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）的目标是提供高水平的 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 提供四项服务。1)线粒体的高分辨率成像。这将包括 分析特定蛋白质重新分布到线粒体、线粒体网络重塑和标志物 线粒体功能（线粒体衍生的活性氧，线粒体膜 电位、肉毒碱代谢物、ATP水平）。2)生物能量学：耗氧量，糖酵解速率，脂肪 酸氧化，细胞ATP动力学测定，机械应力下ETC复合物分析 挑战细胞3)生物样品中PTM的分析。将纯化单个蛋白质以进行含量测定 体内PTM。为了分析PTM，我们将利用质谱法结合检测， 通过串联质谱法对修饰的特定位点进行定量。4)代谢组学分析 在培养细胞、肺组织和来自动物和患有先天性心脏病的儿童的血浆中的单个代谢物 导致肺血流（PBF）变化的先天性心脏病。核心B将增强 通过确保所有线粒体、PTM和代谢组学分析， 是标准化的，并以统一的标准化方式执行，并由经验丰富， 训练有素的人员。核心B由具有多年经验的经验丰富的调查人员管理 使用这些技术。他们已经开发或改造了其中的许多技术，并将其应用于 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 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.