Summary of Study ST003544
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 PR002180. The data can be accessed directly via it's Project DOI: 10.21228/M8TV67 This work is supported by NIH grant, U2C- DK119886. See: https://www.metabolomicsworkbench.org/about/howtocite.php
| Study ID | ST003544 |
| Study Title | Metabolomics of Ndufs4 KO human induced pluripotent stem cells (iPSCs) |
| Study Summary | Mitochondrial diseases, often linked to complex I (CI) defects, lack curative treatments. High-throughput drug screening using human-relevant platforms is crucial for identifying new therapeutics. Induced pluripotent stem cell (iPSC) and CRISPR technologies offer a powerful tool for this purpose. While typically differentiated into disease-relevant cell types, recent studies support the use of undifferentiated iPSCs for drug discovery. Here, we developed and characterized NDUFS4 KO iPSCs in their pluripotent state. Metabolomic profiling revealed a distinct phenotype in NDUFS4 KO iPSCs, predominantly associated with an elevated NADH/NAD+ ratio, consistent with alterations observed in other models of mitochondrial dysfunction. These findings underscore the potential of iPSCs for early-stage, high-throughput therapeutic screening in mitochondrial diseases. |
| Institute | North-West University |
| Last Name | Louw |
| First Name | Roan |
| Address | Hofman Street, Potchefstroom, North-West, 2520, South Africa |
| Roan.Louw@nwu.ac.za | |
| Phone | +27182994074 |
| Submit Date | 2024-10-06 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | mzML,cdf,d |
| Analysis Type Detail | GC-MS/LC-MS |
| Release Date | 2025-10-06 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR002180 |
| Project DOI: | doi: 10.21228/M8TV67 |
| Project Title: | Metabolomics of Ndufs4 KO human induced pluripotent stem cells (iPSCs) |
| Project Type: | Multi-platform metabolomics analysis |
| Project Summary: | Mitochondrial diseases, often linked to complex I (CI) defects, lack curative treatments. High-throughput drug screening using human-relevant platforms is crucial for identifying new therapeutics. Induced pluripotent stem cell (iPSC) and CRISPR technologies offer a powerful tool for this purpose. While typically differentiated into disease-relevant cell types, recent studies support the use of undifferentiated iPSCs for drug discovery. The aim of this project was to develop and characterize NDUFS4 KO iPSCs in their pluripotent state. The metabolic profile of Ndufs4 KO human induced pluripotent stem cells (iPSCs) were compared to that of isogenic controls using multi-platform metabolomics, consisting of targeted LC-MS/MS, targeted GC-MS/MS, and untargeted GC-TOFMS analyses. Metabolic profiling revealed a distinct phenotype in NDUFS4 KO iPSCs, predominantly associated with an elevated NADH/NAD+ ratio, consistent with alterations observed in other models of mitochondrial dysfunction. These findings underscore the potential of iPSCs for early-stage, high-throughput therapeutic screening in mitochondrial diseases. |
| Institute: | North-West University |
| Last Name: | Louw |
| First Name: | Roan |
| Address: | Hofman Street, Potchefstroom, North-West, 2520, South Africa |
| Email: | Roan.Louw@nwu.ac.za |
| Phone: | +27182994074 |
Subject:
| Subject ID: | SU003673 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Species Group: | Mammals |
Factors:
Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)
| mb_sample_id | local_sample_id | Genotype |
|---|---|---|
| SA387723 | KO5_2 | KO |
| SA387724 | KO5_1 | KO |
| SA387725 | KO5_3 | KO |
| SA387726 | KO5_4 | KO |
| SA387727 | KO5_5 | KO |
| SA387728 | WT7_1 | WT |
| SA387729 | WT7_2 | WT |
| SA387730 | WT7_3 | WT |
| SA387731 | WT7_4 | WT |
| SA387732 | WT7_5 | WT |
| Showing results 1 to 10 of 10 |
Collection:
| Collection ID: | CO003666 |
| Collection Summary: | Five replicate samples per genotype (i.e. distinct cultures in parallel) were prepared for each metabolic analysis. Each cell pellet, harvested from a T25 culture flasks, was washed three times with chilled PBS before being quenched in cold HPLC-grade methanol, with subsequent addition of an internal standard mixture in cold HPLC-grade water. Thereafter, samples were homogenized using a vibration mill (30 Hz, 1 min) and incubated on ice (10 min) after the addition of cold HPLC-grade chloroform. Solvents were added in a ratio of 3:1:1, methanol/water/chloroform, as required for a modified monophasic Bligh-Dyer extraction. After centrifugation at 12 000 ×g (10 min, 4°C) supernatants were aliquoted into 2 mL glass vials. |
| Sample Type: | Cultured cells |
Treatment:
| Treatment ID: | TR003682 |
| Treatment Summary: | N/A |
Sample Preparation:
| Sampleprep ID: | SP003680 |
| Sampleprep Summary: | Samples were derivatized by butylation on the day of analysis. To butylate the dried extracts, 300 μL of freshly prepared 1-butanol:acetyl chloride (4:1, v/v) was added and samples were incubated at 50°C for 60 min. Thereafter, the samples were evaporated to dryness under a gentle stream of nitrogen at 37 °C. The samples were then reconstituted in 100 μL of water:acetonitrile (50:50, v/v), containing 0.1% formic acid and vortex mixed. Finally, the total volume was transferred to 250 μL tapered glass inserts placed in 2 mL vials and loaded onto the autosampler for analysis. |
Combined analysis:
| Analysis ID | AN005820 | AN005821 | AN005822 |
|---|---|---|---|
| Chromatography ID | CH004421 | CH004422 | CH004423 |
| MS ID | MS005540 | MS005541 | MS005542 |
| Analysis type | MS | MS | MS |
| Chromatography type | Reversed phase | GC | GC |
| Chromatography system | Agilent 1200 Infinity | Agilent 7890B | Agilent 8890 |
| Column | Agilent C18 Zorbax SB-AQ (100 x 2.1mm, 1.8um) | Restek Rxi-1MS (30m x 0.32mm x 0.25μm) | Restek Rxi-5MS (29.69m x 0.25mm,0.25um) |
| MS Type | ESI | EI | EI |
| MS instrument type | Triple quadrupole | Triple quadrupole | GC-TOF |
| MS instrument name | Agilent 6410 QQQ | Agilent 7010B | LECO Pegasus BT |
| Ion Mode | POSITIVE | POSITIVE | POSITIVE |
| Units | Normalised peak areas | Normalised peak areas |
Chromatography:
| Chromatography ID: | CH004421 |
| Instrument Name: | Agilent 1200 Infinity |
| Column Name: | Agilent C18 Zorbax SB-AQ (100 x 2.1mm, 1.8um) |
| Column Temperature: | 45°C |
| Flow Gradient: | 95 % of solvent A held for 0.2 min, increased to 25 % of solvent B over 1.8 min, held for 5 min, increased to 90 % of solvent B over 0.5 min, held for 1.6 min, increased to 95 % of solvent B over 2.9 min, decreased to 5 % of solvent B over 1 min, and re-equilibrated for 3 min to give a total run time of 16 min. |
| Flow Rate: | For the first 9 min of the run, the mobile phase flow rate was set at 0.3 mL/min, after which it was increased to 0.4 mL/min in a span of 0.1 min and maintained at this level for the subsequent 3.9 min of the run |
| Solvent A: | 100% Water; 0.1 % Formic acid |
| Solvent B: | 100% Acetonitrile; 0.1 % Formic acid |
| Chromatography Type: | Reversed phase |
| Chromatography ID: | CH004422 |
| Instrument Name: | Agilent 7890B |
| Column Name: | Restek Rxi-1MS (30m x 0.32mm x 0.25μm) |
| Column Temperature: | 70°C for 1 min; followed by a ramp at 12°C/min until reaching 135°C, held for 1.5 min; a ramp at 2°C/min until reaching 145°C, held for 1.5 min; and a ramp of 23°C/min until reaching a final temperature of 300°C, held for 1 min |
| Flow Gradient: | N/A |
| Flow Rate: | 1 mL/min |
| Solvent A: | N/A |
| Solvent B: | N/A |
| Chromatography Type: | GC |
| Chromatography ID: | CH004423 |
| Instrument Name: | Agilent 8890 |
| Column Name: | Restek Rxi-5MS (29.69m x 0.25mm,0.25um) |
| Column Temperature: | 70°C for 1 min, followed by a ramp at 10°C/min until reaching a final temperature of 320°C, held for 3 min |
| Flow Gradient: | N/A |
| Flow Rate: | 1 mL/min |
| Solvent A: | N/A |
| Solvent B: | N/A |
| Chromatography Type: | GC |
MS:
| MS ID: | MS005540 |
| Analysis ID: | AN005820 |
| Instrument Name: | Agilent 6410 QQQ |
| Instrument Type: | Triple quadrupole |
| MS Type: | ESI |
| MS Comments: | Amino acid and acylcarnitine species were analyzed by dynamic multiple reaction monitoring of the transition from precursor to product ions at associated optimized collision energies, and fragmentor voltages using Agilent Masshunter. To achieve the highest confidence level in metabolite identities, two unique transitions were monitored per metabolite, thus, allowing spectral and retention time matching |
| Ion Mode: | POSITIVE |
| MS ID: | MS005541 |
| Analysis ID: | AN005821 |
| Instrument Name: | Agilent 7010B |
| Instrument Type: | Triple quadrupole |
| MS Type: | EI |
| MS Comments: | Redox-related organic acids were analysed by multiple reaction monitoring of the transition from precursor to product ions at associated optimized collision energies, and fragmentor voltages using Agilent Masshunter. To increase analytical sensitivity, dMRMs were divided into three methods (identical chromatography), thus requiring three separate sample injections to analyse all compounds.To achieve the highest confidence level in metabolite identities, two unique transitions were monitored per metabolite, thus, allowing spectral and retention time matching |
| Ion Mode: | POSITIVE |
| MS ID: | MS005542 |
| Analysis ID: | AN005822 |
| Instrument Name: | LECO Pegasus BT |
| Instrument Type: | GC-TOF |
| MS Type: | EI |
| MS Comments: | Mass spectra (50–850 m/z) were acquired at a scan rate of 20 spectra/s after a 300-s solvent delay.The ChromaTOF software (LECO Corp., version 4.51) was used for data acquisition, processing, and extraction. Peaks with similar mass spectra and retention times were aligned using the Statistical Compare package. |
| Ion Mode: | POSITIVE |
