#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