#METABOLOMICS WORKBENCH Fernandez_MFG_20200312_115357 DATATRACK_ID:1939 STUDY_ID:ST001328 ANALYSIS_ID:AN002214 PROJECT_ID:PR000906 VERSION 1 CREATED_ON March 18, 2020, 10:34 am #PROJECT PR:PROJECT_TITLE Pulmonary Tuberculosis Mice Project PR:PROJECT_TYPE Natural disease progression assessment PR:PROJECT_SUMMARY The goal of this multiplatform, non-targeted metabolomics study was to explore PR:PROJECT_SUMMARY the metabolic alterations occurring during the natural progression of pulmonary PR:PROJECT_SUMMARY tuberculosis in a murine model of disease (C57BL/6 genotype). For this purpose, PR:PROJECT_SUMMARY we used gas chromatography, capillary electrophoresis, and reversed-phase liquid PR:PROJECT_SUMMARY chromatography coupled to high-resolution mass analyzers (GC-EI-QTOF/MS, PR:PROJECT_SUMMARY CE-ESI(+)-QTOF/MS, LC-ESI(+)-QTOF/MS and LC-ESI(-)-QTOF/MS to analyze lung PR:PROJECT_SUMMARY extracts of age and sex-matched uninfected mice (UW, n=4), Mycobacterium PR:PROJECT_SUMMARY tuberculosis-infected mice at 4 weeks post-infection (4W, n=4) and Mycobacterium PR:PROJECT_SUMMARY tuberculosis-infected mice at 9 weeks post-infection. All data were acquired in PR:PROJECT_SUMMARY MS1 mode, following a canonical non-targeted workflow. The potential benefit for PR:PROJECT_SUMMARY the general research community arising from the combination of these analyses is PR:PROJECT_SUMMARY to generate mass spectrometry data which has not been previously described for PR:PROJECT_SUMMARY this model of the disease. This data may serve as a foundation for the PR:PROJECT_SUMMARY validation of observations, data modeling, and biochemical interpretation of PR:PROJECT_SUMMARY MS-based metabolic changes found in the comparisons between the different sample PR:PROJECT_SUMMARY groups PR:INSTITUTE Universidad San Pablo-CEU, CEU Universities PR:DEPARTMENT Departamento de Quimica y Bioquimica PR:LABORATORY Centro de Metabolomica y Bioanalisis (CEMBIO) PR:LAST_NAME Fernandez Garcia PR:FIRST_NAME Miguel PR:ADDRESS Facultad de Farmacia, Universidad San Pablo-CEU, CEU Universities, Urbanización PR:ADDRESS Montepríncipe, 28660 Boadilla del Monte, Spain PR:EMAIL miguel.fernandezgarcia@ceu.es PR:PHONE +34690090778 #STUDY ST:STUDY_TITLE Multiplatform, non-targeted analysis of lung extracts of uninfected and ST:STUDY_TITLE Mtb-infected C57BL/6 mice at 4 and 9 weeks p.i. ST:STUDY_SUMMARY The goal of this multiplatform, non-targeted metabolomics study was to explore ST:STUDY_SUMMARY the metabolic alterations occurring during the natural progression of pulmonary ST:STUDY_SUMMARY tuberculosis in a murine model of disease (C57BL/6 genotype). For this purpose, ST:STUDY_SUMMARY we used gas chromatography, capillary electrophoresis, and reversed-phase liquid ST:STUDY_SUMMARY chromatography coupled to high-resolution mass analyzers (GC-EI-QTOF/MS, ST:STUDY_SUMMARY CE-ESI(+)-QTOF/MS, LC-ESI(+)-QTOF/MS and LC-ESI(-)-QTOF/MS to analyze lung ST:STUDY_SUMMARY extracts of age and sex-matched uninfected mice (UW, n=4), Mycobacterium ST:STUDY_SUMMARY tuberculosis-infected mice at 4 weeks post-infection (4W, n=4) and Mycobacterium ST:STUDY_SUMMARY tuberculosis-infected mice at 9 weeks post-infection. All data were acquired in ST:STUDY_SUMMARY MS1 mode, following a canonical non-targeted workflow. ST:INSTITUTE Universidad San Pablo-CEU, CEU Universities ST:DEPARTMENT Departamento de Quimica y Bioquimica ST:LABORATORY Centro de Metabolomica y Bioanalisis (CEMBIO) ST:LAST_NAME Fernandez Garcia ST:FIRST_NAME Miguel ST:ADDRESS Facultad de Farmacia, Universidad San Pablo-CEU, CEU Universities, Urbanización ST:ADDRESS Montepríncipe, 28660 Boadilla del Monte, Spain ST:EMAIL miguel.fernandezgarcia@ceu.es ST:PHONE +34690090778 ST:NUM_GROUPS 3 ST:TOTAL_SUBJECTS 13 ST:NUM_MALES 6 ST:NUM_FEMALES 7 #SUBJECT SU:SUBJECT_TYPE Mammal SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENOTYPE_STRAIN C57BL/6 SU:AGE_OR_AGE_RANGE 8-10 weeks old SU:GENDER Male and female SU:ANIMAL_FEED ad libitum SU:ANIMAL_WATER ad libitum #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 Mouse #1 CEMS_UW1 Pulmonary TB status:Control - uninfected Sex=Female; Analytical platform=CE-ESI(+)-TOF/MS; RAW_FILE_NAME=CEMS_UW1.d SUBJECT_SAMPLE_FACTORS Mouse #2 CEMS_UW2 Pulmonary TB status:Control - uninfected Sex=Female; Analytical platform=CE-ESI(+)-TOF/MS; RAW_FILE_NAME=CEMS_UW2.d SUBJECT_SAMPLE_FACTORS Mouse #3 CEMS_UW3 Pulmonary TB status:Control - uninfected Sex=Male; Analytical platform=CE-ESI(+)-TOF/MS; RAW_FILE_NAME=CEMS_UW3.d SUBJECT_SAMPLE_FACTORS Mouse #4 CEMS_UW4 Pulmonary TB status:Control - uninfected Sex=Male; Analytical platform=CE-ESI(+)-TOF/MS; RAW_FILE_NAME=CEMS_UW4.d SUBJECT_SAMPLE_FACTORS Mouse #5 CEMS_4W1 Pulmonary TB status:4 weeks post-infection Sex=Female; Analytical platform=CE-ESI(+)-TOF/MS; RAW_FILE_NAME=CEMS_4W1.d SUBJECT_SAMPLE_FACTORS Mouse #6 CEMS_4W2 Pulmonary TB status:4 weeks post-infection Sex=Female; Analytical platform=CE-ESI(+)-TOF/MS; RAW_FILE_NAME=CEMS_4W2.d SUBJECT_SAMPLE_FACTORS Mouse #7 CEMS_4W3 Pulmonary TB status:4 weeks post-infection Sex=Male; Analytical platform=CE-ESI(+)-TOF/MS; RAW_FILE_NAME=CEMS_4W3.d SUBJECT_SAMPLE_FACTORS Mouse #8 CEMS_4W4 Pulmonary TB status:4 weeks post-infection Sex=Male; Analytical platform=CE-ESI(+)-TOF/MS; RAW_FILE_NAME=CEMS_4W4.d SUBJECT_SAMPLE_FACTORS Mouse #9 CEMS_9W1 Pulmonary TB status:9 weeks post-infection Sex=Female; Analytical platform=CE-ESI(+)-TOF/MS; RAW_FILE_NAME=CEMS_9W1.d SUBJECT_SAMPLE_FACTORS Mouse #10 CEMS_9W2 Pulmonary TB status:9 weeks post-infection Sex=Female; Analytical platform=CE-ESI(+)-TOF/MS; RAW_FILE_NAME=CEMS_9W2.d SUBJECT_SAMPLE_FACTORS Mouse #11 CEMS_9W3 Pulmonary TB status:9 weeks post-infection Sex=Female; Analytical platform=CE-ESI(+)-TOF/MS; RAW_FILE_NAME=CEMS_9W3.d SUBJECT_SAMPLE_FACTORS Mouse #12 CEMS_9W4 Pulmonary TB status:9 weeks post-infection Sex=Male; Analytical platform=CE-ESI(+)-TOF/MS; RAW_FILE_NAME=CEMS_9W4.d SUBJECT_SAMPLE_FACTORS Mouse #13 CEMS_9W5 Pulmonary TB status:9 weeks post-infection Sex=Male; Analytical platform=CE-ESI(+)-TOF/MS; RAW_FILE_NAME=CEMS_9W5.d SUBJECT_SAMPLE_FACTORS Mouse #1 GCMS_UW1 Pulmonary TB status:Control - uninfected Sex=Female; Analytical platform=GC-EI-QTOF/MS; RAW_FILE_NAME=GCMS_UW1.d SUBJECT_SAMPLE_FACTORS Mouse #2 GCMS_UW2 Pulmonary TB status:Control - uninfected Sex=Female; Analytical platform=GC-EI-QTOF/MS; RAW_FILE_NAME=GCMS_UW2.d SUBJECT_SAMPLE_FACTORS Mouse #3 GCMS_UW3 Pulmonary TB status:Control - uninfected Sex=Male; Analytical platform=GC-EI-QTOF/MS; RAW_FILE_NAME=GCMS_UW3.d SUBJECT_SAMPLE_FACTORS Mouse #4 GCMS_UW4 Pulmonary TB status:Control - uninfected Sex=Male; Analytical platform=GC-EI-QTOF/MS; RAW_FILE_NAME=GCMS_UW4.d SUBJECT_SAMPLE_FACTORS Mouse #5 GCMS_4W1 Pulmonary TB status:4 weeks post-infection Sex=Female; Analytical platform=GC-EI-QTOF/MS; RAW_FILE_NAME=GCMS_4W1.d SUBJECT_SAMPLE_FACTORS Mouse #6 GCMS_4W2 Pulmonary TB status:4 weeks post-infection Sex=Female; Analytical platform=GC-EI-QTOF/MS; RAW_FILE_NAME=GCMS_4W2.d SUBJECT_SAMPLE_FACTORS Mouse #7 GCMS_4W3 Pulmonary TB status:4 weeks post-infection Sex=Male; Analytical platform=GC-EI-QTOF/MS; RAW_FILE_NAME=GCMS_4W3.d SUBJECT_SAMPLE_FACTORS Mouse #8 GCMS_4W4 Pulmonary TB status:4 weeks post-infection Sex=Male; Analytical platform=GC-EI-QTOF/MS; RAW_FILE_NAME=GCMS_4W4.d SUBJECT_SAMPLE_FACTORS Mouse #9 GCMS_9W1 Pulmonary TB status:9 weeks post-infection Sex=Female; Analytical platform=GC-EI-QTOF/MS; RAW_FILE_NAME=GCMS_9W1.d SUBJECT_SAMPLE_FACTORS Mouse #10 GCMS_9W2 Pulmonary TB status:9 weeks post-infection Sex=Female; Analytical platform=GC-EI-QTOF/MS; RAW_FILE_NAME=GCMS_9W2.d SUBJECT_SAMPLE_FACTORS Mouse #11 GCMS_9W3 Pulmonary TB status:9 weeks post-infection Sex=Female; Analytical platform=GC-EI-QTOF/MS; RAW_FILE_NAME=GCMS_9W3.d SUBJECT_SAMPLE_FACTORS Mouse #12 GCMS_9W4 Pulmonary TB status:9 weeks post-infection Sex=Male; Analytical platform=GC-EI-QTOF/MS; RAW_FILE_NAME=GCMS_9W4.d SUBJECT_SAMPLE_FACTORS Mouse #13 GCMS_9W5 Pulmonary TB status:9 weeks post-infection Sex=Male; Analytical platform=GC-EI-QTOF/MS; RAW_FILE_NAME=GCMS_9W5.d SUBJECT_SAMPLE_FACTORS Mouse #1 LCMSpos_UW1 Pulmonary TB status:Control - uninfected Sex=Female; Analytical platform=LC-ESI(+)-QTOF/MS; RAW_FILE_NAME=LCMSpos_UW1.d SUBJECT_SAMPLE_FACTORS Mouse #2 LCMSpos_UW2 Pulmonary TB status:Control - uninfected Sex=Female; Analytical platform=LC-ESI(+)-QTOF/MS; RAW_FILE_NAME=LCMSpos_UW2.d SUBJECT_SAMPLE_FACTORS Mouse #3 LCMSpos_UW3 Pulmonary TB status:Control - uninfected Sex=Male; Analytical platform=LC-ESI(+)-QTOF/MS; RAW_FILE_NAME=LCMSpos_UW3.d SUBJECT_SAMPLE_FACTORS Mouse #4 LCMSpos_UW4 Pulmonary TB status:Control - uninfected Sex=Male; Analytical platform=LC-ESI(+)-QTOF/MS; RAW_FILE_NAME=LCMSpos_UW4.d SUBJECT_SAMPLE_FACTORS Mouse #5 LCMSpos_4W1 Pulmonary TB status:4 weeks post-infection Sex=Female; Analytical platform=LC-ESI(+)-QTOF/MS; RAW_FILE_NAME=LCMSpos_4W1.d SUBJECT_SAMPLE_FACTORS Mouse #6 LCMSpos_4W2 Pulmonary TB status:4 weeks post-infection Sex=Female; Analytical platform=LC-ESI(+)-QTOF/MS; RAW_FILE_NAME=LCMSpos_4W2.d SUBJECT_SAMPLE_FACTORS Mouse #7 LCMSpos_4W3 Pulmonary TB status:4 weeks post-infection Sex=Male; Analytical platform=LC-ESI(+)-QTOF/MS; RAW_FILE_NAME=LCMSpos_4W3.d SUBJECT_SAMPLE_FACTORS Mouse #8 LCMSpos_4W4 Pulmonary TB status:4 weeks post-infection Sex=Male; Analytical platform=LC-ESI(+)-QTOF/MS; RAW_FILE_NAME=LCMSpos_4W4.d SUBJECT_SAMPLE_FACTORS Mouse #9 LCMSpos_9W1 Pulmonary TB status:9 weeks post-infection Sex=Female; Analytical platform=LC-ESI(+)-QTOF/MS; RAW_FILE_NAME=LCMSpos_9W1.d SUBJECT_SAMPLE_FACTORS Mouse #10 LCMSpos_9W2 Pulmonary TB status:9 weeks post-infection Sex=Female; Analytical platform=LC-ESI(+)-QTOF/MS; RAW_FILE_NAME=LCMSpos_9W2.d SUBJECT_SAMPLE_FACTORS Mouse #11 LCMSpos_9W3 Pulmonary TB status:9 weeks post-infection Sex=Female; Analytical platform=LC-ESI(+)-QTOF/MS; RAW_FILE_NAME=LCMSpos_9W3.d SUBJECT_SAMPLE_FACTORS Mouse #12 LCMSpos_9W4 Pulmonary TB status:9 weeks post-infection Sex=Male; Analytical platform=LC-ESI(+)-QTOF/MS; RAW_FILE_NAME=LCMSpos_9W4.d SUBJECT_SAMPLE_FACTORS Mouse #13 LCMSpos_9W5 Pulmonary TB status:9 weeks post-infection Sex=Male; Analytical platform=LC-ESI(+)-QTOF/MS; RAW_FILE_NAME=LCMSpos_9W5.d SUBJECT_SAMPLE_FACTORS Mouse #1 LCMSneg_UW1 Pulmonary TB status:Control - uninfected Sex=Female; Analytical platform=LC-ESI(-)-QTOF/MS; RAW_FILE_NAME=LCMSneg_UW1.d SUBJECT_SAMPLE_FACTORS Mouse #2 LCMSneg_UW2 Pulmonary TB status:Control - uninfected Sex=Female; Analytical platform=LC-ESI(-)-QTOF/MS; RAW_FILE_NAME=LCMSneg_UW2.d SUBJECT_SAMPLE_FACTORS Mouse #3 LCMSneg_UW3 Pulmonary TB status:Control - uninfected Sex=Male; Analytical platform=LC-ESI(-)-QTOF/MS; RAW_FILE_NAME=LCMSneg_UW3.d SUBJECT_SAMPLE_FACTORS Mouse #4 LCMSneg_UW4 Pulmonary TB status:Control - uninfected Sex=Male; Analytical platform=LC-ESI(-)-QTOF/MS; RAW_FILE_NAME=LCMSneg_UW4.d SUBJECT_SAMPLE_FACTORS Mouse #5 LCMSneg_4W1 Pulmonary TB status:4 weeks post-infection Sex=Female; Analytical platform=LC-ESI(-)-QTOF/MS; RAW_FILE_NAME=LCMSneg_4W1.d SUBJECT_SAMPLE_FACTORS Mouse #6 LCMSneg_4W2 Pulmonary TB status:4 weeks post-infection Sex=Female; Analytical platform=LC-ESI(-)-QTOF/MS; RAW_FILE_NAME=LCMSneg_4W2.d SUBJECT_SAMPLE_FACTORS Mouse #7 LCMSneg_4W3 Pulmonary TB status:4 weeks post-infection Sex=Male; Analytical platform=LC-ESI(-)-QTOF/MS; RAW_FILE_NAME=LCMSneg_4W3.d SUBJECT_SAMPLE_FACTORS Mouse #8 LCMSneg_4W4 Pulmonary TB status:4 weeks post-infection Sex=Male; Analytical platform=LC-ESI(-)-QTOF/MS; RAW_FILE_NAME=LCMSneg_4W4.d SUBJECT_SAMPLE_FACTORS Mouse #9 LCMSneg_9W1 Pulmonary TB status:9 weeks post-infection Sex=Female; Analytical platform=LC-ESI(-)-QTOF/MS; RAW_FILE_NAME=LCMSneg_9W1.d SUBJECT_SAMPLE_FACTORS Mouse #10 LCMSneg_9W2 Pulmonary TB status:9 weeks post-infection Sex=Female; Analytical platform=LC-ESI(-)-QTOF/MS; RAW_FILE_NAME=LCMSneg_9W2.d SUBJECT_SAMPLE_FACTORS Mouse #11 LCMSneg_9W3 Pulmonary TB status:9 weeks post-infection Sex=Female; Analytical platform=LC-ESI(-)-QTOF/MS; RAW_FILE_NAME=LCMSneg_9W3.d SUBJECT_SAMPLE_FACTORS Mouse #12 LCMSneg_9W4 Pulmonary TB status:9 weeks post-infection Sex=Male; Analytical platform=LC-ESI(-)-QTOF/MS; RAW_FILE_NAME=LCMSneg_9W4.d SUBJECT_SAMPLE_FACTORS Mouse #13 LCMSneg_9W5 Pulmonary TB status:9 weeks post-infection Sex=Male; Analytical platform=LC-ESI(-)-QTOF/MS; RAW_FILE_NAME=LCMSneg_9W5.d #COLLECTION CO:COLLECTION_SUMMARY Both male and female age-matched C57BL/6 mice (8-10 weeks old) were infected CO:COLLECTION_SUMMARY with Mtb H37Rv in animal BSL-3 laboratory and monitored with food and water ad CO:COLLECTION_SUMMARY libitum. Mice were sacrificed by anesthesia with isoflurane followed by gentle CO:COLLECTION_SUMMARY cervical dislocation as approved by institutional Animal Protocol Number (APN): CO:COLLECTION_SUMMARY 08591. Mice experimental procedures were approved by the Institutional Animal CO:COLLECTION_SUMMARY Care and Use Committee (IACUC) at the University of Alabama at Birmingham, USA. CO:COLLECTION_SUMMARY For mice studies, we adhered as per national/international regulation of CO:COLLECTION_SUMMARY “Public Health Service Policy on Humane Care and Use of Laboratory Animals” CO:COLLECTION_SUMMARY (NIH), and “Animal Welfare Act and Animal Welfare Regulations” (USDA). Mouse CO:COLLECTION_SUMMARY genotype was confirmed by PCR and Western blotting. Mtb H37Rv was grown at 37 CO:COLLECTION_SUMMARY °C with shaking in BD Difco Middlebrook 7H9 media supplemented with 0.2 % CO:COLLECTION_SUMMARY glycerol, ADS (Albumin, Dextrose, NaCl) with 0.02 % tyloxapol. Mice were CO:COLLECTION_SUMMARY infected with 5 x 104 Mtb H37Rv via the intratracheal route. Lungs were CO:COLLECTION_SUMMARY collected from uninfected (n = 4), and Mtb-infected mice at 4- and 9-weeks CO:COLLECTION_SUMMARY post-infection (n = 4 and n = 5, respectively), and stored immediately at -80 CO:COLLECTION_SUMMARY °C for further processing and metabolite extraction. CO:SAMPLE_TYPE Lung CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY Mice were infected with 5 x 104 Mtb H37Rv via the intratracheal route and zero TR:TREATMENT_SUMMARY timepoint and monitored for disease progression until lung collection at 4 or 9 TR:TREATMENT_SUMMARY weeks post-infection TR:TREATMENT Infection TR:TREATMENT_ROUTE Intratracheal TR:TREATMENT_DOSE 5 x 10^4 Mtb H37Rv #SAMPLEPREP SP:SAMPLEPREP_SUMMARY After lung collection, 1 mL of 50 % methanol was added to 100 mg of Mtb-infected SP:SAMPLEPREP_SUMMARY or uninfected lung tissue and homogenized in a dounce homogenizer to prepare a SP:SAMPLEPREP_SUMMARY uniform suspension. For CE-TOF/MS, 200 uL of homogenate were mixed with 200 L SP:SAMPLEPREP_SUMMARY of 0.2 M formic acid and vortexed for 2 min. The samples were cleared by SP:SAMPLEPREP_SUMMARY centrifugation at 16000 x g for 10 min at 4 °C and the supernatant was SP:SAMPLEPREP_SUMMARY filter-sterilized using 0.22 um spin-X columns (Sigma). For GC-QTOF/MS and SP:SAMPLEPREP_SUMMARY LC-QTOF/MS, 200 uL of each sample homogenate were mixed with 800 L of 80:20 SP:SAMPLEPREP_SUMMARY methanol:MTBE (Methyl tert-butyl ether) and vortexed for 2 min. Metabolites were SP:SAMPLEPREP_SUMMARY then extracted for 1 h with shaking at room temperature and then centrifuged at SP:SAMPLEPREP_SUMMARY 4000 x g at 20 °C for 20 min. Supernatants were sterile filtered using 0.22 um SP:SAMPLEPREP_SUMMARY Spin-X columns. All samples were passed through a Millipore filter (30-kDa SP:SAMPLEPREP_SUMMARY cutoff) to remove large proteins. Samples were dried under high vacuum and SP:SAMPLEPREP_SUMMARY stored at -80 °C until further platform-specific processing and analysis. For SP:SAMPLEPREP_SUMMARY CE-ESI(+)-TOF/MS analysis, The dried samples were resuspended in Milli-Q water SP:SAMPLEPREP_SUMMARY containing 0.1 mM formic acid and 0.2 mM methionine sulfone (internal standard) SP:SAMPLEPREP_SUMMARY (Sigma-Aldrich, Germany) by vortexing for 1 min. After subsequent centrifugation SP:SAMPLEPREP_SUMMARY (12600 x g, 15 min), the resulting clear solution was analyzed. For GC-QTOF/MS SP:SAMPLEPREP_SUMMARY analysis, The above described dried samples were re-suspended in 450 µL of SP:SAMPLEPREP_SUMMARY MeOH:H2O:MTBE (74:10:16) and after centrifugation at 12600 x g, 15 min at 4 °C, SP:SAMPLEPREP_SUMMARY the supernatant was transferred to a vial with insert and evaporated to dryness SP:SAMPLEPREP_SUMMARY under high vacuum. The obtained dried extracts were derivatized by a MPS SP:SAMPLEPREP_SUMMARY autosampler for GC/MS analysis as previously described (Fiehn O., 2006). For SP:SAMPLEPREP_SUMMARY LC-QTOF/MS analysis, The above described dried samples were resuspended in 200 SP:SAMPLEPREP_SUMMARY µL of methanol:water:MTBE (7.4:1:1.6), vortexed for 1.5 h and centrifuged (4000 SP:SAMPLEPREP_SUMMARY x g, 10 min, 4 °C). Clear solutions were analyzed. SP:SAMPLE_DERIVATIZATION In GC-EI-QTOF/MS analyses, Briefly, aldehyde and keto groups were first SP:SAMPLE_DERIVATIZATION converted to O-methyloximes by reaction with 10 µL pyridine containing 15 mg/mL SP:SAMPLE_DERIVATIZATION O-methoxyamine (Sigma-Aldrich, Germany) for 60 min at 70 °C. In a second step, SP:SAMPLE_DERIVATIZATION acid hydrogen-containing metabolites were trimethylsilylated by reaction with 10 SP:SAMPLE_DERIVATIZATION µL N,O-Bis(trimethylsilyl)trifluoroacetamide (BSTFA) (Sigma-Aldrich, Germany) SP:SAMPLE_DERIVATIZATION to enhance the GC/MS metabolite coverage #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY The separation occurred in a fused-silica capillary (Agilent Technologies) CH:CHROMATOGRAPHY_SUMMARY (total length, 96 cm; i.d., 50 μm) under normal polarity with a background CH:CHROMATOGRAPHY_SUMMARY electrolyte containing 1.0 M formic acid in 10 % (v/v) methanol at 20 °C. CH:CHROMATOGRAPHY_SUMMARY Sheath liquid (6 µL·min-1) was methanol/water (1/1, v/v) containing 1.0 mM CH:CHROMATOGRAPHY_SUMMARY formic acid with two reference masses to allow correction and high mass CH:CHROMATOGRAPHY_SUMMARY resolution in the MS. Samples were hydrodynamically injected at 50 mbar for 35 s CH:CHROMATOGRAPHY_SUMMARY and stacked by injecting background electrolyte at 100 mbar for 10 s. CH:CHROMATOGRAPHY_TYPE CE CH:INSTRUMENT_NAME Agilent 7100 CE CH:COLUMN_NAME Agilent bare-fused silica capillary (96 cm x 50 um i.d.) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6224 TOF MS:INSTRUMENT_TYPE TOF MS:MS_TYPE ESI MS:ION_MODE POSITIVE MS:MS_COMMENTS The optimized MS parameters were: fragmentor 125 V, Skimmer 65 V, octopole 750 MS:MS_COMMENTS V, nebulizer pressure 10 psi, drying gas temperature at 200 °C and flow rate MS:MS_COMMENTS 10.0 L·min-1. The capillary voltage was 3500 V. Data were acquired in positive MS:MS_COMMENTS electrospray ionization (ESI) mode with a full scan from m/z 50 to 1000 at a MS:MS_COMMENTS rate of 1 spectra·s-1. The resulting CE-TOF/MS data files were cleaned of MS:MS_COMMENTS background noise and unrelated ions by the Batch Recursive Feature Extraction MS:MS_COMMENTS tool with Agilent MassHunter Profinder version B.06.00 software. Data were MS:MS_COMMENTS extracted using a data mining algorithm based on the software. MS:CAPILLARY_VOLTAGE 3.5 kV MS:DRY_GAS_FLOW 10 L·min-1 MS:OCTPOLE_VOLTAGE 750 V MS:MS_RESULTS_FILE ST001328_AN002214_Results.txt UNITS:Abundance Has m/z:Neutral masses Has RT:Yes RT units:Minutes #END