Metabolomics, the quantitative analysis of small molecules produced in metabolic processes, is providing new insights into diseases that have traditionally lacked predictive biomarkers. A prime example is Parkinson's disease, an increasingly prevalent neurodegenerative disorder characterized by multifactorial origins and diverse symptoms, now benefiting from these innovations.
In an exclusive Separation Science webinar, researcher Luojiao Huang reveals an innovative method for acquiring intact and partial forms of metabolites labeled with stable carbon-13 and nitrogen-15 isotopes. Discover how this technique, developed in Thomas Hankemeier’s group at the University of Leiden and recently published in Analytical Chemistry, has led to new insights into abnormal metabolic pathways involved in Parkinson’s disease and the identification of disease-specific metabolic fingerprints.
Stable Isotope Labeling in Metabolite Analysis
While metabolites offer researchers a means to quantify and understand human health complexities, their levels in the body are not constant. They can decrease through various biological mechanisms or increase via synthesis and recycling. This dynamic nature makes metabolites both challenging and crucial in research and the development of predictive health models.
Stable isotope labeling, paired with advanced mass spectrometry (MS), provides a powerful approach for studying metabolite dynamics using mass isotopologues—chemically identical molecules differentiated by their isotopic composition. Introducing isotope-labeled nutrients, such as 13C-enriched glucose, into biological systems enables researchers to track nutrient fragmentation through mass isotopologue distributions. This method efficiently determines metabolite concentrations and metabolic flux rates.
Tandem mass spectrometry (MS/MS) methods, especially multiple reaction monitoring (MRM), help link mass isotopologue distributions to metabolic functions and pathways in the body. MRM accurately identifies isotope labels in ions, but with larger metabolites, the process becomes more complex. This can lead to fewer data points and reduced sensitivity and accuracy, especially for less abundant isotopologues.
Higher resolution versions of MS/MS techniques, including sequential windowed acquisition of all theoretical mass spectra (SWATH) and parallel reaction monitoring (PRM), offer unique advantages in metabolite analysis. SWATH fragments all ions within a set mass range, regardless of their abundance, while PRM, employing high-resolution MS, records the complete product-ion spectrum for specific labeled metabolite isotopologues, significantly reducing transition pairs. Both methods achieve lower cycle times than MRM, enabling broader metabolite spectrum capture and improving the detection of compounds, especially those at lower concentrations.
Enhancing MS Duty Cycles
The researchers’ approach centers on a PRM method (also called MRM High-Resolution) that uses high-performance liquid chromatography, including columns with zwitterionic stationary phases functionalized with phosphorylcholine, to enhance the separation of metabolites. This approach was fine-tuned for the effective profiling of 40 vital metabolites, pivotal in primary carbon metabolism, glutathione metabolism, and purine and pyrimidine metabolism.
Their technique is further augmented by the Zeno trap, a novel form of linear ion trap designed to capture and accumulate ions before they are introduced into the TOF analyzer. This trapping mechanism allows for a more concentrated sample of ions to be analyzed, which inherently increases detection sensitivity and accuracy. The trap's design also minimizes ion fragmentation and scattering, which can be a concern in quadrupole detectors.
A further benefit of Zeno trapping is its role in enhancing the 'duty cycle' efficiency with which the MS can analyze samples. By gathering and holding onto ions before they are released in a pulse toward the detector, the MS can operate at optimal efficiency and ensure thorough analysis of even trace amounts of ions.
The research team tested different methods with and without Zeno trapping enabled to analyze labeled metabolite isotopologues, both intact and fragmented. Their HILIC-Zeno MRM High-Resolution method proved superior in detecting isotopologues. It enhanced the signal for 13C-glutamate precursors by nearly five times and their fragments by almost eight times compared to the SWATH method. For 15N-glutamate, the increase was about seven times for precursors and over eight times for fragments.
By tracing the labeled atoms, we were able to reconstruct the cell-type and condition-specific pathways of glutathione metabolism in healthy and perturbed mid-brain neurons
Deciphering Glutathione's Role in Parkinson's
Huang's team used their method to investigate glutathione metabolism, vital for brain health and often implicated in Parkinson's disease. Using human-derived midbrain neurons as a Parkinson's model, they fed the cells 13C-glucose or 15N-glutamine. The study showed that these isotopes were integrated into glutathione's components, or its precursor metabolites including glutamate and glycine, both in placement and amount, confirming their role in glutathione synthesis.
Researchers exposed the neuronal model to rotenone, a neurotoxin that induces symptoms similar to Parkinson's disease, to study changes in carbon and glutathione metabolism under neurodegenerative stress. Treatment with rotenone led to a marked decrease in both glutathione and its oxidized form, pointing to impaired production or maintenance and heightened risk of cellular damage. The results also suggest rotenone interferes with key enzymes needed for glutathione synthesis, worsening its decline.
Metabolic Breakthroughs Ahead
Huang's work sheds light on how rotenone impacts glutathione metabolism, deepening our grasp of environmental toxins in neurodegenerative diseases. She also proposes further possibilities for method improvements, such as triggering MS/MS events based on an intensity-dependent selection of precursor ions and thereby engaging more key metabolites over the pathway modulation as method targets.
“By tracing the labeled 13C and 15N atoms in the moieties of metabolite isotopologues, we were able to reconstruct the cell-type and condition-specific pathways of glutathione metabolism in healthy and perturbed mid-brain neurons,” explains Huang. “The quantitative isotopologue analysis greatly contributed to elucidating how glutathione metabolism regulation responds to rotenone perturbation, and this method holds potential for other pathways and reactions too.”
Discover more insights and details about this work by viewing the original webinar on Separation Science.
Adam Dickie is a science writer for Separation Science. He can be reached at adickie@sepscience.com.
Luojiao Huang
Metabolomics and Analytics Centre, Leiden Academic Centre for Drug Research, Leiden University, Leiden
Luojiao Huang studied Pharmacy for her bachelor’s and master's degrees, developing ambient mass spectrometry imaging methods for molecular pathological diagnosis of thyroid tumors. To explore new analytical possibilities for understanding disease mechanisms, she began her PhD research at Leiden University. Her project aims to characterize the metabolic dysregulation related to Parkinson's disease neurodegeneration. To address this, she uses high-throughput and quantitative metabolomics, assisted with stable isotope tracing, for studying in vitro neuronal models. She is also developing an automated data analysis pipeline for metabolic flux analysis to monitor metabolic network activity.