Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA097593

Local Sample ID:	WD3a
Subject ID:	SU001413
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606

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Subject:

Subject ID:	SU001413
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
WD3a	SA097593	FL013661	sensitive	genotype
WD3a	SA097593	FL013661	DMSO	treatment

Collection:

Collection ID:	CO001408
Collection Summary:	Single-cell derived BRAF-mutated SKMEL5 subclones were derived as previously described (Paudel et al., 2018). BRAF-mutated melanoma cells (SKMEL5, WM88), including the SKMEL5 subclones, NRAS-mutated melanoma cells (SKMEL2), and NF1-mutated melanoma cells (MeWO) were grown and cultured in Dulbecco’s modified Eagle’s medium and Ham’s F-12 media (DMEM:F12, 1:1, Cat. No. 11330-032). Media were obtained from Gibco (Grand Island, NY), and supplemented with 10% fetal bovine serum. All cells were cultured in humidified incubators that were CO2 and temperature (37oC) controlled. Cells were passaged 1–2 times per week and were maintained as exponentially growing cultures for a maximum of less than 20 passages. All cells were tested for mycoplasma, and tested negative.
Sample Type:	Tumor cells

Treatment:

Treatment ID:	TR001428
Treatment Summary:	PLX4720 (Cat. No. S1152) and vemurafenib (Cat. No. S1267) were obtained from Selleckchem (Houston, TX). Dabrafenib (Cat No. HY-14660), (1S,3R)-RSL3 (Cat No. HY-100218A), Ferrostatin-1 (FER1, Cat No. HY-100579), Erastin (Cat No. HY-15763), (E)-Daporinad (FK866) (Cat No. HY-50876) were obtained from MedChem Express (Monmouth Junction, NJ) and solubilized in dimethyl sulfoxide (DMSO) at a stock concentration of 10 mM. Powdered L-Buthionine-sulfoximine (BSO) (Product No. B2515), powdered Diphenyleneiodonium chloride (DPI) (Product No. D2926), and powdered L-Glutathione reduced (GSH) were obtained from Sigma-Aldrich. BSO, and GSH was freshly made at a stock concentration of 100 mM in H2O, while DPI was solubilized in DMSO at a stock concentration of 10mM. CellRoxTM DeepRed Reagent (Cat No. C10422) for oxidative stress detection was obtained from ThermoFisher Scientific. Acetonitrile (AcCN) (Cat No. A955-1), Methanol (MeOH) (Cat No. A456-1) and water (Cat No. W6-1), Optima LC-MS grade, for the mass spectrometry analysis were obtained from ThermoFisher Scientific.

Treatment ID:

TR001428

Treatment Summary:

PLX4720 (Cat. No. S1152) and vemurafenib (Cat. No. S1267) were obtained from Selleckchem (Houston, TX). Dabrafenib (Cat No. HY-14660), (1S,3R)-RSL3 (Cat No. HY-100218A), Ferrostatin-1 (FER1, Cat No. HY-100579), Erastin (Cat No. HY-15763), (E)-Daporinad (FK866) (Cat No. HY-50876) were obtained from MedChem Express (Monmouth Junction, NJ) and solubilized in dimethyl sulfoxide (DMSO) at a stock concentration of 10 mM. Powdered L-Buthionine-sulfoximine (BSO) (Product No. B2515), powdered Diphenyleneiodonium chloride (DPI) (Product No. D2926), and powdered L-Glutathione reduced (GSH) were obtained from Sigma-Aldrich. BSO, and GSH was freshly made at a stock concentration of 100 mM in H2O, while DPI was solubilized in DMSO at a stock concentration of 10mM. CellRoxTM DeepRed Reagent (Cat No. C10422) for oxidative stress detection was obtained from ThermoFisher Scientific. Acetonitrile (AcCN) (Cat No. A955-1), Methanol (MeOH) (Cat No. A456-1) and water (Cat No. W6-1), Optima LC-MS grade, for the mass spectrometry analysis were obtained from ThermoFisher Scientific.

Sample Preparation:

Sampleprep ID:	SP001421
Sampleprep Summary:	The combined global untargeted-targeted metabolomic analysis used BRAF-mutated melanoma cells (WM88, SKMEL5-SC10) treated with either DMSO or 8μM PLX4720 for 24 hrs. Cell pellet samples were lysed using 400µL ice cold lysis buffer (1:1:2, ACN:MeOH:Ammonium Bicarbonate 0.1M, pH 8.0, LC-MS grade) and vortexed well until the cells mixed well with the solvent. Each sample was sonicated using a probe tip sonicator, 10 pulses at 30% power, cooling down in ice between samples. A BCA protein assay was used to determine the protein concentration for each individual sample, and adjusted to a total amount of protein of 200µg total protein in 200 µL of lysis buffer. Heavy labeled standard molecules, Phenylalanine-D8 (CDN Isotopes, Quebec, CA), and Biotin-D2(Cambridge Isotope Laboratories, Inc., MA, USA), were added to each sample to assess sample handling steps. Samples were subjected to protein precipitation by addition of 800µL of ice cold methanol (4x by volume), then incubated at -80°C overnight. Samples were centrifuged at 10,000 rpm for 10 min to eliminate precipitated proteins and the metabolite containing supernatant was dried in vacuo and stored at -80°C until LC-MS analysis.

Sampleprep ID:

SP001421

Sampleprep Summary:

The combined global untargeted-targeted metabolomic analysis used BRAF-mutated melanoma cells (WM88, SKMEL5-SC10) treated with either DMSO or 8μM PLX4720 for 24 hrs. Cell pellet samples were lysed using 400µL ice cold lysis buffer (1:1:2, ACN:MeOH:Ammonium Bicarbonate 0.1M, pH 8.0, LC-MS grade) and vortexed well until the cells mixed well with the solvent. Each sample was sonicated using a probe tip sonicator, 10 pulses at 30% power, cooling down in ice between samples. A BCA protein assay was used to determine the protein concentration for each individual sample, and adjusted to a total amount of protein of 200µg total protein in 200 µL of lysis buffer. Heavy labeled standard molecules, Phenylalanine-D8 (CDN Isotopes, Quebec, CA), and Biotin-D2(Cambridge Isotope Laboratories, Inc., MA, USA), were added to each sample to assess sample handling steps. Samples were subjected to protein precipitation by addition of 800µL of ice cold methanol (4x by volume), then incubated at -80°C overnight. Samples were centrifuged at 10,000 rpm for 10 min to eliminate precipitated proteins and the metabolite containing supernatant was dried in vacuo and stored at -80°C until LC-MS analysis.

Combined analysis:

Analysis ID	AN002233
Analysis type	MS
Chromatography type	HILIC
Chromatography system	Thermo Vanquish UHPLC
Column	SeQuant ZIC-HILIC (100 x 2.1mm,3.5um)
MS Type	ESI
MS instrument type	Orbitrap
MS instrument name	Thermo Q Exactive HF hybrid Orbitrap
Ion Mode	NEGATIVE
Units	peak area

Chromatography:

Chromatography ID:	CH001639
Chromatography Summary:	Vanquish UHPLC binary system and autosampler
Instrument Name:	Thermo Vanquish UHPLC
Column Name:	SeQuant ZIC-HILIC (100 x 2.1mm,3.5um)
Chromatography Type:	HILIC

MS:

MS ID:	MS002079
Analysis ID:	AN002233
Instrument Name:	Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	LC-MS/MS raw data were imported, processed, normalized and reviewed using Progenesis QI v.2.1 (Non-linear Dynamics, Newcastle, UK). All FMS, DDA and PRM sample runs were chromatographically aligned against a QC reference run. Following peak picking, unique spectral features (retention time and m/z pairs) were grouped based on adducts and isotopes, and individual features or metabolites were normalized to all features. Further filtering was carried out by removing features or metabolites that had >30% coefficient of variance.
Ion Mode:	NEGATIVE