#METABOLOMICS WORKBENCH jgengatharan_20240726_090613 DATATRACK_ID:5035 STUDY_ID:ST003353 ANALYSIS_ID:AN005494 PROJECT_ID:PR002085 VERSION 1 CREATED_ON July 26, 2024, 9:25 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 long-chain base of sphingolipids ST:STUDY_TITLE in Huh7 cells. ST:STUDY_SUMMARY We analyzed hydrolyzed long-chain bases (LCBs) in Huh7 cells treated with ST:STUDY_SUMMARY BSA-oleate-d9 or BSA-elaidate-d17 in the presence or absence of myriocin, a ST:STUDY_SUMMARY pharmacological inhibitor of SPT, the initial rate-limiting enzyme of ST:STUDY_SUMMARY sphingolipid biosynthesis. 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_oleated9_1 Sample source:Huh7 cells | Treatment:BSA-oleate-d9 + DMSO RAW_FILE_NAME(Raw file name)=Huh7_oleated9_1.d SUBJECT_SAMPLE_FACTORS - Huh7_oleated9_2 Sample source:Huh7 cells | Treatment:BSA-oleate-d9 + DMSO RAW_FILE_NAME(Raw file name)=Huh7_oleated9_2.d SUBJECT_SAMPLE_FACTORS - Huh7_oleated9_3 Sample source:Huh7 cells | Treatment:BSA-oleate-d9 + DMSO RAW_FILE_NAME(Raw file name)=Huh7_oleated9_3.d SUBJECT_SAMPLE_FACTORS - Huh7_oleated9myr_1 Sample source:Huh7 cells | Treatment:BSA-oleate-d9 + myriocin RAW_FILE_NAME(Raw file name)=Huh7_oleated9myr_1.d SUBJECT_SAMPLE_FACTORS - Huh7_oleated9myr_2 Sample source:Huh7 cells | Treatment:BSA-oleate-d9 + myriocin RAW_FILE_NAME(Raw file name)=Huh7_oleated9myr_2.d SUBJECT_SAMPLE_FACTORS - Huh7_oleated9myr_3 Sample source:Huh7 cells | Treatment:BSA-oleate-d9 + myriocin RAW_FILE_NAME(Raw file name)=Huh7_oleated9myr_3.d SUBJECT_SAMPLE_FACTORS - Huh7_elaidated17_1 Sample source:Huh7 cells | Treatment:BSA-elaidate-d17 + DMSO RAW_FILE_NAME(Raw file name)=Huh7_elaidated17_1.d SUBJECT_SAMPLE_FACTORS - Huh7_elaidated17_2 Sample source:Huh7 cells | Treatment:BSA-elaidate-d17 + DMSO RAW_FILE_NAME(Raw file name)=Huh7_elaidated17_2.d SUBJECT_SAMPLE_FACTORS - Huh7_elaidated17_3 Sample source:Huh7 cells | Treatment:BSA-elaidate-d17 + DMSO RAW_FILE_NAME(Raw file name)=Huh7_elaidated17_3.d SUBJECT_SAMPLE_FACTORS - Huh7_elaidated17myr_1 Sample source:Huh7 cells | Treatment:BSA-elaidate-d17 + myriocin RAW_FILE_NAME(Raw file name)=Huh7_elaidated17myr_1.d SUBJECT_SAMPLE_FACTORS - Huh7_elaidated17myr_2 Sample source:Huh7 cells | Treatment:BSA-elaidate-d17 + myriocin RAW_FILE_NAME(Raw file name)=Huh7_elaidated17myr_2.d SUBJECT_SAMPLE_FACTORS - Huh7_elaidated17myr_3 Sample source:Huh7 cells | Treatment:BSA-elaidate-d17 + myriocin RAW_FILE_NAME(Raw file name)=Huh7_elaidated17myr_3.d #COLLECTION CO:COLLECTION_SUMMARY Cells (~400,000) were spiked with internal standards sphinganine-d7 (Avanti CO:COLLECTION_SUMMARY Polar Lipids, Cat# 860658) and sphingosine-d7 (Avanti Polar Lipids, Cat# 860657) CO:COLLECTION_SUMMARY and were scraped with 0.5 mL methanol. 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) were spiked with internal standards sphinganine-d7 (Avanti SP:SAMPLEPREP_SUMMARY Polar Lipids, Cat# 860658) and sphingosine-d7 (Avanti Polar Lipids, Cat# 860657) SP:SAMPLEPREP_SUMMARY and were scraped with 0.5 mL methanol. Homogenate aliquot of 50 µL was taken to SP:SAMPLEPREP_SUMMARY determine protein content using the BCA protein assay (Thermo Fisher SP:SAMPLEPREP_SUMMARY Scientific). Samples were placed on a mixer for 1 hr at 37°C and then SP:SAMPLEPREP_SUMMARY centrifuged for 5 min at 16,000g. The MeOH was supernatant transferred to a new SP:SAMPLEPREP_SUMMARY Eppendorf tube and hydrolyzed for 16 hr at 65°C. 100 µL 10M KOH, 625 µL SP:SAMPLEPREP_SUMMARY chloroform, 100 µL 2N NH4OH, and 500 µL alkaline water were added to samples SP:SAMPLEPREP_SUMMARY followed by vortexing for 5 min and centrifugation for 5 min at 16,000g. The SP:SAMPLEPREP_SUMMARY lower organic phase was washed 3 times with alkaline water and dried under air. SP:SAMPLEPREP_SUMMARY After dried extracts were resuspended in 60 µl Buffer B, 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 Hypersil GOLD C18 Selectivity (100 x 2.1 mm, 1.9 µm) CH:SOLVENT_A 60% methanol 40% water; 0.1% formic acid; 5 mM ammonium formate CH:SOLVENT_B 100% methanol; 0.1% formic acid; 5 mM ammonium formate CH:FLOW_GRADIENT 0 min, 40%B; 0.5 min, 40%B; 16 min, 100%B; 25.5 min, 100%B; 26 min, 40%B CH:FLOW_RATE 0.2 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 Huh7_oleated9_1 Huh7_oleated9_2 Huh7_oleated9_3 Huh7_oleated9myr_1 Huh7_oleated9myr_2 Huh7_oleated9myr_3 Huh7_elaidated17_1 Huh7_elaidated17_2 Huh7_elaidated17_3 Huh7_elaidated17myr_1 Huh7_elaidated17myr_2 Huh7_elaidated17myr_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 SA d18:0 d7 57224 85231 66114 57026 63944 80951 77591 75105 73433 65509 70716 77692 SA d20:1-d9 14144 15889 9695 1201 0 0 0 0 0 0 SA d20:1-d17 0 0 0 0 0 0 92169 92288 90111 2524 2052 1843 SO d18:1 d7 541345 875261 714109 650010 748182 967901 809445 798830 842267 742360 768750 869004 SO d20:2-d9 39094 39130 34248 2244 2099 0 0 0 0 0 SO d20:2-d17 0 0 0 0 725442 735261 654806 22293 22522 25005 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Precursor Ion Product Ion SA d18:0 d7 309.4 291.5 SA d20:1-d9 337.4 319.4 SA d20:1-d17 345.4 327.4 SO d18:1 d7 307.3 289.4 SO d20:2-d9 335.4 317.4 SO d20:2-d17 343.4 325.4 METABOLITES_END #END