#METABOLOMICS WORKBENCH jgengatharan_20240726_093132 DATATRACK_ID:5036 STUDY_ID:ST003354 ANALYSIS_ID:AN005495 PROJECT_ID: VERSION 1 CREATED_ON 07-30-2024 #PROJECT #STUDY #SUBJECT #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data #COLLECTION #TREATMENT #SAMPLEPREP #CHROMATOGRAPHY #ANALYSIS #END #METABOLOMICS WORKBENCH jgengatharan_20240726_093132 DATATRACK_ID:5036 STUDY_ID:ST003354 ANALYSIS_ID:AN005495 PROJECT_ID:PR002085 VERSION 1 CREATED_ON 07-30-2024 #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 Serine palmitoyltransferase (SPT), the initial rate-limiting enzyme of PR:PROJECT_SUMMARY 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 PR:DOI http://dx.doi.org/10.21228/M83R6P #STUDY ST:STUDY_TITLE Incorporation of co-treated oleate-d9 and elaidate-d17 in long-chain base of ST:STUDY_TITLE sphingolipids in Huh7 cells. ST:STUDY_SUMMARY We analyzed hydrolyzed long-chain bases (LCBs) in Huh7 cells treated with a ST:STUDY_SUMMARY combination of BSA-oleate-d9 and BSA-elaidate-d17. We aimed to confirm the ST:STUDY_SUMMARY incorporation of oleate and elaidate in the LCB of sphingolipids using their ST:STUDY_SUMMARY deuterated versions and put them both in direct competition for SPT. 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 ST:SUBMIT_DATE 2024-07-26 #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]Additional sample data SUBJECT_SAMPLE_FACTORS - Huh7_oleated9elaidated17_1 Sample source:Huh7 cells | Treatment:BSA-oleate-d9 + BSA-elaidate-d17 RAW_FILE_NAME(Raw file name)=Huh7_oleated9elaidated17_1.d SUBJECT_SAMPLE_FACTORS - Huh7_oleated9elaidated17_2 Sample source:Huh7 cells | Treatment:BSA-oleate-d9 + BSA-elaidate-d17 RAW_FILE_NAME(Raw file name)=Huh7_oleated9elaidated17_2.d SUBJECT_SAMPLE_FACTORS - Huh7_oleated9elaidated17_3 Sample source:Huh7 cells | Treatment:BSA-oleate-d9 + BSA-elaidate-d17 RAW_FILE_NAME(Raw file name)=Huh7_oleated9elaidated17_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 a combination of 50 µM BSA-oleate-d9 and 50 µM TR:TREATMENT_SUMMARY BSA-elaidate-d17 for 48 hours in 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:INSTRUMENT_NAME Agilent 1260 Infinity II CH:COLUMN_NAME Thermo Hypersil GOLD C18 Selectivity (100 x 2.1 mm, 1.9 µm) CH:COLUMN_TEMPERATURE 40°C 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: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:CHROMATOGRAPHY_TYPE Reversed phase #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6460 QQQ MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:MS_COMMENTS Long-chain base (LCB) species were analyzed by multiple reaction monitoring of MS:MS_COMMENTS the 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:ION_MODE POSITIVE #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Peak area MS_METABOLITE_DATA_START Samples Huh7_oleated9elaidated17_1 Huh7_oleated9elaidated17_2 Huh7_oleated9elaidated17_3 Factors Sample source:Huh7 cells | Treatment:BSA-oleate-d9 + BSA-elaidate-d17 Sample source:Huh7 cells | Treatment:BSA-oleate-d9 + BSA-elaidate-d17 Sample source:Huh7 cells | Treatment:BSA-oleate-d9 + BSA-elaidate-d17 SA d18:0 d7 88652.0000 63277.0000 63481.0000 SA d20:1-d17 60081.0000 43025.0000 42070.0000 SA d20:1-d9 2647.0000 3100.0000 2473.0000 SO d18:1 d7 887646.0000 693947.0000 672659.0000 SO d20:2-d17 525415.0000 399006.0000 381920.0000 SO d20:2-d9 11749.0000 4988.0000 5532.0000 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name pubchem_id inchi_key kegg_id other_id other_id_type ri ri_type moverz_quant SA d18:0 d7 SA d20:1-d17 SA d20:1-d9 SO d18:1 d7 SO d20:2-d17 SO d20:2-d9 METABOLITES_END #END