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Proteomics of Cell Death via 2-D Microfluidic Profiling

通过二维微流控分析进行细胞死亡的蛋白质组学

基本信息

批准号：
7935869
负责人：
Don L DeVoe
金额：
$ 23.65万
依托单位：
UNIV OF MARYLAND, COLLEGE PARK
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-09-30 至 2010-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7935869
关键词：
Address Animals Apoptosis Autoimmunity Automation Bioinformatics Biological Markers Biological Models Biotechnology Blood capillaries Capillary Electrophoresis Caspase Cell Death Cells Clinical Comparative Study Confocal Microscopy Coupled Cysteine Protease DNA-Binding Proteins Data Data Analyses Databases Defect Deposition Detection Development Disease Drosophila genus Drosophila melanogaster Embryo Embryo Deaths Engineering Escherichia coli Family Fluorescence Fluorescence Microscopy Gel Gene Expression Profile Gene Proteins Genetic Genetic Transcription Goals Human Human Resources Image Image Analysis Individual Institutes Investigation Isoelectric Focusing Isoelectric Point Laboratories Lasers Lead Liquid Chromatography Malignant Neoplasms Manuals Maryland Mass Spectrum Analysis Measurement Methodology Methods Microfabrication Microfluidics Modeling Modification Molecular Weight Operative Surgical Procedures Organism Pathway interactions Pattern Peptide Hydrolases Performance Phase Phosphorylation Physiological Plastics Play Polymers Post-Translational Protein Processing Process Protein Family Proteins Proteolysis Proteome Proteomics RNA Reproducibility Research Research Personnel Resolution Role Salivary Glands Sampling Screening procedure Sodium Dodecyl Sulfate Specimen Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization System Technology Text Time Two-Dimensional Polyacrylamide Gel Electrophoresis Universities Validation Variant Washington Work base capillary cell type clinical application college experience gel electrophoresis image processing improved innovation insight instrumentation interest liquid chromatography mass spectrometry mRNA Differential Displays meetings nanofluidic nanoscale novel protein expression protein profiling prototype simulation technology development tool virtual web site

项目摘要

Programmed cell death plays an important role during animal development, and defects in this process result in a variety of human disorders including cancer and autoimmunity. A family of cysteine proteases, called Caspases, are conserved throughout animals and function to dismantle cells during programmed cell death by proteolysis. The goal of this project is to develop, optimize, and apply new multidimensional microfluidics technology for the rapid profiling of protein modifications based on changes in isoelectric point (pi) and molecular weight (MW) during programmed cell death, and identification of modified proteins via mass spectrometry. By using the fruit fly Drosophila melanogaster as a model system, these studies will explore pathways and identify biomarkers associated with Caspase activation during cell death in developing animals which will provide important insight into human cell death pathways. This challenge will be addressed through the development and application of a microfluidic platform capable of ultra-high-throughput multidimensional protein separation, followed by extremely sensitive protein quantification and identification, enabling effective screening of protein modifications. By offering significant reductions in sample requirements, the platforms will also serve to greatly improve the efficiency of Drosophila proteomic studies, and provide important benefits for downstream clinical applications of the technology. The proposed research will couple our team's expertise in programmed cell death studies and bioinformatics with experience in the development of capillary electrophoresis, microfluidic, and mass spectrometry proteomic instrumentation. Dr. DeVoe (Univ. of Maryland) will lead the project as PI, and take responsibility for overall coordination between the personnel and organizations involved in the research. He will be the leader for all activities involving microfabrication, micro and nanofluidics, and system engineering. Dr. Lee (Univ. of Maryland) will direct the activities in protein separation development and mass spectrometry analysis. Dr. Baehrecke (Univ. of Maryland Biotechnology Institute) will lead the investigation of the programmed cell death studies, and analysis of the resulting protein profiling data. Dr. Rudnick (Calibrant Biosystems) will work in concert with Drs. Baehrecke and Lee to develop and apply bioinformatics tools relevant to the programmed cell death studies. Dr. English (Univ. of Maryland) will collaborate with Drs. DeVoe and Lee on the development and implementation of ultrasensitive confocal microscopy systems for nanofluidic separation platforms. Dr. Ivory (Washington State Univ.) will work with Dr. DeVoe to develop electrokinetic simulations to be employed in optimizing the microfluidic separation systems in order to meet the stated performance goals for ultra-high-throughput protein profiling.

细胞程序性死亡在动物发育过程中扮演着重要的角色，而这一过程中的缺陷会导致包括癌症和自身免疫在内的各种人类疾病。半胱氨酸蛋白酶家族被称为半胱氨酸蛋白酶，在动物体内是保守的，在细胞程序性死亡过程中通过蛋白分解来分解细胞。该项目的目标是开发、优化和应用新的多维微流控技术，以基于细胞程序性死亡过程中等电点(Pi)和相对分子质量(MW)的变化来快速分析蛋白质修饰，并通过质谱学鉴定修饰蛋白质。通过以果蝇黑腹果蝇为模型系统，这些研究将探索与发育中动物细胞死亡过程中Caspase激活相关的途径和生物标志物，这将为了解人类细胞死亡途径提供重要的见解。这一挑战将通过开发和应用能够超高通量多维蛋白质分离的微流控平台来解决，随后将进行极其敏感的蛋白质定量和鉴定，从而能够有效地筛选蛋白质修饰。通过大幅减少样品需求，这些平台还将大大提高果蝇蛋白质组研究的效率，并为该技术的下游临床应用提供重要好处。这项拟议的研究将把我们团队在程序性细胞死亡研究和生物信息学方面的专业知识与毛细管电泳法、微流控和质谱仪蛋白质组仪器的开发经验结合起来。DeVoe博士(大学)将以PI的身份领导该项目，并负责参与研究的人员和组织之间的整体协调。他将领导所有涉及微制造、微和纳米流体以及系统工程的活动。李博士(大学将指导蛋白质分离开发和质谱分析方面的活动。Baehrecke博士(大学马里兰州生物技术研究所)将领导对程序性细胞死亡研究的调查，并分析产生的蛋白质图谱数据。Rudnick博士(校准生物系统)将与Baehrecke博士和Lee博士合作，开发和应用与程序化细胞死亡研究相关的生物信息学工具。英格利希博士(大学)将与DeVoe博士和Lee博士合作开发和实施用于纳米流体分离平台的超灵敏共聚焦显微镜系统。象牙博士(华盛顿州大学)将与DeVoe博士合作开发电动模拟，用于优化微流控分离系统，以满足超高通量蛋白质图谱所述的性能目标。