Proteomics/Metabolism Core

项目总结 ‘蛋白质组学和新陈代谢组学’核心C将由比尔吉特·希林博士管理，他是一名独立的 巴克研究所的教员。核心C将为衰老相关样本提供最先进的分析 从所有三个项目中。希林博士也是巴克大学质谱学(MS)设施的主任 将领导蛋白质组学和代谢组学核心，并提供所有基础设施，协调MS 对所有参与的项目领导和调查人员进行采购和培训。核心设备非常齐全 共有七个现代质谱学系统和四个质谱仪专门用于 蛋白质组学研究和工作流程。核心C的目标是提供跨每个组件的公共平台 设计、执行和分析实验的项目，这些实验涉及到关键的分泌分子 衰老细胞产生的信号调节因子以及对AD模型的后续影响，包括诱导 多能干细胞(IPS)和类器官。为了实现本计划项目的目标，Core C将致力于 努力做以下工作：i)确定衰老相关分泌表型中存在的蛋白质 (SASP)来自大脑特定的衰老细胞，II)测定SASP和来自 衰老的星形胶质细胞和小胶质细胞或衰老样神经元及其对神经元的影响，III)研究 来自IPSC AD模型的有机物和共培养物，IV)分析代谢物并测量NAD水平 最后v)决定tau翻译后修饰的动态变化。核心C 专门创新、开发和实施先进的蛋白质分析技术，包括定量 蛋白质组学、蛋白质分泌体和外切体的测定以及监测疾病的PTM状态 蛋白质，以促进基础生物学和生物医学研究。为了支持这个PPG的项目和目标， Core C将提供各种不同的质谱学工作流程，包括依赖数据的采集 (DDA)用于蛋白质发现和鉴定，以及与数据无关的获取(DIA或SWATH) SASP和细胞内蛋白质组的同步准确相对综合采样 量化。我们将使用我们的正交四极飞行时间质谱仪，TripleTOF 6600 和5600(SCIEX)。我们还订购了一台Orbitrap Eclipse Tribrid(Thermo)质谱计，使 以非常高的分辨率、灵敏度、动态范围进行深入的蛋白质组覆盖，还提供了出色的 多路复用功能。我们的专业知识和仪器也特别适合分析SASP 在衰老过程中可能会受到动态影响。持续的系统适宜性和质量控制分析 以提供最高水平的数据重复性和科学严谨性。希林博士的实验室和C核心 将为数据分析和生物信息/统计处理提供所有必要的基础设施和专业知识 结果的结果。我们将与Core A的生物信息学专家密切互动，进行多组学系统范围的分析。

PROJECT SUMMARY The ‘Proteomics and Metabolomics’ Core C will be under the administration of Dr. Birgit Schilling, an independent faculty member at the Buck Institute. Core C will provide state-of-the-art analysis for senescence-related samples from all three Projects. Dr. Schilling, who is also the Director of the Mass Spectrometry (MS) Facility at the Buck Institute, will direct the Proteomics and Metabolomics Core and provide all infrastructure, coordination of MS acquisitions, and training for all participating project leaders and investigators. The Core is very well-equipped with a total of seven modern mass spectrometric systems, and four mass spectrometers are dedicated to proteomics research and workflows. The objective of Core C is to provide a common platform across each Project for the design, execution and analysis of experiments that involve secreted molecules that are key signaling regulators generated by senescent cells and subsequent effects on our AD models, including induced pluripotent stem cells (iPS) and organoids. To achieve the goals of this Program Project, Core C will devote its efforts to the following work: i) determine proteins present in the senescence-associated secretory phenotype (SASP) from brain specific senescent cells, ii) determine the components of SASP and exosomes from senescent astrocytes and microglia or senescent-like neurons and their effects on neurons, iii) investigate organoids and co-cultures from iPSC models of AD, iv) analyze metabolites and measure NAD levels of senescent cells, and finally v) determine dynamic changes of post-translational modifications of tau. Core C specifically innovates, develops, and implements advanced protein analytical technologies, including quantitative proteomics, determination of protein secretomes and exosomes, as well as monitoring the PTM status of disease proteins, to advance basic biological and biomedical research. To support the projects and aims of this PPG, Core C will offer a variety of different mass spectrometric workflows, including data-dependent acquisitions (DDA) for protein discovery and identification, and data-independent acquisitions (DIA or SWATH) for comprehensive sampling of the SASP and intracellular proteomes with simultaneous accurate relative quantification. We will use our orthogonal Quadrupole Time-of-Flight mass spectrometers, the TripleTOF 6600 and 5600 (SCIEX). We also just ordered an Orbitrap Eclipse Tribrid (Thermo) mass spectrometer enabling very deep proteome coverage at very high resolution, sensitivity, dynamic range and also offering excellent multiplexing capabilities. Our expertise and instrumentation are also particularly well suited for analysis of SASP that may be dynamically affected during senescence. Continuous system suitability and quality control assays are acquired to provide highest levels of data reproducibility and scientific rigor. Dr. Schilling’s lab and Core C will provide all necessary infrastructure and expertise for data analysis and bioinformatic/statistical processing of the results. We will closely interact with bioinformatics experts in Core A for multi-omics system-wide analysis.