Quantitative sub-cellular acyl-CoA analysis reveals distinct nuclear regulation

Metabolism is highly compartmentalized within cells, and the sub-cellular distribution of metabolites determines their use. Quantitative sub-cellular metabolomic measurements can yield crucial insights into the roles of metabolites in cellular processes. Yet, these analyses are subject to multiple confounding factors in sample preparation. We developed table Isotope abeling of ssential nutrients in cell ulture - ub-cellular ractionation (SILEC-SF), which uses rigorous internal standard controls that are present throughout fractionation and processing to quantify metabolites in sub-cellular compartments by liquid chromatography-mass spectrometry (LC-MS). Focusing on the analysis of acyl-Coenzyme A thioester metabolites (acyl-CoAs), SILEC-SF was tested in a range of sample types from cell lines to mouse and human tissues. Its utility was further validated by analysis of mitochondrial versus cytosolic acyl-CoAs in the well-defined compartmentalized metabolic response to hypoxia. We then applied the method to investigate metabolic responses in the cytosol and nucleus. Within the cytosol, we found that the mevalonate pathway intermediate 3-Hydroxy-3-methylglutaryl-CoA (HMG-CoA) is exquisitely sensitive to acetyl-CoA supply. The nucleus has been an exceptionally challenging compartment in which to quantify metabolites, due in part to its permeability. We applied the SILEC-SF method to nuclei, identifying that the nuclear acyl-CoA profile is distinct from the cytosolic compartment, with notable nuclear enrichment of propionyl-CoA. Altogether, we present the SILEC-SF method as a flexible approach for quantitative sub-cellular metabolic analyses.