 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.

描述（由申请人提供）：高通量化学分析对于生物医学研究的进展至关重要。高通量用于药物发现，药物开发，表征组合合成反应，诊断和新的生物技术，例如蛋白质的定向演化。目前的高通量化学分析以多孔板上进行的荧光测定为主。这种方法具有重要的局限性，包括误报以及将荧光变化变化为生化反应的成本和时间。作为高通量分析方法，质谱法（MS）和电泳是高度有希望的，可以通过直接检测到参与反应涉及的生化物质来最少或无需标记的要求。它们还通过在一个测定中解决多种化合物来提供多重多路复用的潜力。开发这些方法进行高通量分析方面已经取得了一些进展。但是，当前的方法无法提供与荧光相当的吞吐量。我们建议将夫妇分割为分段，其中样品被划分为不可分化的流体中的纳米液滴，直接将其直接划分为MS和电泳。这种方法将允许分析率仅消耗样品的纳米含量10 Hz。后者很重要，因为与筛查相关的成本降低。由于没有高通量样品制备就无法实现快速分析，因此我们还建议开发新的方法来制备MS和电泳样品，包括高通量提取。这些新方法将应用于筛选新型蛋白质靶标的调节剂，这些蛋白质目前没有可用的高通量测定。我们将靶向HSP70-蛋白质复合物的形成，Sirtuin 5 desuccinylase，Sirtuin 6脱乙酰基酶和细胞色素P450 CYP2E1活性。这些蛋白质分别与阿尔茨海默氏病，2型糖尿病，癌症和酒精肝病有关。这项工作的意义在于，它为高通量化学分析提供了强大的新技术，以高度微型化的规模增加，以提高药物开发的步伐并降低成本。它还将确定几种新型蛋白质靶标的化学铅。重要的创新包括用于MS和电泳的分段流样品，高通量样品制备以及开发新型蛋白质靶标的测定法。