#METABOLOMICS WORKBENCH kuppal2_20170621_093022 DATATRACK_ID:1069 STUDY_ID:ST000657 ANALYSIS_ID:AN001003 PROJECT_ID:PR000462 VERSION 1 CREATED_ON June 28, 2017, 11:01 am #PROJECT PR:PROJECT_TITLE Metabolomics analysis of different mouse APC subsets from lung and spleen of WT PR:PROJECT_TITLE and mTOR-APC-KO PR:PROJECT_SUMMARY Antigen presenting cells from WT or mTOR-APC-KO mice were sorted from lung and PR:PROJECT_SUMMARY spleens and subjected to metabolomics analysis. PR:INSTITUTE Emory University PR:DEPARTMENT Department of Medicine PR:LABORATORY Clinical Biomarkers Laboratory PR:LAST_NAME Li; Gardinassi PR:FIRST_NAME Shuzhao; Luiz PR:ADDRESS Whitehead Biomedical Research Building, Suit 205, 615 Michael Street, Atlanta, PR:ADDRESS GA 30322 PR:EMAIL shuzhao.li@gmail.com or luiz.gardinassi@emory.edu PR:PHONE 404-712-2988 #STUDY ST:STUDY_TITLE mTOR regulates metabolic adaptation of APCs in the lung microenvironment and ST:STUDY_TITLE controls the outcome of allergic inflammation ST:STUDY_SUMMARY Antigen presenting cells (APCs) occupy diverse anatomical tissues, but their ST:STUDY_SUMMARY tissue-restricted homeostasis remains poorly understood. Here, working in mouse ST:STUDY_SUMMARY models of inflammation, we found that mTOR-dependent metabolic adaptation was ST:STUDY_SUMMARY required at discrete locations. mTOR was dispensable for DC homeostasis in ST:STUDY_SUMMARY secondary lymphoid tissues, but necessary to regulate cellular metabolism and ST:STUDY_SUMMARY accumulation of CD103+ DCs and alveolar macrophages in lung. Moreover, whilst ST:STUDY_SUMMARY numbers of mTOR-deficient lung CD11b+ DCs were not changed, they were ST:STUDY_SUMMARY metabolically reprogrammed to skew allergic inflammation from eosinophilic Th2 ST:STUDY_SUMMARY to neutrophilic Th17 polarity. The mechanism for this change was independent of ST:STUDY_SUMMARY translational control, but dependent on inflammatory DC which produced IL-23 and ST:STUDY_SUMMARY increased fatty acid oxidation. mTOR therefore mediates metabolic adaptation of ST:STUDY_SUMMARY APCs in distinct tissues, influencing the immunological character of allergic ST:STUDY_SUMMARY inflammation. ST:INSTITUTE Emory University ST:LAST_NAME Li; Gardinassi ST:FIRST_NAME Shuzhao; Luiz ST:ADDRESS Emory Woodruff Memorial Research Building, 1639 Pierce Dr NE, Atlanta, GA 30322 ST:EMAIL shuzhao.li@gmail.com; luiz.gardinassi@emory.edu ST:PHONE 404-712-2988 or 404-727-5091 #SUBJECT SU:SUBJECT_TYPE cells SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN WT or mTOR-APC-KO/C57BL6 #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data SUBJECT_SAMPLE_FACTORS 1162014_CD8a_Spleen_B6_1 VT_150922_001 Genotype:WT | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 1162014_CD8a_Spleen_B6_3 VT_150922_003 Genotype:WT | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 1162014_CD8a_Spleen_B6_5 VT_150922_005 Genotype:WT | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 1162014_CD8a 2_Spleen_B6_1 VT_150922_007 Genotype:WT | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 1162014_CD8a 2_Spleen_B6_3 VT_150922_009 Genotype:WT | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 1162014_CD8a 2_Spleen_B6_5 VT_150922_011 Genotype:WT | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 1162014_CD8a 3_Spleen_B6_1 VT_150922_013 Genotype:WT | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 1162014_CD8a 3_Spleen_B6_3 VT_150922_015 Genotype:WT | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 1162014_CD8a 3_Spleen_B6_5 VT_150922_017 Genotype:WT | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 12172014_CD8a_Spleen_mTOR DC-KO_1 VT_150922_019 Genotype:mTOR-APC-KO | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 12172014_CD8a_Spleen_mTOR DC-KO_3 VT_150922_021 Genotype:mTOR-APC-KO | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 12172014_CD8a_Spleen_mTOR DC-KO_5 VT_150922_023 Genotype:mTOR-APC-KO | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 162014_CD8a 1_Spleen_mTOR DC-KO_1 VT_150922_025 Genotype:mTOR-APC-KO | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 162014_CD8a 1_Spleen_mTOR DC-KO_3 VT_150922_027 Genotype:mTOR-APC-KO | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 162014_CD8a 1_Spleen_mTOR DC-KO_5 VT_150922_029 Genotype:mTOR-APC-KO | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 162014_CD8a 2_Spleen_mTOR DC-KO_1 VT_150922_031 Genotype:mTOR-APC-KO | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 162014_CD8a 2_Spleen_mTOR DC-KO_3 VT_150922_033 Genotype:mTOR-APC-KO | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 162014_CD8a 2_Spleen_mTOR DC-KO_5 VT_150922_035 Genotype:mTOR-APC-KO | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 1162014_CD11b_Spleen_B6_1 VT_150922_037 Genotype:WT | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 1162014_CD11b_Spleen_B6_3 VT_150922_039 Genotype:WT | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 1162014_CD11b_Spleen_B6_5 VT_150922_041 Genotype:WT | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 1162014_CD11b 2_Spleen_B6_1 VT_150922_043 Genotype:WT | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 1162014_CD11b 2_Spleen_B6_3 VT_150922_045 Genotype:WT | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 1162014_CD11b 2_Spleen_B6_5 VT_150922_047 Genotype:WT | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 1162014_CD11b 3_Spleen_B6_1 VT_150922_049 Genotype:WT | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 1162014_CD11b 3_Spleen_B6_3 VT_150922_051 Genotype:WT | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 1162014_CD11b 3_Spleen_B6_5 VT_150922_053 Genotype:WT | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 12172014_CD11b_Spleen_mTOR DC-KO_1 VT_150922_055 Genotype:mTOR-APC-KO | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 12172014_CD11b_Spleen_mTOR DC-KO_3 VT_150922_057 Genotype:mTOR-APC-KO | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 12172014_CD11b_Spleen_mTOR DC-KO_5 VT_150922_059 Genotype:mTOR-APC-KO | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 162014_CD11b 1_Spleen_mTOR DC-KO_1 VT_150922_061 Genotype:mTOR-APC-KO | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 162014_CD11b 1_Spleen_mTOR DC-KO_3 VT_150922_063 Genotype:mTOR-APC-KO | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 162014_CD11b 1_Spleen_mTOR DC-KO_5 VT_150922_065 Genotype:mTOR-APC-KO | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 162014_CD11b 2_Spleen_mTOR DC-KO_1 VT_150922_067 Genotype:mTOR-APC-KO | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 162014_CD11b 2_Spleen_mTOR DC-KO_3 VT_150922_069 Genotype:mTOR-APC-KO | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 162014_CD11b 2_Spleen_mTOR DC-KO_5 VT_150922_071 Genotype:mTOR-APC-KO | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 10162014_AM 1_Lung_B6_1 VT_150922_073 Genotype:WT | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 10162014_AM 1_Lung_B6_3 VT_150922_075 Genotype:WT | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 10162014_AM 1_Lung_B6_5 VT_150922_077 Genotype:WT | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 10162014_AM 2_Lung_B6_1 VT_150922_079 Genotype:WT | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 10162014_AM 2_Lung_B6_3 VT_150922_081 Genotype:WT | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 10162014_AM 2_Lung_B6_5 VT_150922_083 Genotype:WT | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 1132014_AM 1_Lung_B6_1 VT_150922_085 Genotype:WT | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 1132014_AM 1_Lung_B6_3 VT_150922_087 Genotype:WT | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 1132014_AM 1_Lung_B6_5 VT_150922_089 Genotype:WT | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 12172014_AM A_Lung_mTOR DC-KO_1 VT_150922_091 Genotype:mTOR-APC-KO | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 12172014_AM A_Lung_mTOR DC-KO_3 VT_150922_093 Genotype:mTOR-APC-KO | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 12172014_AM A_Lung_mTOR DC-KO_5 VT_150922_095 Genotype:mTOR-APC-KO | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 12172014_AM B_Lung_mTOR DC-KO_1 VT_150922_097 Genotype:mTOR-APC-KO | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 12172014_AM B_Lung_mTOR DC-KO_3 VT_150922_099 Genotype:mTOR-APC-KO | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 12172014_AM B_Lung_mTOR DC-KO_5 VT_150922_101 Genotype:mTOR-APC-KO | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 162014_AM A_Lung_mTOR DC-KO_1 VT_150922_103 Genotype:mTOR-APC-KO | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 162014_AM A_Lung_mTOR DC-KO_3 VT_150922_105 Genotype:mTOR-APC-KO | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 162014_AM A_Lung_mTOR DC-KO_5 VT_150922_107 Genotype:mTOR-APC-KO | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 10162014_CD11b_Lung_B6_1 VT_150922_109 Genotype:WT | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 10162014_CD11b_Lung_B6_3 VT_150922_111 Genotype:WT | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 10162014_CD11b_Lung_B6_5 VT_150922_113 Genotype:WT | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 1132014_CD11b 1_Lung_B6_1 VT_150922_115 Genotype:WT | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 1132014_CD11b 1_Lung_B6_3 VT_150922_117 Genotype:WT | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 1132014_CD11b 1_Lung_B6_5 VT_150922_119 Genotype:WT | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 1132014_CD11b 2_Lung_B6_1 VT_150922_121 Genotype:WT | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 1132014_CD11b 2_Lung_B6_3 VT_150922_123 Genotype:WT | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 1132014_CD11b 2_Lung_B6_5 VT_150922_125 Genotype:WT | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 12172014_CD11b A_Lung_mTOR DC-KO_1 VT_150922_127 Genotype:mTOR-APC-KO | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 12172014_CD11b A_Lung_mTOR DC-KO_3 VT_150922_129 Genotype:mTOR-APC-KO | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 12172014_CD11b A_Lung_mTOR DC-KO_5 VT_150922_131 Genotype:mTOR-APC-KO | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 12172014_CD11b B_Lung_mTOR DC-KO_1 VT_150922_133 Genotype:mTOR-APC-KO | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 12172014_CD11b B_Lung_mTOR DC-KO_3 VT_150922_135 Genotype:mTOR-APC-KO | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 12172014_CD11b B_Lung_mTOR DC-KO_5 VT_150922_137 Genotype:mTOR-APC-KO | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 162014_CD11b B_Lung_mTOR DC-KO_1 VT_150922_139 Genotype:mTOR-APC-KO | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 162014_CD11b B_Lung_mTOR DC-KO_3 VT_150922_141 Genotype:mTOR-APC-KO | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 162014_CD11b B_Lung_mTOR DC-KO_5 VT_150922_143 Genotype:mTOR-APC-KO | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 10162014_CD103_Lung_B6_1 VT_150922_145 Genotype:WT | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 10162014_CD103_Lung_B6_3 VT_150922_147 Genotype:WT | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 10162014_CD103_Lung_B6_5 VT_150922_149 Genotype:WT | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 1132014_CD103 1_Lung_B6_1 VT_150922_151 Genotype:WT | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 1132014_CD103 1_Lung_B6_3 VT_150922_153 Genotype:WT | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 1132014_CD103 1_Lung_B6_5 VT_150922_155 Genotype:WT | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 1132014_CD103 2_Lung_B6_1 VT_150922_157 Genotype:WT | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 1132014_CD103 2_Lung_B6_3 VT_150922_159 Genotype:WT | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 1132014_CD103 2_Lung_B6_5 VT_150922_161 Genotype:WT | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 12172014_CD103+ A&B_Lung_mTOR DC-KO_1 VT_150922_163 Genotype:mTOR-APC-KO | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 12172014_CD103+ A&B_Lung_mTOR DC-KO_3 VT_150922_165 Genotype:mTOR-APC-KO | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 12172014_CD103+ A&B_Lung_mTOR DC-KO_5 VT_150922_167 Genotype:mTOR-APC-KO | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 162014_CD103+ A&B_Lung_mTOR DC-KO_1 VT_150922_169 Genotype:mTOR-APC-KO | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 162014_CD103+ A&B_Lung_mTOR DC-KO_3 VT_150922_171 Genotype:mTOR-APC-KO | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 162014_CD103+ A&B_Lung_mTOR DC-KO_5 VT_150922_173 Genotype:mTOR-APC-KO | HDM_stimulated:No SUBJECT_SAMPLE_FACTORS 3192015_DC WT 1_HDM Lung_mTOR ctrl_1 VT_150922_175 Genotype:WT | HDM_stimulated:YES SUBJECT_SAMPLE_FACTORS 3192015_DC WT 1_HDM Lung_mTOR ctrl_3 VT_150922_177 Genotype:WT | HDM_stimulated:YES SUBJECT_SAMPLE_FACTORS 3192015_DC WT 1_HDM Lung_mTOR ctrl_5 VT_150922_179 Genotype:WT | HDM_stimulated:YES SUBJECT_SAMPLE_FACTORS 3192015_DC WT 2_HDM Lung_mTOR ctrl_1 VT_150922_181 Genotype:WT | HDM_stimulated:YES SUBJECT_SAMPLE_FACTORS 3192015_DC WT 2_HDM Lung_mTOR ctrl_3 VT_150922_183 Genotype:WT | HDM_stimulated:YES SUBJECT_SAMPLE_FACTORS 3192015_DC WT 2_HDM Lung_mTOR ctrl_5 VT_150922_185 Genotype:WT | HDM_stimulated:YES SUBJECT_SAMPLE_FACTORS 3192015_DC WT 3_HDM Lung_mTOR ctrl_1 VT_150922_187 Genotype:WT | HDM_stimulated:YES SUBJECT_SAMPLE_FACTORS 3192015_DC WT 3_HDM Lung_mTOR ctrl_3 VT_150922_189 Genotype:WT | HDM_stimulated:YES SUBJECT_SAMPLE_FACTORS 3192015_DC WT 3_HDM Lung_mTOR ctrl_5 VT_150922_191 Genotype:WT | HDM_stimulated:YES SUBJECT_SAMPLE_FACTORS 3192015_DC KO 1_HDM Lung_mTOR DCKO_1 VT_150922_193 Genotype:mTOR-APC-KO | HDM_stimulated:YES SUBJECT_SAMPLE_FACTORS 3192015_DC KO 1_HDM Lung_mTOR DCKO_3 VT_150922_195 Genotype:mTOR-APC-KO | HDM_stimulated:YES SUBJECT_SAMPLE_FACTORS 3192015_DC KO 1_HDM Lung_mTOR DCKO_5 VT_150922_197 Genotype:mTOR-APC-KO | HDM_stimulated:YES SUBJECT_SAMPLE_FACTORS 3192015_DC KO 2_HDM Lung_mTOR DCKO_1 VT_150922_199 Genotype:mTOR-APC-KO | HDM_stimulated:YES SUBJECT_SAMPLE_FACTORS 3192015_DC KO 2_HDM Lung_mTOR DCKO_3 VT_150922_201 Genotype:mTOR-APC-KO | HDM_stimulated:YES SUBJECT_SAMPLE_FACTORS 3192015_DC KO 2_HDM Lung_mTOR DCKO_5 VT_150922_203 Genotype:mTOR-APC-KO | HDM_stimulated:YES SUBJECT_SAMPLE_FACTORS 3192015_DC KO 3_HDM Lung_mTOR DCKO_1 VT_150922_205 Genotype:mTOR-APC-KO | HDM_stimulated:YES SUBJECT_SAMPLE_FACTORS 3192015_DC KO 3_HDM Lung_mTOR DCKO_3 VT_150922_207 Genotype:mTOR-APC-KO | HDM_stimulated:YES SUBJECT_SAMPLE_FACTORS 3192015_DC KO 3_HDM Lung_mTOR DCKO_5 VT_150922_209 Genotype:mTOR-APC-KO | HDM_stimulated:YES #COLLECTION CO:COLLECTION_SUMMARY Lungs and Spleen of WT or mTOR-APC-KO mice were aseptically removed. Spleens CO:COLLECTION_SUMMARY were finely chopped with a scalpel in 6-well plates, and digested with CO:COLLECTION_SUMMARY 1-1.5mg/mL collagenase type 4 (Worthington Biochemical Corporation) dissolved in CO:COLLECTION_SUMMARY HBSS. Digestion was performed for 30 minutes at 37°C, and reactions were CO:COLLECTION_SUMMARY stopped with addition of 2mM EDTA. Digestions were passed through 40μm cell CO:COLLECTION_SUMMARY strainers to obtain a homogenous cell suspension. To obtain lung suspensions, CO:COLLECTION_SUMMARY mice were sacrificed with CO2 and perfused with 5mL PBS injected into the right CO:COLLECTION_SUMMARY ventricle. Lungs were dissected into gentleMACS™ C tubes (Miltenyi) and CO:COLLECTION_SUMMARY dissociated using a gentleMACS™ octo dissociator using the pre-set program CO:COLLECTION_SUMMARY m_lung_01_02. Dissociated lung was digested with 1-1.5mg/mL collagenase type 4 CO:COLLECTION_SUMMARY for 30 minutes at 37°C. Lungs were next homogenized with gentleMACS™ octo CO:COLLECTION_SUMMARY dissociator program m_lung_02_01, reactions were stopped with 2mM EDTA and CO:COLLECTION_SUMMARY digestions passed through 40μm cell strainers. Cells were pelleted CO:COLLECTION_SUMMARY (1500-2000rpm/300- 500xg for 5 minutes). Red blood cells were lysed with 1mL Ack CO:COLLECTION_SUMMARY lysing buffer (Lonza) for 1 minute and lysis was stopped by addition of ≥ 5 CO:COLLECTION_SUMMARY volumes of HBSS. Cells were washed once to obtain single cell suspensions. APCs CO:COLLECTION_SUMMARY were enriched with CD11c and CD11b microbeads and MACS manual separation CO:COLLECTION_SUMMARY (Miltenyi), according to the manufacturer’s instructions. Enriched cells were CO:COLLECTION_SUMMARY stained with live/dead Aqua or Via-Probe™ (BD) and with surface antibody CO:COLLECTION_SUMMARY cocktails. Populations were further purified to ≥95% purity with a BD FACSAria CO:COLLECTION_SUMMARY cell sorter. CO:SAMPLE_TYPE Sorted cells #TREATMENT TR:TREATMENT_SUMMARY No treatment #SAMPLEPREP SP:SAMPLEPREP_SUMMARY Acetonitrile (2:1, v/v) was added to cell extracts, wih 3.25ul internal SP:SAMPLEPREP_SUMMARY standard. Proteins were removed by centrifugation at 14,000g for 10 min at 4 SP:SAMPLEPREP_SUMMARY °C. The supernatant was transferred into autosampler vials for LC-MS analysis. SP:PROCESSING_METHOD precipitation of protein, centrifuge, and remove supernatant SP:PROCESSING_STORAGE_CONDITIONS on ice SP:EXTRACTION_METHOD 1:2 sample:acetonitrile SP:EXTRACT_STORAGE supernatant pipette into autosampler vials for mass spectrometer SP:SAMPLE_SPIKING internal standards: [13C6]-D-glucose, [15N]-indole, [15N,13C5]-L-methionine, SP:SAMPLE_SPIKING [2-15N]-L-lysine dihydrochloride, [13C5]-L-glutamic acid, [15N]-L-tyrosine, SP:SAMPLE_SPIKING [15N2]-uracil, [3,3-13C2]-cystine, [trimethyl-13C3]-caffeine, [U-13C5, SP:SAMPLE_SPIKING U-15N2]-L-glutamine #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY Untargeted analysis with HILIC positive mode. CH:CHROMATOGRAPHY_TYPE HILIC MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap CH:COLUMN_NAME Thermo Accucore HILIC (100 x 2.1mm, 2.6um) CH:FLOW_GRADIENT A: H2O, B: acetonitrile (ACN), C: 2% formic acid in H20. Flow: 0-2min, CH:FLOW_GRADIENT 10%A-80%B-10%C; 2-7min, 80%A-10%B-10%C; 7-10min, 80%A-10%B-10%C. CH:FLOW_RATE .350 ul/min CH:COLUMN_TEMPERATURE 30 CH:METHODS_FILENAME Metab_HFQE_Setup.docx CH:INTERNAL_STANDARD internal standards: [13C6]-D-glucose, [15N]-indole, [15N,13C5]-L-methionine, CH:INTERNAL_STANDARD [2-15N]-L-lysine dihydrochloride, [13C5]-L-glutamic acid, [15N]-L-tyrosine, CH:INTERNAL_STANDARD [15N2]-uracil, [3,3-13C2]-cystine, [trimethyl-13C3]-caffeine, [U-13C5, CH:INTERNAL_STANDARD U-15N2]-L-glutamine CH:SAMPLE_INJECTION 10ul CH:SAMPLING_CONE HESI probe with S-lens combination for ESI CH:ANALYTICAL_TIME 10min CH:WEAK_WASH_SOLVENT_NAME water with 10% methanol #ANALYSIS AN:ANALYSIS_TYPE MS AN:LABORATORY_NAME Clinical Biomarkers Laboratory AN:OPERATOR_NAME ViLinh Tran AN:DETECTOR_TYPE orbitrap AN:SOFTWARE_VERSION Thermo Excalibur 2.2 AN:ACQUISITION_DATE 9/23/2015-9/25/2015 AN:DATA_FORMAT .raw, .cdf #MS MS:MS_COMMENTS - MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:CAPILLARY_TEMPERATURE 300C MS:CAPILLARY_VOLTAGE 44V MS:IONIZATION electrospray ionization MS:MASS_ACCURACY 10ppm MS:SPRAY_VOLTAGE 3.5 kV MS:MS_RESULTS_FILE ST000657_AN001003_Results.txt UNITS:au #END