Summary of Study ST003959
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 PR002482. The data can be accessed directly via it's Project DOI: 10.21228/M8TC38 This work is supported by NIH grant, U2C- DK119886. See: https://www.metabolomicsworkbench.org/about/howtocite.php
| Study ID | ST003959 |
| Study Title | Steady state levels of polar metabolites HEK293T, SDHBKO and UQCRC1KO compared to WT cells. |
| Study Summary | This study aimed to investigate changes in relative abundance of polar metabolites in cells with OXPHOS dysfunction using targeted GC-MS analysis. Steady-state analysis of polar metabolites by gas chromatography-mass spectrometry (GC-MS) in HEK293T UQCRC1KO and SDHBKO cell lines enabled the relative quantification of TCA intermediates. The results revealed a significant increase in succinate and a similar behavior in respect to the abundances of other TCA metabolites as was observed in SDHBKO. |
| Institute | University of Melbourne |
| Last Name | Roopasingam |
| First Name | Kugapreethan |
| Address | 30 Flemington Rd, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, VIC, Australia. |
| k.roopasingam@unimelb.edu.au | |
| Phone | 0434297212 |
| Submit Date | 2025-05-29 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | gqd |
| Analysis Type Detail | GC-MS |
| Release Date | 2025-06-11 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR002482 |
| Project DOI: | doi: 10.21228/M8TC38 |
| Project Title: | Complex II assembly drives metabolic adaptation to OXPHOS dysfunction. |
| Project Summary: | During acute oxidative phosphorylation (OXPHOS) dysfunction, the reverse activity of succinate dehydrogenase (Complex II) maintains the redox state of Coenzyme Q by utilizing either fumarate or oxygen as terminal electron acceptors. The tendency for one over another has been suggested to be tissue-specific, but the underlying mechanism and consequence of this is unknown. Using quantitative proteomics to screen a panel of HEK293T knockout cell lines, we identified an increase in SDHAF2 protein, a Complex II assembly factor that enhances the flavination of catalytic subunit SDHA, as critical for metabolic adaptation during OXPHOS stress in HEK293T cells. Loss of SDHAF2 during Complex III inhibition resulted in a reduction in Complex II F-site derived reactive oxygen species (ROS), a severe growth impairment, and a net reductive TCA cycle driven by an inability of mitochondria to support additional Complex II assembly. This in turn leads to use of fumarate as terminal electron acceptor at the cost of a ROS-mediated switch to glycolysis. Cell lines adapted to glycolysis did not accumulate SDHAF2 upon OXPHOS stress and exhibited a net reductive TCA cycle and mild growth phenotypes with or without SDHAF2 being present. Thus, our study reveals how Complex II assembly controls a balance between protection of the Q-pool and ROS-meditated signaling during oxidative stress in cells reliant on mitochondrial OXPHOS. |
| Institute: | University of Melbourne |
| Last Name: | Roopasingam |
| First Name: | Kugapreethan |
| Address: | 30 Flemington Rd, Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, VIC, Australia. |
| Email: | k.roopasingam@unimelb.edu.au |
| Phone: | 0434297212 |
Subject:
| Subject ID: | SU004096 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
Factors:
Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)
| mb_sample_id | local_sample_id | Sample source | Treatment |
|---|---|---|---|
| SA452749 | BKO_001_UNK-0022_13032023_22 | SDHBKO_001 | Knock out |
| SA452750 | BKO_002_UNK-0032_13032023_32 | SDHBKO_002 | Knock out |
| SA452751 | BKO_003_UNK-0026_13032023_26 | SDHBKO_003 | Knock out |
| SA452752 | BKO_004_UNK-0017_13032023_17 | SDHBKO_004 | Knock out |
| SA452753 | BKO_005_UNK-0015_13032023_15 | SDHBKO_005 | Knock out |
| SA452754 | BKO_006_UNK-0007_13032023_7 | SDHBKO_006 | Knock out |
| SA452755 | UQKO_001_UNK-0027_13032023_27 | UQCRC1KO_001 | Knock out |
| SA452756 | UQKO_002_UNK-0030_13032023_30 | UQCRC1KO_002 | Knock out |
| SA452757 | UQKO_003_UNK-0029_13032023_29 | UQCRC1KO_003 | Knock out |
| SA452758 | UQKO_004_UNK-0006_13032023_6 | UQCRC1KO_004 | Knock out |
| SA452759 | UQKO_005_UNK-0008_13032023_8 | UQCRC1KO_005 | Knock out |
| SA452760 | UQKO_006_UNK-0028_13032023_28 | UQCRC1KO_006 | Knock out |
| SA452761 | WT_001_UNK-0009_13032023_9 | Wild type_001 | Control |
| SA452762 | WT_002_UNK-0033_13032023_33 | Wild type_002 | Control |
| SA452763 | WT_003_UNK-0023_13032023_23 | Wild type_003 | Control |
| SA452764 | WT_004_UNK-0019_13032023_19 | Wild type_004 | Control |
| SA452765 | WT_005_UNK-0020_13032023_20 | Wild type_005 | Control |
| SA452766 | WT_006_UNK-0012_13032023_12 | Wild type_006 | Control |
| Showing results 1 to 18 of 18 |
Collection:
| Collection ID: | CO004089 |
| Collection Summary: | HEK293T cells were cultured in Dulbecco's Modified Eagle Medium (DMEM, Thermo Fisher Scientific) supplemented with 10 % (v/v) fetal bovine serum (FBS, CellSera), 50 µg/mL uridine (Sigma), and a mixture of 100 µg/mL streptomycin and 100 units/mL penicillin (Thermo Scientific). |
| Sample Type: | Embryonic cells |
Treatment:
| Treatment ID: | TR004105 |
| Treatment Summary: | HEK293T knockouts for SDHB and UQCRC1 were made using the CRISPR-Cas9 mediated technology as described in Stroud, D. A. et al. Nature 538, 123–126 (2016). |
Sample Preparation:
| Sampleprep ID: | SP004102 |
| Sampleprep Summary: | For steady-state metabolite profiling, polar metabolites were extracted from snap-frozen cells using 600 µL of HPLC grade methanol:chloroform mixture (9:1; [v/v]), along with internal standards (1.66μM 13C5,15N-valine, 1.66μM 13C6-sorbitol), as described above. The clarified supernatants and pooled biological quality controls (PBQC’s) were dried using a CentriVap concentrator (Labconco). |
Combined analysis:
| Analysis ID | AN006507 |
|---|---|
| Chromatography ID | CH004942 |
| MS ID | MS006206 |
| Analysis type | MS |
| Chromatography type | GC |
| Chromatography system | Shimadzu GC-2010 |
| Column | Agilent DB-5 GC column (30 m x 0.25 mm, 1 µm) |
| MS Type | EI |
| MS instrument type | Triple quadrupole |
| MS instrument name | Shimadzu TQ8050NX |
| Ion Mode | POSITIVE |
| Units | Relative abundance |
Chromatography:
| Chromatography ID: | CH004942 |
| Instrument Name: | Shimadzu GC-2010 |
| Column Name: | Agilent DB-5 GC column (30 m x 0.25 mm, 1 µm) |
| Column Temperature: | 100℃ |
| Flow Gradient: | N/A |
| Flow Rate: | 1 mL/min |
| Solvent A: | N/A |
| Solvent B: | N/A |
| Chromatography Type: | GC |
MS:
| MS ID: | MS006206 |
| Analysis ID: | AN006507 |
| Instrument Name: | Shimadzu TQ8050NX |
| Instrument Type: | Triple quadrupole |
| MS Type: | EI |
| MS Comments: | The GC-MS system used comprised of an AOC6000 autosampler, a 2030 Shimadzu gas chromatograph and a TQ8050NX triple quadrupole mass spectrometer (Shimadzu, Japan)with an electron ionisation source(-70eV). The mass spectrometer was tuned according to the manufacturer’s recommendations using tris-(perfluorobutyl)-amine (CF43). GC-MS was performed on a 30m Agilent DB-5 column with 0.25mm internal diameter column and 1µm film thickness. The injection temperature (inlet) was set at 280°C, the MS transfer line at 280°C and the ion source adjusted to 200°C. Helium was used as the carrier gas at a flow rate of 1 mL/min and argon gas was used in the collision cell to generate the MRM product ion. The analysis of the derivatised samples was performed under the following oven temperature program; 100°C start temperature, hold for 4 minutes, followed by a 10°C/min oven temperature ramp to 320°C with a following final hold for 11 minutes. |
| Ion Mode: | POSITIVE |
