Summary of Study ST002220

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.

Perform statistical analysis  |  Show all samples  |  Show named metabolites  |  Download named metabolite data  
Download mwTab file (text)   |  Download mwTab file(JSON)   |  Download data files (Contains raw data)
Study IDST002220
Study TitleCatabolism of branched-chain amino acids (BCAAs) in renal cells HK2 and 786-O
Study SummaryThe objective of this experiment is to compare the catabolism of branched-chain amino acids (BCAAs) in human renal epithelial cell line HK2 versus ccRCC cell lines 786-O, 786-M1A and 786-M2A using 13C6-labelled leucine and isoleucine stable isotope tracers. To this end, we incubated the above cell lines with 13C6-leucine and 13C6-isoleucine in Plasmax media for 27h. Data were generated from 5 independent cultures. This is Part I of the study and the experimental number is MS42.
Institute
CECAD Research Center
Last NameYang
First NameMing
AddressJoseph-Stelzmann-Straße 26, CECAD Research Center, Köln, Koeln, 50931, Germany
Emailming.yang@uni-koeln.de
Phone+4922147884306
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:SU002306
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
SA211952MS42-13786-M1A 13C6-leucine+13C6-isoleucine
SA211953MS42-14786-M1A 13C6-leucine+13C6-isoleucine
SA211954MS42-15786-M1A 13C6-leucine+13C6-isoleucine
SA211955MS42-12786-M1A 13C6-leucine+13C6-isoleucine
SA211956MS42-11786-M1A 13C6-leucine+13C6-isoleucine
SA211957MS42-18786-M2A 13C6-leucine+13C6-isoleucine
SA211958MS42-19786-M2A 13C6-leucine+13C6-isoleucine
SA211959MS42-16786-M2A 13C6-leucine+13C6-isoleucine
SA211960MS42-20786-M2A 13C6-leucine+13C6-isoleucine
SA211961MS42-17786-M2A 13C6-leucine+13C6-isoleucine
SA211962MS42-07786-O 13C6-leucine+13C6-isoleucine
SA211963MS42-06786-O 13C6-leucine+13C6-isoleucine
SA211964MS42-08786-O 13C6-leucine+13C6-isoleucine
SA211965MS42-09786-O 13C6-leucine+13C6-isoleucine
SA211966MS42-10786-O 13C6-leucine+13C6-isoleucine
SA211967MS42-02HK2 13C6-leucine+13C6-isoleucine
SA211968MS42-04HK2 13C6-leucine+13C6-isoleucine
SA211969MS42-01HK2 13C6-leucine+13C6-isoleucine
SA211970MS42-05HK2 13C6-leucine+13C6-isoleucine
SA211971MS42-03HK2 13C6-leucine+13C6-isoleucine
Showing results 1 to 20 of 20

Collection:

Collection ID:CO002299
Collection Summary:2x105 cells were plated onto 6-well plates in Plasmax media (5 replicates for each cell type). The day after, fresh media containing 13C6-leucine and 13C6-isoleucine for 27h was applied. 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:TR002318
Treatment Summary:Cells were cultured in Plasmax media supplemented with 2.5% FBS in the presence of 13C6-leucine and 13C6-isoleucine for 27 hours.

Sample Preparation:

Sampleprep ID:SP002312
Sampleprep Summary:Metabolite extraction solution (50% methanol, 30% acetonitrile, 20% ultrapure water, 5 µM final concentration valine-d8) was added to each well after the washes in PBS following the proportion of 1ml 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 AN003629
Analysis type MS
Chromatography type HILIC
Chromatography system Dionex Ultimate 3000 UHPLC
Column SeQuant ZIC-pHILIC
MS Type ESI
MS instrument type Orbitrap
MS instrument name Thermo Q Exactive Orbitrap
Ion Mode UNSPECIFIED
Units peak area

Chromatography:

Chromatography ID:CH002684
Chromatography Summary:Chromatographic separation of polar 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; 17.1-22.5 min: hold at 80% B. Samples were randomized and analysed with LC–MS in a blinded manner with an injection volume was 5 µl. Pooled samples were generated from an equal mixture of all individual samples and analysed interspersed at regular intervals within sample sequence as a quality control.
Instrument Name:Dionex Ultimate 3000 UHPLC
Column Name:SeQuant ZIC-pHILIC
Chromatography Type:HILIC

MS:

MS ID:MS003380
Analysis ID:AN003629
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Metabolites were measured with a Thermo Scientific Q Exactive Hybrid Quadrupole-Orbitrap Mass spectrometer (HRMS) coupled to a Dionex Ultimate 3000 UHPLC. The mass spectrometer was operated in full-scan, polarity-switching mode, with the spray voltage set to +4.5 kV/-3.5 kV, the heated capillary held at 320 °C, and the auxiliary gas heater held at 280 °C. The sheath gas flow was set to 55 units, the auxiliary gas flow was set to 15 units, and the sweep gas flow was set to 0 unit. HRMS data acquisition was performed in a range of m/z = 70–900, with the resolution set at 70,000, the AGC target at 1 × 106, and the maximum injection time (Max IT) at 120 ms. 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 through instrument analysis. The normalized areas were used as variables for further statistical data analysis.
Ion Mode:UNSPECIFIED
  logo