Isotope tracing untargeted metabolomics is an advanced approach that builds on traditional untargeted metabolomics by enabling the dynamic tracking of nutrient utilization and metabolic flux. By introducing stable isotope-labeled substrates—such as 13C-labeled compounds—into biological systems, this method reveals how nutrients are processed and routed through various metabolic pathways, offering insights into hidden or alternative metabolic activities.

In our lab, we employ 13C-labeled glucose, 13C-labeled fatty acids, and 13C-labeled ketones to trace carbon flow through central metabolic pathways, including glycolysis, the TCA cycle, fatty acid oxidation, and ketone metabolism. Using high-resolution mass spectrometry (Q Exactive™ Orbitrap instrument), we capture labeled metabolite distributions and uncover pathway activity and connectivity that static profiling alone cannot reveal.

This dynamic approach allows us to investigate metabolic flexibility and rewiring in response to environmental cues, disease states, or experimental perturbations. While our current focus includes select 13C-labeled nutrients, the platform supports a wide range of labeled substrates, enabling broad and customizable exploration of metabolic networks across diverse biological systems.

See more:

• Queathem et al. Sci Adv. 2025 Jan 31;11(5):eads0535.  "Ketogenesis supports hepatic polyunsaturated fatty acid homeostasis via fatty acid elongation."

• Nelson et al. Int J Mol Sci. 2025 Feb 25;26(5):1976. "Deciphering Colorectal Cancer-Hepatocyte Interactions: A Multiomics Platform for Interrogation of Metabolic Crosstalk in the Liver-Tumor Microenvironment."

• Puchalska et al. iScience. 2018 Nov 30:9:298-313. "Isotope Tracing Untargeted Metabolomics Reveals Macrophage Polarization-State-Specific Metabolic Coordination across Intracellular Compartments."

Targeted lipidomics is a high-throughput and precision analytical approach designed to directly quantify predefined lipid species with high sensitivity and specificity. It is particularly well-suited for profiling complex lipidomes in lipid-rich tissues such as the brain, liver, and adipose tissue, where lipids play crucial structural and signaling roles. Unlike untargeted methods, targeted lipidomics enables absolute or relative quantification of known lipid classes, making it ideal for hypothesis-driven studies and biomarker validation.

In our lab, we utilize a shotgun lipidomics strategy, employing a TSQ triple quadrupole mass spectrometer hyphenated with an Advion automated nanoelectrospray infusion robot. This setup allows for direct infusion of lipid extracts without chromatographic separation, significantly increasing throughput while maintaining quantitative robustness. The method is optimized for sensitivity, reproducibility, and minimal sample preparation, enabling efficient processing of large sample cohorts.

Our platform supports the quantification of a wide array of lipid classes, including triacylglycerols (TAG), phosphatidylcholines (PC), phosphatidylethanolamines (PE), phosphatidylserines (PS), phosphatidylinositols (PI), cholesterol, free fatty acids, sphingomyelins (SM), ceramides, cardiolipins (CL), phosphatidylglycerols (PG), and phosphatidic acids (PA). This comprehensive coverage enables detailed insights into lipid metabolism, membrane remodeling, and lipid-mediated signaling in health and disease.

See more:

• d'Avignon, JCI Insight. 2018 Jun 21;3(12):e99762. "Hepatic ketogenic insufficiency reprograms hepatic glycogen metabolism and the lipidome."

• Queathem et al. Sci Adv. 2025 Jan 31;11(5):eads0535.  "Ketogenesis supports hepatic polyunsaturated fatty acid homeostasis via fatty acid elongation."

Metabolic flux analysis (MFA) is a powerful systems-level approach used to quantify the rates at which metabolites flow through biochemical pathways. By integrating stable isotope labeling with precise analytical measurements, MFA provides dynamic, quantitative insights into cellular metabolism that go beyond static metabolite concentrations. This enables the identification of active metabolic routes, pathway bottlenecks, and compensatory fluxes under various physiological or experimental conditions.

In our lab, we perform MFA using both nuclear magnetic resonance (NMR) and a newly acquired GC Orbitrap Exploris 240 mass spectrometer. NMR offers robust, quantitative analysis of isotope incorporation patterns in central carbon metabolites with excellent reproducibility, while the high-resolution GC-Orbitrap system allows for detailed isotopologue profiling of volatile and derivatized metabolites with superior mass accuracy and sensitivity. Together, these complementary platforms provide a comprehensive view of carbon flux through glycolysis, the TCA cycle, anaplerotic reactions, and amino acid metabolism.

This dual-technology approach enhances our ability to model and reconstruct metabolic networks in various biological contexts, including in vitro systems and in vivo tissues. It is especially valuable for studying metabolic rewiring in response to disease, drug treatments, or nutrient changes, and serves as a cornerstone for deeper mechanistic understanding of metabolic regulation.

See more:

• Queathem et al. J Clin Invest. 2025 Apr 24:e191021. "Ketogenesis mitigates metabolic dysfunction-associated steatotic liver disease through mechanisms that extend beyond fat oxidation."

• Stagg et al. Mol Metab. 2021 Nov:53:101269.  "Diminished ketone interconversion, hepatic TCA cycle flux, and glucose production in D-β-hydroxybutyrate dehydrogenase hepatocyte-deficient mice."

• d'Avignon et al., JCI Insight. 2018 Jun 21;3(12):e99762. "Hepatic ketogenic insufficiency reprograms hepatic glycogen metabolism and the lipidome."