#METABOLOMICS WORKBENCH Carol_Glez_20220422_081600 DATATRACK_ID:3215 STUDY_ID:ST002150 ANALYSIS_ID:AN003521 PROJECT_ID:PR001363 VERSION 1 CREATED_ON April 24, 2022, 5:47 pm #PROJECT PR:PROJECT_TITLE Sphingomyelin depletion inhibits CXCR4 dynamics and CXCL12-mediated directed PR:PROJECT_TITLE cell migration in human T cells PR:PROJECT_SUMMARY Sphingolipids, ceramides and cholesterol are integral components of cellular PR:PROJECT_SUMMARY membranes, and they also play important roles in signal transduction by PR:PROJECT_SUMMARY regulating the dynamics of membrane receptors through their effects on membrane PR:PROJECT_SUMMARY fluidity. Here, we combined biochemical and functional assays with PR:PROJECT_SUMMARY single-molecule dynamic approaches to demonstrate that the local lipid PR:PROJECT_SUMMARY environment regulates CXCR4 organization and function and modulates PR:PROJECT_SUMMARY chemokine-triggered directed cell migration. Prolonged treatment of T cells with PR:PROJECT_SUMMARY neutral sphingomyelinase promoted the complete and sustained breakdown of PR:PROJECT_SUMMARY sphingomyelins and the accumulation of the corresponding ceramides, which PR:PROJECT_SUMMARY altered both membrane fluidity and CXCR4 nanoclustering and dynamics. Under PR:PROJECT_SUMMARY these conditions CXCR4 retained some CXCL12-mediated signaling activity but PR:PROJECT_SUMMARY failed to promote efficient directed cell migration. Our data underscore a PR:PROJECT_SUMMARY critical role for the local lipid composition at the cell membrane in regulating PR:PROJECT_SUMMARY the lateral mobility of chemokine receptors, and their ability to dynamically PR:PROJECT_SUMMARY increase receptor density at the leading edge to promote efficient cell PR:PROJECT_SUMMARY migration. PR:INSTITUTE Universidad CEU San Pablo PR:DEPARTMENT Center of Metabolomics and Bioanalysis PR:LAST_NAME Gonzalez-Riano PR:FIRST_NAME Carolina PR:ADDRESS km 0, Universidad CEU-San Pablo Urbanización Montepríncipe. M-501 PR:EMAIL carolina.gonzalezriano@ceu.es PR:PHONE 646251045 #STUDY ST:STUDY_TITLE Sphingomyelin depletion inhibits CXCR4 dynamics and CXCL12-mediated directed ST:STUDY_TITLE cell migration in human T cells ST:STUDY_SUMMARY Sphingolipids, ceramides and cholesterol are integral components of cellular ST:STUDY_SUMMARY membranes, and they also play important roles in signal transduction by ST:STUDY_SUMMARY regulating the dynamics of membrane receptors through their effects on membrane ST:STUDY_SUMMARY fluidity. Here, we combined biochemical and functional assays with ST:STUDY_SUMMARY single-molecule dynamic approaches to demonstrate that the local lipid ST:STUDY_SUMMARY environment regulates CXCR4 organization and function and modulates ST:STUDY_SUMMARY chemokine-triggered directed cell migration. Prolonged treatment of T cells with ST:STUDY_SUMMARY neutral sphingomyelinase promoted the complete and sustained breakdown of ST:STUDY_SUMMARY sphingomyelins and the accumulation of the corresponding ceramides, which ST:STUDY_SUMMARY altered both membrane fluidity and CXCR4 nanoclustering and dynamics. Under ST:STUDY_SUMMARY these conditions CXCR4 retained some CXCL12-mediated signaling activity but ST:STUDY_SUMMARY failed to promote efficient directed cell migration. Our data underscore a ST:STUDY_SUMMARY critical role for the local lipid composition at the cell membrane in regulating ST:STUDY_SUMMARY the lateral mobility of chemokine receptors, and their ability to dynamically ST:STUDY_SUMMARY increase receptor density at the leading edge to promote efficient cell ST:STUDY_SUMMARY migration ST:INSTITUTE Universidad CEU San Pablo ST:LAST_NAME Gonzalez-Riano ST:FIRST_NAME Carolina ST:ADDRESS km 0, Universidad CEU-San Pablo Urbanización Montepríncipe. M-501 ST:EMAIL carolina.gonzalezriano@ceu.es ST:PHONE 646251045 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 #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 Blasto_Control_1 Blasto_Control_1 Factor1:CONTROL RAW_FILE_NAME=Blasto_Control_1 SUBJECT_SAMPLE_FACTORS Blasto_Control_4 Blasto_Control_4 Factor1:CONTROL RAW_FILE_NAME=Blasto_Control_4 SUBJECT_SAMPLE_FACTORS Blasto_Control_5 Blasto_Control_5 Factor1:CONTROL RAW_FILE_NAME=Blasto_Control_5 SUBJECT_SAMPLE_FACTORS Blasto_Control_7 Blasto_Control_7 Factor1:CONTROL RAW_FILE_NAME=Blasto_Control_7 SUBJECT_SAMPLE_FACTORS Blasto_Control_8 Blasto_Control_8 Factor1:CONTROL RAW_FILE_NAME=Blasto_Control_8 SUBJECT_SAMPLE_FACTORS Jurkat_Control_2 Jurkat_Control_2 Factor1:CONTROL RAW_FILE_NAME=Jurkat_Control_2 SUBJECT_SAMPLE_FACTORS Jurkat_Control_5 Jurkat_Control_5 Factor1:CONTROL RAW_FILE_NAME=Jurkat_Control_5 SUBJECT_SAMPLE_FACTORS Jurkat_Control_6 Jurkat_Control_6 Factor1:CONTROL RAW_FILE_NAME=Jurkat_Control_6 SUBJECT_SAMPLE_FACTORS Jurkat_Control_8 Jurkat_Control_8 Factor1:CONTROL RAW_FILE_NAME=Jurkat_Control_8 SUBJECT_SAMPLE_FACTORS Jurkat_Control_9 Jurkat_Control_9 Factor1:CONTROL RAW_FILE_NAME=Jurkat_Control_9 SUBJECT_SAMPLE_FACTORS Blasto_SMasa_10 Blasto_SMasa_10 Factor1:CASE RAW_FILE_NAME=Blasto_SMasa_10 SUBJECT_SAMPLE_FACTORS Blasto_SMasa_2 Blasto_SMasa_2 Factor1:CASE RAW_FILE_NAME=Blasto_SMasa_2 SUBJECT_SAMPLE_FACTORS Blasto_SMasa_3 Blasto_SMasa_3 Factor1:CASE RAW_FILE_NAME=Blasto_SMasa_3 SUBJECT_SAMPLE_FACTORS Blasto_SMasa_6 Blasto_SMasa_6 Factor1:CASE RAW_FILE_NAME=Blasto_SMasa_6 SUBJECT_SAMPLE_FACTORS Blasto_SMasa_9 Blasto_SMasa_9 Factor1:CASE RAW_FILE_NAME=Blasto_SMasa_9 SUBJECT_SAMPLE_FACTORS Jurkat_SMasa_1 Jurkat_SMasa_1 Factor1:CASE RAW_FILE_NAME=Jurkat_SMasa_1 SUBJECT_SAMPLE_FACTORS Jurkat_SMase_10 Jurkat_SMase_10 Factor1:CASE RAW_FILE_NAME=Jurkat_SMase_10 SUBJECT_SAMPLE_FACTORS Jurkat_SMasa_3 Jurkat_SMasa_3 Factor1:CASE RAW_FILE_NAME=Jurkat_SMasa_3 SUBJECT_SAMPLE_FACTORS Jurkat_SMasa_4 Jurkat_SMasa_4 Factor1:CASE RAW_FILE_NAME=Jurkat_SMasa_4 SUBJECT_SAMPLE_FACTORS Jurkat_SMasa_7 Jurkat_SMasa_7 Factor1:CASE RAW_FILE_NAME=Jurkat_SMasa_7 SUBJECT_SAMPLE_FACTORS QC_1_B QC_1_B Factor1:QC RAW_FILE_NAME=QC_1_B SUBJECT_SAMPLE_FACTORS QC_2_B QC_2_B Factor1:QC RAW_FILE_NAME=QC_2_B SUBJECT_SAMPLE_FACTORS QC_3_B QC_3_B Factor1:QC RAW_FILE_NAME=QC_3_B SUBJECT_SAMPLE_FACTORS QC_1_J_1 QC_1_J_1 Factor1:QC RAW_FILE_NAME=QC_1_J_1 SUBJECT_SAMPLE_FACTORS QC_1_J_2 QC_1_J_2 Factor1:QC RAW_FILE_NAME=QC_1_J_2 SUBJECT_SAMPLE_FACTORS QC_2_J QC_2_J Factor1:QC RAW_FILE_NAME=QC_2_J SUBJECT_SAMPLE_FACTORS QC_3_J QC_3_J Factor1:QC RAW_FILE_NAME=QC_3_J #COLLECTION CO:COLLECTION_SUMMARY HEK-293T cells were obtained from the ATCC (CRL-11268) and human Jurkat leukemia CO:COLLECTION_SUMMARY CD4+ cells were kindly donated by Dr. J. Alcamí (Centro Nacional de CO:COLLECTION_SUMMARY Microbiología, Instituto de Salud Carlos III, Madrid, Spain). When needed, CO:COLLECTION_SUMMARY Jurkat cells lacking endogenous CXCR4 expression (Jurkat-/-) were transiently CO:COLLECTION_SUMMARY transfected with CXCR4-AcGFP (20 µg; JK-/-X4) using a BioRad electroporator (20 CO:COLLECTION_SUMMARY × 106 cells/400 µL RPMI 1640 with 10% fetal calf serum) and analyzed 24 hours CO:COLLECTION_SUMMARY later. Human peripheral blood mononuclear cells were isolated from buffy coats CO:COLLECTION_SUMMARY by centrifugation through FicollPaque PLUS density gradients (GE Healthcare, CO:COLLECTION_SUMMARY Wakuesha, WI) at 760 × g for 30 minutes at room temperature (RT). They were CO:COLLECTION_SUMMARY then in vitro activated with 20 U/mL of IL-2 (Teceleukin; Roche, Nutley, NJ) and CO:COLLECTION_SUMMARY 5 µg/mL phytohemagglutinin PHA (Roche) to generate T cell blasts. CO:SAMPLE_TYPE HEK cells #TREATMENT TR:TREATMENT_SUMMARY For lipid extraction, cell pellets were mixed with 200 µL of cold (-20°C) TR:TREATMENT_SUMMARY methanol:water (1:1, v/v) and sonicated with an ultrasonic homogenizer (UP200S, TR:TREATMENT_SUMMARY Hielscher Ultrasound Technology, HIELSCHER GmbH, Chamerau, Germany) for 16 TR:TREATMENT_SUMMARY bursts (0.5 second pulse) at 80% amplitude. Homogenates (100 µL) were mixed TR:TREATMENT_SUMMARY with 320 µL of cold (-20°C) methanol containing 1.6 ppm of sphinganine (d17:0) TR:TREATMENT_SUMMARY as the internal standard. Samples were then vortex-mixed for 2 minutes, followed TR:TREATMENT_SUMMARY by the addition of 80 µL of methyl tert-butyl ether. Subsequently, samples were TR:TREATMENT_SUMMARY vortex-mixed (1 hour, RT). After centrifugation (16,000 × g, 15°C, 10 TR:TREATMENT_SUMMARY minutes), samples were used for ultra-high performance liquid chromatography TR:TREATMENT_SUMMARY (UHPLC; Agilent 1290 Infinity II, Agilent Technologies Inc., Santa Clara, CA) TR:TREATMENT_SUMMARY coupled with (ESI) quadrupole time-of-flight (QTOF) mass spectrometry (MS) TR:TREATMENT_SUMMARY (Agilent 6546): 100 µL of each sample was divided between two UHPLC-MS vials TR:TREATMENT_SUMMARY with inserts (50 µL/each) for direct injection into the system for LC-MS TR:TREATMENT_SUMMARY analyses in positive and negative ionization modes. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY For lipid extraction from Jurkat and T cell blasts, cell pellets were mixed with SP:SAMPLEPREP_SUMMARY 200 µL of cold (-20°C) methanol:water (1:1, v/v) and sonicated with an SP:SAMPLEPREP_SUMMARY ultrasonic homogenizer (UP200S, Hielscher Ultrasound Technology, HIELSCHER GmbH, SP:SAMPLEPREP_SUMMARY Chamerau, Germany) for 16 bursts (0.5 second pulse) at 80% amplitude. SP:SAMPLEPREP_SUMMARY Homogenates (100 µL) were mixed with 320 µL of cold (-20°C) methanol SP:SAMPLEPREP_SUMMARY containing 1.6 ppm of sphinganine (d17:0) as the internal standard. Samples were SP:SAMPLEPREP_SUMMARY then vortex-mixed for 2 minutes, followed by the addition of 80 µL of methyl SP:SAMPLEPREP_SUMMARY tert-butyl ether. Subsequently, samples were vortex-mixed (1 hour, RT). After SP:SAMPLEPREP_SUMMARY centrifugation (16,000 × g, 15°C, 10 minutes), samples were used for SP:SAMPLEPREP_SUMMARY ultra-high performance liquid chromatography (UHPLC; Agilent 1290 Infinity II, SP:SAMPLEPREP_SUMMARY Agilent Technologies Inc., Santa Clara, CA) coupled with (ESI) quadrupole SP:SAMPLEPREP_SUMMARY time-of-flight (QTOF) mass spectrometry (MS) (Agilent 6546): 100 µL of each SP:SAMPLEPREP_SUMMARY sample was divided between two UHPLC-MS vials with inserts (50 µL/each) for SP:SAMPLEPREP_SUMMARY direct injection into the system for LC-MS analyses in positive and negative SP:SAMPLEPREP_SUMMARY ionization modes. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY RP-UHPLC-ESI(-)-QTOF MS CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 1290 Infinity II CH:COLUMN_NAME Agilent InfinityLab Poroshell 120 EC–C18, 3.0 × 5 mm, 2.7 μm #ANALYSIS AN:ANALYSIS_TYPE MS AN:LABORATORY_NAME CEMBIO AN:OPERATOR_NAME Carolina Gonzalez Riano #MS MS:INSTRUMENT_NAME Agilent 6546 QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS The Agilent 6545 QTOF mass spectrometer equipped with a dual AJS ESI ion source MS:MS_COMMENTS was set with the following parameters: 150 V fragmentor, 65 V skimmer, 3500 V MS:MS_COMMENTS capillary voltage, 750 V octopole radio frequency voltage, 10 L/min nebulizer MS:MS_COMMENTS gas flow, 200 °C gas temperature, 50 psi nebulizer gas pressure, 12 L/min MS:MS_COMMENTS sheath gas flow, and 300 °C sheath gas temperature. Data were collected in MS:MS_COMMENTS positive and negative ESI modes in separate runs, operated in full scan mode MS:MS_COMMENTS from 50 to 1800 m/z with a scan rate of 3 spectra/s. A solution consisting of MS:MS_COMMENTS two reference mass compounds were used throughout the whole analysis: purine MS:MS_COMMENTS (C5H4N4) at m/z 121.0509 for the positive and m/z 119.0363 for the negative MS:MS_COMMENTS ionization modes; and HP-0921 (C18H18O6N3P3F24) at m/z 922.0098 for the positive MS:MS_COMMENTS and m/z 980.0163 (HP-0921+acetate) for the negative ionization modes. These MS:MS_COMMENTS masses were continuously infused into the system through an Agilent 1260 Iso MS:MS_COMMENTS Pump at a 1 mL/min (split ratio 1:100) to provide a constant mass correction. MS:MS_COMMENTS Ten Iterative-MS/MS runs were performed for both ion modes at the end of the MS:MS_COMMENTS analytical run. They were operated with an MS and MS/MS scan rates of 3 MS:MS_COMMENTS spectra/s, 40–1800 m/z mass window, a narrow (∼ 1.3 amu) MS/MS isolation MS:MS_COMMENTS width, 3 precursors per cycle, and 5000 counts and 0.001% of MS/MS threshold. MS:MS_COMMENTS Five iterative-MS/MS runs were set with a collision energy of 20 eV, and the MS:MS_COMMENTS subsequent five runs were performed at 40 eV. References masses and contaminants MS:MS_COMMENTS detected in blank samples were excluded from the analysis to avoid inclusion in MS:MS_COMMENTS the iterative-MS/MS. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS AREA MS_METABOLITE_DATA_START Samples Blasto_Control_1 Blasto_Control_4 Blasto_Control_5 Blasto_Control_7 Blasto_Control_8 Jurkat_Control_2 Jurkat_Control_5 Jurkat_Control_6 Jurkat_Control_8 Jurkat_Control_9 Blasto_SMasa_10 Blasto_SMasa_2 Blasto_SMasa_3 Blasto_SMasa_6 Blasto_SMasa_9 Jurkat_SMasa_1 Jurkat_SMase_10 Jurkat_SMasa_3 Jurkat_SMasa_4 Jurkat_SMasa_7 QC_1_B QC_2_B QC_3_B QC_1_J_1 QC_1_J_2 QC_2_J QC_3_J Factors Factor1:CONTROL Factor1:CONTROL Factor1:CONTROL Factor1:CONTROL Factor1:CONTROL Factor1:CONTROL Factor1:CONTROL Factor1:CONTROL Factor1:CONTROL Factor1:CONTROL Factor1:CASE Factor1:CASE Factor1:CASE Factor1:CASE Factor1:CASE Factor1:CASE Factor1:CASE Factor1:CASE Factor1:CASE Factor1:CASE Factor1:QC Factor1:QC Factor1:QC Factor1:QC Factor1:QC Factor1:QC Factor1:QC C17 Sphinganine (IS) 152773 161940 156418 161832 161620 162736 158280 163579 163365 163913 159417 161851 148734 155813 158618 156785 157368 153040 155340 159222 172102 167977 164710 162746 160591 150996 157577 Cer(d18:2/20:0) 108803 99428 99904 71461 78978 298284 613332 603305 315202 299860 46013 56858 47191 54340 69013 187402 210682 188542 204649 205363 216606 227099 209052 102783 103362 129697 128019 Cer(d18:1(4E)/16:0(2OH)) 6596 4747 5766 2976 33094 40921 41520 37870 29846 19060 16462 9928 13034 10965 64769 64999 69749 71295 52132 21317 21461 23709 51535 55299 47140 48094 Cer(d18:1/17:0) 204861 158810 151645 107276 108250 928638 1759152 1800699 1025605 911608 129565 128106 117340 132710 115018 789466 831048 889222 935549 733113 741723 743247 736321 563416 572126 518129 529122 Cer(d18:0/18:0) 241657 249065 352615 254597 296338 379979 983165 944837 401851 389987 348429 339743 316595 366747 364906 355683 394270 369974 350815 395239 474760 473403 452095 386139 357022 364460 369539 Cer(d18:2/18:0) 210216 135437 129484 85083 94529 525312 811933 818740 576500 530395 189240 173999 155256 190001 170436 632888 684586 672139 682806 646684 446764 428776 426221 485730 477560 449052 465672 Cer(d18:1/24:0) 2745599 3066197 3673738 2626194 2633555 8911384 15602200 15843935 9828343 8250025 3538705 3392508 3186685 3721550 3786918 8798892 8880535 9030210 8416382 9466109 7889315 7804183 7534823 6500992 6485373 6670869 6530221 Cer(d18:1/15:0) 356717 195543 224051 119822 119758 1455970 2014907 2025031 1602361 1474448 414985 415324 400052 448342 371665 3004870 2974576 3657459 3775982 2972442 1017086 1007972 1010609 2146406 2117813 1834861 2020579 Cer(d18:1/16:0) 8977866 4945032 7044093 4014194 4165972 54818402 66735019 61815670 60948339 56494827 10407646 9798034 9016903 10884874 9185952 78384245 58939445 93624854 92895046 83876224 33975488 37113992 35380967 56572630 58735984 18936027 58172325 Cer(d18:1/14:0) 733437 348986 519874 241677 244170 2420907 3094424 3053361 2753291 2512338 609389 608610 637154 740374 612448 3915639 3645859 4649720 4844287 3935361 1689950 1672636 1665719 2750955 2886734 2573343 2735025 Cer(d18:2/16:0) 874386 449302 514673 245984 264589 2023805 2767746 2702932 2260267 1982886 1546538 1535582 1510887 1703273 1446314 7171738 6353944 8053316 7869668 6671579 1494188 1474874 1417455 4914531 4976877 4604871 4623885 Cer(d18:0/14:0) 79519 48515 63284 46516 48719 112998 334756 311804 124145 117942 157043 163728 135470 175624 164416 266202 297419 295551 289916 270596 143539 136783 136149 247749 251225 224194 243437 Cer(d18:0/16:0) 548826 412924 638125 625395 576055 2312887 5639659 5352999 2507784 2454728 2633777 3145959 2492821 2263730 2085440 6542492 7202670 7565866 7278779 7325012 2184094 2165816 2152093 5539731 5552929 4955684 5360111 Cer(d18:1/18:0) 497052 440720 450095 343278 367946 2469398 4953077 5179745 2736800 2400051 287879 261049 232478 295281 278368 1691734 1602116 1884403 1817750 1806137 2018885 1988621 1947265 1150558 1193859 1084008 1077693 Cer(d18:1/20:0) 378049 358545 440533 341994 344663 1493496 2883095 2879445 1608417 1552680 249952 231595 216475 266324 245060 831138 845069 880210 852773 876615 1307183 1301167 1243560 587276 585425 559762 580072 Cer(d18:1/22:0) 1091079 1174031 1351635 1013094 1057315 4073411 7105389 7024168 4490210 4026700 1008799 867988 884005 1029497 1049137 3234048 3632948 3794986 3500933 4043973 3412301 3621268 3513453 2497612 2480235 2320105 2298374 Cer(d18:0/22:0) 362271 378596 477121 489397 517428 597132 1623436 1585207 615833 612039 551657 613308 518850 510581 513685 514065 569350 563564 507126 616403 753793 755077 764016 584056 565135 546694 525642 Cer(d18:1/24:1(15Z)) 5887817 6043485 6473211 5489300 5535261 25264050 37412556 37333776 28113834 23424444 6904605 6813667 6314527 7125698 6805909 21265977 26514573 29263890 26345730 30283052 18933903 21703009 20584869 18384317 18872471 16828428 16766543 Cer(d18:0/24:0) 469625 501655 803123 748972 735611 858327 2764010 2585616 909975 801805 1239045 1469670 1297610 1110675 1082847 838334 875045 914779 824805 979264 1242457 1192913 1195774 989313 965340 1085942 1037171 Cer(d18:2/22:0) 590471 563459 708456 489333 519855 2365929 4025553 3995712 2597349 2481110 621987 557570 546933 656883 614412 2104836 2201994 2267325 2280525 2342809 1936759 1932519 1866103 1517072 1523090 1462001 1507337 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Retention time (min) units Formula Mass C17 Sphinganine (IS) 1.92 peak area C17 H37 N O2 287.2822 Cer(d18:2/20:0) 11.61 peak area C38 H73 N O3 591.5585 Cer(d18:1(4E)/16:0(2OH)) 6.46 peak area C34 H67 N O4 553.5061 Cer(d18:1/17:0) 10.43 peak area C35 H69 N O3 551.5272 Cer(d18:0/18:0) 11.71 peak area C36 H73 N O3 567.5584 Cer(d18:2/18:0) 9.65 peak area C36 H69 N O3 563.5268 Cer(d18:1/24:0) 12.58 peak area C42 H83 N O3 649.6376 Cer(d18:1/15:0) 8.11 peak area C33 H65 N O3 523.4964 Cer(d18:1/16:0) 9.15 peak area C34 H67 N O3 537.5124 Cer(d18:1/14:0) 7.26 peak area C32 H63 N O3 509.4816 Cer(d18:2/16:0) 7.55 peak area C34 H65 N O3 535.4964 Cer(d18:0/14:0) 7.84 peak area C32 H65 N O3 511.4958 Cer(d18:0/16:0) 10.03 peak area C34 H69 N O3 539.5276 Cer(d18:1/18:0) 11.46 peak area C36 H71 N O3 565.5432 Cer(d18:1/20:0) 11.96 peak area C38 H75 N O3 593.5743 Cer(d18:1/22:0) 12.26 peak area C40 H79 N O3 621.606 Cer(d18:0/22:0) 12.38 peak area C40 H81 N O3 623.6209 Cer(d18:1/24:1(15Z)) 12.26 peak area C42 H81 N O3 647.6221 Cer(d18:0/24:0) 12.72 peak area C42 H85 N O3 651.6525 Cer(d18:2/22:0) 11.97 peak area C40 H77 N O3 619.5899 METABOLITES_END #END