Summary of Study ST001210
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 PR000813. The data can be accessed directly via it's Project DOI: 10.21228/M8KX27 This work is supported by NIH grant, U2C- DK119886.
See: https://www.metabolomicsworkbench.org/about/howtocite.php
Study ID | ST001210 |
Study Title | Comprehensive UHPLC-MS/MS lipidomics profiling to study effects of betulin on keratinocytes |
Study Summary | Lipidomics analysis of betulin in human primary keratinocytes to monitor alterations in the lipid profiles induced by treatment with betulin |
Institute | Eberhard Karls University of Tübingen |
Department | Institute of Pharmaceutical Sciences |
Laboratory | Pharmaceutical (Bio-)Analysis |
Last Name | Calderon |
First Name | Carlos |
Address | Auf der Morgenstelle 8 (Haus B), D-72076, Tuebingen, baden württemberg, 72076, Germany |
carlos.calderon@uni-tuebingen.de | |
Phone | +49 (0)7071 29 74009 |
Submit Date | 2019-07-05 |
Num Groups | 2 |
Total Subjects | 20 |
Study Comments | Eberhard-Karls-University Tuebingen |
Raw Data Available | Yes |
Raw Data File Type(s) | wiff |
Analysis Type Detail | LC-MS |
Release Date | 2019-09-23 |
Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
Project ID: | PR000813 |
Project DOI: | doi: 10.21228/M8KX27 |
Project Title: | Comprehensive UHPLC-MS/MS lipidomics profiling to study effects of betulin on keratinocytes |
Project Type: | Lipid profiling of Keratinocytes |
Project Summary: | Lipidomics analysis of betulin in human primary keratinocytes to monitor alterations in the lipid profiles induced by treatment with betulin. |
Institute: | Eberhard Karls University of Tübingen |
Department: | Institute of Pharmaceutical Sciences |
Laboratory: | Pharmaceutical (Bio-)Analysis |
Last Name: | Calderon |
First Name: | Carlos |
Address: | Auf der Morgenstelle 8 (Haus B), D-72076, Tuebingen, baden württemberg, 72076, Germany |
Email: | carlos.calderon@uni-tuebingen.de |
Phone: | +49 (0)7071 29 74009 |
Contributors: | Malgorzata Cebo, Irmgard Merfort, Michael Lämmerhofer |
Subject:
Subject ID: | SU001277 |
Subject Type: | Cultured cells |
Subject Species: | Homo sapiens |
Taxonomy ID: | 9606 |
Species Group: | Mammals |
Factors:
Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)
mb_sample_id | local_sample_id | Treatment |
---|---|---|
SA085190 | B9 | Betulin |
SA085191 | B10 | Betulin |
SA085192 | B1 | Betulin |
SA085193 | B7 | Betulin |
SA085194 | B8 | Betulin |
SA085195 | B6 | Betulin |
SA085196 | B2 | Betulin |
SA085197 | B4 | Betulin |
SA085198 | B3 | Betulin |
SA085199 | B5 | Betulin |
SA085200 | K8 | Control |
SA085201 | K9 | Control |
SA085202 | K10 | Control |
SA085203 | K7 | Control |
SA085204 | K1 | Control |
SA085205 | K2 | Control |
SA085206 | K3 | Control |
SA085207 | K4 | Control |
SA085208 | K5 | Control |
SA085209 | K6 | Control |
Showing results 1 to 20 of 20 |
Collection:
Collection ID: | CO001271 |
Collection Summary: | Human immortalized keratinocytes are cultivated in Keratinocyte SFM supplemented with rhEGF, BPE and 1 % v/v penicillin/streptomycin at 5 % CO2 and 37 °C. Cells were split when they reached a confluency of 80 %. Passage 3 or 4 were used. Cells were plated in 20 Petri dishes (10 cm2), 1.0 million cells/ per dish |
Collection Protocol Filename: | ccalcas_Extraction_and_treatment.pdf |
Sample Type: | Keratinocytes |
Treatment:
Treatment ID: | TR001292 |
Treatment Summary: | Cells were split when they reached a confluency of 80 %. Passage 3 or 4 were used. Cells were plated in 20 Petri dishes (10 cm2, 1 x 106 cells/ per dish) and cultivated for 4 days in 10 mL of the above mentioned medium for adherence. 10 dishes were incubated with 1.95 µM betulin (10 µL of the 1.95 mM stock solution in DMSO) for 8 hrs prior to removal of the medium and the remaining ones were used as control. Control samples were treated with 10 µL DMSO. The concentration of 1.95 µM of betulin was used, as this concentration has shown effects in our previous studies on the molecular wound healing effect [1]. On day 4, cells were incubated with 3 ml of trypsin (0.05 %) at 37°C. After 5 min cells were washed by adding 7 mL medium. The suspension was transferred into 15 mL falcon tubes and centrifuged for 5 min at 4°C and 1.200 rpm, respectively. The supernatant was withdrawn and the remaining cell pellets were washed and then frozen at -20°C in the falcon tubes until extraction. |
Treatment Protocol Filename: | ccalcas_Extraction_and_treatment.pdf |
Sample Preparation:
Sampleprep ID: | SP001285 |
Sampleprep Summary: | Lipid extraction was performed with IPA:H2O (90:10 v/v). Briefly, a 5% (v/v) SPLASH Lipidomix solution was prepared in MeOH and then 50 µL of the diluted solution was added to the cell pellet. Next, 4.95 mL of IPA:H2O (90:10 v/v) were added. Samples were vortexed (30 s), sonicated (2 min) and vortexed again (30 s) to disrupt the pellet. Incubation on ice was continued on a shaker for 1h (500 rpm, 60 min). Samples were centrifuged (3500 rcf, 10 min) and the supernatant was transferred to 15 mL falcon tubes. Samples were dried in an evaporator for 10 hours under nitrogen protection. Dried extract was resuspended in 100 µL of methanol containing odd-chained lipid standards LPC 17:1 and PC 17:0-20:4 at 500 ng/mL and 125 ng/mL, respectively. Sonication (2 min) and vortexing (30 s) were applied to ensure that lipids were not stuck to the surface of the extraction container. Then, samples were centrifuged (3500 rcf, 10 min) and the supernatant was transferred to vials for LC-MS measurements. Quality control (QC) sample was prepared by pooling together 15 µL aliquot of each sample. Samples were measured randomly and QC samples were run at the beginning, at the end and every fifth sample during the sequence. |
Sampleprep Protocol Filename: | ccalcas_Extraction_and_treatment.pdf |
Combined analysis:
Analysis ID | AN002014 | AN002015 |
---|---|---|
Analysis type | MS | MS |
Chromatography type | Reversed phase | Reversed phase |
Chromatography system | Agilent 1290 | Agilent 1290 |
Column | Waters ACQUITY UPLC CSH C18 (100 x 2.1mm,1.7um,130Å) | Waters ACQUITY UPLC CSH C18 (100 x 2.1mm,1.7um,130Å) |
MS Type | ESI | ESI |
MS instrument type | Triple TOF | Triple TOF |
MS instrument name | ABI Sciex 5600 TripleTOF | ABI Sciex 5600 TripleTOF |
Ion Mode | POSITIVE | NEGATIVE |
Units | Normalized intensities | Normalized intensities |
Chromatography:
Chromatography ID: | CH001457 |
Chromatography Summary: | Analyses were performed by Agilent 1290 Series UHPLC instrument coupled to Sciex TripleTOF 5600+ MS with duospray source and Pal HTC-XS autosampler from CTC. Positive and negative ESI ionization were used in separate LC-MS runs. Conditions: Acquity UPLC CSH C18 (130Å, 1.7 µm, 2.1 mm X 100 mm) column was utilized with Acquity UPLC CSH C18 VanGuard pre-column (130Å, 1.7 µm, 2.1 mm X 5 mm). The mobile phase was composed of 10 mM ammonium formate and 0.1 % formic acid dissolved in 60:40 ACN:H2O (v/v) (A) and 90:10 (v/v) IPA:ACN (B). |
Methods Filename: | ccalcas_LC-MS.pdf |
Instrument Name: | Agilent 1290 |
Column Name: | Waters ACQUITY UPLC CSH C18 (100 x 2.1mm,1.7um,130Å) |
Column Temperature: | 65 |
Flow Rate: | 600 uL/min |
Solvent A: | 100% water; 5 mM ammonium acetate, pH 9.9 |
Solvent B: | 100% acetonitrile |
Chromatography Type: | Reversed phase |
MS:
MS ID: | MS001867 |
Analysis ID: | AN002014 |
Instrument Name: | ABI Sciex 5600 TripleTOF |
Instrument Type: | Triple TOF |
MS Type: | ESI |
MS Comments: | SWATH adquisition |
Ion Mode: | POSITIVE |
Analysis Protocol File: | ccalcas_LC-MS.pdf |
MS ID: | MS001868 |
Analysis ID: | AN002015 |
Instrument Name: | ABI Sciex 5600 TripleTOF |
Instrument Type: | Triple TOF |
MS Type: | ESI |
MS Comments: | SWATH adquisition |
Ion Mode: | NEGATIVE |
Analysis Protocol File: | ccalcas_LC-MS.pdf |