#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