Scientific Core 2:Mass Spectrometry and Biophysics

科学核心2（SC2）。项目总结/摘要 质谱和生物物理方法在本计划项目各个方面的应用是 这个研究项目的独特之处科学核心2（SC2）的目的是提供质量 光谱和生物物理支持的研究人员在程序项目。的主要目标 SC2旨在提供可靠的、最先进的技术，用于准确和可重现的 大分子相互作用和蛋白质物理性质。为了实现这一目标，核心服务于 所有三个项目的共同需要，研究蛋白质特性，构象变化， 蛋白质相互作用第二个主要目的是质谱检测和定量 小分子，包括生物活性的氧化脂质和氧化氨基酸，它们是氧化脂质的报告分子。 流程. SC2是分析、结构和建模/计算方面的集中专业领域 生物物理学核心不仅提供了仪器和试剂，更重要的是提供了 在建立方法、分析和解释结果以及保证质量方面的经验和专业知识 控制核心由两个主要的子组件组成，它们具有不同但高度互补的方法， 方法：质谱分析子组件和生物物理学子组件。SC2将提供 用于通过表面等离子体共振光谱法检测直接相互作用的专业知识和试剂， 报告了结合的结合和解离速率以及解离常数KD的准确测定。这 方法是补充等温滴定量热法的热力学特性， 大分子组装体SC2的质谱分析子组件将有助于识别 通过氢-氘交换与质谱联用（HDX-MS）分析蛋白质-蛋白质相互作用位点， 一个强大的工具，也提供了蛋白质动力学的信息。这一方法得到两个补充 生物物理子组件提供的方法-福斯特共振能量转移和系留 人工蛋白酶程序（“Fe-BABE”）。核心提供蛋白质建模及其 使用已知结构，蛋白质-蛋白质对接方法和分子动力学模拟的相互作用。 质谱法在蛋白质组学分析中提供了一种强有力的发现工具，可用于鉴定蛋白质 以及翻译后修饰。替代质谱仪器将用于 检测和定量小分子，包括生物活性氧化脂质和修饰氨基酸 它们是氧化应激的特异性报告者。SC2将提供多种服务，包括 试剂，协助探针设计和构建，实验设计和执行，以及数据 分析和解释结果。教育部分是这一核心的额外力量。所有 SC2提供的方法需要高度专业化的仪器、技术专长和经验， 通常可在现代细胞或分子生物学实验室中获得;此外，一些方法需要 不熟悉的计算分析方法。因此，质谱和生物物理学核心提供了关键的 为项目的成功所必需的能力。

Scientific Core 2 (SC2). Project Summary/Abstract The application of mass spectrometry and biophysical approaches to various facets of this Program Project is a unique aspect of this research program. The purpose of Scientific Core 2 (SC2) is to provide both mass spectrometric and biophysical support for the investigators within the Program Project. A principal objective of SC2 is to make available robust, state-of-the-art techniques for accurate and reproducible measurement of macromolecular interactions and protein physical properties. Addressing this objective, the Core serves the common need of all three Projects for investigation of protein characteristics, conformational changes, and protein-protein interactions. A second principal objective is mass spectrometric detection and quantification of small molecules, including bioactive, oxidized lipids and oxidized amino acids that are reporters of oxidative processes. SC2 serves as a centralized area of expertise in analytical, structural, and modeling/computational biophysics. The Core provides not only access to instrumentation and reagents, but more importantly provides experience and expertise in setting up the methods, analyzing and interpreting the results, and assuring quality control. The Core consists of two major sub-components with different but highly complementary methods and approaches: a Mass Spectrometry Sub-component and a Biophysics Sub-component. SC2 will provide expertise and reagents for detection of direct interaction by surface plasmon resonance spectrometry that reports accurate determinations of on- and off-rates of binding, as well as dissociation constants, KD. This method is complemented by isothermal titration calorimetry for thermodynamic characterization of macromolecular assemblies. The Mass Spectrometry Sub-component of SC2 will facilitate identification of protein-protein interaction sites by hydrogen-deuterium exchange coupled with mass spectrometry (HDX-MS), a powerful tool that also provides information on protein dynamics. This approach is supplemented by two methods provided by the Biophysics Sub-component - Förster resonance energy transfer and the tethered artificial protease procedure (“Fe-BABE”). The Core provides expertise in modeling of proteins and their interactions using known structures, protein-protein docking methods, and molecular dynamics simulations. Mass spectrometry provides a powerful discovery tool in proteomic analysis useful in identification of proteins as well as post-translational modifications. Alternative mass spectrometry instrumentation will be used for detection and quantification of small molecules, including bioactive oxidized lipids and modified amino acids that are specific reporters of oxidative stress. SC2 will provide a diversity of services including provision of reagents, assistance in probe design and construction, design and performance of experiments, and data analysis and interpretation of results. An educational component is an additional strength of this Core. All methods provided by SC2 require highly specialized instrumentation, technical expertise, and experience not generally available in a modern cell or molecular biology laboratory; additionally, some methods require unfamiliar computational analysis methods. Thus, the Mass Spectrometry and Biophysics Core provides critical capabilities essential for the success of the Program Project.