{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST002364","ANALYSIS_ID":"AN003859","VERSION":"1","CREATED_ON":"November 28, 2022, 10:06 pm"},

"PROJECT":{"PROJECT_TITLE":"[U-13C]glucose 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]glucose for 4-6 hours. Intracellular glucose-derived glutamate, serine 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]glucose 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]glucose. Intracellular glucose-derived glutamate, serine 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-Gluc_Got1_Wt_01.d"}
},
{
"Subject ID":"-",
"Sample ID":"Got1_Wt_02",
"Factors":{"Treatment":"GOT1_WT"},
"Additional sample data":{"RAW_FILE_NAME":"13C-Gluc_Got1_Wt_02.d"}
},
{
"Subject ID":"-",
"Sample ID":"Got1_Wt_03",
"Factors":{"Treatment":"GOT1_WT"},
"Additional sample data":{"RAW_FILE_NAME":"13C-Gluc_Got1_Wt_03.d"}
},
{
"Subject ID":"-",
"Sample ID":"Got1_KO_01",
"Factors":{"Treatment":"GOT1_KO"},
"Additional sample data":{"RAW_FILE_NAME":"13C-Gluc_Got1_KO_01.d"}
},
{
"Subject ID":"-",
"Sample ID":"Got1_KO_02",
"Factors":{"Treatment":"GOT1_KO"},
"Additional sample data":{"RAW_FILE_NAME":"13C-Gluc_Got1_KO_02.d"}
},
{
"Subject ID":"-",
"Sample ID":"Got1_KO_03",
"Factors":{"Treatment":"GOT1_KO"},
"Additional sample data":{"RAW_FILE_NAME":"13C-Gluc_Got1_KO_03.d"}
},
{
"Subject ID":"-",
"Sample ID":"Glud1_Wt_01",
"Factors":{"Treatment":"GLUD1_WT"},
"Additional sample data":{"RAW_FILE_NAME":"13C-Gluc_Glud1_Wt_01.d"}
},
{
"Subject ID":"-",
"Sample ID":"Glud1_Wt_02",
"Factors":{"Treatment":"GLUD1_WT"},
"Additional sample data":{"RAW_FILE_NAME":"13C-Gluc_Glud1_Wt_02.d"}
},
{
"Subject ID":"-",
"Sample ID":"Glud1_Wt_03",
"Factors":{"Treatment":"GLUD1_WT"},
"Additional sample data":{"RAW_FILE_NAME":"13C-Gluc_Glud1_Wt_03.d"}
},
{
"Subject ID":"-",
"Sample ID":"Glud1_KO_01",
"Factors":{"Treatment":"GLUD1_KO"},
"Additional sample data":{"RAW_FILE_NAME":"13C-Gluc_Glud1_KO_01.d"}
},
{
"Subject ID":"-",
"Sample ID":"Glud1_KO_02",
"Factors":{"Treatment":"GLUD1_KO"},
"Additional sample data":{"RAW_FILE_NAME":"13C-Gluc_Glud1_KO_02.d"}
},
{
"Subject ID":"-",
"Sample ID":"Glud1_KO_03",
"Factors":{"Treatment":"GLUD1_KO"},
"Additional sample data":{"RAW_FILE_NAME":"13C-Gluc_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 11 mM [U-13C]glucose 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":"10285159.12","Got1_Wt_02":"10306480.5","Got1_Wt_03":"9240850.12","Got1_KO_01":"5584220.68","Got1_KO_02":"5280384.06","Got1_KO_03":"4866645.71","Glud1_Wt_01":"12538912.08","Glud1_Wt_02":"12262365.85","Glud1_Wt_03":"11671083.5","Glud1_KO_01":"11809447.16","Glud1_KO_02":"11619371.62","Glud1_KO_03":"11719981.49"},{"Metabolite":"Glutamate_M+1","Got1_Wt_01":"851113.07","Got1_Wt_02":"849611.71","Got1_Wt_03":"779928.25","Got1_KO_01":"525205.75","Got1_KO_02":"520164.2","Got1_KO_03":"474629.75","Glud1_Wt_01":"1007094.49","Glud1_Wt_02":"973278.58","Glud1_Wt_03":"933642.56","Glud1_KO_01":"955421.94","Glud1_KO_02":"952486.81","Glud1_KO_03":"955784.2"},{"Metabolite":"Glutamate_M+2","Got1_Wt_01":"2201341.6","Got1_Wt_02":"2289307.65","Got1_Wt_03":"2291818.68","Got1_KO_01":"1178774.31","Got1_KO_02":"1144788.51","Got1_KO_03":"1108808.24","Glud1_Wt_01":"2672429.91","Glud1_Wt_02":"2625398.95","Glud1_Wt_03":"2632125.93","Glud1_KO_01":"2682270.6","Glud1_KO_02":"2866309.41","Glud1_KO_03":"2962091.63"},{"Metabolite":"Glutamate_M+3","Got1_Wt_01":"640809.38","Got1_Wt_02":"645109.59","Got1_Wt_03":"613033.94","Got1_KO_01":"277420.26","Got1_KO_02":"274609.25","Got1_KO_03":"265232.17","Glud1_Wt_01":"773210.22","Glud1_Wt_02":"736280.73","Glud1_Wt_03":"737092.05","Glud1_KO_01":"838387.79","Glud1_KO_02":"830336.84","Glud1_KO_03":"912093.17"},{"Metabolite":"Glutamate_M+4","Got1_Wt_01":"692784.8","Got1_Wt_02":"707526.57","Got1_Wt_03":"730485","Got1_KO_01":"229758.41","Got1_KO_02":"216661.88","Got1_KO_03":"213752.38","Glud1_Wt_01":"860851.71","Glud1_Wt_02":"834992.27","Glud1_Wt_03":"827501.23","Glud1_KO_01":"935251.8","Glud1_KO_02":"962823.01","Glud1_KO_03":"1055208.47"},{"Metabolite":"Glutamate_M+5","Got1_Wt_01":"256815.68","Got1_Wt_02":"250191.32","Got1_Wt_03":"246172.37","Got1_KO_01":"38797.47","Got1_KO_02":"34425.53","Got1_KO_03":"33540.84","Glud1_Wt_01":"314822.93","Glud1_Wt_02":"291205.36","Glud1_Wt_03":"288372.95","Glud1_KO_01":"345383.46","Glud1_KO_02":"356763.14","Glud1_KO_03":"406551.15"},{"Metabolite":"Serine_M+0","Got1_Wt_01":"68117.64","Got1_Wt_02":"68284.91","Got1_Wt_03":"65265.2","Got1_KO_01":"60388.47","Got1_KO_02":"60862.1","Got1_KO_03":"64623.36","Glud1_Wt_01":"78868.46","Glud1_Wt_02":"78176.09","Glud1_Wt_03":"79918.41","Glud1_KO_01":"81203.26","Glud1_KO_02":"90482.89","Glud1_KO_03":"88905.73"},{"Metabolite":"Serine_M+1","Got1_Wt_01":"12079.31","Got1_Wt_02":"7759.52","Got1_Wt_03":"9343.68","Got1_KO_01":"1550.64","Got1_KO_02":"3427.46","Got1_KO_03":"1736.59","Glud1_Wt_01":"10528.03","Glud1_Wt_02":"13531.79","Glud1_Wt_03":"12167.2","Glud1_KO_01":"15122.96","Glud1_KO_02":"10302.91","Glud1_KO_03":"10198.52"},{"Metabolite":"Serine_M+2","Got1_Wt_01":"4669.42","Got1_Wt_02":"6103.66","Got1_Wt_03":"4623.34","Got1_KO_01":"1172.51","Got1_KO_02":"1391.97","Got1_KO_03":"1473.78","Glud1_Wt_01":"7116.43","Glud1_Wt_02":"5113.16","Glud1_Wt_03":"7607.19","Glud1_KO_01":"7000.81","Glud1_KO_02":"6895.32","Glud1_KO_03":"7112.12"},{"Metabolite":"Serine_M+3","Got1_Wt_01":"11950.23","Got1_Wt_02":"11075.95","Got1_Wt_03":"11946.66","Got1_KO_01":"5709.28","Got1_KO_02":"6787.3","Got1_KO_03":"5562.68","Glud1_Wt_01":"12699.25","Glud1_Wt_02":"11340.44","Glud1_Wt_03":"9274.44","Glud1_KO_01":"12419.84","Glud1_KO_02":"11637.29","Glud1_KO_03":"13236.54"},{"Metabolite":"α-Ketoglutarate_M+0","Got1_Wt_01":"191447.35","Got1_Wt_02":"194321.94","Got1_Wt_03":"175786.36","Got1_KO_01":"110221.47","Got1_KO_02":"101399.25","Got1_KO_03":"84973.99","Glud1_Wt_01":"243084.45","Glud1_Wt_02":"239583.08","Glud1_Wt_03":"211576.6","Glud1_KO_01":"198005.85","Glud1_KO_02":"203767.73","Glud1_KO_03":"212564.09"},{"Metabolite":"α-Ketoglutarate_M+1","Got1_Wt_01":"15464.74","Got1_Wt_02":"15998.73","Got1_Wt_03":"13922.72","Got1_KO_01":"9724.23","Got1_KO_02":"10340.28","Got1_KO_03":"6711.39","Glud1_Wt_01":"20014.16","Glud1_Wt_02":"15115.49","Glud1_Wt_03":"14645.23","Glud1_KO_01":"16125.94","Glud1_KO_02":"15782","Glud1_KO_03":"17908.98"},{"Metabolite":"α-Ketoglutarate_M+2","Got1_Wt_01":"42786.23","Got1_Wt_02":"45487.64","Got1_Wt_03":"45454.37","Got1_KO_01":"25375.54","Got1_KO_02":"25833.5","Got1_KO_03":"24811.49","Glud1_Wt_01":"59522.45","Glud1_Wt_02":"56247.62","Glud1_Wt_03":"51456.81","Glud1_KO_01":"51204.43","Glud1_KO_02":"51140.17","Glud1_KO_03":"50777.56"},{"Metabolite":"α-Ketoglutarate_M+3","Got1_Wt_01":"11309.62","Got1_Wt_02":"12895.68","Got1_Wt_03":"13246.42","Got1_KO_01":"6670.19","Got1_KO_02":"4444.71","Got1_KO_03":"4294.95","Glud1_Wt_01":"14401.27","Glud1_Wt_02":"14797.44","Glud1_Wt_03":"13261.63","Glud1_KO_01":"12425.94","Glud1_KO_02":"15228.57","Glud1_KO_03":"17108.28"},{"Metabolite":"α-Ketoglutarate_M+4","Got1_Wt_01":"16065.73","Got1_Wt_02":"16032","Got1_Wt_03":"16030.49","Got1_KO_01":"5891.69","Got1_KO_02":"5142.05","Got1_KO_03":"6023.04","Glud1_Wt_01":"20273.37","Glud1_Wt_02":"20796.35","Glud1_Wt_03":"17670.45","Glud1_KO_01":"18994.07","Glud1_KO_02":"21657.28","Glud1_KO_03":"23603.71"},{"Metabolite":"α-Ketoglutarate_M+5","Got1_Wt_01":"5067.18","Got1_Wt_02":"4341.9","Got1_Wt_03":"5157.36","Got1_KO_01":"106.1","Got1_KO_02":"587.76","Got1_KO_03":"1053.62","Glud1_Wt_01":"6178.45","Glud1_Wt_02":"5909.68","Glud1_Wt_03":"4502.39","Glud1_KO_01":"6215.19","Glud1_KO_02":"7446.66","Glud1_KO_03":"7664.7"}],

"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":"Serine_M+0","quantified m/z":"104.0353","PubChem ID":"5951","KEGG ID":"C00716"},{"Metabolite":"Serine_M+1","quantified m/z":"105.0387","PubChem ID":"5951","KEGG ID":"C00716"},{"Metabolite":"Serine_M+2","quantified m/z":"106.0420","PubChem ID":"5951","KEGG ID":"C00716"},{"Metabolite":"Serine_M+3","quantified m/z":"107.0454","PubChem ID":"5951","KEGG ID":"C00716"},{"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"}]
}

}