Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA097584

Local Sample ID:	SP3a
Subject ID:	SU001413
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606

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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.