#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