#METABOLOMICS WORKBENCH jgengatharan_20240726_094854 DATATRACK_ID:5037 STUDY_ID:ST003355 ANALYSIS_ID:AN005496 PROJECT_ID:PR002085 VERSION 1 CREATED_ON July 26, 2024, 9:56 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 two types of cis and trans fatty acids 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 ST:STUDY_SUMMARY various BSA-oleate, BSA-elaidate, BSA-cis-vaccenate, or BSA-trans-vaccenate. 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_EtOH_1 Sample source:Huh7 cells | Treatment:BSA-EtOH RAW_FILE_NAME(Raw file name)=Huh7_EtOH_1.d SUBJECT_SAMPLE_FACTORS - Huh7_EtOH_2 Sample source:Huh7 cells | Treatment:BSA-EtOH RAW_FILE_NAME(Raw file name)=Huh7_EtOH_2.d SUBJECT_SAMPLE_FACTORS - Huh7_EtOH_3 Sample source:Huh7 cells | Treatment:BSA-EtOH RAW_FILE_NAME(Raw file name)=Huh7_EtOH_3.d SUBJECT_SAMPLE_FACTORS - Huh7_oleic_1 Sample source:Huh7 cells | Treatment:BSA-oleate RAW_FILE_NAME(Raw file name)=Huh7_oleic_1.d SUBJECT_SAMPLE_FACTORS - Huh7_oleic_2 Sample source:Huh7 cells | Treatment:BSA-oleate RAW_FILE_NAME(Raw file name)=Huh7_oleic_2.d SUBJECT_SAMPLE_FACTORS - Huh7_oleic_3 Sample source:Huh7 cells | Treatment:BSA-oleate RAW_FILE_NAME(Raw file name)=Huh7_oleic_3.d SUBJECT_SAMPLE_FACTORS - Huh7_elaidic_1 Sample source:Huh7 cells | Treatment:BSA-elaidate RAW_FILE_NAME(Raw file name)=Huh7_elaidic_1.d SUBJECT_SAMPLE_FACTORS - Huh7_elaidic_2 Sample source:Huh7 cells | Treatment:BSA-elaidate RAW_FILE_NAME(Raw file name)=Huh7_elaidic_2.d SUBJECT_SAMPLE_FACTORS - Huh7_elaidic_3 Sample source:Huh7 cells | Treatment:BSA-elaidate RAW_FILE_NAME(Raw file name)=Huh7_elaidic_3.d SUBJECT_SAMPLE_FACTORS - Huh7_cisvacc_1 Sample source:Huh7 cells | Treatment:BSA-cis-vaccenate RAW_FILE_NAME(Raw file name)=Huh7_cisvacc_1.d SUBJECT_SAMPLE_FACTORS - Huh7_cisvacc_2 Sample source:Huh7 cells | Treatment:BSA-cis-vaccenate RAW_FILE_NAME(Raw file name)=Huh7_cisvacc_2.d SUBJECT_SAMPLE_FACTORS - Huh7_cisvacc_3 Sample source:Huh7 cells | Treatment:BSA-cis-vaccenate RAW_FILE_NAME(Raw file name)=Huh7_cisvacc_3.d SUBJECT_SAMPLE_FACTORS - Huh7_transvacc_1 Sample source:Huh7 cells | Treatment:BSA-trans-vaccenate RAW_FILE_NAME(Raw file name)=Huh7_transvacc_1.d SUBJECT_SAMPLE_FACTORS - Huh7_transvacc_2 Sample source:Huh7 cells | Treatment:BSA-trans-vaccenate RAW_FILE_NAME(Raw file name)=Huh7_transvacc_2.d SUBJECT_SAMPLE_FACTORS - Huh7_transvacc_3 Sample source:Huh7 cells | Treatment:BSA-trans-vaccenate RAW_FILE_NAME(Raw file name)=Huh7_transvacc_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 100 µM BSA-oleate, BSA-elaidate, TR:TREATMENT_SUMMARY BSA-cis-vaccenate, BSA-trans-vaccenate, or BSA-EtOH as a vehicle for 48 hours. #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_EtOH_1 Huh7_EtOH_2 Huh7_EtOH_3 Huh7_oleic_1 Huh7_oleic_2 Huh7_oleic_3 Huh7_elaidic_1 Huh7_elaidic_2 Huh7_elaidic_3 Huh7_cisvacc_1 Huh7_cisvacc_2 Huh7_cisvacc_3 Huh7_transvacc_1 Huh7_transvacc_2 Huh7_transvacc_3 Factors Sample source:Huh7 cells | Treatment:BSA-EtOH Sample source:Huh7 cells | Treatment:BSA-EtOH Sample source:Huh7 cells | Treatment:BSA-EtOH Sample source:Huh7 cells | Treatment:BSA-oleate Sample source:Huh7 cells | Treatment:BSA-oleate Sample source:Huh7 cells | Treatment:BSA-oleate Sample source:Huh7 cells | Treatment:BSA-elaidate Sample source:Huh7 cells | Treatment:BSA-elaidate Sample source:Huh7 cells | Treatment:BSA-elaidate Sample source:Huh7 cells | Treatment:BSA-cis-vaccenate Sample source:Huh7 cells | Treatment:BSA-cis-vaccenate Sample source:Huh7 cells | Treatment:BSA-cis-vaccenate Sample source:Huh7 cells | Treatment:BSA-trans-vaccenate Sample source:Huh7 cells | Treatment:BSA-trans-vaccenate Sample source:Huh7 cells | Treatment:BSA-trans-vaccenate SA d18:0 d7 149081 189978 189263 122580 136384 154983 142166 152666 156630 132043 146621 157655 128395 176833 185405 SA d20:1 (Z) 5822 10719 9326 40746 56216 52367 36964 46047 48172 SA d20:1 (E) 720103 571585 650979 240153 347250 335170 SO d18:1 d7 1453710 1682572 1782968 1238556 1294792 1468317 1386042 1433918 1463094 1270773 1558246 1585092 1290386 1638055 1658240 SO d20:2 (Z) 53136 75655 59417 99280 149768 136392 139010 177032 190938 SO d20:2 (E) 2781968 2248759 2460327 1241831 1557452 1582213 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Precursor Ion Product Ion SA d18:0 d7 309.4 291.5 SA d20:1 (Z) 328.4 310.4 SA d20:1 (E) 328.4 310.4 SO d18:1 d7 307.3 289.4 SO d20:2 (Z) 326.4 308.4 SO d20:2 (E) 326.4 308.4 METABOLITES_END #END