#METABOLOMICS WORKBENCH nsuhasj_20230625_185514 DATATRACK_ID:4112 STUDY_ID:ST002743 ANALYSIS_ID:AN004447 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 CE-TOFMS MS:INSTRUMENT_TYPE Other MS:MS_TYPE ESI MS:ION_MODE POSITIVE 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 Urea N.D. N.D. N.D. N.D. N.D. N.D. Gly 24307.2 22607.3 21737.1 9857.7 10531.9 10572.5 Putrescine N.D. N.D. N.D. N.D. N.D. N.D. Ala 41203.7 38676.5 40866.5 11938.4 11644.3 11543.4 Beta-Ala 422.1 428.3 423.1 1977.6 1877.6 2113.9 Sarcosine 224.5 129.5 209.1 N.D. N.D. N.D. N,N-Dimethylglycine N.D. N.D. N.D. N.D. N.D. N.D. Gamma-Aminobutyric acid 250.0 238.6 274.4 527.0 452.8 468.6 Choline 127.7 90.7 52.5 858.6 842.8 795.2 Ser 41175.2 38109.1 38737.3 13256.0 13219.5 12711.0 Carnosine N.D. N.D. N.D. 1183.7 1137.9 1258.7 Creatinine 227.0 224.2 242.6 456.7 313.9 342.8 Pro 17069.0 16121.9 17025.3 13000.1 11737.3 11132.3 Val 16022.6 14944.9 16280.9 7668.2 6689.5 6315.9 Betaine 429.0 417.3 417.3 N.D. 184.1 187.3 Homoserine 23.6 24.8 24.4 N.D. N.D. N.D. Thr 72006.1 65802.5 65521.4 18867.8 18793.6 17106.3 Betaine aldehyde N.D. N.D. N.D. N.D. N.D. N.D. Cys N.D. N.D. N.D. N.D. N.D. N.D. Hydroxyproline 1001.8 909.0 940.6 642.2 560.8 571.5 Creatine 3846.0 3768.5 4270.7 22335.2 18029.6 18300.5 Leu 23711.8 23523.0 23828.8 8223.7 8006.9 7463.5 Ile 15459.3 15474.3 15853.3 6871.8 6553.0 6036.0 Asn 973.3 1005.4 1074.4 576.5 476.9 501.9 Ornithine 178.8 185.8 196.4 302.1 253.7 271.2 Asp 15632.0 15511.5 15818.9 3632.8 3454.4 3153.5 Homocysteine N.D. N.D. N.D. N.D. N.D. N.D. Adenine N.D. N.D. N.D. N.D. N.D. N.D. Hypoxanthine N.D. N.D. N.D. 253.9 242.1 252.3 Spermidine 68.4 45.1 43.2 N.D. N.D. N.D. Gln 22904.3 22961.9 23511.8 4120.1 3821.3 4147.9 Lys 1550.7 1441.7 1443.2 1061.2 910.5 928.8 Glu 126774.1 124192.7 130893.5 69337.8 67267.4 64394.4 Met 91.7 210.7 74.8 N.D. N.D. N.D. Guanine N.D. N.D. N.D. N.D. N.D. N.D. His 8159.1 7417.6 7001.5 2452.4 2272.4 2345.2 Carnitine 47.6 54.7 68.0 213.6 175.1 161.3 Phe 11686.6 10972.6 11209.7 3545.7 3489.9 3340.1 Arg 1192.2 1128.8 1141.6 535.3 433.8 436.1 Citrulline 348.1 353.2 350.7 150.4 114.6 132.3 Tyr 10158.3 9696.5 10487.8 3178.2 3107.7 2833.0 S-Adenosylhomocysteine N.D. N.D. N.D. N.D. N.D. N.D. Spermine 16.4 N.D. N.D. N.D. N.D. N.D. Trp 260.4 287.5 320.8 147.1 N.D. 102.9 Cystathionine N.D. N.D. N.D. N.D. N.D. N.D. Adenosine 104.7 87.7 99.9 119.9 103.7 116.7 Inosine N.D. N.D. N.D. N.D. 544.2 649.8 Guanosine N.D. N.D. N.D. N.D. N.D. N.D. Argininosuccinic acid 83.8 97.7 98.5 N.D. N.D. N.D. Glutathione (GSSG) 3308.3 3245.4 3437.3 4507.3 3799.0 3736.9 Glutathione (GSH) 492.8 475.2 485.8 1029.6 853.6 916.7 S-Adenosylmethionine 187.2 190.9 170.0 181.5 160.3 170.8 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name PubChem ID HMDB ID Urea 1176 HMDB0000294 Gly 750 HMDB0000123 Putrescine 1045 HMDB0001414 Ala 602 "HMDB0000161,HMDB0001310" Beta-Ala 239 HMDB0000056 Sarcosine 1088 HMDB0000271 N,N-Dimethylglycine 673 HMDB0000092 Gamma-Aminobutyric acid 119 HMDB0000112 Choline 305 HMDB0000097 Ser 617 "HMDB0000187,HMDB0003406" Carnosine 439224 HMDB0000033 Creatinine 588 HMDB0000562 Pro 614 "HMDB0000162,HMDB0003411" Val 1182 HMDB0000883 Betaine 247 HMDB0000043 Homoserine 12647 HMDB0000719 Thr 6288 HMDB0000167 Betaine aldehyde 249 HMDB0001252 Cys 594 "HMDB0000574,HMDB0003417" Hydroxyproline 5810 HMDB0000725 Creatine 586 HMDB0000064 Leu 857 HMDB0000687 Ile 791 HMDB0000172 Asn 236 "HMDB0000168,HMDB0033780" Ornithine 389 "HMDB0000214,HMDB0003374" Asp 424 "HMDB0000191,HMDB0006483" Homocysteine 778 HMDB0000742 Adenine 190 HMDB0000034 Hypoxanthine 790 HMDB0000157 Spermidine 1102 HMDB0001257 Gln 738 "HMDB0000641,HMDB0003423" Lys 866 "HMDB0000182,HMDB0003405" Glu 611 "HMDB0000148,HMDB0003339" Met 876 HMDB0000696 Guanine 764 HMDB0000132 His 773 HMDB0000177 Carnitine 85 HMDB0000062 Phe 994 HMDB0000159 Arg 6322 "HMDB0000517,HMDB0003416" Citrulline 9750 HMDB0000904 Tyr 1153 HMDB0000158 S-Adenosylhomocysteine 439155 HMDB0000939 Spermine 1103 HMDB0001256 Trp 1148 HMDB0000929 Cystathionine 834 HMDB0000099 Adenosine 60961 HMDB0000050 Inosine 6021 HMDB0000195 Guanosine 6802 HMDB0000133 Argininosuccinic acid 16950 HMDB0000052 Glutathione (GSSG) 65359 HMDB0003337 Glutathione (GSH) 124886 HMDB0000125 S-Adenosylmethionine 34755 HMDB0001185 METABOLITES_END #END