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