#METABOLOMICS WORKBENCH prats1988_20210404_005908 DATATRACK_ID:2563 STUDY_ID:ST001779 ANALYSIS_ID:AN002888 PROJECT_ID:PR001132 VERSION 1 CREATED_ON May 8, 2021, 11:06 am #PROJECT PR:PROJECT_TYPE Untargeted quantitative metabolomics analysis PR:PROJECT_SUMMARY A549 lung cancer cells were treated in vitro with control, 0.5 mM ATP and 10 PR:PROJECT_SUMMARY ng/ml TGF-beta for 2, 6 and 12 hours The untargeted metabolomics data was PR:PROJECT_SUMMARY generated from these studies. PR:INSTITUTE Ohio University PR:DEPARTMENT Biological Sciences PR:LABORATORY Dr. Xiaozhuo Chen, Edison biotechnology Institute PR:FIRST_NAME Pratik PR:ADDRESS Room 425, Parks Hall, College of Pharmacy, Ohio State University, Columbus Ohio. PR:ADDRESS 43210 PR:EMAIL ps774614@ohio.edu PR:PHONE 7406033801 PR:FUNDING_SOURCE This study was funded by NIH R15 grant CA242177-01 to Dr. Xiaozhuo Chen. PR:PROJECT_TITLE Untargeted metabolomics analysis of A549 cancer cells treated in control, 0.5 mM PR:PROJECT_TITLE ATP and 10 ng/ml TGF-beta for 2, 6 and 12 hours PR:LAST_NAME Shriwas #STUDY ST:STUDY_TITLE Untargeted Metabolomics analysis of A549 treated with 0.5 mM extracellular ATP ST:STUDY_TITLE and 10 ng/ml TGF-beta ST:STUDY_SUMMARY Control, 0.5 mM extracellular ATP and 10 ng/ml TGF-beta were used to treated 5 ST:STUDY_SUMMARY million A549 lung cancer cells in vitro for 2, 6 and 12 hours. The untargeted ST:STUDY_SUMMARY metabolomics analysis was performed on the cell lysates. The main objective of ST:STUDY_SUMMARY the study was to determine changes in metabolite abundances in lung cancer after ST:STUDY_SUMMARY treatment with extracellular ATP and TGF-beta (a known EMT inducer). ST:INSTITUTE Ohio University ST:DEPARTMENT Biological Sciences ST:LABORATORY Dr. Xiaozhuo Chen, Edison biotechnology Institute ST:LAST_NAME Shriwas ST:FIRST_NAME Pratik ST:ADDRESS Room 425, Parks Hall, College of Pharmacy, Ohio State University, Columbus Ohio. ST:ADDRESS 43210 ST:EMAIL ps774614@ohio.edu ST:NUM_GROUPS 7 ST:STUDY_TYPE Untargeted metabolomics analysis in lung cancer cells ST:PHONE 7406033801 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:CELL_BIOSOURCE_OR_SUPPLIER ATCC SU:CELL_STRAIN_DETAILS Human Lung epithelial A549 cancer cells SU:CELL_PASSAGE_NUMBER 10 SU:CELL_COUNTS 5 million cells #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 - Control_1 Treatment:Control RAW_FILE_NAME=ControlA_1.mzxml SUBJECT_SAMPLE_FACTORS - Control_2 Treatment:Control RAW_FILE_NAME=ControlA_2.mzxml SUBJECT_SAMPLE_FACTORS - Control_3 Treatment:Control RAW_FILE_NAME=ControlB_1.mzxml SUBJECT_SAMPLE_FACTORS - Control_4 Treatment:Control RAW_FILE_NAME=ControlB_1.mzxml SUBJECT_SAMPLE_FACTORS - 2h ATP_1 Treatment:2h ATP RAW_FILE_NAME=2hATP1_1.mzxml SUBJECT_SAMPLE_FACTORS - 2h ATP_2 Treatment:2h ATP RAW_FILE_NAME=2hATP1_2.mzxml SUBJECT_SAMPLE_FACTORS - 2h ATP_3 Treatment:2h ATP RAW_FILE_NAME=2hATP2_1.mzxml SUBJECT_SAMPLE_FACTORS - 2h ATP_4 Treatment:2h ATP RAW_FILE_NAME=2hATP2_2.mzxml SUBJECT_SAMPLE_FACTORS - 2h ATP_5 Treatment:2h ATP RAW_FILE_NAME=2hATP3_1.mzxml SUBJECT_SAMPLE_FACTORS - 2h ATP_6 Treatment:2h ATP RAW_FILE_NAME=2hATP3_2.mzxml SUBJECT_SAMPLE_FACTORS - 6h ATP_1 Treatment:6h ATP RAW_FILE_NAME=6hATP1_1.mzxml SUBJECT_SAMPLE_FACTORS - 6h ATP_2 Treatment:6h ATP RAW_FILE_NAME=6hATP1_2.mzxml SUBJECT_SAMPLE_FACTORS - 6h ATP_3 Treatment:6h ATP RAW_FILE_NAME=6hATP2_1.mzxml SUBJECT_SAMPLE_FACTORS - 6h ATP_4 Treatment:6h ATP RAW_FILE_NAME=6hATP2_2.mzxml SUBJECT_SAMPLE_FACTORS - 12h ATP_1 Treatment:12h ATP RAW_FILE_NAME=12hATP1_1.mzxml SUBJECT_SAMPLE_FACTORS - 12h ATP_2 Treatment:12h ATP RAW_FILE_NAME=12hATP1_2.mzxml SUBJECT_SAMPLE_FACTORS - 12h ATP_3 Treatment:12h ATP RAW_FILE_NAME=12hATP2_1.mzxml SUBJECT_SAMPLE_FACTORS - 12h ATP_4 Treatment:12h ATP RAW_FILE_NAME=12hATP2_2.mzxml SUBJECT_SAMPLE_FACTORS - 12h ATP_5 Treatment:12h ATP RAW_FILE_NAME=12hATP3_1.mzxml SUBJECT_SAMPLE_FACTORS - 12h ATP_6 Treatment:12h ATP RAW_FILE_NAME=12hATP3_2.mzxml SUBJECT_SAMPLE_FACTORS - 2h TGF-β_1 Treatment:2h TGF-β RAW_FILE_NAME=2hTGF1_1.mzxml SUBJECT_SAMPLE_FACTORS - 2h TGF-β_2 Treatment:2h TGF-β RAW_FILE_NAME=2hTGF1_2.mzxml SUBJECT_SAMPLE_FACTORS - 2h TGF-β_3 Treatment:2h TGF-β RAW_FILE_NAME=2hTGF2_1.mzxml SUBJECT_SAMPLE_FACTORS - 2h TGF-β_4 Treatment:2h TGF-β RAW_FILE_NAME=2hTGF2_2.mzxml SUBJECT_SAMPLE_FACTORS - 2h TGF-β_5 Treatment:2h TGF-β RAW_FILE_NAME=2hTGF3_1.mzxml SUBJECT_SAMPLE_FACTORS - 2h TGF-β_6 Treatment:2h TGF-β RAW_FILE_NAME=2hTGF3_2.mzxml SUBJECT_SAMPLE_FACTORS - 6h TGF-β_1 Treatment:6h TGF-β RAW_FILE_NAME=6hTGF1_1.mzxml SUBJECT_SAMPLE_FACTORS - 6h TGF-β_2 Treatment:6h TGF-β RAW_FILE_NAME=6hTGF1_2.mzxml SUBJECT_SAMPLE_FACTORS - 6h TGF-β_3 Treatment:6h TGF-β RAW_FILE_NAME=6hTGF2_1.mzxml SUBJECT_SAMPLE_FACTORS - 6h TGF-β_4 Treatment:6h TGF-β RAW_FILE_NAME=6hTGF2_2.mzxml SUBJECT_SAMPLE_FACTORS - 12h TGF-β_1 Treatment:12h TGF-β RAW_FILE_NAME=12hTGF1_1.mzxml SUBJECT_SAMPLE_FACTORS - 12h TGF-β_2 Treatment:12h TGF-β RAW_FILE_NAME=12hTGF1_2.mzxml SUBJECT_SAMPLE_FACTORS - 12h TGF-β_3 Treatment:12h TGF-β RAW_FILE_NAME=12hTGF2_1.mzxml SUBJECT_SAMPLE_FACTORS - 12h TGF-β_4 Treatment:12h TGF-β RAW_FILE_NAME=12hTGF2_2.mzxml SUBJECT_SAMPLE_FACTORS - 12h TGF-β_5 Treatment:12h TGF-β RAW_FILE_NAME=12hTGF3_1.mzxml SUBJECT_SAMPLE_FACTORS - 12h TGF-β_6 Treatment:12h TGF-β RAW_FILE_NAME=12hTGF3_2.mzxml #COLLECTION CO:COLLECTION_SUMMARY A549 lung cancer cells were treated with control, ATP, TGF-beta and then CO:COLLECTION_SUMMARY collected according to sample preparation protocol. CO:COLLECTION_PROTOCOL_FILENAME Collection protocol CO:SAMPLE_TYPE Lung CO:COLLECTION_METHOD Ice cold methanol (80%) using cell scrappers CO:COLLECTION_LOCATION 150mm dish CO:COLLECTION_FREQUENCY Once CO:COLLECTION_DURATION 1-2 sec CO:VOLUMEORAMOUNT_COLLECTED 1 ml CO:STORAGE_CONDITIONS -80℃ CO:COLLECTION_VIALS Polypropylene 1.5 ml tubes CO:STORAGE_VIALS Polypropylene 1.5 ml CO:COLLECTION_TUBE_TEMP 4C CO:TISSUE_CELL_QUANTITY_TAKEN 5 million #TREATMENT TR:TREATMENT_SUMMARY 5 million A549 cells were treated with control (no treatment), 0.5 mM ATP or 10 TR:TREATMENT_SUMMARY ng/ml TGF-beta for 2, 6 and 12 hours. TR:TREATMENT In vitro treatment with small molecules TR:TREATMENT_DOSEVOLUME 10 ml DMEM media containing appropriate compounds TR:CELL_STORAGE 37C; 5% CO2 incubator TR:CELL_GROWTH_CONTAINER 150 mm Dish tissue culture treated TR:CELL_MEDIA DMEM (10% FBS; 1% Pen/Strep) TR:CELL_HARVESTING after 2, 6 and 12 hours TR:CELL_PCT_CONFLUENCE 80% TR:CELL_MEDIA_LASTCHANGED NA TR:TREATMENT_COMPOUND 0.5 mM ATP and 10 ng/ml TGF-beta #SAMPLEPREP SP:SAMPLEPREP_SUMMARY 5 × 106 A549 cells were treated with control (no treatment), 0.5 mM ATP or 10 SP:SAMPLEPREP_SUMMARY ng/ml TGF-beta for 2, 6 and 12 hours. After treatment, cells were washed twice SP:SAMPLEPREP_SUMMARY with deionized water and polar metabolites were then extracted with SP:SAMPLEPREP_SUMMARY cryogenically cold 80% methanol/water mixture. LC-MS grade water, methanol, and SP:SAMPLEPREP_SUMMARY acetonitrile (Fischer Scientific, PA, USA) were used. Methanol-extracted samples SP:SAMPLEPREP_SUMMARY were then sonicated in cycles of sonication phase and rest phase for 10 minutes SP:SAMPLEPREP_SUMMARY (5 second sonication phase and 10 seconds halt). The samples were then SP:SAMPLEPREP_SUMMARY centrifuged at 13,000 rpm for 10 minutes and supernatant was then collected. SP:SAMPLEPREP_SUMMARY Supernatants collected from in vitro and in vivo extraction were then SP:SAMPLEPREP_SUMMARY lyophilized. Briefly, the supernatant was then lyophilized by using a speed SP:SAMPLEPREP_SUMMARY vacuum evaporator. The samples were then dissolved into a mixture of SP:SAMPLEPREP_SUMMARY acetonitrile/water (1:1; v/v). SP:SAMPLEPREP_PROTOCOL_FILENAME Sample preparation protocol SP:PROCESSING_METHOD CEll scrapping Quenching SP:PROCESSING_STORAGE_CONDITIONS -80℃ SP:EXTRACTION_METHOD Quenching with Ice cold methanol SP:EXTRACT_ENRICHMENT Speed vaccum evaporator SP:EXTRACT_STORAGE -80℃ SP:SAMPLE_RESUSPENSION Acetonitrile/water (1:1) #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY The entire LC/MS-MS experiment was performed in the Campus Chemical CH:CHROMATOGRAPHY_SUMMARY Instrumentation Center’s Mass Spectrometry and Proteomics facility at The Ohio CH:CHROMATOGRAPHY_SUMMARY State University. Lyophilized samples were dissolved in equal amounts of LC-MS CH:CHROMATOGRAPHY_SUMMARY grade water and acetonitrile and run with LC/MS-MS analysis, using an untargeted CH:CHROMATOGRAPHY_SUMMARY metabolomics approach by utilizing Agilent Q-TOF 6545 mass spectrometer CH:CHROMATOGRAPHY_SUMMARY connected to an Agilent 1290 UHPLC system with a Poroshell 120 SB-C18 (2 x 100 CH:CHROMATOGRAPHY_SUMMARY mm, 2.7 µm particle size) column. The LC gradient consisted of solvent A, H2O CH:CHROMATOGRAPHY_SUMMARY with 0.1 % Formic acid, and solvent B, 100 % acetonitrile at a 200 µL/min flow CH:CHROMATOGRAPHY_SUMMARY rate with an initial 2 % solvent B with a linear ramp to 95 % B at 15 min, CH:CHROMATOGRAPHY_SUMMARY holding at 95% B for 1 minutes, and back to 2 % B from 16 min and equilibration CH:CHROMATOGRAPHY_SUMMARY of 2 % B until min 32. A 5 µL volume sample was injected for each run and the CH:CHROMATOGRAPHY_SUMMARY top 5 ions were selected for data-dependent analysis with a 15 second exclusion CH:CHROMATOGRAPHY_SUMMARY window. CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 1290 CH:COLUMN_NAME Poroshell 120 SB-C18 (2.1 x 100 mm x 2.7 µm) CH:FLOW_GRADIENT an initial 2 % solvent B with a linear ramp to 95 % B at 15 min, holding at 95% CH:FLOW_GRADIENT B for 1 minutes, and back to 2 % B from 16 min and equilibration of 2 % B until CH:FLOW_GRADIENT min 32. CH:FLOW_RATE 200 µL/min CH:COLUMN_TEMPERATURE 40 CH:SOLVENT_A H2O with 0.1 % Formic acid CH:SOLVENT_B 100 % acetonitrile #ANALYSIS AN:ANALYSIS_TYPE MS AN:LABORATORY_NAME Campus Center for Chemical Instrumentation (CCIC) #MS MS:INSTRUMENT_NAME Agilent 6545 QTOF MS:INSTRUMENT_TYPE QTOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS Masshunter software was used to collect the raw data. Progenesis was used for MS:MS_COMMENTS peak intergration. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS normalized MS_METABOLITE_DATA_START Samples Control_1 Control_2 Control_3 Control_4 2h ATP_1 2h ATP_2 2h ATP_3 2h ATP_4 2h TGF-β_1 2h TGF-β_2 2h TGF-β_3 2h TGF-β_4 Factors Treatment:Control Treatment:Control Treatment:Control Treatment:Control Treatment:2h ATP Treatment:2h ATP Treatment:2h ATP Treatment:2h ATP Treatment:2h TGF-β Treatment:2h TGF-β Treatment:2h TGF-β Treatment:2h TGF-β glucose-6-phosphate 102979.3174 95099.67725 169225.4087 194364.2848 142345.3552 123460.2078 123023.109 119684.3112 59423.01716 52552.15518 57348.77976 66727.4978 gamma-Glutamylcysteine 106200.9029 111657.9312 145486.5678 160580.0895 28451.09293 32751.30622 58632.56987 47255.0099 4651.684556 3226.595579 4912.753233 3554.641448 L-Cysteine 143279.0567 139255.5089 229898.7066 97083.09029 36521.82124 86218.81787 46444.96421 12387.98658 11749.49594 17776.8443 17987.98454 NADPH 899.3351172 3250.756112 2149.439816 906.8343734 1553.171532 2322.815674 1020.819744 2858.617641 1234.217026 1469.710669 1342.879962 1726.900041 NADP 25430.75568 35891.25176 27447.82833 17634.48117 34987.29924 33126.94661 22456.62328 29629.96277 22232.23451 24060.41113 23148.89253 21383.44907 Ubiquinone-2 957.9189286 1036.762959 437.9648284 377.1326976 278.1060856 475.9302029 628.114387 433.0856195 447.4160761 362.6247398 658.8495932 QH(2) 17105.33318 7099.353003 29271.29429 41445.37858 39683.52244 75805.15361 61366.16093 4228.279119 3585.672182 2148.902116 6774.171271 Guanosine triphosphate 1119.444524 899.0160947 496.2612172 501.994448 805.5061377 459.8842034 395.8217624 236.0024761 6307.963919 8525.178851 8502.192867 6208.768129 NADH 12983.28892 15231.76412 8204.008583 15335.04874 16455.91694 18838.24167 21647.6 20259.1694 17904.41933 16249.72582 20410.58666 33012.77186 FADH 1770.178308 4238.917001 1347.455445 1704.475493 1936.681228 1244.248591 2704.605031 3115.99094 5832.545752 11748.24209 5871.909317 2083.517754 Sphingosine 1-phosphate 44709.64106 59937.90217 48113.36454 111886.0248 77291.01023 117531.4062 101045.3927 108943.7019 82055.12193 Sphingosine 370904.4219 1599826.709 236812.8422 189881.6439 113495.854 104878.4702 184609.9763 260548.8452 195231.089 Sphinganine 17326.17368 27034.60726 6836.958729 4100.10731 4581.469327 5019.052169 985.6085801 3852.990169 2983.474302 Sphinganine 1-phosphate 15565.27036 15868.32095 14969.36669 31171.96214 31943.50697 33673.36749 17924.35933 19093.36799 24000.93178 Choline 787.8240036 1621.194477 3767.779458 3550.339187 3559.402373 3232.930367 1166.68219 1245.251183 1991.634849 PGP(16:0/16:0) 3620.624188 4532.942908 5849.090226 52411.56384 11081.43711 16621.27282 40891.70646 16142.49826 36780.25224 CDP-DG(16:0/16:0) 8832.547528 4773.215017 5241.315741 2795.827532 2653.382588 3571.393825 1792.02473 2180.495334 848.5393192 CerP(d18:1/12:0) 774.5729885 322.0608539 637.0527935 1317.925062 1135.041644 932.1830646 673.9284138 1430.47275 696.0295746 Dihydroceramide 67780.53513 29793.09816 38044.23758 49263.54581 38499.07602 45912.79527 87707.31406 67813.52302 61678.82656 Arachidonyl-CoA 509.2674814 678.541006 437.0566543 3601.950809 2806.106393 1516.036512 2131.086522 2984.53387 3379.96452 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name KEGG ID HMDB ID PUBCHEM ID Mode retention time m/z glucose-6-phosphate C00092 HMDB0001401 5958 Positive 10.28 259.0203538 gamma-Glutamylcysteine C00669 HMDB0001049 123938 Positive 3.56 249.056088 L-Cysteine C00097 HMDB0000574 5862 Positive 1.40 122.0261672 NADPH C0005 HMDB0000221 5884 Positive 10.99 783.0640344 NADP C00006 HMDB0000217 5886 Positive 9.14 784.0747519 Ubiquinone-2 C00399 HMDB0006709 5280346 Positive 10.87 637.345942 QH(2) C00390 HMDB0059661 447920 Positive 9.82 291.0997952 Guanosine triphosphate C00044 HMDB0001273 6830 Positive 5.34 523.9958502 NADH C000)4 HMDB0001487 439153 Positive 8.86 704.0773637 FADH C01352 HMDB0001197 446013 Positive 12.44 826.1013232 Sphingosine 1-phosphate C06124 HMDB0000277 5353956 Positive 9.52 402.2316347 Sphingosine C00319 HMDB0000252 5353955 Positive 14.42 300.2888506 Sphinganine C00836 HMDB0000269 91486 Positive 13.72 302.3042431 Sphinganine 1-phosphate C01120 HMDB0001383 644260 Positive 9.24 763.5595787 Choline C00114 HMDB0000097 305 Positive 10.21 143.0693893 PGP(16:0/16:0) NA HMDB0013472 49859598 Positive 13.48 841.4543001 CDP-DG(16:0/16:0) NA HMDB0006968 24779567 Positive 11.31 954.4894967 CerP(d18:1/12:0) NA HMDB0010699 5283580 Positive 24.07 1123.836277 Dihydroceramide C12126 HMDB0006752 16755624 Positive 10.98 659.5975016 Arachidonyl-CoA C02249 HMDB0006523 16061152 Positive 13.54 1054.345472 METABOLITES_END #END