Summary of Study ST002157

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001370. The data can be accessed directly via it's Project DOI: 10.21228/M8N12W This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

Study ID	ST002157
Study Title	Effect of long-term exposure to graphene on skin cell metabolism
Study Summary	Graphene-derived materials are a family of nanomaterials with multiple potential applications in different fields such as biomedicine. It is therefore essential to understand their interaction with cellular barriers such as skin. In this work we evaluated the metabolic changes in human skin cells (HaCaT) exposed to different GRMs for 7 and 30 days. Objectives Endogenous metabolic profiles of control and graphene-treated keratinocytes have been studied using ultra-high performance liquid chromatography – mass spectrometry (UHPLC-MS). Keratinocytes were treated with graphene oxide (GO) from two different suppliers and with few layer graphene (FLG). Samples were collected one week and one month after the start of the treatment. The general aim of the project was to evaluate potential metabolic differences between: 1) Graphene-treated keratinocytes and control keratinocytes at one week; 2) Graphene-treated keratinocytes and control keratinocytes at one month; 3) Control keratinocytes at 1 month and 1 week; 4) Graphene-treated keratinocytes at 1 month and 1 week. Experimental Procedures A successful metabolic profiling experiment relies on the ability to determine changes in an organism’s biofluid or tissue complement of metabolites. Mass spectrometry coupled to ultra-high performance liquid chromatography (UHPLC-MS) is well suited to such analyses due to its high sensitivity, large coverage over different classes of metabolites, high throughput capacity, and wide dynamic range. In this study, one UHPLC-MS based platform was used to analyse endogenous analytes for inclusion in subsequent statistical analysis procedures used to study metabolic differences between the groups of samples. Results The oxidation degree and size of the GRMs is determinant in the effect on cell metabolism, as well as the exposure time. Thus, one of the materials used generated a change in the energy metabolism of the cells, significantly increasing the level of different Krebs cycle metabolites.
Institute	University of Castilla-La Mancha
Department	Medical Sciences
Laboratory	Group of Oxidative Stress and Neurodegeneration
Last Name	Frontinan
First Name	Javier
Address	F. Medicina Camino de Moledores s/n 13071
Email	javier.frontinan@uclm.es
Phone	+34656967979
Submit Date	2022-04-25
Num Groups	8
Raw Data Available	Yes
Raw Data File Type(s)	raw(Waters)
Analysis Type Detail	LC-MS
Release Date	2022-06-01
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR001370
Project DOI:	doi: 10.21228/M8N12W
Project Title:	Effect of long-term exposure to graphene on skin cell metabolism
Project Type:	MS HaCaT Cells
Project Summary:	Graphene-derived materials are a family of nanomaterials with multiple potential applications in different fields such as biomedicine. It is therefore essential to understand their interaction with cellular barriers such as skin. In this work we evaluated the metabolic changes in human skin cells (HaCaT) exposed to different GRMs for 7 and 30 days. Objectives Endogenous metabolic profiles of control and graphene-treated keratinocytes have been studied using ultra-high performance liquid chromatography – mass spectrometry (UHPLC-MS). Keratinocytes were treated with graphene oxide (GO) from two different suppliers and with few layer graphene (FLG). Samples were collected one week and one month after the start of the treatment. The general aim of the project was to evaluate potential metabolic differences between: 1) Graphene-treated keratinocytes and control keratinocytes at one week; 2) Graphene-treated keratinocytes and control keratinocytes at one month; 3) Control keratinocytes at 1 month and 1 week; 4) Graphene-treated keratinocytes at 1 month and 1 week. Experimental Procedures A successful metabolic profiling experiment relies on the ability to determine changes in an organism’s biofluid or tissue complement of metabolites. Mass spectrometry coupled to ultra-high performance liquid chromatography (UHPLC-MS) is well suited to such analyses due to its high sensitivity, large coverage over different classes of metabolites, high throughput capacity, and wide dynamic range. In this study, one UHPLC-MS based platform was used to analyse endogenous analytes for inclusion in subsequent statistical analysis procedures used to study metabolic differences between the groups of samples.
Institute:	University of Castilla-La Mancha
Department:	Medical Sciences
Laboratory:	Group of Oxidative Stress and Neurodegeneration
Last Name:	Frontiñán
First Name:	Javier
Address:	F. Medicina Camino de Moledores s/n 13071
Email:	javier.frontinan@uclm.es
Phone:	+34656967979
Funding Source:	Graphene Flagship (EU)

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:

Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id	local_sample_id	Exposure time	Nanomaterial	Replicate
SA206929	190905_19_0009_UCLM_CCM_059	Month	C	N1
SA206928	190905_19_0009_UCLM_CCM_043	Month	C-	N1
SA206930	190905_19_0009_UCLM_CCM_049	Month	C	N2
SA206931	190905_19_0009_UCLM_CCM_050	Month	C	N3
SA206932	190905_19_0009_UCLM_CCM_030	Month	C	N4
SA206933	190905_19_0009_UCLM_CCM_060	Month	C	N5
SA206935	190905_19_0009_UCLM_CCM_024	Month	FLG	N1
SA206934	190905_19_0009_UCLM_CCM_062	Month	FLG-	N1
SA206936	190905_19_0009_UCLM_CCM_053	Month	FLG	N2
SA206937	190905_19_0009_UCLM_CCM_052	Month	FLG	N3
SA206938	190905_19_0009_UCLM_CCM_013	Month	FLG	N4
SA206939	190905_19_0009_UCLM_CCM_038	Month	FLG	N5
SA206941	190905_19_0009_UCLM_CCM_042	Month	GO g	N1
SA206940	190905_19_0009_UCLM_CCM_031	Month	GO g-	N1
SA206942	190905_19_0009_UCLM_CCM_028	Month	GO g	N2
SA206943	190905_19_0009_UCLM_CCM_064	Month	GO g	N3
SA206944	190905_19_0009_UCLM_CCM_022	Month	GO g	N4
SA206945	190905_19_0009_UCLM_CCM_035	Month	GO g	N5
SA206947	190905_19_0009_UCLM_CCM_041	Month	GO	N1
SA206946	190905_19_0009_UCLM_CCM_018	Month	GO-	N1
SA206948	190905_19_0009_UCLM_CCM_029	Month	GO	N2
SA206949	190905_19_0009_UCLM_CCM_069	Month	GO	N3
SA206950	190905_19_0009_UCLM_CCM_055	Month	GO	N4
SA206951	190905_19_0009_UCLM_CCM_019	Month	GO	N5
SA206905	190905_19_0009_UCLM_CCM_001	-	-	-
SA206906	190905_19_0009_UCLM_CCM_037	-	-	-
SA206907	190905_19_0009_UCLM_CCM_027	-	-	-
SA206908	190905_19_0009_UCLM_CCM_026	-	-	-
SA206909	190905_19_0009_UCLM_CCM_017	-	-	-
SA206910	190905_19_0009_UCLM_CCM_046	-	-	-
SA206911	190905_19_0009_UCLM_CCM_047	-	-	-
SA206912	190905_19_0009_UCLM_CCM_067	-	-	-
SA206913	190905_19_0009_UCLM_CCM_066	-	-	-
SA206914	190905_19_0009_UCLM_CCM_057	-	-	-
SA206915	190905_19_0009_UCLM_CCM_056	-	-	-
SA206916	190905_19_0009_UCLM_CCM_016	-	-	-
SA206917	190905_19_0009_UCLM_CCM_036	-	-	-
SA206918	190905_19_0009_UCLM_CCM_007	-	-	-
SA206919	190905_19_0009_UCLM_CCM_008	-	-	-
SA206920	190905_19_0009_UCLM_CCM_009	-	-	-
SA206921	190905_19_0009_UCLM_CCM_010	-	-	-
SA206922	190905_19_0009_UCLM_CCM_006	-	-	-
SA206923	190905_19_0009_UCLM_CCM_005	-	-	-
SA206924	190905_19_0009_UCLM_CCM_002	-	-	-
SA206925	190905_19_0009_UCLM_CCM_003	-	-	-
SA206926	190905_19_0009_UCLM_CCM_004	-	-	-
SA206927	190905_19_0009_UCLM_CCM_011	-	-	-
SA206953	190905_19_0009_UCLM_CCM_020	Week	C	N1
SA206952	190905_19_0009_UCLM_CCM_048	Week	C-	N1
SA206954	190905_19_0009_UCLM_CCM_070	Week	C	N2
SA206955	190905_19_0009_UCLM_CCM_061	Week	C	N3
SA206956	190905_19_0009_UCLM_CCM_025	Week	C	N4
SA206957	190905_19_0009_UCLM_CCM_039	Week	C	N5
SA206959	190905_19_0009_UCLM_CCM_014	Week	FLG	N1
SA206958	190905_19_0009_UCLM_CCM_032	Week	FLG-	N1
SA206960	190905_19_0009_UCLM_CCM_054	Week	FLG	N2
SA206961	190905_19_0009_UCLM_CCM_044	Week	FLG	N3
SA206962	190905_19_0009_UCLM_CCM_058	Week	FLG	N4
SA206963	190905_19_0009_UCLM_CCM_051	Week	FLG	N5
SA206965	190905_19_0009_UCLM_CCM_023	Week	GO g	N1
SA206964	190905_19_0009_UCLM_CCM_021	Week	GO g-	N1
SA206966	190905_19_0009_UCLM_CCM_065	Week	GO g	N2
SA206967	190905_19_0009_UCLM_CCM_068	Week	GO g	N3
SA206968	190905_19_0009_UCLM_CCM_034	Week	GO g	N4
SA206969	190905_19_0009_UCLM_CCM_033	Week	GO g	N5
SA206971	190905_19_0009_UCLM_CCM_012	Week	GO	N1
SA206970	190905_19_0009_UCLM_CCM_045	Week	GO-	N1
SA206972	190905_19_0009_UCLM_CCM_071	Week	GO	N2
SA206973	190905_19_0009_UCLM_CCM_015	Week	GO	N3
SA206974	190905_19_0009_UCLM_CCM_040	Week	GO	N4
SA206975	190905_19_0009_UCLM_CCM_063	Week	GO	N5

Showing results 1 to 71 of 71

Showing results 1 to 71 of 71

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