#METABOLOMICS WORKBENCH kurtafungo_20251126_162629 DATATRACK_ID:6772 STUDY_ID:ST004411 ANALYSIS_ID:AN007377 PROJECT_ID:PR002787 VERSION 1 CREATED_ON December 1, 2025, 3:39 pm #PROJECT PR:PROJECT_TITLE Uncoupling de novo pyrimidine biosynthesis from mitochondrial electron transport PR:PROJECT_TITLE by ectopic expression of cytosolic DHODH (tracing experiments) PR:PROJECT_TYPE Synthetic biology PR:PROJECT_SUMMARY Dihydroorotate dehydrogenase (DHODH) is an enzyme involved in the biosynthesis PR:PROJECT_SUMMARY of pyrimidine nucleotides. In most eukaryotes, this enzyme is bound to the inner PR:PROJECT_SUMMARY mitochondrial membrane, where it couples the synthesis of orotate with the PR:PROJECT_SUMMARY reduction of ubiquinone. As ubiquinone must be regenerated by respiratory PR:PROJECT_SUMMARY complex III, pyrimidine biosynthesis and cellular respiration are tightly PR:PROJECT_SUMMARY coupled. Consequently, inhibition of respiration leads to cessation of DNA PR:PROJECT_SUMMARY synthesis and impairs cell proliferation. We show that expression of PR:PROJECT_SUMMARY Saccharomyces cerevisiae URA1 gene (ScURA) in mammalian cells uncouples PR:PROJECT_SUMMARY biosynthesis of pyrimidines from mitochondrial electron transport. ScURA forms a PR:PROJECT_SUMMARY homodimer in the cytosol that uses fumarate instead of ubiquinone as the PR:PROJECT_SUMMARY electron acceptor, enabling oxygen-independent pyrimidine biosynthesis. Cells PR:PROJECT_SUMMARY expressing ScURA are resistant to drugs that inhibit complex III and the PR:PROJECT_SUMMARY mitochondrial ribosome. ScURA enables the growth of mtDNA-lacking ρ0 cells in PR:PROJECT_SUMMARY uridine-deficient medium and ameliorates the phenotype of cellular models of PR:PROJECT_SUMMARY mitochondrial diseases. This genetic tool uncovers the contribution of PR:PROJECT_SUMMARY pyrimidine biosynthesis to the phenotypes arising from electron transport chain PR:PROJECT_SUMMARY defects. PR:INSTITUTE Centro Nacional de Investigaciones Cardiovasculares Carlos III PR:LABORATORY Genoxphos PR:LAST_NAME Curtabbi PR:FIRST_NAME Andrea PR:ADDRESS C. de Melchor Fernández Almagro, 3, Fuencarral-El Pardo, 28029 Madrid, Spain PR:EMAIL andrea.curtabbi@gmail.com PR:PHONE +34 914 53 12 00 #STUDY ST:STUDY_TITLE Uncoupling de novo pyrimidine biosynthesis from mitochondrial electron transport ST:STUDY_TITLE by ectopic expression of cytosolic DHODH (tracing experiments) ST:STUDY_TYPE Semi-targeted metaolomics ST:STUDY_SUMMARY Dihydroorotate dehydrogenase (DHODH) is an enzyme involved in the biosynthesis ST:STUDY_SUMMARY of pyrimidine nucleotides. In most eukaryotes, this enzyme is bound to the inner ST:STUDY_SUMMARY mitochondrial membrane, where it couples the synthesis of orotate with the ST:STUDY_SUMMARY reduction of ubiquinone. As ubiquinone must be regenerated by respiratory ST:STUDY_SUMMARY complex III, pyrimidine biosynthesis and cellular respiration are tightly ST:STUDY_SUMMARY coupled. Consequently, inhibition of respiration leads to cessation of DNA ST:STUDY_SUMMARY synthesis and impairs cell proliferation. We show that expression of ST:STUDY_SUMMARY Saccharomyces cerevisiae URA1 gene (ScURA) in mammalian cells uncouples ST:STUDY_SUMMARY biosynthesis of pyrimidines from mitochondrial electron transport. ScURA forms a ST:STUDY_SUMMARY homodimer in the cytosol that uses fumarate instead of ubiquinone as the ST:STUDY_SUMMARY electron acceptor, enabling oxygen-independent pyrimidine biosynthesis. Cells ST:STUDY_SUMMARY expressing ScURA are resistant to drugs that inhibit complex III and the ST:STUDY_SUMMARY mitochondrial ribosome. ScURA enables the growth of mtDNA-lacking ρ0 cells in ST:STUDY_SUMMARY uridine-deficient medium and ameliorates the phenotype of cellular models of ST:STUDY_SUMMARY mitochondrial diseases. This genetic tool uncovers the contribution of ST:STUDY_SUMMARY pyrimidine biosynthesis to the phenotypes arising from electron transport chain ST:STUDY_SUMMARY defects. For metabolomics experiments we 143B cells, expressing ScURA, treated ST:STUDY_SUMMARY with either DHODH or complex III inhibitors for 48 hours. Tracers (C- or N- ST:STUDY_SUMMARY labeled Glutamine, were added after 24 hours. All conditions had 3 independent ST:STUDY_SUMMARY biological replicates. ST:INSTITUTE UCSF ST:LAST_NAME Curtabbi ST:FIRST_NAME Andrea ST:ADDRESS 1450 3rd St, San Francisco, San Francisco, CA, 94158, USA ST:EMAIL andrea.curtabbi@gmail.com ST:PHONE +34 914 53 12 00 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:GENDER Female #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - ACu02_001 Tracer:15N_glutamine | Genotype:WT | Treatment:Control | Sample source:143B_cultured_cells Sample_code=143B_wt_control_15N_glutamine_tracing_1; Biological Replicate=1; Extraction Buffer volume=97; Exact Volume in Vial=85; RAW_FILE_NAME(Raw_data)=ACu02_001.mzML SUBJECT_SAMPLE_FACTORS - ACu02_002 Tracer:15N_glutamine | Genotype:WT | Treatment:Control | Sample source:143B_cultured_cells Sample_code=143B_wt_control_15N_glutamine_tracing_2; Biological Replicate=2; Extraction Buffer volume=97; Exact Volume in Vial=85; RAW_FILE_NAME(Raw_data)=ACu02_002.mzML SUBJECT_SAMPLE_FACTORS - ACu02_003 Tracer:15N_glutamine | Genotype:WT | Treatment:Control | Sample source:143B_cultured_cells Sample_code=143B_wt_control_15N_glutamine_tracing_3; Biological Replicate=3; Extraction Buffer volume=97; Exact Volume in Vial=85; RAW_FILE_NAME(Raw_data)=ACu02_003.mzML SUBJECT_SAMPLE_FACTORS - ACu02_004 Tracer:15N_glutamine | Genotype:ScURA | Treatment:Control | Sample source:143B_cultured_cells Sample_code=143B_ScURA_control_15N_glutamine_tracing_1; Biological Replicate=1; Extraction Buffer volume=430; Exact Volume in Vial=400; RAW_FILE_NAME(Raw_data)=ACu02_004.mzML SUBJECT_SAMPLE_FACTORS - ACu02_005 Tracer:15N_glutamine | Genotype:ScURA | Treatment:Control | Sample source:143B_cultured_cells Sample_code=143B_ScURA_control_15N_glutamine_tracing_2; Biological Replicate=2; Extraction Buffer volume=430; Exact Volume in Vial=400; RAW_FILE_NAME(Raw_data)=ACu02_005.mzML SUBJECT_SAMPLE_FACTORS - ACu02_006 Tracer:15N_glutamine | Genotype:ScURA | Treatment:Control | Sample source:143B_cultured_cells Sample_code=143B_ScURA_control_15N_glutamine_tracing_3; Biological Replicate=3; Extraction Buffer volume=430; Exact Volume in Vial=400; RAW_FILE_NAME(Raw_data)=ACu02_006.mzML SUBJECT_SAMPLE_FACTORS - ACu02_007 Tracer:15N_glutamine | Genotype:WT | Treatment:Brequinar | Sample source:143B_cultured_cells Sample_code=143B_wt_brequinar_15N_glutamine_tracing_1; Biological Replicate=1; Extraction Buffer volume=63; Exact Volume in Vial=55; RAW_FILE_NAME(Raw_data)=ACu02_007.mzML SUBJECT_SAMPLE_FACTORS - ACu02_008 Tracer:15N_glutamine | Genotype:WT | Treatment:Brequinar | Sample source:143B_cultured_cells Sample_code=143B_wt_brequinar_15N_glutamine_tracing_2; Biological Replicate=2; Extraction Buffer volume=63; Exact Volume in Vial=55; RAW_FILE_NAME(Raw_data)=ACu02_008.mzML SUBJECT_SAMPLE_FACTORS - ACu02_009 Tracer:15N_glutamine | Genotype:WT | Treatment:Brequinar | Sample source:143B_cultured_cells Sample_code=143B_wt_brequinar_15N_glutamine_tracing_3; Biological Replicate=3; Extraction Buffer volume=63; Exact Volume in Vial=55; RAW_FILE_NAME(Raw_data)=ACu02_009.mzML SUBJECT_SAMPLE_FACTORS - ACu02_010 Tracer:15N_glutamine | Genotype:ScURA | Treatment:Brequinar | Sample source:143B_cultured_cells Sample_code=143B_ScURA_brequinar_15N_glutamine_tracing_1; Biological Replicate=1; Extraction Buffer volume=399; Exact Volume in Vial=360; RAW_FILE_NAME(Raw_data)=ACu02_010.mzML SUBJECT_SAMPLE_FACTORS - ACu02_011 Tracer:15N_glutamine | Genotype:ScURA | Treatment:Brequinar | Sample source:143B_cultured_cells Sample_code=143B_ScURA_brequinar_15N_glutamine_tracing_2; Biological Replicate=2; Extraction Buffer volume=399; Exact Volume in Vial=360; RAW_FILE_NAME(Raw_data)=ACu02_011.mzML SUBJECT_SAMPLE_FACTORS - ACu02_012 Tracer:15N_glutamine | Genotype:ScURA | Treatment:Brequinar | Sample source:143B_cultured_cells Sample_code=143B_ScURA_brequinar_15N_glutamine_tracing_3; Biological Replicate=3; Extraction Buffer volume=399; Exact Volume in Vial=360; RAW_FILE_NAME(Raw_data)=ACu02_012.mzML SUBJECT_SAMPLE_FACTORS - ACu02_013 Tracer:15N_glutamine | Genotype:WT | Treatment:Antimycin_Myxothiazol | Sample source:143B_cultured_cells Sample_code=143B_wt_antimycin_15N_glutamine_tracing_1; Biological Replicate=1; Extraction Buffer volume=62; Exact Volume in Vial=55; RAW_FILE_NAME(Raw_data)=ACu02_013.mzML SUBJECT_SAMPLE_FACTORS - ACu02_014 Tracer:15N_glutamine | Genotype:WT | Treatment:Antimycin_Myxothiazol | Sample source:143B_cultured_cells Sample_code=143B_wt_antimycin_15N_glutamine_tracing_2; Biological Replicate=2; Extraction Buffer volume=62; Exact Volume in Vial=55; RAW_FILE_NAME(Raw_data)=ACu02_014.mzML SUBJECT_SAMPLE_FACTORS - ACu02_015 Tracer:15N_glutamine | Genotype:WT | Treatment:Antimycin_Myxothiazol | Sample source:143B_cultured_cells Sample_code=143B_wt_antimycin_15N_glutamine_tracing_3; Biological Replicate=3; Extraction Buffer volume=62; Exact Volume in Vial=55; RAW_FILE_NAME(Raw_data)=ACu02_015.mzML SUBJECT_SAMPLE_FACTORS - ACu02_016 Tracer:15N_glutamine | Genotype:ScURA | Treatment:Antimycin_Myxothiazol | Sample source:143B_cultured_cells Sample_code=143B_ScURA_antimycin_15N_glutamine_tracing_1; Biological Replicate=1; Extraction Buffer volume=232; Exact Volume in Vial=200; RAW_FILE_NAME(Raw_data)=ACu02_016.mzML SUBJECT_SAMPLE_FACTORS - ACu02_017 Tracer:15N_glutamine | Genotype:ScURA | Treatment:Antimycin_Myxothiazol | Sample source:143B_cultured_cells Sample_code=143B_ScURA_antimycin_15N_glutamine_tracing_2; Biological Replicate=2; Extraction Buffer volume=232; Exact Volume in Vial=200; RAW_FILE_NAME(Raw_data)=ACu02_017.mzML SUBJECT_SAMPLE_FACTORS - ACu02_018 Tracer:15N_glutamine | Genotype:ScURA | Treatment:Antimycin_Myxothiazol | Sample source:143B_cultured_cells Sample_code=143B_ScURA_antimycin_15N_glutamine_tracing_3; Biological Replicate=3; Extraction Buffer volume=232; Exact Volume in Vial=200; RAW_FILE_NAME(Raw_data)=ACu02_018.mzML SUBJECT_SAMPLE_FACTORS - ACu02_019 Tracer:13C_glutamine | Genotype:WT | Treatment:Control | Sample source:143B_cultured_cells Sample_code=143B_wt_control_13C_glutamine_tracing_1; Biological Replicate=1; Extraction Buffer volume=97; Exact Volume in Vial=85; RAW_FILE_NAME(Raw_data)=ACu02_019.mzML SUBJECT_SAMPLE_FACTORS - ACu02_020 Tracer:13C_glutamine | Genotype:WT | Treatment:Control | Sample source:143B_cultured_cells Sample_code=143B_wt_control_13C_glutamine_tracing_2; Biological Replicate=2; Extraction Buffer volume=97; Exact Volume in Vial=85; RAW_FILE_NAME(Raw_data)=ACu02_020.mzML SUBJECT_SAMPLE_FACTORS - ACu02_021 Tracer:13C_glutamine | Genotype:WT | Treatment:Control | Sample source:143B_cultured_cells Sample_code=143B_wt_control_13C_glutamine_tracing_3; Biological Replicate=3; Extraction Buffer volume=97; Exact Volume in Vial=85; RAW_FILE_NAME(Raw_data)=ACu02_021.mzML SUBJECT_SAMPLE_FACTORS - ACu02_022 Tracer:13C_glutamine | Genotype:ScURA | Treatment:Control | Sample source:143B_cultured_cells Sample_code=143B_ScURA_control_13C_glutamine_tracing_1; Biological Replicate=1; Extraction Buffer volume=430; Exact Volume in Vial=400; RAW_FILE_NAME(Raw_data)=ACu02_022.mzML SUBJECT_SAMPLE_FACTORS - ACu02_023 Tracer:13C_glutamine | Genotype:ScURA | Treatment:Control | Sample source:143B_cultured_cells Sample_code=143B_ScURA_control_13C_glutamine_tracing_2; Biological Replicate=2; Extraction Buffer volume=430; Exact Volume in Vial=400; RAW_FILE_NAME(Raw_data)=ACu02_023.mzML SUBJECT_SAMPLE_FACTORS - ACu02_024 Tracer:13C_glutamine | Genotype:ScURA | Treatment:Control | Sample source:143B_cultured_cells Sample_code=143B_ScURA_control_13C_glutamine_tracing_3; Biological Replicate=3; Extraction Buffer volume=430; Exact Volume in Vial=400; RAW_FILE_NAME(Raw_data)=ACu02_024.mzML SUBJECT_SAMPLE_FACTORS - ACu02_025 Tracer:13C_glutamine | Genotype:WT | Treatment:Brequinar | Sample source:143B_cultured_cells Sample_code=143B_wt_brequinar_13C_glutamine_tracing_1; Biological Replicate=1; Extraction Buffer volume=63; Exact Volume in Vial=55; RAW_FILE_NAME(Raw_data)=ACu02_025.mzML SUBJECT_SAMPLE_FACTORS - ACu02_026 Tracer:13C_glutamine | Genotype:WT | Treatment:Brequinar | Sample source:143B_cultured_cells Sample_code=143B_wt_brequinar_13C_glutamine_tracing_2; Biological Replicate=2; Extraction Buffer volume=63; Exact Volume in Vial=55; RAW_FILE_NAME(Raw_data)=ACu02_026.mzML SUBJECT_SAMPLE_FACTORS - ACu02_027 Tracer:13C_glutamine | Genotype:WT | Treatment:Brequinar | Sample source:143B_cultured_cells Sample_code=143B_wt_brequinar_13C_glutamine_tracing_3; Biological Replicate=3; Extraction Buffer volume=63; Exact Volume in Vial=55; RAW_FILE_NAME(Raw_data)=ACu02_027.mzML SUBJECT_SAMPLE_FACTORS - ACu02_028 Tracer:13C_glutamine | Genotype:ScURA | Treatment:Brequinar | Sample source:143B_cultured_cells Sample_code=143B_ScURA_brequinar_13C_glutamine_tracing_1; Biological Replicate=1; Extraction Buffer volume=399; Exact Volume in Vial=360; RAW_FILE_NAME(Raw_data)=ACu02_028.mzML SUBJECT_SAMPLE_FACTORS - ACu02_029 Tracer:13C_glutamine | Genotype:ScURA | Treatment:Brequinar | Sample source:143B_cultured_cells Sample_code=143B_ScURA_brequinar_13C_glutamine_tracing_2; Biological Replicate=2; Extraction Buffer volume=399; Exact Volume in Vial=360; RAW_FILE_NAME(Raw_data)=ACu02_029.mzML SUBJECT_SAMPLE_FACTORS - ACu02_030 Tracer:13C_glutamine | Genotype:ScURA | Treatment:Brequinar | Sample source:143B_cultured_cells Sample_code=143B_ScURA_brequinar_13C_glutamine_tracing_3; Biological Replicate=3; Extraction Buffer volume=399; Exact Volume in Vial=360; RAW_FILE_NAME(Raw_data)=ACu02_030.mzML SUBJECT_SAMPLE_FACTORS - ACu02_031 Tracer:13C_glutamine | Genotype:WT | Treatment:Antimycin_Myxothiazol | Sample source:143B_cultured_cells Sample_code=143B_wt_antimycin_13C_glutamine_tracing_1; Biological Replicate=1; Extraction Buffer volume=62; Exact Volume in Vial=55; RAW_FILE_NAME(Raw_data)=ACu02_031.mzML SUBJECT_SAMPLE_FACTORS - ACu02_032 Tracer:13C_glutamine | Genotype:WT | Treatment:Antimycin_Myxothiazol | Sample source:143B_cultured_cells Sample_code=143B_wt_antimycin_13C_glutamine_tracing_2; Biological Replicate=2; Extraction Buffer volume=62; Exact Volume in Vial=55; RAW_FILE_NAME(Raw_data)=ACu02_032.mzML SUBJECT_SAMPLE_FACTORS - ACu02_033 Tracer:13C_glutamine | Genotype:WT | Treatment:Antimycin_Myxothiazol | Sample source:143B_cultured_cells Sample_code=143B_wt_antimycin_13C_glutamine_tracing_3; Biological Replicate=3; Extraction Buffer volume=62; Exact Volume in Vial=55; RAW_FILE_NAME(Raw_data)=ACu02_033.mzML SUBJECT_SAMPLE_FACTORS - ACu02_034 Tracer:13C_glutamine | Genotype:ScURA | Treatment:Antimycin_Myxothiazol | Sample source:143B_cultured_cells Sample_code=143B_ScURA_antimycin_13C_glutamine_tracing_1; Biological Replicate=1; Extraction Buffer volume=232; Exact Volume in Vial=200; RAW_FILE_NAME(Raw_data)=ACu02_034.mzML SUBJECT_SAMPLE_FACTORS - ACu02_035 Tracer:13C_glutamine | Genotype:ScURA | Treatment:Antimycin_Myxothiazol | Sample source:143B_cultured_cells Sample_code=143B_ScURA_antimycin_13C_glutamine_tracing_2; Biological Replicate=2; Extraction Buffer volume=232; Exact Volume in Vial=200; RAW_FILE_NAME(Raw_data)=ACu02_035.mzML SUBJECT_SAMPLE_FACTORS - ACu02_036 Tracer:13C_glutamine | Genotype:ScURA | Treatment:Antimycin_Myxothiazol | Sample source:143B_cultured_cells Sample_code=143B_ScURA_antimycin_13C_glutamine_tracing_3; Biological Replicate=3; Extraction Buffer volume=232; Exact Volume in Vial=200; RAW_FILE_NAME(Raw_data)=ACu02_036.mzML #COLLECTION CO:COLLECTION_SUMMARY Samples obtained directly from cell culture. Cells were grown in complete DMEM CO:COLLECTION_SUMMARY medium (D5796, containing 4500 mg/L glucose, 2 mM l-glutamine) supplemented with CO:COLLECTION_SUMMARY dialyzed 10 % fetal bovine serum (FBS, Sigma F7524), 1 % penicillin-streptomycin CO:COLLECTION_SUMMARY (PenStrep, Lonza), at 37 °C in an atmosphere of 5 % CO2/95 % air. The day of CO:COLLECTION_SUMMARY the experiment metabolite extraction buffer was added directly to the cells (see CO:COLLECTION_SUMMARY Sampleprep section). CO:SAMPLE_TYPE Cultured cells #TREATMENT TR:TREATMENT_SUMMARY Complex III inhibitors (antimycin/myxothiazol 1 uM each) dissolved in EtOh were TR:TREATMENT_SUMMARY added to cell growth medium for 48 hours before metabolite extraction. Control TR:TREATMENT_SUMMARY cells were treated with the same amount of EtOh. Alternatively brequinar (1 uM) TR:TREATMENT_SUMMARY was used to inhibit DHODH. 24 hours before extraction, 2 mM of TR:TREATMENT_SUMMARY [α-¹⁵N]glutamine or [U-¹³C5]glutamine was added to the media for heavy TR:TREATMENT_SUMMARY isotope labeling. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Metabolites were extracted in Metabolite Extraction Buffer (MEB, 50% methanol, SP:SAMPLEPREP_SUMMARY 30% acetonitrile, 20% water, all LC-MS grade, valine-d8 5 μM, CK isotopes, SP:SAMPLEPREP_SUMMARY DLM-488). Cells were washed with PBS, 500 μl of cold MEB per million of cells SP:SAMPLEPREP_SUMMARY were added to the wells and the plate was immediately put at -80°C for 20 SP:SAMPLEPREP_SUMMARY minutes. The cell lysate/extraction buffer solution mixture was shake at maximum SP:SAMPLEPREP_SUMMARY speed for 15 minutes at 4°C, centrifuged at 13000g for 30 minutes at 4°C and SP:SAMPLEPREP_SUMMARY the supernatant was collected in a HPLC vial. A pooled sample was prepared by SP:SAMPLEPREP_SUMMARY taking 15 μl from each individual sample. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Metabolites were separated on a Millipore SeQuant ZIC-pHILIC analytical column CH:CHROMATOGRAPHY_SUMMARY (5 µm, 2.1 × 150 mm) coupled to a guard column (2.1 × 20 mm, 5 µm). The CH:CHROMATOGRAPHY_SUMMARY mobile phase consisted of Solvent A (20 mM ammonium carbonate with 2.5 µM CH:CHROMATOGRAPHY_SUMMARY medronic acid, adjusted to pH 9.7 with ammonium hydroxide) and Solvent B CH:CHROMATOGRAPHY_SUMMARY (acetonitrile/water, 95:5, v/v). The column oven was maintained at 40°C, and CH:CHROMATOGRAPHY_SUMMARY the autosampler was kept at 4 °C. Chromatography was performed at a flow rate CH:CHROMATOGRAPHY_SUMMARY of 0.20 mL/min with the following gradient program: 0–2 min, 85% B; 2–14 CH:CHROMATOGRAPHY_SUMMARY min, linear decrease to 30% B; 14–15 min, isocratic at 30% B; 15–17.1 min, CH:CHROMATOGRAPHY_SUMMARY re-equilibration to 85% B; and 17.1–23 min, hold at 85% B. Metabolite CH:CHROMATOGRAPHY_SUMMARY quantification was performed on a Vanquish Horizon UHPLC system coupled to CH:CHROMATOGRAPHY_SUMMARY Orbitrap Exploris 120/240 mass spectrometer (Thermo Fisher Scientific) equipped CH:CHROMATOGRAPHY_SUMMARY with a heated electrospray ionization (HESI) source. Ionization parameters were CH:CHROMATOGRAPHY_SUMMARY set to +3.5 kV for positive mode and −2.8 kV for negative mode, with an RF CH:CHROMATOGRAPHY_SUMMARY lens setting of 70, a heated capillary temperature of 320 C, and an auxiliary CH:CHROMATOGRAPHY_SUMMARY gas heater temperature of 280 C. Sheath gas flow was set to 40, auxiliary gas to CH:CHROMATOGRAPHY_SUMMARY 15, and sweep gas was disabled. For MS1 acquisition, data were collected in full CH:CHROMATOGRAPHY_SUMMARY scan mode over a mass range of m/z 70–900, using a standard automatic gain CH:CHROMATOGRAPHY_SUMMARY control (AGC) target with an automatically determined maximum injection time. CH:CHROMATOGRAPHY_SUMMARY Data were acquired with polarity switching at an Orbitrap resolution of 120,000. CH:CHROMATOGRAPHY_SUMMARY Untargeted metabolite profiling was performed using the AcquireX Deep Scan CH:CHROMATOGRAPHY_SUMMARY workflow with iterative data-dependent acquisition on pooled samples. Full scans CH:CHROMATOGRAPHY_SUMMARY were acquired at a resolution of 60,000, with MS/MS fragmentation at 30,000 CH:CHROMATOGRAPHY_SUMMARY resolution and a minimum intensity threshold of 5.0 × 10³. Dynamic exclusion CH:CHROMATOGRAPHY_SUMMARY was triggered after a single event (10 s, ±5 ppm), and precursors were isolated CH:CHROMATOGRAPHY_SUMMARY with a 1.2 m/z window. Stepped HCD energies of 30, 50, and 150 were applied, CH:CHROMATOGRAPHY_SUMMARY with mild trapping enabled to improve signal quality. CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME SeQuant ZIC-HILIC (150 x 2.1mm,5um) CH:SOLVENT_A 20 mM ammonium carbonate with 2.5 µM medronic acid, adjusted to pH 9.7 with CH:SOLVENT_A ammonium hydroxide CH:SOLVENT_B acetonitrile/water, 95:5, v/v CH:FLOW_GRADIENT 0–2 min, 85% B; 2–14 min, linear decrease to 30% B; 14–15 min, isocratic CH:FLOW_GRADIENT at 30% B; 15–17.1 min, re-equilibration to 85% B; and 17.1–23 min, hold at CH:FLOW_GRADIENT 85% B. CH:FLOW_RATE 0.20 mL/min CH:COLUMN_TEMPERATURE 40°C #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Orbitrap Exploris 240 MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS Metabolites were separated on a Millipore SeQuant ZIC-pHILIC analytical column MS:MS_COMMENTS (5 µm, 2.1 × 150 mm) coupled to a guard column (2.1 × 20 mm, 5 µm). The MS:MS_COMMENTS mobile phase consisted of Solvent A (20 mM ammonium carbonate with 2.5 µM MS:MS_COMMENTS medronic acid, adjusted to pH 9.7 with ammonium hydroxide) and Solvent B MS:MS_COMMENTS (acetonitrile/water, 95:5, v/v). The column oven was maintained at 40°C, and MS:MS_COMMENTS the autosampler was kept at 4 °C. Chromatography was performed at a flow rate MS:MS_COMMENTS of 0.20 mL/min with the following gradient program: 0–2 min, 85% B; 2–14 MS:MS_COMMENTS min, linear decrease to 30% B; 14–15 min, isocratic at 30% B; 15–17.1 min, MS:MS_COMMENTS re-equilibration to 85% B; and 17.1–23 min, hold at 85% B. Metabolite MS:MS_COMMENTS quantification was performed on a Vanquish Horizon UHPLC system coupled to MS:MS_COMMENTS Orbitrap Exploris 120/240 mass spectrometer (Thermo Fisher Scientific) equipped MS:MS_COMMENTS with a heated electrospray ionization (HESI) source. Ionization parameters were MS:MS_COMMENTS set to +3.5 kV for positive mode and −2.8 kV for negative mode, with an RF MS:MS_COMMENTS lens setting of 70, a heated capillary temperature of 320 C, and an auxiliary MS:MS_COMMENTS gas heater temperature of 280 C. Sheath gas flow was set to 40, auxiliary gas to MS:MS_COMMENTS 15, and sweep gas was disabled. For MS1 acquisition, data were collected in full MS:MS_COMMENTS scan mode over a mass range of m/z 70–900, using a standard automatic gain MS:MS_COMMENTS control (AGC) target with an automatically determined maximum injection time. MS:MS_COMMENTS Data were acquired with polarity switching at an Orbitrap resolution of 120,000. MS:MS_COMMENTS Untargeted metabolite profiling was performed using the AcquireX Deep Scan MS:MS_COMMENTS workflow with iterative data-dependent acquisition on pooled samples. Full scans MS:MS_COMMENTS were acquired at a resolution of 60,000, with MS/MS fragmentation at 30,000 MS:MS_COMMENTS resolution and a minimum intensity threshold of 5.0 × 10³. Dynamic exclusion MS:MS_COMMENTS was triggered after a single event (10 s, ±5 ppm), and precursors were isolated MS:MS_COMMENTS with a 1.2 m/z window. Stepped HCD energies of 30, 50, and 150 were applied, MS:MS_COMMENTS with mild trapping enabled to improve signal quality. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Peak Area MS_METABOLITE_DATA_START Samples ACu02_001 ACu02_002 ACu02_003 ACu02_004 ACu02_005 ACu02_006 ACu02_007 ACu02_008 ACu02_009 ACu02_010 ACu02_011 ACu02_012 ACu02_013 ACu02_014 ACu02_015 ACu02_016 ACu02_017 ACu02_018 ACu02_019 ACu02_020 ACu02_021 ACu02_022 ACu02_023 ACu02_024 ACu02_025 ACu02_026 ACu02_027 ACu02_028 ACu02_029 ACu02_030 ACu02_031 ACu02_032 ACu02_033 ACu02_034 ACu02_035 ACu02_036 Factors Tracer:15N_glutamine | Genotype:WT | Treatment:Control | Sample source:143B_cultured_cells Tracer:15N_glutamine | Genotype:WT | Treatment:Control | Sample source:143B_cultured_cells Tracer:15N_glutamine | Genotype:WT | Treatment:Control | Sample source:143B_cultured_cells Tracer:15N_glutamine | Genotype:ScURA | Treatment:Control | Sample source:143B_cultured_cells Tracer:15N_glutamine | Genotype:ScURA | Treatment:Control | Sample source:143B_cultured_cells Tracer:15N_glutamine | Genotype:ScURA | Treatment:Control | Sample source:143B_cultured_cells Tracer:15N_glutamine | Genotype:WT | Treatment:Brequinar | Sample source:143B_cultured_cells Tracer:15N_glutamine | Genotype:WT | Treatment:Brequinar | Sample source:143B_cultured_cells Tracer:15N_glutamine | Genotype:WT | Treatment:Brequinar | Sample source:143B_cultured_cells Tracer:15N_glutamine | Genotype:ScURA | Treatment:Brequinar | Sample source:143B_cultured_cells Tracer:15N_glutamine | Genotype:ScURA | Treatment:Brequinar | Sample source:143B_cultured_cells Tracer:15N_glutamine | Genotype:ScURA | Treatment:Brequinar | Sample source:143B_cultured_cells Tracer:15N_glutamine | Genotype:WT | Treatment:Antimycin_Myxothiazol | Sample source:143B_cultured_cells Tracer:15N_glutamine | Genotype:WT | Treatment:Antimycin_Myxothiazol | Sample source:143B_cultured_cells Tracer:15N_glutamine | Genotype:WT | Treatment:Antimycin_Myxothiazol | Sample source:143B_cultured_cells Tracer:15N_glutamine | Genotype:ScURA | Treatment:Antimycin_Myxothiazol | Sample source:143B_cultured_cells Tracer:15N_glutamine | Genotype:ScURA | Treatment:Antimycin_Myxothiazol | Sample source:143B_cultured_cells Tracer:15N_glutamine | Genotype:ScURA | Treatment:Antimycin_Myxothiazol | Sample source:143B_cultured_cells Tracer:13C_glutamine | Genotype:WT | Treatment:Control | Sample source:143B_cultured_cells Tracer:13C_glutamine | Genotype:WT | Treatment:Control | Sample source:143B_cultured_cells Tracer:13C_glutamine | Genotype:WT | Treatment:Control | Sample source:143B_cultured_cells Tracer:13C_glutamine | Genotype:ScURA | Treatment:Control | Sample source:143B_cultured_cells Tracer:13C_glutamine | Genotype:ScURA | Treatment:Control | Sample source:143B_cultured_cells Tracer:13C_glutamine | Genotype:ScURA | Treatment:Control | Sample source:143B_cultured_cells Tracer:13C_glutamine | Genotype:WT | Treatment:Brequinar | Sample source:143B_cultured_cells Tracer:13C_glutamine | Genotype:WT | Treatment:Brequinar | Sample source:143B_cultured_cells Tracer:13C_glutamine | Genotype:WT | Treatment:Brequinar | Sample source:143B_cultured_cells Tracer:13C_glutamine | Genotype:ScURA | Treatment:Brequinar | Sample source:143B_cultured_cells Tracer:13C_glutamine | Genotype:ScURA | Treatment:Brequinar | Sample source:143B_cultured_cells Tracer:13C_glutamine | Genotype:ScURA | Treatment:Brequinar | Sample source:143B_cultured_cells Tracer:13C_glutamine | Genotype:WT | Treatment:Antimycin_Myxothiazol | Sample source:143B_cultured_cells Tracer:13C_glutamine | Genotype:WT | Treatment:Antimycin_Myxothiazol | Sample source:143B_cultured_cells Tracer:13C_glutamine | Genotype:WT | Treatment:Antimycin_Myxothiazol | Sample source:143B_cultured_cells Tracer:13C_glutamine | Genotype:ScURA | Treatment:Antimycin_Myxothiazol | Sample source:143B_cultured_cells Tracer:13C_glutamine | Genotype:ScURA | Treatment:Antimycin_Myxothiazol | Sample source:143B_cultured_cells Tracer:13C_glutamine | Genotype:ScURA | Treatment:Antimycin_Myxothiazol | Sample source:143B_cultured_cells ATP_C10H16N5O13P3_C12 PARENT 342814233.5 360038799.2 390679080.7 235805310.5 217384545.6 231226988.6 351961344.7 319534301.9 352043644.8 270003302 252246942.9 265244854.4 284994833 343675958.9 287770302 414140119.4 467971531.5 427631378.2 ATP_C10H16N5O13P3_N15-label-1 115737482.7 129068052.6 150871380.6 100733888.7 97538707.26 96148636.74 96964103.8 88529247.47 102752594.6 113406019 120580392.7 119169347.8 68681058.63 95844724.13 84110726.2 163159348.2 194441277.6 181191129.9 ATP_C10H16N5O13P3_N15-label-2 6971937.817 8406546.735 10853789.27 8522907.065 8919878.009 8269345.21 5957992.04 5637326.842 7233504.963 9720280.178 11893068.98 10029659.49 3649923.619 6616782.363 6663362.243 13167392.93 15851234.98 16909924.69 aspartate_C4H7NO4_C12 PARENT_15N 260334334.6 242904461.8 283552969.4 154457196.7 175357436.3 119743362.5 462912662.9 450469492.5 494151826.3 114548677 163937492.7 200059418.1 104527599.1 117478055.8 96266683.02 47561007.46 69345602.42 47575201.03 aspartate_C4H7NO4_N15-label-1 39897521.6 42562515.88 49664147.53 31371850.61 33818936.66 22610417.77 83433359.35 81786948.35 94273149.92 22334278.65 37171349.68 42226044.43 16725049.95 17356319.37 17104786.27 7731475.891 11611086.64 6757357.828 glutamine_C5H10N2O3_C12 PARENT_15N 8294234411 8052375396 8721635150 7150558621 7054349814 7528760531 8101230572 8702896231 7352353775 7991301987 7844204128 7439629248 6996505065 6647089396 6330975580 9339074832 8886502248 9143599824 glutamine_C5H10N2O3_N15-label-1 3765674831 3861712986 4512364780 3273311614 3386782039 3443520383 3843109074 4145110515 3765857211 3647076429 3930241861 3497389320 2944177107 3290503995 3354055827 4287530642 4190108654 4468282200 glutamine_C5H10N2O3_N15-label-2 12763199.89 13596874.21 14996748.26 12406036.39 11551102 11956023.54 12773773.35 14408464.88 13304774.61 12567393.1 13215818.33 11314226.63 10498918.39 10717489.28 11732050.17 14995058.66 14432722.82 15399962.31 CTP_C9H16N3O14P3_C12 PARENT_15N 47039630.76 48176430.18 57101334.37 26709706.44 24051562.37 26604984.09 2223287.241 1904645.196 2186366.053 32711015.07 31653846.38 34625714.43 1825664.674 1639673.504 1663144.607 23743943.87 25816050.68 21131669.66 CTP_C9H16N3O14P3_N15-label-1 5278582.728 6129593.021 7823153.267 4082868.865 3994829.923 3929642.405 0 0 0 5045736.901 5636379.1 5844718.038 0 4813.216224 0 3069455.363 3438305.527 3012783.553 CTP_C9H16N3O14P3_N15-label-2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 GTP_C10H16N5O14P3_C12 PARENT 70978535.73 71676629.72 90004288.88 31152707.7 29172000.32 29489744.96 74403605.95 73642832.01 87234780.66 40605638.7 39001397.54 43313390.32 63363927.28 75436636.92 76464020.12 97233369.09 118004811.2 89645253.72 GTP_C10H16N5O14P3_N15-label-1 11147182.11 11725926.25 16665960.29 6048011.623 6122367.119 5757595.09 5882520.175 6042037.998 7577513.21 8462248.061 8986762.572 9353693.146 3518074.665 5836755.414 6403100.428 18647709.82 23970770.14 18286750.79 GTP_C10H16N5O14P3_N15-label-2 7775.238 0 11940.87547 0 0 0 0 0 6828.473738 0 0 0 0 150913.5529 0 0 0 0 UTP_C9H15N2O15P3_C12 PARENT_15N 36341809.36 37891190.01 44309876.68 25382548.77 22095575.48 23943649.14 3722162.367 3392075.86 4283459.724 26208901.96 26821868.73 29682506.11 1902658.918 1785495.638 2062793.946 22080793.62 24290488.35 20173056.43 UTP_C9H15N2O15P3_N15-label-1 4682307.299 5507897.691 6292175.395 4430520.263 3453072.757 4243661.016 25451.11687 0 0 3226276.911 5204202.565 5332672.258 0 6981.282818 0 3201343.839 3218591.164 2738779.239 UTP_C9H15N2O15P3_N15-label-2 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2-oxoglutarate_C5H6O5_C12 PARENT 4078762.377 2397137.698 3926742.849 41798618.4 30812006.68 24103335.98 850374.1572 325307.3615 224757.9632 49180141.8 30665907.43 34898021.3 2379524.742 6550239.385 3514366.759 48745390.15 93725744.75 47215717.89 2-oxoglutarate_C5H6O5_C13-label-1 0 0 0 515501.7699 417625.007 194276.557 0 0 0 1577019.762 1317815.624 1457552.989 0 0 0 581968.666 3081105.246 0 2-oxoglutarate_C5H6O5_C13-label-2 0 0 244130.6278 0 0 263744.8291 186275.8944 0 0 0 0 214477.9769 0 0 0 0 194707.7705 478011.4486 2-oxoglutarate_C5H6O5_C13-label-3 0 0 0 1575889.213 1280567.209 729944.3941 0 0 0 1551390.438 1074371.209 1275938.653 0 0 0 181935.2377 0 0 2-oxoglutarate_C5H6O5_C13-label-4 0 0 0 0 0 0 0 216522.1553 0 0 0 0 0 0 0 0 302677.4664 0 2-oxoglutarate_C5H6O5_C13-label-5 503042.3024 1095792.685 844048.6747 14705401.48 11831855.42 8413620.635 224632.9477 0 0 15125174.44 12409202.31 12690998.31 0 1750598.351 1160646.078 23499011.92 52121962.12 22811214.97 aspartate_C4H7NO4_C12 PARENT_13C 77177964.6 83865445.87 82928653.02 70320438.59 65421813.63 74209006.89 124849276.8 146222226 144583046 63594771.67 62219671.43 71796013.11 19169787.72 17093288.65 20753079.04 17243143.71 17980295.04 15287566.37 aspartate_C4H7NO4_C13-label-1 4467060.659 5149446.873 4386798.626 3428607.496 3493004.603 4273593.197 5135591.911 6512263.733 6977065.539 3047116.807 3527159.932 4120616.161 535096.5061 317949.1465 722209.6884 432952.1784 601839.9959 356028.0136 aspartate_C4H7NO4_C13-label-2 3971481.985 5196855.923 4934886.827 4378367.252 3519538.511 4369206.725 2206585.499 2947956.161 3065012.694 3482785.246 3862234.575 4295633.218 54541.22637 26582.64912 31710.74222 46547.96112 94928.43901 18642.64148 aspartate_C4H7NO4_C13-label-3 2253055.023 3485030.257 2700243.947 2967727.798 3224775.502 3581375.455 8043649.689 8528345.994 10135575.65 2587733.083 3116806.393 3480572.772 2331312.187 1979247.248 2734931.653 3637107.24 4678267.337 3110161.872 aspartate_C4H7NO4_C13-label-4 18963353.7 22756535.9 23054809.19 19190510.54 19044301.4 20916430.78 26126887.41 27932478.86 32919266.06 17022121.78 17925019.84 19325950.66 24820.04836 0 37782.86096 128506.8489 180329.4697 88950.49718 citrate_C6H8O7_C12 PARENT 1423150223 922869013.5 1236983786 1019729200 965906274.5 631465610.1 901319942.8 647403464.1 553247269.6 1045906609 668141628.9 993307637.6 801661188.5 1493237652 696006781.7 544688899.4 517507331.1 422919193.1 citrate_C6H8O7_C13-label-1 90317895.37 60578376.76 83738984.61 71443520.27 75135152.94 40655282.06 50381936.12 33072349.13 30496250.49 76743571.95 44749724.17 70432659.71 42776446.63 80806201.56 36306692.62 26241379.83 23757364.83 17525342.41 citrate_C6H8O7_C13-label-2 26054593.22 30051497.77 43526137.77 43287976.67 47102654.1 29630117.91 1268011.089 274840.3006 2422871.876 41304866.4 28600866.08 45633772.48 0 0 0 287344.0265 0 236895.695 citrate_C6H8O7_C13-label-3 23394703.15 28503957.12 34896322.69 48062031.39 50514112.29 35810496.76 4800545.923 3519808.746 10219884.91 41415163.43 34083732.38 46115792.98 0 0 0 772954.5736 610889.5291 941763.5726 citrate_C6H8O7_C13-label-4 115838783.1 115939412.8 177265747.4 153654320.8 169593895.9 104728732.4 13371810.47 10155409.26 21618024.03 135233529.2 112859278.9 152822242.8 1172263.946 261336.4803 460036.8548 4242059.697 1852894.384 1412579.616 citrate_C6H8O7_C13-label-5 15684339.3 12379681.08 19379372.03 48658697.76 50818981.78 34068142.86 19831703.36 19242583.39 30917151.85 39766264.5 29686305.86 41340958.62 12493832.58 10282114.06 13257406.84 91868062.92 108980701.6 100874172.8 citrate_C6H8O7_C13-label-6 0 0 0 0 0 207366.6674 0 0 0 0 223963.4616 0 209959.6663 0 185685.0789 0 0 233207.561 glutamate_C5H9NO4_C12 PARENT 1290320417 1252211170 1330434982 1356402075 1310120481 1374717496 952224345.7 955713078.7 1063452623 1363916743 1228926869 1337565473 868820133.7 851558745.5 914707674.4 1539650772 1693956333 1553417442 glutamate_C5H9NO4_C13-label-1 86799000.51 84356222.55 90658502.06 88557157.32 87980264.78 91183392.87 54521918.39 54571156.5 59572596.2 92401761.26 82273566.8 91567151.09 45819425.97 46875634.29 51129390.54 82939327.72 90540791.63 87512349.46 glutamate_C5H9NO4_C13-label-2 18422860.51 19468689.96 21656258.46 22622471.41 22554449.11 22735472.82 6841168.609 7420578 8658531.128 22999028.21 22084230.36 24628241.14 246839.6188 217325.2121 299129.0426 1764899.487 1401376.929 1782835.475 glutamate_C5H9NO4_C13-label-3 103081467.7 107059560.1 115556325.3 105843387.3 107958413.6 110502749 32006706.46 32743715.53 38057952.12 103755124.4 106170578.7 108386618.2 127859.3989 0 89205.44201 388783.5447 1508484.201 1027744.561 glutamate_C5H9NO4_C13-label-4 21374247.65 22566178.09 24093245.82 24173939.82 24625879.64 24917246.49 17003969.01 18013114.19 20092546.3 21992354.24 21848757.29 22649035.82 16274784.97 18375684.47 19105655.49 32125022.59 36340491.5 33290405.21 glutamate_C5H9NO4_C13-label-5 507667972.3 541910783 579637910.7 577021731.3 585465699.2 597511209.3 444620962.9 472635081.8 540349509.2 545623627.6 548754452 562325881.6 453504295.4 484305845.5 530314495.9 853397532.1 1001902710 838884510.5 glutamine_C5H10N2O3_C12 PARENT_13C 8634214346 7417284992 7647013877 7036510225 6653487026 6966456653 7219658602 6751254874 6827529090 7294266409 6511567083 7110953444 6548769942 8303171795 6522315239 7929895246 8363103450 7620989022 glutamine_C5H10N2O3_C13-label-1 490866448.7 422861788.6 432734129.3 400582561.6 373770969.1 394457057.2 410951296.2 378192128.4 387015511.2 415559218.8 369927044.5 398984174.9 368003615.7 466584740.3 371446806.4 447326482.9 471056065.9 432940141.4 glutamine_C5H10N2O3_C13-label-2 9154892.968 7424243.152 7698381.672 5964460.529 8008583.043 7911231.242 7734879.71 7309795.243 7234290.039 8308989.759 8145485.762 7627427.694 6142324.201 7971980.381 6690426.035 7550574.836 9189129.335 7590068.132 glutamine_C5H10N2O3_C13-label-3 5802618.103 6465435.132 6398722.489 6466428.954 7342121.988 7121679.483 4288806.582 6053001.553 5876472.69 6829102.734 7554003.967 7065168.48 1717017.069 2602875.825 1730073.498 2097642.364 2966317.656 2382104.444 glutamine_C5H10N2O3_C13-label-4 186563736.6 176500226 184079032.7 154284103 157607522.8 158304717.5 156117421.8 154186277.6 156306782.3 158729320.8 154987928.2 159120337.6 138146154.2 192312933.3 152298626.2 177139962.3 198122374.9 168723259 glutamine_C5H10N2O3_C13-label-5 4657731292 4382085299 4539812963 3830112166 3885542215 3883656975 3801313415 3802015378 3919213906 3880083262 3815909537 3929456385 3378402266 4752028573 3762181461 4438783413 4936203093 4119105707 malate_C4H6O5_C12 PARENT 116427126.5 123464012.5 131961242.1 377892926.5 416512247.1 405449856.9 72094740.69 74678856.96 86367424.23 371654221.3 360753264 381629643.4 15696702.68 22560621.82 18521160.81 173978924.9 273452605.4 184842789.3 malate_C4H6O5_C13-label-1 2447036.405 2969458.054 4060009.679 16973073.75 19872533.94 20784730.06 0 1479817.636 760426.9511 19275589.66 18304875.7 20404777.65 0 0 0 3752566.197 7648848.999 4789867.446 malate_C4H6O5_C13-label-2 3484619.741 3884968.209 4210297.779 19827501.46 20459807.32 21892729.66 0 0 0 16549022.27 22463511.66 21625582.44 0 0 0 921364.5885 3186472.783 0 malate_C4H6O5_C13-label-3 2022043.478 4105511.74 3498314.858 18728774.6 18583115.61 21340186 1600387.873 1559323.829 4055628.246 14707491.15 19476888.62 18785399.27 0 0 0 37467738.19 68469313.28 40152636.73 malate_C4H6O5_C13-label-4 19736603.38 26367062.32 27872784.22 93725513.79 113858076.1 103404606.7 13627021.52 11942818.9 14939132.8 86556341.28 94846165.09 91653302.74 0 0 0 0 2443662.334 684858.593 2-HG_C5H8O5_C12 PARENT 5674626.266 7891022.95 7954014.203 27825876.63 28307091.68 27113457.86 7434625.023 10232640 9610409.982 30849835.46 27879974.02 30413536.27 35072286.12 29238706.26 37889726.86 160053316.3 228933070.2 176476634.2 2-HG_C5H8O5_C13-label-1 183935.2121 0 0 203370.3701 451214.2335 0 0 0 0 258198.4774 242161.2827 241917.1791 452230.9964 370914.255 204124.2021 6006600.286 8466408.843 6503970.45 2-HG_C5H8O5_C13-label-2 209056.2606 0 0 265655.7985 0 0 0 0 0 163703.3622 0 0 0 0 0 0 0 0 2-HG_C5H8O5_C13-label-3 318993.6068 236221.6461 841662.5887 228083.5277 292277.5178 235148.5578 217397.0915 233868.5038 0 1405543.371 330224.5243 201766.5681 203804.9615 0 0 234771.3596 616571.6889 242412.9698 2-HG_C5H8O5_C13-label-4 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 2142287.552 1262059.976 2-HG_C5H8O5_C13-label-5 355813.3706 1035711.712 940561.2501 9568507.09 9232017.423 11131751.84 1115059.868 933579.1296 2427742.147 10982714.22 11896815.91 10199922.22 15398318.16 15204941.64 19877981.87 90669056.08 131655842.4 94511889.89 UTP_C9H15N2O15P3_C12 PARENT_13C 30385694.87 24070939.52 34835452.92 29034103.91 29058829.65 24094156.29 3864571.759 1959987.537 2040590.882 27602066 23260982.07 29752965.22 3702586.415 5079802.697 2339815.729 20256467.51 14804089.78 16096604.51 UTP_C9H15N2O15P3_C13-label-1 3842154.044 3256358.885 4605262.086 3939837.464 4085658.445 3150555.602 347180.2439 161153.8715 99195.93262 3545905.322 3006426.412 3992709.803 307592.3278 412374.944 143370.4759 1791170.337 1252689.79 1422266.637 UTP_C9H15N2O15P3_C13-label-2 1758224.073 1409230.626 2072306.386 1967590.026 2203555.521 1698030.323 39226.4087 0 0 1871163.729 1594919.353 2195633.243 27054.38983 55944.43774 9010.051228 2559682.93 1871630.609 2035078.841 UTP_C9H15N2O15P3_C13-label-3 6888818.611 6060956.077 8898087.393 7834236.873 8489237.216 6525689.946 140606.8116 60279.08982 28357.02019 7141386.62 6566950.417 7932359.344 0 12469.12489 21542.13528 2663090.942 2098740.109 2091408.233 UTP_C9H15N2O15P3_C13-label-4 394592.8781 348186.8563 539180.041 522294.8572 440313.8743 367912.9335 0 0 0 422980.388 374981.471 485297.4012 0 0 0 141286.8099 18930.29715 102689.5566 UTP_C9H15N2O15P3_C13-label-5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 UTP_C9H15N2O15P3_C13-label-6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 UTP_C9H15N2O15P3_C13-label-7 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 UTP_C9H15N2O15P3_C13-label-8 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 UTP_C9H15N2O15P3_C13-label-9 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 Carbamoyl aspartate_C5H8N2O5_C12 PARENT 84369.30204 165913.0172 876233.7877 0 6551.017559 0 299276698.1 305980734.5 352657854 10501.60426 14607.89316 0 180244778.5 170195519.4 191097348 35781.15946 67059.12115 8163.603722 Carbamoyl aspartate_C5H8N2O5_C13-label-1 0 0 0 0 0 0 18152843.01 19017665.65 21960037.41 0 0 0 9908619.458 9182023.576 10475361.93 0 0 0 Carbamoyl aspartate_C5H8N2O5_C13-label-2 0 0 0 0 0 0 7466677.826 8276825.21 9937302.005 0 0 0 1109711.787 1253200.015 1400318.125 0 0 0 Carbamoyl aspartate_C5H8N2O5_C13-label-3 0 0 11489.61637 0 0 0 19293296.65 21631492.56 25064667.2 0 0 0 26121848.24 27110275.06 31197183.06 0 0 0 Carbamoyl aspartate_C5H8N2O5_C13-label-4 25733.75351 11465.31802 10261.17187 0 0 0 67697342.33 73982889.08 86580913.83 0 0 0 1484881.036 1652615.102 1880613.298 0 0 0 Carbamoyl aspartate_C5H8N2O5_C13-label-5 0 0 0 0 0 0 604440.2005 662915.2284 884056.382 0 0 5881.687231 0 0 0 0 0 0 CTP_C9H16N3O14P3_C12 PARENT_13C 35327952.36 30766061.46 42563513.45 34851489.51 34343038.99 24685896.27 2146359.882 1748323.968 1902807.245 31476529.81 26286988.73 35735245.91 1398834.547 2010217.881 1482654.483 23118848.14 17083207.23 18849092.6 CTP_C9H16N3O14P3_C13-label-1 4384615.603 3718800.371 5336034.336 4674679.155 4532927.514 3172848.353 113140.0484 60822.53782 53391.81176 3933507.304 3518397.685 4796276.55 44892.17752 55571.26691 38431.10748 1990858.52 1512765.73 1724385.564 CTP_C9H16N3O14P3_C13-label-2 1825334.367 1707012.122 2280973.109 2289551.641 2374166.565 1642081.113 7760.230698 0 0 2089498.939 1838682.033 2395916.534 0 0 0 2392918.261 1970130.175 2093895.53 CTP_C9H16N3O14P3_C13-label-3 7246380.421 6938071.163 9927399.511 8885365.245 9426054.494 6386358.367 27953.63757 0 0 7785987.478 7057265.726 9220738.086 8255.617488 0 0 2488773.123 2011750.617 2146265.972 CTP_C9H16N3O14P3_C13-label-4 396741.5159 347311.6806 536446.4998 476852.051 480058.5952 368419.8151 0 0 0 450092.8999 393364.3535 553240.8522 0 0 0 111891.8478 86541.75179 69851.00414 CTP_C9H16N3O14P3_C13-label-5 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 CTP_C9H16N3O14P3_C13-label-6 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 CTP_C9H16N3O14P3_C13-label-7 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 CTP_C9H16N3O14P3_C13-label-8 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 CTP_C9H16N3O14P3_C13-label-9 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 0 succinate_C4H6O4_C12 PARENT 57346207.57 59006701.31 52497834.39 31862517.72 33088661.98 36902521.11 47999643.61 58234360.28 50297487.4 33009660.49 32726712.03 35500383.81 179269877.9 177747817.3 193319958.1 431847128.6 503274628.5 423468982.5 succinate_C4H6O4_C13-label-1 0 0 0 0 364346.3347 0 0 0 166100.3192 0 0 0 2928463.364 3311094.298 3741947.023 13654573.89 18404032.66 12332279.14 succinate_C4H6O4_C13-label-2 262956.0326 0 0 0 193281.5037 0 202076.5727 0 0 0 206320.8213 0 0 0 0 199137.7622 464359.5467 268731.0445 succinate_C4H6O4_C13-label-3 0 0 0 0 0 0 0 0 0 0 0 0 887820.909 395093.5594 1710523.977 33017390.84 40196495.04 32128231.06 succinate_C4H6O4_C13-label-4 4966840.033 2502378.041 2792061.551 1643600.871 1510648.328 5064432.603 719973.0467 0 1744684.925 2118009.32 2322796.528 1499499.462 49405406.87 60096638.32 66186001.42 127823026.8 152922213.4 123067976.1 dihydroorotate_C5H6N2O4_C12 PARENT 222039.1161 210708.965 720175.4265 0 0 0 1414410305 1343939172 1670121019 0 0 0 1054676041 1093442793 946499262.6 176742.6295 0 160248.6006 dihydroorotate_C5H6N2O4_C13-label-1 186040.3409 211028.9514 0 0 0 0 72670358.9 66749691.56 84744199.19 207817.6757 0 0 23041598.61 43337596.6 26836628.47 263186.1056 184171.7158 0 dihydroorotate_C5H6N2O4_C13-label-2 0 0 0 0 0 0 26376733.08 28616181.29 34604768.78 0 0 0 2056487.032 1862083.946 400721.0376 0 0 0 dihydroorotate_C5H6N2O4_C13-label-3 0 0 0 0 0 0 81811952.31 78975533.36 106633734.9 0 0 0 141377869.6 161694361.7 141400778.6 0 0 0 dihydroorotate_C5H6N2O4_C13-label-4 0 198230.8913 0 0 0 0 301432025.8 307378425.7 388607121.9 0 0 0 1136556.638 2427792.141 2136870.36 0 0 0 dihydroorotate_C5H6N2O4_C13-label-5 0 0 0 0 0 0 284279.0811 0 200338.3008 0 0 0 0 0 0 0 0 220985.7732 CAR 2:0_C9H17NO4_C12 PARENT 33503695.2 31564367.26 34883861.4 12335137.92 10903293.89 13008742.81 50759428.07 50597193.87 61266394.13 12189011.63 11055409.47 12584467.42 61687966.64 57384799.67 63799813.03 38069203.29 24872372.07 21312427.38 CAR 2:0_C9H17NO4_C13-label-1 2690654.95 2446388.513 2842605.921 710192.3883 726993.3025 902677.9137 4482775.001 4522025.789 5841380.535 754698.6795 597920.6495 762591.5625 5632111.069 4824094.708 5612376.38 2894200.216 1925304.382 1667940.357 CAR 2:0_C9H17NO4_C13-label-2 19381.37126 16963.76498 22060.77545 0 10905.55882 0 383965.1766 312712.2818 594908.1121 0 0 12000.47773 2120405.812 2157027.39 2665526.193 1400904.936 783826.4123 679939.4738 2-SC_C7H11NO6S_C12 PARENT 514959.8726 425977.3094 668079.9071 1069043.306 1003378.689 736858.7511 430927.5253 531609.9034 444171.5242 893418.8825 690630.8165 1149675.35 143086.2091 194556.3556 94092.2115 914395.0787 847858.9158 896295.1711 2-SC_C7H11NO6S_C13-label-1 0 11090.75028 14754.37259 39306.38883 45129.76046 22894.5724 7242.617798 0 0 46064.29515 7462.480955 41136.84483 0 0 0 15153.06101 10171.74175 16092.78438 2-SC_C7H11NO6S_C13-label-2 0 0 6403.583315 11572.85419 13971.57127 0 0 0 0 0 0 0 0 0 0 0 0 0 2-SC_C7H11NO6S_C13-label-3 6129.459834 0 0 0 0 0 0 0 0 7382.83596 0 7162.150176 0 0 0 120431.2036 125630.5141 97071.39755 2-SC_C7H11NO6S_C13-label-4 80379.30186 17564.38422 107199.4716 184216.4846 236780.8772 160520.4362 22243.45546 7714.907615 8113.995759 139348.5223 121927.6271 171199.7223 0 0 0 7261.246783 0 6143.543757 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name ATP_C10H16N5O13P3_C12 PARENT ATP_C10H16N5O13P3_N15-label-1 ATP_C10H16N5O13P3_N15-label-2 aspartate_C4H7NO4_C12 PARENT_15N aspartate_C4H7NO4_N15-label-1 glutamine_C5H10N2O3_C12 PARENT_15N glutamine_C5H10N2O3_N15-label-1 glutamine_C5H10N2O3_N15-label-2 CTP_C9H16N3O14P3_C12 PARENT_15N CTP_C9H16N3O14P3_N15-label-1 CTP_C9H16N3O14P3_N15-label-2 GTP_C10H16N5O14P3_C12 PARENT GTP_C10H16N5O14P3_N15-label-1 GTP_C10H16N5O14P3_N15-label-2 UTP_C9H15N2O15P3_C12 PARENT_15N UTP_C9H15N2O15P3_N15-label-1 UTP_C9H15N2O15P3_N15-label-2 2-oxoglutarate_C5H6O5_C12 PARENT 2-oxoglutarate_C5H6O5_C13-label-1 2-oxoglutarate_C5H6O5_C13-label-2 2-oxoglutarate_C5H6O5_C13-label-3 2-oxoglutarate_C5H6O5_C13-label-4 2-oxoglutarate_C5H6O5_C13-label-5 aspartate_C4H7NO4_C12 PARENT_13C aspartate_C4H7NO4_C13-label-1 aspartate_C4H7NO4_C13-label-2 aspartate_C4H7NO4_C13-label-3 aspartate_C4H7NO4_C13-label-4 citrate_C6H8O7_C12 PARENT citrate_C6H8O7_C13-label-1 citrate_C6H8O7_C13-label-2 citrate_C6H8O7_C13-label-3 citrate_C6H8O7_C13-label-4 citrate_C6H8O7_C13-label-5 citrate_C6H8O7_C13-label-6 glutamate_C5H9NO4_C12 PARENT glutamate_C5H9NO4_C13-label-1 glutamate_C5H9NO4_C13-label-2 glutamate_C5H9NO4_C13-label-3 glutamate_C5H9NO4_C13-label-4 glutamate_C5H9NO4_C13-label-5 glutamine_C5H10N2O3_C12 PARENT_13C glutamine_C5H10N2O3_C13-label-1 glutamine_C5H10N2O3_C13-label-2 glutamine_C5H10N2O3_C13-label-3 glutamine_C5H10N2O3_C13-label-4 glutamine_C5H10N2O3_C13-label-5 malate_C4H6O5_C12 PARENT malate_C4H6O5_C13-label-1 malate_C4H6O5_C13-label-2 malate_C4H6O5_C13-label-3 malate_C4H6O5_C13-label-4 2-HG_C5H8O5_C12 PARENT 2-HG_C5H8O5_C13-label-1 2-HG_C5H8O5_C13-label-2 2-HG_C5H8O5_C13-label-3 2-HG_C5H8O5_C13-label-4 2-HG_C5H8O5_C13-label-5 UTP_C9H15N2O15P3_C12 PARENT_13C UTP_C9H15N2O15P3_C13-label-1 UTP_C9H15N2O15P3_C13-label-2 UTP_C9H15N2O15P3_C13-label-3 UTP_C9H15N2O15P3_C13-label-4 UTP_C9H15N2O15P3_C13-label-5 UTP_C9H15N2O15P3_C13-label-6 UTP_C9H15N2O15P3_C13-label-7 UTP_C9H15N2O15P3_C13-label-8 UTP_C9H15N2O15P3_C13-label-9 Carbamoyl aspartate_C5H8N2O5_C12 PARENT Carbamoyl aspartate_C5H8N2O5_C13-label-1 Carbamoyl aspartate_C5H8N2O5_C13-label-2 Carbamoyl aspartate_C5H8N2O5_C13-label-3 Carbamoyl aspartate_C5H8N2O5_C13-label-4 Carbamoyl aspartate_C5H8N2O5_C13-label-5 CTP_C9H16N3O14P3_C12 PARENT_13C CTP_C9H16N3O14P3_C13-label-1 CTP_C9H16N3O14P3_C13-label-2 CTP_C9H16N3O14P3_C13-label-3 CTP_C9H16N3O14P3_C13-label-4 CTP_C9H16N3O14P3_C13-label-5 CTP_C9H16N3O14P3_C13-label-6 CTP_C9H16N3O14P3_C13-label-7 CTP_C9H16N3O14P3_C13-label-8 CTP_C9H16N3O14P3_C13-label-9 succinate_C4H6O4_C12 PARENT succinate_C4H6O4_C13-label-1 succinate_C4H6O4_C13-label-2 succinate_C4H6O4_C13-label-3 succinate_C4H6O4_C13-label-4 dihydroorotate_C5H6N2O4_C12 PARENT dihydroorotate_C5H6N2O4_C13-label-1 dihydroorotate_C5H6N2O4_C13-label-2 dihydroorotate_C5H6N2O4_C13-label-3 dihydroorotate_C5H6N2O4_C13-label-4 dihydroorotate_C5H6N2O4_C13-label-5 CAR 2:0_C9H17NO4_C12 PARENT CAR 2:0_C9H17NO4_C13-label-1 CAR 2:0_C9H17NO4_C13-label-2 2-SC_C7H11NO6S_C12 PARENT 2-SC_C7H11NO6S_C13-label-1 2-SC_C7H11NO6S_C13-label-2 2-SC_C7H11NO6S_C13-label-3 2-SC_C7H11NO6S_C13-label-4 METABOLITES_END #END