Summary of Study ST002225
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 | ST002225 |
| Study Title | Time sensitive contribution of the BCAA catabolism to the TCA cycle carbons in HK2, 786-O, OS-RC-2 and RFX-631 |
| Study Summary | The 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. |
| 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: | SU002311 |
| 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 | Treatment duration |
|---|---|---|---|---|
| SA212194 | MS52-14 | 786-M1A | 13C6-leucine+13C6-isoleucine | 10 mins |
| SA212195 | MS52-15 | 786-M1A | 13C6-leucine+13C6-isoleucine | 10 mins |
| SA212196 | MS52-17 | 786-M1A | 13C6-leucine+13C6-isoleucine | 10 mins |
| SA212197 | MS52-18 | 786-M1A | 13C6-leucine+13C6-isoleucine | 10 mins |
| SA212198 | MS52-13 | 786-M1A | 13C6-leucine+13C6-isoleucine | 10 mins |
| SA212199 | MS52-16 | 786-M1A | 13C6-leucine+13C6-isoleucine | 10 mins |
| SA212188 | MS52-54 | 786-M1A | 13C6-leucine+13C6-isoleucine | 1 hour |
| SA212189 | MS52-52 | 786-M1A | 13C6-leucine+13C6-isoleucine | 1 hour |
| SA212190 | MS52-53 | 786-M1A | 13C6-leucine+13C6-isoleucine | 1 hour |
| SA212191 | MS52-51 | 786-M1A | 13C6-leucine+13C6-isoleucine | 1 hour |
| SA212192 | MS52-50 | 786-M1A | 13C6-leucine+13C6-isoleucine | 1 hour |
| SA212193 | MS52-49 | 786-M1A | 13C6-leucine+13C6-isoleucine | 1 hour |
| SA212200 | MS52-88 | 786-M1A | 13C6-leucine+13C6-isoleucine | 3 hours |
| SA212201 | MS52-89 | 786-M1A | 13C6-leucine+13C6-isoleucine | 3 hours |
| SA212202 | MS52-85 | 786-M1A | 13C6-leucine+13C6-isoleucine | 3 hours |
| SA212203 | MS52-87 | 786-M1A | 13C6-leucine+13C6-isoleucine | 3 hours |
| SA212204 | MS52-90 | 786-M1A | 13C6-leucine+13C6-isoleucine | 3 hours |
| SA212205 | MS52-86 | 786-M1A | 13C6-leucine+13C6-isoleucine | 3 hours |
| SA212212 | MS52-07 | 786-O | 13C6-leucine+13C6-isoleucine | 10 mins |
| SA212213 | MS52-11 | 786-O | 13C6-leucine+13C6-isoleucine | 10 mins |
| SA212214 | MS52-10 | 786-O | 13C6-leucine+13C6-isoleucine | 10 mins |
| SA212215 | MS52-09 | 786-O | 13C6-leucine+13C6-isoleucine | 10 mins |
| SA212216 | MS52-08 | 786-O | 13C6-leucine+13C6-isoleucine | 10 mins |
| SA212217 | MS52-12 | 786-O | 13C6-leucine+13C6-isoleucine | 10 mins |
| SA212206 | MS52-43 | 786-O | 13C6-leucine+13C6-isoleucine | 1 hour |
| SA212207 | MS52-48 | 786-O | 13C6-leucine+13C6-isoleucine | 1 hour |
| SA212208 | MS52-45 | 786-O | 13C6-leucine+13C6-isoleucine | 1 hour |
| SA212209 | MS52-46 | 786-O | 13C6-leucine+13C6-isoleucine | 1 hour |
| SA212210 | MS52-47 | 786-O | 13C6-leucine+13C6-isoleucine | 1 hour |
| SA212211 | MS52-44 | 786-O | 13C6-leucine+13C6-isoleucine | 1 hour |
| SA212218 | MS52-84 | 786-O | 13C6-leucine+13C6-isoleucine | 3 hours |
| SA212219 | MS52-79 | 786-O | 13C6-leucine+13C6-isoleucine | 3 hours |
| SA212220 | MS52-80 | 786-O | 13C6-leucine+13C6-isoleucine | 3 hours |
| SA212221 | MS52-81 | 786-O | 13C6-leucine+13C6-isoleucine | 3 hours |
| SA212222 | MS52-83 | 786-O | 13C6-leucine+13C6-isoleucine | 3 hours |
| SA212223 | MS52-82 | 786-O | 13C6-leucine+13C6-isoleucine | 3 hours |
| SA212230 | MS52-01 | HK2 | 13C6-leucine+13C6-isoleucine | 10 mins |
| SA212231 | MS52-02 | HK2 | 13C6-leucine+13C6-isoleucine | 10 mins |
| SA212232 | MS52-06 | HK2 | 13C6-leucine+13C6-isoleucine | 10 mins |
| SA212233 | MS52-05 | HK2 | 13C6-leucine+13C6-isoleucine | 10 mins |
| SA212234 | MS52-03 | HK2 | 13C6-leucine+13C6-isoleucine | 10 mins |
| SA212235 | MS52-04 | HK2 | 13C6-leucine+13C6-isoleucine | 10 mins |
| SA212224 | MS52-42 | HK2 | 13C6-leucine+13C6-isoleucine | 1 hour |
| SA212225 | MS52-41 | HK2 | 13C6-leucine+13C6-isoleucine | 1 hour |
| SA212226 | MS52-37 | HK2 | 13C6-leucine+13C6-isoleucine | 1 hour |
| SA212227 | MS52-40 | HK2 | 13C6-leucine+13C6-isoleucine | 1 hour |
| SA212228 | MS52-38 | HK2 | 13C6-leucine+13C6-isoleucine | 1 hour |
| SA212229 | MS52-39 | HK2 | 13C6-leucine+13C6-isoleucine | 1 hour |
| SA212236 | MS52-78 | HK2 | 13C6-leucine+13C6-isoleucine | 3 hours |
| SA212237 | MS52-76 | HK2 | 13C6-leucine+13C6-isoleucine | 3 hours |
| SA212238 | MS52-77 | HK2 | 13C6-leucine+13C6-isoleucine | 3 hours |
| SA212239 | MS52-74 | HK2 | 13C6-leucine+13C6-isoleucine | 3 hours |
| SA212240 | MS52-75 | HK2 | 13C6-leucine+13C6-isoleucine | 3 hours |
| SA212241 | MS52-73 | HK2 | 13C6-leucine+13C6-isoleucine | 3 hours |
| SA212248 | MS52-28 | OSLM1B | 13C6-leucine+13C6-isoleucine | 10 mins |
| SA212249 | MS52-25 | OSLM1B | 13C6-leucine+13C6-isoleucine | 10 mins |
| SA212250 | MS52-29 | OSLM1B | 13C6-leucine+13C6-isoleucine | 10 mins |
| SA212251 | MS52-30 | OSLM1B | 13C6-leucine+13C6-isoleucine | 10 mins |
| SA212252 | MS52-26 | OSLM1B | 13C6-leucine+13C6-isoleucine | 10 mins |
| SA212253 | MS52-27 | OSLM1B | 13C6-leucine+13C6-isoleucine | 10 mins |
| SA212242 | MS52-63 | OSLM1B | 13C6-leucine+13C6-isoleucine | 1 hour |
| SA212243 | MS52-64 | OSLM1B | 13C6-leucine+13C6-isoleucine | 1 hour |
| SA212244 | MS52-65 | OSLM1B | 13C6-leucine+13C6-isoleucine | 1 hour |
| SA212245 | MS52-66 | OSLM1B | 13C6-leucine+13C6-isoleucine | 1 hour |
| SA212246 | MS52-62 | OSLM1B | 13C6-leucine+13C6-isoleucine | 1 hour |
| SA212247 | MS52-61 | OSLM1B | 13C6-leucine+13C6-isoleucine | 1 hour |
| SA212254 | MS52-102 | OSLM1B | 13C6-leucine+13C6-isoleucine | 3 hours |
| SA212255 | MS52-97 | OSLM1B | 13C6-leucine+13C6-isoleucine | 3 hours |
| SA212256 | MS52-98 | OSLM1B | 13C6-leucine+13C6-isoleucine | 3 hours |
| SA212257 | MS52-99 | OSLM1B | 13C6-leucine+13C6-isoleucine | 3 hours |
| SA212258 | MS52-100 | OSLM1B | 13C6-leucine+13C6-isoleucine | 3 hours |
| SA212259 | MS52-101 | OSLM1B | 13C6-leucine+13C6-isoleucine | 3 hours |
| SA212266 | MS52-23 | OSRC2 | 13C6-leucine+13C6-isoleucine | 10 mins |
| SA212267 | MS52-24 | OSRC2 | 13C6-leucine+13C6-isoleucine | 10 mins |
| SA212268 | MS52-20 | OSRC2 | 13C6-leucine+13C6-isoleucine | 10 mins |
| SA212269 | MS52-19 | OSRC2 | 13C6-leucine+13C6-isoleucine | 10 mins |
| SA212270 | MS52-21 | OSRC2 | 13C6-leucine+13C6-isoleucine | 10 mins |
| SA212271 | MS52-22 | OSRC2 | 13C6-leucine+13C6-isoleucine | 10 mins |
| SA212260 | MS52-55 | OSRC2 | 13C6-leucine+13C6-isoleucine | 1 hour |
| SA212261 | MS52-58 | OSRC2 | 13C6-leucine+13C6-isoleucine | 1 hour |
| SA212262 | MS52-57 | OSRC2 | 13C6-leucine+13C6-isoleucine | 1 hour |
| SA212263 | MS52-56 | OSRC2 | 13C6-leucine+13C6-isoleucine | 1 hour |
| SA212264 | MS52-60 | OSRC2 | 13C6-leucine+13C6-isoleucine | 1 hour |
| SA212265 | MS52-59 | OSRC2 | 13C6-leucine+13C6-isoleucine | 1 hour |
| SA212272 | MS52-93 | OSRC2 | 13C6-leucine+13C6-isoleucine | 3 hours |
| SA212273 | MS52-94 | OSRC2 | 13C6-leucine+13C6-isoleucine | 3 hours |
| SA212274 | MS52-96 | OSRC2 | 13C6-leucine+13C6-isoleucine | 3 hours |
| SA212275 | MS52-91 | OSRC2 | 13C6-leucine+13C6-isoleucine | 3 hours |
| SA212276 | MS52-95 | OSRC2 | 13C6-leucine+13C6-isoleucine | 3 hours |
| SA212277 | MS52-92 | OSRC2 | 13C6-leucine+13C6-isoleucine | 3 hours |
| SA212284 | MS52-36 | RFX631 | 13C6-leucine+13C6-isoleucine | 10 mins |
| SA212285 | MS52-32 | RFX631 | 13C6-leucine+13C6-isoleucine | 10 mins |
| SA212286 | MS52-33 | RFX631 | 13C6-leucine+13C6-isoleucine | 10 mins |
| SA212287 | MS52-34 | RFX631 | 13C6-leucine+13C6-isoleucine | 10 mins |
| SA212288 | MS52-31 | RFX631 | 13C6-leucine+13C6-isoleucine | 10 mins |
| SA212289 | MS52-35 | RFX631 | 13C6-leucine+13C6-isoleucine | 10 mins |
| SA212278 | MS52-72 | RFX631 | 13C6-leucine+13C6-isoleucine | 1 hour |
| SA212279 | MS52-69 | RFX631 | 13C6-leucine+13C6-isoleucine | 1 hour |
| SA212280 | MS52-70 | RFX631 | 13C6-leucine+13C6-isoleucine | 1 hour |
| SA212281 | MS52-71 | RFX631 | 13C6-leucine+13C6-isoleucine | 1 hour |
Collection:
| 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:
| 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 Type | ESI |
| MS instrument type | Orbitrap |
| MS instrument name | Thermo Q Exactive Orbitrap |
| Ion Mode | UNSPECIFIED |
| Units | peak area |
Chromatography:
| 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:
| MS ID: | MS003385 |
| Analysis ID: | AN003634 |
| 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 |
