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.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
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.
| Study ID | ST002221 |
| Study Title | Glutaminolysis contribution to the carbon backbone of aspartate through ATP Citrate Lyase (ACLY) in ccRCC |
| Study Summary | The 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 Name | Yang |
| First Name | Ming |
| Address | Joseph-Stelzmann-Straße 26, Köln, Koeln, 50931, Germany |
| ming.yang@uni-koeln.de | |
| Phone | 4922147884306 |
| Submit Date | 2022-07-15 |
| Raw Data Available | Yes |
| Raw Data File Type(s) | raw(Thermo) |
| Analysis Type Detail | LC-MS |
| Release Date | 2022-08-03 |
| Release Version | 1 |
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: | 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 |
|---|---|---|---|
| SA211972 | MS58_042 | 786M1A | BMS 10μM |
| SA211973 | MS58_041 | 786M1A | BMS 10μM |
| SA211974 | MS58_043 | 786M1A | BMS 10μM |
| SA211975 | MS58_044 | 786M1A | BMS 10μM |
| SA211976 | MS58_045 | 786M1A | BMS 10μM |
| SA211977 | MS58_037 | 786M1A | BMS 5μM |
| SA211978 | MS58_038 | 786M1A | BMS 5μM |
| SA211979 | MS58_036 | 786M1A | BMS 5μM |
| SA211980 | MS58_039 | 786M1A | BMS 5μM |
| SA211981 | MS58_040 | 786M1A | BMS 5μM |
| SA211982 | MS58_033 | 786M1A | DMSO |
| SA211983 | MS58_031 | 786M1A | DMSO |
| SA211984 | MS58_032 | 786M1A | DMSO |
| SA211985 | MS58_034 | 786M1A | DMSO |
| SA211986 | MS58_035 | 786M1A | DMSO |
| SA211987 | MS58_027 | 786O | BMS 10μM |
| SA211988 | MS58_026 | 786O | BMS 10μM |
| SA211989 | MS58_028 | 786O | BMS 10μM |
| SA211990 | MS58_029 | 786O | BMS 10μM |
| SA211991 | MS58_030 | 786O | BMS 10μM |
| SA211992 | MS58_025 | 786O | BMS 5μM |
| SA211993 | MS58_024 | 786O | BMS 5μM |
| SA211994 | MS58_023 | 786O | BMS 5μM |
| SA211995 | MS58_022 | 786O | BMS 5μM |
| SA211996 | MS58_021 | 786O | BMS 5μM |
| SA211997 | MS58_017 | 786O | DMSO |
| SA211998 | MS58_016 | 786O | DMSO |
| SA211999 | MS58_018 | 786O | DMSO |
| SA212000 | MS58_019 | 786O | DMSO |
| SA212001 | MS58_020 | 786O | DMSO |
| SA212002 | MS58_011 | HK2 | BMS 10μM |
| SA212003 | MS58_012 | HK2 | BMS 10μM |
| SA212004 | MS58_013 | HK2 | BMS 10μM |
| SA212005 | MS58_015 | HK2 | BMS 10μM |
| SA212006 | MS58_014 | HK2 | BMS 10μM |
| SA212007 | MS58_006 | HK2 | BMS 5μM |
| SA212008 | MS58_007 | HK2 | BMS 5μM |
| SA212009 | MS58_008 | HK2 | BMS 5μM |
| SA212010 | MS58_010 | HK2 | BMS 5μM |
| SA212011 | MS58_009 | HK2 | BMS 5μM |
| SA212012 | MS58_002 | HK2 | DMSO |
| SA212013 | MS58_003 | HK2 | DMSO |
| SA212014 | MS58_005 | HK2 | DMSO |
| SA212015 | MS58_001 | HK2 | DMSO |
| SA212016 | MS58_004 | HK2 | 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 |
| 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 |
