#METABOLOMICS WORKBENCH Wei_Xu_20221128_202535 DATATRACK_ID:3604 STUDY_ID:ST002363 ANALYSIS_ID:AN003858 PROJECT_ID:PR001518
VERSION             	1
CREATED_ON             	November 28, 2022, 9:06 pm
#PROJECT
PR:PROJECT_TITLE                 	[U-13C]glucose tracing in NT, AOA or EGCG treated activated CD8+ T cells
PR:PROJECT_TYPE                  	MS quantifying intracellular glutamate levels
PR:PROJECT_SUMMARY               	CD8+ T cells were activated with plate-bound anti-CD3 and soluble anti-CD28 for
PR:PROJECT_SUMMARY               	24 hours without (NT), or with AOA (250uM), or EGCG (500uM) treatment. CD8+ T
PR:PROJECT_SUMMARY               	cells were pulsed with [U-13C]glucose for 4-6 hours. Intracellular
PR:PROJECT_SUMMARY               	glucose-derived glutamate 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 NT, AOA or EGCG treated activated CD8+ T cells
ST:STUDY_SUMMARY                 	CD8+ T cells were activated with plate-bound anti-CD3 and soluble anti-CD28 for
ST:STUDY_SUMMARY                 	24 hours without (NT), or with AOA (250uM) or EGCG (500uM) treatment. CD8+ T
ST:STUDY_SUMMARY                 	cells were pulsed with [U-13C]glucose. Intracellular glucose-derived glutamate
ST:STUDY_SUMMARY                 	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           	-	NT_01	Treatment:NT	RAW_FILE_NAME=NT_01.d
SUBJECT_SAMPLE_FACTORS           	-	NT_02	Treatment:NT	RAW_FILE_NAME=NT_02.d
SUBJECT_SAMPLE_FACTORS           	-	NT_03	Treatment:NT	RAW_FILE_NAME=NT_03.d
SUBJECT_SAMPLE_FACTORS           	-	AOA_01	Treatment:AOA	RAW_FILE_NAME=AOA_01.d
SUBJECT_SAMPLE_FACTORS           	-	AOA_02	Treatment:AOA	RAW_FILE_NAME=AOA_02.d
SUBJECT_SAMPLE_FACTORS           	-	AOA_03	Treatment:AOA	RAW_FILE_NAME=AOA_03.d
SUBJECT_SAMPLE_FACTORS           	-	EGCG_01	Treatment:EGCG	RAW_FILE_NAME=EGCG_01.d
SUBJECT_SAMPLE_FACTORS           	-	EGCG_02	Treatment:EGCG	RAW_FILE_NAME=EGCG_02.d
SUBJECT_SAMPLE_FACTORS           	-	EGCG_03	Treatment:EGCG	RAW_FILE_NAME=EGCG_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 C57BL/6J mice.
TR:TREATMENT_SUMMARY             	Cells were activated with plate-bound anti-CD3 and soluble anti-CD28 for 24
TR:TREATMENT_SUMMARY             	hours, without (NT) or with AOA (250uM) or EGCG (500uM) treatment. CD8+ T cells
TR:TREATMENT_SUMMARY             	were counted and resuspended in full media containing 11 mM [U-13C]glucose at 2
TR:TREATMENT_SUMMARY             	E6 mL-1. Normal FBS was substituted with dialyzed FBS. Cells were collected for
TR:TREATMENT_SUMMARY             	LC-MS analysis 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	NT_01	NT_02	NT_03	AOA_01	AOA_02	AOA_03	EGCG_01	EGCG_02	EGCG_03
Factors	Treatment:NT	Treatment:NT	Treatment:NT	Treatment:AOA	Treatment:AOA	Treatment:AOA	Treatment:EGCG	Treatment:EGCG	Treatment:EGCG
Glutamate_M+0	2036085.45	1815686.58	1813901.61	2504717.50	2508358.22	2461829.93	1417735.11	1399433.95	1389672.53
Glutamate_M+1	227399.47	207516.61	211090.50	276069.97	276462.01	272267.67	195881.28	189509.47	189698.54
Glutamate_M+2	810852.18	739183.82	745987.17	532309.33	533958.57	523701.98	634148.21	611219.03	619890.84
Glutamate_M+3	298382.72	281753.93	285766.90	192912.45	195599.37	186144.30	274890.26	260687.42	262786.69
Glutamate_M+4	319896.17	290737.33	291325.82	155589.95	157530.83	150357.27	262497.90	257852.98	258426.41
Glutamate_M+5	132661.13	120190.82	119767.01	49244.20	50366.26	47535.26	111032.37	110785.47	111162.82
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
METABOLITES_END
#END