#METABOLOMICS WORKBENCH jgengatharan_20240729_084003 DATATRACK_ID:5043 STUDY_ID:ST003357 ANALYSIS_ID:AN005499 PROJECT_ID:PR002085 VERSION 1 CREATED_ON July 29, 2024, 9:19 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_C8SL1_palm1 Sample source:Huh7 cells | Treatment:BSA-palmitate RAW_FILE_NAME(Raw file name)=Huh7cells_C8SL1_palm1.d SUBJECT_SAMPLE_FACTORS - Huh7cells_C8SL1_palm2 Sample source:Huh7 cells | Treatment:BSA-palmitate RAW_FILE_NAME(Raw file name)=Huh7cells_C8SL1_palm2.d SUBJECT_SAMPLE_FACTORS - Huh7cells_C8SL1_palm3 Sample source:Huh7 cells | Treatment:BSA-palmitate RAW_FILE_NAME(Raw file name)=Huh7cells_C8SL1_palm3.d SUBJECT_SAMPLE_FACTORS - Huh7cells_C8SL1_stear1 Sample source:Huh7 cells | Treatment:BSA-stearate RAW_FILE_NAME(Raw file name)=Huh7cells_C8SL1_stear1.d SUBJECT_SAMPLE_FACTORS - Huh7cells_C8SL1_stear2 Sample source:Huh7 cells | Treatment:BSA-stearate RAW_FILE_NAME(Raw file name)=Huh7cells_C8SL1_stear2.d SUBJECT_SAMPLE_FACTORS - Huh7cells_C8SL1_stear3 Sample source:Huh7 cells | Treatment:BSA-stearate RAW_FILE_NAME(Raw file name)=Huh7cells_C8SL1_stear3.d SUBJECT_SAMPLE_FACTORS - Huh7cells_C8SL1_d9oleic1 Sample source:Huh7 cells | Treatment:BSA-oleate-d9 RAW_FILE_NAME(Raw file name)=Huh7cells_C8SL1_d9oleic1.d SUBJECT_SAMPLE_FACTORS - Huh7cells_C8SL1_d9oleic2 Sample source:Huh7 cells | Treatment:BSA-oleate-d9 RAW_FILE_NAME(Raw file name)=Huh7cells_C8SL1_d9oleic2.d SUBJECT_SAMPLE_FACTORS - Huh7cells_C8SL1_d9oleic3 Sample source:Huh7 cells | Treatment:BSA-oleate-d9 RAW_FILE_NAME(Raw file name)=Huh7cells_C8SL1_d9oleic3.d SUBJECT_SAMPLE_FACTORS - Huh7cells_C8SL1_d17elaidic1 Sample source:Huh7 cells | Treatment:BSA-elaidate-d17 RAW_FILE_NAME(Raw file name)=Huh7cells_C8SL1_d17elaidic1.d SUBJECT_SAMPLE_FACTORS - Huh7cells_C8SL1_d17elaidic2 Sample source:Huh7 cells | Treatment:BSA-elaidate-d17 RAW_FILE_NAME(Raw file name)=Huh7cells_C8SL1_d17elaidic2.d SUBJECT_SAMPLE_FACTORS - Huh7cells_C8SL1_d17elaidic3 Sample source:Huh7 cells | Treatment:BSA-elaidate-d17 RAW_FILE_NAME(Raw file name)=Huh7cells_C8SL1_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_C8SL1_palm1 Huh7cells_C8SL1_palm2 Huh7cells_C8SL1_palm3 Huh7cells_C8SL1_stear1 Huh7cells_C8SL1_stear2 Huh7cells_C8SL1_stear3 Huh7cells_C8SL1_d9oleic1 Huh7cells_C8SL1_d9oleic2 Huh7cells_C8SL1_d9oleic3 Huh7cells_C8SL1_d17elaidic1 Huh7cells_C8SL1_d17elaidic2 Huh7cells_C8SL1_d17elaidic3 Factors Sample source:Huh7 cells | Treatment:BSA-palmitate Sample source:Huh7 cells | Treatment:BSA-palmitate Sample source:Huh7 cells | Treatment:BSA-palmitate Sample source:Huh7 cells | Treatment:BSA-stearate Sample source:Huh7 cells | Treatment:BSA-stearate Sample source:Huh7 cells | Treatment:BSA-stearate 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 Cer d18:1-d7/15:0 525218 446725 411765 454416 440945 471932 454226 408923 454826 461643 569454 442282 Cer d18:1/16:0 91900 76303 79211 62743 68424 69261 28903 29272 33637 6041 7614 7929 Cer d18:1/16:1-d17 3187 4774 2689 Cer d18:1/18:0 17185 9105 7574 54346 56516 52494 1034 Cer d18:1/18:1-d17 2963 4890 2022 Cer d18:1/20:0 17170 19540 13751 17746 14730 12407 2144 1389 2562 Cer d18:1/20:1-d17 1748 1539 Cer d18:1/22:0 203530 174340 77639 54524 70181 74347 9015 8578 12692 5384 7340 3117 Cer d18:1/22:1 11465 17583 5928 1732 2029 1750 1809 Cer d18:1/22:1-d17 6506 4967 5609 Cer d18:1/23:0 25162 30382 9869 5378 5831 10701 4998 3140 5929 2455 2986 1625 Cer d18:1/24:0 331322 336022 194693 136598 152435 151544 58148 41074 82173 19551 22573 19344 Cer d18:1/24:1 231763 233087 110529 42861 62022 57637 24019 26460 39003 23627 25053 20045 Cer d18:1/24:1-d17 49327 40753 30917 Cer d18:1/24:1-d9 138545 99638 171543 Cer d18:1/25:0 13386 10629 3211 2971 2459 5148 2783 1026 4763 2299 1516 1430 Cer d18:1/26:0 13687 11494 5895 3649 3791 5081 2590 3924 1826 2013 1640 Cer d18:1/26:1 25941 27031 22252 4064 5061 5063 4555 1976 5411 1093 1607 2276 Cer d18:1/26:1-d17 2609 2250 1225 Cer d18:1/26:1-d9 10424 6073 10610 Cer d20:1/18:0 61582 36622 34790 Cer d20:1/20:0 7060 13253 15594 Cer d20:1/22:0 9361 9037 6203 97100 111614 122585 Cer d20:1/23:0 1526 5733 13957 6828 Cer d20:1/24:0 22791 11648 7492 82755 130853 186253 3542 8957 2521 2150 2079 Cer d20:1/24:1 13554 10444 6950 61146 65945 66184 3843 1387 5145 2356 2169 Cer d20:1/24:1-d17 2004 2799 1482 Cer d20:1/24:1-d9 13947 9106 22442 Cer d20:1/25:0 1006 1591 1821 1977 4020 1015 Cer d20:1/26:0 1744 2189 2940 4272 4233 2058 1526 1535 Cer d20:1/26:1 1595 2235 1863 1495 1351 2017 1761 Cer d20:2-d17/18:1-d17 1543 1030 1369 Cer d20:2-d17/22:1-d17 3736 2654 2674 Cer d20:2-d17/24:0 5386 4546 4434 Cer d20:2-d17/24:1 4778 6219 4778 Cer d20:2-d17/24:1-d17 12672 8356 13870 Gluc-Cer d18:1-d7/15:0 63672 14881 13286 27582 27410 29492 29800 66073 67746 62661 96031 53725 Hex-Cer d20:2-d17/24:0 1263 1818 1088 Hex-Cer d20:2-d17/24:1-d17 1898 1108 1007 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Precusor Ion Product Ion Cer d18:1-d7/15:0 531.5 271.4 Cer d18:1/16:0 538.5 264.4 Cer d18:1/16:1-d17 553.5 264.4 Cer d18:1/18:0 566.6 264.4 Cer d18:1/18:1-d17 581.5 264.4 Cer d18:1/20:0 594.6 264.4 Cer d18:1/20:1-d17 609.6 264.4 Cer d18:1/22:0 622.6 264.4 Cer d18:1/22:1 620.6 264.4 Cer d18:1/22:1-d17 637.6 264.4 Cer d18:1/23:0 636.6 264.4 Cer d18:1/24:0 650.6 264.4 Cer d18:1/24:1 648.6 264.4 Cer d18:1/24:1-d17 665.6 264.4 Cer d18:1/24:1-d9 657.6 264.4 Cer d18:1/25:0 664.7 264.4 Cer d18:1/26:0 678.7 264.4 Cer d18:1/26:1 676.7 264.4 Cer d18:1/26:1-d17 693.7 264.4 Cer d18:1/26:1-d9 685.7 264.4 Cer d20:1/18:0 594.6 292.4 Cer d20:1/20:0 622.6 292.4 Cer d20:1/22:0 650.6 292.4 Cer d20:1/23:0 664.7 292.4 Cer d20:1/24:0 678.7 292.4 Cer d20:1/24:1 676.7 292.4 Cer d20:1/24:1-d17 693.7 292.4 Cer d20:1/24:1-d9 685.7 292.4 Cer d20:1/25:0 692.7 292.4 Cer d20:1/26:0 706.7 292.4 Cer d20:1/26:1 704.7 292.4 Cer d20:2-d17/18:1-d17 624.6 307.4 Cer d20:2-d17/22:1-d17 680.6 307.4 Cer d20:2-d17/24:0 693.6 307.4 Cer d20:2-d17/24:1 691.6 307.4 Cer d20:2-d17/24:1-d17 708.6 307.4 Gluc-Cer d18:1-d7/15:0 693.6 271.2 Hex-Cer d20:2-d17/24:0 855.7 307.4 Hex-Cer d20:2-d17/24:1-d17 870.7 307.4 METABOLITES_END #END