#METABOLOMICS WORKBENCH Carol_Glez_20220422_081600 DATATRACK_ID:3215 STUDY_ID:ST002150 ANALYSIS_ID:AN003520 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 POSITIVE 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) 10276650 10091210 10048161 10081603 11423967 11093067 11055900 10696921 10846303 10624577 9513550 9915491 10725081 9900391 10859222 11207130 10767055 10310720 10167611 10782159 12090679 10176391 11074844 11202153 10799677 10755133 10906130 SM(d18:1/14:0) 180107 213341 178403 185941 178429 258461 274896 243526 246167 242867 0 0 0 0 0 0 0 0 0 0 118824 120493 112571 149871 140842 152696 146897 SM(d18:0/14:0) 38479 43947 20484 19051 17810 33809 30176 28830 30870 30397 0 0 0 0 0 0 0 0 0 0 17449 13316 16713 16756 19296 16930 14936 SM(d18:1/15:0) 197291 211578 180548 183819 173422 280540 285702 275788 284145 260718 0 0 0 0 0 0 0 0 0 0 125590 122777 128881 174664 178656 172536 171021 SM(d18:2/16:0) 297158 339907 297017 297358 303080 527133 529749 501647 489358 486210 0 0 0 0 0 0 0 0 0 0 232462 239742 197190 335501 338109 306761 319388 SM(d18:1/16:0) 3622483 3990003 3282781 3432842 3454173 5734854 6130684 5642264 5646034 5536511 0 0 0 0 0 0 0 0 0 0 1886788 1829607 1867880 2967258 2876214 2857950 2897812 SM(d18:0/16:0) 457726 493455 248743 271578 267250 594817 608753 586933 578303 555740 0 0 0 0 0 0 0 0 0 0 202558 196788 191463 318812 325877 303971 316228 SM(d18:1/17:0) 114160 137212 96141 91157 124630 103664 108347 77686 104573 103572 0 0 0 0 0 0 0 0 0 0 82490 77118 66626 59396 37367 76205 71005 SM(d18:1/16:0(2OH)) 7919 9738 11214 9613 11461 15398 18305 15065 14344 15634 0 0 0 0 0 0 0 0 0 0 6114 7562 6203 9897 7562 10496 8366 SM(d18:2/18:0) 218261 233050 212949 188586 200632 199314 191083 193914 191134 188535 0 0 0 0 0 0 0 0 0 0 257330 262091 251502 188627 182473 184921 192596 SM(d18:1/18:0) 307399 317422 211032 194685 196958 163588 160719 160869 166286 163241 0 0 0 0 0 0 0 0 0 0 177206 169700 168100 124642 121041 109873 120329 SM(d18:0/18:0) 96537 99135 54396 56140 53721 71171 67428 67940 63945 64468 0 0 0 0 0 0 0 0 0 0 55223 53916 55508 45333 44383 45090 46141 SM(d18:2/20:0) 87783 91987 57921 56597 49410 60942 52149 54132 51574 48324 0 0 0 0 0 0 0 0 0 0 42632 41541 44347 44728 38972 38992 40623 SM(d18:1/20:0) 217841 220294 191704 173196 170216 190181 191485 183801 183458 186547 0 0 0 0 0 0 0 0 0 0 135672 138025 115748 71989 69884 61222 61237 SM(d18:2/22:0) 135478 144718 110778 110429 111363 107091 104245 100183 101992 105355 0 0 0 0 0 0 0 0 0 0 98245 99540 86330 77611 81222 74426 76701 SM(d18:1/22:0) 496145 506317 398760 392660 393451 298683 292583 277529 297867 285386 0 0 0 0 0 0 0 0 0 0 318581 308856 293505 233940 235473 234136 223042 SM(d18:0/22:0) 146236 148816 113441 112066 112995 109484 95075 101736 99948 97539 0 0 0 0 0 0 0 0 0 0 116094 112870 116050 90021 92307 90011 80237 SM(d18:2/24:1) 554677 583981 345723 357222 346398 212608 234544 222686 227909 214915 0 0 0 0 0 0 0 0 0 0 270156 256835 261422 145085 141262 143607 142186 SM(d18:1/24:1(15Z)) 2010251 2189870 1443051 1434232 1452560 1258465 1316838 1219227 1276623 1227195 0 0 0 0 0 0 0 0 0 0 1079286 1017159 1007848 761917 785473 766486 747596 SM(d18:1/24:0) 635079 683166 455024 441607 450913 417090 407837 396611 384943 381870 0 0 0 0 0 0 0 0 0 0 355504 339177 339434 248906 253323 243781 237594 SM(d18:0/24:0) 165667 173434 108834 102075 108414 103734 85671 94070 90513 86884 0 0 0 0 0 0 0 0 0 0 96558 96230 93718 81928 72726 74450 76719 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Retention time (min) units Formula Mass C17 Sphinganine (IS) 1.91 peak area C17 H37 N O2 287.2829 SM(d18:1/14:0) 5.88 peak area C37 H75 N2 O6 P 674.5361 SM(d18:0/14:0) 6.34 peak area C37 H77 N2 O6 P 676.5513 SM(d18:1/15:0) 6.48 peak area C38 H77 N2 O6 P 688.5512 SM(d18:2/16:0) 6.11 peak area C39 H77 N2 O6 P 700.5515 SM(d18:1/16:0) 7.18 peak area C39 H79 N2 O6 P 702.568 SM(d18:0/16:0) 7.83 peak area C39 H81 N2 O6 P 704.5829 SM(d18:1/17:0) 8.03 peak area C40 H81 N2 O6 P 716.582 SM(d18:1/16:0(2OH)) 5.42 peak area C39 H79 N2 O7 P 718.5616 SM(d18:2/18:0) 7.5 peak area C41 H81 N2 O6 P 728.583 SM(d18:1/18:0) 9.06 peak area C41 H83 N2 O6 P 730.5984 SM(d18:0/18:0) 9.95 peak area C41 H85 N2 O6 P 732.6143 SM(d18:2/20:0) 9.48 peak area C43 H85 N2 O6 P 756.6137 SM(d18:1/20:0) 11.38 peak area C43 H87 N2 O6 P 758.6292 SM(d18:2/22:0) 11.43 peak area C45 H89 N2 O6 P 784.6448 SM(d18:1/22:0) 11.91 peak area C45 H91 N2 O6 P 786.6614 SM(d18:0/22:0) 12.03 peak area C45 H93 N2 O6 P 788.6767 SM(d18:2/24:1) 11.58 peak area C47 H91 N2 O6 P 810.6607 SM(d18:1/24:1(15Z)) 11.91 peak area C47 H93 N2 O6 P 812.6775 SM(d18:1/24:0) 12.2 peak area C47 H95 N2 O6 P 814.6926 SM(d18:0/24:0) 12.32 peak area C47 H97 N2 O6 P 816.7087 METABOLITES_END #END