Absolute protein quantification allows differentiation of cell-specific metabolic routes and functions.

Total protein approach (TPA) is a proteomic method that 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. All MS data have been deposited in the ProteomeXchange with identifier PXD001352 (http://proteomecentral.proteomexchange.org/dataset/PXD001352).