High-Resolution Metabolomics with Acyl-CoA Profiling Reveals Widespread Remodeling in Response to Diet

The availability of acyl-Coenzyme A (acyl-CoA) thioester compounds affects numerous cellular functions including autophagy, lipid oxidation and synthesis, and post-translational modifications. Consequently, the acyl-CoA level changes tend to be associated with other metabolic alterations that regulate these critical cellular functions. Despite their biological importance, this class of metabolites remains difficult to detect and quantify using current analytical methods. Here we show a universal method for metabolomics that allows for the detection of an expansive set of acyl-CoA compounds and hundreds of other cellular metabolites. We apply this method to profile the dynamics of acyl-CoA compounds and corresponding alterations in metabolism across the metabolic network in response to high fat feeding in mice. We identified targeted metabolites (>50) and untargeted features (>1000) with significant changes (FDR < 0.05) in response to diet. A substantial extent of this metabolic remodeling exhibited correlated changes in acyl-CoA metabolism with acyl-carnitine metabolism and other features of the metabolic network that together can lead to the discovery of biomarkers of acyl-CoA metabolism. These findings show a robust acyl-CoA profiling method and identify coordinated changes of acyl-CoA metabolism in response to nutritional stress.Thioester compounds containing acyl-coenzyme A (acyl-CoA)¹ are key metabolites in intermediary metabolism. The most prominent of which is acetyl-CoA whose levels regulate critical cellular processes such as energy metabolism, protein acetylation, lipid synthesis and catabolism, and even autophagy (1–4). Other acyl-CoA compounds are also increasingly appreciated as playing important roles in diverse cellular processes (5–8). These compounds are generated from multiple pathways, such as glycolysis, the citric acid cycle (TCA cycle), beta-oxidation, and branched chain amino acid catabolism. As the acyl group carrier, acyl-CoA can partake in chemical reactions on proteins including histones resulting in mediation of chromatin biology. Therefore, considerable effort has been spent on developing methods for acyl-CoA and corresponding acyl protein modification measurements (9–17). Liquid chromatography coupled to mass spectrometry (LC-MS) is the most frequently used method for small molecule analysis in large part because of superior sensitivity. Moreover, LC-MS analysis can handle a broad range of complex biological mixtures and the analysis is relatively easier compared with many other methods, such as NMR, scintillation counting, and UV detection.Reversed phase LC coupled to a triple quadrupole mass spectrometer has been frequently used as for targeted measurements of specific acyl-CoA compounds, because acyl-CoA compounds undergo a common fragmentation, the neutral loss of adenosine diphosphate, which is the basis of multiple reaction monitoring for acyl-CoA measurements. Especially when stable isotope labeled acyl-CoA standards are used, this method has shown high accuracy and precision (11, 14). However, these methods involve several laborious steps of sample purification and enrichment before LC-MS analysis, such as solid phase extraction, which in addition to often being time- and cost-prohibitive, can also result in poor sensitivity and accuracy because of imperfect metabolite recovery. Moreover, reversed phase ion-paired chromatography coupled to high-resolution MS has also been used for short, medium, and long chain acyl-CoA identification or quantification with the help of stable isotope labeled standards (10, 13). However, these methods were also developed with limited coverage of metabolites, and the quantitative capacity without using stable isotope labeled standards was not evaluated.We therefore developed a novel method for sensitive, rapid, and quantitative acyl-CoA profiling, with a compatible sample preparation procedure that has been previously shown for polar metabolite analysis (18). The method involves LC-MS using reversed phase chromatographic separation coupled to a high-resolution Orbitrap mass spectrometer with label free quantitation. With a single liquid extraction from a few milligrams of tissue, followed by three separate chromatography methods, a broad coverage of metabolites is achieved, which is especially valuable when sample availability is limited.To show the utility of our approach, we considered the alterations in the metabolic network that accompany high fat (HF) feeding. Conditions of high fat feeding that induce nutritional stress are shown to induce global changes in enzymes in metabolism (19, 20), but a comprehensive assessment of the global alterations in metabolism that remains include possible remodeling of acyl-CoA metabolism remain unknown. We reasoned that under such a condition, a dynamic response involving alterations in acyl-CoA levels along with the rest of the metabolome may be observed. This remodeling could also be associated with acyl-carnitine metabolism that often serves as both a readout of acyl-CoA metabolism and other features of metabolism status. Propionyl-CoA that is mainly generated from branched chain amino acid (BCAAs) catabolism and has been implicated in contributing to insulin resistance (21, 22), exhibits large changes. We applied our method to understand the metabolic changes that accompany HF feeding in mouse liver (23). We identify acyl-CoA compounds with dramatic changes after administration of a HF diet. Hierarchical clustering and principle component analysis (PCA) of metabolites measured in liver tissue show further diet-dependent metabolic profiling changes. Moreover, measurements of acyl-carnitine compounds have been used to reflect acyl-CoA levels (24), but the correlation between these two species has not been studied. Our method with coverage of both acyl-carnitine and acyl-CoA enabled us to evaluate acyl-carnitine as a biomarker of acyl-CoA status. In turn, we were able to confirm many relationships between acyl-CoA and acyl-carnitine levels but also discovered several unexpected relationships as well.