Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA329896

Local Sample ID:	Pfizer-516-8545
Subject ID:	SU003153
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Gender:	Male

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Sample Preparation:

Sampleprep ID:	SP003159
Sampleprep Summary:	we used chloroform/methanol extraction protocol to increase the coverage of the extracted metabolites. At first, the samples (cells and buffer) were transferred into Eppendorf tubes then centrifuged at 14000 rpm for 5 min. Afterward, the buffer was discarded, and the cells were preserved. To each sample, 400 µL of the mixture containing one protease inhibitor tablet and 10 mL of lysis buffer was added. Following rest for 10 minutes, samples were transferred to 10 mL tubes, vortexed for 2–4 minutes, and sonicated with a COPLEY probe-sonicator (QSONICA SONICATOR, USA) for 30 seconds while utilizing a 30 % amplifier in an ice bath. The samples were then transferred to Eppendorf tubes and centrifuged for 5 minutes at 14000 rpm. The supernatant was then transferred to another Eppendorf, and 400 µL of methanol and 300 µL of chloroform were added. Following that, the samples were vortexed for 30 seconds and centrifuged for 5 minutes at 14000 rpm. After that, two metabolite-containing layers are obtained, after transferring the upper layer of each sample to glass vials, 400 µl of methanol was added, followed by vertexing and centrifugation. The remaining supernatant was transferred to the same glass vials used before for the drying step, with the remaining protein pellets being air-dried for proteomics. A dried metabolomics sample was resuspended in 200 µL (0.1% formic acid in water) and injected into HPLC to be analysed by Q-TOF MS.In summary, After dividing the samples into 100 µL portions in Eppendorf tubes, 300 µL of methanol from Wunstorfer Strasse, Seelze, Germany, was introduced. The tubes were subsequently vortexed and placed in an incubator at -20°C for two hours. Following incubation, the samples underwent another round of vortexing and were centrifuged for 15 minutes at 14000 rpm. The resulting supernatant was evaporated at 35 to 40 °C through speed vacuum evaporation. To assess the analysis's repeatability, a quality control (QC) sample was created by pooling the same volume of each sample (10µl). The extracted samples were then resuspended in 100 µL of Honeywell's LC-MS CHROMASOLV's 0.1% formic acid in Deionized Water (Wunstorfer Strasse, Seelze, Germany). Following that, 100 µL of the prepared sample was collected in an insert inside LC glass vials after filtration through a 0.45µm hydrophilic nylon syringe filter for LC-MS/MS analysis.

Sampleprep ID:

SP003159

Sampleprep Summary:

we used chloroform/methanol extraction protocol to increase the coverage of the extracted metabolites. At first, the samples (cells and buffer) were transferred into Eppendorf tubes then centrifuged at 14000 rpm for 5 min. Afterward, the buffer was discarded, and the cells were preserved. To each sample, 400 µL of the mixture containing one protease inhibitor tablet and 10 mL of lysis buffer was added. Following rest for 10 minutes, samples were transferred to 10 mL tubes, vortexed for 2–4 minutes, and sonicated with a COPLEY probe-sonicator (QSONICA SONICATOR, USA) for 30 seconds while utilizing a 30 % amplifier in an ice bath. The samples were then transferred to Eppendorf tubes and centrifuged for 5 minutes at 14000 rpm. The supernatant was then transferred to another Eppendorf, and 400 µL of methanol and 300 µL of chloroform were added. Following that, the samples were vortexed for 30 seconds and centrifuged for 5 minutes at 14000 rpm. After that, two metabolite-containing layers are obtained, after transferring the upper layer of each sample to glass vials, 400 µl of methanol was added, followed by vertexing and centrifugation. The remaining supernatant was transferred to the same glass vials used before for the drying step, with the remaining protein pellets being air-dried for proteomics. A dried metabolomics sample was resuspended in 200 µL (0.1% formic acid in water) and injected into HPLC to be analysed by Q-TOF MS.In summary, After dividing the samples into 100 µL portions in Eppendorf tubes, 300 µL of methanol from Wunstorfer Strasse, Seelze, Germany, was introduced. The tubes were subsequently vortexed and placed in an incubator at -20°C for two hours. Following incubation, the samples underwent another round of vortexing and were centrifuged for 15 minutes at 14000 rpm. The resulting supernatant was evaporated at 35 to 40 °C through speed vacuum evaporation. To assess the analysis's repeatability, a quality control (QC) sample was created by pooling the same volume of each sample (10µl). The extracted samples were then resuspended in 100 µL of Honeywell's LC-MS CHROMASOLV's 0.1% formic acid in Deionized Water (Wunstorfer Strasse, Seelze, Germany). Following that, 100 µL of the prepared sample was collected in an insert inside LC glass vials after filtration through a 0.45µm hydrophilic nylon syringe filter for LC-MS/MS analysis.