Core B: Proteomics and Protein Analysis Core

项目摘要 核心B将为PPG项目提供蛋白质组学和蛋白质分析服务。在最近的融资中， 在一个周期内，核心B的蛋白质分析组件已显著扩展，以满足个人的需求 项目在PPG。除了基于质谱（MS）的足迹技术用于绘制 对于先前申请中提出的蛋白质-蛋白质和蛋白质-核酸相互作用，我们增加了 以下新服务的核心：1）AKTA快速蛋白质液相色谱（FPLC），其中发挥了 在获得纯化的重组蛋白用于生物化学/生物物理表征中的关键作用 项目3和原项目2的抗体生产;以及2）EnSpire多模式酶标仪，以提供 一种高灵敏度、快速和稳健的蛋白质-蛋白质和蛋白质-核酸监测的新服务 交互.基于MS的蛋白质组学方法使我们能够识别新的蛋白质-蛋白质相互作用， 翻译后修饰，因此有助于推进以前的项目的研究 1和2中所示。上述技术将继续发挥重要作用， 在这次竞争性更新中提出的PPG项目的研究目标。特别是， 蛋白质组学和蛋白质分析核心是：目标1：进行基于MS的蛋白质足迹实验， 在大的生物相关核蛋白和蛋白质-蛋白质的背景下鉴定接触氨基酸 目标2：开发EnSpire多模式酶标仪在生化分析中的新应用 使用均相时间分辨的蛋白质-核酸和蛋白质-蛋白质相互作用的表征 基于荧光能量转移（HTRF）、AlphaLISA和AlphaScreen（ALPHA）的技术;目标3： 使用AKTA-FPLC提供柱色谱服务，以协助各个PPG项目分离低浓度的 丰富的天然蛋白质-蛋白质复合物从哺乳动物细胞和纯化mg量的难溶性 重组E.大肠杆菌进行后续的生物化学和生物物理表征;目的4： 进行基于MS的蛋白质组学实验，以确定相互作用的伙伴和蛋白质翻译后 修改.总的来说，核心B将提供广泛的蛋白质分析服务。重要的是， 这些服务将用于所有四个项目编制小组项目。

PROJECT SUMMARY Core B will provide proteomics and protein analysis services for PPG projects. Throughout the last funding cycle, the protein analysis component of Core B has significantly expanded to meet the needs of the individual projects in the PPG. In addition to mass spectrometry (MS)-based footprinting technology for the mapping of protein-protein and protein-nucleic acid interactions proposed in the previous application, we have added the following new services to the Core: 1) the AKTA-fast protein liquid chromatography (FPLC), which has played a critical role in obtaining purified recombinant proteins for biochemical/biophysical characterization for former Project 3 and antibody production for former Project 2; and 2) the EnSpire multimode plate reader to provide a new service for highly sensitive, rapid and robust monitoring of protein-protein and protein-nucleic acid interactions. MS-based proteomic approaches enabled us to identify new protein-protein interactions and posttranslational modifications, and have thus been instrumental for advancing the research of former Projects 1 and 2, respectively. The above technologies will continue to play an important role in accomplishing the research objectives of the PPG projects proposed in this competitive renewal. In particular, Specific Aims of the Proteomics and Protein Analysis Core are: Aim 1: to perform MS-based protein footprinting experiments to identify contact amino acids in the context of large biologically relevant nucleoprotein and protein-protein complexes; Aim 2: to develop novel applications of the EnSpire multimode plate reader for biochemical characterization of protein-nucleic acid and protein-protein interactions using homogeneous time-resolved fluorescence energy transfer (HTRF), AlphaLISA, and AlphaScreen (ALPHA) based technologies; Aim 3: to provide a column chromatography service using AKTA-FPLC to assist individual PPG projects to isolate low abundant native protein-protein complexes from mammalian cells and purify mg quantities of poorly soluble recombinant proteins from E. coli for their subsequent biochemical and biophysical characterization; Aim 4: to conduct MS-based proteomics experiments to identify interacting partners and protein posttranslational modifications. Taken together, Core B will provide the wide range of protein analysis services. Importantly, these services will be utilized by all four PPG projects.