#METABOLOMICS WORKBENCH jgengatharan_20240730_135434 DATATRACK_ID:5055 STUDY_ID:ST003372 ANALYSIS_ID:AN005524 PROJECT_ID:PR002085 VERSION 1 CREATED_ON July 30, 2024, 1:58 pm #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 broader lipidome of Huh7 cells. ST:STUDY_SUMMARY We analyzed several lipid classes including LPC, LPE, PC, PE, DG, and TG in Huh7 ST:STUDY_SUMMARY cells treated with BSA-oleate-d9 or BSA-elaidate-d17 to determine their ST:STUDY_SUMMARY incorporation into the broader lipidome. 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 - Huh7_C30Lip2_oleicd9_1 Sample source:Huh7 cells | Treatment:BSA-oleate-d9 RAW_FILE_NAME(Raw file name)=Huh7_C30Lip2_oleicd9_1.d SUBJECT_SAMPLE_FACTORS - Huh7_C30Lip2_oleicd9_2 Sample source:Huh7 cells | Treatment:BSA-oleate-d9 RAW_FILE_NAME(Raw file name)=Huh7_C30Lip2_oleicd9_2.d SUBJECT_SAMPLE_FACTORS - Huh7_C30Lip2_oleicd9_3 Sample source:Huh7 cells | Treatment:BSA-oleate-d9 RAW_FILE_NAME(Raw file name)=Huh7_C30Lip2_oleicd9_3.d SUBJECT_SAMPLE_FACTORS - Huh7_C30Lip2_elaidicd17_1 Sample source:Huh7 cells | Treatment:BSA-elaidate-d17 RAW_FILE_NAME(Raw file name)=Huh7_C30Lip2_elaidicd17_1.d SUBJECT_SAMPLE_FACTORS - Huh7_C30Lip2_elaidicd17_2 Sample source:Huh7 cells | Treatment:BSA-elaidate-d17 RAW_FILE_NAME(Raw file name)=Huh7_C30Lip2_elaidicd17_2.d SUBJECT_SAMPLE_FACTORS - Huh7_C30Lip2_elaidicd17_3 Sample source:Huh7 cells | Treatment:BSA-elaidate-d17 RAW_FILE_NAME(Raw file name)=Huh7_C30Lip2_elaidicd17_3.d #COLLECTION CO:COLLECTION_SUMMARY Cells (~400,000 cells)were spiked with 1 µg of each of the following internal CO:COLLECTION_SUMMARY standards: 15:0-18:1(d7) phosphatidylcholine (Avanti Polar Lipids, Cat #791637), CO:COLLECTION_SUMMARY 15:0-18:1(d7) phosphatidylethanolamine (Avanti Polar Lipids, Cat #791638), CO:COLLECTION_SUMMARY 18:1(d7) lysophosphatidylcholine (Avanti Polar Lipids, Cat#791643), 18:1(d7) CO:COLLECTION_SUMMARY lysophosphatidylethanolamine (Avanti Polar Lipids, Cat #791644), 15:0-18:1(d7) CO:COLLECTION_SUMMARY diacylglycerol (Avanti Polar Lipids, Cat #791647), 15:0-18:1(d7)-15:0 CO:COLLECTION_SUMMARY triacylglycerol (Avanti Polar Lipids, Cat #791648). Cells were scraped with 0.5 CO:COLLECTION_SUMMARY 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 1 µg of each of the following internal SP:SAMPLEPREP_SUMMARY standards: 15:0-18:1(d7) phosphatidylcholine (Avanti Polar Lipids, Cat #791637), SP:SAMPLEPREP_SUMMARY 15:0-18:1(d7) phosphatidylethanolamine (Avanti Polar Lipids, Cat #791638), SP:SAMPLEPREP_SUMMARY 18:1(d7) lysophosphatidylcholine (Avanti Polar Lipids, Cat#791643), 18:1(d7) SP:SAMPLEPREP_SUMMARY lysophosphatidylethanolamine (Avanti Polar Lipids, Cat #791644), 15:0-18:1(d7) SP:SAMPLEPREP_SUMMARY diacylglycerol (Avanti Polar Lipids, Cat #791647), 15:0-18:1(d7)-15:0 SP:SAMPLEPREP_SUMMARY triacylglycerol (Avanti Polar Lipids, Cat #791648). Cells were scraped with 0.5 SP:SAMPLEPREP_SUMMARY mL methanol and 0.5 mL H2O. Homogenate aliquot of 100 µL was taken to determine SP:SAMPLEPREP_SUMMARY protein content using the BCA protein assay (Thermo Fisher Scientific). The SP:SAMPLEPREP_SUMMARY remaining homogenate was transferred to a new Eppendorf tube and 1 mL chloroform SP:SAMPLEPREP_SUMMARY was added. For media, 0.5 mL methanol, 0.5 mL H2O, and 1 mL chloroform were SP:SAMPLEPREP_SUMMARY added directly. Samples were vortexed for 5 min and centrifuged for 5 min at 4 SP:SAMPLEPREP_SUMMARY ˚C at 15,000g. The organic phase was collected and 2 μL of formic acid was SP:SAMPLEPREP_SUMMARY added to the remaining polar phase which was re-extracted with 1 mL of SP:SAMPLEPREP_SUMMARY chloroform. Combined organic phases were dried under nitrogen. After dried SP:SAMPLEPREP_SUMMARY extracts were resuspended in 60 µl 65:30:5 ACN: IPA:H2O, 5 µL of sample was SP:SAMPLEPREP_SUMMARY injected. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 1260 Infinity II CH:COLUMN_NAME Thermo Accucore C30 (150 x 2.1mm,2.6um) CH:SOLVENT_A 60% acetonitrile, 40% water; 0.1% formic acid; 10 mM ammonium formate CH:SOLVENT_B 90% isopropanol, 10% acetonitrile; 0.1% formic acid; 10 mM ammonium formate CH:FLOW_GRADIENT 0 min, 30% B; 3 min, 30% B; 8 min, 43% B; 9 min, 50% B; 18 min, 90% B; 26 min, CH:FLOW_GRADIENT 99% B; 30 min, 99%B; 36 min, 30% B CH:FLOW_RATE 0 min, 0.1 ml/min; 8 min, 0.2 ml/min CH:COLUMN_TEMPERATURE 40˚C #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 Lipid species were analyzed by multiple reaction monitoring of the transition MS:MS_COMMENTS from precursor to product ions at associated optimized collision energies, and MS:MS_COMMENTS fragmentor voltages using Agilent Masshunter. The m/z values of the precursor MS:MS_COMMENTS and product ions are provided in the metabolite metadata section. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Peak area MS_METABOLITE_DATA_START Samples Huh7_C30Lip2_oleicd9_1 Huh7_C30Lip2_oleicd9_2 Huh7_C30Lip2_oleicd9_3 Huh7_C30Lip2_elaidicd17_1 Huh7_C30Lip2_elaidicd17_2 Huh7_C30Lip2_elaidicd17_3 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 DG 15:0/18:1-d7 30737 10728 22411 9898 21890 45925 DG 18:1-d17_18:1 0 0 0 2736 1490 5415 DG 18:1-d17_18:1-d17 0 0 0 20113 18407 14428 DG 18:1-d9_18:1 12917 11206 9265 0 0 0 DG 18:1-d9_18:1-d9 37964 29442 30177 0 0 0 TG 15:0/18:1-d7/15:0 277268 200036 51463 52941 154999 173346 TG 50:2-d34 (dC32:1-d17) 0 0 0 839525 1427547 813922 TG 52:2-d18 (dC34:1-d9) 827266 879628 752650 0 0 0 TG 52:2-d34 (dC34:1-d17) 0 0 0 84472 1093279 101593 TG 52:3-d18 (dC34:2-d9) 614823 595128 597463 0 0 0 TG 52:3-d27 (dC34:2-d18) 403701 403150 445166 0 0 0 TG 52:3-d34 (dC34:2-d17) 0 0 0 835710 2288528 2271478 TG 52:3-d51 (dC34:2-d34) 0 0 0 217848 1036289 604686 TG 54:2-d34 (dC36:1-d17) 0 0 0 19193 574518 33673 TG 54:3-d18 (dC36:2-d9) 770524 857878 932934 0 0 0 TG 54:3-d27 (dC36:2-d18) 10200762 3318728 2527687 0 0 0 TG 54:3-d34 (dC36:2-d17) 0 0 0 948031 1759145 1497614 TG 54:3-d51 (dC36:2-d34) 0 0 0 336756 5626350 2246677 TG 54:4-d34 (dC36:3-d17) 0 0 0 313987 374057 421849 TG 56:2-d18 (dC38:1-d9) 659584 588723 442650 0 0 0 TG 56:3-d27 (dC38:2-d18) 676179 654568 646440 0 0 0 TG 56:3-d34 (dC38:2-d17) 0 0 0 645948 204624 139645 TG 56:3-d51 (dC38:2-d34) 0 0 0 94009 188847 42967 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Precursor Ion Product Ion DG 15:0/18:1-d7 605.6 299 DG 18:1-d17_18:1 655.5 356 DG 18:1-d17_18:1-d17 672.5 356 DG 18:1-d9_18:1 647.5 348 DG 18:1-d9_18:1-d9 656.5 348 TG 15:0/18:1-d7/15:0 829.8 523.5 TG 50:2-d34 (dC32:1-d17) 882.8 566.5 TG 52:2-d18 (dC34:1-d9) 894.8 586.5 TG 52:2-d34 (dC34:1-d17) 910.8 594.5 TG 52:3-d18 (dC34:2-d9) 892.8 584.5 TG 52:3-d27 (dC34:2-d18) 901.8 593.5 TG 52:3-d34 (dC34:2-d17) 908.8 592.5 TG 52:3-d51 (dC34:2-d34) 925.8 609.5 TG 54:2-d34 (dC36:1-d17) 938.9 622.6 TG 54:3-d18 (dC36:2-d9) 920.9 612.6 TG 54:3-d27 (dC36:2-d18) 929.9 621.6 TG 54:3-d34 (dC36:2-d17) 936.9 620.6 TG 54:3-d51 (dC36:2-d34) 953.9 637.6 TG 54:4-d34 (dC36:3-d17) 934.8 618.5 TG 56:2-d18 (dC38:1-d9) 950.9 642.5 TG 56:3-d27 (dC38:2-d18) 957.9 649.5 TG 56:3-d34 (dC38:2-d17) 964.9 648.5 TG 56:3-d51 (dC38:2-d34) 981.9 665.5 METABOLITES_END #END