Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA174792

Local Sample ID:	HepG2 cells-30V
Subject ID:	SU001950
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606

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Subject:

Subject ID:	SU001950
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
HepG2 cells-30V	SA174792	FL021469	Hep G2 cells	Source
HepG2 cells-30V	SA174792	FL021469	30V	MS2 CE

Collection:

Collection ID:	CO001943
Collection Summary:	NIST SRM 1950 was purchased from NIST, and HepG2 cells were purchased from the Chinese Tissue Culture Collections (CTCC, Shanghai, China). Urine was collected from volunteers. Six- to eight-week-old male C57BL/6 mice were fed a regular standard breeding diet and housed under standard pathogen-free (SPF) conditions. Faecal samples and liver tissue were collected and immediately stored at -80 °C for further analysis.
Sample Type:	Blood (plasma), Urine, HepG2 cells
Storage Conditions:	-80℃

Treatment:

Treatment ID:	TR001962
Treatment Summary:	No treatment

Sample Preparation:

Sampleprep ID:	SP001956
Sampleprep Summary:	Plasma (NIST SRM 1950): A 240 μL volume of Mix-IS-ACN was added to 60 μL of NIST SRM 1950 for protein precipitation. Then, the sample was vortexed for 60 s and centrifuged for 10 min at 15,000 g and 4 °C. Then, 250 μL of supernatant was transferred to a new centrifuge tube for lyophilization. The residue was reconstituted with 60 μL of 90% H2O/CH3OH (v/v). Urine: A 60 μL sample of urine was centrifuged for 10 min at 15,000 g and 4 °C, and the supernatant was transferred to a new centrifuge tube. Then, 30 μL Mix-IS-MeOH was added to the same centrifuge tube, and the mixture was thoroughly combined on a vortex mixer for 60 s. HepG2: HepG2 cells were purchased from the Chinese Tissue Culture Collections (CTCC, Shanghai, China) and cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco), 100 IU/ml penicillin and 100 µg/ml streptomycin (Gibco). These cells were cultured at 37 °C in a humidified 5% CO2 atmosphere and treated within 24 hours after the confluence of cells reached 80%. The culture media of the cells was removed, and the cells were washed three times in phosphate-buffered saline (PBS). Then, the cells were quickly frozen in liquid nitrogen and stored at -80 °C before extraction. Then, 1 mL 80% CH3OH/H2O (v/v) was added to the cells, and the cells were scraped with extraction solvent. Both the extract and cell/debris suspension were transferred to a clean tube and thoroughly mixed on a vortex mixer for 120 s. Ultrasonication at 30 Hz for 2 × 10 s was performed. After being allowed to settle for 10 min, the cell extraction solution was centrifuged for 15 min at 15,000 g and 4 °C. Then, 800 μL of supernatant was transferred to a new centrifuge tube for lyophilization. The residue was reconstituted with 40 μL of Mix-IS-MeOH.

Sampleprep ID:

SP001956

Sampleprep Summary:

Plasma (NIST SRM 1950): A 240 μL volume of Mix-IS-ACN was added to 60 μL of NIST SRM 1950 for protein precipitation. Then, the sample was vortexed for 60 s and centrifuged for 10 min at 15,000 g and 4 °C. Then, 250 μL of supernatant was transferred to a new centrifuge tube for lyophilization. The residue was reconstituted with 60 μL of 90% H2O/CH3OH (v/v). Urine: A 60 μL sample of urine was centrifuged for 10 min at 15,000 g and 4 °C, and the supernatant was transferred to a new centrifuge tube. Then, 30 μL Mix-IS-MeOH was added to the same centrifuge tube, and the mixture was thoroughly combined on a vortex mixer for 60 s. HepG2: HepG2 cells were purchased from the Chinese Tissue Culture Collections (CTCC, Shanghai, China) and cultured in Dulbecco’s modified Eagle’s medium (DMEM, Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco), 100 IU/ml penicillin and 100 µg/ml streptomycin (Gibco). These cells were cultured at 37 °C in a humidified 5% CO2 atmosphere and treated within 24 hours after the confluence of cells reached 80%. The culture media of the cells was removed, and the cells were washed three times in phosphate-buffered saline (PBS). Then, the cells were quickly frozen in liquid nitrogen and stored at -80 °C before extraction. Then, 1 mL 80% CH3OH/H2O (v/v) was added to the cells, and the cells were scraped with extraction solvent. Both the extract and cell/debris suspension were transferred to a clean tube and thoroughly mixed on a vortex mixer for 120 s. Ultrasonication at 30 Hz for 2 × 10 s was performed. After being allowed to settle for 10 min, the cell extraction solution was centrifuged for 15 min at 15,000 g and 4 °C. Then, 800 μL of supernatant was transferred to a new centrifuge tube for lyophilization. The residue was reconstituted with 40 μL of Mix-IS-MeOH.

Combined analysis:

Analysis ID	AN003035
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Waters Acquity
Column	Waters Acquity BEH C8 (100 x 2.1mm,1.7um)
MS Type	ESI
MS instrument type	QTOF
MS instrument name	ABI Sciex 5600+ TripleTOF
Ion Mode	POSITIVE
Units	peak area

Chromatography:

Chromatography ID:	CH002248
Instrument Name:	Waters Acquity
Column Name:	Waters Acquity BEH C8 (100 x 2.1mm,1.7um)
Column Temperature:	50
Flow Rate:	0.35 mL/min
Chromatography Type:	Reversed phase

MS:

MS ID:	MS002822
Analysis ID:	AN003035
Instrument Name:	ABI Sciex 5600+ TripleTOF
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	The spray voltage was 5.5 kV and the source temperature was 550 °C, Curtain gas, gas 1 and gas 2 were set at 35, 55, and 55 psi, respectively. The data was processed by XCMS, MS-DIAL, MZmine 2 and MetEx.
Ion Mode:	POSITIVE