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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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:SU002307
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
SA211972MS58_042786M1A BMS 10μM
SA211973MS58_041786M1A BMS 10μM
SA211974MS58_043786M1A BMS 10μM
SA211975MS58_044786M1A BMS 10μM
SA211976MS58_045786M1A BMS 10μM
SA211977MS58_037786M1A BMS 5μM
SA211978MS58_038786M1A BMS 5μM
SA211979MS58_036786M1A BMS 5μM
SA211980MS58_039786M1A BMS 5μM
SA211981MS58_040786M1A BMS 5μM
SA211982MS58_033786M1A DMSO
SA211983MS58_031786M1A DMSO
SA211984MS58_032786M1A DMSO
SA211985MS58_034786M1A DMSO
SA211986MS58_035786M1A DMSO
SA211987MS58_027786O BMS 10μM
SA211988MS58_026786O BMS 10μM
SA211989MS58_028786O BMS 10μM
SA211990MS58_029786O BMS 10μM
SA211991MS58_030786O BMS 10μM
SA211992MS58_025786O BMS 5μM
SA211993MS58_024786O BMS 5μM
SA211994MS58_023786O BMS 5μM
SA211995MS58_022786O BMS 5μM
SA211996MS58_021786O BMS 5μM
SA211997MS58_017786O DMSO
SA211998MS58_016786O DMSO
SA211999MS58_018786O DMSO
SA212000MS58_019786O DMSO
SA212001MS58_020786O DMSO
SA212002MS58_011HK2 BMS 10μM
SA212003MS58_012HK2 BMS 10μM
SA212004MS58_013HK2 BMS 10μM
SA212005MS58_015HK2 BMS 10μM
SA212006MS58_014HK2 BMS 10μM
SA212007MS58_006HK2 BMS 5μM
SA212008MS58_007HK2 BMS 5μM
SA212009MS58_008HK2 BMS 5μM
SA212010MS58_010HK2 BMS 5μM
SA212011MS58_009HK2 BMS 5μM
SA212012MS58_002HK2 DMSO
SA212013MS58_003HK2 DMSO
SA212014MS58_005HK2 DMSO
SA212015MS58_001HK2 DMSO
SA212016MS58_004HK2 DMSO
Showing results 1 to 45 of 45

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
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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