Summary of Study ST004322
This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR002736. The data can be accessed directly via it's Project DOI: 10.21228/M80P0S This work is supported by NIH grant, U2C- DK119886.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.
| Study ID | ST004322 |
| Study Title | Evidence for glutathione-mediated protection against cPGA-induced acylation in human HCT116 DJ-1 knockout cells. |
| Study Summary | This study investigates the role of glutathione (GSH) in protecting cells from acylation damage caused by the reactive metabolite cyclic 3-phosphoglyceric anhydride (cPGA), which forms spontaneously from 1,3-bisphosphoglycerate. To investigate the contribution of GSH to cPGA detoxification, HCT116 DJ-1 knockout cells were treated with the GSH synthesis inhibitor buthionine sulfoximine (BSO) at two concentrations (10 µM and 100 µM) for 24 hours. Metabolic profiling revealed a concentration-dependent increase in N-glyceroyl glutamine levels upon GSH depletion. This accumulation indicates that GSH plays a key role in detoxifying cPGA and preventing its acylation of cellular biomolecules. The study included three biological replicates per condition, and metabolite identification was verified by matching retention time and MS/MS fragmentation with an authentic N-glyceroyl glutamine standard. |
| Institute | National Laboratory Astana |
| Last Name | Akhmadi |
| First Name | Aizhan |
| Address | Kabanbay Batyr Ave 53, Astana, Kazakhstan |
| aizhan.tkirova@nu.edu.kz | |
| Phone | +77172694682 |
| Submit Date | 2025-10-21 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | mzXML |
| Analysis Type Detail | LC-MS |
| Release Date | 2025-11-20 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR002736 |
| Project DOI: | doi: 10.21228/M80P0S |
| Project Title: | Kinetic analysis of cellular fates of cyclic 3-phosphoglyceric anhydride reveals a crucial role of DJ-1 and glutathione in protecting biomolecules from acylation. |
| Project Summary: | A spontaneous cyclization of 1,3-bisphosphoglycerate produces a highly reactive cyclic 3- phosphoglyceric anhydride (cPGA) which damages cellular nucleophiles through indiscriminate acylation. Although evidence suggests that most of the cPGA is inactivated by DJ-1, a highly efficient cPGA hydrolase, it is not known what percentage of cPGA is inactivated by DJ-1 and how the remaining cPGA is distributed in reactions with major cellular nucleophiles. Here, we use a kinetic approach to model cellular fates of cPGA in HCT116 cells. Quantitative assessment of DJ-1 activity and reactivity of cellular nucleophiles toward cPGA indicates that up to 99% of intracellular cPGA is inactivated by DJ-1. Unexpectedly, more than half of cPGA that escapes the inactivation by DJ-1 is predicted to react with the thiol group of glutathione (GSH) to produce a corresponding thioester S-D-3-phosphoglyceroyl glutathione (pgGS). We found that pgGS is unstable and decomposes back to GSH and cPGA with a half-life of 80 minutes providing DJ-1 with another chance to inactivate cPGA. Apart from spontaneous decomposition, pgGS is efficiently hydrolyzed by Glyoxalase II (GlxII), therefore a significant fraction of cPGA that escapes hydrolysis by DJ-1 is trapped by GSH and subsequently detoxified. Experiments with DJ-1-null cells revealed that depletion of GSH causes a multi-fold increase in cellular level of N-glyceroyl glutamine confirming that a reversible formation of pgGS in reaction of cPGA with GSH serves as a second line of defense against acylation of biomolecules by cPGA. |
| Institute: | National Laboratory Astana |
| Last Name: | Akhmadi |
| First Name: | Aizhan |
| Address: | Kabanbay Batyr Ave 53, Astana, Kazakhstan |
| Email: | aizhan.tkirova@nu.edu.kz |
| Phone: | +77172694682 |
| Project Comments: | I |
Subject:
| Subject ID: | SU004477 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Genotype Strain: | HCT116 |
Factors:
Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)
| mb_sample_id | local_sample_id | Sample source | Genotype | Treatment |
|---|---|---|---|---|
| SA505930 | BSO100_r1 | HCT116 cells | DJ-1 knock-out | 100 uM Buthionine sulfoximine |
| SA505931 | BSO100_r2 | HCT116 cells | DJ-1 knock-out | 100 uM Buthionine sulfoximine |
| SA505932 | BSO100_r3 | HCT116 cells | DJ-1 knock-out | 100 uM Buthionine sulfoximine |
| SA505927 | BSO10_r1 | HCT116 cells | DJ-1 knock-out | 10 uM Buthionine sulfoximine |
| SA505928 | BSO10_r2 | HCT116 cells | DJ-1 knock-out | 10 uM Buthionine sulfoximine |
| SA505929 | BSO10_r3 | HCT116 cells | DJ-1 knock-out | 10 uM Buthionine sulfoximine |
| SA505933 | Сtrl_r1 | HCT116 cells | DJ-1 knock-out | Сontrol |
| SA505934 | Сtrl_r2 | HCT116 cells | DJ-1 knock-out | Сontrol |
| SA505935 | Сtrl_r3 | HCT116 cells | DJ-1 knock-out | Сontrol |
| Showing results 1 to 9 of 9 |
Collection:
| Collection ID: | CO004470 |
| Collection Summary: | HCT116 DJ-1 knockout cells were cultured under standard conditions in appropriate growth medium until approximately 80% confluence. Following treatment with buthionine sulfoximine (BSO) or control (no BSO) for 24 hours, cells were rapidly washed twice with ice-cold PBS. Cellular metabolites were extracted with 80% methanol, concentrated using a speed vacuum concentrator, and stored at -80 °C until LC-MS analysis. Three biological replicates were collected for each treatment condition. |
| Sample Type: | Cultured cells |
| Storage Conditions: | -80℃ |
Treatment:
| Treatment ID: | TR004486 |
| Treatment Summary: | Cells were treated with the glutathione synthesis inhibitor buthionine sulfoximine (BSO) to deplete intracellular GSH levels. Treatments included two BSO concentrations: 10 µM (low inhibition) and 100 µM (strong inhibition). Cells were incubated for 24 hours under each condition. Untreated cells served as controls. The experiment was designed to evaluate the effect of GSH depletion on the formation of N-glyceroyl glutamine and to assess the role of GSH in detoxification of cyclic 3-phosphoglyceric anhydride (cPGA). Three biological replicates were analyzed for each treatment group. |
| Treatment Dose: | 10 μM and 100 μM |
Sample Preparation:
| Sampleprep ID: | SP004483 |
| Sampleprep Summary: | Cellular metabolites were extracted with 1 mL of 80% methanol. The suspension was vortexed at 4°C for 10 min and centrifuged at 22,000xg for 10 min. The supernatant was dried at speed vac and resuspended in 100 µL of 80% methanol before the LC-MS analysis. |
| Extract Storage: | -80℃ |
Chromatography:
| Chromatography ID: | CH005470 |
| Instrument Name: | Bruker Q-TOF Impact II VIP |
| Column Name: | Thermo Hypersil GOLD PEI (100 x 2.1 mm, 5 μm) |
| Column Temperature: | 30°C |
| Flow Gradient: | The gradient program was as follows: 0-10 min, 80% to 70% B; 10–18 min, 70% to 40% B; 18-20.5 min, 40% to 30% B; and 20.5-22 min, 30% to 80% B. |
| Flow Rate: | 0.3 mL/min |
| Solvent A: | 100% Water; 5 mM Ammonium formate; 0.1% Formic acid |
| Solvent B: | 100% Acetonitrile; 0.1% Formic acid |
| Chromatography Type: | HILIC |
Analysis:
| Analysis ID: | AN007201 |
| Analysis Type: | MS |
| Chromatography ID: | CH005470 |
| Num Factors: | 3 |
| Num Metabolites: | 2 |
| Units: | peak area |
