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MB Sample ID: SA258071
| Local Sample ID: | 2021-11-29_015_G2_E2 |
| Subject ID: | SU002668 |
| Subject Type: | Human |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Gender: | Male |
Select appropriate tab below to view additional metadata details:
Subject:
| Subject ID: | SU002668 |
| Subject Type: | Human |
| Subject Species: | Homo sapiens |
| Taxonomy ID: | 9606 |
| Gender: | Male |
Factors:
| Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
|---|---|---|---|---|
| 2021-11-29_015_G2_E2 | SA258071 | FL033136 | 2.8G_DHT_60min | Treatment |
Collection:
| Collection ID: | CO002661 |
| Collection Summary: | Following the incubation period, tubes were briefly centrifuged at low speed and media was withdrawn. 150mM ammonium acetate in water were added to rinse residual media (150mM ammonium acetate is an osmolarity-matching, mass spectrometry compatible rinse buffer with near-neutral pH). Tubes were centrifuged, supernatant was withdrawn, and they were frozen by immersion in liquid nitrogen and stored at -80C prior to extraction. |
| Sample Type: | Pancreas |
| Volumeoramount Collected: | 500-1000uL |
| Storage Conditions: | -80℃ |
| Storage Vials: | 1ml Eppendorf Tubes |
| Collection Tube Temp: | -40C |
| Additives: | Ammonium acetate 150mM |
Treatment:
| Treatment ID: | TR002680 |
| Treatment Summary: | Human islets were treated overnight with 10nM DHT or EtOH in a humidified incubator containing 4% CO2 at 37°C and then batched in groups of 100 islets in microcentrifuge tubes and treated for either 30 or 60 minutes with media containing either 2.8mM or 16.7 mM 13C6 glucose; the same DHT or EtOH concentrations were maintained. |
| Treatment Compound: | DHT |
| Treatment Dose: | 10nM |
| Treatment Dosevolume: | 1ul per 1ml media |
| Treatment Doseduration: | 24 hours prior to experiment |
| Treatment Vehicle: | Ethanol |
Sample Preparation:
| Sampleprep ID: | SP002674 |
| Sampleprep Summary: | Sample prep summary: Islet samples were extracted in 200μL of ice-cold 8:1:1 methanol:water:chloroform, with tissue disruption aided by immediate probe sonication for 20 seconds with a Branson 450 Sonifier set to output level 2, duty cycle 20%. The disrupted samples were allowed to incubate on ice for 10 minutes and were then centrifuged for 5 min at 14,000 xg to pellet cell debris. 180μL of supernatant were dried under a gentle stream of nitrogen gas at room temperature, and were then reconstituted in 45μL of 4:1 water:methanol and transferred to autosampler vials for analysis. Extraction method: Single-phase extraction using 8:1:1 methanol:water:chloroform Extract storage: -80°C until extraction; 4°C after reconstitution |
| Processing Storage Conditions: | -80℃ |
| Extraction Method: | Single-phase extraction using 8:1:1 methanol:water:chloroform |
| Extract Storage: | -80℃ |
| Sample Resuspension: | -80°C until extraction; 4°C after reconstitution |
Combined analysis:
| Analysis ID | AN004228 |
|---|---|
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | Agilent 6530 qTOF |
| Column | Agilent Zorbax Extend C18 (150 x 2.1mm, 1.8um) with matched guard column |
| MS Type | ESI |
| MS instrument type | QTOF |
| MS instrument name | Agilent 6530 QTOF |
| Ion Mode | NEGATIVE |
| Units | EicCoreArea/Peak Area |
Chromatography:
| Chromatography ID: | CH003137 |
| Methods Filename: | experiment_metadata |
| Instrument Name: | Agilent 6530 qTOF |
| Column Name: | Agilent Zorbax Extend C18 (150 x 2.1mm, 1.8um) with matched guard column |
| Column Temperature: | 35°C |
| Flow Gradient: | 0-2min hold 100/0/0 (%A/%B/%C); 2-12 min linear to 1/99/0, 12-20 min hold 1/99/0, 20-20.5 min linear to 5/0/95, 20.5-26.1 5/0/95, 26.1-26.3 min linear to 100/0/0, 26.3-33 min hold 100/0/0 |
| Flow Rate: | 0-20.5 min hold 0.25mL/min, [18 min switch flow direction to backflush], 20.5-21.5 linear to 0.4mL/min, 21.5-22.3 linear to 0.8 mL/min, [26 min switch flow direction to forward] 26-26.1 linear to 0.6mL/min, 26.1-26.3 linear to 0.4 mL/min, 26.3-31.8 min hold 0.4mL/min, 31.8-32 min linear to 0.25mL/min |
| Solvent A: | 97% water/3% methanol; 10 mM tributylamine; 5mM acetic acid |
| Solvent B: | 100% methanol; 10 mM tributylamine; 5mM acetic acid |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS003975 |
| Analysis ID: | AN004228 |
| Instrument Name: | Agilent 6530 QTOF |
| Instrument Type: | QTOF |
| MS Type: | ESI |
| MS Comments: | MS acquisition was performed in negative ion mode, scan rate 2 spectra/sec, mass range 50-1200 m/z. Source parameters were: drying gas temperature 250°C, drying gas flow rate 13 L/min, nebulizer pressure 35 psig, sheath gas temp 325°C and flow 12 l/min, capillary voltage 3500V, internal reference mass correction enabled. Data analysis was performed using Agilent Profinder 10.0 software in batch isotopologue extraction mode with automated natural isotope abundance correction enabled. Compound identification was performed by matching accurate mass and retention time to those of authentic standards analyzed using the same method. |
| Ion Mode: | NEGATIVE |
| Capillary Voltage: | 3500V |
| Dry Gas Flow: | 13L/min |
| Dry Gas Temp: | 250 C |
| Fragment Voltage: | 150V |
| Nebulizer: | 35 psig |
