Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA271292

Local Sample ID:	P Control 1A-01_7_1_3213
Subject ID:	SU002808
Subject Type:	Human
Subject Species:	Homo sapiens
Taxonomy ID:	9606

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Sample Preparation:

Sampleprep ID:	SP002814
Sampleprep Summary:	Two million cells from both cell lines (A549-P and A549-R) were seeded in T75 cell culture flasks. Following culturing the cells, they were collected by trypsin and a pellet of 3x106 cells was stored at -80 ℃ for further analysis. Each cell line was prepared in triplicates for the metabolomics and proteomics studies. Frozen A549-P and A549-R cells pellets were centrifuged at 14000 rpm for 5 min at 4 ℃ to separate them from Phosphate Buffered Saline (PBS) buffer. The pellets were suspended in a lysis buffer (10 mM Tris) and protease inhibitor cocktail tablets. The lysed cells were kept on ice for 10 min, and then each sample was vortexed for 30 s. After that, the samples were sonicated in an ice bath using a Q500 sonicator from QSonica Sonicator (Fairfield, Connecticut, USA) at 30% amp for 30 s. Then, the samples were centrifuged at 14000 rpm for 5 min at 4℃, 400 µL of methanol was added to the supernatant, followed by 300 µL of chloroform and vortexed for 30 seconds, and then centrifuged at 14000 rpm for 5 min at 4 ℃. The resulting aqueous biphasic solution's upper layer was transferred carefully into LC vials without touching the white disk for metabolites analysis. For proteins extraction, the generated white disk and the lower layer were washed with 300 µL of methanol and vortexed vigorously, and then centrifuged at 14000 rpm for 3 min at 4 ℃. After that, the protein pellet was precipitated and allowed to dry by keeping the tubes opened for 5 min. Then a denaturation buffer was used to re-suspend the pellets (10 mM Tris, 6 M Urea, 2 M Thiourea, pH 8) and the concentration of precipitated proteins was determined using the modified Bradford assay. The resulted supernatant from the previous centrifugation, which contains metabolites, was transferred to the previously collected metabolites in the LC vials. The metabolites samples were dried at 45 ℃ using an EZ-2 Plus evaporator from GeneVac (Ipswich, UK), and then the dehydrated extracts were reconstituted in 200 µL 0.1% formic acid (FA) in water and vortexed for 2 min. Finally, the samples were filtered using a hydrophilic syringe filter with a pore size of 0.45 µm and were ready to be analyzed using Q-TOF MS.

Sampleprep ID:

SP002814

Sampleprep Summary:

Two million cells from both cell lines (A549-P and A549-R) were seeded in T75 cell culture flasks. Following culturing the cells, they were collected by trypsin and a pellet of 3x106 cells was stored at -80 ℃ for further analysis. Each cell line was prepared in triplicates for the metabolomics and proteomics studies. Frozen A549-P and A549-R cells pellets were centrifuged at 14000 rpm for 5 min at 4 ℃ to separate them from Phosphate Buffered Saline (PBS) buffer. The pellets were suspended in a lysis buffer (10 mM Tris) and protease inhibitor cocktail tablets. The lysed cells were kept on ice for 10 min, and then each sample was vortexed for 30 s. After that, the samples were sonicated in an ice bath using a Q500 sonicator from QSonica Sonicator (Fairfield, Connecticut, USA) at 30% amp for 30 s. Then, the samples were centrifuged at 14000 rpm for 5 min at 4℃, 400 µL of methanol was added to the supernatant, followed by 300 µL of chloroform and vortexed for 30 seconds, and then centrifuged at 14000 rpm for 5 min at 4 ℃. The resulting aqueous biphasic solution's upper layer was transferred carefully into LC vials without touching the white disk for metabolites analysis. For proteins extraction, the generated white disk and the lower layer were washed with 300 µL of methanol and vortexed vigorously, and then centrifuged at 14000 rpm for 3 min at 4 ℃. After that, the protein pellet was precipitated and allowed to dry by keeping the tubes opened for 5 min. Then a denaturation buffer was used to re-suspend the pellets (10 mM Tris, 6 M Urea, 2 M Thiourea, pH 8) and the concentration of precipitated proteins was determined using the modified Bradford assay. The resulted supernatant from the previous centrifugation, which contains metabolites, was transferred to the previously collected metabolites in the LC vials. The metabolites samples were dried at 45 ℃ using an EZ-2 Plus evaporator from GeneVac (Ipswich, UK), and then the dehydrated extracts were reconstituted in 200 µL 0.1% formic acid (FA) in water and vortexed for 2 min. Finally, the samples were filtered using a hydrophilic syringe filter with a pore size of 0.45 µm and were ready to be analyzed using Q-TOF MS.