Reconstruction of Glutathione Metabolism in the Neuronal Model of Rotenone-Induced Neurodegeneration Using Mass Isotopologue Analysis with Hydrophilic Interaction Liquid Chromatography-Zeno High-Resolution Multiple Reaction Monitoring

Accurate reconstruction of metabolic pathways is an important
prerequisite
for interpreting metabolomics changes and understanding the diverse
biological processes in disease models. A tracer-based metabolomics
strategy utilizes stable isotope-labeled precursors to resolve complex
pathways by tracing the labeled atom(s) to downstream metabolites
through enzymatic reactions. Isotope enrichment analysis is informative
and achieved by counting total labeled atoms and acquiring the mass
isotopologue distribution (MID) of the intact metabolite. However,
quantitative analysis of labeled metabolite substructures/moieties
(MS2 fragments) can offer more valuable insights into the
reaction connections through measuring metabolite transformation.
In order to acquire the isotopic labeling information at the intact
metabolite and moiety level simultaneously, we developed a method
that couples hydrophilic interaction liquid chromatography (HILIC)
with Zeno trap-enabled high-resolution multiple reaction monitoring
(MRMHR). The method enabled accurate and reproducible MID
quantification for intact metabolites as well as their fragmented
moieties, with notably high sensitivity in the MS2 fragmentation
mode based on the measurement of 13C- or 15N-labeled
cellular samples. The method was applied to human-induced pluripotent
stem cell-derived neurons to trace the fate of 13C/15N atoms from D-13C6-glucose/L-15N2-glutamine added to the media. With the MID
analysis of both intact metabolites and fragmented moieties, we validated
the pathway reconstruction of de novo glutathione synthesis in mid-brain
neurons. We discovered increased glutathione oxidization from both
basal and newly synthesized glutathione pools under neuronal oxidative
stress. Furthermore, the significantly decreased de novo glutathione
synthesis was investigated and associated with altered activities
of several key enzymes, as evidenced by suppressed glutamate supply
via glucose metabolism and a diminished flux of glutathione synthetic
reaction in the neuronal model of rotenone-induced neurodegeneration.