#METABOLOMICS WORKBENCH Sethjparker_20210526_151006 DATATRACK_ID:2657 STUDY_ID:ST001860 ANALYSIS_ID:AN003015 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                      	NEGATIVE
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_AN003015_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)
2,3-Diphosphoglyceric acid	17275	17275	17275	17275	17275	17275	17275	17275	17275
3-Hydroxybutyric acid	5062673.5	5062673.5	5062673.5	5062673.5	5062673.5	5062673.5	5062673.5	5062673.5	5062673.5
3-Phosphoglyceric acid	15357	15357	15357	16519	15357	40346	16609	15357	15357
4,5-Dihydroorotic acid	14092.5	14092.5	14092.5	14092.5	14092.5	14092.5	14092.5	14092.5	14092.5
5-Phosphoribosylamine	10000	10000	10000	10000	10000	10000	10000	10000	10000
6-Phosphogluconic acid	13584	13584	13584	13584	13584	13584	13584	13584	13584
Acetoacetic acid	118683.5	118683.5	118683.5	145633	119737	135244	118683.5	118683.5	118683.5
Adenosine diphosphate ribose	10784.5	14258	31551	24888	10784.5	28702	21753	10784.5	14325
Adenosine triphosphate	37495	39765	37495	53000	37495	51688	63410	37495	46029
Adenylsuccinic acid	10000	10000	10000	10000	10000	10000	10000	10000	10000
ADP	65959	65959	65959	72885	65959	91215	66212	65959	65959
Allantoin	21376	28825	23860	21673	23964	23974	35266	37202	26125
Aminoadipic acid	742392	1060873	1429260	1342694	1153650	1840811	1466279	1212977	1407958
Argininosuccinic acid	11388.5	11388.5	11388.5	11388.5	11388.5	18863	56743	33117	48657
Carbamoyl phosphate	15266.5	15266.5	15266.5	15266.5	15266.5	15266.5	15266.5	15266.5	15266.5
cis-Aconitic acid	2621500	3199978	3386901	4383227	3075470	4408516	6663232	4442668	6280930
Citric acid	321442	321442	321442	709935	321442	1074635	1104618	409391	1022127
Cyclic AMP	10836	10000	16708	15508	12625	18128	17182	14512	13517
Cytidine triphosphate	11834.5	11834.5	11834.5	11834.5	11834.5	11834.5	11834.5	11834.5	11834.5
D-2-Hydroxyglutaric acid	648087	886967	1105866	1302175	1041870	1588352	4405415	2849337	3546333
D-Glucose	13571387	15191066	19322880	17496420	16404799	20517378	19872596	16799792	18797498
D-Glyceraldehyde 3-phosphate	12416	12416	12416	12416	12416	12416	12416	12416	12416
Dihydroxyacetone phosphate	79909	151295	168670	226576	148612	271372	165205	90656	145496
D-Ribose 5-phosphate	31148	45577	40980	93005	69127	233703	51027	42972	38067
D-Sedoheptulose 7-phosphate	19148	20329	24697	30448	17479	39671	42906	27184	33467
dTDP	11114.5	11114.5	11114.5	11114.5	11114.5	11114.5	11114.5	11114.5	11114.5
Fructose 1,6-bisphosphate	10694	10694	10694	10694	10694	10694	10694	10694	10694
Fructose 6-phosphate	81197	124112	104465	178023	134126	264854	157833	81297	122811
Fumaric acid	1289357	1586562	1901462	2440619	1717420	4549376	9587739	7340844	8993491
Glucose 6-phosphate	293965	492685	452541	576833	441372	874516	571215	349059	734045
Glycerol 3-phosphate	660813	1028120	964804	1479194	1101668	2395761	1264464	724946	1163591
Guanosine diphosphate	13233.5	13233.5	13233.5	13233.5	13233.5	13233.5	13233.5	13233.5	13233.5
Guanosine triphosphate	10000	10000	10000	10000	10000	10000	10000	10000	10000
Homocysteine	10859	10859	10859	10859	10859	10859	10859	10859	10859
L-Aspartic acid	13356314	15692725	16806956	17417370	14968908	15250917	37506916	31578560	36945512
L-Cystine	340929	561551	825225	233902	143976	203740	162077	108146	152598
L-Lactic acid	20807254	26330298	31058576	33302772	27757884	39588552	28390524	19413534	21473202
L-Malic acid	22371478	26684776	30691290	43317656	29832890	90153440	136096544	105104104	130430264
L-Threonine	15203128	20347308	22476232	22888044	19128272	26705572	25337638	20540124	24229506
N-Acetylglutamine	54066	68500	85165	109445	74306	92703	130613	75512	89500
N-Acetylserine	8337286	11074774	12591269	13463451	11290738	15901635	14170906	10589457	13094227
NADH	70993	82283	95091	142511	104551	11948	126564	74169	60815
NADPH	10645.5	10645.5	10645.5	10645.5	10645.5	10645.5	10645.5	10645.5	10645.5
Nicotinic acid	48707	48707	48707	48707	48707	48707	48707	48707	48707
Orotic acid	45901	51473	117510	72909	69498	116169	78979	55203	97010
Oxoadipic acid	18393	18393	18393	18393	18393	18393	55752	50058	62886
Oxoglutaric acid	4720589	4720589	5059704	171147760	159098528	190401920	127153976	84432136	106727296
Palmitic acid	10526122	13231931	11742567	8990748	15755418	22264104	27769432	25574588	13953207
Phenol	2344528	2344528	2344528	2344528	2344528	2344528	2344528	2344528	2344528
Phosphocreatine	286097	497357	516780	625545	394151	750660	538857	287075	550842
Phosphoenolpyruvic acid	43643.5	56022	43643.5	105790	43643.5	333222	66464	43643.5	51931
Phosphoribosyl pyrophosphate	10000	10000	10000	10000	10000	10000	10000	10000	10000
Pyruvic acid	254417	177077	309257	537984	519382	3138472	455341	528608	649926
Ribose 1-phosphate	12713.5	12713.5	12713.5	12713.5	12713.5	12713.5	12713.5	12713.5	12829
Saccharopine	10616.5	10616.5	10616.5	10616.5	10616.5	10616.5	16793	10616.5	14219
S-Adenosylhomocysteine	10000	10000	10461	11415	10000	13629	16716	10000	10000
Succinic acid	1122269	1385384	1789396	10965860	8824452	10106919	8674890	5603230	7633037
Taurine	16144191	20892426	23251224	24990640	21162420	28297166	26740744	20496016	22861624
Thymidine	17531	17531	17531	17531	17531	17531	17531	17531	17531
Ureidopropionic acid	122232	155982	158605	202272	125042	408878	449946	375439	497404
Ureidosuccinic acid	62706	163360	177173	168165	84389	205076	155782	83280	126486
Uridine	321690	1810280	405614	749337	613062	2186774	3472786	948926	373451
Uridine 5'-monophosphate	92105	145958	167906	239069	93800	156500	54126.5	54126.5	158269
Uridine diphosphate glucose	293758	527328	569136	731860	524568	794808	661641	443422	575454
Uridine triphosphate	20178	20178	20178	20178	20178	22249	22934	20178	24245
Xanthine	61450	66346	92926	159477	66972	6953216	82868	54178	67823
Xanthosine	10643.5	10643.5	10643.5	10643.5	10643.5	34607	10643.5	10643.5	10643.5
Xanthylic acid	10000	10000	10000	10000	10000	10000	10000	10000	10000
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name
2,3-Diphosphoglyceric acid
3-Hydroxybutyric acid
3-Phosphoglyceric acid
4,5-Dihydroorotic acid
5-Phosphoribosylamine
6-Phosphogluconic acid
Acetoacetic acid
Adenosine diphosphate ribose
Adenosine triphosphate
Adenylsuccinic acid
ADP
Allantoin
Aminoadipic acid
Argininosuccinic acid
Carbamoyl phosphate
cis-Aconitic acid
Citric acid
Cyclic AMP
Cytidine triphosphate
D-2-Hydroxyglutaric acid
D-Glucose
D-Glyceraldehyde 3-phosphate
Dihydroxyacetone phosphate
D-Ribose 5-phosphate
D-Sedoheptulose 7-phosphate
dTDP
Fructose 1,6-bisphosphate
Fructose 6-phosphate
Fumaric acid
Glucose 6-phosphate
Glycerol 3-phosphate
Guanosine diphosphate
Guanosine triphosphate
Homocysteine
L-Aspartic acid
L-Cystine
L-Lactic acid
L-Malic acid
L-Threonine
N-Acetylglutamine
N-Acetylserine
NADH
NADPH
Nicotinic acid
Orotic acid
Oxoadipic acid
Oxoglutaric acid
Palmitic acid
Phenol
Phosphocreatine
Phosphoenolpyruvic acid
Phosphoribosyl pyrophosphate
Pyruvic acid
Ribose 1-phosphate
Saccharopine
S-Adenosylhomocysteine
Succinic acid
Taurine
Thymidine
Ureidopropionic acid
Ureidosuccinic acid
Uridine
Uridine 5'-monophosphate
Uridine diphosphate glucose
Uridine triphosphate
Xanthine
Xanthosine
Xanthylic acid
METABOLITES_END
#END