Quantitative sub-cellular acyl-CoA analysis reveals distinct nuclear regulation [article]

Sophie Trefely, Katharina Huber, Joyce Liu, Jay Singh, Mary Doan, Claudia D. Lovell, Michael Noji, Eliana von Krusenstiern, Helen Jiang, Anna Bostwick, Luke Izzo, Steven Zhao (+6 others)
2020 bioRxiv   pre-print
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 Stable Isotope Labeling of Essential nutrients in cell Culture - Sub-cellular Fractionation (SILEC-SF), which uses rigorous internal standard
more » ... nternal 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.
doi:10.1101/2020.07.30.229468 fatcat:ajvcrnv5t5bplp536awhpuwbn4