Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA080931

Local Sample ID:	0.1_NC_72h_2
Subject ID:	SU001235
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Gender:	Not applicable

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Subject:

Subject ID:	SU001235
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Gender:	Not applicable

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
0.1_NC_72h_2	SA080931	FL012368	72	hours
0.1_NC_72h_2	SA080931	FL012368	-	Doxorubicin dosage(μM)

Collection:

Collection ID:	CO001229
Collection Summary:	Cells were washed with cold PBS twice and then permeabilized with cold 80% methanol followed by a 20-minute incubation at -80℃. The cell lysates were centrifuges at 14000g for 5 mins and the supernatant were collected and evaporated to dryness.
Sample Type:	Cultured cells

Treatment:

Treatment ID:	TR001250
Treatment Summary:	MCF-7 cells were exposed to 10 μM doxorubicin for 4 hr followed by subsequent withdrawal of the drug. Meanwhile, MCF-7 cells continuously exposed to a low dosage of doxorubicin at 0.1 μM for 96 hr. Doxorubicin-treated cells and control cells were collected at different time.

Sample Preparation:

Sampleprep ID:	SP001243
Sampleprep Summary:	MCF-7 cells were harvested and permeabilized with cold (80% (v/v) methanol: water solution followed by a 20-min incubation at -80 ℃. A stock solution of 4-Chloro-DL-phenylalanine was pre-spiked into the methanol aqueous buffer to a final concentration of 1.5 μg/mL, which subsequently served as an internal standard (IS). The cell lysates were centrifuged at 14,000g for 5 min at 4 ℃, and the resultant supernatant were collected and evaporated to dryness. The samples were reconstituted in 100 μL of water (LC-MS grade) before analysis on an HPLC−MS/MS, and 20 μL was used for analysis. For each group of samples subjected to metabolomics analysis, five biological replicates were prepared.

Sampleprep ID:

SP001243

Sampleprep Summary:

MCF-7 cells were harvested and permeabilized with cold (80% (v/v) methanol: water solution followed by a 20-min incubation at -80 ℃. A stock solution of 4-Chloro-DL-phenylalanine was pre-spiked into the methanol aqueous buffer to a final concentration of 1.5 μg/mL, which subsequently served as an internal standard (IS). The cell lysates were centrifuged at 14,000g for 5 min at 4 ℃, and the resultant supernatant were collected and evaporated to dryness. The samples were reconstituted in 100 μL of water (LC-MS grade) before analysis on an HPLC−MS/MS, and 20 μL was used for analysis. For each group of samples subjected to metabolomics analysis, five biological replicates were prepared.

Combined analysis:

Analysis ID	AN001935
Analysis type	MS
Chromatography type	HILIC
Chromatography system	Shimadzu LC-30A
Column	Waters XBridge Amide (100 x 4.6mm,3.5um)
MS Type	ESI
MS instrument type	QTOF
MS instrument name	ABI Sciex 5600 TripleTOF
Ion Mode	NEGATIVE
Units	normalized peak area

Chromatography:

Chromatography ID:	CH001404
Chromatography Summary:	The mobile phase consisted of solvent A (5 mM ammonium acetate, pH=9, and 5% acetonitrile) and solvent B (acetonitrile). The gradient was set as follows: 0–3 min, 85% B; 3–6 min, 85–30% B; 6–15 min, 30–2% B; 15–18 min, 2% B; 18–19 min, 2–85% B; 19–26 min, 85% B. The flow rate was set at 0.4 mL/min.
Instrument Name:	Shimadzu LC-30A
Column Name:	Waters XBridge Amide (100 x 4.6mm,3.5um)
Flow Gradient:	0-3 min, 85% B; 3-6 min, 85-30% B; 6-15 min, 30-2% B; 15-18 min, 2% B; 18-19 min, 2-85% B; 19-26 min, 85% B.
Flow Rate:	0.4 mL/min
Solvent A:	5% acetonitrile/95% water; 5 mM ammonium acetate, pH 9
Solvent B:	100% acetonitrile
Chromatography Type:	HILIC

MS:

MS ID:	MS001791
Analysis ID:	AN001935
Instrument Name:	ABI Sciex 5600 TripleTOF
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	The MS data was acquired in the negative ion mode using a data-dependent acquisition approach. The electrospray ionization (ESI) source conditions were used as follows: TOF MS scan, m/z 50 – 1000 Da; Product ion scan, m/z 50 – 900 Da; Ion Source Gas 1 (Gas 1), 50 psi; Ion Source Gas 2 (Gas 2), 30 psi; Curtain gas, 30 psi; Source temperature, 500 ℃; Ion Spray Voltage Floating, -4500 V; Declustering potential (DP), -100V; Collision energy (CE), -35 V; CE spread, 10 V. MS data acquisition was controlled by Analyst TF 1.6.1 (AB SCIEX, Framingham, MA, USA). The accurate mass was calibrated by Calibration Delivery System.
Ion Mode:	NEGATIVE