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MB Sample ID: SA229479
| Local Sample ID: | CTR day 200 2 |
| Subject ID: | SU002414 |
| Subject Type: | Cultured cells |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU002414 |
| 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 |
|---|---|---|---|---|
| CTR day 200 2 | SA229479 | FL028988 | CTR day 200 | Group |
Collection:
| Collection ID: | CO002407 |
| Collection Summary: | Midbrain organoids of day 40, 100 and 200 had a media change 2h prior collection. 5 organoids spheres were added to 1.5 ml tubes and washed in cold PBS twice. The tubes were them frozen in liquid nitrogen. |
| Collection Protocol Filename: | metabolomics_methods.pdf |
| Sample Type: | Brain |
Treatment:
| Treatment ID: | TR002426 |
| Treatment Summary: | Organoids bearing PARK7/DJ1 KO mutation were used together with isogenic CTR cell lines (BJSIPS) |
Sample Preparation:
| Sampleprep ID: | SP002420 |
| Sampleprep Summary: | After colection,the organoids were extracted in 1 mL/well of 80:20 (v/v) methanol:water with 0.01 ng/mL Val-d8 and Phe-d8 internal extraction standards. extraction solvent was dried under nitrogen gas and metabolite samples were stored at −80 °C until analysis. |
| Sampleprep Protocol Filename: | metabolomics_methods.pdf |
Combined analysis:
| Analysis ID | AN003796 |
|---|---|
| Analysis type | MS |
| Chromatography type | HILIC |
| Chromatography system | Dionex Ultimate 3000TM |
| Column | Millipore(TM) ZIC-pHILIC |
| MS Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Q Exactive HF hybrid Orbitrap |
| Ion Mode | POSITIVE |
| Units | intensity |
Chromatography:
| Chromatography ID: | CH002809 |
| Chromatography Summary: | The LC column was a Millipore TMZIC-pHILIC (2.1x150 mm, 5μm) coupled to a Dionex Ultimate 3000TM system and the column oven temperature was set to 25oC for the gradient elution. A flow rate of 100μL/min was used with the 10 mM ammonium carbonate in water (A), pH 9.0, and acetonitrile (B). The gradient profile was 80-20% B (0-30min), 20-80% B (30-31min), 80-80% B (31-42min). Injection volume was set to 2 μL for all analyses (42min total run time per injection). |
| Methods Filename: | metabolomics_methods.pdf |
| Instrument Name: | Dionex Ultimate 3000TM |
| Column Name: | Millipore(TM) ZIC-pHILIC |
| Column Temperature: | 25 |
| Flow Gradient: | 80-20% B (0-30min), 20-80% B (30-31min), 80-80% B (31-42min). |
| Flow Rate: | 100µL/min |
| Solvent A: | 100% water; 10 mM ammonium carbonate, pH 9.0 |
| Solvent B: | 100% acetonitrile |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS003538 |
| Analysis ID: | AN003796 |
| Instrument Name: | Thermo Q Exactive HF hybrid Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | Method duration was 30 min with polarity switching data-dependent Top 5method for both positive and negative modes. Spray voltage for both positive and negative modes was 3.5kV and the capillary temperature was set to 320 ⁰C with a sheath gas rate of 35, aux gas of 10, and max spray current of 100 μA. The full MS scan for both polarities utilized 120,000 resolution with an AGC target of 3e6 and a maximum IT of 100 ms, and the scan range was from 67-1000 m/z. Tandem MS spectra for both positive and negative modes used a resolution of 15,000, AGC target of 1 e5, maximum IT of 50 ms, isolation window of 0.4 m/z, isolation offset of 0.1 m/z, fixed first mass of 50 m/z, and 3-way multiplexed normalized collision energies (nCE) of 10, 35, 80. The minimum AGC target was 1e4 with an intensity threshold of 2e5. All data were acquired in profile mode. |
| Ion Mode: | POSITIVE |
| Analysis Protocol File: | metabolomics_methods.pdf |
