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MB Sample ID: SA173302
Local Sample ID: | C_C.HGF2.BeiR-2 (50uM_3-oxo-Δ4-LCA_pH7) |
Subject ID: | SU001929 |
Subject Type: | Bacteria |
Subject Species: | Bacteria |
Select appropriate tab below to view additional metadata details:
Subject:
Subject ID: | SU001929 |
Subject Type: | Bacteria |
Subject Species: | Bacteria |
Factors:
Local Sample ID | MB Sample ID | Factor Level ID | Level Value | Factor Name |
---|---|---|---|---|
C_C.HGF2.BeiR-2 (50uM_3-oxo-Δ4-LCA_pH7) | SA173302 | FL020932 | C_C.HGF2.BeiR (50uM_3-oxo-Δ4-LCA_pH7) | Subject |
Collection:
Collection ID: | CO001922 |
Collection Summary: | After 48 hr of anaerobic incubation at 37 °C, culture supernatants were collected and stored at -80 °C until sample preparation for the analysis. |
Sample Type: | Bacterial cells |
Storage Conditions: | -80℃ |
Treatment:
Treatment ID: | TR001941 |
Treatment Summary: | Under anaerobic conditions, isolated bacteria strains were cultured together with 50 μM of CDCA, 3-oxo-Δ4-LCA, or LCA to screen for their bile acid metabolism in a 96-deep well plate covered with a gas-permeable membrane. 20 μL of bacterial culture in exponential to stationary phase was inoculated into 1 mL of Wilkins-Chalgren Anaerobe (WCA) media adjusted to pH 7 (using MOPS buffer solution) or pH 9 (TAPS buffer solution). |
Sample Preparation:
Sampleprep ID: | SP001935 |
Sampleprep Summary: | 100 μL of culture supernatant was transferred into a screw-cap glass vial containing 10 μL of deuterium-labelled internal standards (d4-CA, d4-CDCA, and d4-LCA, 1 nmol/mL each). 400 μL of water was added and sonicated for 10 min, then applied onto the solid-phase extraction cartridge (Agilent Bond Elut C18, 100 mg/1 mL, preconditioned with 1 mL of methanol and 3 mL of water). The cartridge was washed with 1 mL of water and captured bile acids were eluted with 1 mL of 90% ethanol. After solvent evaporation, the remaining residue was dissolved in 100 μL of 50% ethanol |
Combined analysis:
Analysis ID | AN003000 |
---|---|
Analysis type | MS |
Chromatography type | Reversed phase |
Chromatography system | Shimadzu CBM20A |
Column | InertSustain C18 |
MS Type | ESI |
MS instrument type | Triple quadrupole |
MS instrument name | Shimadzu LCMS-8040 |
Ion Mode | UNSPECIFIED |
Units | uM |
Chromatography:
Chromatography ID: | CH002225 |
Chromatography Summary: | 2 μL of the solution was injected to LC/ESI-MS/MS (LC-MS8040 tandem mass spectrometer, equipped with an ESI probe and Nexera X2 ultra-high-pressure liquid chromatography system; Shimadzu). A separation column, InertSustain C18 (150 mm × 2.1 mm ID, 2 μm particle size; GL Sciences Inc.), was utilized at 40 °C. Mixture A (10 mM ammonium acetate, 0.01% formic acid, and 20% acetonitrile) and mixture B (30% acetonitrile and 70% methanol) were used as the eluent, and the separation was carried out by linear gradient elution at a flow rate of 0.2 mL/min. The mobile phase composition was gradually changed as follows: Mixture A:B (80:20, v/v) for 0.1 min, (48:52, v/v) for 0.1-1 min, (30:70, v/v) for 1-27 min, (0:100, v/v) for 27-27.1 min, (0:100, v/v) for 27.1-33 min, (80:20, v/v) for 33-33.1 min, and (80:20 v/v) for 33.1-83 min. The total run time was 38 min. |
Instrument Name: | Shimadzu CBM20A |
Column Name: | InertSustain C18 |
Column Temperature: | 40 |
Flow Gradient: | The mobile phase composition was gradually changed as follows: Mixture A:B (80:20, v/v) for 0.1 min, (48:52, v/v) for 0.1-1 min, (30:70, v/v) for 1-27 min, (0:100, v/v) for 27-27.1 min, (0:100, v/v) for 27.1-33 min, (80:20, v/v) for 33-33.1 min, and (80:20 v/v) for 33.1-83 min. The total run time was 38 min. |
Flow Rate: | 0.2ml/min |
Solvent A: | 20% acetonitrile/80% water; 0.01% formic acid; 10mM ammonium acetate |
Solvent B: | 30% acetonitrile/70% methanol |
Chromatography Type: | Reversed phase |
MS:
MS ID: | MS002789 |
Analysis ID: | AN003000 |
Instrument Name: | Shimadzu LCMS-8040 |
Instrument Type: | Triple quadrupole |
MS Type: | ESI |
MS Comments: | The following MS parameters were used: spray voltage; 3,000V, heating block temperature; 400°C, nebulizing gas flow; 3 L/min, drying gas flow; 15 L/min, interface temperature 300°C, collision gas (argon) pressure; 230 kPa, collision energy; negative (11 to -35 eV); and positive (-16 to -19 eV) ion modes. Samples were analysed and quantified using LabSolutions Insight LC-MS software (Shimadzu). |
Ion Mode: | UNSPECIFIED |