Summary of Study ST002307

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 PR001479. The data can be accessed directly via it's Project DOI: 10.21228/M8JM7Q 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	ST002307
Study Title	Metabolomics profiling of the secondary fractions 4 of bacterial culture supernatants.
Study Type	MS quantitative analysis
Study Summary	Separate LC-MS runs of AhR-active and inactive HPLC fractions of bacterial culture supernatants were used to identify differentially expressed compounds in the active bacterial strains.
Institute	University of Connecticut
Department	Chemistry
Laboratory	Yao Lab
Last Name	Tian
First Name	Huidi
Address	55 N. Eagleville Road, Unit 3060, Storrs CT 06269
Email	huidi.tian@uconn.edu
Phone	8606341143
Submit Date	2022-10-03
Raw Data Available	Yes
Raw Data File Type(s)	mzML
Analysis Type Detail	LC-MS
Release Date	2024-10-04
Release Version	1

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Project:

Project ID:	PR001479
Project DOI:	doi: 10.21228/M8JM7Q
Project Title:	Metabolomics Discovery of Aryl Hydrocarbon Receptor Activating Metabolites from the Human Microbiota
Project Type:	Bacteria supernatant
Project Summary:	The aryl hydrocarbon receptor (AhR) is a transcription factor that regulates gene expression upon activation by small molecules. It plays a significant role in the innate immune recognition of bacteria and response to exogenous molecules in the human host. By stimulating host immune cells with microbiota metabolites, the AhR signaling enables microbiota-dependent induction, training, and function of the host immune system. AhR is a potential target for developing therapeutics to treat myalgic encephalomyelitis/chronic fatigue syndrome (ME/CFS), cancer, and aging-related diseases. A variety of bioactive molecules can act as AhR agonists, including the metabolites and derivatives of indole and tryptophan. However, given the ligand-binding versatility of AhR, new methods are needed to discover novel AhR agonists. Herein, we report an analytical workflow for the deep discovery of AhR agonists from the secreted metabolome of bacteria. It is efficient to involve the activity measurement in the early stages of discovering new AhR ligands. Moreover, utilization of the AhR-chaperone complex in live cells by the AhR activity assay can mitigate the need for purifying the complex and allows for the deep discovery of low-concentration activators.
Institute:	University of Connecticut
Department:	Chemistry
Laboratory:	Yao Lab
Last Name:	Tian
First Name:	Huidi
Address:	55 N. Eagleville Road, Unit 3060, Storrs CT 06269
Email:	huidi.tian@uconn.edu
Phone:	8606341143
Funding Source:	NIH

Subject:

Subject ID:	SU002393
Subject Type:	Bacteria
Subject Species:	Bacillus megaterium; Enterococcus faecium
Taxonomy ID:	1404;1352

Factors:

Subject type: Bacteria; Subject species: Bacillus megaterium; Enterococcus faecium (Factor headings shown in green)

mb_sample_id	local_sample_id	Bacteria	AhR_activity
SA226728	MSR_F4_2-1	Bacillus megaterium	Active
SA226729	MSR_F4_2-2	Bacillus megaterium	Active
SA226730	MSR_F4_3-2	Bacillus megaterium	Active
SA226731	MSR_F4_1-2	Bacillus megaterium	Active
SA226732	MSR_F4_3-1	Bacillus megaterium	Active
SA226733	MSR_F4_1-1	Bacillus megaterium	Active
SA226734	SAM_F4_2-1	Enterococcus faecium	Inactive
SA226735	SAM_F4_1-2	Enterococcus faecium	Inactive
SA226736	SAM_F4_2-2	Enterococcus faecium	Inactive
SA226737	SAM_F4_3-1	Enterococcus faecium	Inactive
SA226738	SAM_F4_3-2	Enterococcus faecium	Inactive
SA226739	SAM_F4_1-1	Enterococcus faecium	Inactive

Showing results 1 to 12 of 12

Showing results 1 to 12 of 12

Collection:

Collection ID:	CO002386
Collection Summary:	Various strains of bacteria were cultured from the microbiota of either healthy volunteers or ME/CFS patients at Jackson Laboratory for Genomic Medicine (Farmington, CT). The derived bacteria were cultured overnight in tryptic soy broth (TSB) media. Cells were pelleted, and the supernatant was filtered through a 0.22-micron filter to prepare cell-free culture supernatants. Supernatants were stored at -80 ℃.
Sample Type:	Bacterial cells
Storage Conditions:	-80℃

Treatment:

Treatment ID:	TR002405
Treatment Summary:	200 µL of MeOH was first pipetted into each column to condition the sorbent. Columns were then centrifuged for 2 minutes at 110 xg. The procedure was repeated three times. Second, 200 µL of water was pipetted into each column to equilibrate the sorbent. Columns were then centrifuged for 2 minutes at 110 xg, and the procedure was repeated three times. Third, the samples were loaded into each prepared column. Each loaded column was placed in a new 2-mL centrifuge tube. Columns were then centrifuged for 2 minutes at 110 xg. Columns were again centrifuged for 2 minutes at 110 xg. The flow-through was collected as the fraction FT. Fourth, 200 µL of water was added to each column to wash the sorbent bed and release very polar metabolites. Each column was placed in a new 2-mL centrifuge tube. Columns were then centrifuged for 2 minutes at 110 xg. The wash was collected as fraction W. Fifth, 200 µL of an elution solvent (80% MeCN in water, v/v) was added to each column. Each column was placed in a new 2-mL centrifuge tube. Columns were then centrifuged for 2 minutes at 110 xg. The eluates were collected as fraction E.

Treatment ID:

TR002405

Treatment Summary:

200 µL of MeOH was first pipetted into each column to condition the sorbent. Columns were then centrifuged for 2 minutes at 110 xg. The procedure was repeated three times. Second, 200 µL of water was pipetted into each column to equilibrate the sorbent. Columns were then centrifuged for 2 minutes at 110 xg, and the procedure was repeated three times. Third, the samples were loaded into each prepared column. Each loaded column was placed in a new 2-mL centrifuge tube. Columns were then centrifuged for 2 minutes at 110 xg. Columns were again centrifuged for 2 minutes at 110 xg. The flow-through was collected as the fraction FT. Fourth, 200 µL of water was added to each column to wash the sorbent bed and release very polar metabolites. Each column was placed in a new 2-mL centrifuge tube. Columns were then centrifuged for 2 minutes at 110 xg. The wash was collected as fraction W. Fifth, 200 µL of an elution solvent (80% MeCN in water, v/v) was added to each column. Each column was placed in a new 2-mL centrifuge tube. Columns were then centrifuged for 2 minutes at 110 xg. The eluates were collected as fraction E.

Sample Preparation:

Sampleprep ID:	SP002399
Sampleprep Summary:	Semi-preparative HPLC was performed on a Shimadzu LC-10A system equipped with binary mobile phase pumps, a high-pressure valve with a 200-μL sample loop for manual injection, a column oven, and a UV-Vis detector (dual channel monitoring). The column was Atlantis T3 prep (10 x 250 mm, 10 µm for particle size, and 20 mL of bed volume; Waters). The column oven was at 40.0 °C. The sample injection volume was 180 µL, and the mobile phase flow rate was 3.000 mL/min. The binary mobile phase system consisted of 0.1% formic acid (FA) in water as solvent A and 0.1% FA in acetonitrile (MeCN) as solvent B. The method profile (% for Solvent B at runtime) was 1% from 0 to 5 minutes, 95% at 25 minutes, 95% from 25 to 29.9 minutes, and 1% at 30 to 35 minutes. The UV-Vis detector was set to monitor 214 nm for universal detection of compounds and 280 nm for detecting aromatic compounds. The full extracts of supernatants were desalted by Targa C18 Macro Spin columns (200 mg of sorbent) and reconstituted using water with a concentration factor of 2.5. In the low-resolution fractionation experiment, starting from 3 minutes, ten large HPLC fractions were collected every 3 minutes, producing 9-mL fractions. In the high-resolution fractionation experiment, starting from 12 minutes, eighteen small HPLC fractions were collected every 30 seconds, producing 1.5-mL fractions. Collected fractions were dried using a vacuum concentrator, followed by lyophilization. The dried fractions were reconstituted in water, transferred to a 96-well assay plate, covered by an aluminum plate seal, and stored at -80 °C.
Processing Storage Conditions:	On ice
Extract Storage:	-80℃

Combined analysis:

Analysis ID	AN003769
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	ACQUITY UPLC H-Class
Column	Waters CORTECS UPLC T3 (150 x 2.1mm,1.6um)
MS Type	ESI
MS instrument type	QTOF
MS instrument name	Waters Synapt-G2-Si
Ion Mode	POSITIVE
Units	peak area

Chromatography:

Chromatography ID:	CH002788
Chromatography Summary:	Lyophilized samples of supernatant full extracts were reconstituted with water and analyzed on an SYNAPT G2-Si mass spectrometer equipped with the electrospray ionization (ESI) source coupled with an ACQUITY UPLC H-Class system (Waters). The MassLynx v4.1 software was used to control the LC-MS system. A reversed-phase column (CORTECS UPLC T3, 2.1 x 150 mm, 1.6 μm particle size, 0.52 mL of bed volume, Waters) was used. The autosampler temperature was 4.0 °C, and the column oven temperature was 40.0 °C. The sample injection volume was 10 µL with a full loop overfilling factor of 2.0. The mobile phase flow rate was 300 µL/min. Solvent A was 0.1 % FA in water, and solvent B was 0.1 % FA in MeCN. The gradient (% for Solvent B at runtime) method was 1% from 0 to 1 minute, 20% at 15 minutes, 90% from 15.1 to 19 minutes, and 1% from 19.1 to 21 minutes. The PDA detector operated at a sampling rate of 20 Hz. The absorption UV-Vis spectrum range was 213 to 500 nm at a resolution of 1.2 nm.
Methods Filename:	F4_chromatography method.docx
Instrument Name:	ACQUITY UPLC H-Class
Column Name:	Waters CORTECS UPLC T3 (150 x 2.1mm,1.6um)
Column Temperature:	40
Flow Gradient:	The gradient (% for Solvent B at runtime) method was 1% from 0 to 1 minute, 20% at 15 minutes, 90% from 15.1 to 19 minutes, and 1% from 19.1 to 21 minutes.
Flow Rate:	300 µL/min
Solvent A:	100% water; 0.1% formic acid
Solvent B:	100% acetonitrile; 0.1% formic acid
Chromatography Type:	Reversed phase

MS:

MS ID:	MS003512
Analysis ID:	AN003769
Instrument Name:	Waters Synapt-G2-Si
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	TOF MS were used to analyze positively charged ions from the full extracts and fractions. Major instrument parameters were: capillary voltage, 3000 V; source temperature, 80 °C; sampling cone, 20 V; source offset, 80 V; desolvation temperature, 150 °C; desolvation nitrogen, 600.0 L/hr; nebulizer nitrogen, 6.5 bar. The lock spray had lock masses of 278.1141 and 556.2771 m/z as internal m/z calibration standards. In the TOF MS mode, the TOF mass analyzer used the high-resolution mode (40,000 FWHM at 956 m/z) with a scan time of 0.1 sec, interscan time of 0.015 sec, and mass range from 100 to 1200 m/z. The data was collected in centroid format. The top nine ions were selected for MS/MS acquisition in the DDA mode. For MS1 survey scans, the TOF mass analyzer used a high-resolution mode with a scan time of 0.1 sec, interscan time of 0.01 sec, and mass range from 100 to 1200 m/z. For data-dependent MS/MS, the quadrupole mass analyzer used unit resolution, the trap collision cell used ramping collision energy from 5 to 65 eV, and TOF mass analyzer used high-resolution mode with a scan time of 0.1 sec, interscan time of 0.015 sec, and mass range from 50 to 1700 m/z. Precursors that triggered MS/MS scans were dynamically excluded within ± 5.0 ppm from repetitive MS/MS scans for 6.0 sec. The data was collected in centroid format. The acquired data was converted with MSConvert41 and processed with MZmine 2.53. The raw data first went through a mass detection with an MS level of 1, and the mass detector was set to the centroid. The mass list was processed by ADAP chromatogram builder with MS level 1, min group size in # of scans of 12, group intensity threshold as 1E3, min highest intensity as 1E3, and m/z tolerance as 1E-4 or 20 ppm. The feature lists were obtained by deisotoping (the same m/z tolerance, retention time tolerance as 0.05 min, maximum charge 1, and most intense as the representative isotope), alignment with the join aligner with the same m/z tolerance and retention time tolerance as 0.1 min, and then gap-filling with the peak finder with 20% intensity tolerance and 0.05 retention tolerance. The feature lists were searched against KEGG with the 20 ppm error allowance. The obtained results were processed in R to generate differential expression plots.
Ion Mode:	POSITIVE