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MB Sample ID: SA211999

Local Sample ID:MS58_018
Subject ID:SU002307
Subject Type:Cultured cells
Subject Species:Homo sapiens
Taxonomy ID:9606

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Subject:

Subject ID:SU002307
Subject Type:Cultured cells
Subject Species:Homo sapiens
Taxonomy ID:9606

Factors:

Local Sample IDMB Sample IDFactor Level IDLevel ValueFactor Name
MS58_018SA211999FL025865786OCell line
MS58_018SA211999FL025865DMSOTreatment

Collection:

Collection ID:CO002300
Collection Summary:2x105 cells were plated onto 6-well plates (5 replicates for each cell type). The day after, the medium was replaced with fresh one containing 13C5 glutamine in the presence of vehicle (DMSO) or ACLY inhibitor (BMS-303141) and further incubated for 8h. Before extraction, cells were counted using CASY cell counter (Omni Life Sciences) using a separate counting plate. After that, cells were washed at room temperature with PBS twice and then kept in a cold bath with dry ice and methanol before adding the metabolite extraction solution.
Sample Type:Cultured cells

Treatment:

Treatment ID:TR002319
Treatment Summary:Cells were cultured in Plasmax media supplemented with 2.5% FBS with 13C5-glutamine (0.65mM), in the presence of DMSO or ACLY inhibitor BMS-303141 (5μM and 10μM)

Sample Preparation:

Sampleprep ID:SP002313
Sampleprep Summary:The day of the extraction, cells were washed at room temperature with PBS twice and then kept on cold bath with dry ice and methanol. Metabolite extraction solution (50% methanol, 30% acetonitrile, 20% ultrapure water, 5 µM final concentration valine-d8) was added to each well following the proportion of 1 ml of extraction solution per million cells. The extracts were scraped and mixed at 4°C for 15 min. After final centrifugation at max speed for 15 min at 4°C, the supernatants were transferred into LC-MS vials.

Combined analysis:

Analysis ID AN003630
Analysis type MS
Chromatography type HILIC
Chromatography system Thermo Vanquish Horizon
Column SeQuant ZIC-pHILIC
MS Type ESI
MS instrument type Orbitrap
MS instrument name Thermo Exploris 240
Ion Mode UNSPECIFIED
Units peak area

Chromatography:

Chromatography ID:CH002685
Chromatography Summary:Chromatographic separation of metabolites was achieved using a Millipore Sequant ZIC-pHILIC analytical column (5 µm, 2.1 × 150 mm) equipped with a 2.1 × 20 mm guard column (both 5 mm particle size) with a binary solvent system. Solvent A was 20 mM ammonium carbonate, 0.05% ammonium hydroxide; Solvent B was acetonitrile. The column oven and autosampler tray were held at 40 °C and 4 °C, respectively. The chromatographic gradient was run at a flow rate of 0.200 mL/min as follows: 0–2 min: 80% B; 2-17 min: linear gradient from 80% B to 20% B; 17-17.1 min: linear gradient from 20% B to 80% B; 17.1-23 min: hold at 80% B. Samples were randomized and the injection volume was 5 µl. A pooled quality control (QC) sample was generated from an equal mixture of all individual samples and analysed interspersed at regular intervals.
Instrument Name:Thermo Vanquish Horizon
Column Name:SeQuant ZIC-pHILIC
Column Temperature:40
Flow Gradient:0-2 min: 80% B; 2-17 min: linear gradient from 80% B to 20% B; 17-17.1 min: linear gradient from 20% B to 80% B; 17.1-23 min: hold at 80% B
Flow Rate:0.200 mL/min
Solvent A:100% water; 20 mM ammonium carbonate; 0.05% ammonium hydroxide
Solvent B:100% acetonitrile
Chromatography Type:HILIC

MS:

MS ID:MS003381
Analysis ID:AN003630
Instrument Name:Thermo Exploris 240
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Chromatographic separation of metabolites was achieved using a Millipore Sequant ZIC-pHILIC analytical column (5 µm, 2.1 × 150 mm) equipped with a 2.1 × 20 mm guard column (both 5 mm particle size) with a binary solvent system. Solvent A was 20 mM ammonium carbonate, 0.05% ammonium hydroxide; Solvent B was acetonitrile. The column oven and autosampler tray were held at 40 °C and 4 °C, respectively. The chromatographic gradient was run at a flow rate of 0.200 mL/min as follows: 0–2 min: 80% B; 2-17 min: linear gradient from 80% B to 20% B; 17-17.1 min: linear gradient from 20% B to 80% B; 17.1-23 min: hold at 80% B. Samples were randomized and the injection volume was 5 µl. A pooled quality control (QC) sample was generated from an equal mixture of all individual samples and analysed interspersed at regular intervals. Metabolites were measured with Vanquish Horizon UHPLC coupled to an Orbitrap Exploris 240 mass spectrometer (both Thermo Fisher Scientific) via a heated electrospray ionization source. The spray voltages were set to +3.5kV/-2.8 kV, RF lens value at 70, the heated capillary held at 320 °C, and the auxiliary gas heater held at 280 °C. The flow rate for sheath gas, aux gas and sweep gas were set to 40, 15 and 0, respectively. Data acquisition was performed in full scan mode with polarity switching at an Orbitrap resolution of 120000, with mass range set to m/z=70-900, AGC target set to standard and maximum injection time (Max IT) set to auto. Metabolite identities were confirmed using two parameters: (1) precursor ion m/z was matched within 5 ppm of theoretical mass predicted by the chemical formula; (2) the retention time of metabolites was within 5% of the retention time of a purified standard run with the same chromatographic method. Chromatogram review and peak area integration were performed using the Thermo Fisher software Tracefinder 5.0 and the peak area for each detected metabolite was normalized against the total ion count (TIC) of that sample to correct any variations introduced from sample handling and instrument analysis. The normalized areas were used as variables for further statistical data analysis. For 13C-tracing analysis, the theoretical masses of 13C isotopes were calculated and added to a library of predicted isotopes in Tracefinder 5.0. These masses were then searched with a 5-ppm tolerance and integrated only if the peak apex showed less than 1% deviation in retention time from the [U-12C] monoisotopic mass in the same chromatogram. The raw data obtained for each isotopologue were corrected for natural isotope abundances using the AccuCor algorithm (https://github.com/lparsons/accucor) before further statistical analysis.
Ion Mode:UNSPECIFIED
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