Summary of Study ST002401

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001547. The data can be accessed directly via it's Project DOI: 10.21228/M8QT3T This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

Study ID	ST002401
Study Title	Human primary astrocytes finger- and footprinting metabolomics indicate biochemical alterations under ayahuasca treatment
Study Summary	Ayahuasca (Aya) is a psychotropic Amazonian beverage formulated from the combination of the Banisteriopsis caapi vine and the Psychotria viridis leaves in a water decoction. Aya is legally used in Latin American countries and used in Brazil for religious, cultural, and therapeutic purposes. Its properties constitute a bio-psycho-social-spiritual model involving effects from β-carboline-derived alkaloids, present in the vine, and N,N-dimethyltryptamine (DMT), a tryptamine-derived alkaloid present in the leaves, which act together in the central nervous system (CNS). Few technical-scientific studies have been conducted to understand the effects of this brew in the metabolism. Therefore, this work aims to investigate an in vitro primary astrocyte lineage model by untargeted metabolomics evaluations of two cellular subfractions: secretome and intracellular content after Aya treatment, where DMT and other β-carbolines were previously quantified. Metabolomics analysis was performed by UHPLC-MS/MS, followed by MS-Dial data processing and statistical analysis to identify metabolites and biochemical alterations related to Aya treatment. Aya doses were applied to the cell cultures considering DMT concentrations of 1, 10 and 20 µM, which are in agreement with non-toxic and toxic DMT threshold assays in primary human astrocyte cells viability
Institute	University of Campinas
Last Name	Zandonadi
First Name	Flavia
Address	Rua Josué de Castro, s/n - Cidade Universitária, Campinas - SP, 13083-970
Email	flazando@unicamp.br
Phone	+551935213038
Submit Date	2022-11-11
Raw Data Available	Yes
Raw Data File Type(s)	mzML
Analysis Type Detail	LC-MS
Release Date	2024-01-16
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR001547
Project DOI:	doi: 10.21228/M8QT3T
Project Title:	Human primary astrocytes finger- and footprinting metabolomics indicate biochemical alterations under ayahuasca treatment
Project Type:	Untargetmetabomics- hilic
Project Summary:	Ayahuasca (Aya) is a psychotropic Amazonian beverage formulated from the combination of the Banisteriopsis caapi vine and the Psychotria viridis leaves in a water decoction. Aya is legally used in Latin American countries and used in Brazil for religious, cultural, and therapeutic purposes. Its properties constitute a bio-psycho-social-spiritual model involving effects from ß-carboline-derived alkaloids, present in the vine, and N,N-dimethyltryptamine (DMT), a tryptamine-derived alkaloid present in the leaves, which act together in the central nervous system (CNS). Few technical-scientific studies have been conducted to understand the effects of this brew in the metabolism. Therefore, this work aims to investigate an in vitro primary astrocyte lineage model by untargeted metabolomics evaluations of two cellular subfractions: secretome and intracellular content after Aya treatment, where DMT and other ß-carbolines were previously quantified. Metabolomics analysis was performed by UHPLC-MS/MS, followed by MS-Dial data processing and statistical analysis to identify metabolites and biochemical alterations related to Aya treatment. Aya doses were applied to the cell cultures considering DMT concentrations of 1, 10 and 20 µM, which are in agreement with non-toxic and toxic DMT threshold assays in primary human astrocyte cells viability.
Institute:	University of Campinas
Department:	Analytical Chemistry Department
Laboratory:	Laboratory of Bioanalytics and Integrated Omics (LABIOmics)
Last Name:	Zandonadi
First Name:	Flavia
Address:	Rua Josué de Castro, s/n - Cidade Universitária, Campinas - SP, 13083-970
Email:	flazando@unicamp.br
Phone:	+551935213038
Funding Source:	Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) [grant number: 88882.305841/2018-01]. Fundação de Amparo à Pesquisa do Estado de São Paulo - FAPESP [grant number: 2018/01525-3] and INCT de Bioanalítica [grant numbers: FAPESP 2014/50867-3 and CNPq 465389/2014-7]

Subject:

Subject ID:	SU002490
Subject Type:	Cultured cells
Subject Species:	Homo sapiens
Taxonomy ID:	9606
Age Or Age Range:	14-36
Cell Passage Number:	4

Factors:

Subject type: Cultured cells; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id	local_sample_id	Treatment
SA239114	E3_Aya10_sec_HILIC_POS	Aya 10 µM DTT-based
SA239115	E3_Aya10_intra_HILIC_POS	Aya 10 µM DTT-based
SA239116	E2_Aya10_sec_HILIC_POS	Aya 10 µM DTT-based
SA239117	E1_Aya10_sec_HILIC_POS	Aya 10 µM DTT-based
SA239118	E1_Aya10_intra_HILIC_POS	Aya 10 µM DTT-based
SA239119	E2_Aya10_intra_HILIC_POS	Aya 10 µM DTT-based
SA239108	E3_Aya1_sec_HILIC_POS	Aya 1 µM DTT-based
SA239109	E2_Aya1_intra_HILIC_POS	Aya 1 µM DTT-based
SA239110	E1_Aya1_sec_HILIC_POS	Aya 1 µM DTT-based
SA239111	E2_Aya1_sec_HILIC_POS	Aya 1 µM DTT-based
SA239112	E3_Aya1_intra_HILIC_POS	Aya 1 µM DTT-based
SA239113	E1_Aya1_intra_HILIC_POS	Aya 1 µM DTT-based
SA239120	E1_Aya20_intra_HILIC_POS	Aya 20 µM DTT-based
SA239121	E2_Aya20_intra_HILIC_POS	Aya 20 µM DTT-based
SA239122	E1_Aya20_sec_HILIC_POS	Aya 20 µM DTT-based
SA239123	E3_Aya20_sec_HILIC_POS	Aya 20 µM DTT-based
SA239124	E3_Aya20_intra_HILIC_POS	Aya 20 µM DTT-based
SA239125	E2_Aya20_sec_HILIC_POS	Aya 20 µM DTT-based
SA239126	E3_AyaDMSO_intra_HILIC_POS	Negative Control
SA239127	E2_AyaDMSO_intra_HILIC_POS	Negative Control
SA239128	E1_AyaDMSO_sec_HILIC_POS	Negative Control
SA239129	E3_AyaDMSO_sec_HILIC_POS	Negative Control
SA239130	E2_AyaDMSO_sec_HILIC_POS	Negative Control
SA239131	E1_AyaDMSO_intra_HILIC_POS	Negative Control
SA239132	E1_Aya0_intra_HILIC_POS	Positive Control
SA239133	E3_Aya0_sec_HILIC_POS	Positive Control
SA239134	E3_Aya0_intra_HILIC_POS	Positive Control
SA239135	E2_Aya0_sec_HILIC_POS	Positive Control
SA239136	E2_Aya0_intra_HILIC_POS	Positive Control
SA239137	E1_Aya0_sec_HILIC_POS	Positive Control
SA239138	QC_1_intra_HILIC_POS	Quality Control
SA239139	QC_20_intra_HILIC_POS	Quality Control
SA239140	QC_10_intra_HILIC_POS	Quality Control
SA239142	QC_10_sec_HILIC_POS	Quality Control Aya 10 µM DTT-based group
SA239141	QC_1_sec_HILIC_POS	Quality Control Aya 1 µM DTT-based group
SA239143	QC_20_sec_HILIC_POS	Quality Control Aya 20 µM DTT-based group

Showing results 1 to 36 of 36

Showing results 1 to 36 of 36

Collection:

Collection ID:	CO002483
Collection Summary:	The astrocyte cells were obtained in collaboration with the Nervous Regeneration Laboratory (Prof. Dr. Alexandre Oliveira), Institute of Biology, UNICAMP, Brazil.
Sample Type:	Astrocytes
Storage Conditions:	-80℃

Treatment:

Treatment ID:	TR002502
Treatment Summary:	Aya doses were applied to the cell cultures considering DMT concentrations of 1, 10 and 20 µM, which are in agreement with non-toxic and toxic DMT threshold assays in primary human astrocyte cells viability.secretome (extracellular fraction) were collected, centrifuged under 500 x g at room temperature (about 27 ºC) for 5 min, stored and conditioned at -80 °C until the time of metabolomics sample preparation. In parallel, to obtain the content of intracellular metabolites, cell monolayers were washed three times with 5 mL of ice-cold PBS to remove any excess of secretome. Subsequently, a mechanical detachment was performed for removal, followed by chemical rupture of the cell wall in 1.2 mL of ice-cold 80 % (v/v) methanol in PBS solution, to obtain an intracellular metabolite suspension. The intracellular fractions, in methanol solution, were stored and conditioned at -80 °C.

Treatment ID:

TR002502

Treatment Summary:

Aya doses were applied to the cell cultures considering DMT concentrations of 1, 10 and 20 µM, which are in agreement with non-toxic and toxic DMT threshold assays in primary human astrocyte cells viability.secretome (extracellular fraction) were collected, centrifuged under 500 x g at room temperature (about 27 ºC) for 5 min, stored and conditioned at -80 °C until the time of metabolomics sample preparation. In parallel, to obtain the content of intracellular metabolites, cell monolayers were washed three times with 5 mL of ice-cold PBS to remove any excess of secretome. Subsequently, a mechanical detachment was performed for removal, followed by chemical rupture of the cell wall in 1.2 mL of ice-cold 80 % (v/v) methanol in PBS solution, to obtain an intracellular metabolite suspension. The intracellular fractions, in methanol solution, were stored and conditioned at -80 °C.

Sample Preparation:

Sampleprep ID:	SP002496
Sampleprep Summary:	For metabolomics analysis, after being defrosted, the secretome samples volumes were reduced to 500 μL by centrifugation in an Amicon® Ultra-2 mL (Millipore) filter, according to the manufacturer specifications. Then, quality control (QC) samples were prepared previously to the protein precipitation. Ice-cold 80 % (v/v) methanol was added to the concentrated secretome, kept at -80 °C, as well as the intracellular fraction, for the protein precipitation step for 24 h. After that period of time, all samples (extra and intracellular metabolite suspensions) were centrifuged at 16,000 x g at 4 °C for 15 min, the supernatants filtered in a 0.22 μm syringe filter, and dried in a vacuum concentrator. For each cellular fraction, 12 samples were included in the analysis, where three (3) experimental samples for each group were identified as Aya0, Aya1, Aya10, or Aya20, corresponding to DMT doses in µmol L-1, and five (5) for QC samples, being four (4) QC related to dose-treatments and one (1) to all treatment groups.
Sampleprep Protocol Filename:	Samplepreparation_zandonadi22.pdf
Processing Storage Conditions:	-80℃
Extract Storage:	On ice

Combined analysis:

Analysis ID	AN003912	AN003913
Analysis type	MS	MS
Chromatography type	HILIC	HILIC
Chromatography system	Waters Acquity	Waters Acquity
Column	Waters Acquity BEH HILIC (150 x 2.1mm, 1.7um)	Waters Acquity BEH HILIC (150 x 2.1mm, 1.7um)
MS Type	ESI	ESI
MS instrument type	QTOF	QTOF
MS instrument name	Waters Xevo-G2-XS	Waters Xevo-G2-XS
Ion Mode	POSITIVE	NEGATIVE
Units	TIC	Abundance

Chromatography:

Chromatography ID:	CH002895
Chromatography Summary:	Acquity BEH Amide column (2.1 mm x 150 mm, 1.7 μm particle size, Waters Corp.) for hydrophilic interaction liquid chromatography (HILIC). The sample injection volume was set to 5 μL and the column temperature was kept at 45 °C. Separation was performed at a 0.4 mL min-1 flow rate under a gradient program in which the mobile phases consisted of: (A) 10 mmol L-1 ammonium acetate in ACN: water (95: 5) and (B) 10 mmol L-1 ammonium acetate in ACN: water (50:50). The gradient started with 1% B for 1 min, increasing to 100% B for 9 min and subsequently returning to 1% B in 0.1 min. Over the next 3.9 min, the column was re-equilibrated before the next injection. Total execution time was 14 min.
Methods Filename:	UntargetHilicmethodXEVO_zandonadi22.pdf
Instrument Name:	Waters Acquity
Column Name:	Waters Acquity BEH HILIC (150 x 2.1mm, 1.7um)
Column Temperature:	45 °C
Flow Gradient:	1 % B for 1 min, increasing to 100% B for 9 min and subsequently returning to 1 % B in 0.1 min. Over the next 3.9 min, the column was re-equilibrated before the next injection. Total execution time was 14 min
Flow Rate:	0.4 mL min-1
Solvent A:	95% acetonitrile/5% water; 10 mM ammonium acetate
Solvent B:	50% acetonitrile/50% water; 10mM ammonium acetate
Chromatography Type:	HILIC

MS:

MS ID:	MS003651
Analysis ID:	AN003912
Instrument Name:	Waters Xevo-G2-XS
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	Detection was performed using a XEVO-G2XS quadrupole time-of-flight (QTOF) mass spectrometer (Waters Corp., Manchester, UK) equipped with an electrospray ionization (ESI) source. Data were collected in both positive and negative ionization modes, separately. The acquisition was performed using data-independent analysis (MSE), with the following parameters: capillary voltage +3.5 kV (positive mode) and -2.5 kV (negative mode), sample cone voltage at 40 kV, source temperature at 140 °C, desolvation temperature of 550 °C, desolvation gas flow of 900 L h−1, and cone gas flow of 10 L h−1 (positive mode) or 50 L h−1 (negative mode). MS data were acquired in the centroid mode from m/z range of 50–1200 Da, using the acquisition rate of 0.5 s per scan. The MSE analysis was operated at 20 V for low collision energy and 50 V for high collision energy. During MS analysis, a leucine enkephalin (Waters®, molecular mass = 555.62; 200 pg μL-1 in 1:1 ACN: H2O) was continuously infused into MS at a flow rate of 30 µL min-1 and the ions [M-H]- = 554.26 and [M+H]+ = 556.27 were used as lock mass for accurate mass measurement. Data acquisition was controlled by MassLynx V4.2 (Waters®). Calibration was performed prior to sample analysis via infusion of 0.5 mmol L-1 sodium formate solution, which was used for calibration procedures, both in positive and negative mode analysis. Samples were randomly analyzed and QC samples composed of pooled samples after resuspension were injected every 8 injections. Data processing and metabolite identification. All MSE datasets were converted from the vendor-specific file format (.raw) into the Analysis Base File format (.abf) using the freely available Reifycs ABF converter (https://www.reifycs.com/AbfConverter/index.html). After conversion, the MS-DIAL software (version 5.0) was used for feature detection, spectral deconvolution, peak identification, and alignment between samples. MassBank databases were used for identification from MS-DIAL metabolomics MSP spectral kit containing ESI-MS and MS/MS (positive; 8,068 and negative; 4,782 compounds). (http://prime.psc.riken.jp/compms/msdial/main.html). The parameters for MS-DIAL were optimized based on their based on developer instructions. The UHPLC-MS raw data were processed with MS-DIAL version 4.8 [30]. Automatic feature detection was performed between 0.3 and 13 min for mass signal extraction between 100 and 1500 Da in positive and negative modes. MS1 and MS2 tolerance were set to 0.01 and 0.4 Da, respectively, in profile mode. For identification, it was used the default of accurate mass tolerance 0.01 Da for MS1 and 0.05 Da for MS2, and identification score cut off 80 %. For the potential adducts, [M+H]+, [M+Na]+, [M+K]+, [M+NH4]+, [2M+H]+, [M+2H]2+, [M+2Na]2+, [M+2K]2+, [M+H+Na]2+, [M+H+K]2+, [M+H+NH4]2+, [M+2Na-H]+, [M+2K-H]+, [M+H-H2O]+, and [M+H-2H2O]+ were considered for positively charged adducts, while [M-H]-, [M+Cl]-, [M-H-H2O]-, [2M-H]-, [M-2H]2-, [M+Na-2H]-, [M+K-2H]-, and [M+FA-H]- were considered for negatively charged. To generate matrix files for downstream statistical analysis, the aligned results were exported by selecting the area raw data matrix, that were restructured by applying a manual exclusion filter for unknown metabolites, and the negative and positive files were combined for statistical analysis in MetaboAnalyst 5.0 [31].
Ion Mode:	POSITIVE

MS ID:	MS003652
Analysis ID:	AN003913
Instrument Name:	Waters Xevo-G2-XS
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	Detection was performed using a XEVO-G2XS quadrupole time-of-flight (QTOF) mass spectrometer (Waters Corp., Manchester, UK) equipped with an electrospray ionization (ESI) source. Data were collected in both positive and negative ionization modes, separately. The acquisition was performed using data-independent analysis (MSE), with the following parameters: capillary voltage +3.5 kV (positive mode) and -2.5 kV (negative mode), sample cone voltage at 40 kV, source temperature at 140 °C, desolvation temperature of 550 °C, desolvation gas flow of 900 L h−1, and cone gas flow of 10 L h−1 (positive mode) or 50 L h−1 (negative mode). MS data were acquired in the centroid mode from m/z range of 50–1200 Da, using the acquisition rate of 0.5 s per scan. The MSE analysis was operated at 20 V for low collision energy and 50 V for high collision energy. During MS analysis, a leucine enkephalin (Waters®, molecular mass = 555.62; 200 pg μL-1 in 1:1 ACN: H2O) was continuously infused into MS at a flow rate of 30 µL min-1 and the ions [M-H]- = 554.26 and [M+H]+ = 556.27 were used as lock mass for accurate mass measurement. Data acquisition was controlled by MassLynx V4.2 (Waters®). Calibration was performed prior to sample analysis via infusion of 0.5 mmol L-1 sodium formate solution, which was used for calibration procedures, both in positive and negative mode analysis. Samples were randomly analyzed and QC samples composed of pooled samples after resuspension were injected every 8 injections. Data processing and metabolite identification. All MSE datasets were converted from the vendor-specific file format (.raw) into the Analysis Base File format (.abf) using the freely available Reifycs ABF converter (https://www.reifycs.com/AbfConverter/index.html). After conversion, the MS-DIAL software (version 5.0) was used for feature detection, spectral deconvolution, peak identification, and alignment between samples. MassBank databases were used for identification from MS-DIAL metabolomics MSP spectral kit containing ESI-MS and MS/MS (positive; 8,068 and negative; 4,782 compounds). (http://prime.psc.riken.jp/compms/msdial/main.html). The parameters for MS-DIAL were optimized based on their based on developer instructions. The UHPLC-MS raw data were processed with MS-DIAL version 4.8 [30]. Automatic feature detection was performed between 0.3 and 13 min for mass signal extraction between 100 and 1500 Da in positive and negative modes. MS1 and MS2 tolerance were set to 0.01 and 0.4 Da, respectively, in profile mode. For identification, it was used the default of accurate mass tolerance 0.01 Da for MS1 and 0.05 Da for MS2, and identification score cut off 80 %. For the potential adducts, [M+H]+, [M+Na]+, [M+K]+, [M+NH4]+, [2M+H]+, [M+2H]2+, [M+2Na]2+, [M+2K]2+, [M+H+Na]2+, [M+H+K]2+, [M+H+NH4]2+, [M+2Na-H]+, [M+2K-H]+, [M+H-H2O]+, and [M+H-2H2O]+ were considered for positively charged adducts, while [M-H]-, [M+Cl]-, [M-H-H2O]-, [2M-H]-, [M-2H]2-, [M+Na-2H]-, [M+K-2H]-, and [M+FA-H]- were considered for negatively charged. To generate matrix files for downstream statistical analysis, the aligned results were exported by selecting the area raw data matrix, that were restructured by applying a manual exclusion filter for unknown metabolites, and the negative and positive files were combined for statistical analysis in MetaboAnalyst 5.0 [31].
Ion Mode:	NEGATIVE