#METABOLOMICS WORKBENCH dcanals_20230530_130039 DATATRACK_ID:4057 STUDY_ID:ST002724 ANALYSIS_ID:AN004415 PROJECT_ID:PR001690
VERSION             	1
CREATED_ON             	June 6, 2023, 9:51 am
#PROJECT
PR:PROJECT_TITLE                 	Ceramides profile in HeLa overexpressing nSMase2 (SMPD3)
PR:PROJECT_TYPE                  	Lipidomics
PR:PROJECT_SUMMARY               	This study aim to determine which species of ceramides are modified by
PR:PROJECT_SUMMARY               	overexpression of the protein neutral sphingomyelinase 2 (nSMase2, gene SMPD3).
PR:PROJECT_SUMMARY               	HeLa cells were transfected with a plasmid containing V5-tagged nSMase2. Lipids
PR:PROJECT_SUMMARY               	were extracted after 24h after transfection and the sphingolipid profile was
PR:PROJECT_SUMMARY               	determined by LC-MS/MS.
PR:INSTITUTE                     	Stony Brook University
PR:DEPARTMENT                    	Medicine
PR:LABORATORY                    	Daniel Canals
PR:LAST_NAME                     	Canals
PR:FIRST_NAME                    	Daniel
PR:ADDRESS                       	100 Nicolls Road, Stony Brook, NY, 11794, USA
PR:EMAIL                         	daniel.canals@stonybrookmedicine.edu
PR:PHONE                         	6312162903
#STUDY
ST:STUDY_TITLE                   	Ceramides profile in HeLa overexpressing nSMase2 (SMPD3)
ST:STUDY_TYPE                    	Lipidomics LC-MS/MS
ST:STUDY_SUMMARY                 	This study aim to determine which species of ceramides are modified by
ST:STUDY_SUMMARY                 	overexpression of the protein neutral sphingomyelinase 2 (nSMase2, gene SMPD3).
ST:STUDY_SUMMARY                 	HeLa cells were transfected with a plasmid containing V5-tagged nSMase2. Lipids
ST:STUDY_SUMMARY                 	were extracted after 24h after transfection and the sphingolipid profile was
ST:STUDY_SUMMARY                 	determined by LC-MS/MS.
ST:INSTITUTE                     	State University of New York
ST:DEPARTMENT                    	MEdicine
ST:LABORATORY                    	Daniel Canals
ST:LAST_NAME                     	Daniel
ST:FIRST_NAME                    	Canals
ST:ADDRESS                       	100 Nicolls Road
ST:EMAIL                         	daniel.canals@stonybrook.edu
ST:PHONE                         	6312162903
#SUBJECT
SU:SUBJECT_TYPE                  	Cultured cells
SU:SUBJECT_SPECIES               	Homo sapiens
SU:TAXONOMY_ID                   	9606
SU:GENDER                        	Female
#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           	-	AO_01	Expression vector:Control	RAW_FILE_NAME=AO_01.mzML
SUBJECT_SAMPLE_FACTORS           	-	AO_02	Expression vector:Control	RAW_FILE_NAME=AO_02.mzML
SUBJECT_SAMPLE_FACTORS           	-	AO_03	Expression vector:Control	RAW_FILE_NAME=AO_03.mzML
SUBJECT_SAMPLE_FACTORS           	-	AO_13	Expression vector:nSMase2-V5	RAW_FILE_NAME=AO_13.mzML
SUBJECT_SAMPLE_FACTORS           	-	AO_14	Expression vector:nSMase2-V5	RAW_FILE_NAME=AO_14.mzML
SUBJECT_SAMPLE_FACTORS           	-	AO_15	Expression vector:nSMase2-V5	RAW_FILE_NAME=AO_15.mzML
#COLLECTION
CO:COLLECTION_SUMMARY            	ATCC HeLa - CCL-2
CO:SAMPLE_TYPE                   	Cultured cells
#TREATMENT
TR:TREATMENT_SUMMARY             	Transfection with SPMD3 gene
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Ceramides were extracted using 2:3 70% Isopropanol: EthylAcetate. See
SP:SAMPLEPREP_SUMMARY            	protocol.docx for details.
SP:PROCESSING_STORAGE_CONDITIONS 	-20℃
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	Thermo Vanquish
CH:COLUMN_NAME                   	Spectra C8SR (150 × 3.0 mm, 3um) (#S-3C8SR-FJ)
CH:SOLVENT_A                     	100% water; 0.2% formic acid ; 1mM ammonium formate
CH:SOLVENT_B                     	100% methanol; 0.2% formic acid ; 1mM ammonium formate
CH:FLOW_GRADIENT                 	0.5ml/min. 70%B. 5 min 90%B, 17 min 99%B, 26min 99%B, 26.5min 70% B. Stop at
CH:FLOW_GRADIENT                 	35min.
CH:FLOW_RATE                     	0.5ml/min
CH:COLUMN_TEMPERATURE            	37
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Thermo Quantiva
MS:INSTRUMENT_TYPE               	Triple quadrupole
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	POSITIVE
MS:MS_COMMENTS                   	Acquisition and Processing were done using TraceFinder Software.
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS	pmols
MS_METABOLITE_DATA_START
Samples	AO_01	AO_02	AO_03	AO_13	AO_14	AO_15
Factors	Expression vector:Control	Expression vector:Control	Expression vector:Control	Expression vector:nSMase2-V5	Expression vector:nSMase2-V5	Expression vector:nSMase2-V5
C12-Cer	0.24	0.21	0.33	0.81	0.75	0.61
C14-Cer_x	5.8	5.06	4.65	21.98	20.07	13.74
C16-Cer_x	40.6	35.07	32.19	143.59	130.61	91.34
C18:1-Cer_x	1.95	1.44	1.42	5.46	5.19	3.88
C18-Cer_x	7.25	6.47	6.27	15.34	13.9	10.95
C20:1-Cer	0.95	0.75	0.75	3.28	3.03	2.28
C20-Cer_x	5.08	4.29	4.52	11.81	11.41	8.74
C22:1-Cer	4.82	4.4	4.3	25.93	23.32	17.44
C22-Cer_x	17.15	16.22	15.66	51.47	46.47	36.32
C24:1-Cer_x	81.88	75.68	75.94	290.75	266.38	214.13
C24-Cer_x	23.49	22.47	23.6	61.3	58.2	47.46
C26:1-Cer	4.08	4.07	4.07	8.72	8.45	7.8
C26-Cer	0.01	-0.05	-0.07	0.35	0.3	0.21
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	RetentionTime	m/z	PubChemID
C12-Cer	12.21	482.5	5283562
C14-Cer_x	13.54	510.5	5282310
C16-Cer_x	14.93	538.5	2498
C18:1-Cer_x	15.3	564.4	5283563
C18-Cer_x	16.3	566.5	5283565
C20:1-Cer	16.6	592.5	249807232
C20-Cer_x	17.6	594.5	5283566  
C22:1-Cer	17.9	620.6	154573071  
C22-Cer_x	18.8	622.6	5283567  
C24:1-Cer_x	19.13	648.7	5283568 
C24-Cer_x	20.18	650.7	5283571
C26:1-Cer	20.3	676.8	154573078  
C26-Cer	20.36	678.7	5283570 
METABOLITES_END
#END