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Quantitative sub-cellular metabolomic measurements can yield crucial insights into the roles of metabolites in cellular processes, but are subject to multiple confounding factors. We developed Stable Isotope Labeling of Essential nutrients in cell Culture - Sub-cellular Fractionation (SILEC-SF), which uses isotope labeled internal standard controls that are present throughout fractionation and processing to quantify acyl-Coenzyme A thioesters in sub-cellular compartments by liquid chromatography-mass spectrometry. We tested SILEC-SF in a range of sample types and examined the compartmentalized responses to oxygen tension, cellular differentiation, and nutrient availability. Application of SILEC-SF to the challenging analysis of the nuclear compartment revealed a nuclear acyl-CoA profile distinct from that of the cytosol, with notable nuclear enrichment of propionyl-CoA. Using isotope tracing we identified the branched chain amino acid (BCAA) isoleucine as a major metabolic source of nuclear propionyl-CoA and histone propionylation, thus revealing a new mechanism of crosstalk between metabolism and the epigenome.

Summary 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 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.

Summary Quantitative subcellular metabolomic measurements can explain the roles of metabolites in cellular processes but are subject to multiple confounding factors. We developed stable isotope labeling of essential nutrients in cell culture-subcellular fractionation (SILEC-SF), which uses isotope-labeled internal standard controls that are present throughout fractionation and processing to quantify acyl-coenzyme A (acyl-CoA) thioesters in subcellular compartments by liquid chromatography-mass spectrometry. We tested SILEC-SF in a range of sample types and examined the compartmentalized responses to oxygen tension, cellular differentiation, and nutrient availability. Application of SILEC-SF to the challenging analysis of the nuclear compartment revealed a nuclear acyl-CoA profile distinct from that of the cytosol, with notable nuclear enrichment of propionyl-CoA. Using isotope tracing, we identified the branched chain amino acid isoleucine as a major metabolic source of nuclear propionyl-CoA and histone propionylation, thus revealing a new mechanism of crosstalk between metabolism and the epigenome.

Metabolism of polyunsaturated fatty acids results in the formation of hydroxylated fatty acids that can be further oxidized by dehydrogenases, often resulting in the formation of electrophilic, α,β-unsaturated ketone containing fatty acids. As electrophiles are associated with redox signaling, we sought to investigate the metabolism of the oxo-fatty acid products in relation to their double bond architecture. Using an untargeted liquid chromatography mass spectrometry approach, we identified mono- and di-saturated products of the arachidonic acid-derived 11-oxoeicosatetraenoic acid (11-oxoETE) and mono-saturated metabolites of 15-oxoETE and docosahexaenoic acid-derived 17-oxodocosahexaenoinc acid (17-oxoDHA) in both human A549 lung carcinoma and umbilical vein endothelial cells. Notably, mono-saturated oxo-fatty acids maintained their electrophilicity as determined by nucleophilic conjugation to glutathione while a second saturation of 11-oxoETE resulted in a loss of electrophilicity. These results would suggest that prostaglandin reductase (PTGR1), known for its reduction of the α,β-unsaturated double bond, was not responsible for the saturation of oxo-fatty acids. Surprisingly, knockdown of PTGR1 expression by shRNA confirmed its participation in the formation of 15-oxoETE and 17-oxoDHA mono-saturated metabolites. Furthermore, overexpression of PTGR1 in A549 cells increased the rate and total amount of oxo-fatty acid saturation. These findings will further facilitate the study of electrophilic fatty acid metabolism and signaling in the context of inflammatory diseases and cancer where they have been shown to have anti-inflammatory and anti-proliferative signaling properties.HighlightsPrimary saturation of electrophilic fatty acids does not abolish biological activity.Prostaglandin reductase 1 reduces double bonds in fatty acids that are structurally similar to 15-keto-prostaglandin E2.Prostaglandin reductase 1 reduces non-carbonyl adjacent double bonds.

Quantification of cellular deoxyribonucleoside mono-(dNMP), di-(dNDP), triphosphates (dNTPs) and related nucleoside metabolites are difficult due to their physiochemical properties and widely varying abundance. Involvement of dNTP metabolism in cellular processes including senescence and pathophysiological processes including cancer and viral infection make dNTP metabolism an important bioanalytical target. We modified a previously developed ion pairing reversed phase chromatography-mass spectrometry method for the simultaneous quantification and 13C isotope tracing of dNTP metabolites. dNMPs, dNDPs, and dNTPs were chromatographically resolved to avoid mis-annotation of in-source fragmentation. We used commercially available 13C15N-stable isotope labeled analogs as internal standards and show that this isotope dilution approach improves analytical figures of merit. At sufficiently high mass resolution achievable on an Orbitrap mass analyzer, stable isotope resolved metabolomics allows simultaneous isotope dilution quantification and 13C isotope tracing from major substrates including 13C-glucose. As a proof of principle, we quantified dNMP, dNDP and dNTP pools from multiple cell lines. We also identified isotopologue enrichment from glucose corresponding to ribose from the pentose-phosphate pathway in dNTP metabolites.