{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST002363","ANALYSIS_ID":"AN003858","VERSION":"1","CREATED_ON":"November 28, 2022, 9:06 pm"},

"PROJECT":{"PROJECT_TITLE":"[U-13C]glucose tracing in NT, AOA or EGCG treated activated CD8+ T cells","PROJECT_TYPE":"MS quantifying intracellular glutamate levels","PROJECT_SUMMARY":"CD8+ T cells were activated with plate-bound anti-CD3 and soluble anti-CD28 for 24 hours without (NT), or with AOA (250uM), or EGCG (500uM) treatment. CD8+ T cells were pulsed with [U-13C]glucose for 4-6 hours. Intracellular glucose-derived glutamate 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 NT, AOA or EGCG treated activated CD8+ T cells","STUDY_SUMMARY":"CD8+ T cells were activated with plate-bound anti-CD3 and soluble anti-CD28 for 24 hours without (NT), or with AOA (250uM) or EGCG (500uM) treatment. CD8+ T cells were pulsed with [U-13C]glucose. Intracellular glucose-derived glutamate 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":"NT_01",
"Factors":{"Treatment":"NT"},
"Additional sample data":{"RAW_FILE_NAME":"NT_01.d"}
},
{
"Subject ID":"-",
"Sample ID":"NT_02",
"Factors":{"Treatment":"NT"},
"Additional sample data":{"RAW_FILE_NAME":"NT_02.d"}
},
{
"Subject ID":"-",
"Sample ID":"NT_03",
"Factors":{"Treatment":"NT"},
"Additional sample data":{"RAW_FILE_NAME":"NT_03.d"}
},
{
"Subject ID":"-",
"Sample ID":"AOA_01",
"Factors":{"Treatment":"AOA"},
"Additional sample data":{"RAW_FILE_NAME":"AOA_01.d"}
},
{
"Subject ID":"-",
"Sample ID":"AOA_02",
"Factors":{"Treatment":"AOA"},
"Additional sample data":{"RAW_FILE_NAME":"AOA_02.d"}
},
{
"Subject ID":"-",
"Sample ID":"AOA_03",
"Factors":{"Treatment":"AOA"},
"Additional sample data":{"RAW_FILE_NAME":"AOA_03.d"}
},
{
"Subject ID":"-",
"Sample ID":"EGCG_01",
"Factors":{"Treatment":"EGCG"},
"Additional sample data":{"RAW_FILE_NAME":"EGCG_01.d"}
},
{
"Subject ID":"-",
"Sample ID":"EGCG_02",
"Factors":{"Treatment":"EGCG"},
"Additional sample data":{"RAW_FILE_NAME":"EGCG_02.d"}
},
{
"Subject ID":"-",
"Sample ID":"EGCG_03",
"Factors":{"Treatment":"EGCG"},
"Additional sample data":{"RAW_FILE_NAME":"EGCG_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 C57BL/6J mice. Cells were activated with plate-bound anti-CD3 and soluble anti-CD28 for 24 hours, without (NT) or with AOA (250uM) or EGCG (500uM) treatment. 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","NT_01":"2036085.45","NT_02":"1815686.58","NT_03":"1813901.61","AOA_01":"2504717.50","AOA_02":"2508358.22","AOA_03":"2461829.93","EGCG_01":"1417735.11","EGCG_02":"1399433.95","EGCG_03":"1389672.53"},{"Metabolite":"Glutamate_M+1","NT_01":"227399.47","NT_02":"207516.61","NT_03":"211090.50","AOA_01":"276069.97","AOA_02":"276462.01","AOA_03":"272267.67","EGCG_01":"195881.28","EGCG_02":"189509.47","EGCG_03":"189698.54"},{"Metabolite":"Glutamate_M+2","NT_01":"810852.18","NT_02":"739183.82","NT_03":"745987.17","AOA_01":"532309.33","AOA_02":"533958.57","AOA_03":"523701.98","EGCG_01":"634148.21","EGCG_02":"611219.03","EGCG_03":"619890.84"},{"Metabolite":"Glutamate_M+3","NT_01":"298382.72","NT_02":"281753.93","NT_03":"285766.90","AOA_01":"192912.45","AOA_02":"195599.37","AOA_03":"186144.30","EGCG_01":"274890.26","EGCG_02":"260687.42","EGCG_03":"262786.69"},{"Metabolite":"Glutamate_M+4","NT_01":"319896.17","NT_02":"290737.33","NT_03":"291325.82","AOA_01":"155589.95","AOA_02":"157530.83","AOA_03":"150357.27","EGCG_01":"262497.90","EGCG_02":"257852.98","EGCG_03":"258426.41"},{"Metabolite":"Glutamate_M+5","NT_01":"132661.13","NT_02":"120190.82","NT_03":"119767.01","AOA_01":"49244.20","AOA_02":"50366.26","AOA_03":"47535.26","EGCG_01":"111032.37","EGCG_02":"110785.47","EGCG_03":"111162.82"}],

"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"}]
}

}