#METABOLOMICS WORKBENCH laura_capolupo_20210210_062234 DATATRACK_ID:2468 STUDY_ID:ST001691 ANALYSIS_ID:AN002761 PROJECT_ID:PR001087
VERSION             	1
CREATED_ON             	February 11, 2021, 2:13 pm
#PROJECT
PR:PROJECT_TITLE                 	Untargeted lipidomics of primary human skin fibroblasts
PR:PROJECT_TYPE                  	MS qualitative untargeted lipidomics of human fibroblasts
PR:PROJECT_SUMMARY               	Untargeted lipidomics of primary human skin fibroblasts to identify their
PR:PROJECT_SUMMARY               	lipidome in positive ion mode
PR:INSTITUTE                     	École polytechnique fédérale de Lausanne (EPFL)
PR:LABORATORY                    	UPDANGELO
PR:LAST_NAME                     	Capolupo
PR:FIRST_NAME                    	Laura
PR:ADDRESS                       	Route Cantonale, Lausanne, Vaud, 1015, Switzerland
PR:EMAIL                         	laura.capolupo@epfl.ch
PR:PHONE                         	+41 21 693 42 79
#STUDY
ST:STUDY_TITLE                   	LC-MS untargeted lipidomics of primary human fibroblasts
ST:STUDY_TYPE                    	LC-MS untargeted lipidomics of fibroblasts
ST:STUDY_SUMMARY                 	We did LC-MS untargeted lipidomics of primary human fibroblasts to have a
ST:STUDY_SUMMARY                 	comprehensive overview of their lipidome in positive ion mode
ST:INSTITUTE                     	École polytechnique fédérale de Lausanne (EPFL)
ST:LAST_NAME                     	Capolupo
ST:FIRST_NAME                    	Laura
ST:ADDRESS                       	Route Cantonale
ST:EMAIL                         	laura.capolupo@epfl.ch
ST:PHONE                         	+41 21 693 42 79
#SUBJECT
SU:SUBJECT_TYPE                  	Cultured cells
SU:SUBJECT_SPECIES               	Homo sapiens
SU:TAXONOMY_ID                   	9606
#SUBJECT_SAMPLE_FACTORS:         	SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data
SUBJECT_SAMPLE_FACTORS           	-	01_01FibroblastSphin1_n1p_A_1	Treatment:methylamine treatment	RAW_FILE_NAME=01_01FibroblastSphin1_n1p_A_1; extraction type=MTBE
SUBJECT_SAMPLE_FACTORS           	-	02_02FibroblastSphin2_n1p_A_1	Treatment:methylamine treatment	RAW_FILE_NAME=02_02FibroblastSphin2_n1p_A_1; extraction type=MTBE
SUBJECT_SAMPLE_FACTORS           	-	03_03FibroblastTotal1_n1p_A_1	Treatment:total lipid extract	RAW_FILE_NAME=03_03FibroblastTotal1_n1p_A_1; extraction type=MTBE
SUBJECT_SAMPLE_FACTORS           	-	04_04FibroblastTotal2_n1p_A_1	Treatment:total lipid extract	RAW_FILE_NAME=04_04FibroblastTotal2_n1p_A_1; extraction type=MTBE
#COLLECTION
CO:COLLECTION_SUMMARY            	Cells were washed with cold PBS, scraped and collected after centrifugation in
CO:COLLECTION_SUMMARY            	-80° untill the MS analysis
CO:SAMPLE_TYPE                   	Fibroblasts
#TREATMENT
TR:TREATMENT_SUMMARY             	No treatment has been done on the cells
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Total lipid extracts were prepared using a standard MTBE protocol followed by a
SP:SAMPLEPREP_SUMMARY            	methylamine treatment for total lipid analysis by mass spectrometry 7. Briefly,
SP:SAMPLEPREP_SUMMARY            	cell pellet was resuspended in 100 μL H2O. 360 μL methanol and 1.2 mL of MTBE
SP:SAMPLEPREP_SUMMARY            	were added and samples were placed for 10 min on a vortex at 4 °C followed by
SP:SAMPLEPREP_SUMMARY            	incubation for 1 h at room temperature on a shaker. Phase separation was induced
SP:SAMPLEPREP_SUMMARY            	by addition of 200 μL of H2O. After 10 min at room temperature, samples were
SP:SAMPLEPREP_SUMMARY            	centrifuged at 1000 g for 10 min. The upper (organic) phase was transferred into
SP:SAMPLEPREP_SUMMARY            	a glass tube and the lower phase was re-extracted with 400 μL artificial upper
SP:SAMPLEPREP_SUMMARY            	phase [MTBE/methanol/H2O (10:3:1.5, v/v/v)]. The combined organic phases were
SP:SAMPLEPREP_SUMMARY            	dried in a vacuum concentrator. Lipids where then resuspended in 500 μL of
SP:SAMPLEPREP_SUMMARY            	CHCl3 and divided in two aliquots for a further methylamine treatment for
SP:SAMPLEPREP_SUMMARY            	sphingo- and glycosphingolipids analysis. In details, 500 μL of freshly
SP:SAMPLEPREP_SUMMARY            	prepared monomethylamine reagent [methylamine/H2O/nbutanol/ methanol (5:3:1:4,
SP:SAMPLEPREP_SUMMARY            	(v/v/v/v)] was added to the dried lipid extract and then incubated at 53 °C for
SP:SAMPLEPREP_SUMMARY            	1 h in a water bath. Lipids were cooled to room temperature and then dried. The
SP:SAMPLEPREP_SUMMARY            	dried lipid extract was then extracted by n-butanol extraction using 300 μL
SP:SAMPLEPREP_SUMMARY            	water-saturated nbutanol and 150 μL H2O. The organic phase was collected, and
SP:SAMPLEPREP_SUMMARY            	the aqueous phase was reextracted twice with 300 μL water-saturated n-butanol.
SP:SAMPLEPREP_SUMMARY            	The organic phases were pooled and dried in a vacuum concentrator.
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_SUMMARY        	Liquid chromatography on a HILIC Column
CH:CHROMATOGRAPHY_TYPE           	HILIC
CH:INSTRUMENT_NAME               	Shimadzu Prominence UFPLC xr system
CH:COLUMN_NAME                   	Kinetex ( 2.6lm, 2.1 × 50 mm2)
CH:METHODS_FILENAME              	laura_capolupo_20210210_062234_PR_CH_Chromatography_methods.pdf
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
AN:ANALYSIS_PROTOCOL_FILE        	laura_capolupo_20210210_062234_PR_MS_MS_methods.pdf
#MS
MS:INSTRUMENT_NAME               	Hybrid Orbitrap Elite
MS:INSTRUMENT_TYPE               	Orbitrap
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	POSITIVE
MS:MS_COMMENTS                   	Check protocol file
MS:MS_RESULTS_FILE               	ST001691_AN002761_Results.txt	UNITS:abundance	Has m/z:Yes	Has RT:Yes	RT units:Minutes
#END