Summary of Study ST002221

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001418. The data can be accessed directly via it's Project DOI: 10.21228/M8F409 This work is supported by NIH grant, U2C- DK119886.

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This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

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Study IDST002221
Study TitleGlutaminolysis contribution to the carbon backbone of aspartate through ATP Citrate Lyase (ACLY) in ccRCC
Study SummaryThe objective of this experiment is to test the contribution of the carbons from glutamine to generation of aspartate via ATP citrate lyase (ACLY) in human epithelial renal cells HK2 and ccRCC cell lines 786-O and 786-M1A. To test this hypothesis, we incubated all cells with 13C5-glutamine in Plasmax media with or without a pharmacological inhibitor of ACLY. This is Part 8 of the study and the experimental number is MS58.
Institute
CECAD Research Center
Last NameYang
First NameMing
AddressJoseph-Stelzmann-Straße 26, Köln, Koeln, 50931, Germany
Emailming.yang@uni-koeln.de
Phone4922147884306
Submit Date2022-07-15
Raw Data AvailableYes
Raw Data File Type(s)raw(Thermo)
Analysis Type DetailLC-MS
Release Date2022-08-03
Release Version1
Ming Yang Ming Yang
https://dx.doi.org/10.21228/M8F409
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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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
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