Protein Chemistry & Biophysics Core

蛋白质化学

• 批准号: 10094245
• 负责人: Stephen M Black
• 金额: $ 19.2万
• 依托单位: UNIVERSITY OF ARIZONA
• 依托单位国家: 美国
• 财政年份: 2018
• 资助国家: 美国
• 起止时间: 2018-02-05 至 2021-04-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/10094245
• 关键词: Adult Respiratory Distress Syndrome; Arizona; Atomic Force Microscopy; Biological; Biological Assay; Cells; Cellular Morphology; Coupled; Cytoskeletal Modeling; Detection; Development; Elasticity; Endothelial Cells; Endothelium; Epigenetic Process; Equipment; Evaluation; Functional disorder; Generations; Genetic; Immunoprecipitation; Inflammation; Laboratories; Mass Spectrum Analysis; Measurement; Mechanical Stress; Mechanics; Mediating; Modification; Post-Translational Protein Processing; Production; Property; Protective Agents; Protein Analysis; Protein Chemistry; Proteins; Receptor Signaling; Recombinant Proteins; Research Personnel; Role; SOX18 gene; Sampling; Services; Site; Sphingosine-1-Phosphate Receptor; Stretching; Structure; Surface Plasmon Resonance; Techniques; Technology; Tissues; Universities; Ventilator-induced lung injury; biophysical chemistry; experience; experimental study; in vivo; insight; mechanical properties; novel; protein protein interaction; receptor; receptor binding; response; tandem mass spectrometry

ABSTRACT: The generation of purified proteins, assessment of protein post-translational modifications (PTMs), protein-protein interactions and evaluation of cellular mechanical properties are integral to the successful completion of the Specific Aims proposed in all three PPG projects. The Protein Chemistry & Biophysics Core will provide services that will support these analyses in all of the PPG Projects. The Specific Aims of Core D are: 1) Generation of purified recombinant proteins; 2) Analysis of protein PTMs in mechanical stress- challenged cells and tissues; 3) Analysis of protein-protein interactions; 4) Analysis of changes in micromechanical properties of endothelial cells (EC) induced by barrier modifying agents. All three Projects will utilize recombinant proteins for PTM and protein interaction analyses. Proteins generated will be available for each project and for “in core” use. Additionally, in this aim, proteins from biological samples will be purified to assay PTMs and protein-protein interactions in vivo. To analyze PTMs, we will utilize a mass spectrometry approach coupled to the detection and quantification of specific sites of modification by tandem mass spectrometry. To enhance the analysis of protein-protein interactions (interactome) required in all three Projects, Core D will utilize surface plasmon resonance (SPR) techniques. In vivo measurements of protein- protein interactions will also be validated using immunoprecipitation–mass spectrometry identification approaches. Core D will also utilize atomic force microscopy (AFM) to evaluate structural and elasticity effects in the endothelium to enable investigators to define the relationship between cell mechanics and cytoskeletal reorganization in response to mechanical stretch and the effects of barrier protective agents. Core D leaders have considerable experience with all of the proposed techniques.

摘要： 纯化蛋白质的生成，蛋白质翻译后修饰（PTM）的评估， 蛋白质-蛋白质相互作用和细胞机械性能的评估是成功的 完成所有三个PPG项目中提出的具体目标。蛋白质化学与生物物理学核心 将在所有PPG项目中提供支持这些分析的服务。核心D的具体目标 是：1）产生纯化的重组蛋白; 2）在机械应力下分析蛋白质PTM- 攻击的细胞和组织; 3）蛋白质-蛋白质相互作用的分析; 4）蛋白质-蛋白质相互作用的变化的分析。 微机械性能的内皮细胞（EC）诱导的屏障改性剂。所有三 项目将利用重组蛋白进行PTM和蛋白质相互作用分析。产生的蛋白质将 可供每个项目和"核心"使用。此外，在这个目标中，来自生物样品的蛋白质将被 纯化以测定体内PTM和蛋白质-蛋白质相互作用。为了分析PTM，我们将利用质量 通过串联质谱法结合特定修饰位点的检测和定量 质谱分析法来为了加强对蛋白质-蛋白质相互作用（相互作用组）的分析， 项目，核心D将利用表面等离子体共振（SPR）技术。蛋白质的体内测量- 还将使用免疫沉淀-质谱鉴定法验证蛋白质相互作用 接近。核心D还将利用原子力显微镜（AFM）来评估结构和弹性效应 使研究人员能够确定细胞力学和细胞骨架之间的关系， 重组响应于机械拉伸和屏障保护剂的作用。核心D领导人 对所有提出的技术都有相当丰富的经验。