Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA027796

Local Sample ID:	NEG_RP_S_25_MCF7
Subject ID:	SU000551
Subject Type:	Cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Subject Comments:	Cell pellets stored at -80˚C.
Cell Counts:	1E 7 cells/pellet
Species Group:	Human

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Subject:

Subject ID:	SU000551
Subject Type:	Cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Subject Comments:	Cell pellets stored at -80˚C.
Cell Counts:	1E 7 cells/pellet
Species Group:	Human

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
NEG_RP_S_25_MCF7	SA027796	FL006904	MCF7 parental	Cell Line

Collection:

Collection ID:	CO000545
Collection Summary:	20 breast cancer cell pellets from four cell lines [MCF7 parental (LSR+), MCF7 Crisper (LSR-), Hs578t parental (LSR-) and Hs578t (LSR++)], with five replicate samples for each cell line were stored at -80˚ until sample preparation.
Sample Type:	Cell pellets
Storage Conditions:	-80˚C

Treatment:

Treatment ID:	TR000565
Treatment Summary:	Cultured in an obesogenic media.

Sample Preparation:

Sampleprep ID:	SP000558
Sampleprep Summary:	Cell pellets were resuspended in volume of ice cold Lipid Extractions Solvent (50 µg/mL) based on biomass. Contents were transferred to new, pre-labeled 2.0 mL LoBind tube with 10-15 ceramic beads, and homogenized using 2 pulses at 2,000 rpm for 30 sec on a MagNA Lyser. Volume of HPLC-grade water (with 0.02 mg/mL Tryptophan-d5 in water) was added based on biomass. Samples were allowed to sit at room temperature for 10 minutes, then centrifuged at 16,000 rcf for 10 min at 10˚C. For reverse phase, transferred the maximum clean volume of the upper aqueous layer to a new, pre-labeled LoBind tube then a 150 µL aliquot was transferred to a new 1.5 mL, LoBind tube for analysis. An additional 50 µL aliquot of each study sample was combined in a 2 mL LoBind tube to make a QC pool, which was vortexed for 30 sec and aliquoted into 5 Total Pool samples (150 µL each), and the remainder used for 1 Equilibrium sample (for column conditioning). Samples were placed at -80˚C for 60 mins and lyophilized to dryness. Samples were reconstituted in H2O/Methanol (95:5 v/v), vortexed at 5,000 rpm for 2 mins and centrifuged at 16,000 rcf for 4 min. 100 µL of each supernatant was transferred to pre-labeled autosampler vials and 5 µL was injected Synapt G2Si ESI-Q-TOF for data acquisition.

Sampleprep ID:

SP000558

Sampleprep Summary:

Cell pellets were resuspended in volume of ice cold Lipid Extractions Solvent (50 µg/mL) based on biomass. Contents were transferred to new, pre-labeled 2.0 mL LoBind tube with 10-15 ceramic beads, and homogenized using 2 pulses at 2,000 rpm for 30 sec on a MagNA Lyser. Volume of HPLC-grade water (with 0.02 mg/mL Tryptophan-d5 in water) was added based on biomass. Samples were allowed to sit at room temperature for 10 minutes, then centrifuged at 16,000 rcf for 10 min at 10˚C. For reverse phase, transferred the maximum clean volume of the upper aqueous layer to a new, pre-labeled LoBind tube then a 150 µL aliquot was transferred to a new 1.5 mL, LoBind tube for analysis. An additional 50 µL aliquot of each study sample was combined in a 2 mL LoBind tube to make a QC pool, which was vortexed for 30 sec and aliquoted into 5 Total Pool samples (150 µL each), and the remainder used for 1 Equilibrium sample (for column conditioning). Samples were placed at -80˚C for 60 mins and lyophilized to dryness. Samples were reconstituted in H2O/Methanol (95:5 v/v), vortexed at 5,000 rpm for 2 mins and centrifuged at 16,000 rcf for 4 min. 100 µL of each supernatant was transferred to pre-labeled autosampler vials and 5 µL was injected Synapt G2Si ESI-Q-TOF for data acquisition.

Combined analysis:

Analysis ID	AN000807
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Waters Acquity
Column	Waters Acquity HSS T3 (100 x 2.1mm,1.8um)
MS Type	ESI
MS instrument type	QTOF
MS instrument name	Waters Synapt G2 Si QTOF
Ion Mode	NEGATIVE
Units	m/z

Chromatography:

Chromatography ID:	CH000582
Chromatography Summary:	Reversed-Phase Gradient Seperation
Methods ID:	RTI-RCMRC-RP
Methods Filename:	RTI-RCMRC-RP
Instrument Name:	Waters Acquity
Column Name:	Waters Acquity HSS T3 (100 x 2.1mm,1.8um)
Column Temperature:	50 °C
Flow Rate:	0.4 mL/min
Internal Standard:	L-Tryptophan-d5
Solvent A:	100% water; 0.1% formic acid
Solvent B:	100% methanol; 0.1% formic acid
Analytical Time:	22 min
Weak Wash Solvent Name:	10% MeOH
Weak Wash Volume:	1000 µL
Strong Wash Solvent Name:	80% MeOH
Strong Wash Volume:	1000 µL
Target Sample Temperature:	8 °C
Sample Loop Size:	10 µL
Sample Syringe Size:	100 µL
Randomization Order:	Yes
Chromatography Type:	Reversed phase

MS:

MS ID:	MS000714
Analysis ID:	AN000807
Instrument Name:	Waters Synapt G2 Si QTOF
Instrument Type:	QTOF
MS Type:	ESI
Ion Mode:	NEGATIVE
Capillary Temperature:	110 °C
Capillary Voltage:	1.0 kV
Collision Energy:	4
Fragmentation Method:	CID
Helium Flow:	180
Ionization:	ES-
Ionization Potential:	5 ppm
Source Temperature:	100 °C
Dataformat:	Continuum
Desolvation Gas Flow:	400 L/Hr
Desolvation Temperature:	400 °C
Resolution Setting:	18000
Scan Range Moverz:	50-1000 m/z
Scanning Cycle:	1.0 s
Tube Lens Voltage:	74