Summary of Study ST002223

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	ST002223
Study Title	Metabolic profiling of mouse tissues and tissue interstitial fluids
Study Summary	Tissue and tissue interstitial fluids was collected from mice of a hybrid C57BL/6J;129/SvJ background of about 12 weeks of age (8 in total) and used to profile the metabolic content. This is Part 6 of a study and the experimental number is MS56.
Institute	CECAD Research Center
Last Name	Yang
First Name	Ming
Address	Joseph-Stelzmann-Straße 26, Köln, Koeln, 50931, Germany
Email	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

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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:	SU002309
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090

Factors:

Subject type: Mammal; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id	local_sample_id	Species	Organ	Matrix
SA212077	MS56-042	mouse	heart	interstitial fluid
SA212078	MS56-062	mouse	heart	interstitial fluid
SA212079	MS56-064	mouse	heart	interstitial fluid
SA212080	MS56-035	mouse	heart	interstitial fluid
SA212081	MS56-063	mouse	heart	interstitial fluid
SA212082	MS56-044	mouse	heart	interstitial fluid
SA212083	MS56-053	mouse	heart	interstitial fluid
SA212084	MS56-055	mouse	heart	interstitial fluid
SA212085	MS56-004	mouse	heart	tissue
SA212086	MS56-021	mouse	heart	tissue
SA212087	MS56-017	mouse	heart	tissue
SA212088	MS56-014	mouse	heart	tissue
SA212089	MS56-023	mouse	heart	tissue
SA212090	MS56-008	mouse	heart	tissue
SA212091	MS56-011	mouse	heart	tissue
SA212092	MS56-006	mouse	heart	tissue
SA212093	MS56-061	mouse	kidney	interstitial fluid
SA212094	MS56-034	mouse	kidney	interstitial fluid
SA212095	MS56-039	mouse	kidney	interstitial fluid
SA212096	MS56-050	mouse	kidney	interstitial fluid
SA212097	MS56-057	mouse	kidney	interstitial fluid
SA212098	MS56-036	mouse	kidney	interstitial fluid
SA212099	MS56-038	mouse	kidney	interstitial fluid
SA212100	MS56-054	mouse	kidney	interstitial fluid
SA212101	MS56-001	mouse	kidney	tissue
SA212102	MS56-026	mouse	kidney	tissue
SA212103	MS56-016	mouse	kidney	tissue
SA212104	MS56-010	mouse	kidney	tissue
SA212105	MS56-012	mouse	kidney	tissue
SA212106	MS56-007	mouse	kidney	tissue
SA212107	MS56-025	mouse	kidney	tissue
SA212108	MS56-003	mouse	kidney	tissue
SA212109	MS56-043	mouse	liver	interstitial fluid
SA212110	MS56-052	mouse	liver	interstitial fluid
SA212111	MS56-049	mouse	liver	interstitial fluid
SA212112	MS56-056	mouse	liver	interstitial fluid
SA212113	MS56-041	mouse	liver	interstitial fluid
SA212114	MS56-040	mouse	liver	interstitial fluid
SA212115	MS56-037	mouse	liver	interstitial fluid
SA212116	MS56-059	mouse	liver	interstitial fluid
SA212117	MS56-031	mouse	liver	tissue
SA212118	MS56-002	mouse	liver	tissue
SA212119	MS56-030	mouse	liver	tissue
SA212120	MS56-027	mouse	liver	tissue
SA212121	MS56-009	mouse	liver	tissue
SA212122	MS56-019	mouse	liver	tissue
SA212123	MS56-013	mouse	liver	tissue
SA212124	MS56-015	mouse	liver	tissue
SA212125	MS56-058	mouse	lung	interstitial fluid
SA212126	MS56-060	mouse	lung	interstitial fluid
SA212127	MS56-033	mouse	lung	interstitial fluid
SA212128	MS56-051	mouse	lung	interstitial fluid
SA212129	MS56-045	mouse	lung	interstitial fluid
SA212130	MS56-046	mouse	lung	interstitial fluid
SA212131	MS56-047	mouse	lung	interstitial fluid
SA212132	MS56-048	mouse	lung	interstitial fluid
SA212133	MS56-018	mouse	lung	tissue
SA212134	MS56-005	mouse	lung	tissue
SA212135	MS56-020	mouse	lung	tissue
SA212136	MS56-032	mouse	lung	tissue
SA212137	MS56-029	mouse	lung	tissue
SA212138	MS56-024	mouse	lung	tissue
SA212139	MS56-022	mouse	lung	tissue

Showing results 1 to 63 of 63

Showing results 1 to 63 of 63

Collection:

Collection ID:	CO002302
Collection Summary:	Mice were culled by cervical dislocation and tissue samples from hearts, livers, kidneys, and lungs were collected and split into two halves. One half was snap frozen in liquid nitrogen and stored at -80°C until further processing. The second half was used for interstitial fluid extraction using a protocol adapted from Sullivan et al. 2019 (https://doi.org/10.7554/eLife.44235)
Sample Type:	Mouse tissues and interstitial fluids

Treatment:

Treatment ID:	TR002321
Treatment Summary:	No further treatment was carried out.

Sample Preparation:

Sampleprep ID:	SP002315
Sampleprep Summary:	For tissue extraction, samples were homogenized in metabolite extraction buffer using the proportion 25 μl/mg of buffer with Precellys Lysing tubes (Bertin Instruments). After that, extracts were kept in the freezer overnight and the following day centrifuged twice at max speed at 4C˚ to remove the protein precipitates. Equal volumes of supernatants were spiked in with 13C arginine (Cambridge Isotopes) for quantification of arginine content. For extraction of the tissue interstitial for interstitial fluid extraction, the organ was washed in saline solution and then a portion was centrifuged at for 10 min at 4°C at 106 x g using 20 µm nylon filters (Spectrum Labs, Waltham, MA, 148134) affixed on top of 2 ml Eppendorf tubes. 1μl of the eluate was extracted in 45μl of extraction buffer and frozen overnight. The following day, all extracted were centrifuged twice at max speed at 4C˚to remove the protein precipitates. Supernatants were finally spiked in with 13C arginine (Cambridge Isotopes) for arginine quantification.

Sampleprep ID:

SP002315

Sampleprep Summary:

For tissue extraction, samples were homogenized in metabolite extraction buffer using the proportion 25 μl/mg of buffer with Precellys Lysing tubes (Bertin Instruments). After that, extracts were kept in the freezer overnight and the following day centrifuged twice at max speed at 4C˚ to remove the protein precipitates. Equal volumes of supernatants were spiked in with 13C arginine (Cambridge Isotopes) for quantification of arginine content. For extraction of the tissue interstitial for interstitial fluid extraction, the organ was washed in saline solution and then a portion was centrifuged at for 10 min at 4°C at 106 x g using 20 µm nylon filters (Spectrum Labs, Waltham, MA, 148134) affixed on top of 2 ml Eppendorf tubes. 1μl of the eluate was extracted in 45μl of extraction buffer and frozen overnight. The following day, all extracted were centrifuged twice at max speed at 4C˚to remove the protein precipitates. Supernatants were finally spiked in with 13C arginine (Cambridge Isotopes) for arginine quantification.

Combined analysis:

Analysis ID	AN003632
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:	CH002687
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:	MS003383
Analysis ID:	AN003632
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