#METABOLOMICS WORKBENCH IsraelPerezChavez_20240504_051440 DATATRACK_ID:4816 STUDY_ID:ST003191 ANALYSIS_ID:AN005239 PROJECT_ID:PR001987 VERSION 1 CREATED_ON May 7, 2024, 5:43 am #PROJECT PR:PROJECT_TITLE Fructose 1,6-bisphosphate quantification in HepG2 liver cancer cell line PR:PROJECT_TYPE LC/MS Quantitative Analysis PR:PROJECT_SUMMARY Quantification of specific fructose 1,6-bisphosphate (FBP) concentrations in PR:PROJECT_SUMMARY HepG2 cells. A minimum of 1 million cells per replicate and condition will be PR:PROJECT_SUMMARY grown and treated. Glucose-starved cells will be either treated with 10 mM PR:PROJECT_SUMMARY glucose, 2.5 μM Oligomycin, 50 mM 2-deoxy-D-glucose (2-DG), or not treated. PR:PROJECT_SUMMARY Cells will be lysed and processed for FBP quantification using an isotope FBP PR:PROJECT_SUMMARY standard by LC/MS. PR:INSTITUTE Vrije Universiteit Brussel PR:DEPARTMENT VIB-VUB Center for Structural Biology PR:LABORATORY RedOx Laboratory PR:LAST_NAME Perez Chavez PR:FIRST_NAME Israel PR:ADDRESS Boulevard de la Plaine 2 PR:EMAIL israel.perez.chavez@vub.be PR:PHONE +32 474160756 #STUDY ST:STUDY_TITLE Fructose 1,6-bisphosphate quantification in HepG2 liver cancer cell line. ST:STUDY_TYPE LC/MS Quantitative Analysis ST:STUDY_SUMMARY Quantification of specific fructose 1,6-bisphosphate (FBP) concentrations in ST:STUDY_SUMMARY HepG2 cells. A minimum of 1 million cells per replicate and condition will be ST:STUDY_SUMMARY grown and treated. Glucose-starved cells will be either treated with 10 mM ST:STUDY_SUMMARY glucose, 2.5 μM Oligomycin, 50 mM 2-deoxy-D-glucose (2-DG), or not treated. ST:STUDY_SUMMARY Cells will be lysed and processed for FBP quantification using an isotope FBP ST:STUDY_SUMMARY standard by LC/MS. ST:INSTITUTE Vrije Universiteit Brussel ST:DEPARTMENT VIB-VUB Center for Structural Biology ST:LABORATORY RedOx Laboratory ST:LAST_NAME Perez Chavez ST:FIRST_NAME Israel ST:ADDRESS Pleinlaan 2, building E, 4th floor ST:EMAIL israel.perez.chavez@vub.be ST:PHONE +32 474160756 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:GENDER Male SU:CELL_STRAIN_DETAILS HepG2 Cells #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 IP01 MCF001881_IP01 Sample source:HepG2 Liver Cancer Cell Line | Treatment:Control RAW_FILE_NAME=MCF001881_IP01.mzML SUBJECT_SAMPLE_FACTORS IP02 MCF001881_IP02 Sample source:HepG2 Liver Cancer Cell Line | Treatment:Control RAW_FILE_NAME=MCF001881_IP02.mzML SUBJECT_SAMPLE_FACTORS IP03 MCF001881_IP03 Sample source:HepG2 Liver Cancer Cell Line | Treatment:Control RAW_FILE_NAME=MCF001881_IP03.mzML SUBJECT_SAMPLE_FACTORS IP04 MCF001881_IP04 Sample source:HepG2 Liver Cancer Cell Line | Treatment:10 mM Glucose RAW_FILE_NAME=MCF001881_IP04.mzML SUBJECT_SAMPLE_FACTORS IP05 MCF001881_IP05 Sample source:HepG2 Liver Cancer Cell Line | Treatment:10 mM Glucose RAW_FILE_NAME=MCF001881_IP05.mzML SUBJECT_SAMPLE_FACTORS IP06 MCF001881_IP06 Sample source:HepG2 Liver Cancer Cell Line | Treatment:10 mM Glucose RAW_FILE_NAME=MCF001881_IP06.mzML SUBJECT_SAMPLE_FACTORS IP07 MCF001881_IP07 Sample source:HepG2 Liver Cancer Cell Line | Treatment:2.5 µM Oligomycin RAW_FILE_NAME=MCF001881_IP07.mzML SUBJECT_SAMPLE_FACTORS IP08 MCF001881_IP08 Sample source:HepG2 Liver Cancer Cell Line | Treatment:2.5 µM Oligomycin RAW_FILE_NAME=MCF001881_IP08.mzML SUBJECT_SAMPLE_FACTORS IP09 MCF001881_IP09 Sample source:HepG2 Liver Cancer Cell Line | Treatment:2.5 µM Oligomycin RAW_FILE_NAME=MCF001881_IP09.mzML SUBJECT_SAMPLE_FACTORS IP10 MCF001881_IP10 Sample source:HepG2 Liver Cancer Cell Line | Treatment:50 mM 2-Deoxy-D-Glucose RAW_FILE_NAME=MCF001881_IP010.mzML SUBJECT_SAMPLE_FACTORS IP11 MCF001881_IP11 Sample source:HepG2 Liver Cancer Cell Line | Treatment:50 mM 2-Deoxy-D-Glucose RAW_FILE_NAME=MCF001881_IP011.mzML SUBJECT_SAMPLE_FACTORS IP12 MCF001881_IP12 Sample source:HepG2 Liver Cancer Cell Line | Treatment:50 mM 2-Deoxy-D-Glucose RAW_FILE_NAME=MCF001881_IP012.mzML #COLLECTION CO:COLLECTION_SUMMARY Cells were trypsinized, washed, counted, centrifuged, resuspended in extraction CO:COLLECTION_SUMMARY buffer, centrifuged, and the supernatant stored at -80°C. CO:COLLECTION_PROTOCOL_FILENAME FBPquantificationMethod.pdf CO:SAMPLE_TYPE HepG2 cells #TREATMENT TR:TREATMENT_SUMMARY Cells were glucose starved for 1 h. Then, it involved the addition of 10 mM TR:TREATMENT_SUMMARY glucose, 2.5 μM of oligomycin, and 50 mM of 2-DG. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY The cell pellet was resuspended in 100 µL of extraction buffer (80% SP:SAMPLEPREP_SUMMARY Methanol/20% mQ water + 2 µM Fructose-1,6-bisphosphate (U-¹³C₆). Cells were SP:SAMPLEPREP_SUMMARY then incubated for 3 min on ice and then spun at 20.000 x g 4°C for 20 min. The SP:SAMPLEPREP_SUMMARY supernatant was collected and stored at -80°C until metabolomics analysis. This SP:SAMPLEPREP_SUMMARY procedure was performed in control, after glucose addition, oligomycin and 2-DG SP:SAMPLEPREP_SUMMARY addition. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Dionex Ultimate 3000 CH:COLUMN_NAME Waters ACQUITY UPLC HSS T3 (150 x 2.1mm,1.8um) CH:SOLVENT_A 95% milliQ Water, 5% Methanol; 10 mM Tertiary Butyl Alcohol and 15 mM acetic acid CH:SOLVENT_B 100% methanol CH:FLOW_GRADIENT 0-13 min: 0% B; 13-14 min: 33.33% B; 14-25 min: 36.4% B CH:FLOW_RATE 0.25 mL/min CH:COLUMN_TEMPERATURE 40℃ #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS The mass spectrometer operated in full scan (range [70.0000-1050.0000]) and AGC MS:MS_COMMENTS target was set at 3.0E+006 using a resolution of 140000. Data collection was MS:MS_COMMENTS performed using the Xcalibur software (Thermo Scientific). The data analyses MS:MS_COMMENTS were performed by integrating the peak areas (El-Maven – Polly - Elucidata). #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS µM MS_METABOLITE_DATA_START Samples MCF001881_IP01 MCF001881_IP02 MCF001881_IP03 MCF001881_IP04 MCF001881_IP05 MCF001881_IP06 MCF001881_IP07 MCF001881_IP08 MCF001881_IP09 MCF001881_IP10 MCF001881_IP11 MCF001881_IP12 Factors Sample source:HepG2 Liver Cancer Cell Line | Treatment:Control Sample source:HepG2 Liver Cancer Cell Line | Treatment:Control Sample source:HepG2 Liver Cancer Cell Line | Treatment:Control Sample source:HepG2 Liver Cancer Cell Line | Treatment:10 mM Glucose Sample source:HepG2 Liver Cancer Cell Line | Treatment:10 mM Glucose Sample source:HepG2 Liver Cancer Cell Line | Treatment:10 mM Glucose Sample source:HepG2 Liver Cancer Cell Line | Treatment:2.5 µM Oligomycin Sample source:HepG2 Liver Cancer Cell Line | Treatment:2.5 µM Oligomycin Sample source:HepG2 Liver Cancer Cell Line | Treatment:2.5 µM Oligomycin Sample source:HepG2 Liver Cancer Cell Line | Treatment:50 mM 2-Deoxy-D-Glucose Sample source:HepG2 Liver Cancer Cell Line | Treatment:50 mM 2-Deoxy-D-Glucose Sample source:HepG2 Liver Cancer Cell Line | Treatment:50 mM 2-Deoxy-D-Glucose Fructose 1,6-bisphosphate 1.187542578 1.123707678 1.484017165 2.507285828 3.474174246 14.20166452 3.836068809 8.328037993 3.876905473 1.108010354 0.858685455 0.770074173 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name PubChem Id KEGG Id Fructose 1,6-bisphosphate 172313 - METABOLITES_END #END