#METABOLOMICS WORKBENCH jgengatharan_20240730_140317 DATATRACK_ID:5056 STUDY_ID:ST003359 ANALYSIS_ID:AN005501 PROJECT_ID:PR002085 VERSION 1 CREATED_ON July 30, 2024, 2:13 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 Secretion of phosphatidylcholine from Huh7 cells treated with oleate-d9 or ST:STUDY_TITLE elaidate-d17 ST:STUDY_SUMMARY We analyzed PC in the fresh and spent media of Huh7 cells treated with ST:STUDY_SUMMARY BSA-oleate-d9 or BSA-elaidate-d17 to determine their incorporation into secreted ST:STUDY_SUMMARY phospholipids. We aimed to determine if SM secretory flux in response to fatty ST:STUDY_SUMMARY acid treatments correlated to PC secretory flux. 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 - Huh7freshmedia_C30Lip1_oleicd9_1 Sample source:Huh7 cells | Condition:BSA-oleate-d9 fresh media RAW_FILE_NAME(Raw file name)=Huh7freshmedia_C30Lip1_oleicd9_1.d SUBJECT_SAMPLE_FACTORS - Huh7freshmedia_C30Lip1_oleicd9_2 Sample source:Huh7 cells | Condition:BSA-oleate-d9 fresh media RAW_FILE_NAME(Raw file name)=Huh7freshmedia_C30Lip1_oleicd9_2.d SUBJECT_SAMPLE_FACTORS - Huh7freshmedia_C30Lip1_oleicd9_3 Sample source:Huh7 cells | Condition:BSA-oleate-d9 fresh media RAW_FILE_NAME(Raw file name)=Huh7freshmedia_C30Lip1_oleicd9_3.d SUBJECT_SAMPLE_FACTORS - Huh7freshmedia_C30Lip1_elaidicd17_1 Sample source:Huh7 cells | Condition:BSA-elaidate-d17 fresh media RAW_FILE_NAME(Raw file name)=Huh7freshmedia_C30Lip1_elaidicd17_1.d SUBJECT_SAMPLE_FACTORS - Huh7freshmedia_C30Lip1_elaidicd17_2 Sample source:Huh7 cells | Condition:BSA-elaidate-d17 fresh media RAW_FILE_NAME(Raw file name)=Huh7freshmedia_C30Lip1_elaidicd17_2.d SUBJECT_SAMPLE_FACTORS - Huh7freshmedia_C30Lip1_elaidicd17_3 Sample source:Huh7 cells | Condition:BSA-elaidate-d17 fresh media RAW_FILE_NAME(Raw file name)=Huh7freshmedia_C30Lip1_elaidicd17_3.d SUBJECT_SAMPLE_FACTORS - Huh7spentmedia_C30Lip1_oleicd9_1 Sample source:Huh7 cells | Condition:BSA-oleate-d9 spent media RAW_FILE_NAME(Raw file name)=Huh7spentmedia_C30Lip1_oleicd9_1.d SUBJECT_SAMPLE_FACTORS - Huh7spentmedia_C30Lip1_oleicd9_2 Sample source:Huh7 cells | Condition:BSA-oleate-d9 spent media RAW_FILE_NAME(Raw file name)=Huh7spentmedia_C30Lip1_oleicd9_2.d SUBJECT_SAMPLE_FACTORS - Huh7spentmedia_C30Lip1_oleicd9_3 Sample source:Huh7 cells | Condition:BSA-oleate-d9 spent media RAW_FILE_NAME(Raw file name)=Huh7spentmedia_C30Lip1_oleicd9_3.d SUBJECT_SAMPLE_FACTORS - Huh7spentmedia_C30Lip1_elaidicd17_1 Sample source:Huh7 cells | Condition:BSA-elaidate-d17 spent media RAW_FILE_NAME(Raw file name)=Huh7spentmedia_C30Lip1_elaidicd17_1.d SUBJECT_SAMPLE_FACTORS - Huh7spentmedia_C30Lip1_elaidicd17_2 Sample source:Huh7 cells | Condition:BSA-elaidate-d17 spent media RAW_FILE_NAME(Raw file name)=Huh7spentmedia_C30Lip1_elaidicd17_2.d SUBJECT_SAMPLE_FACTORS - Huh7spentmedia_C30Lip1_elaidicd17_3 Sample source:Huh7 cells | Condition:BSA-elaidate-d17 spent media RAW_FILE_NAME(Raw file name)=Huh7spentmedia_C30Lip1_elaidicd17_3.d #COLLECTION CO:COLLECTION_SUMMARY 0.75 ml of fresh media or 1.5 ml of spent media was evaporated under vacuum at CO:COLLECTION_SUMMARY 4°C and resuspended into 0.1 mL of 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 Media was spiked with 1 µg of each of the following internal standards: SP:SAMPLEPREP_SUMMARY 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). 0.5 mL methanol, 0.5 mL H2O, SP:SAMPLEPREP_SUMMARY and 1 mL chloroform were added directly. Samples were vortexed for 5 min and SP:SAMPLEPREP_SUMMARY centrifuged for 5 min at 4 ˚C at 15,000g. The organic phase was collected and 2 SP:SAMPLEPREP_SUMMARY μL of formic acid was added to the remaining polar phase which was re-extracted SP:SAMPLEPREP_SUMMARY with 1 mL of chloroform. Combined organic phases were dried under nitrogen. SP:SAMPLEPREP_SUMMARY After dried extracts were resuspended in 60 µl 65:30:5 ACN: IPA:H2O, 5 µL of SP:SAMPLEPREP_SUMMARY sample was 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 Huh7freshmedia_C30Lip1_oleicd9_1 Huh7freshmedia_C30Lip1_oleicd9_2 Huh7freshmedia_C30Lip1_oleicd9_3 Huh7freshmedia_C30Lip1_elaidicd17_1 Huh7freshmedia_C30Lip1_elaidicd17_2 Huh7freshmedia_C30Lip1_elaidicd17_3 Huh7spentmedia_C30Lip1_oleicd9_1 Huh7spentmedia_C30Lip1_oleicd9_2 Huh7spentmedia_C30Lip1_oleicd9_3 Huh7spentmedia_C30Lip1_elaidicd17_1 Huh7spentmedia_C30Lip1_elaidicd17_2 Huh7spentmedia_C30Lip1_elaidicd17_3 Factors Sample source:Huh7 cells | Condition:BSA-oleate-d9 fresh media Sample source:Huh7 cells | Condition:BSA-oleate-d9 fresh media Sample source:Huh7 cells | Condition:BSA-oleate-d9 fresh media Sample source:Huh7 cells | Condition:BSA-elaidate-d17 fresh media Sample source:Huh7 cells | Condition:BSA-elaidate-d17 fresh media Sample source:Huh7 cells | Condition:BSA-elaidate-d17 fresh media Sample source:Huh7 cells | Condition:BSA-oleate-d9 spent media Sample source:Huh7 cells | Condition:BSA-oleate-d9 spent media Sample source:Huh7 cells | Condition:BSA-oleate-d9 spent media Sample source:Huh7 cells | Condition:BSA-elaidate-d17 spent media Sample source:Huh7 cells | Condition:BSA-elaidate-d17 spent media Sample source:Huh7 cells | Condition:BSA-elaidate-d17 spent media PC 15:0/18:1-d7 81383175 88835637 84729706 83389915 83315693 80814374 93636587 106897335 111767788 76636397 84444281 83050299 PC 32:1-d17 0 0 0 1901143 1898421 1729688 PC 32:1-d9 1696404 1218549 1974406 0 0 0 PC 32:2-d17 0 0 0 840438 827319 789921 PC 32:2-d18 172943 166300 154317 0 0 0 PC 32:2-d34 0 0 0 1767353 1632725 1682575 PC 32:2-d9 317118 360388 364530 0 0 0 PC 34:1-d17 0 0 0 2396877 3025871 2895191 PC 34:1-d9 11852845 10533606 11248589 0 0 0 PC 34:2-d17 0 0 0 2643543 2602482 2645455 PC 34:2-d18 1674879 2009714 1793939 0 0 0 PC 34:2-d34 0 0 0 1105298 1693059 1314481 PC 34:2-d9 2243929 2035276 1732967 0 0 0 PC 34:3-d17 0 0 0 138640 71935 136765 PC 34:3-d9 145382 109613 137895 0 0 0 PC 36:1-d17 0 0 0 228178 210754 265548 PC 36:1-d9 3139082 3280142 2802272 0 0 0 PC 36:2-d17 0 0 0 4870165 4855987 3965524 PC 36:2-d18 28328604 27455902 31697975 0 0 0 PC 36:2-d34 0 0 0 8456316 7961174 9109602 PC 36:2-d9 6240945 5626160 6220206 0 0 0 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Precursor Ion Product Ion PC 15:0/18:1-d7 753.6 184.1 PC 32:1-d17 749.6 184.1 PC 32:1-d9 741.6 184.1 PC 32:2-d17 747.6 184.1 PC 32:2-d18 748.6 184.1 PC 32:2-d34 764.6 184.1 PC 32:2-d9 739.6 184.1 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 34:3-d17 773.6 184.1 PC 34:3-d9 765.6 184.1 PC 36:1-d17 805.6 184.1 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 METABOLITES_END #END