Mass Spectrometry Based Subcellular Coenzyme Analysis

Abstract

Metabolism in eukaryotes relies on compartmentalization of processes between sub‐cellular compartments. Our objective was to develop, test, and apply methods that can quantitatively measure families of metabolites within distinct sub‐cellular compartments in eukaryotic cells. We created Stable Isotope Labeling of Essential nutrients in Cell culture ‐ Subcellular Fractionation (SILEC‐SF) with the essential precursors of the major cellular coenzymes, Coenzyme A and NAD to incorporate a 13C,15N‐label into the families of each coenzyme present within cells. Using multiple fractionation techniques coupled to liquid chromatography‐high resolution mass spectrometry we quantify distinct cytoplasmic, mitochondrial, and nuclear pools within eukaryotic cells. We successfully applied these methods to cells and human tissue demonstrating distinct compartmental metabolic changes by pathway in genetic models of compartmentalized metabolism, in adipocyte differentiation and in changing oxygen tension. This confirmed orthogonal measurements of subcellular metabolism but revealed unexpected localizations and enrichments of certain metabolite pools.