Mitochondrial Imaging, PTM Analysis and Metabolism Core

摘要 线粒体成像、PTM分析和代谢核心(核心B)的目标是提供高- 优质的最先进的服务，以支持所有三个PPG项目的分析。在这种情况下，核心B将 通过提供四项服务来定义。1)线粒体高分辨率成像。这将包括 线粒体特异性蛋白质重分布、线粒体网络重构和标志物的分析 线粒体功能(线粒体衍生的活性氧物种、线粒体膜 潜力、肉碱代谢物、三磷酸腺苷水平)。2)生物能量学：耗氧量、糖酵解率、脂肪 机械应力中的酸氧化、细胞ATP动力学测定等综合分析 挑战细胞。3)生物样品中PTMS的分析。单独的蛋白质将被提纯以进行检测 体内的PTMS。为了分析PTMS，我们将利用质谱学方法结合检测和 串联质谱法对修饰的特定部位进行定量。4)新陈代谢分析 来自动物和儿童的培养细胞、肺组织和血浆中的个别代谢物 导致肺血流量(PBF)改变的先天性心脏病。核心B将加强 通过确保所有线粒体、PTM和代谢组学分析 都是标准化的，并以统一标准化的方式执行，并由经验丰富的 训练有素的人员。核心B由有多年经验的有经验的调查人员负责 使用这些技术。他们已经开发或调整了其中的许多技术，并将它们应用于 在体外和体内系统中进行生理学研究。作为中央专用分析资源核心 B将确保以具有成本效益的方式进行这些分析。

ABSTRACT The goal of the Mitochondrial Imaging, PTM analysis, and Metabolism Core (Core B) is to provide a high- quality state-of-the art service to support analyses in all three of the PPG Projects. In this context, Core B will be defined by providing four services. 1) High resolution imaging of mitochondria. This will include analyses of specific protein redistribution to mitochondria, mitochondrial network remodeling, and markers of mitochondrial function (mitochondrial derived reactive oxygen species, mitochondrial membrane potential, carnitine metabolites, ATP levels). 2) Bioenergetics: oxygen consumption, rate of glycolysis, fatty acid oxidation, measurement of cellular ATP kinetics, ETC complex analyses in mechanical stress challenged cells. 3) The analysis of PTMs in biological samples. Individual proteins will be purified to assay PTMs 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. 4) Metabolomic analyses of individual metabolites in cultured cells, lung tissue, and plasma from animals and children born with congenital heart disease that results in changes in pulmonary blood flow (PBF). Core B will enhance the scientific work for all three projects by assuring that all mitochondrial, PTM, and metabolomic analyses are standardized and performed in a uniform standardized manner, and by highly experienced and trained personnel. Core B is run by experienced investigators who have had many years of experience using these techniques. They have developed or adapted many of these techniques and applied them to perform physiological studies in in vitro and in vivo systems. As a central dedicated analytical resource Core B will ensure these analyses are carried out in a cost-efficient manner.