Protein Chemistry & Biophysics Core

摘要： 纯化蛋白质的产生，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不可或缺的一部分 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的核心 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 1)纯化重组蛋白的制备；2)机械应力下蛋白质PTM的分析。 挑战细胞和组织；3)分析蛋白质-蛋白质相互作用；4)分析 屏障修饰剂诱导内皮细胞的微观力学特性。三个都是 项目将利用重组蛋白质进行PTM和蛋白质相互作用分析。产生的蛋白质将是 可用于每个项目和“核心”使用。此外，在这个目标中，生物样本中的蛋白质将被 纯化后用于体内PTMS和蛋白质-蛋白质相互作用的检测。为了分析PTMS，我们将利用质量 串联检测和定量特定修饰位点的光谱分析方法 质谱学。为了增强对所有三种方法中所需的蛋白质-蛋白质相互作用(交互作用组)的分析 项目中，核心D将利用表面等离子激元共振(SPR)技术。蛋白质的活体测量- 蛋白质的相互作用也将通过免疫沉淀-质谱学鉴定来验证。 接近了。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 机械拉伸反应的重组和屏障保护剂的影响。核心D领导者 对所有提议的技术都有相当的经验。

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.