Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA217817

Local Sample ID:	A02-02_58_1_1700
Subject ID:	SU002359
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Gender:	Not applicable
Cell Biosource Or Supplier:	Sigma-Aldrich (St. Louis, MO)
Cell Strain Details:	Hep3B

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Subject:

Subject ID:	SU002359
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Gender:	Not applicable
Cell Biosource Or Supplier:	Sigma-Aldrich (St. Louis, MO)
Cell Strain Details:	Hep3B

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
A02-02_58_1_1700	SA217817	FL026443	Parental	Phenotype

Collection:

Collection ID:	CO002352
Collection Summary:	Cell lines and culture conditions: The Hep3B cell line used in this study was purchased from Sigma-Aldrich (St. Louis, MO). Sorafenib-resistant Hep3B cells were developed from Hep3B cell lines One million Hep3B cells were seeded in T75 flasks and cultured in Dulbecco's Modified Eagle Medium (DMEM) as monolayers. The medium was sup-plemented with 10% fetal bovine serum (FBS) (Sigma Aldrich, St. Louis, #F2442-500ML) and 1% penicillin/streptomycin (Sigma Aldrich, St. Louis, #P4333). Cultures were then incubated in 5% CO2 humidified atmosphere at 37°C for 3-4 days until 70-80% confluency was reached. Aseptic techniques were applied to avoid poten-tial contamination, and the confluency and contamination were checked routinely.
Sample Type:	Cultured cells

Treatment:

Treatment ID:	TR002371
Treatment Summary:	Generation of resistant Hep3B cell line: Resistant Hep3B cells were developed in clinically relevant models. In T-75 flasks, Hep3B cells were seeded overnight and incubated at 37°C. The cells were then treated with sorafenib at the 10 % inhibitory concentration (IC10) (0.4µM) of sorafenib (Biovision, Milpitas, CA, USA #BAY 43-9006) to mimic the clinically consumed low dose of chemotherapeutic drugs by a cancer patient who is then exposed to escalating doses over time. To develop resistance, the survived cells were transplanted to a new flask and then treated with sorafenib at escalating concentrations over six months. Then, sorafenib at concentration IC10 (0.4µM) was persistently retained in the culture media to ensure and maintain resistance. MTT cell viability assay was performed each month to validate the resistance behavior of the cells.

Treatment ID:

TR002371

Treatment Summary:

Generation of resistant Hep3B cell line: Resistant Hep3B cells were developed in clinically relevant models. In T-75 flasks, Hep3B cells were seeded overnight and incubated at 37°C. The cells were then treated with sorafenib at the 10 % inhibitory concentration (IC10) (0.4µM) of sorafenib (Biovision, Milpitas, CA, USA #BAY 43-9006) to mimic the clinically consumed low dose of chemotherapeutic drugs by a cancer patient who is then exposed to escalating doses over time. To develop resistance, the survived cells were transplanted to a new flask and then treated with sorafenib at escalating concentrations over six months. Then, sorafenib at concentration IC10 (0.4µM) was persistently retained in the culture media to ensure and maintain resistance. MTT cell viability assay was performed each month to validate the resistance behavior of the cells.

Sample Preparation:

Sampleprep ID:	SP002365
Sampleprep Summary:	Metabolite extraction: For metabolomics, three biological replicates were used. An equal number of cells were utilized for each sample. The cells were centrifuged separately at 15000 rpm for 10 min to separate the cells and cell-free supernatants. The supernatants were dis-carded, and to each cell pellet (~3x 106), 1 mL of 0.1% formic acid in methanol was added, followed by vertexing for 2 min x 4, interrupted by 15 min incubation on ice. This is followed by sonication using a COPLEY probe-sonicator (QSONICA SONI-CATOR, USA) for 30 seconds with 30% amplifier in an ice bath. Subsequently, the cel-lular debris of each sample was collected by centrifugation at 15000 rpm for 10 min at 4°C. The supernatants were separated and dried at 37 ± 1 °C using EZ-2 Plus (Gene-Vac-Ipswich, UK). Finally, 200 µL of 0.1% formic acid in water was used to resuspend the dried samples, which were then vortexed for 2 min, filtered by a 0.45µm hydro-philic Nylon Syringe Filter and transferred to 200 μL (micro-inserts) in LC vials. The quality control (QC) sample was prepared by pooling the same volume (10 μL) of each sample, and all samples were placed in the autosampler at a temperature set at 4℃ and analyzed with UHPLC-QTOF-MS.

Sampleprep ID:

SP002365

Sampleprep Summary:

Metabolite extraction: For metabolomics, three biological replicates were used. An equal number of cells were utilized for each sample. The cells were centrifuged separately at 15000 rpm for 10 min to separate the cells and cell-free supernatants. The supernatants were dis-carded, and to each cell pellet (~3x 106), 1 mL of 0.1% formic acid in methanol was added, followed by vertexing for 2 min x 4, interrupted by 15 min incubation on ice. This is followed by sonication using a COPLEY probe-sonicator (QSONICA SONI-CATOR, USA) for 30 seconds with 30% amplifier in an ice bath. Subsequently, the cel-lular debris of each sample was collected by centrifugation at 15000 rpm for 10 min at 4°C. The supernatants were separated and dried at 37 ± 1 °C using EZ-2 Plus (Gene-Vac-Ipswich, UK). Finally, 200 µL of 0.1% formic acid in water was used to resuspend the dried samples, which were then vortexed for 2 min, filtered by a 0.45µm hydro-philic Nylon Syringe Filter and transferred to 200 μL (micro-inserts) in LC vials. The quality control (QC) sample was prepared by pooling the same volume (10 μL) of each sample, and all samples were placed in the autosampler at a temperature set at 4℃ and analyzed with UHPLC-QTOF-MS.

Combined analysis:

Analysis ID	AN003715
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	UHPLC-QTOF-MS
Column	Hamilton Intensity Solo 2 C18 (100 mm x 2.1 mm,1.8um)
MS Type	ESI
MS instrument type	Trapped Ion Mobility Q-TOF
MS instrument name	Bruker timsTOF
Ion Mode	POSITIVE
Units	AU

Chromatography:

Chromatography ID:	CH002752
Chromatography Summary:	The Elute UHPLC and Q-TOF Mass Spectrometer (Bruker, Bremen, Germany) were utilized for metabolite and peptide detection. The Elute HPG 1300 pumps, Elute Au-tosampler (Bruker, Bremen, Germany), and Hamilton® Intensity Solo 2 C18 column (100 mm x 2.1 mm, 1.8 m beads) were employed using reversed-phase chromatog-raphy. Solvents used for separation were 0.1 % FA in LC grade water (solvent A) and 0.1 % FA in ACN (solvent B). Each metabolite and protein extract were analyzed in duplicate. For metabolomics analysis, the column was kept at 35°C, and each sample was injected twice with an in-jection volume of 10 µL. Sample elution was performed in 30 min gradient starting with 1% ACN for 2 min and then ramped to 99% ACN within 15 min. After that, 99% ACN was kept for 3 min, and then the re-equilibration to 1% ACN was done for 10 min. The flow rate was 0.25 mL/min for 20 min and then 0.35 mL/min for 8.3 min and then the flow rate set at 0.25 mL/min for 1.7 min.
Instrument Name:	UHPLC-QTOF-MS
Column Name:	Hamilton Intensity Solo 2 C18 (100 mm x 2.1 mm,1.8um)
Column Temperature:	35°C
Flow Gradient:	Sample elution was performed in 30 min gradient starting with 1% ACN for 2 min and then ramped to 99% ACN within 15 min. After that, 99% ACN was kept for 3 min, and then the re-equilibration to 1% ACN was done for 10 min
Flow Rate:	was 0.25 mL/min for 20 min and then 0.35 mL/min for 8.3 min and then the flow rate set at 0.25 mL/min for 1.7 min.
Solvent A:	100% water; 0.1% formic acid
Solvent B:	100% acetonitrile; 0.1% formic acid
Chromatography Type:	Reversed phase

MS:

MS ID:	MS003464
Analysis ID:	AN003715
Instrument Name:	Bruker timsTOF
Instrument Type:	Trapped Ion Mobility Q-TOF
MS Type:	ESI
MS Comments:	For MS2 acquisition in metabolomics analysis, the collision energy was fluctuated between 100-250% of 20 eV and end plate offset of 500V. The acquisition was in two sections: auto MS scan for the calibrant sodium formate in 0-0.3 min, and auto MS/MS for fragmentation, in 0.3 to 30 min. Positive mode at 12 Hz was performed in both acquisition sections . The scan range was 20 to 1300 m/z, the precursor ion’s width of ±0.5, the precursors number of 3 , the cycle time of 0.5 s econds, and the threshold of 400 counts. After three spectra, active exclusion was performed and released after 0.2 min. A timsTOF (Bruker, Bremen, Germany) with an Apollo II electrospray ionization (ESI) source was utilized for the MS analysis with the following parameters: the nebulizer pressure was 2.2 bar, the drying gas flow rate was 10 L/min, the drying tempera-ture was 220°C, and the capillary voltage was 4500 V. In the first 0.3 min of each LC-MS/MS run, the external cali-brant, sodium formate, was injected. Mass calibration was done prior to analysis ac-cording to the manufacturer’s recommendations using external mass calibration (10 mM sodium formate calibrant solution). The performance of the column and the mass spectrometer was tested using a test mixture of (TRX-2101/RT-28-calibrants for Bruker T-ReX LC-QTOF solution from Nova Medical Testing Inc.) to check the performance of reversed-phase liquid chromatography (RPLC) separation and perform multipoint re-tention time calibration, and (TRX-3112-R/MS Certified Human serum for Bruker T-ReX LC-QTOF solution from Nova Medical Testing Inc.) to check the performance of sample preparation protocols as well as LC-MS instruments. This product is prepared from pooled human blood.
Ion Mode:	POSITIVE