PROTEIN IDENTIFICATION AND LOCALIZATION CORE

蛋白质鉴定和定位核心

基本信息

批准号：
8588313
负责人：
JOHN A WILLIAMS
金额：
$ 16.09万
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：
资助国家：
美国
起止时间：
至
项目状态：
未结题

来源：
https://reporter.nih.gov/project-details/8588313
关键词：
3-Dimensional Actins Affect Affinity Algorithms Amylases Animals Antibodies Area Attention Biological Biomedical Research Blood capillaries Cancer Center Cell Cycle Cell Fraction Cell Line Cell Nucleus Cells Cellular biology Chemicals Chemistry Citrates Clinical Investigator Collection Color Complex Computer software Computers Computing Methodologies Confocal Microscopy Consultations Coomassie blue Copper Core Protein Coupled Cryoultramicrotomy Cybernetics Data Data Analyses Data Files Databases Dependence Development Developmental Cell Biology Devices Diabetes Mellitus Dimensions Disease Dyes Educational process of instructing Electron Microscopy Endocytosis Energy Transfer Enzymes Equipment Equipment and supply inventories Evaluation F-Actin Fees Fixatives Fluorescence Microscopy Fluorescence Resonance Energy Transfer Fluorescent Antibody Technique Fluorescent Probes Frequencies Frozen Sections Fura-2 Gel Genome Mappings Genomics Goals Gold Hand High Pressure Liquid Chromatography Hormones Hour Housing Human Resources Image Image Analysis Imagery Immunofluorescence Immunologic Immunohistochemistry Individual Informatics Instruction Isotopes Label Laboratories Laboratory Research Laboratory Scientists Laser Scanning Confocal Microscopy Lasers Lead Life Light Location Mammalian Cell Manuals Maps Mass Spectrum Analysis Measurement Measures Methodology Methods Michigan Microscope Microscopic Microscopy Mission Mitochondria Modeling Modification Molecular Molecular Biology Monitor Morphology Nature Nickel Nuclear Organelles Organism Paraffin Embedding Pathology Peptide Hydrolases Peptide Synthesis Peptides Perfusion Phase Phosphopeptides Phosphoproteins Phosphorylated Peptide Phosphorylation Phosphorylation Site Physiological Pilot Projects Plant Resins Post-Translational Modification Site Post-Translational Protein Processing Procedures Process Property Protein Chemistry Proteins Proteome Proteomics Protocols documentation Publications Publishing Recipe Research Research Design Research Personnel Research Training Resolution Resources Sampling Series Services Signal Pathway Silver Site Source Specimen Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization Spottings Staging Staining method Stains Structure System Techniques Time Tissues Tolonium chloride Training Transmission Electron Microscopy Tube Universities Walkers base capillary cell fixation cell fixing cell type cellular imaging cost cryostat design digital digital imaging epon experience fluorescence imaging fluorescence microscope fluorophore gel electrophoresis immunoaffinity chromatography immunocytochemistry improved innovation instrument instrumentation interest mass spectrometer member novel operation pH gradient protein complex ratiometric reconstruction research facility research study response sample fixation scaffold screening small molecule sodium-binding benzofuran isophthalate spurr resin synthetic peptide tandem mass spectrometry tissue preparation tool uranyl acetate vector zirconium oxide

项目摘要

A. DEFINITION The Protein Identification and Localization (PIL) Core is a restructured core that has evolved from aspects of two previous cores, the Cell Biology and Cell Imaging Core which focused on cellular imaging and the Peptides and Proteomics Core which had a dual service of providing synthetic peptides and identifying proteins by mass spectrometry. While cell biological methods are now standard in most laboratories and peptide synthesis can be obtained both from University and commercial sources, what was unique about the two previous cores was their synergy in identifying and localizing proteins. These aspects are preserved and enhanced by the structure of the new PIL Core and build on the longstanding collaborative interactions between Drs. Williams, Andrews and Ernst. They also build on the fact that this type of research requires complex and expensive equipment not available in individual laboratories. The PIL Core is designed to provide information on the identity of proteins by mass spectrometry, either as individual proteins purified in an investigator's laboratory and usually separated as a band or spot by gel electrophoresis or globally as a mixture of proteins in a protein complex or organelle. Information is also be provided on post-translational modifications of proteins and on quantitative changes in protein content. Once a protein is identified, the Core can provide information on the localization of the protein in live and fixed cells and their changes over time and in response to physiological and pathophysiological perturbation. It is centered around microscopic imaging and quantitative analysis of digital information obtained primarily by laser scanning confocal microscopy (LSCM) and multiwavelength fluorescence imaging, although traditional electron microscopy of fixed and embedded specimens is also available for fine structure analysis. Modern biomedical research is characterized both by its interdisciplinary nature and by its dependence on increasingly sophisticated instrumentation and informatics. Although the goal is to look at a tissue, disease, or organism as a complex integrated system, we are still developing an understanding of the structure and function of the components which are often cell type dependent. With the mapping of the genome, attention has shifted to the more complex world of the proteome. Complete inventories of most mammalian protein complexes or organelles are yet to be completed and they often vary between cell types, during the cell cycle, and during various stages of development. Thus identification of proteins, their post-translational modifications, dynamic interactions and cellular localization, all in time and space and in a quantitative manner, is a daunting task that almost all laboratory scientists in the GI Peptide Center encounter in their research. These questions can be approached through the tools of the PIL Core, very often in concert with the Molecular Biology Core and national genomic resources such as the NCBI. The new PIL Core is built around expertise in the director's laboratories, and the availability of sophisticated instrumentation in three existing facilities, the Michigan Proteome Core, the Morphology and Imaging Laboratory (MIL) of the Department of Cell and Developmental Biology and the Michigan Diabetes Research and Training Center (MDRTC) which maintain facilities discussed in detail below for sophisticated mass spectrometry and high resolution microscopy. This arrangement is facilitated by the fact that Dr Andrews directs the Michigan Proteome Core, Dr Williams directs the Microscopy and Image Analysis Laboratory of the MDRTC and Dr Ernst has a long standing affiliation with the MIL. All three entities have established personnel and a recharge structure and are currently being use by GI Center members. While the Protein Identification and Localization Core will make use of the available highly sophisticated instrumentation, it also is built around the over 90 years of proteomic, cell biology and imaging expertise of Drs. Williams, Andrews and Ernst which has been primarily devoted to GI Tissues. Experienced Core staff, including Drs. Walker, Strahler and Mr. Nelson, will directly assist Center Investigators and carry out the procedures of the Core. All Core personnel will demonstrate and teach techniques to GI Center investigators, trainees and technicians. Finally another mission of the Core is to initiate, implement and disseminate new and innovative techniques in proteomics and cell imaging.

A. 定义蛋白质识别和定位 (PIL) 核心是一个重组核心，由之前两个核心的各个方面，即细胞生物学和细胞成像核心，重点关注细胞成像以及肽和蛋白质组学核心，具有提供合成的双重服务肽并通过质谱法鉴定蛋白质。虽然现在细胞生物学方法大多数实验室的标准，肽合成可以从大学和大学获得商业来源，前两个核心的独特之处在于它们在识别和定位蛋白质。新 PIL 核心的结构保留并增强了这些方面并以博士之间的长期合作互动为基础。威廉姆斯、安德鲁斯和恩斯特。他们还建立在这样一个事实之上：此类研究需要复杂且昂贵的设备，而不是可在各个实验室获得。 PIL Core 旨在提供有关身份的信息通过质谱分析蛋白质，或者作为在研究人员实验室纯化的单个蛋白质，通常通过凝胶电泳分离为条带或斑点，或者总体上作为蛋白质混合物分离蛋白质复合物或细胞器。还提供了有关翻译后修饰的信息蛋白质和蛋白质含量的定量变化。一旦蛋白质被识别，Core 就可以提供有关活细胞和固定细胞中蛋白质的定位及其随时间变化的信息以及对生理和病理生理扰动的反应。它以微观为中心主要通过激光扫描共焦获得的数字信息的成像和定量分析显微镜（LSCM）和多波长荧光成像，尽管传统的电子固定和嵌入标本的显微镜检查也可用于精细结构分析。现代生物医学研究的特点是跨学科性和依赖性日益复杂的仪器仪表和信息学。虽然目标是观察组织，疾病或有机体作为一个复杂的集成系统，我们仍在发展对疾病的理解组件的结构和功能通常依赖于细胞类型。随着映射的随着基因组的出现，注意力已经转移到更复杂的蛋白质组世界。完整库存大多数哺乳动物蛋白质复合物或细胞器尚未完成，它们通常在细胞类型、细胞周期和发育的各个阶段。从而识别出蛋白质、它们的翻译后修饰、动态相互作用和细胞定位，全部及时和空间并以定量的方式，是一项艰巨的任务，几乎所有 GI 的实验室科学家都肽中心在他们的研究中遇到了。这些问题可以通过以下工具来解决 PIL 核心，通常与分子生物学核心和国家基因组资源相结合例如 NCBI。新的 PIL 核心是围绕主任实验室的专业知识以及可用的三个现有设施（密歇根蛋白质组核心、形态学）拥有先进的仪器细胞与发育生物学系和密歇根大学成像实验室 (MIL) 糖尿病研究和培训中心 (MDRTC)，其维护的设施将在下面详细讨论复杂的质谱分析和高分辨率显微镜。这项安排是由事实上，安德鲁斯博士指导密歇根蛋白质组核心，威廉姆斯博士指导显微镜和 MDRTC 的图像分析实验室和 Ernst 博士与 MIL 有着长期的合作关系。全部三个实体已建立人员和补给结构，目前正在由 GI 使用中心成员。虽然蛋白质识别和定位核心将利用现有的高度复杂的它也是围绕 90 多年的蛋白质组学、细胞生物学和成像技术而构建的博士的专业知识。 Williams、Andrews 和 Ernst 主要致力于胃肠道组织。经验丰富的核心员工，包括博士。沃克、斯特拉勒和尼尔森先生将直接协助中心调查人员并执行核心程序。所有核心人员将进行演示和教学向 GI 中心研究人员、实习生和技术人员提供技术。最后，核心的另一个使命是发起、实施和传播蛋白质组学和细胞成像领域的新技术和创新技术。