#METABOLOMICS WORKBENCH nsuhasj_20230625_185514 DATATRACK_ID:4112 STUDY_ID:ST002743 ANALYSIS_ID:AN004448 PROJECT_ID:PR001708
VERSION             	1
CREATED_ON             	June 26, 2023, 10:29 am
#PROJECT
PR:PROJECT_TITLE                 	Metabolomics comparison of lung fibroblasts from Pteropus alecto and Homo
PR:PROJECT_TITLE                 	sapiens
PR:PROJECT_TYPE                  	MS quantitative analysis
PR:PROJECT_SUMMARY               	Targeted Metabolomics was performed to identify differences in central carbon
PR:PROJECT_SUMMARY               	metabolism between lung fibroblasts from black flying fox fruit bat Pteropus
PR:PROJECT_SUMMARY               	alecto (PaLung cells) and Humans (WI-38 cells).
PR:INSTITUTE                     	Duke-NUS Medical School
PR:LAST_NAME                     	Jagannathan
PR:FIRST_NAME                    	Narendra Suhas
PR:ADDRESS                       	Centre for Computational Biology, 8 College Road, Singapore, Singapore, 169857,
PR:ADDRESS                       	Singapore
PR:EMAIL                         	gmsnsja@nus.edu.sg
PR:PHONE                         	(65) 66015073
PR:PROJECT_COMMENTS              	Metabolomics was performed by commissioning to Human Metabolome Technologies
PR:PROJECT_COMMENTS              	(HMT) service.
PR:CONTRIBUTORS                  	Javier Yu Peng Koh, Yoko Itahana, Koji Itahana, Lisa Tucker-Kellogg
#STUDY
ST:STUDY_TITLE                   	Metabolomics comparison of lung fibroblasts from Pteropus alecto and Homo
ST:STUDY_TITLE                   	sapiens
ST:STUDY_TYPE                    	MS Quantitative analysis
ST:STUDY_SUMMARY                 	Targeted Metabolomics was performed to identify differences in central carbon
ST:STUDY_SUMMARY                 	metabolism between lung fibroblasts from black flying fox fruit bat Pteropus
ST:STUDY_SUMMARY                 	alecto (PaLung cells) and Humans (WI-38 cells). For both PaLung and WI-38 cells,
ST:STUDY_SUMMARY                 	metabolomics measurements were carried out for n = 3 biological replicates.
ST:INSTITUTE                     	Duke-NUS Medical School
ST:LAST_NAME                     	Jagannathan
ST:FIRST_NAME                    	Narendra Suhas
ST:ADDRESS                       	8 College Road, Singapore, Singapore, 169857, Singapore
ST:EMAIL                         	gmsnsja@nus.edu.sg
ST:PHONE                         	(65) 66015073
#SUBJECT
SU:SUBJECT_TYPE                  	Cultured cells
SU:SUBJECT_SPECIES               	Pteropus alecto / Homo sapiens
#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           	-	WI38-1	Genotype:Wild-type | Species:H. sapiens	
SUBJECT_SAMPLE_FACTORS           	-	WI38-2	Genotype:Wild-type | Species:H. sapiens	
SUBJECT_SAMPLE_FACTORS           	-	WI38-3	Genotype:Wild-type | Species:H. sapiens	
SUBJECT_SAMPLE_FACTORS           	-	PaLung-1	Genotype:Wild-type | Species:P. alecto	
SUBJECT_SAMPLE_FACTORS           	-	PaLung-2	Genotype:Wild-type | Species:P. alecto	
SUBJECT_SAMPLE_FACTORS           	-	PaLung-3	Genotype:Wild-type | Species:P. alecto	
#COLLECTION
CO:COLLECTION_SUMMARY            	WI-38 and PaLung cells were seeded in a 100 mm dish and harvested 2 days after
CO:COLLECTION_SUMMARY            	seeding according to the protocol provided by Human Metabolome Technologies (HMT
CO:COLLECTION_SUMMARY            	Japan).
CO:SAMPLE_TYPE                   	Cultured fibroblasts
#TREATMENT
TR:TREATMENT_SUMMARY             	PaLung and WI-38 cells were washed with 5% (w/w) mannitol twice and fixed with
TR:TREATMENT_SUMMARY             	methanol containing the internal standard solution. The internal standard
TR:TREATMENT_SUMMARY             	solution was provided by HMT. Lysates were centrifuged at 2,300 x g for 5 min at
TR:TREATMENT_SUMMARY             	4°C, and supernatants were filtered using the provided filter units by
TR:TREATMENT_SUMMARY             	centrifuging at 9,100 × g for 3 hours at 4 °C. Filtrates were evaporated
TR:TREATMENT_SUMMARY             	by SpeedVac Concentrator SPD1010 (Thermo Fisher Scientific) and submitted to HMT
TR:TREATMENT_SUMMARY             	for analysis. The total cell numbers at the time of harvest were about 1.4 x 107
TR:TREATMENT_SUMMARY             	cells for each cell line. Targeted quantitative analysis was performed by HMT
TR:TREATMENT_SUMMARY             	using capillary electrophoresis mass spectrometry (CE-TOFMS and CE-QqQMS).
TR:TREATMENT_SUMMARY             	Absolute abundances (normalized by cell numbers) were obtained for a total of
TR:TREATMENT_SUMMARY             	116 metabolites (54 and 62 metabolites in the cation and anion modes,
TR:TREATMENT_SUMMARY             	respectively).
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	PaLung and WI-38 cells were washed with 5% (w/w) mannitol twice and fixed with
SP:SAMPLEPREP_SUMMARY            	methanol containing the internal standard solution. The internal standard
SP:SAMPLEPREP_SUMMARY            	solution was provided by HMT. Lysates were centrifuged at 2,300 x g for 5 min at
SP:SAMPLEPREP_SUMMARY            	4°C, and supernatants were filtered using the provided filter units by
SP:SAMPLEPREP_SUMMARY            	centrifuging at 9,100 × g for 3 hours at 4 °C. Filtrates were evaporated
SP:SAMPLEPREP_SUMMARY            	by SpeedVac Concentrator SPD1010 (Thermo Fisher Scientific) and submitted to HMT
SP:SAMPLEPREP_SUMMARY            	for analysis. The total cell numbers at the time of harvest were about 1.4 x 107
SP:SAMPLEPREP_SUMMARY            	cells for each cell line. Targeted quantitative analysis was performed by HMT
SP:SAMPLEPREP_SUMMARY            	using capillary electrophoresis mass spectrometry (CE-TOFMS and CE-QqQMS).
SP:SAMPLEPREP_SUMMARY            	Absolute abundances (normalized by cell numbers) were obtained for a total of
SP:SAMPLEPREP_SUMMARY            	116 metabolites (54 and 62 metabolites in the cation and anion modes,
SP:SAMPLEPREP_SUMMARY            	respectively).
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_TYPE           	CE
CH:INSTRUMENT_NAME               	Agilent CE
CH:COLUMN_NAME                   	Fused silica capillary, i.d. 50 μm × 80 cm
CH:SOLVENT_A                     	NA
CH:SOLVENT_B                     	NA
CH:FLOW_GRADIENT                 	NA
CH:FLOW_RATE                     	NA
CH:COLUMN_TEMPERATURE            	NA
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Agilent 6460 QQQ
MS:INSTRUMENT_TYPE               	Triple quadrupole
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	NEGATIVE
MS:MS_COMMENTS                   	Peaks detected in CE-TOFMS analysis were extracted using automatic integration
MS:MS_COMMENTS                   	software (MasterHands ver.2.18.0.1 developed at Keio University) (4) and those
MS:MS_COMMENTS                   	in CE-QqQMS analysis were extracted using automatic integration software
MS:MS_COMMENTS                   	(MassHunter Quantitative Analysis B.06.00 Agilent Technologies, Santa Clara, CA,
MS:MS_COMMENTS                   	USA) in order to obtain peak information, which includes m/z, migration time
MS:MS_COMMENTS                   	(MT), and peak area.
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS	pmol/10^6 cells
MS_METABOLITE_DATA_START
Samples	WI38-1	WI38-2	WI38-3	PaLung-1	PaLung-2	PaLung-3
Factors	Genotype:Wild-type | Species:H. sapiens	Genotype:Wild-type | Species:H. sapiens	Genotype:Wild-type | Species:H. sapiens	Genotype:Wild-type | Species:P. alecto	Genotype:Wild-type | Species:P. alecto	Genotype:Wild-type | Species:P. alecto
NAD+	2352.3	2395.1	2493.1	1198.4	1233.3	1114.4
cAMP	4.0	4.7	4.2	3.1	2.2	3.2
cGMP	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.
NADH	83.7	79.8	82.9	141.1	N.D.	N.D.
Xanthine	47.7	39.5	44.3	34.1	33.8	28.4
ADP-ribose	59.7	59.4	58.9	42.2	58.3	53.2
Mevalonic acid	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.
UDP-glucose	1814.4	1851.3	1700.4	1543.9	1581.4	1627.8
Uric acid	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.
NADP+	49.2	51.5	53.8	67.2	57.1	60.3
IMP	19.5	22.0	18.3	450.7	626.1	633.9
Sedoheptulose 7-phosphate	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.
Glucose 6-phosphate	677.8	645.1	532.4	109.9	94.6	93.9
Fructose 6-phosphate	206.6	186.9	174.5	139.1	112.4	131.8
Fructose 1-phosphate	49.3	50.0	33.9	N.D.	N.D.	N.D.
Galactose 1-phosphate	39.7	28.7	26.7	16.4	13.8	14.8
Glucose 1-phosphate	48.7	47.5	34.0	N.D.	N.D.	N.D.
Acetoacetyl CoA	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.
Acetyl CoA	29.1	24.8	26.0	18.0	17.7	22.0
Folic acid	1.9	2.0	2.1	3.1	2.3	1.8
Ribose 5-phosphate	91.6	84.5	68.3	72.8	65.2	65.5
CoA	8.1	7.3	7.1	11.6	11.1	10.9
Ribose 1-phosphate	16.9	27.9	32.4	21.8	23.3	27.9
Ribulose 5-phosphate	73.1	75.3	75.3	54.6	47.7	46.5
Xylulose 5-phosphate	83.6	64.8	60.0	N.D.	N.D.	N.D.
Erythrose 4-phosphate	15.8	21.6	21.9	18.3	20.1	17.8
HMG CoA	6.7	5.9	5.8	10.1	9.3	10.3
Glyceraldehyde 3-phosphate	245.6	200.3	178.4	552.2	713.4	624.6
NADPH	N.D.	53.2	56.3	95.5	N.D.	N.D.
Malonyl CoA	6.4	7.8	8.5	N.D.	N.D.	N.D.
Phosphocreatine	491.7	401.2	300.7	758.5	620.9	822.5
XMP	3.3	2.9	3.1	3.9	3.5	6.4
Dihydroxyacetone phosphate	1353.0	1247.5	1094.4	1653.0	1552.8	1543.0
Adenylosuccinic acid	3.9	4.0	4.0	13.3	14.1	15.5
Fructose 1,6-diphosphate	3937.2	3916.2	3681.5	5263.5	5740.5	5406.5
6-Phosphogluconic acid	232.5	219.9	194.5	76.2	61.6	55.9
N-Carbamoylaspartic acid	25.6	22.1	20.6	10.0	12.1	9.8
PRPP	68.3	64.5	65.6	39.9	37.2	37.3
2-Phosphoglyceric acid	17.1	19.1	21.7	20.7	24.8	19.4
2,3-Diphosphoglyceric acid	455.6	496.6	485.1	1173.3	1261.9	1261.4
3-Phosphoglyceric acid	186.5	211.6	261.9	243.8	207.2	200.5
Phosphoenolpyruvic acid	N.D.	N.D.	0.4	N.D.	N.D.	N.D.
GMP	25.8	33.4	26.1	85.1	88.8	92.6
AMP	97.3	94.4	91.6	305.9	275.6	236.6
2-Oxoisovaleric acid	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.
GDP	113.4	120.9	126.8	199.2	185.3	154.1
Lactic acid	25643.1	24251.0	27589.3	12548.4	11242.2	10670.2
ADP	665.6	693.4	776.7	1164.2	1128.9	1038.1
GTP	2419.3	2316.7	2346.1	1210.6	1245.4	1241.1
Glyoxylic acid	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.
ATP	9143.2	9287.4	9368.9	3897.0	3711.5	3952.4
Glycerol 3-phosphate	308.7	276.1	310.6	350.0	343.3	386.2
Glycolic acid	N.D.	100.51	N.D.	N.D.	391.61	272.07
Pyruvic acid	231.18	180.98	201.04	292.33	292.64	318.94
N-Acetylglutamic acid	25.70	31.71	30.08	27.62	19.69	19.61
2-Hydroxyglutaric acid	39.89	33.46	39.89	50.07	55.38	52.45
Carbamoylphosphate	N.D.	N.D.	N.D.	N.D.	N.D.	74.10
Succinic acid	492.57	478.27	548.23	381.15	354.22	352.24
Malic acid	2354.65	2448.25	2736.21	211.14	265.53	293.28
2-Oxoglutaric acid	N.D.	N.D.	N.D.	N.D.	N.D.	N.D.
Fumaric acid	462.06	452.50	507.20	73.30	57.21	71.54
Citric acid	2478.15	2270.38	2411.20	1339.78	1389.65	1332.35
cis-Aconitic acid	57.65	51.17	53.06	24.50	18.19	18.61
Isocitric acid	49.89	63.22	89.90	N.D.	N.D.	N.D.
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	PubChem ID	HMDB ID
NAD+	5893	HMDB0000902
cAMP	6076	HMDB0000058
cGMP	24316	HMDB0001314
NADH	439153	HMDB0001487
Xanthine	1188	HMDB0000292
ADP-ribose	445794	HMDB0001178
Mevalonic acid	134965	HMDB0000227
UDP-glucose	8629	HMDB0000286
Uric acid	1175	HMDB0000289
NADP+	5886	HMDB0000217
IMP	8582	HMDB0000175
Sedoheptulose 7-phosphate	165007	HMDB0001068
Glucose 6-phosphate	5958	HMDB0001401
Fructose 6-phosphate	603	HMDB0000124
Fructose 1-phosphate	439394	HMDB0001076
Galactose 1-phosphate	123912	HMDB0000645
Glucose 1-phosphate	65533	HMDB0001586
Acetoacetyl CoA	92153	HMDB0001484
Acetyl CoA	444493	HMDB0001206
Folic acid	6037	HMDB0000121
Ribose 5-phosphate	439167	HMDB0001548
CoA	87642	HMDB0001423
Ribose 1-phosphate	439236	HMDB0001489
Ribulose 5-phosphate	439184	HMDB0000618
Xylulose 5-phosphate	439190	HMDB0000868
Erythrose 4-phosphate	122357	HMDB0001321
HMG CoA	445127	HMDB0001375
Glyceraldehyde 3-phosphate	729	HMDB0001112
NADPH	5884	HMDB0000221
Malonyl CoA	644066	HMDB0001175
Phosphocreatine	9548602	HMDB0001511
XMP	73323	HMDB0001554
Dihydroxyacetone phosphate	668	HMDB0001473
Adenylosuccinic acid	447145	HMDB0000536
Fructose 1,6-diphosphate	172313	HMDB0001058
6-Phosphogluconic acid	91493	HMDB0001316
N-Carbamoylaspartic acid	93072	HMDB0000828
PRPP	7339	HMDB0000280
2-Phosphoglyceric acid	439278	HMDB0003391
2,3-Diphosphoglyceric acid	186004	HMDB0001294
3-Phosphoglyceric acid	439183	HMDB0000807
Phosphoenolpyruvic acid	1005	HMDB0000263
GMP	6804	HMDB0001397
AMP	6083	HMDB0000045
2-Oxoisovaleric acid	49	HMDB0000019
GDP	8977	HMDB0001201
Lactic acid	612	"HMDB0000190,HMDB0001311"
ADP	6022	HMDB0001341
GTP	6830	HMDB0001273
Glyoxylic acid	760	HMDB0000119
ATP	5957	HMDB0000538
Glycerol 3-phosphate	439162	HMDB0000126
Glycolic acid	757	HMDB0000115
Pyruvic acid	1060	HMDB0000243
N-Acetylglutamic acid	70914	HMDB0001138
2-Hydroxyglutaric acid	43	"HMDB0000606,HMDB0000694"
Carbamoylphosphate	278	HMDB0001096
Succinic acid	1110	HMDB0000254
Malic acid	525	"HMDB0000156,HMDB0000744"
2-Oxoglutaric acid	51	HMDB0000208
Fumaric acid	444972	HMDB0000134
Citric acid	311	HMDB0000094
cis-Aconitic acid	643757	HMDB0000072
Isocitric acid	1198	HMDB0000193
METABOLITES_END
#END