{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST001210","ANALYSIS_ID":"AN002014","VERSION":"1","CREATED_ON":"July 12, 2019, 12:01 pm"},

"PROJECT":{"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 Tuebingen","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"},

"STUDY":{"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 Tuebingen","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","NUM_GROUPS":"2","TOTAL_SUBJECTS":"20"},

"SUBJECT":{"SUBJECT_TYPE":"Cultured cells","SUBJECT_SPECIES":"Homo sapiens","TAXONOMY_ID":"9606"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"B1",
"Factors":{"Treatment":"Betulin"}
},
{
"Subject ID":"-",
"Sample ID":"B2",
"Factors":{"Treatment":"Betulin"}
},
{
"Subject ID":"-",
"Sample ID":"B3",
"Factors":{"Treatment":"Betulin"}
},
{
"Subject ID":"-",
"Sample ID":"B4",
"Factors":{"Treatment":"Betulin"}
},
{
"Subject ID":"-",
"Sample ID":"B5",
"Factors":{"Treatment":"Betulin"}
},
{
"Subject ID":"-",
"Sample ID":"B6",
"Factors":{"Treatment":"Betulin"}
},
{
"Subject ID":"-",
"Sample ID":"B7",
"Factors":{"Treatment":"Betulin"}
},
{
"Subject ID":"-",
"Sample ID":"B8",
"Factors":{"Treatment":"Betulin"}
},
{
"Subject ID":"-",
"Sample ID":"B9",
"Factors":{"Treatment":"Betulin"}
},
{
"Subject ID":"-",
"Sample ID":"B10",
"Factors":{"Treatment":"Betulin"}
},
{
"Subject ID":"-",
"Sample ID":"K1",
"Factors":{"Treatment":"Control"}
},
{
"Subject ID":"-",
"Sample ID":"K2",
"Factors":{"Treatment":"Control"}
},
{
"Subject ID":"-",
"Sample ID":"K3",
"Factors":{"Treatment":"Control"}
},
{
"Subject ID":"-",
"Sample ID":"K4",
"Factors":{"Treatment":"Control"}
},
{
"Subject ID":"-",
"Sample ID":"K5",
"Factors":{"Treatment":"Control"}
},
{
"Subject ID":"-",
"Sample ID":"K6",
"Factors":{"Treatment":"Control"}
},
{
"Subject ID":"-",
"Sample ID":"K7",
"Factors":{"Treatment":"Control"}
},
{
"Subject ID":"-",
"Sample ID":"K8",
"Factors":{"Treatment":"Control"}
},
{
"Subject ID":"-",
"Sample ID":"K9",
"Factors":{"Treatment":"Control"}
},
{
"Subject ID":"-",
"Sample ID":"K10",
"Factors":{"Treatment":"Control"}
}
],
"COLLECTION":{"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_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"},

"SAMPLEPREP":{"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"},

"CHROMATOGRAPHY":{"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).","CHROMATOGRAPHY_TYPE":"Reversed phase","INSTRUMENT_NAME":"Agilent 1290","COLUMN_NAME":"Acquity UPLC CSH C18 (130Ã…, 1.7 µm, 2.1 mm X 100 mm)","METHODS_FILENAME":"ccalcas_Extraction_and_treatment.pdf"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS","ANALYSIS_PROTOCOL_FILE":"ccalcas_LC-MS.pdf"},

"MS":{"INSTRUMENT_NAME":"ABI Sciex 5600 TripleTOF","INSTRUMENT_TYPE":"QTOF","MS_TYPE":"ESI","ION_MODE":"POSITIVE","MS_COMMENTS":"SWATH adquisition"},

"MS_METABOLITE_DATA":{
"Units":"Normalized intensities",

"Data":[{"Metabolite":"ACar 14:0","B1":"0.110425595","B2":"0.074936284","B3":"0.101203068","B4":"0.094815603","B5":"0.100724155","B6":"0.080493537","B7":"0.112362797","B8":"0.086266926","B9":"0.111288258","B10":"0.131698527","K1":"0.06975144","K2":"0.043076439","K3":"0.077640738","K4":"0.08070851","K5":"0.082927637","K6":"0.071724067","K7":"0.061404756","K8":"0.064941244","K9":"0.071564469","K10":"0.063122885"},{"Metabolite":"ACar 14:1","B1":"0.017474228","B2":"0.011456362","B3":"0.011105685","B4":"0.01262623","B5":"0.014420554","B6":"0.017979638","B7":"0.014450884","B8":"0.015884855","B9":"0.019366057","B10":"0.020510168","K1":"0.010636904","K2":"0.006652562","K3":"0.013308702","K4":"0.012894309","K5":"0.01829078","K6":"0.007884802","K7":"0.009272207","K8":"0.009521775","K9":"0.004718598","K10":"0.011097646"},{"Metabolite":"ACar 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mode":"TOF"},{"Metabolite":"DAG 38:3 (2)","Retention times":"7.48","quantified m/z":"664.5880","Adduct":"[M+NH4]+","Pos/Neg":"Pos","MS mode":"TOF"},{"Metabolite":"DAG 38:3 (3)","Retention times":"7.63","quantified m/z":"664.5880","Adduct":"[M+NH4]+","Pos/Neg":"Pos","MS mode":"TOF"},{"Metabolite":"DAG 38:4 (1)","Retention times":"6.84","quantified m/z":"662.5723","Adduct":"[M+NH4]+","Pos/Neg":"Pos","MS mode":"TOF"},{"Metabolite":"DAG 38:4 (2)","Retention times":"6.96","quantified m/z":"662.5723","Adduct":"[M+NH4]+","Pos/Neg":"Pos","MS mode":"TOF"},{"Metabolite":"DAG 38:4 (3)","Retention times":"7.15","quantified m/z":"662.5723","Adduct":"[M+NH4]+","Pos/Neg":"Pos","MS mode":"TOF"},{"Metabolite":"DAG 40:1","Retention times":"9.02","quantified m/z":"696.6506","Adduct":"[M+NH4]+","Pos/Neg":"Pos","MS mode":"TOF"},{"Metabolite":"DAG 40:2","Retention times":"8.50","quantified m/z":"694.6349","Adduct":"[M+NH4]+","Pos/Neg":"Pos","MS mode":"TOF"},{"Metabolite":"DAG 40:3","Retention 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mode":"TOF"},{"Metabolite":"DAG 52:1","Retention times":"11.89","quantified m/z":"864.8384","Adduct":"[M+NH4]+","Pos/Neg":"Pos","MS mode":"TOF"},{"Metabolite":"DAG 52:2","Retention times":"11.69","quantified m/z":"862.8227","Adduct":"[M+NH4]+","Pos/Neg":"Pos","MS mode":"TOF"},{"Metabolite":"DAG 52:3","Retention times":"11.36","quantified m/z":"860.8071","Adduct":"[M+NH4]+","Pos/Neg":"Pos","MS mode":"TOF"},{"Metabolite":"DAG 54:1","Retention times":"12.00","quantified m/z":"892.8697","Adduct":"[M+NH4]+","Pos/Neg":"Pos","MS mode":"TOF"},{"Metabolite":"DAG 54:2","Retention times":"11.88","quantified m/z":"890.8540","Adduct":"[M+NH4]+","Pos/Neg":"Pos","MS mode":"TOF"},{"Metabolite":"DAG 54:3","Retention times":"11.72","quantified m/z":"888.8384","Adduct":"[M+NH4]+","Pos/Neg":"Pos","MS mode":"TOF"},{"Metabolite":"HexCer d32:1","Retention times":"4.48","quantified m/z":"672.5414","Adduct":"[M+H]+","Pos/Neg":"Pos","MS mode":"TOF"},{"Metabolite":"HexCer d34:1","Retention 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m/z":"840.7292","Adduct":"[M+H]+","Pos/Neg":"Pos","MS mode":"TOF"},{"Metabolite":"HexCer d44:2","Retention times":"7.87","quantified m/z":"838.7136","Adduct":"[M+H]+","Pos/Neg":"Pos","MS mode":"TOF"},{"Metabolite":"HexCer d44:3","Retention times":"7.29","quantified m/z":"836.6979","Adduct":"[M+H]+","Pos/Neg":"Pos","MS mode":"TOF"},{"Metabolite":"HexCer d46:1","Retention times":"9.12","quantified m/z":"868.7605","Adduct":"[M+H]+","Pos/Neg":"Pos","MS mode":"TOF"},{"Metabolite":"HexCer d46:2","Retention times":"8.50","quantified m/z":"866.7449","Adduct":"[M+H]+","Pos/Neg":"Pos","MS mode":"TOF"},{"Metabolite":"HexCer d48:2","Retention times":"9.07","quantified m/z":"894.7762","Adduct":"[M+H]+","Pos/Neg":"Pos","MS mode":"TOF"},{"Metabolite":"HexCer d50:2","Retention times":"9.62","quantified m/z":"922.8075","Adduct":"[M+H]+","Pos/Neg":"Pos","MS mode":"TOF"},{"Metabolite":"LPC 14:0 (1)","Retention times":"1.00","quantified m/z":"468.3090","Adduct":"[M+H]+","Pos/Neg":"Pos","MS mode":"TOF"},{"Metabolite":"LPC 14:0 (2)","Retention times":"0.92","quantified m/z":"468.3090","Adduct":"[M+H]+","Pos/Neg":"Pos","MS mode":"TOF"},{"Metabolite":"LPC 16:0 (1)","Retention times":"1.53","quantified m/z":"496.3403","Adduct":"[M+H]+","Pos/Neg":"Pos","MS mode":"TOF"},{"Metabolite":"LPC 16:0 (2)","Retention times":"1.39","quantified m/z":"496.3403","Adduct":"[M+H]+","Pos/Neg":"Pos","MS mode":"TOF"},{"Metabolite":"LPC 16:1","Retention times":"1.11","quantified m/z":"494.3247","Adduct":"[M+H]+","Pos/Neg":"Pos","MS mode":"TOF"},{"Metabolite":"LPC 18:0 (1)","Retention times":"2.32","quantified m/z":"524.3716","Adduct":"[M+H]+","Pos/Neg":"Pos","MS mode":"TOF"},{"Metabolite":"LPC 18:0 (2)","Retention times":"2.14","quantified m/z":"524.3716","Adduct":"[M+H]+","Pos/Neg":"Pos","MS mode":"TOF"},{"Metabolite":"LPC 18:1 (1)","Retention times":"1.68","quantified m/z":"522.3560","Adduct":"[M+H]+","Pos/Neg":"Pos","MS mode":"TOF"},{"Metabolite":"LPC 18:1 (2)","Retention times":"1.54","quantified m/z":"522.3560","Adduct":"[M+H]+","Pos/Neg":"Pos","MS mode":"TOF"},{"Metabolite":"LPC 18:2","Retention times":"1.27","quantified m/z":"520.3403","Adduct":"[M+H]+","Pos/Neg":"Pos","MS mode":"TOF"},{"Metabolite":"LPC 20:0","Retention times":"3.09","quantified m/z":"552.4029","Adduct":"[M+H]+","Pos/Neg":"Pos","MS mode":"TOF"},{"Metabolite":"LPC 20:1","Retention times":"2.47","quantified m/z":"550.3873","Adduct":"[M+H]+","Pos/Neg":"Pos","MS mode":"TOF"},{"Metabolite":"LPC 20:2","Retention times":"1.89","quantified m/z":"548.3716","Adduct":"[M+H]+","Pos/Neg":"Pos","MS mode":"TOF"},{"Metabolite":"LPC 20:3 (1)","Retention times":"1.61","quantified m/z":"546.3560","Adduct":"[M+H]+","Pos/Neg":"Pos","MS mode":"TOF"},{"Metabolite":"LPC 20:3 (2)","Retention times":"1.50","quantified m/z":"546.3560","Adduct":"[M+H]+","Pos/Neg":"Pos","MS mode":"TOF"},{"Metabolite":"LPC 22:0","Retention times":"3.61","quantified m/z":"580.4342","Adduct":"[M+H]+","Pos/Neg":"Pos","MS mode":"TOF"},{"Metabolite":"LPC 22:1","Retention times":"3.15","quantified m/z":"578.4186","Adduct":"[M+H]+","Pos/Neg":"Pos","MS mode":"TOF"},{"Metabolite":"LPC 24:0","Retention times":"4.18","quantified m/z":"608.4655","Adduct":"[M+H]+","Pos/Neg":"Pos","MS mode":"TOF"},{"Metabolite":"LPC 24:1","Retention times":"3.62","quantified m/z":"606.4499","Adduct":"[M+H]+","Pos/Neg":"Pos","MS mode":"TOF"},{"Metabolite":"LPC 24:2","Retention times":"3.23","quantified m/z":"604.4342","Adduct":"[M+H]+","Pos/Neg":"Pos","MS mode":"TOF"},{"Metabolite":"LPC 26:0","Retention times":"4.82","quantified m/z":"636.4968","Adduct":"[M+H]+","Pos/Neg":"Pos","MS mode":"TOF"},{"Metabolite":"LPC 26:1","Retention times":"4.18","quantified m/z":"634.4812","Adduct":"[M+H]+","Pos/Neg":"Pos","MS mode":"TOF"},{"Metabolite":"LPC 26:2","Retention times":"3.69","quantified m/z":"632.4655","Adduct":"[M+H]+","Pos/Neg":"Pos","MS mode":"TOF"},{"Metabolite":"LPE 16:0 (1)","Retention times":"1.55","quantified m/z":"454.2934","Adduct":"[M+H]+","Pos/Neg":"Pos","MS mode":"TOF"},{"Metabolite":"LPE 16:0 (2)","Retention times":"1.42","quantified m/z":"454.2934","Adduct":"[M+H]+","Pos/Neg":"Pos","MS mode":"TOF"},{"Metabolite":"LPE 16:1 (1)","Retention times":"1.12","quantified m/z":"452.2777","Adduct":"[M+H]+","Pos/Neg":"Pos","MS mode":"TOF"},{"Metabolite":"LPE 18:0 (1)","Retention times":"2.37","quantified m/z":"482.3247","Adduct":"[M+H]+","Pos/Neg":"Pos","MS mode":"TOF"},{"Metabolite":"LPE 18:0 (2)","Retention times":"2.19","quantified m/z":"482.3247","Adduct":"[M+H]+","Pos/Neg":"Pos","MS mode":"TOF"},{"Metabolite":"LPE 18:1 (1)","Retention times":"1.71","quantified m/z":"480.3090","Adduct":"[M+H]+","Pos/Neg":"Pos","MS mode":"TOF"},{"Metabolite":"LPE 18:1 (2)","Retention times":"1.57","quantified m/z":"480.3090","Adduct":"[M+H]+","Pos/Neg":"Pos","MS mode":"TOF"},{"Metabolite":"LPE 20:1 (1)","Retention times":"2.53","quantified m/z":"508.3403","Adduct":"[M+H]+","Pos/Neg":"Pos","MS mode":"TOF"},{"Metabolite":"LPE 20:2","Retention times":"1.92","quantified m/z":"506.3247","Adduct":"[M+H]+","Pos/Neg":"Pos","MS mode":"TOF"},{"Metabolite":"LPE 22:1 (1)","Retention times":"3.18","quantified m/z":"536.3716","Adduct":"[M+H]+","Pos/Neg":"Pos","MS mode":"TOF"},{"Metabolite":"LPE 22:2 (1)","Retention times":"2.71","quantified m/z":"534.3560","Adduct":"[M+H]+","Pos/Neg":"Pos","MS mode":"TOF"},{"Metabolite":"LPE 22:3 (1)","Retention times":"2.26","quantified m/z":"532.3403","Adduct":"[M+H]+","Pos/Neg":"Pos","MS mode":"TOF"},{"Metabolite":"LPE 22:3 (2)","Retention times":"2.13","quantified m/z":"532.3403","Adduct":"[M+H]+","Pos/Neg":"Pos","MS mode":"TOF"},{"Metabolite":"LPE 24:1","Retention times":"3.67","quantified m/z":"564.4029","Adduct":"[M+H]+","Pos/Neg":"Pos","MS mode":"TOF"},{"Metabolite":"LPE 26:1","Retention times":"4.14","quantified m/z":"592.4342","Adduct":"[M+H]+","Pos/Neg":"Pos","MS mode":"TOF"},{"Metabolite":"LPE 26:2","Retention times":"3.74","quantified 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}

}