PROTEIN IDENTIFICATION AND LOCALIZATION CORE

蛋白质鉴定和定位核心

• 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
• 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

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）核心是一个重组的核心， 前两个核心的方面，细胞生物学和细胞成像核心，重点是细胞 成像和肽和蛋白质组学核心，它有双重服务，提供合成 肽和通过质谱鉴定蛋白质。虽然细胞生物学方法现在 大多数实验室的标准，肽合成可以从大学和 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。新的PIL核心结构保留并增强了这些方面 并建立在威廉姆斯博士、安德鲁斯博士和恩斯特博士之间长期的合作互动基础上。 他们还建立在这样一个事实上，即这种类型的研究需要复杂和昂贵的设备， 可在个别实验室使用。PIL核心旨在提供有关身份的信息， 蛋白质，或者作为在研究者的实验室中纯化的单个蛋白质， 通常通过凝胶电泳分离为条带或斑点，或者作为蛋白质的混合物在凝胶电泳中整体分离。 蛋白质复合物或细胞器。还提供了关于蛋白质的翻译后修饰的信息。 蛋白质和蛋白质含量的定量变化。一旦蛋白质被识别，核心可以 提供活细胞和固定细胞中蛋白质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响应。它以微观为中心 主要通过激光扫描共聚焦获得的数字信息的成像和定量分析 虽然传统的电子显微镜（LSCM）和多波长荧光成像， 固定和包埋样品的显微镜也可用于精细结构分析。 现代生物医学研究的特点是它的跨学科性质和依赖性 越来越复杂的仪器和信息学。虽然目标是观察组织， 疾病，或有机体作为一个复杂的综合系统，我们仍然在发展的理解， 这些组分的结构和功能通常依赖于细胞类型。通过映射 随着基因组的发展，人们的注意力已经转移到更复杂的蛋白质组世界。量的完整清单 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，在细胞周期中，以及在发育的各个阶段。因此， 蛋白质，它们的翻译后修饰，动态相互作用和细胞定位， 和空间，并以定量的方式，是一项艰巨的任务，几乎所有的实验室科学家在GI 肽中心在他们的研究中遇到。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 如NCBI。 新的PIL核心是围绕主任实验室的专业知识而建立的， 在三个现有的设施，密歇根州蛋白质组核心，形态复杂的仪器 和成像实验室（MIL）的细胞和发育生物学系和密歇根大学 糖尿病研究和培训中心（MDRTC），该中心维护以下详细讨论的设施， 精密的质谱和高分辨率显微镜。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， MDRTC的图像分析实验室和Ernst博士与MIL有着长期的联系。所有 三个实体已经建立了人员和补给结构，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 仪器，它也是建立在超过90年的蛋白质组学，细胞生物学和成像 威廉姆斯、安德鲁斯和恩斯特博士的专业知识，主要致力于胃肠道组织。 经验丰富的核心员工，包括步行者博士、Strahler和纳尔逊先生，将直接协助中心 调查员并执行核心程序。所有核心人员将进行演示和教学 技术GI中心的研究人员，学员和技术人员。最后，内核的另一个使命是 发起、实施和传播蛋白质组学和细胞成像方面的新技术和创新技术。