A Novel Approach for Real-time Proteomics in Live Cells

活细胞实时蛋白质组学的新方法

• 批准号: 8518416
• 负责人: Vaidyanathan Subramaniam
• 金额: $ 27.5万
• 依托单位: POWERHOUSE PROTEOMIC SYSTEMS, LLC
• 依托单位国家: 美国
• 财政年份: 2010
• 资助国家: 美国
• 起止时间: 2010-09-16 至 2015-07-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8518416
• 关键词: Biological Assay; Biological Process; Cell Line; Cell physiology; Cells; Development; Disease; ES Cell Line; Embryo; Flow Cytometry; Fluorescence; Fluorescence Microscopy; Gene Proteins; Generations; Genes; Goals; Green Fluorescent Proteins; Histocompatibility Testing; Individual; Libraries; Life; Location; Molecular; Molecular Profiling; Monitor; Mus; Pattern; Phase; Positioning Attribute; Preclinical Drug Evaluation; Proteins; Proteomics; Reporter; Research; Research Personnel; Resources; Set protein; Small Business Innovation Research Grant; System; Techniques; Therapeutic Agents; Time; Transgenic Mice; base; design; embryonic stem cell; human disease; in vivo; interest; novel; novel strategies; protein expression; public health relevance; stem; therapeutic target; tool

DESCRIPTION (provided by applicant): The ultimate goal of this SBIR project is to develop a novel proteomic system that will enable the real- time expression profiling of proteins in live murine embryonic stem (mES) cells and in differentiated cells derived there from. The first step involves the establishment of a library of fluorescent trap mES cell lines, each of which will harbor a single fluorescently-tagged protein. The fluorescence in each cell of a particular clonal line will serve as a reporter for the expression and subcellular localization of the tagged protein in that cell line. Such a library of clonal fluorescently-tagged lines can be subjected to quantitative fluorescence microscopy in multiwell live-cell arrays, to enable the simultaneous real-time expression profiling of multiple proteins in live cells under various culture conditions. In Phase I we demonstrated the feasibility of establishing a large and diverse protein-trap library using our approach. This proposed Phase II effort will be focused generating a library of at 1500 unique fluorescent protein-trap mES cell lines. Individual cell lines from such a library of fluorescently protein-trap mES lines can also be used as versatile protein-specific research tools as they can be manipulated in culture into different tissue-types or used for the generation of transgenic mice when desired. Many also these cell lines can also be used to design novel powerful cell-based assays. Therefore this proposed Phase II project will result in the generation of a versatile and diverse proteomic research resource in mES cells. PUBLIC HEALTH RELEVANCE: The ultimate aim of this SBIR project is to develop a system that will enable researchers to monitor the location and levels of hundreds of proteins simultaneously in living cells. Such a system will be useful in researching a wide variety of biological processes, including understanding the molecular basis of a number of human diseases. Further, such a system can be applied to the identification therapeutic targets and the creation of powerful drug-screening platforms to identify potential therapeutic agents that may cure these diseases.

描述（由申请人提供）：该SBIR项目的最终目标是开发一种新型的蛋白质组学系统，该系统将使蛋白质在活鼠胚胎茎（MES）细胞（MES）细胞中以及从那里得出的分化细胞中进行实时表达分析。第一步涉及建立一个荧光陷阱MES细胞系的库，每条库将带有单个荧光标记的蛋白质。特定克隆线的每个细胞中的荧光将作为该细胞系中标记蛋白的表达和亚细胞定位的报告。这种克隆荧光系列的库可以在多井活细胞阵列中进行定量荧光显微镜，以使在各种培养物条件下活细胞中多个蛋白质的同时实时表达分析。在第一阶段，我们证明了使用我们的方法建立大型且多样化的蛋白质陷阱文库的可行性。该提出的II期努力将集中于1500个独特的荧光蛋白陷阱MES细胞系的库。来自这种荧光蛋白陷阱MES系的单个细胞系也可以用作多功能蛋白质特异性研究工具，因为它们可以在培养中被操纵为不同的组织类型，也可以在需要时用于生成转基因小鼠。许多这些细胞系也可以用于设计新型的基于细胞的测定。因此，该提出的II期项目将导致在MES细胞中产生多种多样的蛋白质组学研究资源。 公共卫生相关性：该SBIR项目的最终目的是开发一种系统，该系统将使研究人员能够同时监测活细胞中数百种蛋白质的位置和水平。这样的系统将在研究各种生物过程中很有用，包括了解许多人类疾病的分子基础。此外，这种系统可以应用于识别治疗靶标和创建强大的药物筛查平台，以识别可能治愈这些疾病的潜在治疗剂。