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MB Sample ID: SA122353
| Local Sample ID: | D5-control2 |
| Subject ID: | SU001515 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU001515 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| D5-control2 | SA122353 | FL014970 | Control | Sample Source |
| D5-control2 | SA122353 | FL014970 | Day5 | Treatment Duration |
| D5-control2 | SA122353 | FL014970 | Control | Sample Source |
| D5-control2 | SA122353 | FL014970 | Day5 | Treatment Duration |
Collection:
| Collection ID: | CO001510 |
| Collection Summary: | 7 control fibroblasts samples and 7 patient-derived fibroblasts samples were collected at day 0 and day 5. Intracellular metabolites were extracted from cells cultured in 6 well plate while acyl-CoA and 5-methyltetrahydrofolate were extracted from cells cultured in 60 mm dish. Intracellular metabolites and acyl-CoA from cells were harvested as described previously using 80% methanol/water as solvent (PMID: 24410464, PMID: 24894601, PMID: 25795660) and dry pellets were stored in -80 °C freezer until ready for LC-MS analysis. For acyl-CoA and 5-methyltetrahydrofolate analysis, dry pellets were reconstituted into 30 μL of sample solvent (water containing 50 mM ammonium acetate), and 10 μL was injected into the LC-MS. For non-acyl-CoA polar metabolite analysis, pellets were reconstituted into 30 μL of sample solvent (water:methanol:acetonitrile, 2:1:1, v/v), and 3 μL was injected into the LC-MS. |
| Sample Type: | Cultured fibroblasts |
Treatment:
| Treatment ID: | TR001530 |
| Treatment Summary: | Patient-derived fibroblasts cultured in standard culture conditions (proliferative condition) and cells cultured for 5 days in low-serum conditions (non-proliferative condition). Patients carry disease-causing mutations in SUCLA2. Controls are fibroblasts from age-matched patients with other mitochondrial diseases. |
Sample Preparation:
| Sampleprep ID: | SP001523 |
| Sampleprep Summary: | Intracellular metabolites and acyl-CoA from cells were harvested as described previously using 80% methanol/water as solvent (PMID: 24410464, PMID: 24894601, PMID: 25795660) and dry pellets were stored in -80 °C freezer until ready for LC-MS analysis. For acyl-CoA and 5-methyltetrahydrofolate analysis, dry pellets were reconstituted into 30 μL of sample solvent (water containing 50 mM ammonium acetate), and 10 μL was injected into the LC-MS. For non-acyl-CoA polar metabolite analysis, pellets were reconstituted into 30 μL of sample solvent (water:methanol:acetonitrile, 2:1:1, v/v), and 3 μL was injected into the LC-MS. |
Combined analysis:
| Analysis ID | AN002407 | AN002408 | AN002409 |
|---|---|---|---|
| Analysis type | MS | MS | MS |
| Chromatography type | HILIC | HILIC | Reversed phase |
| Chromatography system | Thermo Dionex Ultimate 3000 | Thermo Dionex Ultimate 3000 | Thermo Dionex Ultimate 3000 |
| Column | Waters Xbridge amide (100 x 2.1mm,3.5um) | Waters Xbridge amide (100 x 2.1mm,3.5um) | Phenomenex Luna C18 (100 x 2.0mm,3um) |
| MS Type | ESI | ESI | ESI |
| MS instrument type | Orbitrap | Orbitrap | Orbitrap |
| MS instrument name | Thermo Exactive Plus Orbitrap | Thermo Exactive Plus Orbitrap | Thermo Q Exactive Plus Orbitrap |
| Ion Mode | POSITIVE | NEGATIVE | POSITIVE |
| Units | peak area | peak area | peak area |
Chromatography:
| Chromatography ID: | CH001769 |
| Chromatography Summary: | HILIC method is for general metabolomics analysis. |
| Instrument Name: | Thermo Dionex Ultimate 3000 |
| Column Name: | Waters Xbridge amide (100 x 2.1mm,3.5um) |
| Chromatography Type: | HILIC |
| Chromatography ID: | CH001770 |
| Chromatography Summary: | RPLC is for acyl-CoA and folate analysis. |
| Instrument Name: | Thermo Dionex Ultimate 3000 |
| Column Name: | Phenomenex Luna C18 (100 x 2.0mm,3um) |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS002248 |
| Analysis ID: | AN002407 |
| Instrument Name: | Thermo Exactive Plus Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | It was operated in the full-scan mode with the resolution set at 70 000 (at m/z 200).LC-MS data were analyzed using Sieve 2.0 (Thermo Scientific), and the integrated area under metabolite peak was used to compare the relative abundance of each metabolite in different samples in the same batch. |
| Ion Mode: | POSITIVE |
| MS ID: | MS002249 |
| Analysis ID: | AN002408 |
| Instrument Name: | Thermo Exactive Plus Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | It was operated in the full-scan mode with the resolution set at 70 000 (at m/z 200).LC-MS data were analyzed using Sieve 2.0 (Thermo Scientific), and the integrated area under metabolite peak was used to compare the relative abundance of each metabolite in different samples in the same batch. |
| Ion Mode: | NEGATIVE |
| MS ID: | MS002250 |
| Analysis ID: | AN002409 |
| Instrument Name: | Thermo Q Exactive Plus Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | It was operated in the full-scan mode with the resolution set at 70 000 (at m/z 200).LC-MS data were analyzed using Sieve 2.0 (Thermo Scientific), and the integrated area under metabolite peak was used to compare the relative abundance of each metabolite in different samples in the same batch. |
| Ion Mode: | POSITIVE |
