Summary of Study ST002225

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench,, 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.


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 IDST002225
Study TitleTime sensitive contribution of the BCAA catabolism to the TCA cycle carbons in HK2, 786-O, OS-RC-2 and RFX-631
Study SummaryThe objective of this experiment is to test the contribution of the branched chain amino acids catabolism to the carbons used in the TCA cycle. To test this hypothesis, we incubated human renal epithelial cells (HK2) and ccRCC cell lines (786-O, 786-M1A, OS-RC-2, OS-LM1, RFX-631) with 13C6-leucine and 13C6-isoleucine in Plasmax media for 10 mins, 1 hour and 3 hours. Data were generated from 5 independent cultures. This is Part 4 of the study and the experiment number is MS52.
CECAD Research Center
Last NameYang
First NameMing
AddressJoseph-Stelzmann-Straße 26, Köln, Koeln, 50931, Germany
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 application/zip

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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


Subject ID:SU002311
Subject Type:Cultured cells
Subject Species:Homo sapiens
Taxonomy ID:9606


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

mb_sample_id local_sample_id Cell line Treatment Treatment duration
SA212194MS52-14786-M1A 13C6-leucine+13C6-isoleucine 10 mins
SA212195MS52-15786-M1A 13C6-leucine+13C6-isoleucine 10 mins
SA212196MS52-17786-M1A 13C6-leucine+13C6-isoleucine 10 mins
SA212197MS52-18786-M1A 13C6-leucine+13C6-isoleucine 10 mins
SA212198MS52-13786-M1A 13C6-leucine+13C6-isoleucine 10 mins
SA212199MS52-16786-M1A 13C6-leucine+13C6-isoleucine 10 mins
SA212188MS52-54786-M1A 13C6-leucine+13C6-isoleucine 1 hour
SA212189MS52-52786-M1A 13C6-leucine+13C6-isoleucine 1 hour
SA212190MS52-53786-M1A 13C6-leucine+13C6-isoleucine 1 hour
SA212191MS52-51786-M1A 13C6-leucine+13C6-isoleucine 1 hour
SA212192MS52-50786-M1A 13C6-leucine+13C6-isoleucine 1 hour
SA212193MS52-49786-M1A 13C6-leucine+13C6-isoleucine 1 hour
SA212200MS52-88786-M1A 13C6-leucine+13C6-isoleucine 3 hours
SA212201MS52-89786-M1A 13C6-leucine+13C6-isoleucine 3 hours
SA212202MS52-85786-M1A 13C6-leucine+13C6-isoleucine 3 hours
SA212203MS52-87786-M1A 13C6-leucine+13C6-isoleucine 3 hours
SA212204MS52-90786-M1A 13C6-leucine+13C6-isoleucine 3 hours
SA212205MS52-86786-M1A 13C6-leucine+13C6-isoleucine 3 hours
SA212212MS52-07786-O 13C6-leucine+13C6-isoleucine 10 mins
SA212213MS52-11786-O 13C6-leucine+13C6-isoleucine 10 mins
SA212214MS52-10786-O 13C6-leucine+13C6-isoleucine 10 mins
SA212215MS52-09786-O 13C6-leucine+13C6-isoleucine 10 mins
SA212216MS52-08786-O 13C6-leucine+13C6-isoleucine 10 mins
SA212217MS52-12786-O 13C6-leucine+13C6-isoleucine 10 mins
SA212206MS52-43786-O 13C6-leucine+13C6-isoleucine 1 hour
SA212207MS52-48786-O 13C6-leucine+13C6-isoleucine 1 hour
SA212208MS52-45786-O 13C6-leucine+13C6-isoleucine 1 hour
SA212209MS52-46786-O 13C6-leucine+13C6-isoleucine 1 hour
SA212210MS52-47786-O 13C6-leucine+13C6-isoleucine 1 hour
SA212211MS52-44786-O 13C6-leucine+13C6-isoleucine 1 hour
SA212218MS52-84786-O 13C6-leucine+13C6-isoleucine 3 hours
SA212219MS52-79786-O 13C6-leucine+13C6-isoleucine 3 hours
SA212220MS52-80786-O 13C6-leucine+13C6-isoleucine 3 hours
SA212221MS52-81786-O 13C6-leucine+13C6-isoleucine 3 hours
SA212222MS52-83786-O 13C6-leucine+13C6-isoleucine 3 hours
SA212223MS52-82786-O 13C6-leucine+13C6-isoleucine 3 hours
SA212230MS52-01HK2 13C6-leucine+13C6-isoleucine 10 mins
SA212231MS52-02HK2 13C6-leucine+13C6-isoleucine 10 mins
SA212232MS52-06HK2 13C6-leucine+13C6-isoleucine 10 mins
SA212233MS52-05HK2 13C6-leucine+13C6-isoleucine 10 mins
SA212234MS52-03HK2 13C6-leucine+13C6-isoleucine 10 mins
SA212235MS52-04HK2 13C6-leucine+13C6-isoleucine 10 mins
SA212224MS52-42HK2 13C6-leucine+13C6-isoleucine 1 hour
SA212225MS52-41HK2 13C6-leucine+13C6-isoleucine 1 hour
SA212226MS52-37HK2 13C6-leucine+13C6-isoleucine 1 hour
SA212227MS52-40HK2 13C6-leucine+13C6-isoleucine 1 hour
SA212228MS52-38HK2 13C6-leucine+13C6-isoleucine 1 hour
SA212229MS52-39HK2 13C6-leucine+13C6-isoleucine 1 hour
SA212236MS52-78HK2 13C6-leucine+13C6-isoleucine 3 hours
SA212237MS52-76HK2 13C6-leucine+13C6-isoleucine 3 hours
SA212238MS52-77HK2 13C6-leucine+13C6-isoleucine 3 hours
SA212239MS52-74HK2 13C6-leucine+13C6-isoleucine 3 hours
SA212240MS52-75HK2 13C6-leucine+13C6-isoleucine 3 hours
SA212241MS52-73HK2 13C6-leucine+13C6-isoleucine 3 hours
SA212248MS52-28OSLM1B 13C6-leucine+13C6-isoleucine 10 mins
SA212249MS52-25OSLM1B 13C6-leucine+13C6-isoleucine 10 mins
SA212250MS52-29OSLM1B 13C6-leucine+13C6-isoleucine 10 mins
SA212251MS52-30OSLM1B 13C6-leucine+13C6-isoleucine 10 mins
SA212252MS52-26OSLM1B 13C6-leucine+13C6-isoleucine 10 mins
SA212253MS52-27OSLM1B 13C6-leucine+13C6-isoleucine 10 mins
SA212242MS52-63OSLM1B 13C6-leucine+13C6-isoleucine 1 hour
SA212243MS52-64OSLM1B 13C6-leucine+13C6-isoleucine 1 hour
SA212244MS52-65OSLM1B 13C6-leucine+13C6-isoleucine 1 hour
SA212245MS52-66OSLM1B 13C6-leucine+13C6-isoleucine 1 hour
SA212246MS52-62OSLM1B 13C6-leucine+13C6-isoleucine 1 hour
SA212247MS52-61OSLM1B 13C6-leucine+13C6-isoleucine 1 hour
SA212254MS52-102OSLM1B 13C6-leucine+13C6-isoleucine 3 hours
SA212255MS52-97OSLM1B 13C6-leucine+13C6-isoleucine 3 hours
SA212256MS52-98OSLM1B 13C6-leucine+13C6-isoleucine 3 hours
SA212257MS52-99OSLM1B 13C6-leucine+13C6-isoleucine 3 hours
SA212258MS52-100OSLM1B 13C6-leucine+13C6-isoleucine 3 hours
SA212259MS52-101OSLM1B 13C6-leucine+13C6-isoleucine 3 hours
SA212266MS52-23OSRC2 13C6-leucine+13C6-isoleucine 10 mins
SA212267MS52-24OSRC2 13C6-leucine+13C6-isoleucine 10 mins
SA212268MS52-20OSRC2 13C6-leucine+13C6-isoleucine 10 mins
SA212269MS52-19OSRC2 13C6-leucine+13C6-isoleucine 10 mins
SA212270MS52-21OSRC2 13C6-leucine+13C6-isoleucine 10 mins
SA212271MS52-22OSRC2 13C6-leucine+13C6-isoleucine 10 mins
SA212260MS52-55OSRC2 13C6-leucine+13C6-isoleucine 1 hour
SA212261MS52-58OSRC2 13C6-leucine+13C6-isoleucine 1 hour
SA212262MS52-57OSRC2 13C6-leucine+13C6-isoleucine 1 hour
SA212263MS52-56OSRC2 13C6-leucine+13C6-isoleucine 1 hour
SA212264MS52-60OSRC2 13C6-leucine+13C6-isoleucine 1 hour
SA212265MS52-59OSRC2 13C6-leucine+13C6-isoleucine 1 hour
SA212272MS52-93OSRC2 13C6-leucine+13C6-isoleucine 3 hours
SA212273MS52-94OSRC2 13C6-leucine+13C6-isoleucine 3 hours
SA212274MS52-96OSRC2 13C6-leucine+13C6-isoleucine 3 hours
SA212275MS52-91OSRC2 13C6-leucine+13C6-isoleucine 3 hours
SA212276MS52-95OSRC2 13C6-leucine+13C6-isoleucine 3 hours
SA212277MS52-92OSRC2 13C6-leucine+13C6-isoleucine 3 hours
SA212284MS52-36RFX631 13C6-leucine+13C6-isoleucine 10 mins
SA212285MS52-32RFX631 13C6-leucine+13C6-isoleucine 10 mins
SA212286MS52-33RFX631 13C6-leucine+13C6-isoleucine 10 mins
SA212287MS52-34RFX631 13C6-leucine+13C6-isoleucine 10 mins
SA212288MS52-31RFX631 13C6-leucine+13C6-isoleucine 10 mins
SA212289MS52-35RFX631 13C6-leucine+13C6-isoleucine 10 mins
SA212278MS52-72RFX631 13C6-leucine+13C6-isoleucine 1 hour
SA212279MS52-69RFX631 13C6-leucine+13C6-isoleucine 1 hour
SA212280MS52-70RFX631 13C6-leucine+13C6-isoleucine 1 hour
SA212281MS52-71RFX631 13C6-leucine+13C6-isoleucine 1 hour
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Collection ID:CO002304
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 13C6-Leucine and 13C6-Isoleucine (obtained from Cambridge Isotopes Laboratories) for 10 mins, 1 hour, 3 hours in Plasmax media. 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 ID:TR002323
Treatment Summary:Cells were cultured in Plasmax mediasupplemented with 2.5% FBS in the presence of 13C6-leucine+ 13C6-isoleucine for the indicated time points.

Sample Preparation:

Sampleprep ID:SP002317
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 AN003634
Analysis type MS
Chromatography type HILIC
Chromatography system Thermo Dionex Ultimate 3000
Column SeQuant ZIC-pHILIC
MS instrument type Orbitrap
MS instrument name Thermo Q Exactive Orbitrap
Units peak area


Chromatography ID:CH002689
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 Dionex Ultimate 3000
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 ID:MS003385
Analysis ID:AN003634
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
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.