#METABOLOMICS WORKBENCH Wei_Xu_20221128_014600 DATATRACK_ID:3600 STUDY_ID:ST002362 ANALYSIS_ID:AN003857 PROJECT_ID:PR001517 VERSION 1 CREATED_ON November 28, 2022, 7:42 pm #PROJECT PR:PROJECT_TITLE [U-13C]glucose tracing in naïve vs. activated CD8+ T cells PR:PROJECT_TYPE MS quantifying intracellular glutamate levels PR:PROJECT_SUMMARY Naïve vs. 24 hr plate-bound anti-CD3 and soluble anti-CD28 activated CD8+ T PR:PROJECT_SUMMARY cells were pulsed with [U-13C]glucose for 4-6 hours. Intracellular PR:PROJECT_SUMMARY glucose-derived glutamate were quantified using MS. 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 [U-13C]glucose tracing in naïve vs. activated CD8+ T cells ST:STUDY_SUMMARY Naïve vs. 24 hr plate-bound anti-CD3 and soluble anti-CD28 activated CD8+ T ST:STUDY_SUMMARY cells were pulsed with [U-13C]glucose. Intracellular glucose-derived glutamate ST:STUDY_SUMMARY levels were quantified using MS. 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 - Naïve_01 Treatment:Naïve RAW_FILE_NAME=Naïve_01.d SUBJECT_SAMPLE_FACTORS - Naïve_02 Treatment:Naïve RAW_FILE_NAME=Naïve_02.d SUBJECT_SAMPLE_FACTORS - Naïve_03 Treatment:Naïve RAW_FILE_NAME=Naïve_03.d SUBJECT_SAMPLE_FACTORS - Act_01 Treatment:Activated RAW_FILE_NAME=Act_01.d SUBJECT_SAMPLE_FACTORS - Act_02 Treatment:Activated RAW_FILE_NAME=Act_02.d SUBJECT_SAMPLE_FACTORS - Act_03 Treatment:Activated RAW_FILE_NAME=Act_03.d #COLLECTION CO:COLLECTION_SUMMARY Cells were spun down and washed once with pre-warmed PBS and metabolites were CO:COLLECTION_SUMMARY immediately extracted or stored at -80℃ until extraction. CO:SAMPLE_TYPE Cultured cells #TREATMENT TR:TREATMENT_SUMMARY CD8+ T cells were isolated from spleens and lymph nodes from C57BL/6J mice. TR:TREATMENT_SUMMARY Cells were left naïve or activated with plate-bound anti-CD3 and soluble TR:TREATMENT_SUMMARY anti-CD28 for 24 hours. CD8+ T cells were counted and resuspended in full media TR:TREATMENT_SUMMARY containing 11 mM [U-13C]glucose at 2 E6 mL-1. Normal FBS was substituted with TR:TREATMENT_SUMMARY dialyzed FBS. Cells were collected for LC-MS analysis 4-6 hrs post incubation. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Cells were spun down and washed once with pre-warmed PBS and metabolites were SP:SAMPLEPREP_SUMMARY immediately extracted by adding methanol:water (80:20, v/v) extraction solution, SP:SAMPLEPREP_SUMMARY sonicated and stored at -80 °C for at least 2 hrs to precipitate the proteins. SP:SAMPLEPREP_SUMMARY Supernatant after centrifugation at 14,000xg for 10 minutes was dried under SP:SAMPLEPREP_SUMMARY nitrogen gas. Metabolites were then reconstituted using ACN:water (50:50, v/v) SP:SAMPLEPREP_SUMMARY overnight at 4 °C. Soluble metabolites after centrifugation at 14,000xg for 10 SP:SAMPLEPREP_SUMMARY minutes were subjected to analysis by liquid chromatography mass spectrometry SP:SAMPLEPREP_SUMMARY (LC-MS). #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 Naïve_01 Naïve_02 Naïve_03 Act_01 Act_02 Act_03 Factors Treatment:Naïve Treatment:Naïve Treatment:Naïve Treatment:Activated Treatment:Activated Treatment:Activated Glutamate_M+0 992518.50 943064.92 1060362.87 2017675.53 2066602.08 2061655.57 Glutamate_M+1 78118.96 74959.89 83320.91 215424.52 224478.31 225659.84 Glutamate_M+2 39769.15 37431.85 39389.25 880301.89 907283.48 948530.04 Glutamate_M+3 5033.04 4088.51 3906.44 333464.10 349071.75 368367.50 Glutamate_M+4 1664.67 1338.11 715.11 375818.20 390894.93 405957.67 Glutamate_M+5 216.28 0.00 0.00 167574.88 178656.15 184188.60 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name quantified m/z PubChem ID KEGG ID Glutamate_M+0 146.0459 33032 C00302 Glutamate_M+1 147.0493 33032 C00302 Glutamate_M+2 148.0526 33032 C00302 Glutamate_M+3 149.0560 33032 C00302 Glutamate_M+4 150.0593 33032 C00302 Glutamate_M+5 151.0627 33032 C00302 METABOLITES_END #END