#METABOLOMICS WORKBENCH Sethjparker_20210526_151006 DATATRACK_ID:2657 STUDY_ID:ST001860 ANALYSIS_ID:AN003016 PROJECT_ID:PR001173 VERSION 1 CREATED_ON July 16, 2021, 12:39 pm #PROJECT PR:PROJECT_TITLE Spontaneous hydrolysis and spurious metabolic properties of α-ketoglutarate PR:PROJECT_TITLE esters PR:PROJECT_TYPE Manuscript PR:PROJECT_SUMMARY α-ketoglutarate (KG), also referred to as 2-oxoglutarate, is a key intermediate PR:PROJECT_SUMMARY of cellular metabolism with pleiotropic functions. Cell-permeable esterified PR:PROJECT_SUMMARY analogs are widely used to study the role of KG in governing bioenergetic and PR:PROJECT_SUMMARY amino acid metabolism and DNA, RNA, and protein hydroxylation reactions, as PR:PROJECT_SUMMARY cellular membranes are thought to be impermeable to KG. Here we show that PR:PROJECT_SUMMARY esterified KG analogs rapidly hydrolyze in aqueous media, yielding KG that, in PR:PROJECT_SUMMARY contrast to prevailing assumptions, can be imported by many cell lines. PR:PROJECT_SUMMARY Esterified KG analogs exhibit spurious KG-independent effects on cellular PR:PROJECT_SUMMARY metabolism, including extracellular acidification, arising from rapid hydrolysis PR:PROJECT_SUMMARY and de-protonation of α-ketoesters, and significant analog-specific inhibitory PR:PROJECT_SUMMARY effects on glycolysis or mitochondrial respiration. In many cell lines, imported PR:PROJECT_SUMMARY KG metabolizes to succinate in the cytosol, and we observe minimal KG PR:PROJECT_SUMMARY utilization for mitochondrial metabolism in normal culture conditions. These PR:PROJECT_SUMMARY findings demonstrate that nuclear and cytosolic KG-dependent reactions may PR:PROJECT_SUMMARY derive KG from functionally distinct subcellular pools and sources. PR:INSTITUTE University of British Columbia PR:DEPARTMENT Biochemistry & Molecular Biology PR:LAST_NAME Parker PR:FIRST_NAME Seth PR:ADDRESS 950 W 28th Ave, Room 2099, Vancouver, British Columbia, Canada V6H 0B3 PR:EMAIL seth.parker@bcchr.ca PR:PHONE 6048753121 #STUDY ST:STUDY_TITLE Spontaneous hydrolysis and spurious metabolic properties of α-ketoglutarate ST:STUDY_TITLE esters ST:STUDY_SUMMARY 8988T cells treated with methyl acetate or 1 mM of alpha-ketoglutarate disodium ST:STUDY_SUMMARY salt or 1 mM of dimethyl-alpha-ketoglutarate for 3 hours prior to rapid ST:STUDY_SUMMARY quenching of metabolism and extraction of metabolites in 80% methanol (-80°C) ST:STUDY_SUMMARY containing internal QC standards. ST:INSTITUTE University of British Columbia ST:LAST_NAME Parker ST:FIRST_NAME Seth ST:ADDRESS 950 W 28th Ave, 2099, Vancouver, British Columbia, Canada V6H 0B3 ST:EMAIL seth.parker@bcchr.ca ST:NUM_GROUPS 3 ST:TOTAL_SUBJECTS 9 ST:NUM_MALES n/a ST:NUM_FEMALES n/a ST:STUDY_TYPE Manuscript ST:PHONE 6048753121 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 SU:CELL_BIOSOURCE_OR_SUPPLIER DSMZ SU:CELL_STRAIN_DETAILS 8988T SU:SUBJECT_COMMENTS pancreatic ductal adenocarcinoma SU:CELL_COUNTS 1-2x10^6 #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 - veh_1 Treatment:vehicle RAW_FILE_NAME=veh_1 SUBJECT_SAMPLE_FACTORS - veh_2 Treatment:vehicle RAW_FILE_NAME=veh_2 SUBJECT_SAMPLE_FACTORS - veh_3 Treatment:vehicle RAW_FILE_NAME=veh_3 SUBJECT_SAMPLE_FACTORS - KG_1 Treatment:KG (1 mM) RAW_FILE_NAME=KG_1 SUBJECT_SAMPLE_FACTORS - KG_2 Treatment:KG (1 mM) RAW_FILE_NAME=KG_2 SUBJECT_SAMPLE_FACTORS - KG_3 Treatment:KG (1 mM) RAW_FILE_NAME=KG_3 SUBJECT_SAMPLE_FACTORS - DMKG_1 Treatment:DMKG (1 mM) RAW_FILE_NAME=DMKG_1 SUBJECT_SAMPLE_FACTORS - DMKG_2 Treatment:DMKG (1 mM) RAW_FILE_NAME=DMKG_2 SUBJECT_SAMPLE_FACTORS - DMKG_3 Treatment:DMKG (1 mM) RAW_FILE_NAME=DMKG_3 #COLLECTION CO:COLLECTION_SUMMARY Metabolites were initially extracted from samples by quickly aspirating the cell CO:COLLECTION_SUMMARY culture media and adding 1 mL of extraction buffer, consisting of 80% methanol CO:COLLECTION_SUMMARY (Fisher Scientific) and 500 nM metabolomics amino acid mix standard (Cambridge CO:COLLECTION_SUMMARY Isotope Laboratories). To effectively scale all harvested samples to equivalent CO:COLLECTION_SUMMARY volumes of extraction buffer, samples were fully dried down by Speedvac (Thermo CO:COLLECTION_SUMMARY Fisher, Waltham, MA) and reconstituted volumetrically by mixing the entire dried CO:COLLECTION_SUMMARY cell pellet sample with 1 mL of 80% methanol without QC standards in 2.0 mL CO:COLLECTION_SUMMARY screw cap vials containing ~100 µL of disruption beads (Research Products CO:COLLECTION_SUMMARY International, Mount Prospect, IL). Samples were scaled to a ratio of 1e6 cells CO:COLLECTION_SUMMARY to 1 mL of extraction solvent with all steps being carried out in a cold room. CO:COLLECTION_SUMMARY Each was homogenized for 10 cycles on a bead blaster homogenizer (Benchmark CO:COLLECTION_SUMMARY Scientific, Edison, NJ). Cycling consisted of a 30 sec homogenization time at 6 CO:COLLECTION_SUMMARY m/s followed by a 30 sec pause. Samples were subsequently spun at 21,000 x g for CO:COLLECTION_SUMMARY 3 min at 4°C. A set volume of each (450 µL) was transferred to a 1.5 mL tube CO:COLLECTION_SUMMARY and dried down by Speedvac concentration. Samples were reconstituted in 50 µL CO:COLLECTION_SUMMARY of Optima LC/MS grade water (Fisher Scientific, Waltham, MA). Samples were CO:COLLECTION_SUMMARY sonicated for 2 mins, then centrifuged at 21,000 x g for 3 min at 4°C. Twenty CO:COLLECTION_SUMMARY microliters were transferred to LC vials containing glass inserts for analysis. CO:COLLECTION_SUMMARY The remaining sample was placed in -80°C for long term storage. CO:SAMPLE_TYPE Cultured cells CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY 8988T cells were treated with methyl acetate, which is used as a vehicle, 1 mM TR:TREATMENT_SUMMARY of alpha-ketoglutarate disodium salt, or 1 mM of dimethyl-alpha-ketoglutarate TR:TREATMENT_SUMMARY (prepared in methyl acetate) for 3 hours in DMEM supplemented with 10% dialyzed TR:TREATMENT_SUMMARY fetal bovine serum and 5% penstrep #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Dried samples were reconstituted in 50 µL of Optima LC/MS grade water (Fisher SP:SAMPLEPREP_SUMMARY Scientific, Waltham, MA). Samples were sonicated for 2 mins, then centrifuged at SP:SAMPLEPREP_SUMMARY 21,000 x g for 3 min at 4°C. Twenty microliters were transferred to LC vials SP:SAMPLEPREP_SUMMARY containing glass inserts for analysis. The remaining sample was placed in -80°C SP:SAMPLEPREP_SUMMARY for long term storage. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Samples were subjected to an LC-MS analysis to detect and quantify known peaks. CH:CHROMATOGRAPHY_SUMMARY A metabolite extraction was carried out on each sample by quickly aspirating CH:CHROMATOGRAPHY_SUMMARY experimental media and adding 1 mL of 80% methanol containing internal QC CH:CHROMATOGRAPHY_SUMMARY standards. The LC column was a MilliporeTM ZIC-pHILIC (2.1 x150 mm, 5 μm) CH:CHROMATOGRAPHY_SUMMARY coupled to a Dionex Ultimate 3000TM system and the column oven temperature was CH:CHROMATOGRAPHY_SUMMARY set to 25oC for the gradient elution. A flow rate of 100 μL/min was used with CH:CHROMATOGRAPHY_SUMMARY the following buffers; A) 10 mM ammonium carbonate in water, pH 9.0, and B) neat CH:CHROMATOGRAPHY_SUMMARY acetonitrile. The gradient profile was as follows; 80-20%B (0-30 min), 20-80%B CH:CHROMATOGRAPHY_SUMMARY (30-31 min), 80-80%B (31-42 min). Injection volume was set to 2 μL for all CH:CHROMATOGRAPHY_SUMMARY analyses (42 min total run time per injection). CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Thermo Dionex Ultimate 3000 CH:COLUMN_NAME SeQuant ZIC- pHILIC (150 x 2.1mm, 5um) CH:FLOW_RATE 100 uL/min CH:COLUMN_TEMPERATURE 25 CH:SOLVENT_A 10 mM ammonium carbonate in water, pH 9.0 CH:SOLVENT_B acetonitrile #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive HF hybrid Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS MS analyses were carried out by coupling the LC system to a Thermo Q Exactive MS:MS_COMMENTS HFTM mass spectrometer operating in heated electrospray ionization mode (HESI). MS:MS_COMMENTS Method duration was 30 min with a polarity switching data-dependent Top 5 method MS:MS_COMMENTS for both positive and negative modes. Spray voltage for both positive and MS:MS_COMMENTS negative modes was 3.5 kV and capillary temperature was set to 320oC with a MS:MS_COMMENTS sheath gas rate of 35, aux gas of 10, and max spray current of 100 μA. The full MS:MS_COMMENTS MS scan for both polarities utilized 120,000 resolution with an AGC target of MS:MS_COMMENTS 3e6 and a maximum IT of 100 ms, and the scan range was from 67-1000 m/z. Tandem MS:MS_COMMENTS MS spectra for both positive and negative mode used a resolution of 15,000, AGC MS:MS_COMMENTS target of 1e5, maximum IT of 50 ms, isolation window of 0.4 m/z, isolation MS:MS_COMMENTS offset of 0.1 m/z, fixed first mass of 50 m/z, and 3-way multiplexed normalized MS:MS_COMMENTS collision energies (nCE) of 10, 30, 80. The minimum AGC target was 1e4 with an MS:MS_COMMENTS intensity threshold of 2e5. All data were acquired in profile mode. The MS:MS_COMMENTS resulting ThermoTM RAW files were read with ThermoFisher CommonCore MS:MS_COMMENTS RawFileReader, and an in-house python script (Skeleton) was used for peak MS:MS_COMMENTS detection and quantification of all internal standards and sample peaks based on MS:MS_COMMENTS a previously established library of metabolite retention times and accurate MS:MS_COMMENTS masses adapted from the Whitehead Institute, and verified with authentic MS:MS_COMMENTS standards and/or high resolution MS/MS spectral manually curated against the MS:MS_COMMENTS NIST14MS/MS and METLIN (2017) tandem mass spectral libraries. MS:MS_RESULTS_FILE ST001860_AN003016_Results.txt UNITS:ion counts Has m/z:Yes Has RT:Yes RT units:Minutes #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS ion counts MS_METABOLITE_DATA_START Samples veh_1 veh_2 veh_3 KG_1 KG_2 KG_3 DMKG_1 DMKG_2 DMKG_3 Factors Treatment:vehicle Treatment:vehicle Treatment:vehicle Treatment:KG (1 mM) Treatment:KG (1 mM) Treatment:KG (1 mM) Treatment:DMKG (1 mM) Treatment:DMKG (1 mM) Treatment:DMKG (1 mM) Choline 115059616 124910208 131269152 137897744 125723368 163344592 146746464 107011584 134810864 L-Carnitine 19673710 29874924 34420632 34776344 27522494 41303960 46803580 31692304 38327672 NAD 741849 1283059 1267461 1772365 1098981 2003639 1818463 1204528 1554624 NADP 17682.5 17682.5 17682.5 17682.5 17682.5 17682.5 17682.5 17682.5 17682.5 1-Methylnicotinamide 33540 33540 33540 33540 33540 33540 33540 33540 33540 4-Hydroxyproline 676894 865509 1047665 787843 816978 980474 1007944 785694 893430 5'-Methylthioadenosine 696831 1503087 2047518 1846815 1447051 2773152 2626496 1669366 2296880 Acetyl-CoA 10000 10000 10000 10000 10000 10000 10000 10000 10000 Acetylcysteine 95203 152445 171255 257765 137116 221391 156072 178789 157420 Adenine 16259498 25201638 28156822 28151896 20872244 29087894 33201872 19688840 23345924 Adenosine 25351948 42133784 26670592 40911804 34938028 19464416 65012204 37852196 27819482 Adenosine monophosphate 114598 274424 260635 294607 116862 265249 73028 73028 250521 Beta-Alanine 446782 446782 446782 474096 446782 446782 446782 446782 446782 Betaine 26708842 32589060 35277420 35828344 31241926 39124040 38536012 32842210 34507180 Butyrylcarnitine 1112232 1834422 2242588 2344046 1751426 2933315 2856718 1796824 2315923 Carnosine 42135 15374.5 15374.5 15374.5 15374.5 15374.5 15374.5 15374.5 15374.5 CDP 13143 13143 13143 13143 13143 13143 13143 13143 13143 Citrulline 751697 561590 473597 3529372 530744 15833396 436323 436323 436323 Creatine 150419264 174162800 188712208 199487184 175832224 225026064 229429264 178608800 204384832 Creatinine 6828964 3526737 4573870 4309227 3593546 6506322 4610216 3533402 4009428 Cytidine 103697 212378 121712 233866 149148 4807023 912246 237084 132736 Cytidine monophosphate 21553 27048 24921 32391 27118 45617 42436 27720 28402 Cytosine 140184 140184 140184 140184 140184 140184 140184 140184 140184 Dihydrofolic acid 10778.5 10778.5 10778.5 10778.5 10778.5 10778.5 10778.5 10778.5 10778.5 FAD 11810.5 11810.5 11810.5 31893 11810.5 11810.5 11810.5 11810.5 11810.5 Folic acid 59337 110743 121368 114533 105036 150367 159157 88539 108510 Glutathione 20840.5 20840.5 20840.5 20840.5 33296 20840.5 20840.5 967161 73463 Glycine 8711905 11479722 12428598 11388022 10158343 13133003 12438314 11043863 12364159 Guanidoacetic acid 191998 169235 182285 221459 163569 387397 217318 171691 233209 Guanine 145507.5 190258 145507.5 659372 145507.5 519946 545243 199366 145507.5 Guanosine 119719 196161 148589 203083 198038 148931 268149 229712 144249 Guanosine monophosphate 15967 15967 15967 15967 15967 15967 15967 15967 15967 Hypotaurine 370668 395318 550540 641354 366178 654660 408947 381266 653964 Hypoxanthine 96359 81022.5 81022.5 172525 81022.5 36108704 119120 81935 81022.5 Inosine 111068 122103 126569 140589 113609 681882 171881 143973 133165 Inosinic acid 10000 10000 10000 10000 10000 10000 10000 10000 10000 L-Acetylcarnitine 5997934 7497806 8608883 10796484 8467374 11496819 3052405 1551751 1769404 L-Alanine 4878953 5256474 5416003 7151127 5443086 19061234 4390061 3456097 4041222 L-Arginine 129146050 129146050 129146050 129146050 129146050 129146050 129146050 129146050 129146050 L-Asparagine 295477 338066 385123 462745 439397 1030582 1107928 926297 1074808 L-Cystathionine 69825 84939 89369 146562 79357 138044 195100 98605 148818 L-Cysteine 11427.5 11427.5 11427.5 11427.5 11427.5 11427.5 11427.5 11427.5 11427.5 L-Glutamic acid 155652624 185090544 204696352 203994544 184163088 234216128 215350720 183725184 199651200 L-Glutamine 987509632 1187409280 1204924288 1228807552 1118130176 1358784256 1383364096 1188801024 1207392640 L-Histidine 222477984 274140192 297152320 284790496 268402576 312768576 292343616 260518032 287365408 L-Isoleucine 327943712 471109536 539576512 542105728 464015136 614817664 575157952 489476480 486693632 L-Kynurenine 71277 118677 196774 140849 135501 195898 179986 117714 163307 L-Leucine 471407744 620270464 709924352 722010176 622099392 822021760 769201728 633569408 700354944 L-Lysine 70620704 81351992 71438784 100574904 78836432 110715776 83842856 63946101 94885744 L-Methionine 201256992 281221600 331845632 314410208 271451744 382040864 338588160 270895072 305705632 L-Phenylalanine 272450880 386117280 470446432 442046432 381494368 516390592 508071232 375790400 422157696 L-Proline 273638752 358998560 382291136 376126912 335457248 474852576 459540288 353039648 410011360 L-Serine 37319604 45215144 52462904 51521912 45146748 62344032 55826300 47417600 53519244 L-Tryptophan 40559800 59680036 73770104 64866504 56888456 86171936 79623864 58870484 68428416 L-Tyrosine 132174744 189834352 220020112 206499872 184015376 248541648 234389120 174368160 204861184 L-Valine 294512608 400630976 433429184 403563456 378056800 506062144 457125792 367388352 420291136 N6-Acetyl-L-lysine 1053885 86912 153567 686503 3019418 1460698 2012349 94565 354962 N-Acetylglutamic acid 696387 1093756 1162969 1444691 1130277 1447156 1103168 920111 867840 N-Acetyl-L-alanine 1501970 2193205 2503034 2841470 2210970 3591204 2938325 2011480 2306898 N-Acetyl-L-methionine 299511 499951 639150 583900 453027 1144491 728772 543478 595526 N-Acetyl-L-phenylalanine 155263.5 155263.5 155263.5 155263.5 155263.5 155263.5 155263.5 155263.5 155263.5 N-Acetylornithine 44533 39952 51165 42020 33955 64760 51370 41191 42139 Niacinamide 147884784 194238640 221535120 205944832 184225696 239834320 243368176 183124288 199330528 O-Phosphoethanolamine 12174 12174 12174 12174 12174 16721 12174 12174 12174 Ornithine 10725298.5 11088351 10725298.5 22605320 10725298.5 36392880 11499996 10725298.5 12332889 Orotidylic acid 10000 10000 10000 15959 10000 10000 10000 10000 10000 Oxidized glutathione 7905098 11234902 11413977 15669883 11801465 17583974 15548888 10825087 14404985 Pantothenic acid 57482720 91449480 112044632 104913016 87508224 127753304 102560696 75176768 81051568 Phosphorylcholine 1142699776 1407257088 1430478592 1553405440 1389457152 1634350976 1572333568 1296004608 1473108864 Phosphoserine 10000 10000 10000 10000 10000 10000 10000 10000 10000 Pipecolic acid 149102.5 149102.5 149102.5 149102.5 149102.5 149102.5 165160 172338 155829 Propionylcarnitine 797635 1275955 1582940 1675712 1429742 2101163 3015406 2104158 2537068 S-Adenosylmethionine 554128 987231 903897 1030049 885721 1360373 1596624 1128412 1313367 Sarcosine 4300904 5172722 5583712 6273128 5203600 6991328 6321376 4559336 5164908 Uracil 34209 108668 27710 57690 59566 119078 230534 67396 31874 Uric acid 10000 10000 10000 10000 10000 10000 10000 10000 10000 Uridine 5'-diphosphate 20919 34676 39149 45760 31829 63021 45525 33987 46334 Uridine diphosphate-N-acetylglucosamine 121648 179520 246628 290900 171733 296929 282007 235335 326077 Valerylcarnitine 21193 25155 26175 25749 18582 46899 36789 20732 32777 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Choline L-Carnitine NAD NADP 1-Methylnicotinamide 4-Hydroxyproline 5'-Methylthioadenosine Acetyl-CoA Acetylcysteine Adenine Adenosine Adenosine monophosphate Beta-Alanine Betaine Butyrylcarnitine Carnosine CDP Citrulline Creatine Creatinine Cytidine Cytidine monophosphate Cytosine Dihydrofolic acid FAD Folic acid Glutathione Glycine Guanidoacetic acid Guanine Guanosine Guanosine monophosphate Hypotaurine Hypoxanthine Inosine Inosinic acid L-Acetylcarnitine L-Alanine L-Arginine L-Asparagine L-Cystathionine L-Cysteine L-Glutamic acid L-Glutamine L-Histidine L-Isoleucine L-Kynurenine L-Leucine L-Lysine L-Methionine L-Phenylalanine L-Proline L-Serine L-Tryptophan L-Tyrosine L-Valine N6-Acetyl-L-lysine N-Acetylglutamic acid N-Acetyl-L-alanine N-Acetyl-L-methionine N-Acetyl-L-phenylalanine N-Acetylornithine Niacinamide O-Phosphoethanolamine Ornithine Orotidylic acid Oxidized glutathione Pantothenic acid Phosphorylcholine Phosphoserine Pipecolic acid Propionylcarnitine S-Adenosylmethionine Sarcosine Uracil Uric acid Uridine 5'-diphosphate Uridine diphosphate-N-acetylglucosamine Valerylcarnitine METABOLITES_END #END