#METABOLOMICS WORKBENCH xz336_20220107_074145 DATATRACK_ID:3027 STUDY_ID:ST002071 ANALYSIS_ID:AN003376 PROJECT_ID:PR001313 VERSION 1 CREATED_ON January 31, 2022, 9:07 am #PROJECT PR:PROJECT_TITLE Metabolic profiling of mouse CD27+ and CD27- gammadelta T cells PR:PROJECT_TYPE Research PR:PROJECT_SUMMARY The objective of this study is to define metabolic profiles of innate mouse PR:PROJECT_SUMMARY CD27+ and CD27- gammadelta T cells. PR:INSTITUTE University of Louisville PR:LAST_NAME Yan PR:FIRST_NAME Jun PR:ADDRESS 505 S Hancock Street, Louisville, KY 40245 PR:EMAIL jun.yan@louisville.edu PR:PHONE 502 852-3628 PR:FUNDING_SOURCE R01AI161106 #STUDY ST:STUDY_TITLE Metabolic profiling of mouse CD27+ and CD27- gammadelta T cells ST:STUDY_SUMMARY Cultured CD27+ and CD27- gammadelta T cells were labeled with 13C-glucose for 24 ST:STUDY_SUMMARY h. Cells were harvested for LC-MS analysis ST:INSTITUTE University of Louisville ST:LAST_NAME Zhang ST:FIRST_NAME Xiang ST:ADDRESS Shumaker Research Bld 349, Louisville, KY 40292 ST:EMAIL xiang.zhang@louisville.edu ST:PHONE 502-852-8878 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:AGE_OR_AGE_RANGE 6-8 weeks old SU:WEIGHT_OR_WEIGHT_RANGE 15-20 grams SU:GENDER Male and female SU:CELL_STRAIN_DETAILS C57Bl/6 #FACTORS #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 - 0412_CD27-_Labeled_1 Cell type:CD27- Label=13C-Glucose; RAW_FILE_NAME=0412_CD27-_Labeled_1_pos.mzXML 0412_CD27-_Labeled_1_neg.mzXML SUBJECT_SAMPLE_FACTORS - 0412_CD27-_Labeled_2 Cell type:CD27- Label=13C-Glucose; RAW_FILE_NAME=0412_CD27-_Labeled_2_pos.mzXML 0412_CD27-_Labeled_2_neg.mzXML SUBJECT_SAMPLE_FACTORS - 0426_CD27-_Labeled_1 Cell type:CD27- Label=13C-Glucose; RAW_FILE_NAME=0426_CD27-_Labeled_1_pos.mzXML 0426_CD27-_Labeled_1_neg.mzXML SUBJECT_SAMPLE_FACTORS - 0426_CD27-_Labeled_2 Cell type:CD27- Label=13C-Glucose; RAW_FILE_NAME=0426_CD27-_Labeled_2_pos.mzXML 0426_CD27-_Labeled_2_neg.mzXML SUBJECT_SAMPLE_FACTORS - 0426_CD27-_Labeled_3 Cell type:CD27- Label=13C-Glucose; RAW_FILE_NAME=0426_CD27-_Labeled_3_pos.mzXML 0426_CD27-_Labeled_3_neg.mzXML SUBJECT_SAMPLE_FACTORS - 0412_CD27+_Labeled_1 Cell type:CD27+ Label=13C-Glucose; RAW_FILE_NAME=0412_CD27+_Labeled_1_pos.mzXML 0412_CD27+_Labeled_1_neg.mzXML SUBJECT_SAMPLE_FACTORS - 0412_CD27+_Labeled_2 Cell type:CD27+ Label=13C-Glucose; RAW_FILE_NAME=0412_CD27+_Labeled_2_pos.mzXML 0412_CD27+_Labeled_2_neg.mzXML SUBJECT_SAMPLE_FACTORS - 0412_CD27+_Labeled_3 Cell type:CD27+ Label=13C-Glucose; RAW_FILE_NAME=0412_CD27+_Labeled_3_pos.mzXML 0412_CD27+_Labeled_3_neg.mzXML SUBJECT_SAMPLE_FACTORS - 0426_CD27+_Labeled_1 Cell type:CD27+ Label=13C-Glucose; RAW_FILE_NAME=0426_CD27+_Labeled_1_pos.mzXML 0426_CD27+_Labeled_1_neg.mzXML SUBJECT_SAMPLE_FACTORS - 0426_CD27+_Labeled_2 Cell type:CD27+ Label=13C-Glucose; RAW_FILE_NAME=0426_CD27+_Labeled_2_pos.mzXML 0426_CD27+_Labeled_2_neg.mzXML SUBJECT_SAMPLE_FACTORS - 0426_CD27+_Labeled_3 Cell type:CD27+ Label=13C-Glucose; RAW_FILE_NAME=0426_CD27+_Labeled_3_pos.mzXML 0426_CD27+_Labeled_3_neg.mzXML #COLLECTION CO:COLLECTION_SUMMARY polarized CD27+ and CD27- γδ T cells were cultured in glucose-free DMEM CO:COLLECTION_SUMMARY supplemented with 10 mM [U-13C]-glucose and 10% dialyzed FBS for 24 h. The cells CO:COLLECTION_SUMMARY were rinsed in cold PBS and quenched using a mixture containing 2 mL CO:COLLECTION_SUMMARY acetonitrile and 1.5 mL H2O. After adding 1 mL chloroform, the sample was CO:COLLECTION_SUMMARY homogenized and centrifuged at 3,000 rpm, 4 oC for 20 min. The top layer was CO:COLLECTION_SUMMARY transferred to a new tube and lyophilized. The dried sample was then dissolved CO:COLLECTION_SUMMARY in 100 µL 20% acetonitrile and vigorously vortex-mixed for 3 min. After CO:COLLECTION_SUMMARY centrifugation at 14,000 rpm, 4oC for 20 min, the supernatant was collected for CO:COLLECTION_SUMMARY two-dimensional liquid chromatography-mass spectrometry (2DLC-MS) analysis. CO:SAMPLE_TYPE T-cells #TREATMENT TR:TREATMENT_SUMMARY Cells were not treated with any drugs #SAMPLEPREP SP:SAMPLEPREP_SUMMARY The cells were rinsed in cold PBS and quenched using a mixture containing 2 mL SP:SAMPLEPREP_SUMMARY acetonitrile and 1.5 mL H2O. After adding 1 mL chloroform, the sample was SP:SAMPLEPREP_SUMMARY homogenized and centrifuged at 3,000 rpm, 4 oC for 20 min. The top layer was SP:SAMPLEPREP_SUMMARY transferred to a new tube and lyophilized. The dried sample was then dissolved SP:SAMPLEPREP_SUMMARY in 100 µL 20% acetonitrile and vigorously vortex-mixed for 3 min. After SP:SAMPLEPREP_SUMMARY centrifugation at 14,000 rpm, 4oC for 20 min, the supernatant was collected for SP:SAMPLEPREP_SUMMARY two-dimensional liquid chromatography-mass spectrometry (2DLC-MS) analysis. All SP:SAMPLEPREP_SUMMARY samples were analyzed on a Thermo Q Exactive HF Hybrid Quadrupole-Orbitrap Mass SP:SAMPLEPREP_SUMMARY Spectrometer coupled with a Thermo DIONEX UltiMate 3000 HPLC system (Thermo SP:SAMPLEPREP_SUMMARY Fisher Scientific, Waltham, MA, USA). The UltiMate 3000 HPLC system was equipped SP:SAMPLEPREP_SUMMARY with a reversed phase chromatography (RPC) column and a hydrophilic interaction SP:SAMPLEPREP_SUMMARY chromatography (HILIC) column. The two columns were configured in parallel 2DLC SP:SAMPLEPREP_SUMMARY mode. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY We use parallel 2DLC-MS, one column is HILIC and another is RP. CH:CHROMATOGRAPHY_TYPE HILIC CH:INSTRUMENT_NAME Q Exactive CH:COLUMN_NAME SeQuant® ZIC®-cHILIC #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Exactive Plus Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS A pooled samples for each group was detected under both positive and negative MS:MS_COMMENTS mode using 20 eV, 40eV and 60 eV. The recorded MS/MS spectrum was used for MS:MS_COMMENTS compound identification. For metabolites quantification, FullMS data of each MS:MS_COMMENTS sample was conducted under both positive and negative mode, XCMS software was MS:MS_COMMENTS used for peak deconvolution and our in-house software, MetSign was used for MS:MS_COMMENTS assignment, alignment, and statistical analysis #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS None MS_METABOLITE_DATA_START Samples 0412_CD27+_Labeled_1 0412_CD27+_Labeled_2 0412_CD27+_Labeled_3 0426_CD27+_Labeled_1 0426_CD27+_Labeled_2 0426_CD27+_Labeled_3 0412_CD27-_Labeled_1 0412_CD27-_Labeled_2 0426_CD27-_Labeled_1 0426_CD27-_Labeled_2 0426_CD27-_Labeled_3 Factors Cell type:CD27+ Cell type:CD27+ Cell type:CD27+ Cell type:CD27+ Cell type:CD27+ Cell type:CD27+ Cell type:CD27- Cell type:CD27- Cell type:CD27- Cell type:CD27- Cell type:CD27- Adenine_None 1883542.359 1390075.56 1267292.014 875544.2591 1353448.343 1374367.088 1167928.024 1011602.372 1143180.318 1227195.543 962247.9627 Adenine_13C_1 523855.9553 395686.8686 368518.2163 355609.5772 558935.7797 542383.7935 181368.9443 156143.9298 307554.354 332864.3522 255672.4616 Adenine_13C_2 207366.0415 151738.9421 131917.9033 155185.6845 238160.7983 237304.4319 58995.06478 55950.79225 139021.7598 163596.6208 128678.531 Adenine_13C_3 56796.07241 38906.01291 36164.54898 52216.89465 92821.176 90102.88584 0 0 70079.76349 78417.96912 61961.33927 alpha-Ketoglutaric acid_None 3077150.219 3325551.08 3407907.242 2345721.707 2062506.117 1737507.101 1431161.104 211082.4813 1882783.41 1650784.361 1304413.495 alpha-Ketoglutaric acid_13C_1 70604.88767 70472.94735 76674.46491 92479.08959 169672.7685 50930.37975 60833.7966 0 65437.16944 55232.91435 38817.38131 alpha-Ketoglutaric acid_13C_2 663083.6322 701449.9098 728201.8168 576799.0853 507701.4157 424688.8123 415855.7632 51699.1433 579523.9032 500770.4388 389882.4389 alpha-Ketoglutaric acid_13C_3 139150.4477 161325.4569 161590.652 137661.5474 121454.5261 100373.8101 124160.5988 0 169727.8323 145298.4159 122246.8401 alpha-Ketoglutaric acid_13C_4 187845.1965 203175.6307 219297.9815 161275.8649 142093.4175 105877.5519 129680.5032 0 213464.2145 185921.0584 143471.1006 alpha-Ketoglutaric acid_13C_5 94982.73972 107074.4871 108247.1835 82749.08296 75028.88527 63552.5135 62821.25249 0 146299.8002 117186.8336 90032.10847 cis-Aconitic acid_None 1165897.368 851675.4871 836904.4607 1035590.132 1086035.931 1085611.594 205543.7034 173203.1087 234391.35 229758.8912 211221.6871 cis-Aconitic acid_13C_1 205691.1424 84851.30038 81531.36143 124774.8095 132456.5204 120577.1749 27898.95595 17959.0134 33463.44104 31487.14285 48489.92059 cis-Aconitic acid_13C_2 671819.9616 503703.4669 480519.7488 683941.6947 707430.9795 695907.8581 142416.855 116160.35 193895.1893 218010.1446 190913.2203 cis-Aconitic acid_13C_3 182268.8424 129777.6499 122738.0517 178544.019 179900.4321 180481.8236 37991.31858 31502.74908 49150.43209 56354.97405 52571.2398 cis-Aconitic acid_13C_4 182261.3573 140241.4319 132988.4882 206001.7495 215125.623 214247.9222 43982.10825 40134.91557 66749.55916 85432.60173 67949.61548 cis-Aconitic acid_13C_5 66510.44547 48455.79553 45638.13994 64718.01506 76480.52933 72947.27343 0 0 0 33203.72057 23089.2366 Citric acid_None 1978133.606 2997591.205 31755312.4 24239795.12 15517242.27 8961476.969 7430870.849 23111.95494 4636375.366 8281500.064 2478536.802 Citric acid_13C_2 240030.6699 4136320.272 17873907.63 15293220.49 14817112.34 5657003.756 3251983.314 0 8505292.33 5361365.503 1980559.442 Citric acid_13C_3 0 1028433.542 2360396.792 4221801.703 3964426.352 1645193.138 449891.4896 0 2300006.091 2608846.932 769012.4372 Citric acid_13C_4 0 1111673.634 3148757.406 4975143.265 4587999.887 1831357.481 2115530.296 0 3075027.53 1247301.79 851373.1158 Citric acid_13C_5 0 402206.6758 1288039.932 1566771.186 729578.8405 777843.2323 190127.8476 0 1058483.812 1814073.749 375295.6447 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name Quantified m/z PubChem ID KEGG ID Adenine_None 134.0471 190 C00147 Adenine_13C_1 135.05038 190 C00147 Adenine_13C_2 136.0538 190 C00147 Adenine_13C_3 137.05704 190 C00147 alpha-Ketoglutaric acid_None 145.01409 164533 C00026 alpha-Ketoglutaric acid_13C_1 146.01749 164533 C00026 alpha-Ketoglutaric acid_13C_2 147.02081 164533 C00026 alpha-Ketoglutaric acid_13C_3 148.02415 164533 C00026 alpha-Ketoglutaric acid_13C_4 149.02753 164533 C00026 alpha-Ketoglutaric acid_13C_5 150.03091 164533 C00026 cis-Aconitic acid_None 173.00895 164533 C00417 cis-Aconitic acid_13C_1 174.01234 643757 C00417 cis-Aconitic acid_13C_2 175.01567 643757 C00417 cis-Aconitic acid_13C_3 176.01898 643757 C00417 cis-Aconitic acid_13C_4 177.0225 643757 C00417 cis-Aconitic acid_13C_5 178.02564 643757 C00417 Citric acid_None 191.01953 311 C00158 Citric acid_13C_2 192.022845 311 C00158 Citric acid_13C_3 193.02616 311 C00158 Citric acid_13C_4 194.02959 311 C00158 Citric acid_13C_5 195.03291 311 C00158 METABOLITES_END #END