#METABOLOMICS WORKBENCH jgengatharan_20240802_094936 DATATRACK_ID:5080 STUDY_ID:ST003377 ANALYSIS_ID:AN005529 PROJECT_ID:PR002085 VERSION 1 CREATED_ON August 2, 2024, 9:59 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 Sphingolipid secretory flux from Huh7 SPTLC3 KO cells ST:STUDY_SUMMARY We analyzed 13C labeling on sphingolipids in fresh and spent media from Huh7 ST:STUDY_SUMMARY control and SPTLC3 KO cells treated with BSA-palmitate and the stable isotope ST:STUDY_SUMMARY tracers [13C]serine and [13C]glycine to measure sphingolipid secretory flux. 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 - Huh7freshmedia_delip_1 Sample source:Huh7 media | Condition:Fresh Media RAW_FILE_NAME(Raw file name)=Huh7freshmedia_delip_1.mzXML SUBJECT_SAMPLE_FACTORS - Huh7freshmedia_delip_2 Sample source:Huh7 media | Condition:Fresh Media RAW_FILE_NAME(Raw file name)=Huh7freshmedia_delip_2.mzXML SUBJECT_SAMPLE_FACTORS - Huh7freshmedia_delip_3 Sample source:Huh7 media | Condition:Fresh Media RAW_FILE_NAME(Raw file name)=Huh7freshmedia_delip_3.mzXML SUBJECT_SAMPLE_FACTORS - Huh7spentmedia_delip_AAVS_1 Sample source:Huh7 media | Condition:AAVS1 Control Spent Media RAW_FILE_NAME(Raw file name)=Huh7spentmedia_delip_AAVS_1.mzXML SUBJECT_SAMPLE_FACTORS - Huh7spentmedia_delip_AAVS_2 Sample source:Huh7 media | Condition:AAVS1 Control Spent Media RAW_FILE_NAME(Raw file name)=Huh7spentmedia_delip_AAVS_2.mzXML SUBJECT_SAMPLE_FACTORS - Huh7spentmedia_delip_AAVS_3 Sample source:Huh7 media | Condition:AAVS1 Control Spent Media RAW_FILE_NAME(Raw file name)=Huh7spentmedia_delip_AAVS_3.mzXML SUBJECT_SAMPLE_FACTORS - Huh7spentmedia_delip_SPT3KO1_1 Sample source:Huh7 media | Condition:SPTLC3 KO #1 Spent Media RAW_FILE_NAME(Raw file name)=Huh7spentmedia_delip_SPT3KO1_1.mzXML SUBJECT_SAMPLE_FACTORS - Huh7spentmedia_delip_SPT3KO1_2 Sample source:Huh7 media | Condition:SPTLC3 KO #1 Spent Media RAW_FILE_NAME(Raw file name)=Huh7spentmedia_delip_SPT3KO1_2.mzXML SUBJECT_SAMPLE_FACTORS - Huh7spentmedia_delip_SPT3KO1_3 Sample source:Huh7 media | Condition:SPTLC3 KO #1 Spent Media RAW_FILE_NAME(Raw file name)=Huh7spentmedia_delip_SPT3KO1_3.mzXML SUBJECT_SAMPLE_FACTORS - Huh7spentmedia_delip_SPT3KO2_1 Sample source:Huh7 media | Condition:SPTLC3 KO #2 Spent Media RAW_FILE_NAME(Raw file name)=Huh7spentmedia_delip_SPT3KO2_1.mzXML SUBJECT_SAMPLE_FACTORS - Huh7spentmedia_delip_SPT3KO2_2 Sample source:Huh7 media | Condition:SPTLC3 KO #2 Spent Media RAW_FILE_NAME(Raw file name)=Huh7spentmedia_delip_SPT3KO2_2.mzXML SUBJECT_SAMPLE_FACTORS - Huh7spentmedia_delip_SPT3KO2_3 Sample source:Huh7 media | Condition:SPTLC3 KO #2 Spent Media RAW_FILE_NAME(Raw file name)=Huh7spentmedia_delip_SPT3KO2_3.mzXML SUBJECT_SAMPLE_FACTORS - Huh7freshmedia_delip_3_MS2 Sample source:Huh7 media | Condition:Fresh Media RAW_FILE_NAME(Raw file name)=Huh7freshmedia_delip_3_MS2.mzXML SUBJECT_SAMPLE_FACTORS - Huh7spentmedia_delip_AAVS_2_MS2 Sample source:Huh7 media | Condition:AAVS1 Control Spent Media RAW_FILE_NAME(Raw file name)=Huh7spentmedia_delip_AAVS_2_MS2.mzXML #COLLECTION CO:COLLECTION_SUMMARY To calculate secretory flux, 0.75 ml of fresh media or 1.5 ml of spent media was CO:COLLECTION_SUMMARY evaporated under vacuum at 4°C and resuspended into 0.1 mL of H2O. CO:SAMPLE_TYPE Culture Media #TREATMENT TR:TREATMENT_SUMMARY To measure sphingomyelin secretory flux, cells were treated with 100 µM TR:TREATMENT_SUMMARY BSA-palmitate and traced with [13C3]serine and [13C2]glycine for 48 hours in TR:TREATMENT_SUMMARY delipidated media to increase the percentage of labeled lipids in spent media. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Media was spiked with the following internal standards: 20 pmol sphinganine-d7 SP:SAMPLEPREP_SUMMARY (Avanti Polar Lipids, Cat# 860658), deoxysphinganine-d3 (Avanti Polar Lipids, SP:SAMPLEPREP_SUMMARY Cat# 860474), 100 pmol d18:0-d7/13:0 dihydroceramide (Avanti Polar Lipids, Cat# SP:SAMPLEPREP_SUMMARY 330726), 200 pmol d18:1-d7/15:0 ceramide (Avanti Polar Lipids, Cat# 860681), 100 SP:SAMPLEPREP_SUMMARY pmol d18:1-d7/15:0 glucosylceramide (Avanti Polar Lipids, Cat# 330729), 100 pmol SP:SAMPLEPREP_SUMMARY d18:1-d7/15:0 lactosylceramide (Avanti Polar Lipids, Cat# 330727), 200 pmol SP:SAMPLEPREP_SUMMARY sphingosine-d7 (Avanti Polar Lipids, Cat# 860657), 200 pmol sphingosine-d7 SP:SAMPLEPREP_SUMMARY (Avanti Polar Lipids, Cat# 860657), and 200 pmol sphingomyelin (d18:1/18:1)-d9 SP:SAMPLEPREP_SUMMARY (Avanti Polar Lipids, Cat#860740). 0.5 mL methanol, 0.5 mL H2O, and 1 mL SP:SAMPLEPREP_SUMMARY chloroform were added directly. Samples were vortexed for 5 min and centrifuged SP:SAMPLEPREP_SUMMARY for 5 min at 4 ˚C at 15,000g. The organic phase was collected and 2 μL of SP:SAMPLEPREP_SUMMARY formic acid was added to the remaining polar phase which was re-extracted with 1 SP:SAMPLEPREP_SUMMARY mL of chloroform. Combined organic phases were dried under nitrogen. After dried SP:SAMPLEPREP_SUMMARY extracts for media were resuspended in 60 µl MeOH, 5 µL of sample was SP:SAMPLEPREP_SUMMARY injected. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Vanquish CH:COLUMN_NAME Phenomenex Kinetex C18 (100 x 2.1mm,1.7um) CH:SOLVENT_A 98% water, 2% methanol; 5 mM ammonium acetate CH:SOLVENT_B 50% methanol, 50% isopropanol; 5 mM ammonium acetate CH:FLOW_GRADIENT 0 min, 30%B; 1 min, 30%B; 2 min, 70%B; 11 min, 95%B; 17 min, 30%B; 21.5 min, CH:FLOW_GRADIENT 30%B; 27 min, 30%B CH:FLOW_RATE 0.2 mL/min CH:COLUMN_TEMPERATURE 35°C #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS Lipids were identified and quantified using EL-MAVEN using exact mass of MS:MS_COMMENTS precursor ion in MS1 chromatogram and product ion in MS2 spectra. The percent MS:MS_COMMENTS isotopologue distribution of each sphingolipid was determined and corrected for MS:MS_COMMENTS natural 13C abundance using in-house algorithms adapted from a previous report. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Peak area MS_METABOLITE_DATA_START Samples Huh7freshmedia_delip_1 Huh7freshmedia_delip_2 Huh7freshmedia_delip_3 Huh7spentmedia_delip_AAVS_1 Huh7spentmedia_delip_AAVS_2 Huh7spentmedia_delip_AAVS_3 Huh7spentmedia_delip_SPT3KO1_1 Huh7spentmedia_delip_SPT3KO1_2 Huh7spentmedia_delip_SPT3KO1_3 Huh7spentmedia_delip_SPT3KO2_1 Huh7spentmedia_delip_SPT3KO2_2 Huh7spentmedia_delip_SPT3KO2_3 Huh7freshmedia_delip_3_MS2 Huh7spentmedia_delip_AAVS_2_MS2 Factors Sample source:Huh7 media | Condition:Fresh Media Sample source:Huh7 media | Condition:Fresh Media Sample source:Huh7 media | Condition:Fresh Media Sample source:Huh7 media | Condition:AAVS1 Control Spent Media Sample source:Huh7 media | Condition:AAVS1 Control Spent Media Sample source:Huh7 media | Condition:AAVS1 Control Spent Media Sample source:Huh7 media | Condition:SPTLC3 KO #1 Spent Media Sample source:Huh7 media | Condition:SPTLC3 KO #1 Spent Media Sample source:Huh7 media | Condition:SPTLC3 KO #1 Spent Media Sample source:Huh7 media | Condition:SPTLC3 KO #2 Spent Media Sample source:Huh7 media | Condition:SPTLC3 KO #2 Spent Media Sample source:Huh7 media | Condition:SPTLC3 KO #2 Spent Media Sample source:Huh7 media | Condition:Fresh Media Sample source:Huh7 media | Condition:AAVS1 Control Spent Media SM d18:1/18:1-d9 942753984 919011648 925193664 1657320064 1803074304 1807801472 1890180224 1945738240 2047715456 1875552512 1913650560 1895164288 SM d32:1 28827968.36 30981279.82 32104802.7 97783582.5 95841637.15 96071648 88302206.38 89985967.94 92911507.31 85864420 86428556.75 86386775.12 SM d34:0 24989616.44 36518601.27 45627269.91 147869951.3 136839490.1 133215234.9 135656487.4 154240511.4 143219311.8 133509805.9 170963998.4 149038160.9 SM d34:1 284674680.9 308052360 466964566 1295340455 1194370458 1181352693 1240105421 1273952654 1226697274 1138886348 1271845393 1170983956 SM d34:2 141369061 149033997 149749202.4 360289353.4 365994085.1 360036003.3 331582615.5 360392282.5 356069256.3 346508546.8 337528505.5 340076883 SM d36:1 24265422.49 26336306.11 153202007.5 118941406 101123349.4 98548403.36 112262015.3 94815581.24 92244546.38 93103217.25 88607482.85 82726822.85 SM d36:2 48609505.5 52963381.56 51860783.98 115425379.3 110524581.7 110518253.8 110789138.2 103647845.9 110613575.9 107523084.3 104755951.8 105992670.4 SM d38:1 7161210.88 14802075 82393184.5 51580571.75 44773177 42171637.5 43004802.25 41219183.5 40817976 34206580 37642302.25 36225229.88 SM d40:1 30577320.12 32503599.28 178020682.8 200989154.4 179224371.2 173465090 184819812.3 168685683.4 156589819.1 151679811.3 163392775.6 146751341.3 SM d40:2 85715095.06 93499006.81 108967373.5 241216544.9 252922491.5 237653096 245459844 232454887.4 239277886.7 235623219.8 224167358 232830258 SM d42:1 44884734.06 62406586.65 294688729.6 255704834.1 267855537.3 268190745 275304883.5 253469732 247964215.9 253090825.7 284443723.4 261774292.7 SM d42:2 336091073.3 365999153.8 815492301.1 1036122455 1000650661 998103227.7 1006801787 934361290 942716246.3 952355521.1 930117079.6 950381170.6 SM d42:3 439768948.3 501210270.4 560209961.8 1127134059 1123166244 1153462419 1105574013 1038833085 1094239330 1098654183 1037578300 1108350714 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name m/z SM d18:1/18:1-d9 738.6443 SM d32:1 675.5434 SM d34:0 705.5893 SM d34:1 703.5742 SM d34:2 701.5585 SM d36:1 731.6054 SM d36:2 729.5897 SM d38:1 759.637 SM d40:1 787.6685 SM d40:2 785.6532 SM d42:1 815.6999 SM d42:2 813.6843 SM d42:3 811.6681 METABOLITES_END #END