MetaboLightsapplication/xmlftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS12791/m_MTBLS12791_LC-MS_negative_hilic_metabolite_profiling_v2_maf.tsvftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS12791/m_MTBLS12791_LC-MS_positive_hilic_metabolite_profiling_v2_maf.tsvftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS12791/a_MTBLS12791_LC-MS_negative_hilic_metabolite_profiling.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS12791/i_Investigation.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS12791/a_MTBLS12791_LC-MS_positive_hilic_metabolite_profiling.txtftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS12791/s_MTBLS12791.txtprimaryOK200ftp://ftp.ebi.ac.uk/pub/databases/metabolights/studies/public/MTBLS12791<p>Metabolite identification and quantification were performed using MZmine software (version 3.53), utilizing an in-house library for matching based on m/z and retention time. Custom database searches were conducted with a tolerance of 10 ppm for m/z and 0.3 minutes for RT, ensuring robust metabolite identification. Adduct and complex searches, peak list filtering, and final peak list exports were executed seamlessly. Peak height values were extracted as CSV files for further processing using the iterative rank-order normalization (IRON) method (Welsh et al. BMC Bioinformatics 2013, 14,153).</p>MetaboLightsPublicLiquid Chromatography MS - positive - HILICLiquid Chromatography MS - negative - HILIC<p>Ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-HRMS) was conducted using a Vanquish UHPLC system interfaced with a Q Exactive HF quadrupole-orbitrap mass spectrometer (Thermo). Chromatographic separation was achieved using an Atlantis Premier BEH Z-HILIC VanGuard FIT column (2.1 mm ID × 150 mm length, 2.5 µm particle size; Waters). Mobile phase A consisted of aqueous 10 mM ammonium carbonate with 0.05% ammonium hydroxide, while mobile phase B was pure acetonitrile. The gradient program was structured as follows: the initial condition was set at 80% B, followed by a linear decrease to 20% B over 13 minutes. The system was remained at 20% B for 2 minutes before swiftly returning to 80% B within 0.1 minutes, concluding with a 4.9-minute re-equilibration period. The total runtime was 20 minutes, with a flow rate of 0.400 mL/min. The autosampler was maintained at 5 °C, while the column temperature was stabilized at 30 °C. A sample injection volume of 2 µL was used for both positive and negative ion mode electrospray ionization. </p>Targeting GSTZ1 sensitizes KRASG12C-mutant lung cancer cells by overcoming glutathione and glycolysis pathway rewiring.Moffitt Cancer CenterJohn Koomenlungmass spectrometry assay<p>H1792 cells were treated with non-targeting siRNA and siGSTZ1 for 24 hours, followed by an additional 72-hour treatment with sotorasib. Cell pellets were washed three times with PBS and stored at -80 °C until use. All procedures were carried out on ice to maintain sample integrity. To initiate protein precipitation, 500 µL of pre-cooled 80% methanol extraction solvent was added to the samples. Following solvent addition, the samples were vortexed thoroughly and centrifuged at 18,800 × g (Microfuge 22R, Beckman Coulter) at 0 °C for 10 minutes. To enhance metabolite extraction, the samples were then incubated in a -80 °C freezer for 30 minutes before undergoing immediate centrifugation again at 18,800 × g at 4 °C for 10 minutes. The resulting supernatant was carefully transferred into a fresh microcentrifuge tube. Meanwhile, the protein pellet was resolubilized in aqueous 20 mM HEPES buffer containing 8 M urea for Bradford assays to quantify protein concentration. Dried metabolites were re-dissolved in 80% methanol containing 10% of the Metabolomics QC kit, ensuring consistency across samples. A 2 µL aliquot of the Metabolomics QC kit metabolite mixture was added to each sample to facilitate downstream analysis.</p>Homo sapienshttps://www.ebi.ac.uk/metabolights/MTBLS12791Uwe Rix. Drug Discovery, Moffitt Cancer Center. 12902 Magnolia Drive Tampa, FL 33612. uwe.rix@moffitt.org. 18137453714.<p>Metabolite identification and quantification were performed using MZmine software (version 3.53), utilizing an in-house library for matching based on m/z and retention time. A batch file streamlined the automation of multiple analytical modules, including centroid mass detection, ADAP chromatogram builder, and deconvolution via local minimum search. Key settings included a minimum group size of five scans and a group intensity threshold set to 1.0 x 104. Smoothing parameters were adjusted to a value of five, and chromatographic thresholding was maintained at 95%. Retention time search parameters ranged from 0.05 to 5 minutes, ensuring precise peak detection. Additional refinements included isotopic peak grouping, peak alignment (weighted 75% for m/z and 25% for RT), duplicate peak filtering, and gap filling. Peak height values were extracted as CSV files for further processing using the iterative rank-order normalization (IRON) method (Welsh et al. BMC Bioinformatics 2013, 14,153).</p>GSTZ1 KDSotorasib txjohn.koomen@moffitt.org<p>The cell line used is a KRAS G12C mutant non small cell lung cancer, H1792 cells. The treatment conditions are: siNT (non-targeting siRNAs, 25 nM), siGSTZ1 (25 nM), siNT+sotorasib (1 micromolar), siGSTZ1+sotorasib (1 micromolar). siRNAs (siNT and siGSTZ1) were incubated with cells for 24h, followed by the addition of sotorasib for an additional 72 hours.</p>MetabolomicsThermo Scientific Vanquish UHPLC Systemlung cancerlunguntargeted analysisuntargeted metabolitesHomo sapiensKRASThermo Scientific Q Exactive HFexperimental sampleThermo Scientific Vanquish UHPLC Systemlung cancerlunguntargeted analysisuntargeted metabolitesHomo sapiensKRASThermo Scientific Q Exactive HFexperimental sample<p>Ultra-high-performance liquid chromatography coupled with high-resolution mass spectrometry (UHPLC-HRMS) was conducted using a Vanquish UHPLC system interfaced with a Q Exactive HF quadrupole-orbitrap mass spectrometer (Thermo). Source and ion transmission parameters included: spray voltage was 3.3 kV, capillary temperature was 320 degrees Celsius, sheath gas flow 50, auxiliary gas flow 10, and S-Lens RF level 50. AGC target was set to 1E6, and maximum injection time at 50 ms.</p><p>Full MS scans were performed separately in both ionization modes, detecting ions ranging from m/z 65 to m/z 900 using resolution 120,000.</p>falseTargeting GSTZ1 sensitizes KRASG12C-mutant lung cancer cells by overcoming glutathione and glycolysis pathway rewiring<p>KRAS mutations are prevalent in lung cancer, but KRAS G12C inhibitors exhibit limited efficacy, partly due to metabolic adaptations, such as enhanced glutathione metabolism and increased glycolysis. Glutathione S-Transferase Zeta 1 (GSTZ1) is a metabolic enzyme that regulates cell metabolism. However, its role in KRAS-driven lung cancer remains underexplored. We recently reported that targeting GSTZ1 significantly enhances the efficacy of FDA-approved KRAS G12C inhibitors in non-small cell lung cancer cells. Untargeted metabolomics now revealed significant alterations in glutathione and glycolytic pathways, marked by lowered lactate levels and increased oxidized glutathione following GSTZ1 ablation. Moreover, pharmacological inhibition of glutathione synthesis and glucose uptake mimicked the sensitizing effects of GSTZ1 targeting. These metabolic shifts were accompanied by increased AMPK phosphorylation and reduced AKT phosphorylation, two key mediators of the response to KRAS G12C inhibition. Our findings establish GSTZ1-induced metabolic and signaling alterations as a novel mechanism of sensitization, positioning GSTZ1 as a potential therapeutic target.</p>2026-05-152025-07-29MTBLS12791