{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST002362","ANALYSIS_ID":"AN003857","VERSION":"1","CREATED_ON":"November 28, 2022, 7:42 pm"},

"PROJECT":{"PROJECT_TITLE":"[U-13C]glucose tracing in naïve vs. activated CD8+ T cells","PROJECT_TYPE":"MS quantifying intracellular glutamate levels","PROJECT_SUMMARY":"Naïve vs. 24 hr plate-bound anti-CD3 and soluble anti-CD28 activated CD8+ T cells were pulsed with [U-13C]glucose for 4-6 hours. Intracellular glucose-derived glutamate 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 naïve vs. activated CD8+ T cells","STUDY_SUMMARY":"Naïve vs. 24 hr plate-bound anti-CD3 and soluble anti-CD28 activated 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":"Naïve_01",
"Factors":{"Treatment":"Naïve"},
"Additional sample data":{"RAW_FILE_NAME":"Naïve_01.d"}
},
{
"Subject ID":"-",
"Sample ID":"Naïve_02",
"Factors":{"Treatment":"Naïve"},
"Additional sample data":{"RAW_FILE_NAME":"Naïve_02.d"}
},
{
"Subject ID":"-",
"Sample ID":"Naïve_03",
"Factors":{"Treatment":"Naïve"},
"Additional sample data":{"RAW_FILE_NAME":"Naïve_03.d"}
},
{
"Subject ID":"-",
"Sample ID":"Act_01",
"Factors":{"Treatment":"Activated"},
"Additional sample data":{"RAW_FILE_NAME":"Act_01.d"}
},
{
"Subject ID":"-",
"Sample ID":"Act_02",
"Factors":{"Treatment":"Activated"},
"Additional sample data":{"RAW_FILE_NAME":"Act_02.d"}
},
{
"Subject ID":"-",
"Sample ID":"Act_03",
"Factors":{"Treatment":"Activated"},
"Additional sample data":{"RAW_FILE_NAME":"Act_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 extraction.","SAMPLE_TYPE":"Cultured cells"},

"TREATMENT":{"TREATMENT_SUMMARY":"CD8+ T cells were isolated from spleens and lymph nodes from C57BL/6J mice. Cells were left naïve or 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","Naïve_01":"992518.50","Naïve_02":"943064.92","Naïve_03":"1060362.87","Act_01":"2017675.53","Act_02":"2066602.08","Act_03":"2061655.57"},{"Metabolite":"Glutamate_M+1","Naïve_01":"78118.96","Naïve_02":"74959.89","Naïve_03":"83320.91","Act_01":"215424.52","Act_02":"224478.31","Act_03":"225659.84"},{"Metabolite":"Glutamate_M+2","Naïve_01":"39769.15","Naïve_02":"37431.85","Naïve_03":"39389.25","Act_01":"880301.89","Act_02":"907283.48","Act_03":"948530.04"},{"Metabolite":"Glutamate_M+3","Naïve_01":"5033.04","Naïve_02":"4088.51","Naïve_03":"3906.44","Act_01":"333464.10","Act_02":"349071.75","Act_03":"368367.50"},{"Metabolite":"Glutamate_M+4","Naïve_01":"1664.67","Naïve_02":"1338.11","Naïve_03":"715.11","Act_01":"375818.20","Act_02":"390894.93","Act_03":"405957.67"},{"Metabolite":"Glutamate_M+5","Naïve_01":"216.28","Naïve_02":"0.00","Naïve_03":"0.00","Act_01":"167574.88","Act_02":"178656.15","Act_03":"184188.60"}],

"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"}]
}

}