Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA227275

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Subject:

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
4a_-1_68_1_446	SA227275	FL027597	COMBINATION	Treatment

Collection:

Collection ID:	CO002398
Collection Summary:	The BT-474 BC cell line utilized in this study was cultured as monolayers in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Sigma Aldrich, St. Louis, MO, USA). All cultures were incubated at 37 °C in a humidified atmosphere of 5% CO2.
Sample Type:	Breast cancer cells
Storage Conditions:	Described in summary

Treatment:

Treatment ID:	TR002417
Treatment Summary:	Triplicate flasks were prepared for each treatment condition for each analysis (metabolomic and proteomic) for a total of 24 flasks. Two million cells were seeded in each 75 cm2 tissue culture flask and incubated for 24 h. The cells were then treated with Tamoxifen (5 μM) and/or Trastuzumab (2.5 μM) for 24 h. These concentrations correspond to the IC50 of these compounds with BT-474 cells, as determined by cytotoxicity assays (data not shown). Control cells were treated with vehicle (dimethyl sulfoxide (DMSO) at 0.5% for 24 h. Following the incubation period, cells were collected by trypsinization and washed twice with phosphate-buffered saline solution (PBS) before re-suspending in 1 mL 1 × PBS for further analysis. Finally, cells were collected as pellets by centrifugation at 1200 rounds per minute (rpm) for 10 min at room temperature. To negate the effect of Circadian rhythms on the response of cells to treatment, cells were kept under the same conditions during the entire incubation period and the cell collection was done concurrently for all samples. In addition, the same number of cells were used for each sample to avoid the effect of variation in cell numbers.
Treatment:	Drugs
Treatment Compound:	Tamoxifen and/or Trastuzumab

Sample Preparation:

Sampleprep ID:	SP002411
Sampleprep Summary:	Sample metabolite extraction A volume of 1 mL of the extraction solvent (methanol+0.1% formic acid) was added to the cell pellets to quench cells. The cells were then vortexed for 2 min to ensure the quantitative extraction of the metabolites and stored on ice for 1 h, during which the samples were vortexed every 15 min. After this, the insoluble cell matrices were collected and transferred to centrifuge tubes, intermittent ultrasonication using the COPLEY sonicator (QSONICA SONICATOR, USA) under 30% amplifier and for 30 s with an ice bath employed throughout the process. Following that, cells debris were then centrifuged (15,000 rpm, 10 min, − 4 °C) and the sample supernatants were collected and transferred to LC vials for drying in the EZ-2 Plus (GeneVac-Ipswich, UK) at 37±1 °C. Dried samples were resuspended with 200 µL (water+0.1% formic acid), and vortexed for 2 min. Finally, the samples were filtered using a hydrophilic Nylon Syringe Filter of 0.45 µm pore size and analyzed by Q-TOF MS
Processing Storage Conditions:	Described in summary
Extract Storage:	Described in summary

Combined analysis:

Chromatography:

Chromatography ID:	CH002800
Chromatography Summary:	Samples were chromatographically separated by inline reversed-phase chromatography using the Elute HPG 1300 pumps and Elute Autosampler (Bruker, Darmstadt, Germany) with solvent A 0.1% FA in HPLC grade water and solvent B 0.1% FA in ACN. A Hamilton Intensity Solo 2 C18 column (100 mm × 2.1 mm, 1.8 μm beads) was maintained at 35 ℃ (metabolomics analyses).
Methods ID:	.
Methods Filename:	.
Chromatography Comments:	.
Instrument Name:	Bruker Elute HPG 1300
Column Name:	Hamilton Intensity Solo 2 C18
Column Pressure:	.
Column Temperature:	35
Flow Gradient:	1%B to 99%B in 15 min
Flow Rate:	250 uL/min
Injection Temperature:	.
Internal Standard:	.
Internal Standard Mt:	.
Retention Index:	.
Retention Time:	.
Sample Injection:	.
Sampling Cone:	.
Solvent A:	100% water; 0.1% formic acid
Solvent B:	100% acetonitrile; 0.1% formic acid
Analytical Time:	.
Capillary Voltage:	.
Migration Time:	.
Oven Temperature:	35C
Preconditioning:	.
Running Buffer:	.
Running Voltage:	.
Sheath Liquid:	.
Time Program:	.
Transferline Temperature:	.
Washing Buffer:	.
Weak Wash Solvent Name:	.
Weak Wash Volume:	.
Strong Wash Solvent Name:	.
Strong Wash Volume:	.
Target Sample Temperature:	.
Sample Loop Size:	.
Sample Syringe Size:	.
Randomization Order:	.
Chromatography Type:	Reversed phase

MS:

MS ID:	MS003529
Analysis ID:	AN003786
Instrument Name:	Bruker timsTOF
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	The MS analysis was performed using a timsTOF (Bruker, Darmstadt, Germany) with Apollo II electrosprayionization (ESI) source. The drying gas was set to flow at 10 L/min and the drying temperature to 220℃ and the nebulizer pressure to 2.2 bar. The capillary voltage was 4500 V and the end plate offset 500 V. For metabolomics 20–1300 m/z. The instrument was operated in auto-MS/MS mode. For metabolomics the collision energy was set to 20 eV, the cycle time to 0.5 s with a relative minimum intensity threshold of 400 counts per thousand and a target intensity of 20,000. Sodium formate was injected as an external calibrant in the first 0.3 min of each LC–MS/MS run.
Ion Mode:	POSITIVE