#METABOLOMICS WORKBENCH flazando_20221111_050439 DATATRACK_ID:3565 STUDY_ID:ST002401 ANALYSIS_ID:AN003913
VERSION                          	1
CREATED_ON                       	02-08-2024
#PROJECT
PR:PROJECT_TITLE                 	Human primary astrocytes finger- and footprinting metabolomics indicate
PR:PROJECT_TITLE                 	biochemical alterations under ayahuasca treatment
PR:PROJECT_TYPE                  	Untargetmetabomics- hilic
PR:PROJECT_SUMMARY               	Ayahuasca (Aya) is a psychotropic Amazonian beverage formulated from the
PR:PROJECT_SUMMARY               	combination of the Banisteriopsis caapi vine and the Psychotria viridis leaves
PR:PROJECT_SUMMARY               	in a water decoction. Aya is legally used in Latin American countries and used
PR:PROJECT_SUMMARY               	in Brazil for religious, cultural, and therapeutic purposes. Its properties
PR:PROJECT_SUMMARY               	constitute a bio-psycho-social-spiritual model involving effects from
PR:PROJECT_SUMMARY               	ß-carboline-derived alkaloids, present in the vine, and N,N-dimethyltryptamine
PR:PROJECT_SUMMARY               	(DMT), a tryptamine-derived alkaloid present in the leaves, which act together
PR:PROJECT_SUMMARY               	in the central nervous system (CNS). Few technical-scientific studies have been
PR:PROJECT_SUMMARY               	conducted to understand the effects of this brew in the metabolism. Therefore,
PR:PROJECT_SUMMARY               	this work aims to investigate an in vitro primary astrocyte lineage model by
PR:PROJECT_SUMMARY               	untargeted metabolomics evaluations of two cellular subfractions: secretome and
PR:PROJECT_SUMMARY               	intracellular content after Aya treatment, where DMT and other ß-carbolines
PR:PROJECT_SUMMARY               	were previously quantified. Metabolomics analysis was performed by UHPLC-MS/MS,
PR:PROJECT_SUMMARY               	followed by MS-Dial data processing and statistical analysis to identify
PR:PROJECT_SUMMARY               	metabolites and biochemical alterations related to Aya treatment. Aya doses were
PR:PROJECT_SUMMARY               	applied to the cell cultures considering DMT concentrations of 1, 10 and 20 µM,
PR:PROJECT_SUMMARY               	which are in agreement with non-toxic and toxic DMT threshold assays in primary
PR:PROJECT_SUMMARY               	human astrocyte cells viability.
PR:INSTITUTE                     	University of Campinas
PR:DEPARTMENT                    	Analytical Chemistry Department
PR:LABORATORY                    	Laboratory of Bioanalytics and Integrated Omics (LABIOmics)
PR:LAST_NAME                     	Zandonadi
PR:FIRST_NAME                    	Flavia
PR:ADDRESS                       	Rua Josué de Castro, s/n - Cidade Universitária, Campinas - SP, 13083-970
PR:EMAIL                         	flazando@unicamp.br
PR:PHONE                         	+551935213038
PR:FUNDING_SOURCE                	Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) [grant
PR:FUNDING_SOURCE                	number: 88882.305841/2018-01]. Fundação de Amparo à Pesquisa do Estado de
PR:FUNDING_SOURCE                	São Paulo - FAPESP [grant number: 2018/01525-3] and INCT de Bioanalítica
PR:FUNDING_SOURCE                	[grant numbers: FAPESP 2014/50867-3 and CNPq 465389/2014-7]
PR:DOI                           	http://dx.doi.org/10.21228/M8QT3T
#STUDY
ST:STUDY_TITLE                   	Human primary astrocytes finger- and footprinting metabolomics indicate
ST:STUDY_TITLE                   	biochemical alterations under ayahuasca treatment
ST:STUDY_SUMMARY                 	Ayahuasca (Aya) is a psychotropic Amazonian beverage formulated from the
ST:STUDY_SUMMARY                 	combination of the Banisteriopsis caapi vine and the Psychotria viridis leaves
ST:STUDY_SUMMARY                 	in a water decoction. Aya is legally used in Latin American countries and used
ST:STUDY_SUMMARY                 	in Brazil for religious, cultural, and therapeutic purposes. Its properties
ST:STUDY_SUMMARY                 	constitute a bio-psycho-social-spiritual model involving effects from
ST:STUDY_SUMMARY                 	β-carboline-derived alkaloids, present in the vine, and N,N-dimethyltryptamine
ST:STUDY_SUMMARY                 	(DMT), a tryptamine-derived alkaloid present in the leaves, which act together
ST:STUDY_SUMMARY                 	in the central nervous system (CNS). Few technical-scientific studies have been
ST:STUDY_SUMMARY                 	conducted to understand the effects of this brew in the metabolism. Therefore,
ST:STUDY_SUMMARY                 	this work aims to investigate an in vitro primary astrocyte lineage model by
ST:STUDY_SUMMARY                 	untargeted metabolomics evaluations of two cellular subfractions: secretome and
ST:STUDY_SUMMARY                 	intracellular content after Aya treatment, where DMT and other β-carbolines
ST:STUDY_SUMMARY                 	were previously quantified. Metabolomics analysis was performed by UHPLC-MS/MS,
ST:STUDY_SUMMARY                 	followed by MS-Dial data processing and statistical analysis to identify
ST:STUDY_SUMMARY                 	metabolites and biochemical alterations related to Aya treatment. Aya doses were
ST:STUDY_SUMMARY                 	applied to the cell cultures considering DMT concentrations of 1, 10 and 20 µM,
ST:STUDY_SUMMARY                 	which are in agreement with non-toxic and toxic DMT threshold assays in primary
ST:STUDY_SUMMARY                 	human astrocyte cells viability
ST:INSTITUTE                     	University of Campinas
ST:LAST_NAME                     	Zandonadi
ST:FIRST_NAME                    	Flavia
ST:ADDRESS                       	Rua Josué de Castro, s/n - Cidade Universitária, Campinas - SP, 13083-970
ST:EMAIL                         	flazando@unicamp.br
ST:PHONE                         	+551935213038
ST:SUBMIT_DATE                   	2022-11-11
#SUBJECT
SU:SUBJECT_TYPE                  	Cultured cells
SU:SUBJECT_SPECIES               	Homo sapiens
SU:TAXONOMY_ID                   	9606
SU:AGE_OR_AGE_RANGE              	14-36
SU:CELL_PASSAGE_NUMBER           	4
#SUBJECT_SAMPLE_FACTORS:         	SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Additional sample data
SUBJECT_SAMPLE_FACTORS           	Intracellular	E1_Aya10_intra_HILIC_POS	Treatment:Aya 10 µM DTT-based	RAW_FILE_NAME=E1_Aya10_intra_HILIC_NEG.mzML; RAW_FILE_NAME=E1_Aya10_intra_HILIC_POS.mzML
SUBJECT_SAMPLE_FACTORS           	Secretome (Extracellular)	E1_Aya10_sec_HILIC_POS	Treatment:Aya 10 µM DTT-based	RAW_FILE_NAME=E1_Aya10_sec_HILIC_NEG.mzML; RAW_FILE_NAME=E1_Aya10_sec_HILIC_POS.mzML
SUBJECT_SAMPLE_FACTORS           	Intracellular	E2_Aya10_intra_HILIC_POS	Treatment:Aya 10 µM DTT-based	RAW_FILE_NAME=E2_Aya10_intra_HILIC_NEG.mzML; RAW_FILE_NAME=E2_Aya10_intra_HILIC_POS.mzML
SUBJECT_SAMPLE_FACTORS           	Secretome (Extracellular)	E2_Aya10_sec_HILIC_POS	Treatment:Aya 10 µM DTT-based	RAW_FILE_NAME=E2_Aya10_sec_HILIC_NEG.mzML; RAW_FILE_NAME=E2_Aya10_sec_HILIC_POS.mzML
SUBJECT_SAMPLE_FACTORS           	Intracellular	E3_Aya10_intra_HILIC_POS	Treatment:Aya 10 µM DTT-based	RAW_FILE_NAME=E3_Aya10_intra_HILIC_NEG.mzML; RAW_FILE_NAME=E3_Aya10_intra_HILIC_POS.mzML
SUBJECT_SAMPLE_FACTORS           	Secretome (Extracellular)	E3_Aya10_sec_HILIC_POS	Treatment:Aya 10 µM DTT-based	RAW_FILE_NAME=E3_Aya10_sec_HILIC_NEG.mzML; RAW_FILE_NAME=E3_Aya10_sec_HILIC_POS.mzML
SUBJECT_SAMPLE_FACTORS           	Intracellular	E1_Aya1_intra_HILIC_POS	Treatment:Aya 1 µM DTT-based	RAW_FILE_NAME=E1_Aya1_intra_HILIC_NEG.mzML; RAW_FILE_NAME=E1_Aya1_intra_HILIC_POS.mzML
SUBJECT_SAMPLE_FACTORS           	Secretome (Extracellular)	E1_Aya1_sec_HILIC_POS	Treatment:Aya 1 µM DTT-based	RAW_FILE_NAME=E1_Aya1_sec_HILIC_NEG.mzML; RAW_FILE_NAME=E1_Aya1_sec_HILIC_POS.mzML
SUBJECT_SAMPLE_FACTORS           	Intracellular	E2_Aya1_intra_HILIC_POS	Treatment:Aya 1 µM DTT-based	RAW_FILE_NAME=E2_Aya1_intra_HILIC_NEG.mzML; RAW_FILE_NAME=E2_Aya1_intra_HILIC_POS.mzML
SUBJECT_SAMPLE_FACTORS           	Secretome (Extracellular)	E2_Aya1_sec_HILIC_POS	Treatment:Aya 1 µM DTT-based	RAW_FILE_NAME=E2_Aya1_sec_HILIC_NEG.mzML; RAW_FILE_NAME=E2_Aya1_sec_HILIC_POS.mzML
SUBJECT_SAMPLE_FACTORS           	Intracellular	E3_Aya1_intra_HILIC_POS	Treatment:Aya 1 µM DTT-based	RAW_FILE_NAME=E3_Aya1_intra_HILIC_NEG.mzML; RAW_FILE_NAME=E3_Aya1_intra_HILIC_POS.mzML
SUBJECT_SAMPLE_FACTORS           	Secretome (Extracellular)	E3_Aya1_sec_HILIC_POS	Treatment:Aya 1 µM DTT-based	RAW_FILE_NAME=E3_Aya1_sec_HILIC_NEG.mzML; RAW_FILE_NAME=E3_Aya1_sec_HILIC_POS.mzML
SUBJECT_SAMPLE_FACTORS           	Intracellular	E1_Aya20_intra_HILIC_POS	Treatment:Aya 20 µM DTT-based	RAW_FILE_NAME=E1_Aya20_intra_HILIC_NEG.mzML; RAW_FILE_NAME=E1_Aya20_intra_HILIC_POS.mzML
SUBJECT_SAMPLE_FACTORS           	Secretome (Extracellular)	E1_Aya20_sec_HILIC_POS	Treatment:Aya 20 µM DTT-based	RAW_FILE_NAME=E1_Aya20_sec_HILIC_NEG.mzML; RAW_FILE_NAME=E1_Aya20_sec_HILIC_POS.mzML
SUBJECT_SAMPLE_FACTORS           	Intracellular	E2_Aya20_intra_HILIC_POS	Treatment:Aya 20 µM DTT-based	RAW_FILE_NAME=E2_Aya20_intra_HILIC_NEG.mzML; RAW_FILE_NAME=E2_Aya20_intra_HILIC_POS.mzML
SUBJECT_SAMPLE_FACTORS           	Secretome (Extracellular)	E2_Aya20_sec_HILIC_POS	Treatment:Aya 20 µM DTT-based	RAW_FILE_NAME=E2_Aya20_sec_HILIC_NEG.mzML; RAW_FILE_NAME=E2_Aya20_sec_HILIC_POS.mzML
SUBJECT_SAMPLE_FACTORS           	Intracellular	E3_Aya20_intra_HILIC_POS	Treatment:Aya 20 µM DTT-based	RAW_FILE_NAME=E3_Aya20_intra_HILIC_NEG.mzML; RAW_FILE_NAME=E3_Aya20_intra_HILIC_POS.mzML
SUBJECT_SAMPLE_FACTORS           	Secretome (Extracellular)	E3_Aya20_sec_HILIC_POS	Treatment:Aya 20 µM DTT-based	RAW_FILE_NAME=E3_Aya20_sec_HILIC_NEG.mzML; RAW_FILE_NAME=E3_Aya20_sec_HILIC_POS.mzML
SUBJECT_SAMPLE_FACTORS           	Intracellular	E1_AyaDMSO_intra_HILIC_POS	Treatment:Negative Control	RAW_FILE_NAME=E1_AyaDMSO_intra_HILIC_NEG.mzML; RAW_FILE_NAME=E1_AyaDMSO_intra_HILIC_POS.mzML
SUBJECT_SAMPLE_FACTORS           	Secretome (Extracellular)	E1_AyaDMSO_sec_HILIC_POS	Treatment:Negative Control	RAW_FILE_NAME=E1_AyaDMSO_sec_HILIC_NEG.mzML; RAW_FILE_NAME=E1_AyaDMSO_sec_HILIC_POS.mzML
SUBJECT_SAMPLE_FACTORS           	Intracellular	E2_AyaDMSO_intra_HILIC_POS	Treatment:Negative Control	RAW_FILE_NAME=E2_AyaDMSO_intra_HILIC_NEG.mzML; RAW_FILE_NAME=E2_AyaDMSO_intra_HILIC_POS.mzML
SUBJECT_SAMPLE_FACTORS           	Secretome (Extracellular)	E2_AyaDMSO_sec_HILIC_POS	Treatment:Negative Control	RAW_FILE_NAME=E2_AyaDMSO_sec_HILIC_NEG.mzML; RAW_FILE_NAME=E2_AyaDMSO_sec_HILIC_POS.mzML
SUBJECT_SAMPLE_FACTORS           	Intracellular	E3_AyaDMSO_intra_HILIC_POS	Treatment:Negative Control	RAW_FILE_NAME=E3_AyaDMSO_intra_HILIC_NEG.mzML; RAW_FILE_NAME=E3_AyaDMSO_intra_HILIC_POS.mzML
SUBJECT_SAMPLE_FACTORS           	Secretome (Extracellular)	E3_AyaDMSO_sec_HILIC_POS	Treatment:Negative Control	RAW_FILE_NAME=E3_AyaDMSO_sec_HILIC_NEG.mzML; RAW_FILE_NAME=E3_AyaDMSO_sec_HILIC_POS.mzML
SUBJECT_SAMPLE_FACTORS           	Intracellular	E1_Aya0_intra_HILIC_POS	Treatment:Positive Control	RAW_FILE_NAME=E1_Aya0_intra_HILIC_NEG.mzML; RAW_FILE_NAME=E1_Aya0_intra_HILIC_POS.mzML
SUBJECT_SAMPLE_FACTORS           	Secretome (Extracellular)	E1_Aya0_sec_HILIC_POS	Treatment:Positive Control	RAW_FILE_NAME=E1_Aya0_sec_HILIC_NEG.mzML; RAW_FILE_NAME=E1_Aya0_sec_HILIC_POS.mzML
SUBJECT_SAMPLE_FACTORS           	Intracellular	E2_Aya0_intra_HILIC_POS	Treatment:Positive Control	RAW_FILE_NAME=E2_Aya0_intra_HILIC_NEG.mzML; RAW_FILE_NAME=E2_Aya0_intra_HILIC_POS.mzML
SUBJECT_SAMPLE_FACTORS           	Secretome (Extracellular)	E2_Aya0_sec_HILIC_POS	Treatment:Positive Control	RAW_FILE_NAME=E2_Aya0_sec_HILIC_NEG.mzML; RAW_FILE_NAME=E2_Aya0_sec_HILIC_POS.mzML
SUBJECT_SAMPLE_FACTORS           	Intracellular	E3_Aya0_intra_HILIC_POS	Treatment:Positive Control	RAW_FILE_NAME=E3_Aya0_intra_HILIC_NEG.mzML; RAW_FILE_NAME=E3_Aya0_intra_HILIC_POS.mzML
SUBJECT_SAMPLE_FACTORS           	Secretome (Extracellular)	E3_Aya0_sec_HILIC_POS	Treatment:Positive Control	RAW_FILE_NAME=E3_Aya0_sec_HILIC_NEG.mzML; RAW_FILE_NAME=E3_Aya0_sec_HILIC_POS.mzML
SUBJECT_SAMPLE_FACTORS           	Intracellular	QC_10_intra_HILIC_POS	Treatment:Quality Control	RAW_FILE_NAME=QC_10_intra_HILIC_NEG.mzML; RAW_FILE_NAME=QC_10_intra_HILIC_POS.mzML
SUBJECT_SAMPLE_FACTORS           	Intracellular	QC_1_intra_HILIC_POS	Treatment:Quality Control	RAW_FILE_NAME=QC_1_intra_HILIC_NEG.mzML; RAW_FILE_NAME=QC_1_intra_HILIC_POS.mzML
SUBJECT_SAMPLE_FACTORS           	Intracellular	QC_20_intra_HILIC_POS	Treatment:Quality Control	RAW_FILE_NAME=QC_20_intra_HILIC_NEG.mzML; RAW_FILE_NAME=QC_20_intra_HILIC_POS.mzML
SUBJECT_SAMPLE_FACTORS           	Secretome (Extracellular)	QC_10_sec_HILIC_POS	Treatment:Quality Control Aya 10 µM DTT-based group	RAW_FILE_NAME=QC_10_sec_HILIC_NEG.mzML; RAW_FILE_NAME=QC_10_sec_HILIC_POS.mzML
SUBJECT_SAMPLE_FACTORS           	Secretome (Extracellular)	QC_1_sec_HILIC_POS	Treatment:Quality Control Aya 1 µM DTT-based group	RAW_FILE_NAME=QC_1_sec_HILIC_NEG.mzML; RAW_FILE_NAME=QC_1_sec_HILIC_POS.mzML
SUBJECT_SAMPLE_FACTORS           	Secretome (Extracellular)	QC_20_sec_HILIC_POS	Treatment:Quality Control Aya 20 µM DTT-based group	RAW_FILE_NAME=QC_20_sec_HILIC_NEG.mzML; RAW_FILE_NAME=QC_20_sec_HILIC_POS.mzML
#COLLECTION
CO:COLLECTION_SUMMARY            	The astrocyte cells were obtained in collaboration with the Nervous Regeneration
CO:COLLECTION_SUMMARY            	Laboratory (Prof. Dr. Alexandre Oliveira), Institute of Biology, UNICAMP,
CO:COLLECTION_SUMMARY            	Brazil.
CO:SAMPLE_TYPE                   	Astrocytes
CO:STORAGE_CONDITIONS            	-80℃
#TREATMENT
TR:TREATMENT_SUMMARY             	Aya doses were applied to the cell cultures considering DMT concentrations of 1,
TR:TREATMENT_SUMMARY             	10 and 20 µM, which are in agreement with non-toxic and toxic DMT threshold
TR:TREATMENT_SUMMARY             	assays in primary human astrocyte cells viability.secretome (extracellular
TR:TREATMENT_SUMMARY             	fraction) were collected, centrifuged under 500 x g at room temperature (about
TR:TREATMENT_SUMMARY             	27 ºC) for 5 min, stored and conditioned at -80 °C until the time of
TR:TREATMENT_SUMMARY             	metabolomics sample preparation. In parallel, to obtain the content of
TR:TREATMENT_SUMMARY             	intracellular metabolites, cell monolayers were washed three times with 5 mL of
TR:TREATMENT_SUMMARY             	ice-cold PBS to remove any excess of secretome. Subsequently, a mechanical
TR:TREATMENT_SUMMARY             	detachment was performed for removal, followed by chemical rupture of the cell
TR:TREATMENT_SUMMARY             	wall in 1.2 mL of ice-cold 80 % (v/v) methanol in PBS solution, to obtain an
TR:TREATMENT_SUMMARY             	intracellular metabolite suspension. The intracellular fractions, in methanol
TR:TREATMENT_SUMMARY             	solution, were stored and conditioned at -80 °C.
#SAMPLEPREP
SP:SAMPLEPREP_SUMMARY            	For metabolomics analysis, after being defrosted, the secretome samples volumes
SP:SAMPLEPREP_SUMMARY            	were reduced to 500 μL by centrifugation in an Amicon® Ultra-2 mL (Millipore)
SP:SAMPLEPREP_SUMMARY            	filter, according to the manufacturer specifications. Then, quality control (QC)
SP:SAMPLEPREP_SUMMARY            	samples were prepared previously to the protein precipitation. Ice-cold 80 %
SP:SAMPLEPREP_SUMMARY            	(v/v) methanol was added to the concentrated secretome, kept at -80 °C, as well
SP:SAMPLEPREP_SUMMARY            	as the intracellular fraction, for the protein precipitation step for 24 h.
SP:SAMPLEPREP_SUMMARY            	After that period of time, all samples (extra and intracellular metabolite
SP:SAMPLEPREP_SUMMARY            	suspensions) were centrifuged at 16,000 x g at 4 °C for 15 min, the
SP:SAMPLEPREP_SUMMARY            	supernatants filtered in a 0.22 μm syringe filter, and dried in a vacuum
SP:SAMPLEPREP_SUMMARY            	concentrator. For each cellular fraction, 12 samples were included in the
SP:SAMPLEPREP_SUMMARY            	analysis, where three (3) experimental samples for each group were identified as
SP:SAMPLEPREP_SUMMARY            	Aya0, Aya1, Aya10, or Aya20, corresponding to DMT doses in µmol L-1, and five
SP:SAMPLEPREP_SUMMARY            	(5) for QC samples, being four (4) QC related to dose-treatments and one (1) to
SP:SAMPLEPREP_SUMMARY            	all treatment groups.
SP:SAMPLEPREP_PROTOCOL_FILENAME  	Samplepreparation_zandonadi22.pdf
SP:PROCESSING_STORAGE_CONDITIONS 	-80℃
SP:EXTRACT_STORAGE               	On ice
#CHROMATOGRAPHY
CH:CHROMATOGRAPHY_SUMMARY        	Acquity BEH Amide column (2.1 mm x 150 mm, 1.7 μm particle size, Waters Corp.)
CH:CHROMATOGRAPHY_SUMMARY        	for hydrophilic interaction liquid chromatography (HILIC). The sample injection
CH:CHROMATOGRAPHY_SUMMARY        	volume was set to 5 μL and the column temperature was kept at 45 °C.
CH:CHROMATOGRAPHY_SUMMARY        	Separation was performed at a 0.4 mL min-1 flow rate under a gradient program in
CH:CHROMATOGRAPHY_SUMMARY        	which the mobile phases consisted of: (A) 10 mmol L-1 ammonium acetate in ACN:
CH:CHROMATOGRAPHY_SUMMARY        	water (95: 5) and (B) 10 mmol L-1 ammonium acetate in ACN: water (50:50). The
CH:CHROMATOGRAPHY_SUMMARY        	gradient started with 1% B for 1 min, increasing to 100% B for 9 min and
CH:CHROMATOGRAPHY_SUMMARY        	subsequently returning to 1% B in 0.1 min. Over the next 3.9 min, the column was
CH:CHROMATOGRAPHY_SUMMARY        	re-equilibrated before the next injection. Total execution time was 14 min.
CH:METHODS_FILENAME              	UntargetHilicmethodXEVO_zandonadi22.pdf
CH:INSTRUMENT_NAME               	Waters Acquity
CH:COLUMN_NAME                   	Waters Acquity BEH HILIC (150 x 2.1mm, 1.7um)
CH:COLUMN_TEMPERATURE            	45 °C
CH:FLOW_GRADIENT                 	1 % B for 1 min, increasing to 100% B for 9 min and subsequently returning to 1
CH:FLOW_GRADIENT                 	% B in 0.1 min. Over the next 3.9 min, the column was re-equilibrated before the
CH:FLOW_GRADIENT                 	next injection. Total execution time was 14 min
CH:FLOW_RATE                     	0.4 mL min-1
CH:SOLVENT_A                     	95% acetonitrile/5% water; 10 mM ammonium acetate
CH:SOLVENT_B                     	50% acetonitrile/50% water; 10mM ammonium acetate
CH:CHROMATOGRAPHY_TYPE           	HILIC
#ANALYSIS
AN:ANALYSIS_TYPE                 	MS
#MS
MS:INSTRUMENT_NAME               	Waters Xevo-G2-XS
MS:INSTRUMENT_TYPE               	QTOF
MS:MS_TYPE                       	ESI
MS:MS_COMMENTS                   	Detection was performed using a XEVO-G2XS quadrupole time-of-flight (QTOF) mass
MS:MS_COMMENTS                   	spectrometer (Waters Corp., Manchester, UK) equipped with an electrospray
MS:MS_COMMENTS                   	ionization (ESI) source. Data were collected in both positive and negative
MS:MS_COMMENTS                   	ionization modes, separately. The acquisition was performed using
MS:MS_COMMENTS                   	data-independent analysis (MSE), with the following parameters: capillary
MS:MS_COMMENTS                   	voltage +3.5 kV (positive mode) and -2.5 kV (negative mode), sample cone voltage
MS:MS_COMMENTS                   	at 40 kV, source temperature at 140 °C, desolvation temperature of 550 °C,
MS:MS_COMMENTS                   	desolvation gas flow of 900 L h−1, and cone gas flow of 10 L h−1 (positive
MS:MS_COMMENTS                   	mode) or 50 L h−1 (negative mode). MS data were acquired in the centroid mode
MS:MS_COMMENTS                   	from m/z range of 50–1200 Da, using the acquisition rate of 0.5 s per scan.
MS:MS_COMMENTS                   	The MSE analysis was operated at 20 V for low collision energy and 50 V for high
MS:MS_COMMENTS                   	collision energy. During MS analysis, a leucine enkephalin (Waters®, molecular
MS:MS_COMMENTS                   	mass = 555.62; 200 pg μL-1 in 1:1 ACN: H2O) was continuously infused into MS at
MS:MS_COMMENTS                   	a flow rate of 30 µL min-1 and the ions [M-H]- = 554.26 and [M+H]+ = 556.27
MS:MS_COMMENTS                   	were used as lock mass for accurate mass measurement. Data acquisition was
MS:MS_COMMENTS                   	controlled by MassLynx V4.2 (Waters®). Calibration was performed prior to
MS:MS_COMMENTS                   	sample analysis via infusion of 0.5 mmol L-1 sodium formate solution, which was
MS:MS_COMMENTS                   	used for calibration procedures, both in positive and negative mode analysis.
MS:MS_COMMENTS                   	Samples were randomly analyzed and QC samples composed of pooled samples after
MS:MS_COMMENTS                   	resuspension were injected every 8 injections. Data processing and metabolite
MS:MS_COMMENTS                   	identification. All MSE datasets were converted from the vendor-specific file
MS:MS_COMMENTS                   	format (.raw) into the Analysis Base File format (.abf) using the freely
MS:MS_COMMENTS                   	available Reifycs ABF converter
MS:MS_COMMENTS                   	(https://www.reifycs.com/AbfConverter/index.html). After conversion, the MS-DIAL
MS:MS_COMMENTS                   	software (version 5.0) was used for feature detection, spectral deconvolution,
MS:MS_COMMENTS                   	peak identification, and alignment between samples. MassBank databases were used
MS:MS_COMMENTS                   	for identification from MS-DIAL metabolomics MSP spectral kit containing ESI-MS
MS:MS_COMMENTS                   	and MS/MS (positive; 8,068 and negative; 4,782 compounds).
MS:MS_COMMENTS                   	(http://prime.psc.riken.jp/compms/msdial/main.html). The parameters for MS-DIAL
MS:MS_COMMENTS                   	were optimized based on their based on developer instructions. The UHPLC-MS raw
MS:MS_COMMENTS                   	data were processed with MS-DIAL version 4.8 [30]. Automatic feature detection
MS:MS_COMMENTS                   	was performed between 0.3 and 13 min for mass signal extraction between 100 and
MS:MS_COMMENTS                   	1500 Da in positive and negative modes. MS1 and MS2 tolerance were set to 0.01
MS:MS_COMMENTS                   	and 0.4 Da, respectively, in profile mode. For identification, it was used the
MS:MS_COMMENTS                   	default of accurate mass tolerance 0.01 Da for MS1 and 0.05 Da for MS2, and
MS:MS_COMMENTS                   	identification score cut off 80 %. For the potential adducts, [M+H]+, [M+Na]+,
MS:MS_COMMENTS                   	[M+K]+, [M+NH4]+, [2M+H]+, [M+2H]2+, [M+2Na]2+, [M+2K]2+, [M+H+Na]2+, [M+H+K]2+,
MS:MS_COMMENTS                   	[M+H+NH4]2+, [M+2Na-H]+, [M+2K-H]+, [M+H-H2O]+, and [M+H-2H2O]+ were considered
MS:MS_COMMENTS                   	for positively charged adducts, while [M-H]-, [M+Cl]-, [M-H-H2O]-, [2M-H]-,
MS:MS_COMMENTS                   	[M-2H]2-, [M+Na-2H]-, [M+K-2H]-, and [M+FA-H]- were considered for negatively
MS:MS_COMMENTS                   	charged. To generate matrix files for downstream statistical analysis, the
MS:MS_COMMENTS                   	aligned results were exported by selecting the area raw data matrix, that were
MS:MS_COMMENTS                   	restructured by applying a manual exclusion filter for unknown metabolites, and
MS:MS_COMMENTS                   	the negative and positive files were combined for statistical analysis in
MS:MS_COMMENTS                   	MetaboAnalyst 5.0 [31].
MS:ION_MODE                      	NEGATIVE
MS:MS_RESULTS_FILE               	ST002401_AN003913_Results.txt	UNITS:Abundance	Has m/z:Yes	Has RT:Yes	RT units:Minutes
#END