Carbon and Nitrogen Positional Isotopomer Determination in Metabolites using Orbitrap IQ-X and Stellar MS - a novel hybrid nominal mass instrument

Importance of the Topic

Flux analysis using stable isotopes reveals metabolic pathway usage under varying conditions, including hypoxia which occurs in many pathological states such as heart attack, stroke and respiratory failure. Determining positional isotopomer distributions of metabolites like AMP enhances understanding of biosynthetic regulation and cellular adaptation to low oxygen.

Goals and Overview of the Study

This work aimed to develop a targeted MS2/MS3 approach for determining carbon and nitrogen positional isotopomers in adenosine monophosphate and to apply it to assess hypoxia induced metabolic changes in a human pancreatic cancer cell model.

Methodology

• Cell culture under normoxic and hypoxic conditions using KP4 cells grown in media containing unlabeled substrates, 13C6 glucose, 15N2 glutamine or both labels
• Separation of polar metabolites by hydrophilic interaction liquid chromatography on a UHPLC system
• High resolution full MS scans followed by targeted HCD and CID MS2 fragmentation of AMP and subsequent MS3 of selected fragments
• Data processing for structural annotation of fragment ions and quantification of isotopologue distributions

Used Instrumentation

• Thermo Scientific Orbitrap IQ-X Tribrid mass spectrometer equipped with HCD and CID fragmentation
• Thermo Scientific Vanquish Horizon UHPLC system with HILIC column
• Thermo Scientific Stellar MS hybrid quadrupole radial ejection linear ion trap for nominal mass targeted MS2/MS3 assays

Main Results and Discussion

• Establishment of a robust targeted MS2/MS3 assay for positional isotopomer analysis of AMP
• Under hypoxia, increased glucose uptake, enhanced anaerobic glycolysis, elevated lactate excretion and reduced glucose oxidation and fatty acid beta oxidation
• Ribose ring labeling from 13C6 glucose was observed in both conditions, indicating consistent carbon incorporation into the sugar moiety
• Nitrogen labeling experiments with 15N2 glutamine revealed retention of unlabeled glycine nitrogen in the adenine ring
• The M+9 isotopologue of AMP showed lower abundance in hypoxic cells, reflecting altered precursor contributions

Benefits and Practical Applications

• Provides detailed positional isotopomer information on nucleotide metabolism
• Enables refined metabolic flux calculations in systems with multiple biosynthetic routes
• Demonstrates feasibility of using nominal mass instruments for targeted isotopomer workflows
• Facilitates studies of metabolic adaptations in disease models and drug response

Future Trends and Potential Applications

• Expansion of targeted MSn assays to other metabolite classes and pathways
• Integration with automated flux modeling and high throughput screening platforms
• Development of standardized workflows for clinical and industrial metabolomics
• Enhanced coupling of nominal mass instruments with machine learning for predictive flux analysis

Conclusion

The presented targeted MS2/MS3 workflow applied to AMP isotopomer analysis offers precise positional labeling data, reveals hypoxia induced metabolic rewiring and underscores the potential of hybrid nominal mass spectrometers for advanced fluxomics studies.