#METABOLOMICS WORKBENCH jgengatharan_20240730_132509 DATATRACK_ID:5053 STUDY_ID:ST003371 ANALYSIS_ID:AN005523 PROJECT_ID:PR002085 VERSION 1 CREATED_ON July 30, 2024, 1:52 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_C30Lip1_oleicd9_1 Sample source:Huh7 cells | Treatment:BSA-oleate-d9 RAW_FILE_NAME(Raw file name)=Huh7_C30Lip1_oleicd9_1.d SUBJECT_SAMPLE_FACTORS - Huh7_C30Lip1_oleicd9_2 Sample source:Huh7 cells | Treatment:BSA-oleate-d9 RAW_FILE_NAME(Raw file name)=Huh7_C30Lip1_oleicd9_2.d SUBJECT_SAMPLE_FACTORS - Huh7_C30Lip1_oleicd9_3 Sample source:Huh7 cells | Treatment:BSA-oleate-d9 RAW_FILE_NAME(Raw file name)=Huh7_C30Lip1_oleicd9_3.d SUBJECT_SAMPLE_FACTORS - Huh7_C30Lip1_elaidicd17_1 Sample source:Huh7 cells | Treatment:BSA-elaidate-d17 RAW_FILE_NAME(Raw file name)=Huh7_C30Lip1_elaidicd17_1.d SUBJECT_SAMPLE_FACTORS - Huh7_C30Lip1_elaidicd17_2 Sample source:Huh7 cells | Treatment:BSA-elaidate-d17 RAW_FILE_NAME(Raw file name)=Huh7_C30Lip1_elaidicd17_2.d SUBJECT_SAMPLE_FACTORS - Huh7_C30Lip1_elaidicd17_3 Sample source:Huh7 cells | Treatment:BSA-elaidate-d17 RAW_FILE_NAME(Raw file name)=Huh7_C30Lip1_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_C30Lip1_oleicd9_1 Huh7_C30Lip1_oleicd9_2 Huh7_C30Lip1_oleicd9_3 Huh7_C30Lip1_elaidicd17_1 Huh7_C30Lip1_elaidicd17_2 Huh7_C30Lip1_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 LPC 18:1-d7 9007710 7871056 6511425 8186781 7278011 8064780 LPC 16:1-d17 0 0 0 340428 330371 318885 LPC 18:1-d17 0 0 0 2516108 2729106 2546011 LPC 18:1-d9 1606944 1689914 2223580 0 0 0 LPC 20:1-d9 12600 13421 18290 0 0 0 LPE 18:1-d7 978294 996634 624139 1001338 919329 936759 LPE 18:1-d17 0 0 0 114365 115650 117027 LPE 18:1-d9 49129 46689 30287 0 0 0 PC 15:0/18:1-d7 51162298 49584456 39608898 31516879 36170360 34235427 PC 32:1-d17 0 0 0 14002965 16256387 15122397 PC 32:2-d34 0 0 0 14739601 16220445 14765212 PC 32:1-d9 17210397 18691827 14855864 0 0 0 PC 32:2-d17 0 0 0 11229639 11777252 9503720 PC 34:1-d17 0 0 0 14937219 17961852 16745976 PC 34:1-d9 70705229 69489912 61454435 0 0 0 PC 34:2-d17 0 0 0 21493093 40514370 39985740 PC 34:2-d18 30265723 29254741 23556344 0 0 0 PC 34:2-d34 0 0 0 26386628 30376560 27406364 PC 34:2-d9 36829616 38415763 32327874 0 0 0 PC 36:1-d9 65164589 65287160 43688784 0 0 0 PC 36:2-d17 0 0 0 29078897 31826370 26972435 PC 36:2-d18 111143069 102962279 101095554 0 0 0 PC 36:2-d34 0 0 0 51531300 55858590 56737131 PC 36:2-d9 39047168 37592611 32400856 0 0 0 PC 36:3-d17 0 0 0 11264850 11467339 12963183 PC 36:3-d9 9693402 12221271 9642547 0 0 0 PE 15:0/18:1-d7 61768 67153 64822 126971 41889 159401 PE 34:2-d17 0 0 0 31132 33288 34801 PE 36:1-d9 18314 71102 11001 0 0 0 PE 36:2-d17 0 0 0 23273 53874 43313 PE 36:2-d18 181422 405830 294478 0 0 0 PE 36:2-d9 25137 26609 33944 0 0 0 PE 36:3-d17 0 0 0 27558 11133 30098 PE 38:2-d18 92098 112859 25112 0 0 0 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Precursor Ion Product Ion LPC 18:1-d7 529.4 184.1 LPC 16:1-d17 511.3 184.1 LPC 18:1-d17 539.3 184.1 LPC 18:1-d9 531.3 184.1 LPC 20:1-d9 559.3 184.1 LPE 18:1-d7 487.3 44.1 LPE 18:1-d17 497.3 44.1 LPE 18:1-d9 489.3 44.1 PC 15:0/18:1-d7 753.6 184.1 PC 32:1-d17 749.6 184.1 PC 32:2-d34 764.6 184.1 PC 32:1-d9 741.6 184.1 PC 32:2-d17 747.6 184.1 PC 34:1-d17 777.6 184.1 PC 34:1-d9 769.6 184.1 PC 34:2-d17 775.6 184.1 PC 34:2-d18 776.6 184.1 PC 34:2-d34 792.6 184.1 PC 34:2-d9 767.6 184.1 PC 36:1-d9 797.6 184.1 PC 36:2-d17 803.6 184.1 PC 36:2-d18 804.6 184.1 PC 36:2-d34 820.6 184.1 PC 36:2-d9 795.6 184.1 PC 36:3-d17 801.6 184.1 PC 36:3-d9 793.6 184.1 PE 15:0/18:1-d7 711.6 570.5 PE 34:2-d17 733.5 592.5 PE 36:1-d9 755.6 614.5 PE 36:2-d17 761.6 620.5 PE 36:2-d18 762.6 621.5 PE 36:2-d9 753.6 612.5 PE 36:3-d17 759.5 618.5 PE 38:2-d18 790.6 649.5 METABOLITES_END #END