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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Subject:

Subject ID:	SU001768
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
01_01FibroblastSphin1_n1p_A_1	SA156655	FL018367	methylamine treatment	Treatment

Collection:

Collection ID:	CO001761
Collection Summary:	Cells were washed with cold PBS, scraped and collected after centrifugation in -80° untill the MS analysis
Sample Type:	Fibroblasts

Treatment:

Treatment ID:	TR001781
Treatment Summary:	No treatment has been done on the cells

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.

Combined analysis:

Analysis ID	AN002761
Analysis type	MS
Chromatography type	HILIC
Chromatography system	Shimadzu Prominence UFPLC xr system
Column	Kinetex (50 x 2.1mm,2.6um)
MS Type	ESI
MS instrument type	Orbitrap
MS instrument name	Hybrid Orbitrap Elite
Ion Mode	POSITIVE
Units	abundance

Chromatography:

Chromatography ID:	CH002041
Chromatography Summary:	Liquid chromatography on a HILIC Column
Methods Filename:	laura_capolupo_20210210_062234_PR_CH_Chromatography_methods.pdf
Instrument Name:	Shimadzu Prominence UFPLC xr system
Column Name:	Kinetex (50 x 2.1mm,2.6um)
Chromatography Type:	HILIC

MS:

MS ID:	MS002558
Analysis ID:	AN002761
Instrument Name:	Hybrid Orbitrap Elite
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Check protocol file
Ion Mode:	POSITIVE
Analysis Protocol File:	laura_capolupo_20210210_062234_PR_CH_Chromatography_methods.pdf