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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 |
Select appropriate tab below to view additional metadata details:
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 |
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 |
