#METABOLOMICS WORKBENCH jgengatharan_20240730_142932 DATATRACK_ID:5058 STUDY_ID:ST003374 ANALYSIS_ID:AN005526 PROJECT_ID:PR002085 VERSION 1 CREATED_ON July 30, 2024, 2:35 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 sphingolipids in Huh7 cells while ST:STUDY_TITLE modulating SPT flux. ST:STUDY_SUMMARY We analyzed sphingolipids in Huh7 cells treated with BSA-oleate-d9 or ST:STUDY_SUMMARY BSA-elaidate-d17 to determine their incorporation into the long-chain base (LCB) ST:STUDY_SUMMARY of intact sphingolipids. We validated their reduction in abundance through ST:STUDY_SUMMARY pharmacological inhibition of SPT with myriocin. 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_delip_SL2_Od9_1 Sample source:Huh7 cells | Treatment:BSA-oleate-d9 + DMSO RAW_FILE_NAME(Raw file name)=Huh7_delip_SL2_Od9_1.d SUBJECT_SAMPLE_FACTORS - Huh7_delip_SL2_Od9_2 Sample source:Huh7 cells | Treatment:BSA-oleate-d9 + DMSO RAW_FILE_NAME(Raw file name)=Huh7_delip_SL2_Od9_2.d SUBJECT_SAMPLE_FACTORS - Huh7_delip_SL2_Od9_3 Sample source:Huh7 cells | Treatment:BSA-oleate-d9 + DMSO RAW_FILE_NAME(Raw file name)=Huh7_delip_SL2_Od9_3.d SUBJECT_SAMPLE_FACTORS - Huh7_delip_SL2_Od9myr_1 Sample source:Huh7 cells | Treatment:BSA-oleate-d9 + myriocin RAW_FILE_NAME(Raw file name)=Huh7_delip_SL2_Od9myr_1.d SUBJECT_SAMPLE_FACTORS - Huh7_delip_SL2_Od9myr_2 Sample source:Huh7 cells | Treatment:BSA-oleate-d9 + myriocin RAW_FILE_NAME(Raw file name)=Huh7_delip_SL2_Od9myr_2.d SUBJECT_SAMPLE_FACTORS - Huh7_delip_SL2_Od9myr_3 Sample source:Huh7 cells | Treatment:BSA-oleate-d9 + myriocin RAW_FILE_NAME(Raw file name)=Huh7_delip_SL2_Od9myr_3.d SUBJECT_SAMPLE_FACTORS - Huh7_delip_SL2_Ed17_1 Sample source:Huh7 cells | Treatment:BSA-elaidate-d17 + DMSO RAW_FILE_NAME(Raw file name)=Huh7_delip_SL2_Ed17_1.d SUBJECT_SAMPLE_FACTORS - Huh7_delip_SL2_Ed17_2 Sample source:Huh7 cells | Treatment:BSA-elaidate-d17 + DMSO RAW_FILE_NAME(Raw file name)=Huh7_delip_SL2_Ed17_2.d SUBJECT_SAMPLE_FACTORS - Huh7_delip_SL2_Ed17_3 Sample source:Huh7 cells | Treatment:BSA-elaidate-d17 + DMSO RAW_FILE_NAME(Raw file name)=Huh7_delip_SL2_Ed17_3.d SUBJECT_SAMPLE_FACTORS - Huh7_delip_SL2_Ed17myr_1 Sample source:Huh7 cells | Treatment:BSA-elaidate-d17 + myriocin RAW_FILE_NAME(Raw file name)=Huh7_delip_SL2_Ed17myr_1.d SUBJECT_SAMPLE_FACTORS - Huh7_delip_SL2_Ed17myr_2 Sample source:Huh7 cells | Treatment:BSA-elaidate-d17 + myriocin RAW_FILE_NAME(Raw file name)=Huh7_delip_SL2_Ed17myr_2.d SUBJECT_SAMPLE_FACTORS - Huh7_delip_SL2_Ed17myr_3 Sample source:Huh7 cells | Treatment:BSA-elaidate-d17 + myriocin RAW_FILE_NAME(Raw file name)=Huh7_delip_SL2_Ed17myr_3.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), 200 pmol sphingosine-d7 (Avanti Polar Lipids, Cat# 860657), and 200 CO:COLLECTION_SUMMARY pmol sphingomyelin (d18:1/18:1)-d9 (Avanti Polar Lipids, Cat#860740). Cells were CO:COLLECTION_SUMMARY 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 with DMSO, 2) 100 µM TR:TREATMENT_SUMMARY BSA-100 µM oleate-d9 with 100 nM myriocin, 3) 100 µM BSA-elaidate-d17 with TR:TREATMENT_SUMMARY DMSO, or 4) 100 µM BSA-elaidate-d17 with 100 nM myriocin for 48 hours in TR:TREATMENT_SUMMARY 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), 200 pmol sphingosine-d7 (Avanti Polar Lipids, Cat# 860657), and 200 SP:SAMPLEPREP_SUMMARY pmol sphingomyelin (d18:1/18:1)-d9 (Avanti Polar Lipids, Cat#860740). Cells were SP:SAMPLEPREP_SUMMARY scraped with 0.5 mL methanol and 0.5 mL H2O. Homogenate aliquot of 100 µL was SP:SAMPLEPREP_SUMMARY taken to determine protein content using the BCA protein assay (Thermo Fisher SP:SAMPLEPREP_SUMMARY Scientific). The remaining homogenate was transferred to a new Eppendorf tube SP:SAMPLEPREP_SUMMARY and 1 mL chloroform was added. For plasma or media, 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 for cells were resuspended in 60 µl Buffer B, 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 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°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 Sphingolipid species were analyzed by multiple reaction monitoring of the MS:MS_COMMENTS transition from precursor to product ions at associated optimized collision MS:MS_COMMENTS energies, and fragmentor voltages using Agilent Masshunter. The m/z values of MS:MS_COMMENTS the precursor 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_delip_SL2_Od9_1 Huh7_delip_SL2_Od9_2 Huh7_delip_SL2_Od9_3 Huh7_delip_SL2_Od9myr_1 Huh7_delip_SL2_Od9myr_2 Huh7_delip_SL2_Od9myr_3 Huh7_delip_SL2_Ed17_1 Huh7_delip_SL2_Ed17_2 Huh7_delip_SL2_Ed17_3 Huh7_delip_SL2_Ed17myr_1 Huh7_delip_SL2_Ed17myr_2 Huh7_delip_SL2_Ed17myr_3 Factors Sample source:Huh7 cells | Treatment:BSA-oleate-d9 + DMSO Sample source:Huh7 cells | Treatment:BSA-oleate-d9 + DMSO Sample source:Huh7 cells | Treatment:BSA-oleate-d9 + DMSO Sample source:Huh7 cells | Treatment:BSA-oleate-d9 + myriocin Sample source:Huh7 cells | Treatment:BSA-oleate-d9 + myriocin Sample source:Huh7 cells | Treatment:BSA-oleate-d9 + myriocin Sample source:Huh7 cells | Treatment:BSA-elaidate-d17 + DMSO Sample source:Huh7 cells | Treatment:BSA-elaidate-d17 + DMSO Sample source:Huh7 cells | Treatment:BSA-elaidate-d17 + DMSO Sample source:Huh7 cells | Treatment:BSA-elaidate-d17 + myriocin Sample source:Huh7 cells | Treatment:BSA-elaidate-d17 + myriocin Sample source:Huh7 cells | Treatment:BSA-elaidate-d17 + myriocin Lac-Cer d18:1-d7/15:0 322506 280582 291250 304474 319527 294250 271877 324117 330521 321274 325545 354832 Lac-Cer d20:2-d17/24:0 0 0 0 0 0 0 1185 1437 1016 0 0 0 SM d(d18:1/18:1)-d9 8360250 8136068 8790683 9079046 9154300 8724479 8227752 8758827 8546790 8976873 9491258 9762315 SM d34:2-d17 6313774 6187961 6083863 1401230 1220445 1293583 SM d34:2-d34 639484 601818 623920 23644 10756 11387 SM d34:3-d34 512965 521786 542802 24185 18783 13346 SM d36:2-d17 3779504 4445123 4358358 770630 336715 477411 SM d36:2-d34 2380693 2203866 2334907 103524 63217 123545 SM d36:2-d9 2277276 2049112 1962058 455121 442062 240826 SM d36:3-d17 492612 432208 476438 247342 235899 278235 SM d36:3-d34 8197420 7670014 7170860 841224 860571 719637 SM d38:2-d17 724747 704220 687159 43906 73620 102739 SM d38:3-d34 2752969 2310390 2608911 697496 674701 741361 SM d40:2-d17 2333539 1852550 2222891 170359 166950 104832 SM d40:2-d9 1287155 1349343 1680551 156914 168977 157556 SM d40:3-d34 1150564 1082272 1208012 526282 633912 451694 SM d42:2-d17 1379136 1188362 1341307 382495 343929 379136 SM d42:2-d9 21334054 20942282 23751455 703915 727243 647825 SM d42:3-d17 694431 635559 684814 104552 107663 115882 SM d42:3-d34 5015487 3824834 4181848 133757 134221 145145 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 SM d(d18:1/18:1)-d9 738.6 193 SM d34:2-d17 718.6 184 SM d34:2-d34 735.6 184 SM d34:3-d34 733.5 184 SM d36:2-d17 746.6 184 SM d36:2-d34 763.6 184 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 d38:3-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-d9 822.7 184 SM d42:3-d17 828.7 184 SM d42:3-d34 845.7 184 METABOLITES_END #END