#METABOLOMICS WORKBENCH SIMR_Core_Facility_20221018_032757 DATATRACK_ID:3518 STUDY_ID:ST002318 ANALYSIS_ID:AN003785 PROJECT_ID:PR001485
VERSION             	1
CREATED_ON             	October 19, 2022, 8:56 am
#PROJECT
PR:PROJECT_TITLE                 	Mass spectroscopy‑based proteomics and metabolomics analysis of
PR:PROJECT_TITLE                 	triple‑positive breast cancer cells treated with tamoxifen and/ or trastuzumab
PR:PROJECT_TYPE                  	LC-MS/MS
PR:PROJECT_SUMMARY               	HER2-enriched breast cancer with high levels of hormone receptor expression,
PR:PROJECT_SUMMARY               	known as "triple positive" breast cancer, may represent a new entity with a
PR:PROJECT_SUMMARY               	relatively favourable prognosis against which the combination of chemotherapy,
PR:PROJECT_SUMMARY               	HER-2 inhibition, and endocrine treatment may be considered overtreatment. We
PR:PROJECT_SUMMARY               	explored the effect of the anticancer drugs tamoxifen and trastuzumab, both
PR:PROJECT_SUMMARY               	separately and in combination, on the integrated proteomic and metabolic profile
PR:PROJECT_SUMMARY               	of "triple positive" breast cancer cells (BT-474). Method We employed
PR:PROJECT_SUMMARY               	ultra-high-performance liquid chromatography-quadrupole time of flight mass
PR:PROJECT_SUMMARY               	spectrometry using a Bruker timsTOF to investigate changes in BT-474 cell line
PR:PROJECT_SUMMARY               	treated with either tamoxifen, trastuzumab or a combination. Differentially
PR:PROJECT_SUMMARY               	abundant metabolites were identified using the Bruker Human Metabolome Database
PR:PROJECT_SUMMARY               	metabolite library and proteins using the Uniprot proteome for Homo sapiens
PR:PROJECT_SUMMARY               	using MetaboScape and MaxQuant, respectively, for identification and
PR:PROJECT_SUMMARY               	quantitation. Results A total of 77 proteins and 85 metabolites were found to
PR:PROJECT_SUMMARY               	significantly differ in abundance in BT-474 treated cells with tamoxifen 5
PR:PROJECT_SUMMARY               	μM/and or trastuzumab 2.5 μM. Findings suggest that by targeting important
PR:PROJECT_SUMMARY               	cellular signalling pathways which regulate cell growth, apoptosis,
PR:PROJECT_SUMMARY               	proliferation, and chemoresistance, these medicines have a considerable
PR:PROJECT_SUMMARY               	anti-growth effect in BT-474 cells. Pathways enriched for dysregulation include
PR:PROJECT_SUMMARY               	RNA splicing, neutrophil degranulation and activation, cellular redox
PR:PROJECT_SUMMARY               	homeostasis, mitochondrial transmembrane transport, ferroptosis and necroptosis,
PR:PROJECT_SUMMARY               	ABC transporters and central carbon metabolism. Conclusion Our findings in
PR:PROJECT_SUMMARY               	protein and metabolite level research revealed that anti-cancer drug therapy had
PR:PROJECT_SUMMARY               	a significant impact on the key signalling pathways and molecular processes in
PR:PROJECT_SUMMARY               	triple positive BT-474 cell lines.
PR:INSTITUTE                     	Sharjah Institute for Medical Research
PR:DEPARTMENT                    	Sharjah Institute for Medical Research
PR:LABORATORY                    	Biomarker Discovery Group
PR:LAST_NAME                     	Facility
PR:FIRST_NAME                    	Core
PR:ADDRESS                       	M32, SIMR, College of Pharmacy, Health Sciences, University of Sharjah, Sharjah,
PR:ADDRESS                       	UAE, Sharjah, 000, United Arab Emirates
PR:EMAIL                         	tims-tof@sharjah.ac.ae
PR:PHONE                         	+971 6 5057656
#STUDY
ST:STUDY_TITLE                   	Mass spectroscopy‑based proteomics and metabolomics analysis of
ST:STUDY_TITLE                   	triple‑positive breast cancer cells treated with trastuzumab
ST:STUDY_SUMMARY                 	HER2-enriched breast cancer with high levels of hormone receptor expression,
ST:STUDY_SUMMARY                 	known as "triple positive" breast cancer, may represent a new entity with a
ST:STUDY_SUMMARY                 	relatively favourable prognosis against which the combination of chemotherapy,
ST:STUDY_SUMMARY                 	HER-2 inhibition, and endocrine treatment may be considered overtreatment. We
ST:STUDY_SUMMARY                 	explored the effect of the anticancer drugs tamoxifen and trastuzumab, both
ST:STUDY_SUMMARY                 	separately and in combination, on the integrated proteomic and metabolic profile
ST:STUDY_SUMMARY                 	of "triple positive" breast cancer cells (BT-474). Method We employed
ST:STUDY_SUMMARY                 	ultra-high-performance liquid chromatography-quadrupole time of flight mass
ST:STUDY_SUMMARY                 	spectrometry using a Bruker timsTOF to investigate changes in BT-474 cell line
ST:STUDY_SUMMARY                 	treated with either tamoxifen, trastuzumab or a combination. Differentially
ST:STUDY_SUMMARY                 	abundant metabolites were identified using the Bruker Human Metabolome Database
ST:STUDY_SUMMARY                 	metabolite library and proteins using the Uniprot proteome for Homo sapiens
ST:STUDY_SUMMARY                 	using MetaboScape and MaxQuant, respectively, for identification and
ST:STUDY_SUMMARY                 	quantitation. Results A total of 77 proteins and 85 metabolites were found to
ST:STUDY_SUMMARY                 	significantly differ in abundance in BT-474 treated cells with tamoxifen 5
ST:STUDY_SUMMARY                 	μM/and or trastuzumab 2.5 μM. Findings suggest that by targeting important
ST:STUDY_SUMMARY                 	cellular signalling pathways which regulate cell growth, apoptosis,
ST:STUDY_SUMMARY                 	proliferation, and chemoresistance, these medicines have a considerable
ST:STUDY_SUMMARY                 	anti-growth effect in BT-474 cells. Pathways enriched for dysregulation include
ST:STUDY_SUMMARY                 	RNA splicing, neutrophil degranulation and activation, cellular redox
ST:STUDY_SUMMARY                 	homeostasis, mitochondrial transmembrane transport, ferroptosis and necroptosis,
ST:STUDY_SUMMARY                 	ABC transporters and central carbon metabolism. Conclusion Our findings in
ST:STUDY_SUMMARY                 	protein and metabolite level research revealed that anti-cancer drug therapy had
ST:STUDY_SUMMARY                 	a significant impact on the key signalling pathways and molecular processes in
ST:STUDY_SUMMARY                 	triple positive BT-474 cell lines.
ST:INSTITUTE                     	University of Sharjah
ST:DEPARTMENT                    	Sharjah Institute for Medical Research
ST:LABORATORY                    	Biomarker Discovery Group
ST:LAST_NAME                     	Soares
ST:FIRST_NAME                    	Nelson
ST:ADDRESS                       	M32, SIMR, College of Pharmacy, Health Sciences, University of Sharjah
ST:EMAIL                         	nsoares@sharjah.ac.ae
ST:PHONE                         	065057656
#SUBJECT
SU:SUBJECT_TYPE                  	Human
SU:SUBJECT_SPECIES               	Homo sapiens
SU:TAXONOMY_ID                   	9606
#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           	-	1A_93_1_328	Treatment:Control	RAW_FILE_NAME=1A''_93_1_328.d
SUBJECT_SAMPLE_FACTORS           	-	1A_93_1_331	Treatment:Control	RAW_FILE_NAME=1A''_93_1_331.d
SUBJECT_SAMPLE_FACTORS           	-	1A_92_1_327	Treatment:Control	RAW_FILE_NAME=1A'_92_1_327.d
SUBJECT_SAMPLE_FACTORS           	-	1A_92_1_330	Treatment:Control	RAW_FILE_NAME=1A'_92_1_330.d
SUBJECT_SAMPLE_FACTORS           	-	1A_91_1_326	Treatment:Control	RAW_FILE_NAME=1A_91_1_326.d
SUBJECT_SAMPLE_FACTORS           	-	1A_91_1_329	Treatment:Control	RAW_FILE_NAME=1A_91_1_329.d
SUBJECT_SAMPLE_FACTORS           	-	3a_-2_93_1_336	Treatment:TRASTUZUMAB	RAW_FILE_NAME=3a"-2_93_1_336.d
SUBJECT_SAMPLE_FACTORS           	-	3a_-7_93_1_386	Treatment:TRASTUZUMAB	RAW_FILE_NAME=3a"-7_93_1_386.d
SUBJECT_SAMPLE_FACTORS           	-	3a_-8_93_1_387	Treatment:TRASTUZUMAB	RAW_FILE_NAME=3a"-8_93_1_387.d
SUBJECT_SAMPLE_FACTORS           	-	3a_-7_92_1_382	Treatment:TRASTUZUMAB	RAW_FILE_NAME=3a'-7_92_1_382.d
SUBJECT_SAMPLE_FACTORS           	-	3a_-8_92_1_383	Treatment:TRASTUZUMAB	RAW_FILE_NAME=3a'-8_92_1_383.d
SUBJECT_SAMPLE_FACTORS           	-	3a-71_91_1_384	Treatment:TRASTUZUMAB	RAW_FILE_NAME=3a-71_91_1_384.d
SUBJECT_SAMPLE_FACTORS           	-	3a-8_91_1_385	Treatment:TRASTUZUMAB	RAW_FILE_NAME=3a-8_91_1_385.d
SUBJECT_SAMPLE_FACTORS           	-	3ar_91_1_333	Treatment:TRASTUZUMAB	RAW_FILE_NAME=3ar_91_1_333.d
#COLLECTION
CO:COLLECTION_SUMMARY            	The BT-474 BC cell line utilized in this study was cultured as monolayers in
CO:COLLECTION_SUMMARY            	DMEM medium supplemented with 10% fetal bovine serum and 1%
CO:COLLECTION_SUMMARY            	penicillin/streptomycin (Sigma Aldrich, St. Louis, MO, USA). All cultures were
CO:COLLECTION_SUMMARY            	incubated at 37 °C in a humidified atmosphere of 5% CO2.
CO:SAMPLE_TYPE                   	Breast cancer cells
CO:STORAGE_CONDITIONS            	Described in summary
#TREATMENT
TR:TREATMENT_SUMMARY             	Triplicate flasks were prepared for each treatment condition for each analysis
TR:TREATMENT_SUMMARY             	(metabolomic and proteomic) for a total of 24 flasks. Two million cells were
TR:TREATMENT_SUMMARY             	seeded in each 75 cm2 tissue culture flask and incubated for 24 h. The cells
TR:TREATMENT_SUMMARY             	were then treated with Tamoxifen (5 μM) and/or Trastuzumab (2.5 μM) for 24 h.
TR:TREATMENT_SUMMARY             	These concentrations correspond to the IC50 of these compounds with BT-474
TR:TREATMENT_SUMMARY             	cells, as determined by cytotoxicity assays (data not shown). Control cells were
TR:TREATMENT_SUMMARY             	treated with vehicle (dimethyl sulfoxide (DMSO) at 0.5% for 24 h. Following the
TR:TREATMENT_SUMMARY             	incubation period, cells were collected by trypsinization and washed twice with
TR:TREATMENT_SUMMARY             	phosphate-buffered saline solution (PBS) before re-suspending in 1 mL 1 × PBS
TR:TREATMENT_SUMMARY             	for further analysis. Finally, cells were collected as pellets by centrifugation
TR:TREATMENT_SUMMARY             	at 1200 rounds per minute (rpm) for 10 min at room temperature. To negate the
TR:TREATMENT_SUMMARY             	effect of Circadian rhythms on the response of cells to treatment, cells were
TR:TREATMENT_SUMMARY             	kept under the same conditions during the entire incubation period and the cell
TR:TREATMENT_SUMMARY             	collection was done concurrently for all samples. In addition, the same number
TR:TREATMENT_SUMMARY             	of cells were used for each sample to avoid the effect of variation in cell
TR:TREATMENT_SUMMARY             	numbers.
TR:TREATMENT                     	Drugs
TR:TREATMENT_COMPOUND            	Tamoxifen and/or Trastuzumab
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Sample metabolite extraction A volume of 1 mL of the extraction solvent
SP:SAMPLEPREP_SUMMARY            	(methanol+0.1% formic acid) was added to the cell pellets to quench cells. The
SP:SAMPLEPREP_SUMMARY            	cells were then vortexed for 2 min to ensure the quantitative extraction of the
SP:SAMPLEPREP_SUMMARY            	metabolites and stored on ice for 1 h, during which the samples were vortexed
SP:SAMPLEPREP_SUMMARY            	every 15 min. After this, the insoluble cell matrices were collected and
SP:SAMPLEPREP_SUMMARY            	transferred to centrifuge tubes, intermittent ultrasonication using the COPLEY
SP:SAMPLEPREP_SUMMARY            	sonicator (QSONICA SONICATOR, USA) under 30% amplifier and for 30 s with an ice
SP:SAMPLEPREP_SUMMARY            	bath employed throughout the process. Following that, cells debris were then
SP:SAMPLEPREP_SUMMARY            	centrifuged (15,000 rpm, 10 min, − 4 °C) and the sample supernatants were
SP:SAMPLEPREP_SUMMARY            	collected and transferred to LC vials for drying in the EZ-2 Plus
SP:SAMPLEPREP_SUMMARY            	(GeneVac-Ipswich, UK) at 37±1 °C. Dried samples were resuspended with 200 µL
SP:SAMPLEPREP_SUMMARY            	(water+0.1% formic acid), and vortexed for 2 min. Finally, the samples were
SP:SAMPLEPREP_SUMMARY            	filtered using a hydrophilic Nylon Syringe Filter of 0.45 µm pore size and
SP:SAMPLEPREP_SUMMARY            	analyzed by Q-TOF MS
SP:PROCESSING_STORAGE_CONDITIONS 	Described in summary
SP:EXTRACT_STORAGE               	Described in summary
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_SUMMARY        	Samples were chromatographically separated by inline reversed-phase
CH:CHROMATOGRAPHY_SUMMARY        	chromatography using the Elute HPG 1300 pumps and Elute Autosampler (Bruker,
CH:CHROMATOGRAPHY_SUMMARY        	Darmstadt, Germany) with solvent A 0.1% FA in HPLC grade water and solvent B
CH:CHROMATOGRAPHY_SUMMARY        	0.1% FA in ACN. A Hamilton Intensity Solo 2 C18 column (100 mm × 2.1 mm, 1.8
CH:CHROMATOGRAPHY_SUMMARY        	μm beads) was maintained at 35 ℃ (metabolomics analyses).
CH:CHROMATOGRAPHY_TYPE           	Reversed phase LC
CH:INSTRUMENT_NAME               	Bruker Elute HPG 1300
CH:COLUMN_NAME                   	Hamilton Intensity Solo 2 C18
CH:FLOW_GRADIENT                 	1%B to 99%B in 15 min
CH:FLOW_RATE                     	250 uL/min
CH:COLUMN_TEMPERATURE            	35
CH:METHODS_FILENAME              	.
CH:SOLVENT_A                     	Water (0.1% Formic Acid)
CH:SOLVENT_B                     	ACN (0.1% Formic Acid)
CH:METHODS_ID                    	.
CH:COLUMN_PRESSURE               	.
CH:INJECTION_TEMPERATURE         	.
CH:INTERNAL_STANDARD             	.
CH:INTERNAL_STANDARD_MT          	.
CH:RETENTION_INDEX               	.
CH:RETENTION_TIME                	.
CH:SAMPLE_INJECTION              	.
CH:SAMPLING_CONE                 	.
CH:ANALYTICAL_TIME               	.
CH:CAPILLARY_VOLTAGE             	.
CH:MIGRATION_TIME                	.
CH:OVEN_TEMPERATURE              	35C
CH:PRECONDITIONING               	.
CH:RUNNING_BUFFER                	.
CH:RUNNING_VOLTAGE               	.
CH:SHEATH_LIQUID                 	.
CH:TIME_PROGRAM                  	.
CH:TRANSFERLINE_TEMPERATURE      	.
CH:WASHING_BUFFER                	.
CH:WEAK_WASH_SOLVENT_NAME        	.
CH:WEAK_WASH_VOLUME              	.
CH:STRONG_WASH_SOLVENT_NAME      	.
CH:STRONG_WASH_VOLUME            	.
CH:TARGET_SAMPLE_TEMPERATURE     	.
CH:SAMPLE_LOOP_SIZE              	.
CH:SAMPLE_SYRINGE_SIZE           	.
CH:RANDOMIZATION_ORDER           	.
CH:CHROMATOGRAPHY_COMMENTS       	.
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Bruker timsTOF
MS:INSTRUMENT_TYPE               	QTOF
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	POSITIVE
MS:MS_COMMENTS                   	The MS analysis was performed using a timsTOF (Bruker, Darmstadt, Germany) with
MS:MS_COMMENTS                   	Apollo II electrosprayionization (ESI) source. The drying gas was set to flow at
MS:MS_COMMENTS                   	10 L/min and the drying temperature to 220℃ and the nebulizer pressure to 2.2
MS:MS_COMMENTS                   	bar. The capillary voltage was 4500 V and the end plate offset 500 V. For
MS:MS_COMMENTS                   	metabolomics 20–1300 m/z. The instrument was operated in auto-MS/MS mode. For
MS:MS_COMMENTS                   	metabolomics the collision energy was set to 20 eV, the cycle time to 0.5 s with
MS:MS_COMMENTS                   	a relative minimum intensity threshold of 400 counts per thousand and a target
MS:MS_COMMENTS                   	intensity of 20,000. Sodium formate was injected as an external calibrant in the
MS:MS_COMMENTS                   	first 0.3 min of each LC–MS/MS run.
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS	AU
MS_METABOLITE_DATA_START
Samples	1A_93_1_328	1A_93_1_331	1A_92_1_327	1A_92_1_330	1A_91_1_326	1A_91_1_329	3a_-2_93_1_336	3a_-7_93_1_386	3a_-8_93_1_387	3a_-7_92_1_382	3a_-8_92_1_383	3a-71_91_1_384	3a-8_91_1_385	3ar_91_1_333
Factors	Treatment:Control	Treatment:Control	Treatment:Control	Treatment:Control	Treatment:Control	Treatment:Control	Treatment:TRASTUZUMAB	Treatment:TRASTUZUMAB	Treatment:TRASTUZUMAB	Treatment:TRASTUZUMAB	Treatment:TRASTUZUMAB	Treatment:TRASTUZUMAB	Treatment:TRASTUZUMAB	Treatment:TRASTUZUMAB
D-Arginine	1678	2032	1866	1960	2126	2186	1766	1434	850	1308	1734	1634	1482	1120
L-Carnitine	2062	2030	1798	2356	1882	2262	1384	1956	1540	1500	1298	1572	2464	1950
Creatine	2148	2514	3084	2904	2892	3080	3326	1284	1452	816	1262	1486	1584	10670
Adenosine 3_,5_-diphosphate	17774	39590	31048	42806	8724	46536	2496	1264	2150	2280	798	1274	1220	732
Cytidine	664	878	888	856	744	988	3618	1412	2114	0	2570	2662	3044	0
L-Acetylcarnitine	19280	17064	16432	17416	13820	17570	23462	8286	13806	184	23240	25666	25818	0
Adenosine monophosphate	110890	101152	110602	106382	137158	104760	43332	23664	25370	628	62608	40546	45662	108
Guanosine monophosphate	6356	7114	7072	6336	20838	6828	3116	1904	2254	262	2910	3142	3630	0
Pyridine	722	1398	1004	1394	1026	956	1756	1146	118	136	740	1670	1032	200
Deoxyadenosine monophosphate	1242	1606	1114	1896	0	1944	0	0	106	0	0	0	0	0
Deoxyguanosine	67130	60510	70696	59562	99992	111320	0	91806	92722	180	79178	120632	136448	127990
Guanosine	12340	11824	12022	10380	17508	16026	426	22058	25632	0	25264	26936	29406	32140
Inosine	14324	11988	12308	11004	16384	15786	0	23446	22520	366	30794	25182	27590	24848
Hypoxanthine	45216	40588	39834	37294	40092	48508	0	56770	62982	0	67776	63876	60374	58366
L-Leucine	1136	1120	1012	1188	700	1018	0	422	460	0	482	430	500	384
Isovalerylcarnitine	22672	20998	19624	20818	0	20210	0	12900	14098	14654	16290	17650	22288	17360
Rutin	1922	2066	2206	0	0	1616	0	0	0	0	0	0	0	0
L-Fucose	766	454	1010	678	120	776	0	1476	1730	1416	852	1442	2084	574
3-Methylindole	574	618	1984	1656	1912	1620	1524	0	0	0	0	400	488	3550
Spermine	43390	42606	37734	42484	47050	36178	12644	8120	9874	13870	11216	15722	15286	10920
L-Glutamic acid	556	1196	1142	1388	574	1368	952	294	84	120	354	216	404	1416
L-Valine	1648	1648	1350	2316	1992	1480	2216	1422	1414	1192	1430	1446	1464	4120
Cytidine monophosphate	2464	2586	2556	2718	2094	3350	728	0	0	512	0	0	0	376
Betaine	0	0	168	166	0	286	194	1880	0	0	1460	1472	1684	0
Uridine 5_-monophosphate	3466	4380	4728	4634	4004	5194	1804	1938	1436	182	1444	1802	1456	1084
Glutathione	472754	387742	463994	435460	469518	487416	316446	231766	271122	3774	304786	377148	379898	0
Pyrrolidonecarboxylic acid	18106	18506	17446	19446	8362	20722	7366	8700	0	366	3320	5958	5078	130
Coumarin	0	0	0	0	2600	0	0	1622	1532	0	0	0	0	0
L-Isoleucine	2060	1970	1452	1704	2652	1440	4710	0	2052	0	1448	1986	1856	0
L-Tyrosine	12872	11892	12656	13046	0	13076	11892	2792	0	0	12952	6928	8222	0
L-Phenylalanine	27126	27804	31070	26546	25402	30608	0	14580	15764	288	18040	21014	20462	25132
2-Ethyl-2-hydroxybutyric acid	1274	1288	1106	1086	1482	908	124	640	626	384	1032	416	354	614
Phosphoric acid	0	0	116	0	356	0	0	1370	0	2804	1724	1182	0	0
Sphingosine	17858	14684	16256	13172	11402	15478	502	140	130	632	114	186	108	278
Aspartyl-lysine	7090	5438	10314	8468	4312	7328	2976	0	0	0	0	0	0	4948
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	Bucket label	RT	Formula	m/z
D-Arginine	174.11168 Da 67.88 s	1.13	C6H14N4O2	175.11896
L-Carnitine	161.10514 Da 74.68 s	1.24	C7H15NO3	162.11242
Creatine	131.06919 Da 81.82 s	1.36	C4H9N3O2	132.07647
Adenosine 3_,5_-diphosphate	427.03082 Da 142.18 s	2.37	C10H15N5O10P2	428.0381
Cytidine	243.08593 Da 142.35 s	2.37	C9H13N3O5	244.09321
L-Acetylcarnitine	203.11597 Da 143.81 s	2.4	C9H17NO4	204.12324
Adenosine monophosphate	347.06396 Da 143.82 s	2.4	C10H14N5O7P	348.07123
Guanosine monophosphate	363.05861 Da 144.51 s	2.41	C10H14N5O8P	364.06588
Pyridine	79.04225 Da 146.66 s	2.44	C5H5N	80.04953
Deoxyadenosine monophosphate	331.06886 Da 149.11 s	2.49	C10H14N5O6P	332.07614
Deoxyguanosine	267.09653 Da 296.83 s	4.95	C10H13N5O4	268.10381
Guanosine	283.09217 Da 303.77 s	5.06	C10H13N5O5	284.09944
Inosine	268.08117 Da 304.47 s	5.07	C10H12N4O5	269.08845
Hypoxanthine	136.03840 Da 304.60 s	5.08	C5H4N4O	137.04568
L-Leucine	131.09459 Da 349.20 s	5.82	C6H13NO2	132.10187
Isovalerylcarnitine	245.16279 Da 404.63 s	6.74	C12H23NO4	246.17007
Rutin	610.15501 Da 442.23 s	7.37	C27H30O16	611.16228
L-Fucose	164.06893 Da 478.05 s	7.97	C6H12O5	165.07621
3-Methylindole	131.07315 Da 886.81 s	14.78	C9H9N	132.08042
Spermine	202.21572 Da 56.90 s	0.95	C10H26N4	203.22299
L-Glutamic acid	147.05309 Da 78.52 s	1.31	C5H9NO4	148.06037
L-Valine	117.07883 Da 79.41 s	1.32	C5H11NO2	118.08611
Cytidine monophosphate	323.05240 Da 82.81 s	1.38	C9H14N3O8P	324.05967
Betaine	117.07888 Da 97.91 s	1.63	C5H11NO2	118.08615
Uridine 5_-monophosphate	324.03661 Da 130.82 s	2.18	C9H13N2O9P	325.04389
Glutathione	307.08374 Da 144.95 s	2.42	C10H17N3O6S	308.09102
Pyrrolidonecarboxylic acid	129.04236 Da 160.74 s	2.68	C5H7NO3	130.04964
Coumarin	146.03654 Da 208.03 s	3.47	C9H6O2	147.04382
L-Isoleucine	131.09475 Da 208.69 s	3.48	C6H13NO2	132.10203
L-Tyrosine	181.07397 Da 228.80 s	3.81	C9H11NO3	182.08125
L-Phenylalanine	165.07888 Da 332.32 s	5.54	C9H11NO2	166.08616
2-Ethyl-2-hydroxybutyric acid	132.07838 Da 369.18 s	6.15	C6H12O3	133.08565
Phosphoric acid	97.97679 Da 562.37 s	9.37	H3O4P	98.98407
Sphingosine	299.28329 Da 745.31 s	12.42	C18H37NO2	282.27997
Aspartyl-lysine	261.13679 Da 980.20 s	16.34	C10H19N3O5	262.14406
METABOLITES_END
#END