#METABOLOMICS WORKBENCH kenanazam_20160912_110837_mwtab.txt DATATRACK_ID:735 STUDY_ID:ST000475 ANALYSIS_ID:AN000740 PROJECT_ID:PR000362 VERSION 1 CREATED_ON September 13, 2016, 2:12 pm #PROJECT PR:PROJECT_TITLE Metabolomic testing of a putative MDH inhibitor PR:PROJECT_SUMMARY Testing for metabolomic changes after treatment of leukemia cells with a PR:PROJECT_SUMMARY putative MDH inhibitor PR:INSTITUTE Case Western Reserve University PR:DEPARTMENT Pathology PR:LABORATORY Foundation and Institutional funds PR:LAST_NAME Wald PR:FIRST_NAME David PR:ADDRESS 2103 Cornell Rd WRB3-530 Cleveland, OH 44106 PR:EMAIL dnw@case.edu PR:PHONE NA #STUDY ST:STUDY_TITLE Metabolomic testing of a putative MDH inhibitor (global) ST:STUDY_TYPE drug treatment (single dose and timepoint) ST:STUDY_SUMMARY HL-60 cells will be treated for 15hr with a putative MDH inhibitor or vehicle ST:STUDY_SUMMARY (n=5 per group) ST:INSTITUTE University of Florida ST:DEPARTMENT SECIM ST:LABORATORY Case Western University ST:LAST_NAME Wald ST:FIRST_NAME David ST:ADDRESS 2103 Cornell Rd WRB3-530 Cleveland, OH 44106 ST:EMAIL dnw@case.edu ST:PHONE 2163685668 ST:NUM_GROUPS 2 ST:TOTAL_SUBJECTS 20 ST:STUDY_COMMENTS Total subjects: 10 for global testing and 10 for targeted testing #SUBJECT SU:SUBJECT_TYPE Human leukemia cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:CELL_STRAIN_DETAILS HL-60 SU:SUBJECT_COMMENTS immortalized SU:CELL_COUNTS 10 million per sample #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS untreat ut1 Treatment:- SUBJECT_SAMPLE_FACTORS untreat ut2 Treatment:- SUBJECT_SAMPLE_FACTORS untreat ut3 Treatment:- SUBJECT_SAMPLE_FACTORS untreat ut4 Treatment:- SUBJECT_SAMPLE_FACTORS untreat ut5 Treatment:- SUBJECT_SAMPLE_FACTORS treat tx1 Treatment:MDH inhib SUBJECT_SAMPLE_FACTORS treat tx2 Treatment:MDH inhib SUBJECT_SAMPLE_FACTORS treat tx3 Treatment:MDH inhib SUBJECT_SAMPLE_FACTORS treat tx4 Treatment:MDH inhib SUBJECT_SAMPLE_FACTORS treat tx5 Treatment:MDH inhib #COLLECTION CO:COLLECTION_SUMMARY Cells were centrifuged to remove growth media/drug and either flash frozen or CO:COLLECTION_SUMMARY washed 1x in PBS. The cells were washed for the targeted assay but not for the CO:COLLECTION_SUMMARY global assay. CO:SAMPLE_TYPE cell CO:STORAGE_CONDITIONS flash frozen CO:TISSUE_CELL_QUANTITY_TAKEN 10 million per sample #TREATMENT TR:TREATMENT_SUMMARY Cells were treated for 15hr with 1 micromolar of drug or vehicle TR:CELL_GROWTH_CONTAINER flasks TR:CELL_GROWTH_RATE ~24hr per division TR:CELL_MEDIA RPMI with 10% FBS TR:CELL_ENVIR_COND 5% CO2 incubator TR:CELL_MEDIA_LASTCHANGED 15 hr #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Amino Acid|Global SP:SAMPLEPREP_PROTOCOL_FILENAME SB_OA_Assay.pdf;Appendix C_Protein_DNA_quant.pdf SP:SAMPLE_SPIKING Appendix A - Internal Standard Prep GLCMS.pdf #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Global CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Thermo Scientific-Dionex Ultimate 3000 CH:COLUMN_NAME ACE Excel 2 C18-PFP (100 x 2.1mm, 2um) CH:METHODS_FILENAME Consolidated_Cellular_Metabolomics_SOP.pdf #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:MS_COMMENTS - MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS Peak area MS_METABOLITE_DATA_START Samples tx2 tx1 ut4 ut1 ut5 tx3 ut3 ut2 tx4 tx5 Factors Treatment:MDH inhib Treatment:MDH inhib Treatment:- Treatment:- Treatment:- Treatment:MDH inhib Treatment:- Treatment:- Treatment:MDH inhib Treatment:MDH inhib 3-tert-Butyladipic acid 3290000.0000 3110000.0000 2850000.0000 3360000.0000 2930000.0000 3580000.0000 3390000.0000 2620000.0000 3400000.0000 3190000.0000 4-oxoproline 14900000.0000 18000000.0000 28900000.0000 31400000.0000 28400000.0000 14200000.0000 39000000.0000 27300000.0000 12900000.0000 14900000.0000 ADP 21800000.0000 27500000.0000 31000000.0000 42300000.0000 28700000.0000 24900000.0000 36000000.0000 27900000.0000 23500000.0000 23000000.0000 ASPARTATE 9310000.0000 12100000.0000 25000000.0000 25500000.0000 20500000.0000 10200000.0000 25500000.0000 23500000.0000 9340000.0000 9540000.0000 C12H22O11-DISACCHARIDE-(6C/6C; GLC-GLC/GLC-FRC/GAL-GLC) 702000.0000 1220000.0000 561000.0000 1330000.0000 1300000.0000 336000.0000 1760000.0000 928000.0000 293000.0000 637000.0000 C6H12O6-HEXOSE/KETOSE/INOSITOL 9260000.0000 7650000.0000 6500000.0000 8830000.0000 8470000.0000 6360000.0000 10500000.0000 7750000.0000 7550000.0000 8640000.0000 CITRATE 11400000.0000 17600000.0000 35400000.0000 42100000.0000 29900000.0000 14100000.0000 43400000.0000 32800000.0000 12800000.0000 12900000.0000 CITRIC ACID-13C6 79600000.0000 84900000.0000 64300000.0000 78800000.0000 70300000.0000 82500000.0000 84100000.0000 58600000.0000 78200000.0000 72700000.0000 CMP 4340000.0000 4540000.0000 5220000.0000 6240000.0000 4960000.0000 5430000.0000 6250000.0000 4940000.0000 4920000.0000 4330000.0000 CREATINE 927000.0000 1710000.0000 7590000.0000 8350000.0000 5970000.0000 1180000.0000 8270000.0000 6320000.0000 1010000.0000 857000.0000 DIHYDROXYACETONE PHOSPHATE 20700000.0000 27600000.0000 27300000.0000 23900000.0000 32400000.0000 20500000.0000 34800000.0000 27900000.0000 19900000.0000 19300000.0000 Dinoterb (herbicide) 2600000.0000 2330000.0000 2150000.0000 2510000.0000 2260000.0000 2590000.0000 2700000.0000 2030000.0000 2690000.0000 2500000.0000 DISACCHARIDE (6C/6C)/2-alpha-D-glucosyl-D-glucose 919000.0000 1010000.0000 1100000.0000 1610000.0000 1710000.0000 816000.0000 2350000.0000 1220000.0000 685000.0000 975000.0000 D-RIBOSE 5-PHOSPHATE/RIBULOSE 5-PHOSPHATE 5240000.0000 6940000.0000 10400000.0000 11100000.0000 10300000.0000 5320000.0000 13200000.0000 10900000.0000 5050000.0000 4900000.0000 Fructose 1,6-bisphosphate 21000000.0000 20900000.0000 4100000.0000 7820000.0000 9150000.0000 24200000.0000 12800000.0000 9250000.0000 24300000.0000 23900000.0000 GLUCOSE/FRUCTOSE_R1 47600000.0000 47800000.0000 70200000.0000 78600000.0000 77400000.0000 39600000.0000 95700000.0000 70200000.0000 37000000.0000 49300000.0000 GLUCOSE/FRUCTOSE_R2 15800000.0000 15900000.0000 21600000.0000 26100000.0000 26100000.0000 13000000.0000 31500000.0000 23400000.0000 12100000.0000 16400000.0000 GLUTATHIONE 39500000.0000 81500000.0000 57200000.0000 63400000.0000 46800000.0000 54900000.0000 59500000.0000 49800000.0000 43000000.0000 38400000.0000 GLYCERALDEHYDE/LACTATE 31300000.0000 38400000.0000 82300000.0000 84000000.0000 72500000.0000 28300000.0000 96100000.0000 77600000.0000 24600000.0000 28300000.0000 GLYCEROL 2-PHOSPHATE/SN-GLYCEROL 3-PHOSPHATE 1860000.0000 2250000.0000 1850000.0000 1910000.0000 1350000.0000 1970000.0000 2230000.0000 1450000.0000 1910000.0000 1720000.0000 GUANINE 1090000.0000 1730000.0000 3200000.0000 3780000.0000 3180000.0000 1240000.0000 3940000.0000 2650000.0000 1230000.0000 1160000.0000 HYPOXANTHINE 9910000.0000 17900000.0000 36700000.0000 38800000.0000 31900000.0000 11600000.0000 36900000.0000 30700000.0000 10100000.0000 9700000.0000 INOSINE 5030000.0000 9540000.0000 28700000.0000 29900000.0000 22900000.0000 6630000.0000 29200000.0000 23500000.0000 5610000.0000 4730000.0000 ISOCITRIC ACID 292000.0000 411000.0000 932000.0000 1210000.0000 936000.0000 322000.0000 1170000.0000 711000.0000 304000.0000 203000.0000 L-GLUTAMIC ACID 29300000.0000 41900000.0000 88200000.0000 92900000.0000 70300000.0000 33500000.0000 94800000.0000 79100000.0000 30300000.0000 29500000.0000 L-GLUTAMINE 6930000.0000 7120000.0000 17100000.0000 17600000.0000 15700000.0000 7160000.0000 19100000.0000 15400000.0000 6670000.0000 6850000.0000 L-Tryptophan-13C11 8340000.0000 9490000.0000 7470000.0000 8920000.0000 8110000.0000 9000000.0000 9630000.0000 6830000.0000 8360000.0000 7880000.0000 LysoPE (18:0) 229000.0000 40700.0000 2000000.0000 3660000.0000 172000.0000 77300.0000 2370000.0000 1840000.0000 1190000.0000 1560000.0000 LysoPE (18:1) 2330000.0000 183000.0000 797000.0000 1120000.0000 425000.0000 577000.0000 993000.0000 758000.0000 7630000.0000 11100000.0000 LysoPE (20:4) 5610000.0000 865000.0000 1500000.0000 2680000.0000 986000.0000 1760000.0000 2030000.0000 1470000.0000 13400000.0000 18600000.0000 MALATE 6780000.0000 12200000.0000 33900000.0000 36400000.0000 24400000.0000 8680000.0000 37200000.0000 28600000.0000 9730000.0000 7890000.0000 Mono-(2-Ethylhexyl) Phthalate 2460000.0000 2150000.0000 1950000.0000 2320000.0000 2140000.0000 2510000.0000 2550000.0000 1910000.0000 2660000.0000 2310000.0000 N-ACETYL-DL-GLUTAMIC ACID 96000.0000 273000.0000 1700000.0000 1940000.0000 1430000.0000 231000.0000 1960000.0000 1490000.0000 172000.0000 160000.0000 N-ACETYL-L-ASPARTIC ACID 4440000.0000 7890000.0000 46700000.0000 53300000.0000 38000000.0000 5820000.0000 52100000.0000 39600000.0000 4940000.0000 4450000.0000 N-ACETYLNEURAMINATE 6460000.0000 8290000.0000 11700000.0000 12400000.0000 11500000.0000 8450000.0000 12200000.0000 9750000.0000 7410000.0000 7120000.0000 PHOSPHOCHOLINE 5210000.0000 7320000.0000 21000000.0000 24100000.0000 19600000.0000 6080000.0000 22400000.0000 19100000.0000 5190000.0000 5300000.0000 SARCOSINE/BETA-ALANINE 461000.0000 838000.0000 3620000.0000 3930000.0000 2900000.0000 569000.0000 3830000.0000 2980000.0000 448000.0000 446000.0000 TAURINE 4350000.0000 9640000.0000 22400000.0000 24100000.0000 17400000.0000 5290000.0000 21000000.0000 20300000.0000 4450000.0000 4010000.0000 Tryptophan-2,3,3-D3 11000000.0000 12600000.0000 9870000.0000 11700000.0000 10800000.0000 11800000.0000 12600000.0000 9240000.0000 10900000.0000 10600000.0000 URATE 1570000.0000 2190000.0000 2340000.0000 3270000.0000 2430000.0000 1310000.0000 3340000.0000 2240000.0000 1210000.0000 1460000.0000 URIDINE-5-MONOPHOSPHATE 8970000.0000 13500000.0000 29600000.0000 34100000.0000 25400000.0000 11500000.0000 34000000.0000 28800000.0000 9310000.0000 8790000.0000 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name retention index quantified m/z PubChem ID KEGG ID 3-tert-Butyladipic acid 9.86 201.1131 5192 4-oxoproline 1.69 128.0352 107541 ADP 1.02 426.0227 6022 ASPARTATE 0.71 132.0304 83887 C12H22O11-DISACCHARIDE-(6C/6C; GLC-GLC/GLC-FRC/GAL-GLC) 0.79 341.1081 C6H12O6-HEXOSE/KETOSE/INOSITOL 0.75 179.0563 CITRATE 1.78 191.0201 311 CITRIC ACID-13C6 1.79 197.0395 16217547 CMP 0.99 322.0453 6131 CREATINE 0.87 130.0621 586 DIHYDROXYACETONE PHOSPHATE 0.71 168.9904 668 Dinoterb (herbicide) 14.31 239.0676 14994 DISACCHARIDE (6C/6C)/2-alpha-D-glucosyl-D-glucose 0.76 377.0856 D-RIBOSE 5-PHOSPHATE/RIBULOSE 5-PHOSPHATE 0.71 229.0114 Fructose 1,6-bisphosphate 0.71 338.9878 10267 GLUCOSE/FRUCTOSE_R1 0.73 215.0333 GLUCOSE/FRUCTOSE_R2 0.73 217.0297 5793 GLUTATHIONE 1.47 306.0769 124886 GLYCERALDEHYDE/LACTATE 1.19 89.0237 GLYCEROL 2-PHOSPHATE/SN-GLYCEROL 3-PHOSPHATE 0.72 171.0063 GUANINE 1.38 150.0422 764 HYPOXANTHINE 1.89 135.0313 790 INOSINE 5.87 267.0729 6021 ISOCITRIC ACID 1.08 191.0200 1198 L-GLUTAMIC ACID 0.75 146.0457 33032 L-GLUTAMINE 0.72 145.0621 5961 L-Tryptophan-13C11 7.61 214.1196 LysoPE (18:0) 14.61 480.3095 LysoPE (18:1) 13.53 478.2943 LysoPE (20:4) 12.97 500.2807 MALATE 1.04 133.0145 92824 Mono-(2-Ethylhexyl) Phthalate 13.54 277.1441 20393 N-ACETYL-DL-GLUTAMIC ACID 2.04 188.0565 185 N-ACETYL-L-ASPARTIC ACID 1.37 174.0410 65065 N-ACETYLNEURAMINATE 0.79 308.0990 439197 PHOSPHOCHOLINE 0.72 218.0351 1014 SARCOSINE/BETA-ALANINE 0.87 88.0400 TAURINE 0.70 124.0075 1123 Tryptophan-2,3,3-D3 7.60 206.1010 URATE 1.73 167.0211 1175 URIDINE-5-MONOPHOSPHATE 1.15 323.0287 6030 METABOLITES_END #END