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

Abstract

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.

Oxygen-Dependent Interactions between the Ruthenium Cage and the Photoreleased Inhibitor in NAMPT-Targeted Photoactivated Chemotherapy

“…The mobile phases were composed of (A) 90% acetonitrile in H 2 O with 5 mM ammonium formate and (B) 10% acetonitrile in H 2 O with 5 mM ammonium formate. Chromatographic separation of analytes was carried out with a gradient elution program at a flow rate of 0.5 mL/min. , Electrospray ionization MS was operated in the negative ion mode, and analytes were monitored in the time-of-flight (ToF) mode at a full scan range of 50–900 m / z . , MultiQuant software (AB SCIEX, version 3.0.1) was used in the quantitative analysis for LC-MS raw data extraction and peak area integration. Internal standards were employed to correct random errors during sample preparation.…”

“…Chromatographic separation of analytes was carried out with a gradient elution program at a flow rate of 0.5 mL/min. 44 , 45 Electrospray ionization MS was operated in the negative ion mode, and analytes were monitored in the time-of-flight (ToF) mode at a full scan range of 50–900 m / z . 44 , 45 MultiQuant software (AB SCIEX, version 3.0.1) was used in the quantitative analysis for LC-MS raw data extraction and peak area integration.…”

“… 44 , 45 Electrospray ionization MS was operated in the negative ion mode, and analytes were monitored in the time-of-flight (ToF) mode at a full scan range of 50–900 m / z . 44 , 45 MultiQuant software (AB SCIEX, version 3.0.1) was used in the quantitative analysis for LC-MS raw data extraction and peak area integration. Internal standards were employed to correct random errors during sample preparation.…”

“…However, this hypothesis had never been tested in a cellular context. Therefore, we conducted a metabolomic analysis to measure the real intracellular level of NAD + in U87MG cells cultured in different conditions (Figure ): either untreated (Ctrl) or treated with Ru-STF31 , Ru-Py , or STF31 , and either kept in the dark (D) or following activation with red light (RL, 21 J/cm 2 ). , According to this analysis, free STF31 significantly decreased NAD + levels in hypoxic cells but the change compared with untreated cells was not statistically significant in normoxic cells. In normoxia, Ru-STF31 in both dark and red light-activated conditions did not affect the amount of NAD + ; however, in hypoxic cells, light-activated Ru-STF31 significantly decreased NAD + levels.…”

Triple acquisition mass spectrometry (TRAM) combining targeted and non-targeted metabolomics in a single run

“…14 The utility of the Zeno function for biological applications has recently been demonstrated by Huang et al in a mass isotopologue distribution study on stem cell-derived neurons. 15 Exploiting the scan speed of up to 133 Hz 12 , data-dependent MS2 acquisition can be tailored to ensure high numbers of MS2 spectra upon each sample injection. Additionally, scheduled MS2 was designed and optimized for MRM-like data evaluation.…”