#METABOLOMICS WORKBENCH Wei_Xu_20221129_180755 DATATRACK_ID:3612 STUDY_ID:ST002372 ANALYSIS_ID:AN003867 PROJECT_ID:PR001525 VERSION 1 CREATED_ON November 29, 2022, 6:41 pm #PROJECT PR:PROJECT_TITLE Extracellular pyruvate secretion by activated CD8+ T cells revealed by PR:PROJECT_TITLE [U-13C]glucose tracing PR:PROJECT_SUMMARY WT OT-I CD8+ T cells were activated in [U-13C]glucose for 24 hours. Blank PR:PROJECT_SUMMARY [U-13C]glucose media or media post cell culture were collected for mass PR:PROJECT_SUMMARY spectrometry analysis. Percent contribution of carbon flux from [U-13C]glucose PR:PROJECT_SUMMARY to pyruvate were analyzed. 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 Extracellular pyruvate secretion by activated CD8+ T cells revealed by ST:STUDY_TITLE [U-13C]glucose tracing ST:STUDY_SUMMARY WT OT-I CD8+ T cells were activated in [U-13C]glucose for 24 hours. Blank ST:STUDY_SUMMARY [U-13C]glucose media or media post cell culture were collected for mass ST:STUDY_SUMMARY spectrometry analysis. Percent contribution of carbon flux from [U-13C]glucose ST:STUDY_SUMMARY to pyruvate were analyzed. 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 - blk_001 Treatment:blank media RAW_FILE_NAME=blk_001.d SUBJECT_SAMPLE_FACTORS - blk_002 Treatment:blank media RAW_FILE_NAME=blk_002.d SUBJECT_SAMPLE_FACTORS - blk_003 Treatment:blank media RAW_FILE_NAME=blk_003.d SUBJECT_SAMPLE_FACTORS - Act_001 Treatment:media post cell culture RAW_FILE_NAME=Act_001.d SUBJECT_SAMPLE_FACTORS - Act_002 Treatment:media post cell culture RAW_FILE_NAME=Act_002.d SUBJECT_SAMPLE_FACTORS - Act_003 Treatment:media post cell culture RAW_FILE_NAME=Act_003.d #COLLECTION CO:COLLECTION_SUMMARY Blank media or media post cell culture were collected and centrifuged at 1200rpm CO:COLLECTION_SUMMARY for 5min. Supernatant were flash frozen in liquid nitrogen until further CO:COLLECTION_SUMMARY analysis. CO:SAMPLE_TYPE Cultured cells #TREATMENT TR:TREATMENT_SUMMARY CD8+ T cells were isolated from spleens and lymph nodes from WT mice. Cells were TR:TREATMENT_SUMMARY activated with plate-bound anti-CD3 and soluble anti-CD28 for 24 hours in the TR:TREATMENT_SUMMARY presence of 11mM [U-13C]glucose. Blank media post 24 hours in the incubator or TR:TREATMENT_SUMMARY media post 24 hr activated CD8+ T cell culture were collected for MS analysis. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY For measurement of extracellular metabolome, 625 µL of media was added to 375 SP:SAMPLEPREP_SUMMARY µL acetonitrile ACN. Samples were stored at -20 °C for at least two hours SP:SAMPLEPREP_SUMMARY followed by centrifugation at 14,000xg to precipitate any proteins. 250 µL of SP:SAMPLEPREP_SUMMARY the supernatant was mixed with 250 µL of water and added to a 3 kDa molecular SP:SAMPLEPREP_SUMMARY weight cut-off filter spin column (Microcon YM-3 Centrifugal Filter, Millipore). SP:SAMPLEPREP_SUMMARY Samples were centrifuged at 14,000xg at 4 °C for 30 min. The flow-through was SP:SAMPLEPREP_SUMMARY saved for LC-MS/MS analysis. #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 blk_001 blk_002 blk_003 Act_001 Act_002 Act_003 Factors Treatment:blank media Treatment:blank media Treatment:blank media Treatment:media post cell culture Treatment:media post cell culture Treatment:media post cell culture Pyruvate_M+0 11494.03 8603.97 11166.14 16120.2 16511.59 14695.48 Pyruvate_M+1 0 0 0 520.94 322.83 338.93 Pyruvate_M+2 0 0 0 0 0 0 Pyruvate_M+3 0 0 0 72436.97 70512.51 70118.68 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name quantified m/z PubChem ID KEGG ID Pyruvate_M+0 87.0088 107735 C00022 Pyruvate_M+1 88.0122 107735 C00022 Pyruvate_M+2 89.0155 107735 C00022 Pyruvate_M+3 90.0189 107735 C00022 METABOLITES_END #END