#METABOLOMICS WORKBENCH Wei_Xu_20221128_211727 DATATRACK_ID:3605 STUDY_ID:ST002364 ANALYSIS_ID:AN003859 PROJECT_ID:PR001519
VERSION             	1
CREATED_ON             	November 28, 2022, 10:06 pm
#PROJECT
PR:PROJECT_TITLE                 	[U-13C]glucose tracing in activated WT or GOT1 or GLUD1 knockout CD8+ T cells
PR:PROJECT_TYPE                  	MS quantifying intracellular glutamate levels
PR:PROJECT_SUMMARY               	WT, GOT1 knockout or GLUD1 knockout CD8+ T cells were activated with plate-bound
PR:PROJECT_SUMMARY               	anti-CD3 and soluble anti-CD28 for 24 hours. CD8+ T cells were pulsed with
PR:PROJECT_SUMMARY               	[U-13C]glucose for 4-6 hours. Intracellular glucose-derived glutamate, serine
PR:PROJECT_SUMMARY               	and α-ketoglutarate levels were quantified using MS.
PR:INSTITUTE                     	Johns Hopkins University
PR:LAST_NAME                     	Xu
PR:FIRST_NAME                    	Wei
PR:ADDRESS                       	1650 Orleans Street, Baltimore, MD 21287, USA.
PR:EMAIL                         	wxu29@jhmi.edu
PR:PHONE                         	443-220-9936
#STUDY
ST:STUDY_TITLE                   	[U-13C]glucose tracing in activated WT, GOT1 or GLUD1 knockout CD8+ T cells
ST:STUDY_SUMMARY                 	WT, GOT1 knockout or GLUD1 knockout CD8+ T cells were activated with plate-bound
ST:STUDY_SUMMARY                 	anti-CD3 and soluble anti-CD28 for 24 hours. CD8+ T cells were pulsed with
ST:STUDY_SUMMARY                 	[U-13C]glucose. Intracellular glucose-derived glutamate, serine and
ST:STUDY_SUMMARY                 	α-ketoglutarate levels were quantified using MS.
ST:INSTITUTE                     	Johns Hopkins University
ST:LAST_NAME                     	Xu
ST:FIRST_NAME                    	Wei
ST:ADDRESS                       	1650 Orleans Street, Baltimore, MD 21287, USA.
ST:EMAIL                         	wxu29@jhmi.edu
ST:PHONE                         	443-220-9936
#SUBJECT
SU:SUBJECT_TYPE                  	Cultured cells
SU:SUBJECT_SPECIES               	Mus musculus
SU:TAXONOMY_ID                   	10090
SU:GENOTYPE_STRAIN               	C57BL/6J
SU:AGE_OR_AGE_RANGE              	6-8 weeks
SU:GENDER                        	Male and female
#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           	-	Got1_Wt_01	Treatment:GOT1_WT	RAW_FILE_NAME=13C-Gluc_Got1_Wt_01.d
SUBJECT_SAMPLE_FACTORS           	-	Got1_Wt_02	Treatment:GOT1_WT	RAW_FILE_NAME=13C-Gluc_Got1_Wt_02.d
SUBJECT_SAMPLE_FACTORS           	-	Got1_Wt_03	Treatment:GOT1_WT	RAW_FILE_NAME=13C-Gluc_Got1_Wt_03.d
SUBJECT_SAMPLE_FACTORS           	-	Got1_KO_01	Treatment:GOT1_KO	RAW_FILE_NAME=13C-Gluc_Got1_KO_01.d
SUBJECT_SAMPLE_FACTORS           	-	Got1_KO_02	Treatment:GOT1_KO	RAW_FILE_NAME=13C-Gluc_Got1_KO_02.d
SUBJECT_SAMPLE_FACTORS           	-	Got1_KO_03	Treatment:GOT1_KO	RAW_FILE_NAME=13C-Gluc_Got1_KO_03.d
SUBJECT_SAMPLE_FACTORS           	-	Glud1_Wt_01	Treatment:GLUD1_WT	RAW_FILE_NAME=13C-Gluc_Glud1_Wt_01.d
SUBJECT_SAMPLE_FACTORS           	-	Glud1_Wt_02	Treatment:GLUD1_WT	RAW_FILE_NAME=13C-Gluc_Glud1_Wt_02.d
SUBJECT_SAMPLE_FACTORS           	-	Glud1_Wt_03	Treatment:GLUD1_WT	RAW_FILE_NAME=13C-Gluc_Glud1_Wt_03.d
SUBJECT_SAMPLE_FACTORS           	-	Glud1_KO_01	Treatment:GLUD1_KO	RAW_FILE_NAME=13C-Gluc_Glud1_KO_01.d
SUBJECT_SAMPLE_FACTORS           	-	Glud1_KO_02	Treatment:GLUD1_KO	RAW_FILE_NAME=13C-Gluc_Glud1_KO_02.d
SUBJECT_SAMPLE_FACTORS           	-	Glud1_KO_03	Treatment:GLUD1_KO	RAW_FILE_NAME=13C-Gluc_Glud1_KO_03.d
#COLLECTION
CO:COLLECTION_SUMMARY            	Cells were spun down and washed once with pre-warmed PBS and metabolites were
CO:COLLECTION_SUMMARY            	immediately extracted or stored at -80℃ until further extraction.
CO:SAMPLE_TYPE                   	Cultured cells
#TREATMENT
TR:TREATMENT_SUMMARY             	CD8+ T cells were isolated from spleens and lymph nodes from WT, T cell
TR:TREATMENT_SUMMARY             	conditional GOT1 or GLUD1 knockout mice. Cells were activated with plate-bound
TR:TREATMENT_SUMMARY             	anti-CD3 and soluble anti-CD28 for 24 hours. CD8+ T cells were counted and
TR:TREATMENT_SUMMARY             	resuspended in full media containing 11 mM [U-13C]glucose at 2 E6 mL-1. Normal
TR:TREATMENT_SUMMARY             	FBS was substituted with dialyzed FBS. Cells were collected for LC-MS analysis
TR:TREATMENT_SUMMARY             	4-6 hrs post incubation.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	Cells were spun down and washed once with pre-warmed PBS and metabolites were
SP:SAMPLEPREP_SUMMARY            	immediately extracted by adding methanol:water (80:20, v/v) extraction solution,
SP:SAMPLEPREP_SUMMARY            	sonicated and stored at -80 °C for at least 2 hrs to precipitate the proteins.
SP:SAMPLEPREP_SUMMARY            	Supernatant after centrifugation at 14,000xg for 10 minutes was dried under
SP:SAMPLEPREP_SUMMARY            	nitrogen gas. Metabolites were then reconstituted using ACN:water (50:50, v/v)
SP:SAMPLEPREP_SUMMARY            	overnight at 4 °C. Soluble metabolites after centrifugation at 14,000xg for 10
SP:SAMPLEPREP_SUMMARY            	minutes were subjected to analysis by liquid chromatography mass spectrometry
SP:SAMPLEPREP_SUMMARY            	(LC-MS).
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_TYPE           	Ion pair
CH:INSTRUMENT_NAME               	Agilent 1290 Infinity
CH:COLUMN_NAME                   	Agilent Zorbax Extend C18, 2.1 x 150 mm, 1.8 μm
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Agilent 6520 QTOF
MS:INSTRUMENT_TYPE               	QTOF
MS:MS_TYPE                       	ESI
MS:ION_MODE                      	NEGATIVE
MS:MS_COMMENTS                   	The optimized ESI Q-TOF parameters for MS experiments were: ion polarity,
MS:MS_COMMENTS                   	negative; gas temperature, 325 °C; drying gas, 10 L min-1; nebulizer pressure,
MS:MS_COMMENTS                   	45 psig; capillary voltage, 4,000 V; fragmentor, 140 V; skimmer, 65 V; mass
MS:MS_COMMENTS                   	range, 50-1100 m/z; acquisition rate, 1.5 spectra s-1; instrument state,
MS:MS_COMMENTS                   	extended dynamic range (1700 m/z, 2 GHz). Spectra were internally mass
MS:MS_COMMENTS                   	calibrated in real time by continuous infusion of a reference mass solution
MS:MS_COMMENTS                   	using an isocratic pump connected to a dual sprayer feeding into an electrospray
MS:MS_COMMENTS                   	ionization source. Data were acquired with MassHunter Acquisition software. A
MS:MS_COMMENTS                   	metabolite database with retention times based on the ion-pairing method was
MS:MS_COMMENTS                   	developed using Agilent MassHunter PCDL manager software. The isotopologue peak
MS:MS_COMMENTS                   	extractions were achieved by Agilent MassHunter Profinder software.
#MS_METABOLITE_DATA
MS_METABOLITE_DATA:UNITS	AUC
MS_METABOLITE_DATA_START
Samples	Got1_Wt_01	Got1_Wt_02	Got1_Wt_03	Got1_KO_01	Got1_KO_02	Got1_KO_03	Glud1_Wt_01	Glud1_Wt_02	Glud1_Wt_03	Glud1_KO_01	Glud1_KO_02	Glud1_KO_03
Factors	Treatment:GOT1_WT	Treatment:GOT1_WT	Treatment:GOT1_WT	Treatment:GOT1_KO	Treatment:GOT1_KO	Treatment:GOT1_KO	Treatment:GLUD1_WT	Treatment:GLUD1_WT	Treatment:GLUD1_WT	Treatment:GLUD1_KO	Treatment:GLUD1_KO	Treatment:GLUD1_KO
Glutamate_M+0	10285159.12	10306480.5	9240850.12	5584220.68	5280384.06	4866645.71	12538912.08	12262365.85	11671083.5	11809447.16	11619371.62	11719981.49
Glutamate_M+1	851113.07	849611.71	779928.25	525205.75	520164.2	474629.75	1007094.49	973278.58	933642.56	955421.94	952486.81	955784.2
Glutamate_M+2	2201341.6	2289307.65	2291818.68	1178774.31	1144788.51	1108808.24	2672429.91	2625398.95	2632125.93	2682270.6	2866309.41	2962091.63
Glutamate_M+3	640809.38	645109.59	613033.94	277420.26	274609.25	265232.17	773210.22	736280.73	737092.05	838387.79	830336.84	912093.17
Glutamate_M+4	692784.8	707526.57	730485	229758.41	216661.88	213752.38	860851.71	834992.27	827501.23	935251.8	962823.01	1055208.47
Glutamate_M+5	256815.68	250191.32	246172.37	38797.47	34425.53	33540.84	314822.93	291205.36	288372.95	345383.46	356763.14	406551.15
Serine_M+0	68117.64	68284.91	65265.2	60388.47	60862.1	64623.36	78868.46	78176.09	79918.41	81203.26	90482.89	88905.73
Serine_M+1	12079.31	7759.52	9343.68	1550.64	3427.46	1736.59	10528.03	13531.79	12167.2	15122.96	10302.91	10198.52
Serine_M+2	4669.42	6103.66	4623.34	1172.51	1391.97	1473.78	7116.43	5113.16	7607.19	7000.81	6895.32	7112.12
Serine_M+3	11950.23	11075.95	11946.66	5709.28	6787.3	5562.68	12699.25	11340.44	9274.44	12419.84	11637.29	13236.54
α-Ketoglutarate_M+0	191447.35	194321.94	175786.36	110221.47	101399.25	84973.99	243084.45	239583.08	211576.6	198005.85	203767.73	212564.09
α-Ketoglutarate_M+1	15464.74	15998.73	13922.72	9724.23	10340.28	6711.39	20014.16	15115.49	14645.23	16125.94	15782	17908.98
α-Ketoglutarate_M+2	42786.23	45487.64	45454.37	25375.54	25833.5	24811.49	59522.45	56247.62	51456.81	51204.43	51140.17	50777.56
α-Ketoglutarate_M+3	11309.62	12895.68	13246.42	6670.19	4444.71	4294.95	14401.27	14797.44	13261.63	12425.94	15228.57	17108.28
α-Ketoglutarate_M+4	16065.73	16032	16030.49	5891.69	5142.05	6023.04	20273.37	20796.35	17670.45	18994.07	21657.28	23603.71
α-Ketoglutarate_M+5	5067.18	4341.9	5157.36	106.1	587.76	1053.62	6178.45	5909.68	4502.39	6215.19	7446.66	7664.7
MS_METABOLITE_DATA_END
#METABOLITES
METABOLITES_START
metabolite_name	quantified m/z	PubChem ID	KEGG ID
Glutamate_M+0	146.0459	33032	C00302
Glutamate_M+1	147.0493	33032	C00302
Glutamate_M+2	148.0526	33032	C00302
Glutamate_M+3	149.0560	33032	C00302
Glutamate_M+4	150.0593	33032	C00302
Glutamate_M+5	151.0627	33032	C00302
Serine_M+0	104.0353	5951	C00716
Serine_M+1	105.0387	5951	C00716
Serine_M+2	106.0420	5951	C00716
Serine_M+3	107.0454	5951	C00716
α-Ketoglutarate_M+0	145.0142	164533	C00026
α-Ketoglutarate_M+1	146.0176	164533	C00026
α-Ketoglutarate_M+2	147.0209	164533	C00026
α-Ketoglutarate_M+3	148.0243	164533	C00026
α-Ketoglutarate_M+4	149.0276	164533	C00026
α-Ketoglutarate_M+5	150.0310	164533	C00026
METABOLITES_END
#END