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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：
8925093
负责人：
ROBERT T KENNEDY
金额：
$ 28.1万
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 2018-07-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/8925093
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、表征组合合成反应、诊断以及蛋白质定向进化等新生物技术。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，包括误报以及将荧光变化构建为生化反应的成本和时间。质谱 (MS) 和电泳作为高通量分析方法是非常有前途的，它们可以通过直接检测反应中涉及的生化物质，在很少或不需要标签的情况下进行。它们还通过在一次测定中解析多种化合物来提供多重分析的潜力。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，当前的方法无法提供与荧光相媲美的通量。我们建议将分段流直接耦合到 MS 和电泳，其中样品在不混溶的流体中被划分为纳升液滴。这种方法将使分析率高达 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了。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。这些新方法将用于筛选目前尚无可用高通量测定的新型蛋白质靶标的调节剂。我们将针对 Hsp70-蛋白复合物的形成、sirtuin 5 脱琥珀酰酶、sirtuin 6 脱乙酰酶和细胞色素 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，它为高度小型化的高通量化学分析提供了强大的新技术，以加快药物开发的步伐并降低成本。它还将鉴定几种新型蛋白质靶标的化学先导物。重要的创新包括用于 MS 和电泳的分段流样品引入、高通量样品制备以及新型蛋白质靶标检测方法的开发。

项目成果

期刊论文数量（6）

专著数量（0）

科研奖励数量（0）

会议论文数量（0）

专利数量（0）

Subsecond electrophoretic separations from droplet samples for screening of enzyme modulators.

DOI：
10.1021/ac502758h
发表时间：
2014-10-21
期刊：
ANALYTICAL CHEMISTRY
影响因子：
7.4
作者：
Guetschow, Erik D.;Steyer, Daniel J.;Kennedy, Robert T.
通讯作者：
Kennedy, Robert T.

Enabling Biocatalysis by High-Throughput Protein Engineering Using Droplet Microfluidics Coupled to Mass Spectrometry.

DOI：
10.1021/acsomega.7b01973
发表时间：
2018-02-28
期刊：
ACS omega
影响因子：
4.1
作者：
Diefenbach XW;Farasat I;Guetschow ED;Welch CJ;Kennedy RT;Sun S;Moore JC
通讯作者：
Moore JC

Droplet electrospray ionization mass spectrometry for high throughput screening for enzyme inhibitors.

DOI：
10.1021/ac502542z
发表时间：
2014-09-16
期刊：
ANALYTICAL CHEMISTRY
影响因子：
7.4
作者：
Sun, Shuwen;Kennedy, Robert T.
通讯作者：
Kennedy, Robert T.

Protein cross-linking capillary electrophoresis at increased throughput for a range of protein-protein interactions.

DOI：
10.1039/c7an02098h
发表时间：
2018-04-16
期刊：
The Analyst
影响因子：
0
作者：
Ouimet CM;Dawod M;Grinias J;Assimon VA;Lodge J;Mapp AK;Gestwicki JE;Kennedy RT
通讯作者：
Kennedy RT

A Label-free Sirtuin 1 Assay based on Droplet-Electrospray Ionization Mass Spectrometry.