Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA080700

Local Sample ID:	13RWPE1_24-3
Subject ID:	SU001232
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606

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Subject:

Subject ID:	SU001232
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
13RWPE1_24-3	SA080700	FL012344	RWPE1	Cell lines
13RWPE1_24-3	SA080700	FL012344	24h	Time points

Collection:

Collection ID:	CO001226
Collection Summary:	cells were grown in RPMI 1640 medium containing 2 g/L glucose with 10% dFBS, and were harvested at 80% confluence. One hour before the isotope switch, the medium was aspirated and replaced with fresh unlabeled medium[1]. Subsequently, VCaP and RWPE-1 were cultured in RPMI 1640 medium with 2 g/L [U−13C]-glucose and 10% dFBS for 0 h, 0.25 h, 4 h, 12 h, 24 h and long-term for 6 days performing thereby 3 passages. 10 cm petri dish with 10 mL of media was used for each sample, and 4 independent biological replicates were prepared for the labeling experiments. Unlabeled samples were treated in the same way with medium containing unlabeled glucose. After the cultures have been grown for the targeted amount of time, the medium was aspirated completely, then cells were washed thrice with 5% D-mannitol solution, followed by inactivation with liquid nitrogen immediately.
Sample Type:	Cultured cells

Treatment:

Treatment ID:	TR001247
Treatment Summary:	One hour before the isotope switch, the medium was aspirated and replaced with fresh unlabeled medium[1]. Subsequently, VCaP and RWPE-1 were cultured in RPMI 1640 medium with 2 g/L [U−13C]-glucose and 10% dFBS for 0 h, 0.25 h, 4 h, 12 h, 24 h and long-term for 6 days performing thereby 3 passages. 10 cm petri dish with 10 mL of media was used for each sample, and 4 independent biological replicates were prepared for the labeling experiments. Unlabeled samples were treated in the same way with medium containing unlabeled glucose.

Treatment ID:

TR001247

Treatment Summary:

One hour before the isotope switch, the medium was aspirated and replaced with fresh unlabeled medium[1]. Subsequently, VCaP and RWPE-1 were cultured in RPMI 1640 medium with 2 g/L [U−13C]-glucose and 10% dFBS for 0 h, 0.25 h, 4 h, 12 h, 24 h and long-term for 6 days performing thereby 3 passages. 10 cm petri dish with 10 mL of media was used for each sample, and 4 independent biological replicates were prepared for the labeling experiments. Unlabeled samples were treated in the same way with medium containing unlabeled glucose.

Sample Preparation:

Sampleprep ID:	SP001240
Sampleprep Summary:	After the cultures have been grown for the targeted amount of time, the medium was aspirated completely, then cells were washed thrice with 5% D-mannitol solution, followed by inactivation with liquid nitrogen immediately. Cells were handled on ice during the preparation. 1 mL of precooled methanol, containing 13.3 μM of MetS and CSA, was added to each cell culture dish. Cells were harvested using a cell scraper, and transferred into a 5 mL tube. Then, 1 mL of chloroform was added and vortexed for 5 min, followed by 400 μL of Milli-Q water and 10 min of vortexing. After 5 min of standing, the mixture was centrifuged to form a two-phase system (10,000 g, 4 °C, 15 min). The upper layer was transferred and centrifugally filtered through a Millipore 5-kDa cutoff filter (USA) (13,000 g, 3h at 4°C). The filtrate was dried and stored at -80 °C. Finally, the upper extract was dissolved in Milli-Q water containing 50 μM of 3-AP, DPA, DHD and TMA for capillary electrophoresis-time of flight mass spectrometry (CE-TOF/MS) analysis.

Sampleprep ID:

SP001240

Sampleprep Summary:

After the cultures have been grown for the targeted amount of time, the medium was aspirated completely, then cells were washed thrice with 5% D-mannitol solution, followed by inactivation with liquid nitrogen immediately. Cells were handled on ice during the preparation. 1 mL of precooled methanol, containing 13.3 μM of MetS and CSA, was added to each cell culture dish. Cells were harvested using a cell scraper, and transferred into a 5 mL tube. Then, 1 mL of chloroform was added and vortexed for 5 min, followed by 400 μL of Milli-Q water and 10 min of vortexing. After 5 min of standing, the mixture was centrifuged to form a two-phase system (10,000 g, 4 °C, 15 min). The upper layer was transferred and centrifugally filtered through a Millipore 5-kDa cutoff filter (USA) (13,000 g, 3h at 4°C). The filtrate was dried and stored at -80 °C. Finally, the upper extract was dissolved in Milli-Q water containing 50 μM of 3-AP, DPA, DHD and TMA for capillary electrophoresis-time of flight mass spectrometry (CE-TOF/MS) analysis.

Combined analysis:

Analysis ID	AN001929	AN001930
Analysis type	MS	MS
Chromatography type	CE	CE
Chromatography system	Agilent 1290 Infinity II	Agilent 1290 Infinity II
Column	a fused silica capillary (50 μm i.d. × 80 cm)	a fused silica capillary (50 μm i.d. × 80 cm)
MS Type	ESI	ESI
MS instrument type	Triple quadrupole	Triple quadrupole
MS instrument name	Agilent 6224 TOF	Agilent 6224 TOF
Ion Mode	POSITIVE	NEGATIVE
Units	Relative intensity after normalization	Relative intensity after normalization

Chromatography:

Chromatography ID:	CH001400
Instrument Name:	Agilent 1290 Infinity II
Column Name:	a fused silica capillary (50 μm i.d. × 80 cm)
Column Temperature:	20
Flow Rate:	a positive voltage of 27 kV
Injection Temperature:	20
Solvent A:	1 M formic acid
Chromatography Type:	CE

Chromatography ID:	CH001401
Instrument Name:	Agilent 1290 Infinity II
Column Name:	a fused silica capillary (50 μm i.d. × 80 cm)
Column Temperature:	20
Flow Rate:	internal pressure of 15 mbar and positive voltage of 30 kV
Injection Temperature:	20
Solvent A:	100% water; 25 mM ammonium acetate; 75 mM ammonium phosphate, pH 8.5
Chromatography Type:	CE

MS:

MS ID:	MS001785
Analysis ID:	AN001929
Instrument Name:	Agilent 6224 TOF
Instrument Type:	Triple quadrupole
MS Type:	ESI
MS Comments:	nebulizer pressure of 5 psig, dry gas temperature of 300 °C, nitrogen flow of 7 L/min, capillary voltage of 4 kV, fragmentor of 105 V, skimmer of 50 V, Oct RFV of 650 V, acquisition rate of 1.5 spectra/s and mass range of 60-1,000 were utilized.
Ion Mode:	POSITIVE

MS ID:	MS001786
Analysis ID:	AN001930
Instrument Name:	Agilent 6224 TOF
Instrument Type:	Triple quadrupole
MS Type:	ESI
MS Comments:	capillary voltage of 3.5 kV, fragmentor of 125 V and mass range of 50-1,000 were set in the TOF/MS analysis. During sample analysis, reference masses were applied to ensure the real-time calibration of exact mass measurement.
Ion Mode:	NEGATIVE