Summary of Study ST002222

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 IDST002222
Study TitleGlutaminolysis contribution to the carbon backbone of aspartate and glutamate in ccRCC
Study SummaryThe objective of this experiment is to test the contribution of the carbons derived from glutamine to the generation of aspartate and glutamate 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 glutaminase CB-839. This is Part 7 of a study and the experimental number is MS57.
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

Select appropriate tab below to view additional metadata details:

Project:

Project ID:PR001418
Project DOI:doi: 10.21228/M8F409
Project Title:Dynamic partitioning of branched-chain amino acids-derived nitrogen supports renal cancer progression
Project Summary:Metabolic reprogramming is critical for tumor initiation and progression. However, the exact impact of specific metabolic changes on cancer progression is poorly understood. Here, we integrate multimodal analyses of primary and metastatic clonally related clear cell renal cancer cells (ccRCC) grown in physiological media to identify key stage-specific metabolic vulnerabilities. We show that a VHL loss-dependent reprogramming of branched-chain amino acid catabolism sustains the de novo biosynthesis of aspartate and arginine enabling tumor cells with the flexibility of partitioning the nitrogen of the amino acids depending on their needs. Importantly, we identify the epigenetic reactivation of argininosuccinate synthase (ASS1), a urea cycle enzyme suppressed in primary ccRCC, as a crucial event for metastatic renal cancer cells to acquire the capability to generate arginine, invade in vitro and metastasize in vivo. Overall, our study uncovers a novel mechanism of metabolic flexibility occurring during ccRCC progression, paving the way for the development of novel stage-specific therapies.
Institute:CECAD Research Center, University Hospital Cologne
Last Name:Yang
First Name:Ming
Address:Joseph-Stelzmann-Straße 26, CECAD Research Center, Köln, Koeln, 50931, Germany
Email:ming.yang@uni-koeln.de
Phone:+4922147884306

Subject:

Subject ID:SU002308
Subject Type:Cultured cells
Subject Species:Homo sapiens
Taxonomy ID:9606

Factors:

Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id local_sample_id Cell line Treatment
SA212017MS57_049786M1A CB839 100nM
SA212018MS57_048786M1A CB839 100nM
SA212019MS57_047786M1A CB839 100nM
SA212020MS57_050786M1A CB839 100nM
SA212021MS57_046786M1A CB839 100nM
SA212022MS57_057786M1A CB839 1μM
SA212023MS57_058786M1A CB839 1μM
SA212024MS57_059786M1A CB839 1μM
SA212025MS57_060786M1A CB839 1μM
SA212026MS57_056786M1A CB839 1μM
SA212027MS57_051786M1A CB839 500nM
SA212028MS57_055786M1A CB839 500nM
SA212029MS57_052786M1A CB839 500nM
SA212030MS57_054786M1A CB839 500nM
SA212031MS57_053786M1A CB839 500nM
SA212032MS57_042786M1A DMSO
SA212033MS57_041786M1A DMSO
SA212034MS57_043786M1A DMSO
SA212035MS57_044786M1A DMSO
SA212036MS57_045786M1A DMSO
SA212037MS57_027786O CB839 100nM
SA212038MS57_028786O CB839 100nM
SA212039MS57_030786O CB839 100nM
SA212040MS57_029786O CB839 100nM
SA212041MS57_026786O CB839 100nM
SA212042MS57_037786O CB839 1μM
SA212043MS57_036786O CB839 1μM
SA212044MS57_038786O CB839 1μM
SA212045MS57_040786O CB839 1μM
SA212046MS57_039786O CB839 1μM
SA212047MS57_035786O CB839 500nM
SA212048MS57_034786O CB839 500nM
SA212049MS57_031786O CB839 500nM
SA212050MS57_033786O CB839 500nM
SA212051MS57_032786O CB839 500nM
SA212052MS57_022786O DMSO
SA212053MS57_021786O DMSO
SA212054MS57_023786O DMSO
SA212055MS57_024786O DMSO
SA212056MS57_025786O DMSO
SA212057MS57_008HK2 CB839 100nM
SA212058MS57_007HK2 CB839 100nM
SA212059MS57_010HK2 CB839 100nM
SA212060MS57_006HK2 CB839 100nM
SA212061MS57_009HK2 CB839 100nM
SA212062MS57_020HK2 CB839 1μM
SA212063MS57_019HK2 CB839 1μM
SA212064MS57_018HK2 CB839 1μM
SA212065MS57_017HK2 CB839 1μM
SA212066MS57_016HK2 CB839 1μM
SA212067MS57_015HK2 CB839 500nM
SA212068MS57_011HK2 CB839 500nM
SA212069MS57_014HK2 CB839 500nM
SA212070MS57_013HK2 CB839 500nM
SA212071MS57_012HK2 CB839 500nM
SA212072MS57_002HK2 DMSO
SA212073MS57_003HK2 DMSO
SA212074MS57_001HK2 DMSO
SA212075MS57_005HK2 DMSO
SA212076MS57_004HK2 DMSO
Showing results 1 to 60 of 60

Collection:

Collection ID:CO002301
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 GLS inhibitor CB-839 (100nM, 500nM or 1μM) and further incubated for 23h. 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:TR002320
Treatment Summary:Cells were cultured in Plasmax media supplemented with 2.5% FBS with 13C5-glutamine (0.65mM), in the presence of DMSO or glutaminase inhibitor CB-839 (100nM, 500nM or 1μM)

Sample Preparation:

Sampleprep ID:SP002314
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 AN003631
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:CH002686
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:MS003382
Analysis ID:AN003631
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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