#METABOLOMICS WORKBENCH aizhantkirova_20251021_072705 DATATRACK_ID:6576 STUDY_ID:ST004322 ANALYSIS_ID:AN007201 PROJECT_ID:PR002736 VERSION 1 CREATED_ON October 28, 2025, 11:01 am #PROJECT PR:PROJECT_TITLE Kinetic analysis of cellular fates of cyclic 3-phosphoglyceric anhydride reveals PR:PROJECT_TITLE a crucial role of DJ-1 and glutathione in protecting biomolecules from PR:PROJECT_TITLE acylation. PR:PROJECT_SUMMARY A spontaneous cyclization of 1,3-bisphosphoglycerate produces a highly reactive PR:PROJECT_SUMMARY cyclic 3- phosphoglyceric anhydride (cPGA) which damages cellular nucleophiles PR:PROJECT_SUMMARY through indiscriminate acylation. Although evidence suggests that most of the PR:PROJECT_SUMMARY cPGA is inactivated by DJ-1, a highly efficient cPGA hydrolase, it is not known PR:PROJECT_SUMMARY what percentage of cPGA is inactivated by DJ-1 and how the remaining cPGA is PR:PROJECT_SUMMARY distributed in reactions with major cellular nucleophiles. Here, we use a PR:PROJECT_SUMMARY kinetic approach to model cellular fates of cPGA in HCT116 cells. Quantitative PR:PROJECT_SUMMARY assessment of DJ-1 activity and reactivity of cellular nucleophiles toward cPGA PR:PROJECT_SUMMARY indicates that up to 99% of intracellular cPGA is inactivated by DJ-1. PR:PROJECT_SUMMARY Unexpectedly, more than half of cPGA that escapes the inactivation by DJ-1 is PR:PROJECT_SUMMARY predicted to react with the thiol group of glutathione (GSH) to produce a PR:PROJECT_SUMMARY corresponding thioester S-D-3-phosphoglyceroyl glutathione (pgGS). We found that PR:PROJECT_SUMMARY pgGS is unstable and decomposes back to GSH and cPGA with a half-life of 80 PR:PROJECT_SUMMARY minutes providing DJ-1 with another chance to inactivate cPGA. Apart from PR:PROJECT_SUMMARY spontaneous decomposition, pgGS is efficiently hydrolyzed by Glyoxalase II PR:PROJECT_SUMMARY (GlxII), therefore a significant fraction of cPGA that escapes hydrolysis by PR:PROJECT_SUMMARY DJ-1 is trapped by GSH and subsequently detoxified. Experiments with DJ-1-null PR:PROJECT_SUMMARY cells revealed that depletion of GSH causes a multi-fold increase in cellular PR:PROJECT_SUMMARY level of N-glyceroyl glutamine confirming that a reversible formation of pgGS in PR:PROJECT_SUMMARY reaction of cPGA with GSH serves as a second line of defense against acylation PR:PROJECT_SUMMARY of biomolecules by cPGA. PR:INSTITUTE National Laboratory Astana PR:LAST_NAME Akhmadi PR:FIRST_NAME Aizhan PR:ADDRESS Kabanbay Batyr Ave 53, Astana, Kazakhstan PR:EMAIL aizhan.tkirova@nu.edu.kz PR:PHONE +77172694682 PR:PROJECT_COMMENTS I #STUDY ST:STUDY_TITLE Evidence for glutathione-mediated protection against cPGA-induced acylation in ST:STUDY_TITLE human HCT116 DJ-1 knockout cells. ST:STUDY_SUMMARY This study investigates the role of glutathione (GSH) in protecting cells from ST:STUDY_SUMMARY acylation damage caused by the reactive metabolite cyclic 3-phosphoglyceric ST:STUDY_SUMMARY anhydride (cPGA), which forms spontaneously from 1,3-bisphosphoglycerate. To ST:STUDY_SUMMARY investigate the contribution of GSH to cPGA detoxification, HCT116 DJ-1 knockout ST:STUDY_SUMMARY cells were treated with the GSH synthesis inhibitor buthionine sulfoximine (BSO) ST:STUDY_SUMMARY at two concentrations (10 µM and 100 µM) for 24 hours. Metabolic profiling ST:STUDY_SUMMARY revealed a concentration-dependent increase in N-glyceroyl glutamine levels upon ST:STUDY_SUMMARY GSH depletion. This accumulation indicates that GSH plays a key role in ST:STUDY_SUMMARY detoxifying cPGA and preventing its acylation of cellular biomolecules. The ST:STUDY_SUMMARY study included three biological replicates per condition, and metabolite ST:STUDY_SUMMARY identification was verified by matching retention time and MS/MS fragmentation ST:STUDY_SUMMARY with an authentic N-glyceroyl glutamine standard. ST:INSTITUTE National Laboratory Astana ST:LAST_NAME Akhmadi ST:FIRST_NAME Aizhan ST:ADDRESS Kabanbay Batyr Ave 53, Astana, Kazakhstan ST:EMAIL aizhan.tkirova@nu.edu.kz ST:PHONE +77172694682 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:GENOTYPE_STRAIN HCT116 #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 - Сtrl_r1 Sample source:HCT116 cells | Genotype:DJ-1 knock-out | Treatment:Сontrol RAW_FILE_NAME(Raw file name)=Сtrl_r1.mzXML SUBJECT_SAMPLE_FACTORS - Сtrl_r2 Sample source:HCT116 cells | Genotype:DJ-1 knock-out | Treatment:Сontrol RAW_FILE_NAME(Raw file name)=Сtrl_r2.mzXML SUBJECT_SAMPLE_FACTORS - Сtrl_r3 Sample source:HCT116 cells | Genotype:DJ-1 knock-out | Treatment:Сontrol RAW_FILE_NAME(Raw file name)=Сtrl_r3.mzXML SUBJECT_SAMPLE_FACTORS - BSO10_r1 Sample source:HCT116 cells | Genotype:DJ-1 knock-out | Treatment:10 uM Buthionine sulfoximine RAW_FILE_NAME(Raw file name)=BSO10_r1.mzXML SUBJECT_SAMPLE_FACTORS - BSO10_r2 Sample source:HCT116 cells | Genotype:DJ-1 knock-out | Treatment:10 uM Buthionine sulfoximine RAW_FILE_NAME(Raw file name)=BSO10_r2.mzXML SUBJECT_SAMPLE_FACTORS - BSO10_r3 Sample source:HCT116 cells | Genotype:DJ-1 knock-out | Treatment:10 uM Buthionine sulfoximine RAW_FILE_NAME(Raw file name)=BSO10_r3.mzXML SUBJECT_SAMPLE_FACTORS - BSO100_r1 Sample source:HCT116 cells | Genotype:DJ-1 knock-out | Treatment:100 uM Buthionine sulfoximine RAW_FILE_NAME(Raw file name)=BSO100_r1.mzXML SUBJECT_SAMPLE_FACTORS - BSO100_r2 Sample source:HCT116 cells | Genotype:DJ-1 knock-out | Treatment:100 uM Buthionine sulfoximine RAW_FILE_NAME(Raw file name)=BSO100_r2.mzXML SUBJECT_SAMPLE_FACTORS - BSO100_r3 Sample source:HCT116 cells | Genotype:DJ-1 knock-out | Treatment:100 uM Buthionine sulfoximine RAW_FILE_NAME(Raw file name)=BSO100_r3.mzXML #COLLECTION CO:COLLECTION_SUMMARY HCT116 DJ-1 knockout cells were cultured under standard conditions in CO:COLLECTION_SUMMARY appropriate growth medium until approximately 80% confluence. Following CO:COLLECTION_SUMMARY treatment with buthionine sulfoximine (BSO) or control (no BSO) for 24 hours, CO:COLLECTION_SUMMARY cells were rapidly washed twice with ice-cold PBS. Cellular metabolites were CO:COLLECTION_SUMMARY extracted with 80% methanol, concentrated using a speed vacuum concentrator, and CO:COLLECTION_SUMMARY stored at -80 °C until LC-MS analysis. Three biological replicates were CO:COLLECTION_SUMMARY collected for each treatment condition. CO:SAMPLE_TYPE Cultured cells CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY Cells were treated with the glutathione synthesis inhibitor buthionine TR:TREATMENT_SUMMARY sulfoximine (BSO) to deplete intracellular GSH levels. Treatments included two TR:TREATMENT_SUMMARY BSO concentrations: 10 µM (low inhibition) and 100 µM (strong inhibition). TR:TREATMENT_SUMMARY Cells were incubated for 24 hours under each condition. Untreated cells served TR:TREATMENT_SUMMARY as controls. The experiment was designed to evaluate the effect of GSH depletion TR:TREATMENT_SUMMARY on the formation of N-glyceroyl glutamine and to assess the role of GSH in TR:TREATMENT_SUMMARY detoxification of cyclic 3-phosphoglyceric anhydride (cPGA). Three biological TR:TREATMENT_SUMMARY replicates were analyzed for each treatment group. TR:TREATMENT_DOSE 10 μM and 100 μM #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Cellular metabolites were extracted with 1 mL of 80% methanol. The suspension SP:SAMPLEPREP_SUMMARY was vortexed at 4°C for 10 min and centrifuged at 22,000xg for 10 min. The SP:SAMPLEPREP_SUMMARY supernatant was dried at speed vac and resuspended in 100 µL of 80% methanol SP:SAMPLEPREP_SUMMARY before the LC-MS analysis. SP:EXTRACT_STORAGE -80℃ #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Bruker Q-TOF Impact II VIP CH:COLUMN_NAME Thermo Hypersil GOLD PEI (100 x 2.1 mm, 5 μm) CH:SOLVENT_A 100% Water; 5 mM Ammonium formate; 0.1% Formic acid CH:SOLVENT_B 100% Acetonitrile; 0.1% Formic acid CH:FLOW_GRADIENT The gradient program was as follows: 0-10 min, 80% to 70% B; 10–18 min, 70% to CH:FLOW_GRADIENT 40% B; 18-20.5 min, 40% to 30% B; and 20.5-22 min, 30% to 80% B. CH:FLOW_RATE 0.3 mL/min CH:COLUMN_TEMPERATURE 30°C #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Bruker Impact II MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS Metabolites were analyzed using a high-resolution mass spectrometer equipped MS:MS_COMMENTS with an electrospray ionization (ESI) source operated in negative ion mode. The MS:MS_COMMENTS ESI parameters were as follows: spray voltage, 3000 V; nebulizer pressure, 3.0 MS:MS_COMMENTS bar; drying gas, 7.0 L/min at 220°C. Spectra were acquired over an m/z range of MS:MS_COMMENTS 50–1300 at a frequency of 2 Hz by averaging 9929 transients. Compound MS:MS_COMMENTS identification was based on accurate m/z values and retention times compared MS:MS_COMMENTS with authentic standards. The areas under the curve of extracted-ion MS:MS_COMMENTS chromatograms of the [M–H]⁻ forms were integrated and normalized to the mean MS:MS_COMMENTS peak areas of all detected metabolites (total ion current). N-glyceroyl MS:MS_COMMENTS glutamine was further characterized by MS/MS fragmentation at collision energies MS:MS_COMMENTS of 5, 10, 20, and 30 eV. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS peak area MS_METABOLITE_DATA_START Samples Сtrl_r1 Сtrl_r2 Сtrl_r3 BSO10_r1 BSO10_r2 BSO10_r3 BSO100_r1 BSO100_r2 BSO100_r3 Factors Sample source:HCT116 cells | Genotype:DJ-1 knock-out | Treatment:Сontrol Sample source:HCT116 cells | Genotype:DJ-1 knock-out | Treatment:Сontrol Sample source:HCT116 cells | Genotype:DJ-1 knock-out | Treatment:Сontrol Sample source:HCT116 cells | Genotype:DJ-1 knock-out | Treatment:10 uM Buthionine sulfoximine Sample source:HCT116 cells | Genotype:DJ-1 knock-out | Treatment:10 uM Buthionine sulfoximine Sample source:HCT116 cells | Genotype:DJ-1 knock-out | Treatment:10 uM Buthionine sulfoximine Sample source:HCT116 cells | Genotype:DJ-1 knock-out | Treatment:100 uM Buthionine sulfoximine Sample source:HCT116 cells | Genotype:DJ-1 knock-out | Treatment:100 uM Buthionine sulfoximine Sample source:HCT116 cells | Genotype:DJ-1 knock-out | Treatment:100 uM Buthionine sulfoximine N-glyceroyl glutamine 79196 94694 44820 329656 299081 527691 282607 126608 576695 Glutathione 36112920 41687388 53812612 14775235 12777270 42386860 7935900 1298721 16404236 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name KEGG ID PubChem ID Retention time (min) Quantitated m/z N-glyceroyl glutamine - - 15.8 233.0775 Glutathione C00051 124886 16.2 306.0765 METABOLITES_END #END