Total protein approach (TPA) is a proteomic method which allows calculation of concentrations of individual proteins and groups of functionally related proteins in any protein mixture without spike-in standards. Using the two step digestion filter aided sample preparation method and LC-MS/MS analysis we generated comprehensive quantitative datasets of mouse intestinal mucosa, liver, red muscle fibers, brain, and of human plasma, erythrocytes, and tumor cells lines. We show that the TPA based quantitative data reflect well-defined and specific physiological functions of different organs and cells, for example nutrient absorption and transport in intestine, amino acid catabolism and bile secretion in liver, and contraction of muscle fibers. Focusing on key metabolic processes we compared metabolic capacities in different tissues and cells. In addition, we demonstrate quantitative differences in the mitochondrial proteomes. Providing insight into the abundances of mitochondrial metabolite transporters we demonstrate that their titers are well tuned to cell specific metabolic requirements. This study provides for the first time a comprehensive overview of the protein hardware mediating metabolism in different mammalian organs and cells. The presented approach can be applied to any other system to study biological processes.