#METABOLOMICS WORKBENCH jgengatharan_20240729_090646 DATATRACK_ID:5044 STUDY_ID:ST003369 ANALYSIS_ID:AN005574 PROJECT_ID:PR002085 VERSION 1 CREATED_ON July 29, 2024, 9:17 am #PROJECT PR:PROJECT_TITLE Altered sphingolipid biosynthetic flux and lipoprotein trafficking contribute to PR:PROJECT_TITLE trans fat-induced atherosclerosis PR:PROJECT_SUMMARY The goal of the project is to determine the role of sphingolipid metabolism in PR:PROJECT_SUMMARY atherosclerosis induced by dietary trans fat. We analyzed lipid metabolites in PR:PROJECT_SUMMARY Huh7 cells following various fatty acid treatments, with specific focus on cis PR:PROJECT_SUMMARY and trans unsaturated fatty acids. Additionally, we analyzed lipid metabolites PR:PROJECT_SUMMARY in plasma and liver of Ldlr-/- mice fed high-fat diets enriched in cis or trans PR:PROJECT_SUMMARY fatty acids in the presence or absence of myriocin, a pharmacological inhibitor PR:PROJECT_SUMMARY of SPT, the initial rate-limiting enzyme of sphingolipid biosynthesis. PR:INSTITUTE Salk Institute for Biological Studies PR:LAST_NAME Gengatharan PR:FIRST_NAME Jivani PR:ADDRESS 10010 N Torrey Pines Rd, La Jolla, CA, 92037, USA PR:EMAIL jivani14@gmail.com PR:PHONE (858) 453-4100 #STUDY ST:STUDY_TITLE Incorporation of oleate-d9 and elaidate-d17 in sphingolipids in Huh7 cells. ST:STUDY_SUMMARY We analyzed sphingolipids in Huh7 cells treated with BSA-oleate-d9 or ST:STUDY_SUMMARY BSA-elaidate-d17. ST:INSTITUTE Salk Institute for Biological Studies ST:LAST_NAME Gengatharan ST:FIRST_NAME Jivani ST:ADDRESS 10010 N Torrey Pines Rd, La Jolla, CA, 92037, USA ST:EMAIL jivani14@gmail.com ST:PHONE (858) 453-4100 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:CELL_STRAIN_DETAILS Huh7 #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 - Huh7cells_C8SL2_d9oleic1 Sample source:Huh7 cells | Treatment:BSA-oleate-d9 RAW_FILE_NAME(Raw file name)=Huh7cells_C8SL2_d9oleic1.d SUBJECT_SAMPLE_FACTORS - Huh7cells_C8SL2_d9oleic2 Sample source:Huh7 cells | Treatment:BSA-oleate-d9 RAW_FILE_NAME(Raw file name)=Huh7cells_C8SL2_d9oleic2.d SUBJECT_SAMPLE_FACTORS - Huh7cells_C8SL2_d9oleic3 Sample source:Huh7 cells | Treatment:BSA-oleate-d9 RAW_FILE_NAME(Raw file name)=Huh7cells_C8SL2_d9oleic3.d SUBJECT_SAMPLE_FACTORS - Huh7cells_C8SL2_d17elaidic1 Sample source:Huh7 cells | Treatment:BSA-elaidate-d17 RAW_FILE_NAME(Raw file name)=Huh7cells_C8SL2_d17elaidic1.d SUBJECT_SAMPLE_FACTORS - Huh7cells_C8SL2_d17elaidic2 Sample source:Huh7 cells | Treatment:BSA-elaidate-d17 RAW_FILE_NAME(Raw file name)=Huh7cells_C8SL2_d17elaidic2.d SUBJECT_SAMPLE_FACTORS - Huh7cells_C8SL2_d17elaidic3 Sample source:Huh7 cells | Treatment:BSA-elaidate-d17 RAW_FILE_NAME(Raw file name)=Huh7cells_C8SL2_d17elaidic3.d #COLLECTION CO:COLLECTION_SUMMARY Cells (~400,000 cells) were spiked with the following internal standards: 20 CO:COLLECTION_SUMMARY pmol sphinganine-d7 (Avanti Polar Lipids, Cat# 860658), deoxysphinganine-d3 CO:COLLECTION_SUMMARY (Avanti Polar Lipids, Cat# 860474), 100 pmol d18:0-d7/13:0 dihydroceramide CO:COLLECTION_SUMMARY (Avanti Polar Lipids, Cat# 330726), 200 pmol d18:1-d7/15:0 ceramide (Avanti CO:COLLECTION_SUMMARY Polar Lipids, Cat# 860681), 100 pmol d18:1-d7/15:0 glucosylceramide (Avanti CO:COLLECTION_SUMMARY Polar Lipids, Cat# 330729), 100 pmol d18:1-d7/15:0 lactosylceramide (Avanti CO:COLLECTION_SUMMARY Polar Lipids, Cat# 330727), 200 pmol sphingosine-d7 (Avanti Polar Lipids, Cat# CO:COLLECTION_SUMMARY 860657). Cells were scraped with 0.5 mL methanol and 0.5 mL H2O. CO:SAMPLE_TYPE Cultured cells #TREATMENT TR:TREATMENT_SUMMARY Huh7 cells were treated with 1) 100 µM BSA-oleate-d9 or 2) 100 µM TR:TREATMENT_SUMMARY BSA-elaidate-d17 for 48 hours in delipidated media. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Cells (~400,000 cells) were spiked with the following internal standards: 20 SP:SAMPLEPREP_SUMMARY pmol sphinganine-d7 (Avanti Polar Lipids, Cat# 860658), deoxysphinganine-d3 SP:SAMPLEPREP_SUMMARY (Avanti Polar Lipids, Cat# 860474), 100 pmol d18:0-d7/13:0 dihydroceramide SP:SAMPLEPREP_SUMMARY (Avanti Polar Lipids, Cat# 330726), 200 pmol d18:1-d7/15:0 ceramide (Avanti SP:SAMPLEPREP_SUMMARY Polar Lipids, Cat# 860681), 100 pmol d18:1-d7/15:0 glucosylceramide (Avanti SP:SAMPLEPREP_SUMMARY Polar Lipids, Cat# 330729), 100 pmol d18:1-d7/15:0 lactosylceramide (Avanti SP:SAMPLEPREP_SUMMARY Polar Lipids, Cat# 330727), 200 pmol sphingosine-d7 (Avanti Polar Lipids, Cat# SP:SAMPLEPREP_SUMMARY 860657). Cells were scraped with 0.5 mL methanol and 0.5 mL H2O. Homogenate SP:SAMPLEPREP_SUMMARY aliquot of 100 µL was taken to determine protein content using the BCA protein SP:SAMPLEPREP_SUMMARY assay (Thermo Fisher Scientific). The remaining homogenate was transferred to a SP:SAMPLEPREP_SUMMARY new Eppendorf tube and 1 mL chloroform was added. For plasma or media, 0.5 mL SP:SAMPLEPREP_SUMMARY methanol, 0.5 mL H2O, and 1 mL chloroform were added directly. Samples were SP:SAMPLEPREP_SUMMARY vortexed for 5 min and centrifuged for 5 min at 4 ˚C at 15,000g. The organic SP:SAMPLEPREP_SUMMARY phase was collected and 2 μL of formic acid was added to the remaining polar SP:SAMPLEPREP_SUMMARY phase which was re-extracted with 1 mL of chloroform. Combined organic phases SP:SAMPLEPREP_SUMMARY were dried under nitrogen. After dried extracts for cells were resuspended in 60 SP:SAMPLEPREP_SUMMARY µl Buffer B, 5 µL of sample was injected. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 1260 Infinity II CH:COLUMN_NAME Peeke Scientific Spectra C8SR (150 x 3.0 mm, 3μm) CH:SOLVENT_A 100% water; 0.2% formic acid; 2 mM ammonium formate CH:SOLVENT_B 100% methanol; 0.2% formic acid; 1 mM ammonium formate CH:FLOW_GRADIENT 0 min, 82% B; 3 min, 82% B; 4 min, 90% B; 18 min, 99% B; 25 min, 99% B; 27 min, CH:FLOW_GRADIENT 82% B; 30 min, 82% B CH:FLOW_RATE 0.5 ml/min CH:COLUMN_TEMPERATURE 40 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6460 QQQ MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS Agilent Masshunter #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Peak area MS_METABOLITE_DATA_START Samples Huh7cells_C8SL2_d9oleic1 Huh7cells_C8SL2_d9oleic2 Huh7cells_C8SL2_d9oleic3 Huh7cells_C8SL2_d17elaidic1 Huh7cells_C8SL2_d17elaidic2 Huh7cells_C8SL2_d17elaidic3 Factors Sample source:Huh7 cells | Treatment:BSA-oleate-d9 Sample source:Huh7 cells | Treatment:BSA-oleate-d9 Sample source:Huh7 cells | Treatment:BSA-oleate-d9 Sample source:Huh7 cells | Treatment:BSA-elaidate-d17 Sample source:Huh7 cells | Treatment:BSA-elaidate-d17 Sample source:Huh7 cells | Treatment:BSA-elaidate-d17 Lac-Cer d18:1-d7/15:0 13801 14258 15786 12552 18506 16908 Lac-Cer d20:2-d17/24:0 1640 1460 1016 Lac-Cer d20:2-d17/24:1-d17 865 1285 1050 SM d34:2-d34 336249 374139 318226 SM d36:2-d17 1766032 1869610 1724339 SM d36:2-d9 346181 323223 337772 SM d36:3-d17 195348 228413 189190 SM d36:3-d34 4461235 4929934 4503866 SM d38:2-d17 416198 465160 370086 SM d38:2-d9 250245 185249 216222 SM d38:3-d34 1717849 1341775 1790553 SM d40:2-d17 1254613 1442670 1213517 SM d40:2-d9 989574 900301 1011671 SM d40:3-d34 1171515 1254900 1100048 SM d42:2-d17 1682811 1964989 1689875 SM d42:2-d34 178456 191907 174251 SM d42:2-d9 10313030 10050197 11405818 SM d42:3-d17 517272 519762 490770 SM d42:3-d34 3444213 3661266 3314513 SM d42:3-d9 403057 283190 500805 SM d44:2-d9 458785 321691 674103 SM d44:3-d17 312567 283395 253109 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Precursor Ion Product Ion Lac-Cer d18:1-d7/15:0 855.7 271.2 Lac-Cer d20:2-d17/24:0 1017.7 307.4 Lac-Cer d20:2-d17/24:1-d17 1032.7 307.4 SM d34:2-d34 735.6 184 SM d36:2-d17 746.6 184 SM d36:2-d9 738.6 184 SM d36:3-d17 744.6 184 SM d36:3-d34 761.6 184 SM d38:2-d17 774.6 184 SM d38:2-d9 766.6 184 SM d38:3-d34 789.6 184 SM d40:2-d17 802.7 184 SM d40:2-d9 794.7 184 SM d40:3-d34 817.6 184 SM d42:2-d17 830.7 184 SM d42:2-d34 847.7 184 SM d42:2-d9 822.7 184 SM d42:3-d17 828.7 184 SM d42:3-d34 845.7 184 SM d42:3-d9 820.7 184 SM d44:2-d9 850.7 184 SM d44:3-d17 856.7 184 METABOLITES_END #END