#METABOLOMICS WORKBENCH jgengatharan_20240802_092947 DATATRACK_ID:5079 STUDY_ID:ST003382 ANALYSIS_ID:AN005540 PROJECT_ID:PR002085 VERSION 1 CREATED_ON August 2, 2024, 9:40 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 biosynthetic flux in Huh7 SPTLC3 KO cells ST:STUDY_SUMMARY We analyzed 13C labeling on sphingolipids in Huh7 control and SPTLC3 KO cells ST:STUDY_SUMMARY treated with BSA-palmitate and the stable isotope tracers [13C]serine and ST:STUDY_SUMMARY [13C]glycine to measure intracellular sphingolipid biosynthetic 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 - Huh7cells_AAVS1_1 Sample source:Huh7 cells | Condition:AAVS1 Control RAW_FILE_NAME(Raw file name)=Huh7cells_AAVS1_1.mzXML SUBJECT_SAMPLE_FACTORS - Huh7cells_AAVS1_2 Sample source:Huh7 cells | Condition:AAVS1 Control RAW_FILE_NAME(Raw file name)=Huh7cells_AAVS1_2.mzXML SUBJECT_SAMPLE_FACTORS - Huh7cells_AAVS1_3 Sample source:Huh7 cells | Condition:AAVS1 Control RAW_FILE_NAME(Raw file name)=Huh7cells_AAVS1_3.mzXML SUBJECT_SAMPLE_FACTORS - Huh7cells_SPT3KO1_1 Sample source:Huh7 cells | Condition:SPTLC3 KO #1 RAW_FILE_NAME(Raw file name)=Huh7cells_SPT3KO1_1.mzXML SUBJECT_SAMPLE_FACTORS - Huh7cells_SPT3KO1_2 Sample source:Huh7 cells | Condition:SPTLC3 KO #1 RAW_FILE_NAME(Raw file name)=Huh7cells_SPT3KO1_2.mzXML SUBJECT_SAMPLE_FACTORS - Huh7cells_SPT3KO1_3 Sample source:Huh7 cells | Condition:SPTLC3 KO #1 RAW_FILE_NAME(Raw file name)=Huh7cells_SPT3KO1_3.mzXML SUBJECT_SAMPLE_FACTORS - Huh7cells_SPT3KO2_1 Sample source:Huh7 cells | Condition:SPTLC3 KO #2 RAW_FILE_NAME(Raw file name)=Huh7cells_SPT3KO2_1.mzXML SUBJECT_SAMPLE_FACTORS - Huh7cells_SPT3KO2_2 Sample source:Huh7 cells | Condition:SPTLC3 KO #2 RAW_FILE_NAME(Raw file name)=Huh7cells_SPT3KO2_2.mzXML SUBJECT_SAMPLE_FACTORS - Huh7cells_SPT3KO2_3 Sample source:Huh7 cells | Condition:SPTLC3 KO #2 RAW_FILE_NAME(Raw file name)=Huh7cells_SPT3KO2_3.mzXML SUBJECT_SAMPLE_FACTORS - Huh7cells_AAVS1_1_MS2 Sample source:Huh7 cells | Condition:AAVS1 Control RAW_FILE_NAME(Raw file name)=Huh7cells_AAVS1_1_MS2.mzXML SUBJECT_SAMPLE_FACTORS - Huh7cells_AAVS1_2_MS2 Sample source:Huh7 cells | Condition:AAVS1 Control RAW_FILE_NAME(Raw file name)=Huh7cells_AAVS1_2_MS2.mzXML #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 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 To measure intracellular sphingolipid biosynthetic flux in Huh7 control and TR:TREATMENT_SUMMARY SPTLC3 KO cell lines, we treated cells with 100 µM BSA-palmitate and performed TR:TREATMENT_SUMMARY stable isotope tracing with a combination [13C3]serine and [13C2]glycine along TR:TREATMENT_SUMMARY for 48 hours. #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 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 MeOH, 5 µL of sample SP:SAMPLEPREP_SUMMARY was 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 Huh7cells_AAVS1_1 Huh7cells_AAVS1_2 Huh7cells_AAVS1_3 Huh7cells_SPT3KO1_1 Huh7cells_SPT3KO1_2 Huh7cells_SPT3KO1_3 Huh7cells_SPT3KO2_1 Huh7cells_SPT3KO2_2 Huh7cells_SPT3KO2_3 Huh7cells_AAVS1_1_MS2 Huh7cells_AAVS1_2_MS2 Factors Sample source:Huh7 cells | Condition:AAVS1 Control Sample source:Huh7 cells | Condition:AAVS1 Control Sample source:Huh7 cells | Condition:AAVS1 Control Sample source:Huh7 cells | Condition:SPTLC3 KO #1 Sample source:Huh7 cells | Condition:SPTLC3 KO #1 Sample source:Huh7 cells | Condition:SPTLC3 KO #1 Sample source:Huh7 cells | Condition:SPTLC3 KO #2 Sample source:Huh7 cells | Condition:SPTLC3 KO #2 Sample source:Huh7 cells | Condition:SPTLC3 KO #2 Sample source:Huh7 cells | Condition:AAVS1 Control Sample source:Huh7 cells | Condition:AAVS1 Control Cer d18:1-d7/15:0 245901808 274480288 299161280 359707104 357491712 339814368 408559488 426615232 446974336 Cer d18:1/16:0 84566619.75 75078057.5 74054898.15 32759578.88 33342974.12 34818504.88 15052104.23 15243881.19 16935000.8 Cer d18:1/20:0 19304147.64 17723962.93 15814885.38 6034629.59 7059507.23 6227834.51 3463904.68 3592550.49 3542433.65 Cer d18:1/22:0 162328754.4 160220725.1 142163776.6 50867342.72 53634888.55 52788283.1 27951480.17 28316473.5 27947502.53 Cer d18:1/23:0 81352819.68 76388397.1 70062096.26 24878465.84 25532627.35 25641150.61 13006129.12 13355080.83 13867486.68 Cer d18:1/24:0 462803212.6 446360808.1 419450223.5 165704208.1 156339294.1 178759486.3 97901897.78 101727639.7 100323788.4 Cer d18:1/24:1 225036288.7 216944723.3 178009868.1 59993310.67 68897556.6 64252678.5 31379200.61 32156850.89 31904406.3 Cer d18:1/25:0 13400375.25 13344205.87 11744342.62 4854944.82 4165492.91 5118297.24 3028332.75 3023446.23 3385721.23 DHCer d18:0-d7/13:0 394242656 485107520 473740128 490412864 500028288 496095616 516705856 484613312 477780480 DHCer d18:0/16:0 28925067 21617189.25 23895022 10990377.25 12296428.71 10643210.45 7098675.69 7822564.94 8675870.5 DHCer d18:0/22:0 48041658.79 46276865.66 40215316.01 10594643.92 11255814.63 11201036.06 6547394.81 5617388.77 6280180.85 DHCer d18:0/23:0 12253899.47 13005075.92 11463848.56 3510143.74 2808023.79 3428494.64 2111101.13 1928876.31 1846879.68 DHCer d18:0/24:0 86389506.53 83113447.26 74257538.37 21628339.32 20967667.04 22720679.16 11861295.48 10903548.67 11537858.25 DHCer d18:0/24:1 38205225.48 31946812.47 27053455.61 6987560.13 7740457.41 8678468.2 3498714.84 3147102.32 3321158.99 DHCer d18:0/26:0 15157056.02 13218663.45 11527289.48 4472898.67 4731866.07 4908155.85 3702517.69 2795873.51 3253794.03 Gluc-Cer d18:1-d7/15:0 178272240 228271072 221469872 230172752 227603440 238491904 219803008 212866448 211627888 Hex-Cer d18:1/16:0 97746424 90235919 89875238 34796180.5 34265523 35720103 18085091.11 25982908.5 20201598.25 Hex-Cer d18:1/22:0 120465628.3 395698277.3 100465955.9 40895372.3 36950351.56 33937189.79 22199636.03 33208672.03 35288240.39 Hex-Cer d18:1/23:0 36803713 34230900 32684732 12924273.91 13221248 13453356.45 7572415.07 6473019.38 7944932.79 Hex-Cer d18:1/24:0 222487411.1 211782120 194850264.4 80675999.81 87101033.12 77976200 46136008 46151544.44 46484691.35 Hex-Cer d18:1/24:1 68930720.25 59155768 53790392 26836828.31 19828780.5 19697288.37 15637556.44 178317477.3 12496601.83 Lac-Cer d18:1-d7/15:0 153898512 207271680 195079104 178705888 184396880 188403552 172101680 175276912 167199344 Lac-Cer d18:1/16:0 130243137.7 126987921 126454106.4 40247736.88 40719926.83 43241360.31 23694310.73 22115604.96 22329778.36 Lac-Cer d18:1/22:0 91367432 78698750.81 74263912.56 23261685.18 22260287.07 22421539.18 12479766.65 13253930.91 14681670.44 Lac-Cer d18:1/23:0 25804821.98 23354264.27 22312578.13 6797650.97 6490672.52 6583364.59 3517756.35 3386219.02 3191843.21 Lac-Cer d18:1/24:0 147628464.9 133679450.7 134013793.4 35224418.66 35209020.82 36455446.66 19189524.21 18159763.9 21038360.85 Lac-Cer d18:1/24:1 44652963.75 41434215.97 40948405 11485003.94 10272562.75 11094053.44 6528498.09 6625068.58 6767328.44 SM d18:1/18:1-d9 799151936 1050647040 993858112 1033570176 1006056192 949632640 1014756864 1039794688 1022041920 SM d32:0 72062077.47 92280620.03 85649229.12 32046247.52 33412925.08 34672610.81 15214186.11 17727699.98 17485994.06 SM d32:1 616450096.8 829013818.9 813192422.8 512301245.5 537161000.8 543328367.8 268958236.4 297798529.7 292257476.8 SM d32:2 19516580.63 22793692.8 22878708.02 20279502.54 17781311.81 19154836.48 12914002.53 16225246.93 17059962.91 SM d34:0 1129099640 1107177293 1024466017 794116257.8 778999487.9 738814567.8 670545480 656023144 670160802.6 SM d34:1 8716247981 8693458206 8346790977 6522009378 6729897929 6663552913 4683327181 5045050285 4781385122 SM d34:2 1319007872 1622033160 1617622251 1128843822 1189296304 1165192066 649530210.3 735783210.7 728687637.2 SM d36:0 77106802.81 61933886 58064155.5 39278264.25 40482782.25 35528218.3 23643714.69 32670325.75 33811451.34 SM d36:1 473152731.4 504374901.5 393612935.8 317598485.6 318116979 349246040 254171195.8 226768481.4 261106104.6 SM d36:2 165238263 171859787 159132654.3 141355612.5 141985619.9 140075098 112268438.2 142644023.7 125688892.4 SM d36:3 5040847.2 5614306.72 6064398.1 5427205.78 4843485.27 4240879.05 2672311.03 3810695.13 3709350.67 SM d38:0 93976386.38 92303056.88 82517399.5 41191270.75 45575197.45 28641328.87 28975735.83 28434495.29 29623792.23 SM d38:1 472617816 475728013.1 363864235.1 254943623 280534526.7 235150606.6 172243478.9 164617549.5 168841650.1 SM d38:2 36872851.75 33529289.5 39086672.25 28402855.5 28801683.5 31216362.79 27360464.25 23895333.12 27540789.89 SM d38:3 103108.3 104421.95 149628.27 126214.51 84606.46 5530158.03 53672.36 240117.69 422214.33 SM d40:0 284590135.7 269330687.8 245198198.6 129087455.8 132728324.2 137618667 81797405.7 78807030.83 85253900.81 SM d40:1 2114060628 1994941074 1878994179 1179501891 1214348901 1154702697 782546913.6 766263890.2 827157607.6 SM d40:2 449490440 488793208 377442491.6 254729946.2 296156544 261426971.8 169874476.5 171432958 173406385.5 SM d40:3 11795847.75 8445719 9447528.44 7290217.22 8801894.5 7039573.26 6641607.5 8223106.25 8676852.75 SM d42:0 183862132.1 171191033 155745404.7 64123969 88013652.53 93740122.46 47147393.49 45845682.37 52157018.4 SM d42:1 2737974351 2604052672 2548623556 1903583884 1966057278 1927884721 1142704044 1174502190 1237376078 SM d42:2 2902836116 2675041529 2530712471 1587382374 1683133134 1601143527 976193548.9 994326234.6 1060408200 SM d42:3 465987341.2 486194257.9 395982572 280781731.1 336036318.9 281600776.2 190681789.9 197596318.9 193010885.8 SM d44:1 103531550.8 104311887.8 93275247.66 86835802.12 93079580.36 92600680.25 51071072.62 47287990.02 53909804.79 SM d44:2 104626838.8 106720625.8 98744934.33 84977416.56 80352788.52 92521493.1 49014353.56 48141147.96 52830476.37 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name m/z Cer d18:1-d7/15:0 531.5477 Cer d18:1/16:0 538.5193 Cer d18:1/20:0 594.582 Cer d18:1/22:0 622.613 Cer d18:1/23:0 636.6288 Cer d18:1/24:0 650.6443 Cer d18:1/24:1 648.629 Cer d18:1/25:0 664.6602 DHCer d18:0-d7/13:0 505.5278 DHCer d18:0/16:0 540.535 DHCer d18:0/22:0 624.6288 DHCer d18:0/23:0 638.6444 DHCer d18:0/24:0 652.6597 DHCer d18:0/24:1 650.6445 DHCer d18:0/26:0 680.6915 Gluc-Cer d18:1-d7/15:0 693.5981 Hex-Cer d18:1/16:0 700.5715 Hex-Cer d18:1/22:0 784.6657 Hex-Cer d18:1/23:0 798.6811 Hex-Cer d18:1/24:0 812.6973 Hex-Cer d18:1/24:1 810.6815 Lac-Cer d18:1-d7/15:0 855.6507 Lac-Cer d18:1/16:0 862.6230 Lac-Cer d18:1/22:0 946.7173 Lac-Cer d18:1/23:0 960.7333 Lac-Cer d18:1/24:0 974.7493 Lac-Cer d18:1/24:1 972.7333 SM d18:1/18:1-d9 738.6443 SM d32:0 667.5592 SM d32:1 675.5434 SM d32:2 705.5893 SM d34:0 703.5742 SM d34:1 701.5585 SM d34:2 699.5431 SM d36:0 733.6218 SM d36:1 731.6054 SM d36:2 729.5897 SM d36:3 727.5748 SM d38:0 761.6531 SM d38:1 759.637 SM d38:2 757.6223 SM d38:3 755.6053 SM d40:0 789.6844 SM d40:1 787.6685 SM d40:2 785.6532 SM d40:3 783.6374 SM d42:0 817.7157 SM d42:1 815.6999 SM d42:2 813.6843 SM d42:3 811.6681 SM d44:1 843.7313 SM d44:2 841.7157 METABOLITES_END #END