#METABOLOMICS WORKBENCH Wei_Xu_20221128_224703 DATATRACK_ID:3608 STUDY_ID:ST002365 ANALYSIS_ID:AN003860 PROJECT_ID:PR001520
VERSION             	1
CREATED_ON             	November 28, 2022, 11:12 pm
#PROJECT
PR:PROJECT_TITLE                 	[U-13C]glutamine 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]glutamine for 4-6 hours. Intracellular glutamine-derived glutamate and
PR:PROJECT_SUMMARY               	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]glutamine 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]glutamine for 4-6 hours. Intracellular glutamine-derived glutamate 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-Gln_Got1_Wt_01.d
SUBJECT_SAMPLE_FACTORS           	-	Got1_Wt_02	Treatment:GOT1_WT	RAW_FILE_NAME=13C-Gln_Got1_Wt_02.d
SUBJECT_SAMPLE_FACTORS           	-	Got1_Wt_03	Treatment:GOT1_WT	RAW_FILE_NAME=13C-Gln_Got1_Wt_03.d
SUBJECT_SAMPLE_FACTORS           	-	Got1_KO_01	Treatment:GOT1_KO	RAW_FILE_NAME=13C-Gln_Got1_KO_01.d
SUBJECT_SAMPLE_FACTORS           	-	Got1_KO_02	Treatment:GOT1_KO	RAW_FILE_NAME=13C-Gln_Got1_KO_02.d
SUBJECT_SAMPLE_FACTORS           	-	Got1_KO_03	Treatment:GOT1_KO	RAW_FILE_NAME=13C-Gln_Got1_KO_03.d
SUBJECT_SAMPLE_FACTORS           	-	Glud1_Wt_01	Treatment:GLUD1_WT	RAW_FILE_NAME=13C-Gln_Glud1_Wt_01.d
SUBJECT_SAMPLE_FACTORS           	-	Glud1_Wt_02	Treatment:GLUD1_WT	RAW_FILE_NAME=13C-Gln_Glud1_Wt_02.d
SUBJECT_SAMPLE_FACTORS           	-	Glud1_Wt_03	Treatment:GLUD1_WT	RAW_FILE_NAME=13C-Gln_Glud1_Wt_03.d
SUBJECT_SAMPLE_FACTORS           	-	Glud1_KO_01	Treatment:GLUD1_KO	RAW_FILE_NAME=13C-Gln_Glud1_KO_01.d
SUBJECT_SAMPLE_FACTORS           	-	Glud1_KO_02	Treatment:GLUD1_KO	RAW_FILE_NAME=13C-Gln_Glud1_KO_02.d
SUBJECT_SAMPLE_FACTORS           	-	Glud1_KO_03	Treatment:GLUD1_KO	RAW_FILE_NAME=13C-Gln_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 4 mM [U-13C]glutamine 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	534836.88	498853.32	473872.75	318101.32	312064.28	324807.72	784154.03	857005.15	784191.31	980927.85	980077.84	991573.11
Glutamate_M+1	322274.83	311435.18	303791.39	159264.14	170329.37	163847.04	463680.39	503651.82	456761.76	539583.02	534228.71	546404.41
Glutamate_M+2	223483.97	219617.72	213630.15	155584.77	161485.19	160207.29	291740.66	314577.2	288615.16	320457.48	320968.41	330838.16
Glutamate_M+3	602413.31	579651.43	587032.17	455233.19	472928.9	469085.63	662994.15	731069.47	669494.73	776876.75	779639.45	808086.84
Glutamate_M+4	111295.53	106454.87	108862.58	113676.34	117834.36	114220.69	104879.4	119767.47	109631.07	113876.5	113382	117103.89
Glutamate_M+5	1955695.46	1900509.45	1931620.33	1606480.11	1619762.29	1558608.02	1705732.91	1942677.11	1813683.03	1963565.29	1949191.82	2026526.61
α-Ketoglutarate_M+0	23175.68	17732.44	23905.92	15360.26	12190.59	12545.54	24248.93	21484.4	18921.92	19968.39	29001.51	28328.55
α-Ketoglutarate_M+1	5806.67	6636.24	4800.44	1444.42	2889.23	1802.64	8265.37	7238.36	7102.7	9825.8	9386.94	9208.44
α-Ketoglutarate_M+2	0	2316.54	0	0	3312.77	0	3793.5	2403.14	3895.99	3509.74	1818.54	976.55
α-Ketoglutarate_M+3	9253.18	10779.38	11564.44	3451.76	5809.96	4687.97	9975.54	11804.84	11127.59	13016.64	13156.77	11762.66
α-Ketoglutarate_M+4	1546.59	993.15	2334.52	1593.98	638.14	943.37	1304.21	2466.99	1188.71	1335.96	869.96	1160.47
α-Ketoglutarate_M+5	31540.37	32340.16	32963.5	14438.09	15844.28	11869.53	26425.42	29688.46	29432.52	28211.29	31299.04	29182.48
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
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