#METABOLOMICS WORKBENCH DavideS_20240228_063403 DATATRACK_ID:4679 STUDY_ID:ST003113 ANALYSIS_ID:AN005100 PROJECT_ID:PR001933
VERSION             	1
CREATED_ON             	February 29, 2024, 3:20 pm
#PROJECT
PR:PROJECT_TITLE                 	Inhibition of Asparagine Synthetase Effectively Retards Polycystic Kidney
PR:PROJECT_TITLE                 	Disease Progression.
PR:PROJECT_SUMMARY               	Polycystic Kidney Disease (PKD) is a genetic disorder characterized by bilateral
PR:PROJECT_SUMMARY               	cyst formation. We showed that PKD cells and kidneys display metabolic
PR:PROJECT_SUMMARY               	alterations, including the Warburg effect and glutaminolysis, sustained in vitro
PR:PROJECT_SUMMARY               	by the enzyme asparagine synthetase (ASNS). Here, we used antisense
PR:PROJECT_SUMMARY               	oligonucleotides (ASO) against ASNS in orthologous and slowly progressive PKD
PR:PROJECT_SUMMARY               	murine models and show that treatment leads to a drastic reduction of total
PR:PROJECT_SUMMARY               	kidney volume (measured by MRI) and a prominent rescue of renal function in the
PR:PROJECT_SUMMARY               	mouse. Mechanistically, the upregulation of an ATF4-ASNS axis in PKD is driven
PR:PROJECT_SUMMARY               	by the amino acid response (AAR) branch of the integrated stress response (ISR).
PR:PROJECT_SUMMARY               	Metabolic profiling of PKD control kidneys treated with ASNS-ASO or Scr-ASO
PR:PROJECT_SUMMARY               	revealed major changes in the mutants, several of which are rescued by ASNS
PR:PROJECT_SUMMARY               	silencing in vivo. Indeed, ASNS drives glutamine-dependent de novo pyrimidine
PR:PROJECT_SUMMARY               	synthesis and proliferation in cystic epithelia. Notably, while several
PR:PROJECT_SUMMARY               	metabolic pathways were completely corrected by ASNS-ASO, glycolysis was only
PR:PROJECT_SUMMARY               	partially restored. Accordingly, combining the glycolytic inhibitor 2DG with
PR:PROJECT_SUMMARY               	ASNS-ASO further improved efficacy. Our studies identify a new therapeutic
PR:PROJECT_SUMMARY               	target and novel metabolic vulnerabilities in PKD. Of interest, in these tracing
PR:PROJECT_SUMMARY               	studies we could confirm that the pyrimidine biosynthesis pathway is increased
PR:PROJECT_SUMMARY               	and rescued by silencing of Asns.
PR:INSTITUTE                     	San Raffaele University
PR:LAST_NAME                     	Stefanoni
PR:FIRST_NAME                    	Davide
PR:ADDRESS                       	Via Olgettina 58, Milan, Milan, 20132, Italy
PR:EMAIL                         	stefanoni.davide@hsr.it
PR:PHONE                         	+393337686005
#STUDY
ST:STUDY_TITLE                   	Inhibition of Asparagine Synthetase Effectively Retards Polycystic Kidney
ST:STUDY_TITLE                   	Disease Progression, investigated with targeted tracing metabolomics analysis in
ST:STUDY_TITLE                   	MEF cells using 15N2-glutamine.
ST:STUDY_SUMMARY                 	Polycystic Kidney Disease (PKD) is a genetic disorder characterized by bilateral
ST:STUDY_SUMMARY                 	cyst formation. We showed that PKD cells and kidneys display metabolic
ST:STUDY_SUMMARY                 	alterations, including the Warburg effect and glutaminolysis, sustained in vitro
ST:STUDY_SUMMARY                 	by the enzyme asparagine synthetase (ASNS). Here, we used antisense
ST:STUDY_SUMMARY                 	oligonucleotides (ASO) against Asns in orthologous and slowly progressive PKD
ST:STUDY_SUMMARY                 	murine models and show that treatment leads to a drastic reduction of total
ST:STUDY_SUMMARY                 	kidney volume (measured by MRI) and a prominent rescue of renal function in the
ST:STUDY_SUMMARY                 	mouse. Mechanistically, the upregulation of an ATF4-ASNS axis in PKD is driven
ST:STUDY_SUMMARY                 	by the amino acid response (AAR) branch of the integrated stress response (ISR).
ST:STUDY_SUMMARY                 	Metabolic profiling of PKD or control kidneys treated with Asns-ASO or Scr-ASO
ST:STUDY_SUMMARY                 	revealed major changes in the mutants, several of which are rescued by Asns
ST:STUDY_SUMMARY                 	silencing in vivo. Indeed, ASNS drives glutamine-dependent de novo pyrimidine
ST:STUDY_SUMMARY                 	synthesis and proliferation in cystic epithelia. Notably, while several
ST:STUDY_SUMMARY                 	metabolic pathways were completely corrected by Asns-ASO, glycolysis was only
ST:STUDY_SUMMARY                 	partially restored. Accordingly, combining the glycolytic inhibitor 2DG with
ST:STUDY_SUMMARY                 	Asns-ASO further improved efficacy. Our studies identify a new therapeutic
ST:STUDY_SUMMARY                 	target and novel metabolic vulnerabilities in PKD. Of interest, in these tracing
ST:STUDY_SUMMARY                 	studies we could confirm that the pyrimidine biosynthesis pathway is increased
ST:STUDY_SUMMARY                 	and rescued by silencing of Asns.
ST:INSTITUTE                     	San Raffaele University
ST:LAST_NAME                     	Stefanoni
ST:FIRST_NAME                    	Davide
ST:ADDRESS                       	Via Olgettina 58, Milan, Milan, 20132, Italy
ST:EMAIL                         	stefanoni.davide@hsr.it
ST:PHONE                         	+393337686005
#SUBJECT
SU:SUBJECT_TYPE                  	Mammal
SU:SUBJECT_SPECIES               	Mus musculus
SU:TAXONOMY_ID                   	10090
#FACTORS
#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           	-	1	Sample source:MEF cells | Factor:Unlabeled_Untreated_11_1	RAW_FILE_NAME(compound)=CA24_06-1
SUBJECT_SAMPLE_FACTORS           	-	2	Sample source:MEF cells | Factor:Unlabeled_Untreated_11_2	RAW_FILE_NAME(compound)=CA24_06-2
SUBJECT_SAMPLE_FACTORS           	-	3	Sample source:MEF cells | Factor:Unlabeled_Untreated_11_3	RAW_FILE_NAME(compound)=CA24_06-3
SUBJECT_SAMPLE_FACTORS           	-	4	Sample source:MEF cells | Factor:Unlabeled_Untreated_11_4	RAW_FILE_NAME(compound)=CA24_06-4
SUBJECT_SAMPLE_FACTORS           	-	5	Sample source:MEF cells | Factor:Unlabeled_Untreated_11_5	RAW_FILE_NAME(compound)=CA24_06-5
SUBJECT_SAMPLE_FACTORS           	-	6	Sample source:MEF cells | Factor:Unlabeled_Untreated_11_6	RAW_FILE_NAME(compound)=CA24_06-6
SUBJECT_SAMPLE_FACTORS           	-	7	Sample source:MEF cells | Factor:Unlabeled_Untreated_14_1	RAW_FILE_NAME(compound)=CA24_06-7
SUBJECT_SAMPLE_FACTORS           	-	8	Sample source:MEF cells | Factor:Unlabeled_Untreated_14_2	RAW_FILE_NAME(compound)=CA24_06-8
SUBJECT_SAMPLE_FACTORS           	-	9	Sample source:MEF cells | Factor:Unlabeled_Untreated_14_3	RAW_FILE_NAME(compound)=CA24_06-9
SUBJECT_SAMPLE_FACTORS           	-	10	Sample source:MEF cells | Factor:Unlabeled_Untreated_14_4	RAW_FILE_NAME(compound)=CA24_06-10
SUBJECT_SAMPLE_FACTORS           	-	11	Sample source:MEF cells | Factor:Unlabeled_Untreated_14_5	RAW_FILE_NAME(compound)=CA24_06-11
SUBJECT_SAMPLE_FACTORS           	-	12	Sample source:MEF cells | Factor:Unlabeled_Untreated_14_6	RAW_FILE_NAME(compound)=CA24_06-12
SUBJECT_SAMPLE_FACTORS           	-	13	Sample source:MEF cells | Factor:Labeled_Untreated_11_1	RAW_FILE_NAME(compound)=CA24_06-13
SUBJECT_SAMPLE_FACTORS           	-	14	Sample source:MEF cells | Factor:Labeled_Untreated_11_2	RAW_FILE_NAME(compound)=CA24_06-14
SUBJECT_SAMPLE_FACTORS           	-	15	Sample source:MEF cells | Factor:Labeled_Untreated_11_3	RAW_FILE_NAME(compound)=CA24_06-15
SUBJECT_SAMPLE_FACTORS           	-	16	Sample source:MEF cells | Factor:Labeled_Untreated_11_4	RAW_FILE_NAME(compound)=CA24_06-16
SUBJECT_SAMPLE_FACTORS           	-	17	Sample source:MEF cells | Factor:Labeled_Untreated_11_5	RAW_FILE_NAME(compound)=CA24_06-17
SUBJECT_SAMPLE_FACTORS           	-	18	Sample source:MEF cells | Factor:Labeled_Untreated_11_6	RAW_FILE_NAME(compound)=CA24_06-18
SUBJECT_SAMPLE_FACTORS           	-	19	Sample source:MEF cells | Factor:Labeled_Untreated_14_1	RAW_FILE_NAME(compound)=CA24_06-19
SUBJECT_SAMPLE_FACTORS           	-	20	Sample source:MEF cells | Factor:Labeled_Untreated_14_2	RAW_FILE_NAME(compound)=CA24_06-20
SUBJECT_SAMPLE_FACTORS           	-	21	Sample source:MEF cells | Factor:Labeled_Untreated_14_3	RAW_FILE_NAME(compound)=CA24_06-21
SUBJECT_SAMPLE_FACTORS           	-	22	Sample source:MEF cells | Factor:Labeled_Untreated_14_4	RAW_FILE_NAME(compound)=CA24_06-22
SUBJECT_SAMPLE_FACTORS           	-	23	Sample source:MEF cells | Factor:Labeled_Untreated_14_5	RAW_FILE_NAME(compound)=CA24_06-23
SUBJECT_SAMPLE_FACTORS           	-	24	Sample source:MEF cells | Factor:Labeled_Untreated_14_6	RAW_FILE_NAME(compound)=CA24_06-24
SUBJECT_SAMPLE_FACTORS           	-	37	Sample source:MEF cells | Factor:Labeled_siASNS_11_1	RAW_FILE_NAME(compound)=CA24_06-37
SUBJECT_SAMPLE_FACTORS           	-	38	Sample source:MEF cells | Factor:Labeled_siASNS_11_2	RAW_FILE_NAME(compound)=CA24_06-38
SUBJECT_SAMPLE_FACTORS           	-	39	Sample source:MEF cells | Factor:Labeled_siASNS_11_3	RAW_FILE_NAME(compound)=CA24_06-39
SUBJECT_SAMPLE_FACTORS           	-	40	Sample source:MEF cells | Factor:Labeled_siASNS_11_4	RAW_FILE_NAME(compound)=CA24_06-40
SUBJECT_SAMPLE_FACTORS           	-	41	Sample source:MEF cells | Factor:Labeled_siASNS_11_5	RAW_FILE_NAME(compound)=CA24_06-41
SUBJECT_SAMPLE_FACTORS           	-	42	Sample source:MEF cells | Factor:Labeled_siASNS_11_6	RAW_FILE_NAME(compound)=CA24_06-42
SUBJECT_SAMPLE_FACTORS           	-	43	Sample source:MEF cells | Factor:Labeled_siASNS_14_1	RAW_FILE_NAME(compound)=CA24_06-43
SUBJECT_SAMPLE_FACTORS           	-	44	Sample source:MEF cells | Factor:Labeled_siASNS_14_2	RAW_FILE_NAME(compound)=CA24_06-44
SUBJECT_SAMPLE_FACTORS           	-	45	Sample source:MEF cells | Factor:Labeled_siASNS_14_3	RAW_FILE_NAME(compound)=CA24_06-45
SUBJECT_SAMPLE_FACTORS           	-	46	Sample source:MEF cells | Factor:Labeled_siASNS_14_4	RAW_FILE_NAME(compound)=CA24_06-46
SUBJECT_SAMPLE_FACTORS           	-	47	Sample source:MEF cells | Factor:Labeled_siASNS_14_5	RAW_FILE_NAME(compound)=CA24_06-47
SUBJECT_SAMPLE_FACTORS           	-	48	Sample source:MEF cells | Factor:Labeled_siASNS_14_6	RAW_FILE_NAME(compound)=CA24_06-48
#COLLECTION
CO:COLLECTION_SUMMARY            	Immortalized Pkd1+/+ and Pkd1−/− MEFs were cultured in either culture media
CO:COLLECTION_SUMMARY            	enriched with and without 15N2-glutamine. Following 24h were collected, pellet
CO:COLLECTION_SUMMARY            	and instantly extracted with metabolomics lysis buffer.
CO:SAMPLE_TYPE                   	Cultured cells
#TREATMENT
TR:TREATMENT_SUMMARY             	Immortalized Pkd1+/+ and Pkd1−/− MEFs were cultured in either culture media
TR:TREATMENT_SUMMARY             	enriched or not with 15N2-glutamine.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	MEF cells samples were extracted in a ratio of 1milion/1mL of ice cold
SP:SAMPLEPREP_SUMMARY            	extraction solution (methanol:acetonitrile:water 5:3:2 v/v/v). Suspensions were
SP:SAMPLEPREP_SUMMARY            	vortexed continuously for 30 min at 4°C. Insoluble material was removed by
SP:SAMPLEPREP_SUMMARY            	centrifugation at 18,000 g for 10 min at 4°C and supernatants were isolated for
SP:SAMPLEPREP_SUMMARY            	metabolomics analysis by UHPLC-MS.
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_SUMMARY        	5MM_POS_ESI
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	Thermo Vanquish
CH:COLUMN_NAME                   	Phenomenex Kinetex C18 (150 x 2.1mm,1.7um)
CH:SOLVENT_A                     	100% water; 0.1% formic acid
CH:SOLVENT_B                     	100% acetonitrile
CH:FLOW_GRADIENT                 	0-0.5 min 5% B, 0.5-1.1 min 5-95% B, 1.1-2.75 min hold at 95% B, 2.75-3 min
CH:FLOW_GRADIENT                 	95-5% B, 3-5 min hold at 5% B.
CH:FLOW_RATE                     	0.450ml/min
CH:COLUMN_TEMPERATURE            	45°C
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
AN:LABORATORY_NAME               	Angelo D'Alessandro Lab
#MS
MS:INSTRUMENT_NAME               	Thermo Q Exactive Orbitrap
MS:INSTRUMENT_TYPE               	Orbitrap
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	POSITIVE
MS:MS_COMMENTS                   	The Q Exactive was run independently in positive and negative ion mode, scanning
MS:MS_COMMENTS                   	using full MS from 125-1500 m/z at 70,000 resolution and top 10 data-dependent
MS:MS_COMMENTS                   	MS2 at 17,500 resolution. Electrospray ionization was achieved with 45 Arb
MS:MS_COMMENTS                   	sheath gas, 25 Arb auxiliary gas, and 4 kV spray voltage. Calibration was
MS:MS_COMMENTS                   	performed prior to the run using the PierceTM Positive and Negative Ion
MS:MS_COMMENTS                   	Calibration Solutions (Thermo Fisher Scientific). Run order of samples was
MS:MS_COMMENTS                   	randomized and technical replicates were injected after every 4 samples to
MS:MS_COMMENTS                   	assess quality control. Metabolite assignments and correction for expected
MS:MS_COMMENTS                   	natural abundances of 13C isotopes were performed using MAVEN.
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS	Peak Area
MS_METABOLITE_DATA_START
Samples	1	2	3	4	5	6	7	8	9	10	11	12	13	14	15	16	17	18	19	20	21	22	23	24	37	38	39	40	41	42	43	44	45	46	47	48
Factors	Sample source:MEF cells | Factor:Unlabeled_Untreated_11_1	Sample source:MEF cells | Factor:Unlabeled_Untreated_11_2	Sample source:MEF cells | Factor:Unlabeled_Untreated_11_3	Sample source:MEF cells | Factor:Unlabeled_Untreated_11_4	Sample source:MEF cells | Factor:Unlabeled_Untreated_11_5	Sample source:MEF cells | Factor:Unlabeled_Untreated_11_6	Sample source:MEF cells | Factor:Unlabeled_Untreated_14_1	Sample source:MEF cells | Factor:Unlabeled_Untreated_14_2	Sample source:MEF cells | Factor:Unlabeled_Untreated_14_3	Sample source:MEF cells | Factor:Unlabeled_Untreated_14_4	Sample source:MEF cells | Factor:Unlabeled_Untreated_14_5	Sample source:MEF cells | Factor:Unlabeled_Untreated_14_6	Sample source:MEF cells | Factor:Labeled_Untreated_11_1	Sample source:MEF cells | Factor:Labeled_Untreated_11_2	Sample source:MEF cells | Factor:Labeled_Untreated_11_3	Sample source:MEF cells | Factor:Labeled_Untreated_11_4	Sample source:MEF cells | Factor:Labeled_Untreated_11_5	Sample source:MEF cells | Factor:Labeled_Untreated_11_6	Sample source:MEF cells | Factor:Labeled_Untreated_14_1	Sample source:MEF cells | Factor:Labeled_Untreated_14_2	Sample source:MEF cells | Factor:Labeled_Untreated_14_3	Sample source:MEF cells | Factor:Labeled_Untreated_14_4	Sample source:MEF cells | Factor:Labeled_Untreated_14_5	Sample source:MEF cells | Factor:Labeled_Untreated_14_6	Sample source:MEF cells | Factor:Labeled_siASNS_11_1	Sample source:MEF cells | Factor:Labeled_siASNS_11_2	Sample source:MEF cells | Factor:Labeled_siASNS_11_3	Sample source:MEF cells | Factor:Labeled_siASNS_11_4	Sample source:MEF cells | Factor:Labeled_siASNS_11_5	Sample source:MEF cells | Factor:Labeled_siASNS_11_6	Sample source:MEF cells | Factor:Labeled_siASNS_14_1	Sample source:MEF cells | Factor:Labeled_siASNS_14_2	Sample source:MEF cells | Factor:Labeled_siASNS_14_3	Sample source:MEF cells | Factor:Labeled_siASNS_14_4	Sample source:MEF cells | Factor:Labeled_siASNS_14_5	Sample source:MEF cells | Factor:Labeled_siASNS_14_6
Cytosine_C12 PARENT	273769.78	427328.34	384665.66	322014.38	412568.25	378301.72	186471.36	321558.41	3126.82	302902.97	275340.91	283378.97	35760.92	45012.78	46522.46	37934.88	54252.36	44077.36	18659.93	19398.63	20635.76	1718.01	103020.02	21733.90	38420.59	45778.47	62389.89	48536.92	59659.39	48028.45	33890.61	49019.06	40232.26	41075.07	40024.24	40359.52
Cytosine_N15-label-1	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	847.42	985.67	1287.21	994.01	1406.40	1076.25	3084.16	4168.79	4131.90	157.16	3323.96	5127.98	665.92	653.35	887.89	799.94	791.56	724.55	586.98	1843.46	1593.96	2148.40	1053.40	2786.00
N-carbamoyl-L-aspartate_C12 PARENT	70096.59	75015.65	80411.03	108302.25	97993.03	110788.91	76196.59	67345.72	17624.87	75534.58	89830.21	85082.47	10542.82	18641.14	23634.35	16480.72	6941.88	17663.41	14758.68	19266.46	6781.26	12074.52	16489.78	11372.47	8710.08	6494.02	15334.67	9410.74	13026.77	11228.59	13292.68	8439.98	13190.40	13893.60	15672.25	9225.72
N-carbamoyl-L-aspartate_N15-label-1	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	2129.79	2883.23	6088.26	2715.13	3708.28	2989.47	11046.89	10342.92	6192.66	141.18	12328.59	10381.61	5815.79	5293.30	8420.35	5830.32	5950.32	8308.47	4096.89	5667.49	3279.13	3168.72	4337.29	3431.16
thymidine_C12 PARENT	842918.69	825068.56	765103.75	886921.81	882046.94	878989.56	1906071.00	2218468.75	167380.30	2280389.00	2258197.00	2379568.00	746333.94	685091.75	852261.44	841343.69	784131.75	802866.00	2320866.00	2375094.25	1925479.00	36501.19	410769.59	2584070.25	1183882.25	1252127.88	1144299.62	1182820.12	1272024.00	1172009.62	2109535.50	2652011.25	2645418.00	2589102.25	2320525.75	2216848.75
thymidine_15N-label-1	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	59.82	3.93	185.96	83.18	90.84	59.28	306.63	588.02	826.90	79.45	283.86	1100.44	51.67	62.66	80.43	40.78	67.98	93.07	171.63	154.67	181.91	194.88	178.59	276.70
UMP_C12 PARENT	147475.73	111093.60	123611.81	145956.84	199034.25	132916.45	170352.12	168352.44	78130.19	174137.48	237272.45	213282.62	28913.75	30442.16	22744.47	32041.56	16615.06	34459.30	10145.96	7476.08	13886.89	3500.96	8273.75	6381.89	10505.11	4939.83	14306.77	11426.52	11239.40	10611.15	34241.55	32720.28	51545.20	49129.14	61429.84	35054.46
UMP_N15-label-1	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	164.19	193.22	123.91	488.03	146.76	472.36	831.73	1996.15	1742.94	1690.49	1477.77	1682.80	133.26	284.51	517.55	354.82	197.46	313.92	666.27	702.49	409.26	797.60	1021.12	371.12
aspartate_C12 PARENT	1719268.62	1845916.50	1633825.62	2146660.75	1863445.62	2360972.75	420063.25	322861.81	965795.69	428296.16	407050.66	420386.34	15044.75	16475.52	16729.68	18672.75	16684.91	17289.12	12708.65	12678.75	12460.76	9176.14	14141.89	9781.47	17631.17	23643.18	23249.29	16445.84	23857.80	23212.75	17876.37	14510.06	186225.07	23142.96	20078.87	16209.16
aspartate_N15-label-1	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	17381.10	14783.94	12780.17	17485.57	17257.26	15370.02	15221.47	13835.54	22734.47	12575.40	13974.88	13484.31	19093.65	21772.36	21439.85	23266.57	21486.17	29929.70	24364.42	10428.56	22801.60	25709.20	19620.58	26994.52
L-Asparagine_C12 PARENT	2198123.00	1885513.00	2224036.00	2197431.00	2572088.00	2054446.00	2559641.00	2388497.00	1007695.00	2659852.00	2485501.00	2737381.00	18604.18	21892.24	21547.16	23057.31	18967.21	20830.48	69908.54	76450.91	64456.53	12095.84	66677.62	75733.15	58059.19	40555.99	49418.81	47154.31	42826.13	47979.90	57949.50	57940.59	59817.54	59809.08	60283.79	53460.44
L-Asparagine_N15-label-1	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	1269.49	2173.94	1428.38	1969.46	2294.73	1494.71	15574.07	13520.13	16415.92	11467.49	10993.08	9774.55	0.00	0.00	0.00	3463.08	0.00	3990.96	474.02	0.00	0.00	731.24	0.00	331.63
Orotate_C12 PARENT	9816.60	8406.08	9173.70	17844.44	6429.07	12822.64	10596.67	18805.90	10303.90	6851.38	10146.91	14667.78	16362.64	18960.75	11511.34	16577.60	13544.82	18085.50	5850.34	14178.06	16168.84	1674.60	6673.57	12498.29	13197.85	8707.96	0.00	22425.94	8658.20	13033.76	5561.77	14673.63	6398.74	19271.30	20769.11	10095.83
Orotate_N15-label-1	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	14.22	21.28	42.73	0.00	33.23	26.26	110.85	454.77	172.78	546.91	136.52	206.69	24.63	24.74	30.98	25.50	0.00	43.04	174.45	173.81	166.71	202.31	160.89	134.75
Orotidine 5--phosphate_C12 PARENT	104336.30	153830.70	102146.40	115514.00	118558.40	94530.07	24027.32	34635.37	9811.17	41553.95	0.00	44679.22	45631.39	36735.96	43350.48	70335.62	55206.76	23827.54	23554.17	30975.88	0.00	0.00	23338.98	15056.96	0.00	0.00	0.00	0.00	0.00	35736.66	12811.79	0.00	0.00	0.00	0.00	0.00
Orotidine 5--phosphate_N15-label-1	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	0.00	774.39	515.57	548.47	772.39	476.11	666.87	488.84	646.44	585.14	243.45	0.00	361.35	216.74	330.43	1461.12	689.79	1104.98	1147.08	629.69	558.91	1312.75	369.58	1124.01	173.69
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	KEGG ID	m/z	r.t.
Cytosine_C12 PARENT	C00380	112.050781	0.7272118
Cytosine_N15-label-1	C00380	113.047829	0.7289864
N-carbamoyl-L-aspartate_C12 PARENT	C00438	177.053192	0.578
N-carbamoyl-L-aspartate_N15-label-1	C00438	178.050192	0.594
thymidine_C12 PARENT	C00214	242.103656	1.7004
thymidine_15N-label-1	C00214	243.100525	1.6869
UMP_C12 PARENT	C00105	325.043152	0.7954
UMP_N15-label-1	C00105	326.040152	0.7956
aspartate_C12 PARENT	C00049	134.044907	0.6478776
aspartate_N15-label-1	C00049	135.042435	0.6460584
L-Asparagine_C12 PARENT	C00152	133.0593	0.6669309
L-Asparagine_N15-label-1	C00152	134.0585	0.6689659
Orotate_C12 PARENT	C00295	157.026	0.71378
Orotate_N15-label-1	C00295	158.0237	0.7279
Orotidine 5--phosphate_C12 PARENT	C01103	369.0278	0.6670325
Orotidine 5--phosphate_N15-label-1	C01103	370.0249	0.6682451
METABOLITES_END
#END