Investigating cellular metabolism on Orbitrap Excidion Pro mass spectrometer by the simultaneous use of differently labeled elemental isotope substrates

Importance of the Topic

Stable isotope labeling in metabolomics (fluxomics) offers unique insights into cellular pathway dynamics, enabling both targeted and untargeted investigations of metabolic networks. By tracking the incorporation of 13C and 15N labels into metabolites, researchers can elucidate pathway connectivity, flux rates, and the impact of environmental factors such as oxygen availability on cancer cell metabolism.

Objectives and Study Overview

This study evaluates the performance of the Orbitrap Excedion Pro hybrid mass spectrometer for simultaneous analysis of 13C- and 15N-labeled substrates in pancreatic cancer cells (Panc02) under normoxic (21% O2) and hypoxic (1.5% O2) conditions. The aim is to demonstrate how enhanced resolution and mass accuracy improve the detection and quantitation of isotopologues across complex metabolite profiles.

Applied Methodology and Instrumentation

Cell Culture and Labeling

• Panc02 cells grown in four media conditions: unlabeled; U-13C glucose; 15N2 glutamine; both U-13C glucose and 15N2 glutamine.
• Oxygen tensions: 21% (normoxia) vs 1.5% (hypoxia).

Metabolite Extraction and Separation

• Cell harvest followed by methanol extraction and sonication.
• Dry-down and re-suspension in 50% acetonitrile.
• Chromatography on a Mixed Mode HILIC-AEX column using a Thermo Scientific Vanquish Horizon UHPLC system.

Mass Spectrometry

• Data acquired on Thermo Scientific Orbitrap Excedion Pro MS, enabling extended dynamic range, enhanced sensitivity, and minimal in-source fragmentation.
• Resolution settings up to 240 000 to resolve fine isotopic patterns.
• Data processed with Thermo Scientific Compound Discoverer 3.4 software for automated isotopologue detection and quantitation.

Main Results and Discussion

High mass accuracy (<1 ppm) and ultra-high resolution (≥240 K) permitted clear separation of closely spaced isotopic peaks in ATP and other central metabolites. Compound Discoverer 3.4 successfully identified differential 13C incorporation into proline under normoxia versus hypoxia, revealing decreased flux through glucose-derived pathways in low-oxygen conditions. The workflow also discriminated contributions from 15N-labeled glutamine, enabling simultaneous multi-tracer analysis.

Contributions and Practical Applications of the Method

- Provides a robust untargeted fluxomics platform combining high resolution and isotope-label specificity.
- Facilitates comprehensive pathway profiling in cancer research and other fields requiring metabolic flux assessment.
- Enhances confidence in metabolite identification by confirming label incorporation patterns.

Future Trends and Potential Applications

Advances in acquisition speed and resolution will further expand untargeted fluxomics to larger sample cohorts. Integration with ion mobility and data-independent acquisition may enable deeper coverage of low-abundance metabolites. Coupling multi-omics data (transcriptomics, proteomics) with fluxomics will refine mechanistic models of cellular adaptation in disease and biotechnology applications.

Conclusion

The Orbitrap Excedion Pro MS, combined with Compound Discoverer 3.4, delivers exceptional mass accuracy and resolution for stable isotope flux analysis. This approach successfully characterizes metabolic adaptations of pancreatic cancer cells under varying oxygen levels, demonstrating its value for both fundamental research and applied clinical or industrial workflows.

Reference

1. Deshpande R., Midha A., Dauly C., Amer B., Jain I., Bird S.S. “Investigating cellular metabolism on Orbitrap Excedion Pro mass spectrometer by the simultaneous use of differently labeled elemental isotope substrates,” Thermo Fisher Scientific Application Note, 2025.