Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA173440

Local Sample ID:	Clostridium symbiosum St.65-1 (50uM_CDCA_pH9)
Subject ID:	SU001929
Subject Type:	Bacteria
Subject Species:	Bacteria

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Subject:

Subject ID:	SU001929
Subject Type:	Bacteria
Subject Species:	Bacteria

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
Clostridium symbiosum St.65-1 (50uM_CDCA_pH9)	SA173440	FL021001	Clostridium symbiosum St.65 (50uM_CDCA_pH9)	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).

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

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