#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