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.
High resolution imaging of the mitochondria, analyses of cell metabolism and PTMs as well as cell, tissue, and plasma metabolites are integral to the success of each of the PPG Projects. These services are best centralized given their inherent difficulty, reliance on specialized and sophisticated equipment and expertise, as well as the requirements of all three projects. Core B will provide these services for all three projects.
