Proteomics of Cell Death via 2-D Micorfluidic Profiling

通过二维微流体分析进行细胞死亡的蛋白质组学

• 批准号: 7120519
• 负责人: Don L DeVoe
• 金额: $ 28.89万
• 依托单位: UNIV OF MARYLAND, COLLEGE PARK
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-09-15 至 2009-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7120519
• 关键词: Drosophilidae; Escherichia coli; apoptosis; bioinformatics; biomarker; biotechnology; confocal scanning microscopy; cysteine endopeptidases; enzyme activity; high throughput technology; isoelectric point; matrix assisted laser desorption ionization; microfluidics; molecular weight; posttranslational modifications; proteomics; reversed phase chromatography; technology /technique development

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.

程序性细胞死亡在动物发育过程中起着重要作用，这一过程中的缺陷会导致各种人类疾病，包括癌症和自身免疫。一个半胱氨酸蛋白酶家族，称为半胱氨酸天冬氨酸蛋白酶，在整个动物中是保守的，并且在通过蛋白水解的程序性细胞死亡期间起拆除细胞的作用。该项目的目标是开发，优化和应用新的多维微流体技术，用于基于程序性细胞死亡期间等电点（pi）和分子量（MW）的变化快速分析蛋白质修饰，并通过质谱法鉴定修饰的蛋白质。通过使用果蝇黑腹果蝇作为模型系统，这些研究将探索发育中动物细胞死亡期间与半胱天冬酶激活相关的途径并识别生物标志物，这将为人类细胞死亡途径提供重要见解。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体平台来解决，然后进行极其灵敏的蛋白质定量和鉴定，从而能够有效筛选蛋白质修饰。通过显著降低样品需求，该平台还将大大提高果蝇蛋白质组学研究的效率，并为该技术的下游临床应用提供重要益处。 拟议的研究将结合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，以及毛细管电泳，微流体和质谱蛋白质组学仪器开发方面的经验。DeVoe博士（马里兰州大学）将作为PI领导该项目，并负责参与研究的人员和组织之间的总体协调。他将领导所有涉及微加工、微纳米流体和系统工程的活动。李博士（马里兰州大学）将指导蛋白质分离开发和质谱分析的活动。Baehrecke博士（马里兰州大学生物技术研究所）将领导程序性细胞死亡研究的调查，并分析所得的蛋白质谱数据。Rudnick博士（Calibrant Biosystems）将与Baehrecke博士和Lee博士合作，开发和应用与程序性细胞死亡研究相关的生物信息学工具。English博士（马里兰州大学）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。Ivory博士（华盛顿州立大学）将与DeVoe博士合作开发电动力学模拟，用于优化微流体分离系统，以满足超高通量蛋白质分析的既定性能目标。