{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST002365","ANALYSIS_ID":"AN003860","VERSION":"1","CREATED_ON":"November 28, 2022, 11:12 pm"},

"PROJECT":{"PROJECT_TITLE":"[U-13C]glutamine tracing in activated WT or GOT1 or GLUD1 knockout CD8+ T cells","PROJECT_TYPE":"MS quantifying intracellular glutamate levels","PROJECT_SUMMARY":"WT, GOT1 knockout or GLUD1 knockout CD8+ T cells were activated with plate-bound anti-CD3 and soluble anti-CD28 for 24 hours. CD8+ T cells were pulsed with [U-13C]glutamine for 4-6 hours. Intracellular glutamine-derived glutamate and α-ketoglutarate levels were quantified using MS.","INSTITUTE":"Johns Hopkins University","LAST_NAME":"Xu","FIRST_NAME":"Wei","ADDRESS":"1650 Orleans Street, Baltimore, MD 21287, USA.","EMAIL":"wxu29@jhmi.edu","PHONE":"443-220-9936"},

"STUDY":{"STUDY_TITLE":"[U-13C]glutamine tracing in activated WT, GOT1 or GLUD1 knockout CD8+ T cells","STUDY_SUMMARY":"WT, GOT1 knockout or GLUD1 knockout CD8+ T cells were activated with plate-bound anti-CD3 and soluble anti-CD28 for 24 hours. CD8+ T cells were pulsed with [U-13C]glutamine for 4-6 hours. Intracellular glutamine-derived glutamate and α-ketoglutarate levels were quantified using MS.","INSTITUTE":"Johns Hopkins University","LAST_NAME":"Xu","FIRST_NAME":"Wei","ADDRESS":"1650 Orleans Street, Baltimore, MD 21287, USA.","EMAIL":"wxu29@jhmi.edu","PHONE":"443-220-9936"},

"SUBJECT":{"SUBJECT_TYPE":"Cultured cells","SUBJECT_SPECIES":"Mus musculus","TAXONOMY_ID":"10090","GENOTYPE_STRAIN":"C57BL/6J","AGE_OR_AGE_RANGE":"6-8 weeks","GENDER":"Male and female"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"Got1_Wt_01",
"Factors":{"Treatment":"GOT1_WT"},
"Additional sample data":{"RAW_FILE_NAME":"13C-Gln_Got1_Wt_01.d"}
},
{
"Subject ID":"-",
"Sample ID":"Got1_Wt_02",
"Factors":{"Treatment":"GOT1_WT"},
"Additional sample data":{"RAW_FILE_NAME":"13C-Gln_Got1_Wt_02.d"}
},
{
"Subject ID":"-",
"Sample ID":"Got1_Wt_03",
"Factors":{"Treatment":"GOT1_WT"},
"Additional sample data":{"RAW_FILE_NAME":"13C-Gln_Got1_Wt_03.d"}
},
{
"Subject ID":"-",
"Sample ID":"Got1_KO_01",
"Factors":{"Treatment":"GOT1_KO"},
"Additional sample data":{"RAW_FILE_NAME":"13C-Gln_Got1_KO_01.d"}
},
{
"Subject ID":"-",
"Sample ID":"Got1_KO_02",
"Factors":{"Treatment":"GOT1_KO"},
"Additional sample data":{"RAW_FILE_NAME":"13C-Gln_Got1_KO_02.d"}
},
{
"Subject ID":"-",
"Sample ID":"Got1_KO_03",
"Factors":{"Treatment":"GOT1_KO"},
"Additional sample data":{"RAW_FILE_NAME":"13C-Gln_Got1_KO_03.d"}
},
{
"Subject ID":"-",
"Sample ID":"Glud1_Wt_01",
"Factors":{"Treatment":"GLUD1_WT"},
"Additional sample data":{"RAW_FILE_NAME":"13C-Gln_Glud1_Wt_01.d"}
},
{
"Subject ID":"-",
"Sample ID":"Glud1_Wt_02",
"Factors":{"Treatment":"GLUD1_WT"},
"Additional sample data":{"RAW_FILE_NAME":"13C-Gln_Glud1_Wt_02.d"}
},
{
"Subject ID":"-",
"Sample ID":"Glud1_Wt_03",
"Factors":{"Treatment":"GLUD1_WT"},
"Additional sample data":{"RAW_FILE_NAME":"13C-Gln_Glud1_Wt_03.d"}
},
{
"Subject ID":"-",
"Sample ID":"Glud1_KO_01",
"Factors":{"Treatment":"GLUD1_KO"},
"Additional sample data":{"RAW_FILE_NAME":"13C-Gln_Glud1_KO_01.d"}
},
{
"Subject ID":"-",
"Sample ID":"Glud1_KO_02",
"Factors":{"Treatment":"GLUD1_KO"},
"Additional sample data":{"RAW_FILE_NAME":"13C-Gln_Glud1_KO_02.d"}
},
{
"Subject ID":"-",
"Sample ID":"Glud1_KO_03",
"Factors":{"Treatment":"GLUD1_KO"},
"Additional sample data":{"RAW_FILE_NAME":"13C-Gln_Glud1_KO_03.d"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"Cells were spun down and washed once with pre-warmed PBS and metabolites were immediately extracted or stored at -80℃ until further extraction.","SAMPLE_TYPE":"Cultured cells"},

"TREATMENT":{"TREATMENT_SUMMARY":"CD8+ T cells were isolated from spleens and lymph nodes from WT, T cell conditional GOT1 or GLUD1 knockout mice. Cells were activated with plate-bound anti-CD3 and soluble anti-CD28 for 24 hours. CD8+ T cells were counted and resuspended in full media containing 4 mM [U-13C]glutamine at 2 E6 mL-1. Normal FBS was substituted with dialyzed FBS. Cells were collected for LC-MS analysis 4-6 hrs post incubation."},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Cells were spun down and washed once with pre-warmed PBS and metabolites were immediately extracted by adding methanol:water (80:20, v/v) extraction solution, sonicated and stored at -80 °C for at least 2 hrs to precipitate the proteins. Supernatant after centrifugation at 14,000xg for 10 minutes was dried under nitrogen gas. Metabolites were then reconstituted using ACN:water (50:50, v/v) overnight at 4 °C. Soluble metabolites after centrifugation at 14,000xg for 10 minutes were subjected to analysis by liquid chromatography mass spectrometry (LC-MS)."},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_TYPE":"Ion pair","INSTRUMENT_NAME":"Agilent 1290 Infinity","COLUMN_NAME":"Agilent Zorbax Extend C18, 2.1 x 150 mm, 1.8 μm"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS"},

"MS":{"INSTRUMENT_NAME":"Agilent 6520 QTOF","INSTRUMENT_TYPE":"QTOF","MS_TYPE":"ESI","ION_MODE":"NEGATIVE","MS_COMMENTS":"The optimized ESI Q-TOF parameters for MS experiments were: ion polarity, negative; gas temperature, 325 °C; drying gas, 10 L min-1; nebulizer pressure, 45 psig; capillary voltage, 4,000 V; fragmentor, 140 V; skimmer, 65 V; mass range, 50-1100 m/z; acquisition rate, 1.5 spectra s-1; instrument state, extended dynamic range (1700 m/z, 2 GHz). Spectra were internally mass calibrated in real time by continuous infusion of a reference mass solution using an isocratic pump connected to a dual sprayer feeding into an electrospray ionization source. Data were acquired with MassHunter Acquisition software. A metabolite database with retention times based on the ion-pairing method was developed using Agilent MassHunter PCDL manager software. The isotopologue peak extractions were achieved by Agilent MassHunter Profinder software."},

"MS_METABOLITE_DATA":{
"Units":"AUC",

"Data":[{"Metabolite":"Glutamate_M+0","Got1_Wt_01":"534836.88","Got1_Wt_02":"498853.32","Got1_Wt_03":"473872.75","Got1_KO_01":"318101.32","Got1_KO_02":"312064.28","Got1_KO_03":"324807.72","Glud1_Wt_01":"784154.03","Glud1_Wt_02":"857005.15","Glud1_Wt_03":"784191.31","Glud1_KO_01":"980927.85","Glud1_KO_02":"980077.84","Glud1_KO_03":"991573.11"},{"Metabolite":"Glutamate_M+1","Got1_Wt_01":"322274.83","Got1_Wt_02":"311435.18","Got1_Wt_03":"303791.39","Got1_KO_01":"159264.14","Got1_KO_02":"170329.37","Got1_KO_03":"163847.04","Glud1_Wt_01":"463680.39","Glud1_Wt_02":"503651.82","Glud1_Wt_03":"456761.76","Glud1_KO_01":"539583.02","Glud1_KO_02":"534228.71","Glud1_KO_03":"546404.41"},{"Metabolite":"Glutamate_M+2","Got1_Wt_01":"223483.97","Got1_Wt_02":"219617.72","Got1_Wt_03":"213630.15","Got1_KO_01":"155584.77","Got1_KO_02":"161485.19","Got1_KO_03":"160207.29","Glud1_Wt_01":"291740.66","Glud1_Wt_02":"314577.2","Glud1_Wt_03":"288615.16","Glud1_KO_01":"320457.48","Glud1_KO_02":"320968.41","Glud1_KO_03":"330838.16"},{"Metabolite":"Glutamate_M+3","Got1_Wt_01":"602413.31","Got1_Wt_02":"579651.43","Got1_Wt_03":"587032.17","Got1_KO_01":"455233.19","Got1_KO_02":"472928.9","Got1_KO_03":"469085.63","Glud1_Wt_01":"662994.15","Glud1_Wt_02":"731069.47","Glud1_Wt_03":"669494.73","Glud1_KO_01":"776876.75","Glud1_KO_02":"779639.45","Glud1_KO_03":"808086.84"},{"Metabolite":"Glutamate_M+4","Got1_Wt_01":"111295.53","Got1_Wt_02":"106454.87","Got1_Wt_03":"108862.58","Got1_KO_01":"113676.34","Got1_KO_02":"117834.36","Got1_KO_03":"114220.69","Glud1_Wt_01":"104879.4","Glud1_Wt_02":"119767.47","Glud1_Wt_03":"109631.07","Glud1_KO_01":"113876.5","Glud1_KO_02":"113382","Glud1_KO_03":"117103.89"},{"Metabolite":"Glutamate_M+5","Got1_Wt_01":"1955695.46","Got1_Wt_02":"1900509.45","Got1_Wt_03":"1931620.33","Got1_KO_01":"1606480.11","Got1_KO_02":"1619762.29","Got1_KO_03":"1558608.02","Glud1_Wt_01":"1705732.91","Glud1_Wt_02":"1942677.11","Glud1_Wt_03":"1813683.03","Glud1_KO_01":"1963565.29","Glud1_KO_02":"1949191.82","Glud1_KO_03":"2026526.61"},{"Metabolite":"α-Ketoglutarate_M+0","Got1_Wt_01":"23175.68","Got1_Wt_02":"17732.44","Got1_Wt_03":"23905.92","Got1_KO_01":"15360.26","Got1_KO_02":"12190.59","Got1_KO_03":"12545.54","Glud1_Wt_01":"24248.93","Glud1_Wt_02":"21484.4","Glud1_Wt_03":"18921.92","Glud1_KO_01":"19968.39","Glud1_KO_02":"29001.51","Glud1_KO_03":"28328.55"},{"Metabolite":"α-Ketoglutarate_M+1","Got1_Wt_01":"5806.67","Got1_Wt_02":"6636.24","Got1_Wt_03":"4800.44","Got1_KO_01":"1444.42","Got1_KO_02":"2889.23","Got1_KO_03":"1802.64","Glud1_Wt_01":"8265.37","Glud1_Wt_02":"7238.36","Glud1_Wt_03":"7102.7","Glud1_KO_01":"9825.8","Glud1_KO_02":"9386.94","Glud1_KO_03":"9208.44"},{"Metabolite":"α-Ketoglutarate_M+2","Got1_Wt_01":"0","Got1_Wt_02":"2316.54","Got1_Wt_03":"0","Got1_KO_01":"0","Got1_KO_02":"3312.77","Got1_KO_03":"0","Glud1_Wt_01":"3793.5","Glud1_Wt_02":"2403.14","Glud1_Wt_03":"3895.99","Glud1_KO_01":"3509.74","Glud1_KO_02":"1818.54","Glud1_KO_03":"976.55"},{"Metabolite":"α-Ketoglutarate_M+3","Got1_Wt_01":"9253.18","Got1_Wt_02":"10779.38","Got1_Wt_03":"11564.44","Got1_KO_01":"3451.76","Got1_KO_02":"5809.96","Got1_KO_03":"4687.97","Glud1_Wt_01":"9975.54","Glud1_Wt_02":"11804.84","Glud1_Wt_03":"11127.59","Glud1_KO_01":"13016.64","Glud1_KO_02":"13156.77","Glud1_KO_03":"11762.66"},{"Metabolite":"α-Ketoglutarate_M+4","Got1_Wt_01":"1546.59","Got1_Wt_02":"993.15","Got1_Wt_03":"2334.52","Got1_KO_01":"1593.98","Got1_KO_02":"638.14","Got1_KO_03":"943.37","Glud1_Wt_01":"1304.21","Glud1_Wt_02":"2466.99","Glud1_Wt_03":"1188.71","Glud1_KO_01":"1335.96","Glud1_KO_02":"869.96","Glud1_KO_03":"1160.47"},{"Metabolite":"α-Ketoglutarate_M+5","Got1_Wt_01":"31540.37","Got1_Wt_02":"32340.16","Got1_Wt_03":"32963.5","Got1_KO_01":"14438.09","Got1_KO_02":"15844.28","Got1_KO_03":"11869.53","Glud1_Wt_01":"26425.42","Glud1_Wt_02":"29688.46","Glud1_Wt_03":"29432.52","Glud1_KO_01":"28211.29","Glud1_KO_02":"31299.04","Glud1_KO_03":"29182.48"}],

"Metabolites":[{"Metabolite":"Glutamate_M+0","quantified m/z":"146.0459","PubChem ID":"33032","KEGG ID":"C00302"},{"Metabolite":"Glutamate_M+1","quantified m/z":"147.0493","PubChem ID":"33032","KEGG ID":"C00302"},{"Metabolite":"Glutamate_M+2","quantified m/z":"148.0526","PubChem ID":"33032","KEGG ID":"C00302"},{"Metabolite":"Glutamate_M+3","quantified m/z":"149.0560","PubChem ID":"33032","KEGG ID":"C00302"},{"Metabolite":"Glutamate_M+4","quantified m/z":"150.0593","PubChem ID":"33032","KEGG ID":"C00302"},{"Metabolite":"Glutamate_M+5","quantified m/z":"151.0627","PubChem ID":"33032","KEGG ID":"C00302"},{"Metabolite":"α-Ketoglutarate_M+0","quantified m/z":"145.0142","PubChem ID":"164533","KEGG ID":"C00026"},{"Metabolite":"α-Ketoglutarate_M+1","quantified m/z":"146.0176","PubChem ID":"164533","KEGG ID":"C00026"},{"Metabolite":"α-Ketoglutarate_M+2","quantified m/z":"147.0209","PubChem ID":"164533","KEGG ID":"C00026"},{"Metabolite":"α-Ketoglutarate_M+3","quantified m/z":"148.0243","PubChem ID":"164533","KEGG ID":"C00026"},{"Metabolite":"α-Ketoglutarate_M+4","quantified m/z":"149.0276","PubChem ID":"164533","KEGG ID":"C00026"},{"Metabolite":"α-Ketoglutarate_M+5","quantified m/z":"150.0310","PubChem ID":"164533","KEGG ID":"C00026"}]
}

}