Summary of Study ST002224

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	ST002224
Study Title	Intracellular metabolic profile of renal cells cultured in Plasmax
Study Summary	The objective of this experiment is to analyse the metabolic profiles of human renal epithelial cells HK2 and ccRCC cell lines 786-O, 786-M1A and 786-M2A in Plasmax media. The experiment was conducted on three different days using cells with different passage numbers. This is Part 5 of a study and the experimental number is MS55.
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

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:	SU002310
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
SA212140	MS55_27	786-M1A	Plasmax + 2.5% FBS
SA212141	MS55_26	786-M1A	Plasmax + 2.5% FBS
SA212142	MS55_12	786-M1A	Plasmax + 2.5% FBS
SA212143	MS55_28	786-M1A	Plasmax + 2.5% FBS
SA212144	MS55_42	786-M1A	Plasmax + 2.5% FBS
SA212145	MS55_44	786-M1A	Plasmax + 2.5% FBS
SA212146	MS55_43	786-M1A	Plasmax + 2.5% FBS
SA212147	MS55_11	786-M1A	Plasmax + 2.5% FBS
SA212148	MS55_41	786-M1A	Plasmax + 2.5% FBS
SA212149	MS55_25	786-M1A	Plasmax + 2.5% FBS
SA212150	MS55_09	786-M1A	Plasmax + 2.5% FBS
SA212151	MS55_10	786-M1A	Plasmax + 2.5% FBS
SA212152	MS55_29	786-M2A	Plasmax + 2.5% FBS
SA212153	MS55_31	786-M2A	Plasmax + 2.5% FBS
SA212154	MS55_32	786-M2A	Plasmax + 2.5% FBS
SA212155	MS55_45	786-M2A	Plasmax + 2.5% FBS
SA212156	MS55_48	786-M2A	Plasmax + 2.5% FBS
SA212157	MS55_47	786-M2A	Plasmax + 2.5% FBS
SA212158	MS55_46	786-M2A	Plasmax + 2.5% FBS
SA212159	MS55_30	786-M2A	Plasmax + 2.5% FBS
SA212160	MS55_14	786-M2A	Plasmax + 2.5% FBS
SA212161	MS55_16	786-M2A	Plasmax + 2.5% FBS
SA212162	MS55_13	786-M2A	Plasmax + 2.5% FBS
SA212163	MS55_15	786-M2A	Plasmax + 2.5% FBS
SA212164	MS55_39	786-O	Plasmax + 2.5% FBS
SA212165	MS55_21	786-O	Plasmax + 2.5% FBS
SA212166	MS55_22	786-O	Plasmax + 2.5% FBS
SA212167	MS55_05	786-O	Plasmax + 2.5% FBS
SA212168	MS55_37	786-O	Plasmax + 2.5% FBS
SA212169	MS55_23	786-O	Plasmax + 2.5% FBS
SA212170	MS55_06	786-O	Plasmax + 2.5% FBS
SA212171	MS55_08	786-O	Plasmax + 2.5% FBS
SA212172	MS55_07	786-O	Plasmax + 2.5% FBS
SA212173	MS55_40	786-O	Plasmax + 2.5% FBS
SA212174	MS55_24	786-O	Plasmax + 2.5% FBS
SA212175	MS55_38	786-O	Plasmax + 2.5% FBS
SA212176	MS55_03	HK2	Plasmax + 2.5% FBS
SA212177	MS55_02	HK2	Plasmax + 2.5% FBS
SA212178	MS55_04	HK2	Plasmax + 2.5% FBS
SA212179	MS55_35	HK2	Plasmax + 2.5% FBS
SA212180	MS55_19	HK2	Plasmax + 2.5% FBS
SA212181	MS55_20	HK2	Plasmax + 2.5% FBS
SA212182	MS55_01	HK2	Plasmax + 2.5% FBS
SA212183	MS55_18	HK2	Plasmax + 2.5% FBS
SA212184	MS55_17	HK2	Plasmax + 2.5% FBS
SA212185	MS55_34	HK2	Plasmax + 2.5% FBS
SA212186	MS55_33	HK2	Plasmax + 2.5% FBS
SA212187	MS55_36	HK2	Plasmax + 2.5% FBS

Showing results 1 to 48 of 48

Showing results 1 to 48 of 48

Collection:

Collection ID:	CO002303
Collection Summary:	2x105 cells were plated the day before onto 6-well plates (5 or 6 replicates for each cell type) and extracted the day after. The experiment was repeated 3 times (N=3). 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:	TR002322
Treatment Summary:	Cells were cultured in Plasmax supplemented with 2.5% FBS. No further treatments were carried out.

Sample Preparation:

Sampleprep ID:	SP002316
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.

Sampleprep ID:

SP002316

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	AN003633
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:	CH002688
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:	MS003384
Analysis ID:	AN003633
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