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.

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Study IDST001210
Study TitleComprehensive UHPLC-MS/MS lipidomics profiling to study effects of betulin on keratinocytes
Study SummaryLipidomics 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
DepartmentInstitute of Pharmaceutical Sciences
LaboratoryPharmaceutical (Bio-)Analysis
Last NameCalderon
First NameCarlos
AddressAuf der Morgenstelle 8 (Haus B), D-72076, Tuebingen, baden württemberg, 72076, Germany
Emailcarlos.calderon@uni-tuebingen.de
Phone+49 (0)7071 29 74009
Submit Date2019-07-05
Num Groups2
Total Subjects20
Study CommentsEberhard-Karls-University Tuebingen
Raw Data AvailableYes
Raw Data File Type(s)wiff
Analysis Type DetailLC-MS
Release Date2019-09-23
Release Version1
Carlos Calderon Carlos Calderon
https://dx.doi.org/10.21228/M8KX27
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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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

Factors:

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

mb_sample_id local_sample_id Treatment
SA085190B9Betulin
SA085191B10Betulin
SA085192B1Betulin
SA085193B7Betulin
SA085194B8Betulin
SA085195B6Betulin
SA085196B2Betulin
SA085197B4Betulin
SA085198B3Betulin
SA085199B5Betulin
SA085200K8Control
SA085201K9Control
SA085202K10Control
SA085203K7Control
SA085204K1Control
SA085205K2Control
SA085206K3Control
SA085207K4Control
SA085208K5Control
SA085209K6Control
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
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