Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA206971

Local Sample ID:	190905_19_0009_UCLM_CCM_012
Subject ID:	SU002243
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Gender:	Male
Cell Biosource Or Supplier:	CLS
Cell Strain Details:	In vitro spontaneously transformed keratinocytes from histologically normal skin
Cell Primary Immortalized:	HaCaT
Cell Passage Number:	Up to the 15th passage

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Subject:

Subject ID:	SU002243
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Gender:	Male
Cell Biosource Or Supplier:	CLS
Cell Strain Details:	In vitro spontaneously transformed keratinocytes from histologically normal skin
Cell Primary Immortalized:	HaCaT
Cell Passage Number:	Up to the 15th passage

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
190905_19_0009_UCLM_CCM_012	SA206971	FL025355	Week	Exposure time
190905_19_0009_UCLM_CCM_012	SA206971	FL025355	GO	Nanomaterial
190905_19_0009_UCLM_CCM_012	SA206971	FL025355	N1	Replicate

Collection:

Collection ID:	CO002236
Collection Summary:	HaCaT cells treated with GO1, GO2 and FLG
Sample Type:	Keratinocytes
Storage Conditions:	-80℃

Treatment:

Treatment ID:	TR002255
Treatment Summary:	Cells were treated with 5 ug/mL of the different GRMs for 7 days and 30 days (1 treatment/week)

Sample Preparation:

Sampleprep ID:	SP002249
Sampleprep Summary:	Keratinocytes were defrosted on ice and proteins were precipitated from the lysed cell samples by adding the extraction solvent spiked with metabolites not detected in unspiked cell extracts. These metabolites, considered as internal standards, were tryptophan-d5, Anthranilic acid-(ring-13C6), Phenylthiohydantoin (PTH)-valine, Glycocholic-2,2,4,4-d4 acid (Sigma Aldrich). Then, cell extracts were incubated at -20 ˚C for 1 hour and after that, samples were vortexed and centrifuged at 18,000 x g for 10 minutes at 4 ºC. Supernatants were collected and kept on ice. A second extraction was performed from the remaining pellets, following the same steps described above. Supernatants obtained from the second extraction were collected and put together with the supernatants of the first extraction. Finally, these supernatants were dried under vacuum, reconstituted in water, resuspended with agitation for 10 minutes, centrifuged at 18,000 x g for 5 minutes at 4ºC, and transferred to vials for UHPLC-MS analysis.

Sampleprep ID:

SP002249

Sampleprep Summary:

Keratinocytes were defrosted on ice and proteins were precipitated from the lysed cell samples by adding the extraction solvent spiked with metabolites not detected in unspiked cell extracts. These metabolites, considered as internal standards, were tryptophan-d5, Anthranilic acid-(ring-13C6), Phenylthiohydantoin (PTH)-valine, Glycocholic-2,2,4,4-d4 acid (Sigma Aldrich). Then, cell extracts were incubated at -20 ˚C for 1 hour and after that, samples were vortexed and centrifuged at 18,000 x g for 10 minutes at 4 ºC. Supernatants were collected and kept on ice. A second extraction was performed from the remaining pellets, following the same steps described above. Supernatants obtained from the second extraction were collected and put together with the supernatants of the first extraction. Finally, these supernatants were dried under vacuum, reconstituted in water, resuspended with agitation for 10 minutes, centrifuged at 18,000 x g for 5 minutes at 4ºC, and transferred to vials for UHPLC-MS analysis.

Combined analysis:

Analysis ID	AN003532
Analysis type	MS
Chromatography type	Ion pair
Chromatography system	Waters Acquity H-Class
Column	Waters Acquity BEH HSS T3 (100 x 2.1mm,1.8um)
MS Type	ESI
MS instrument type	Time of flight
MS instrument name	Waters LCT premier
Ion Mode	NEGATIVE
Units	Log2 (fold-change)

Chromatography:

Chromatography ID:	CH002609
Chromatography Summary:	Chromatography was performed on a 1.0-mm internal diameter x 150 mm Acquity HSS T3 1.7 µm column (Waters Corp., Milford, MA) using an ACQUITY UPLC system (Waters Corp.). The column was maintained at 40 ºC. Samples (2µL) were injected onto the column at a flow rate of 100 µL/min, for a total run time of 25 min. The following linear elution gradient was used: 100% solvent A (10mM tributylamine + 15mM acetic acid + 0.2% methanol in water), to which solvent B (methanol) was added incrementally to reach a concentration of 4% B after 1.5 min, increasing to 20% B at 3 min, 25% B at 8 min, 50% B at 10 min, 55% at 15 min, and 100% B over the next 5 min, and returning to the initial composition over the final 5 min.
Instrument Name:	Waters Acquity H-Class
Column Name:	Waters Acquity BEH HSS T3 (100 x 2.1mm,1.8um)
Chromatography Type:	Ion pair

MS:

MS ID:	MS003290
Analysis ID:	AN003532
Instrument Name:	Waters LCT premier
Instrument Type:	Time of flight
MS Type:	ESI
MS Comments:	The eluent was introduced into the mass spectrometer (LCT Premier (Waters Corp.) by electrospray ionization, with capillary and cone voltages set in the negative ion mode to 2800 and 100 V, respectively. The nebulization gas was set to 600 L/hour and 300ºC. The cone gas was fixed at 50 L/hour, and the source temperature was maintained at 120ºC. Centroid data were acquired over the mass range of 50-1000 Da, using an accumulation time of 0.2 seconds per spectrum. All spectra were mass corrected in real time by reference to leucine enkephalin, infused at 10 µL/minute through an independent reference electrospray, sampled every 10 seconds.
Ion Mode:	NEGATIVE