#METABOLOMICS WORKBENCH juliehaines_20220624_080435_mwtab.txt DATATRACK_ID:3317 STUDY_ID:ST002214 ANALYSIS_ID:AN003622 PROJECT_ID:PR001414
VERSION             	1
CREATED_ON             	July 12, 2022, 10:40 am
#PROJECT
PR:PROJECT_TITLE                 	The effects of PKM2 modulation and hypoxia on the metabolic landscape of
PR:PROJECT_TITLE                 	Alzheimer patient-derived induced neurons
PR:PROJECT_SUMMARY               	We have obtained fibroblast cultures from old adult human non-demented control
PR:PROJECT_SUMMARY               	donors and Alzheimer patients (AD). The fibroblasts were reprogrammed into
PR:PROJECT_SUMMARY               	directly induced neurons (iNs) to serve as an adult-like and age-equivalent
PR:PROJECT_SUMMARY               	model for aging and neurodegeneration. Their response to PKM2 modulation
PR:PROJECT_SUMMARY               	(shikonin 10 µM or PKM2 overexpression) and hypoxia (CoDo treatment) were
PR:PROJECT_SUMMARY               	assessed.
PR:INSTITUTE                     	University of Colorado Denver
PR:LABORATORY                    	Lab of Angelo D'Alessandro in collaboration with lab of Jerome Mertens (Univ of
PR:LABORATORY                    	Innsbruck)
PR:LAST_NAME                     	Haines
PR:FIRST_NAME                    	Julie
PR:ADDRESS                       	12801 E 17th Ave, Room 1303, Aurora, Colorado, 80045, USA
PR:EMAIL                         	julie.haines@cuanschutz.edu
PR:PHONE                         	3037243339
#STUDY
ST:STUDY_TITLE                   	The effects of PKM2 modulation and hypoxia on the metabolic landscape of
ST:STUDY_TITLE                   	Alzheimer patient-derived induced neurons
ST:STUDY_SUMMARY                 	We have obtained fibroblast cultures from old adult human non-demented control
ST:STUDY_SUMMARY                 	donors and Alzheimer patients (AD). The fibroblasts were reprogrammed into
ST:STUDY_SUMMARY                 	directly induced neurons (iNs) to serve as an adult-like and age-equivalent
ST:STUDY_SUMMARY                 	model for aging and neurodegeneration. Their response to PKM2 modulation
ST:STUDY_SUMMARY                 	(shikonin 10 µM or PKM2 overexpression) and hypoxia (CoDo treatment) were
ST:STUDY_SUMMARY                 	assessed.
ST:INSTITUTE                     	University of Colorado Denver
ST:LAST_NAME                     	Haines
ST:FIRST_NAME                    	Julie
ST:ADDRESS                       	12801 E 17th Ave, Room 1303, Aurora, Colorado, 80045, USA
ST:EMAIL                         	julie.haines@cuanschutz.edu
ST:PHONE                         	3037243339
#SUBJECT
SU:SUBJECT_TYPE                  	Human
SU:SUBJECT_SPECIES               	Homo sapiens
SU:TAXONOMY_ID                   	9606
SU:AGE_OR_AGE_RANGE              	50-90 yrs
#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           	-	3131-C	Condition:Alzheimer patient | Treatment:DMSO	RAW_FILE_NAME=MR36-1
SUBJECT_SAMPLE_FACTORS           	-	3131-S	Condition:Alzheimer patient | Treatment:Shikonin	RAW_FILE_NAME=MR36-3
SUBJECT_SAMPLE_FACTORS           	-	3053-C	Condition:Alzheimer patient | Treatment:DMSO	RAW_FILE_NAME=MR36-5
SUBJECT_SAMPLE_FACTORS           	-	3053-S	Condition:Alzheimer patient | Treatment:Shikonin	RAW_FILE_NAME=MR36-7
SUBJECT_SAMPLE_FACTORS           	-	8020-C	Condition:Alzheimer patient | Treatment:DMSO	RAW_FILE_NAME=MR36-9
SUBJECT_SAMPLE_FACTORS           	-	8020-S	Condition:Alzheimer patient | Treatment:Shikonin	RAW_FILE_NAME=MR36-11
SUBJECT_SAMPLE_FACTORS           	-	2800-C	Condition:Alzheimer patient | Treatment:DMSO	RAW_FILE_NAME=MR36-13
SUBJECT_SAMPLE_FACTORS           	-	2800-S	Condition:Alzheimer patient | Treatment:Shikonin	RAW_FILE_NAME=MR36-15
SUBJECT_SAMPLE_FACTORS           	-	2608-C	Condition:non-demented control | Treatment:GFP-overexpression	RAW_FILE_NAME=MR36-17
SUBJECT_SAMPLE_FACTORS           	-	2608-OE	Condition:non-demented control | Treatment:PKM2-overexpression	RAW_FILE_NAME=MR36-18
SUBJECT_SAMPLE_FACTORS           	-	8011-C	Condition:non-demented control | Treatment:GFP-overexpression	RAW_FILE_NAME=MR36-19
SUBJECT_SAMPLE_FACTORS           	-	8011-OE	Condition:non-demented control | Treatment:PKM2-overexpression	RAW_FILE_NAME=MR36-20
SUBJECT_SAMPLE_FACTORS           	-	8011-DFO	Condition:non-demented control | Treatment:CoDo Treatment	RAW_FILE_NAME=MR36-21
SUBJECT_SAMPLE_FACTORS           	-	2785-C	Condition:non-demented control | Treatment:GFP-overexpression	RAW_FILE_NAME=MR36-22
SUBJECT_SAMPLE_FACTORS           	-	2785-OE	Condition:non-demented control | Treatment:PKM2-overexpression	RAW_FILE_NAME=MR36-23
SUBJECT_SAMPLE_FACTORS           	-	2785-DFO	Condition:non-demented control | Treatment:CoDo Treatment	RAW_FILE_NAME=MR36-24
#COLLECTION
CO:COLLECTION_SUMMARY            	For Metabolome, iNs were flow cytometry-isolated 21 days of conversion. iNs were
CO:COLLECTION_SUMMARY            	sorted based on the expression of the PSA-NCAM surface markers using PSA-NCAM-PE
CO:COLLECTION_SUMMARY            	(Miltenyi) and re-plated on Geltrex-coated wells. The following day, iNs were
CO:COLLECTION_SUMMARY            	treated with 2.5 mM DGlucose-C13 for 6 hours and harvested using Tryple.
CO:COLLECTION_SUMMARY            	Supernatant and cell pellets were shock frozen in liquid nitrogen.
CO:SAMPLE_TYPE                   	Biopsy
#TREATMENT
TR:TREATMENT_SUMMARY             	After two weeks of conversion, cells were treated for 10 days with shikonin (10
TR:TREATMENT_SUMMARY             	µM, ChemCruz) before FACS sorting. Control iNs were FACS-sorted and plated on
TR:TREATMENT_SUMMARY             	Geltrex-coated wells before transduction with pLVXTP-EGFP-PKM2 or pLVXTP-EGFP.
TR:TREATMENT_SUMMARY             	Around 60 % of all neurons were successfully transduced. Five days after
TR:TREATMENT_SUMMARY             	transduction, iNs were harvested for MS-analysis. Alternatively, cells were
TR:TREATMENT_SUMMARY             	treated with CoDo for 6 hours before harvest.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Metabolites from frozen cell pellets were extracted at 150,000 cells/mL in cold
SP:SAMPLEPREP_SUMMARY            	5:3:2 MeOH:acetonitrile:water. Samples were vortexed 30 min at 4 degrees C then
SP:SAMPLEPREP_SUMMARY            	supernatants clarified by centrifugation (10 min, 10,000 g, 4 degrees C).
SP:SAMPLEPREP_SUMMARY            	Supernatants were split into two equal tubes and gently dried using a vacuum
SP:SAMPLEPREP_SUMMARY            	concentrator. One set of residues were resuspended in 12 uL of 0.1% formic acid
SP:SAMPLEPREP_SUMMARY            	(for positive ion mode). The other set of residues were resuspended in 12 uL of
SP:SAMPLEPREP_SUMMARY            	1 mM ammonium acetate (for negative ion mode).
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_SUMMARY        	Negative C18 cells
CH:CHROMATOGRAPHY_TYPE           	Reversed phase
CH:INSTRUMENT_NAME               	Thermo Vanquish
CH:COLUMN_NAME                   	Phenomenex Kinetex C18 2.1 x 150 mm, 1.7 um
CH:SAMPLE_INJECTION              	10 uL
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Thermo Q Exactive Orbitrap
MS:INSTRUMENT_TYPE               	Orbitrap
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	NEGATIVE
MS:MS_COMMENTS                   	Resolution 70,000, scan range 65-900 m/z, maximum injection time 200 ms,
MS:MS_COMMENTS                   	microscans 2, automatic gain control (AGC) 3 x 10^6 ions, source voltage 4.0 kV,
MS:MS_COMMENTS                   	capillary temperature 320 C, and sheath gas 45, auxiliary gas 15, and sweep gas
MS:MS_COMMENTS                   	0 (all nitrogen). Data converted to mzXML using RawConverter. Metabolites were
MS:MS_COMMENTS                   	annotated and integrated using Maven in conjunction with the KEGG database.
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS	peak area
MS_METABOLITE_DATA_START
Samples	3131-C	3053-C	8020-C	2800-C	2608-C	8011-C	2785-C	3131-S	3053-S	8020-S	2800-S	2608-OE	8011-OE	2785-OE	8011-DFO	2785-DFO
Factors	Condition:Alzheimer patient | Treatment:DMSO	Condition:Alzheimer patient | Treatment:DMSO	Condition:Alzheimer patient | Treatment:DMSO	Condition:Alzheimer patient | Treatment:DMSO	Condition:non-demented control | Treatment:GFP-overexpression	Condition:non-demented control | Treatment:GFP-overexpression	Condition:non-demented control | Treatment:GFP-overexpression	Condition:Alzheimer patient | Treatment:Shikonin	Condition:Alzheimer patient | Treatment:Shikonin	Condition:Alzheimer patient | Treatment:Shikonin	Condition:Alzheimer patient | Treatment:Shikonin	Condition:non-demented control | Treatment:PKM2-overexpression	Condition:non-demented control | Treatment:PKM2-overexpression	Condition:non-demented control | Treatment:PKM2-overexpression	Condition:non-demented control | Treatment:CoDo Treatment	Condition:non-demented control | Treatment:CoDo Treatment
dAMP	8698.363	7426.417	8423.361	7044.199	9537.36	8682.341	7659.17	17786.48	16565.23	9340.69	5567.291	10290.04	20328.4	12761.64	4939.935	8523.719
dCMP	18106.84	12643.76	18691.69	13855.19	18535.58	19082.22	17449.67	28185.16	26349.17	22559.44	9480.604	16186.61	36955.8	36406.25	6288.346	0
2',3'-Cyclic CMP	15536.93	12155.62	18338.95	7597.142	15392	24252.85	14159.78	26469.65	25856.12	23985.45	12885.97	9775.735	38799.95	43326.52	3296.335	0
UDP-glucose	9225.375	4387.281	14858.42	5246.401	10953.94	9931.653	4131.906	18422.71	10495.27	11047.78	8744.188	10123.17	21301.77	13193.1	0	0
Phosphate	63421910	50400140	74532060	91823960	94394330	49109160	68457760	66822090	57828630	98450790	52413810	146029400	88570180	83214020	199778400	193063000
Diphosphate	7107586	5027406	9217669	9813512	12212620	5307097	7544398	7003068	5972100	11565840	6021240	19258740	9363520	10060660	26835490	25344520
D-Glucose	362824.8	483750.1	353864.9	288063.7	245480.1	420020.3	367578.4	480316.8	510257.4	390168.8	305124.4	299636.6	754697.2	390516.4	93525.4	62192.28
D-Glyceraldehyde 3-phosphate/Glycerone phosphate	156736.9	62239.52	192731.8	77514.23	129983.7	80303.42	89817.5	137813.1	152200.2	40691.15	128698.7	174803.4	614163.4	49632.51	6558.595	4170.484
2/3-Phospho-D-glycerate	156736.9	62239.52	192731.8	77514.23	129983.7	80303.42	89817.5	137813.1	152200.2	128698.7	40691.15	174803.4	614163.4	49632.51	6558.595	4170.484
Phosphoenolpyruvate	25019.55	12221.58	23240.05	18224.95	23803.34	13100.15	9300.866	25474.38	30062.85	6585.779	8797.708	34063.74	127171.5	5064.1	2067.09	0
Lactate	1538848	2782565	2332704	6177034	1919105	7293848	2385349	2961188	4349398	1957746	3672666	1845232	1886110	2527975	981915.1	1546692
Maltose	8907.681	36746.84	32818.59	53051.46	46331.81	31827.07	50712.95	312253	39016.46	46981.57	10031.48	34361.03	9674.745	23153.16	18553.48	34158.29
Maltotriose	14739.79	17038.73	16602.95	74746.49	13389.44	10652.63	15625.31	17800.7	11304.02	11018.87	13554.23	14290.98	15460.59	11214.57	13524.79	18232.32
Mannitol	63967.51	146251.4	45167.46	156700	37267.49	128331.9	45524.84	105580.2	166284.8	58045.91	29092.58	40337.45	98778.48	81625.46	30020.98	78348.6
Citrate	491482.3	623103.8	861660.3	2793952	1330968	1027024	921850.6	900203.4	1213030	1083270	491126	1004159	1104433	1071646	874627	914965.7
2-Oxoglutaramate	10160.83	15135.68	9182.937	22655.79	24100.81	11942.21	13286.21	26958.18	20294.9	13291.42	13262.75	10315.8	16016.4	25197	59782.79	19539.13
Succinate	242844.2	432249.6	198636.7	567978.2	299422.7	278764	170777.7	364957.2	335984.6	241483.2	189284.1	228697.2	327206.7	344245.4	247010.5	360944.3
Fumarate	16756.59	41482.12	23797.42	41503.58	32056.89	22280.22	16754.75	21464.97	60352.2	34980.46	26742.44	28178.72	27808.57	27122.15	7027.039	14166.9
Malate	328949.8	499910.1	644982.4	964666.7	669392.2	391439.4	362706	583184.3	517380.4	672316.3	331348.8	589609.4	645829.5	453728.1	267034.9	472282.4
Oxaloacetate	62740.75	45201.44	36387.04	48787.06	39116.63	35646.02	46600.53	49050.53	35933.69	42086.34	32584.35	48240.18	45890.05	38515	57477.17	59357.68
Sedoheptulose 1-phosphate	34684.73	9958.817	35681.03	31807.46	168357.6	24243.72	12627.79	35356.29	79948.9	4103.978	4510.142	9242.829	295663	8033.564	0	0
alpha-D-Ribose 1-phosphate	864939.9	366432.6	961099	447715.6	782573.5	553002.4	692805.9	624716.2	569977.8	466067	478638.1	1048346	1048907	299725.4	101299.6	87787.12
Ascorbate	14221.64	25792.08	28204.79	77755.91	30722.13	52766.27	25270	26043.91	32120.09	29079.84	20683.59	15771.74	27778.61	22293.92	39480.23	120073.5
L-gamma-Glutamyl-L-hypoglycin	13946.91	10204.89	17300.56	13531.73	19814.07	20289.55	17955.01	19594.85	11439.27	14159.84	12286.83	23473.17	39502.73	30854.24	18074.15	2395.416
Dimethylglycine	13181.13	17507.52	18664.62	1592.291	16267.04	21076.16	13788.66	17278.8	21930.33	6862.195	14007.77	19346.25	64850.25	35010.08	1194.109	2013.915
3-Phosphonooxypyruvate	1849.787	5150.528	5411.963	4983.985	3808.336	5329.268	4859.75	5595.26	4772.292	5092.737	4739.385	4179.087	4810.162	4995.352	6854.706	8227.251
N-Acetylneuraminate	11825.52	10246.44	22044.76	21505.99	20016.22	39462.93	19626.59	17569.73	25198.38	14194.28	14647.03	5741.222	38512.47	39934.63	0	0
UDP-N-acetyl-D-glucosamine	24047.63	36975.72	58595.38	20700.61	48019.64	37858.76	31875.4	49008.14	56247.55	65534.56	15853.27	80684.46	153271.6	54611.36	0	0
N-Acetyl-L-citrulline	43956.15	27426.72	26590.95	9558.133	20484.1	55412.38	32917.23	46666.07	37028.39	14949.95	44497.91	10552.13	74765.34	52252.12	11603.95	8395.821
N-Acetylornithine	37873.98	18764.28	36389.91	24474.92	41736.43	65050.83	33872.22	54646.31	25240.8	33880.23	32464.95	34070.92	101761.9	99722.99	9927.365	4565.348
Pantetheine	14049.11	8444.029	19467.54	13512.84	9229.919	24772.02	18895.65	25360.6	12619.37	23237.97	14434.16	17215.01	32142.45	41765.82	4227.843	796.2341
L-Selenomethionine	26550.42	40358.24	53032.58	58354.97	80622.57	36016.67	22450.91	45837.82	32091.68	94012.16	30168.66	65502.61	63790.61	72109.26	54877.45	47008.91
Nonanoic acid (pelargonate)	973017.2	965740.6	816486.5	1168327	1092306	544304.8	1435196	1689896	913470.9	620789.6	727703.1	1188157	1041023	671845.7	1224851	1622174
Decanoic acid (caprate)	515447.3	262240.7	348070.6	452774.7	476374.6	257495.2	601805.6	661072.2	519495.1	262682.7	287700	514184.1	421086	296774.2	549629.6	600450.7
Dodecanoic acid	489058.3	296921.4	354690	450488.6	359052.2	282186.5	533820.6	523372.8	1576561	340770	205678.5	594543.3	367925.7	543020.1	946812.5	875483.5
Tetradecanoic acid	622386.7	592002.2	651915.8	555347.1	522350.7	488031.2	581228.5	568931.5	623334.2	553863.6	475342.9	788588.2	546169.8	522165.8	2559952	2427063
Hexadecanoic acid	6513356	6294962	6175384	8714241	5364646	4372932	6550848	5931772	5021934	5981080	6238626	9215445	6617094	6231094	36604080	15727020
Octadecanoic acid	3652742	3590058	3951184	5952124	4804864	2516195	3453004	4106652	3314861	4005914	4041477	4979340	4101822	3360525	18652760	11110600
Tetradecenoic acid	29595.47	22524.65	21391.42	25363.59	19475.61	20549.46	28965.26	34548.71	123225.3	20000.72	15457.99	32834.68	21510.67	35350.77	53447.89	65338.08
Octadecenoic acid	179504.3	151499.3	250286.3	361962.6	246065.1	159211.1	180250.4	174919.5	159764.4	256415.3	230628.8	300227.7	597113.4	311866.5	2600651	464724.5
Linoleate	63057.97	43510.89	143725.6	139973.5	92145.45	38341	61657.95	50754.21	44995.18	134499.9	51433.38	333496.9	273339.7	154572.3	1254432	199063.8
Octadecatrienoic acid	11382.21	9322.453	28587.92	24756.79	15921.75	5592.601	15787.93	9697.188	15224.05	21407.66	8306.858	86279.88	55281.98	31070.21	216364.4	47154.47
Eicosatetraenoic acid	8760.267	5295.031	17426.12	24746.65	7552.434	3172.496	6410.102	6791.636	1235.863	11854.66	4555.685	37358.14	41373.67	9581.407	326403	12672.74
Eicosapentaenoic acid	4367.532	1806.235	5402.795	5622.183	3163.541	1754.145	4654.617	3232.155	934.6623	4852.628	1741.498	31137.88	15179.28	9201.031	93785.48	4668.346
Docosahexaenoic acid	3531.895	1198.58	5171.113	9207.46	1573.638	2199.593	2697.753	2363.851	0	6570.996	3557.484	14702.51	29496.41	3672.418	160822.1	2569.011
(8Z-11Z-14Z)-Icosatrienoic acid	6504.195	3652.789	20950.91	21601.06	11862.54	2728.598	5750.969	6126.607	1014.936	30821.55	9181.758	26933.54	53704.68	10425.79	273532.8	4842.954
(7Z-10Z-13Z-16Z-19Z)-Docosa-7-10-13-16-19-pentaenoic acid	3774.3	819.452	9358.396	12289.09	4828.871	2282.767	2583.198	3117.139	0	17277.91	6980.5	15669.27	35230.24	4052.182	253661	1831.109
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	KEGG ID	mz	r.t.
dAMP	C00360	330.057	0.8938249
dCMP	C00239	306.054	0.8841972
2',3'-Cyclic CMP	C02354	304.0387	0.8926612
UDP-glucose	C00029	565.0472	0.5639161
Phosphate	C00009	96.96803	0.5931287
Diphosphate	C00013	172.2786	0.5697691
D-Glucose	C00031	179.0554	0.6441681
D-Glyceraldehyde 3-phosphate/Glycerone phosphate	C00118	184.9846	0.5634706
2/3-Phospho-D-glycerate	C00631	184.9846	0.5634706
Phosphoenolpyruvate	C00074	166.9736	0.5888274
Lactate	C01432	89.02291	0.6447554
Maltose	C00208	341.1087	0.6883896
Maltotriose	C01835	503.1624	2.033332
Mannitol	C00392	181.0706	0.7418652
Citrate	C00158	191.0188	0.5733267
2-Oxoglutaramate	C00940	145.0137	0.5812235
Succinate	C00042	117.0179	0.5672373
Fumarate	C00122	115.0022	0.5814553
Malate	C00149	133.0129	0.5606962
Oxaloacetate	C00036	131.0003	0.584277
Sedoheptulose 1-phosphate	C06222	289.0329	0.5822244
alpha-D-Ribose 1-phosphate	C00620	229.0112	0.5781442
Ascorbate	C00072	170.5368	0.5791757
L-gamma-Glutamyl-L-hypoglycin	C08280	269.1117	0.9075518
Dimethylglycine	C01026	102.0545	0.6244575
3-Phosphonooxypyruvate	C03232	182.9668	0.8649063
N-Acetylneuraminate	C00270	308.0985	0.618627
UDP-N-acetyl-D-glucosamine	C00043	606.074	0.5645219
N-Acetyl-L-citrulline	C15532	216.0981	0.6439784
N-Acetylornithine	C00437	173.0921	0.786604
Pantetheine	C00831	277.1221	1.736557
L-Selenomethionine	C05335	195.9913	0.7191679
Nonanoic acid (pelargonate)	C01601	157.1222	2.099991
Decanoic acid (caprate)	C01571	171.1381	2.174107
Dodecanoic acid	C02679	199.1695	2.338359
Tetradecanoic acid	C06424	227.2011	2.571939
Hexadecanoic acid	C00249	255.2327	2.935052
Octadecanoic acid	C01530	283.2639	3.429194
Tetradecenoic acid	C08322	225.1855	2.379286
Octadecenoic acid	C00712	281.2484	2.963366
Linoleate	C01595	279.2328	2.655937
Octadecatrienoic acid	C06427	269.7249	2.464637
Eicosatetraenoic acid	C00219	303.2328	2.556659
Eicosapentaenoic acid	C06428	301.2172	2.404701
Docosahexaenoic acid	C06429	327.2331	2.470542
(8Z-11Z-14Z)-Icosatrienoic acid	C03242	305.2487	2.725766
(7Z-10Z-13Z-16Z-19Z)-Docosa-7-10-13-16-19-pentaenoic acid	C16513	329.249	2.577192
METABOLITES_END
#END