#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