#METABOLOMICS WORKBENCH jgengatharan_20240730_142213 DATATRACK_ID:5057 STUDY_ID:ST003373 ANALYSIS_ID:AN005525 PROJECT_ID:PR002085 VERSION 1 CREATED_ON July 30, 2024, 2:29 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_SL1_Od9_1 Sample source:Huh7 cells | Treatment:BSA-oleate-d9 + DMSO RAW_FILE_NAME(Raw file name)=Huh7_delip_SL1_Od9_1.d SUBJECT_SAMPLE_FACTORS - Huh7_delip_SL1_Od9_2 Sample source:Huh7 cells | Treatment:BSA-oleate-d9 + DMSO RAW_FILE_NAME(Raw file name)=Huh7_delip_SL1_Od9_2.d SUBJECT_SAMPLE_FACTORS - Huh7_delip_SL1_Od9_3 Sample source:Huh7 cells | Treatment:BSA-oleate-d9 + DMSO RAW_FILE_NAME(Raw file name)=Huh7_delip_SL1_Od9_3.d SUBJECT_SAMPLE_FACTORS - Huh7_delip_SL1_Od9myr_1 Sample source:Huh7 cells | Treatment:BSA-oleate-d9 + myriocin RAW_FILE_NAME(Raw file name)=Huh7_delip_SL1_Od9myr_1.d SUBJECT_SAMPLE_FACTORS - Huh7_delip_SL1_Od9myr_2 Sample source:Huh7 cells | Treatment:BSA-oleate-d9 + myriocin RAW_FILE_NAME(Raw file name)=Huh7_delip_SL1_Od9myr_2.d SUBJECT_SAMPLE_FACTORS - Huh7_delip_SL1_Od9myr_3 Sample source:Huh7 cells | Treatment:BSA-oleate-d9 + myriocin RAW_FILE_NAME(Raw file name)=Huh7_delip_SL1_Od9myr_3.d SUBJECT_SAMPLE_FACTORS - Huh7_delip_SL1_Ed17_1 Sample source:Huh7 cells | Treatment:BSA-elaidate-d17 + DMSO RAW_FILE_NAME(Raw file name)=Huh7_delip_SL1_Ed17_1.d SUBJECT_SAMPLE_FACTORS - Huh7_delip_SL1_Ed17_2 Sample source:Huh7 cells | Treatment:BSA-elaidate-d17 + DMSO RAW_FILE_NAME(Raw file name)=Huh7_delip_SL1_Ed17_2.d SUBJECT_SAMPLE_FACTORS - Huh7_delip_SL1_Ed17_3 Sample source:Huh7 cells | Treatment:BSA-elaidate-d17 + DMSO RAW_FILE_NAME(Raw file name)=Huh7_delip_SL1_Ed17_3.d SUBJECT_SAMPLE_FACTORS - Huh7_delip_SL1_Ed17myr_1 Sample source:Huh7 cells | Treatment:BSA-elaidate-d17 + myriocin RAW_FILE_NAME(Raw file name)=Huh7_delip_SL1_Ed17myr_1.d SUBJECT_SAMPLE_FACTORS - Huh7_delip_SL1_Ed17myr_2 Sample source:Huh7 cells | Treatment:BSA-elaidate-d17 + myriocin RAW_FILE_NAME(Raw file name)=Huh7_delip_SL1_Ed17myr_2.d SUBJECT_SAMPLE_FACTORS - Huh7_delip_SL1_Ed17myr_3 Sample source:Huh7 cells | Treatment:BSA-elaidate-d17 + myriocin RAW_FILE_NAME(Raw file name)=Huh7_delip_SL1_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 d18:1/18:1-d9 sphingomyelin (Avanti Polar Lipids, Cat# 791649) or 200 pmol CO:COLLECTION_SUMMARY 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 d18:1/18:1-d9 sphingomyelin (Avanti Polar Lipids, Cat# 791649) or 200 pmol SP:SAMPLEPREP_SUMMARY 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_SL1_Od9_1 Huh7_delip_SL1_Od9_2 Huh7_delip_SL1_Od9_3 Huh7_delip_SL1_Od9myr_1 Huh7_delip_SL1_Od9myr_2 Huh7_delip_SL1_Od9myr_3 Huh7_delip_SL1_Ed17_1 Huh7_delip_SL1_Ed17_2 Huh7_delip_SL1_Ed17_3 Huh7_delip_SL1_Ed17myr_1 Huh7_delip_SL1_Ed17myr_2 Huh7_delip_SL1_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 Cer d18:1-d7/15:0 2005739 1690413 1820713 2652621 2580746 2898124 1746168 2076586 1864612 2096203 2464482 2661620 Cer d20:2-d17/18:1-d17 3574 1988 3818 Cer d20:2-d17/22:0 4400 2075 1940 Cer d20:2-d17/22:1 937 2618 1545 Cer d20:2-d17/22:1-d17 3146 3890 3968 Cer d20:2-d17/24:0 3224 5980 4157 Cer d20:2-d17/24:1 5770 4878 6807 Cer d20:2-d17/24:1-d17 8905 7999 7836 Gluc-Cer d18:1-d7/15:0 395013 345502 370367 224477 359383 352413 360433 469754 442026 459585 422974 428587 Hex-Cer d20:2-d17/22:1-d17 2121 864 1226 Hex-Cer d20:2-d17/24:1-d17 904 1226 1366 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Precursor Ion Product Ion Cer d18:1-d7/15:0 531.5 271.4 Cer d20:2-d17/18:1-d17 624.6 307.4 Cer d20:2-d17/22:0 665.6 307.4 Cer d20:2-d17/22:1 663.6 307.4 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-d17/22:1-d17 842.7 307.4 Hex-Cer d20:2-d17/24:1-d17 870.7 307.4 METABOLITES_END #END