Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA156655

Local Sample ID:	01_01FibroblastSphin1_n1p_A_1
Subject ID:	SU001768
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606

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Sample Preparation:

Sampleprep ID:	SP001774
Sampleprep Summary:	Total lipid extracts were prepared using a standard MTBE protocol followed by a methylamine treatment for total lipid analysis by mass spectrometry 7. Briefly, cell pellet was resuspended in 100 μL H2O. 360 μL methanol and 1.2 mL of MTBE were added and samples were placed for 10 min on a vortex at 4 °C followed by incubation for 1 h at room temperature on a shaker. Phase separation was induced by addition of 200 μL of H2O. After 10 min at room temperature, samples were centrifuged at 1000 g for 10 min. The upper (organic) phase was transferred into a glass tube and the lower phase was re-extracted with 400 μL artificial upper phase [MTBE/methanol/H2O (10:3:1.5, v/v/v)]. The combined organic phases were dried in a vacuum concentrator. Lipids where then resuspended in 500 μL of CHCl3 and divided in two aliquots for a further methylamine treatment for sphingo- and glycosphingolipids analysis. In details, 500 μL of freshly prepared monomethylamine reagent [methylamine/H2O/nbutanol/ methanol (5:3:1:4, (v/v/v/v)] was added to the dried lipid extract and then incubated at 53 °C for 1 h in a water bath. Lipids were cooled to room temperature and then dried. The dried lipid extract was then extracted by n-butanol extraction using 300 μL water-saturated nbutanol and 150 μL H2O. The organic phase was collected, and the aqueous phase was reextracted twice with 300 μL water-saturated n-butanol. The organic phases were pooled and dried in a vacuum concentrator.

Sampleprep ID:

SP001774

Sampleprep Summary:

Total lipid extracts were prepared using a standard MTBE protocol followed by a methylamine treatment for total lipid analysis by mass spectrometry 7. Briefly, cell pellet was resuspended in 100 μL H2O. 360 μL methanol and 1.2 mL of MTBE were added and samples were placed for 10 min on a vortex at 4 °C followed by incubation for 1 h at room temperature on a shaker. Phase separation was induced by addition of 200 μL of H2O. After 10 min at room temperature, samples were centrifuged at 1000 g for 10 min. The upper (organic) phase was transferred into a glass tube and the lower phase was re-extracted with 400 μL artificial upper phase [MTBE/methanol/H2O (10:3:1.5, v/v/v)]. The combined organic phases were dried in a vacuum concentrator. Lipids where then resuspended in 500 μL of CHCl3 and divided in two aliquots for a further methylamine treatment for sphingo- and glycosphingolipids analysis. In details, 500 μL of freshly prepared monomethylamine reagent [methylamine/H2O/nbutanol/ methanol (5:3:1:4, (v/v/v/v)] was added to the dried lipid extract and then incubated at 53 °C for 1 h in a water bath. Lipids were cooled to room temperature and then dried. The dried lipid extract was then extracted by n-butanol extraction using 300 μL water-saturated nbutanol and 150 μL H2O. The organic phase was collected, and the aqueous phase was reextracted twice with 300 μL water-saturated n-butanol. The organic phases were pooled and dried in a vacuum concentrator.