Summary of Study ST002308

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 PR001820. The data can be accessed directly via it's Project DOI: 10.21228/M8G13Q This work is supported by NIH grant, U2C- DK119886. See: https://www.metabolomicsworkbench.org/about/howtocite.php

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Study IDST002308
Study TitleMetabolomics profiling of full extracts and fractions of bacterial culture supernatants.
Study TypeMSMS quantitative analysis
Study SummaryTargeted quantitation of select indole compounds in supernatant full extracts was performed on a quadrupole orbitrap mass spectrometer.
Institute
University of Connecticut
DepartmentChemistry
LaboratoryYao Lab
Last NameTian
First NameHuidi
Address55 N. Eagleville Road, Unit 3060, Storrs CT 06269
Emailhuidi.tian@uconn.edu
Phone8606341143
Submit Date2022-10-03
Raw Data AvailableYes
Raw Data File Type(s)mzML
Analysis Type DetailLC-MS
Release Date2025-10-05
Release Version1
Huidi Tian Huidi Tian
https://dx.doi.org/10.21228/M8G13Q
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR001820
Project DOI:doi: 10.21228/M8G13Q
Project Title:Metabolomics Discovery of Aryl Hydrocarbon Receptor Activating Metabolites from the Human Microbiota (Targeted)
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. We also describe a method of targeted discovery of AhR-activating metabolites and report a correlation analysis of the AhR activity with the concentration of endogenous metabolites that identified significant common AhR activators shared by different strains of bacteria in the human microbiome. Principal component analysis of relative concentrations of indole compounds clusters different bacteria species, providing a possible means for evaluating the regulation of the bacteria indole pathway.
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-NINDS and NIH-NIAID

Subject:

Subject ID:SU002394
Subject Type:Bacteria
Subject Species:Species specified in the sample list
Species Group:Bacteria

Factors:

Subject type: Bacteria; Subject species: Species specified in the sample list (Factor headings shown in green)

mb_sample_id local_sample_id Bacterial strain Concentration
SA226740B2_5xC_2Bacillus megaterium 5 times concentrated
SA226741B2_5xC_1Bacillus megaterium 5 times concentrated
SA226742B1_5xC_3Bacillus megaterium 5 times concentrated
SA226743B2_5xC_3Bacillus megaterium 5 times concentrated
SA226744B3_5xC_1Bacillus megaterium 5 times concentrated
SA226745B3_5xC_3Bacillus megaterium 5 times concentrated
SA226746B3_5xC_2Bacillus megaterium 5 times concentrated
SA226747B1_5xC_1Bacillus megaterium 5 times concentrated
SA226748B1_5xC_2Bacillus megaterium 5 times concentrated
SA226749B3_5xD_2Bacillus megaterium 5 times diluted
SA226750B3_5xD_1Bacillus megaterium 5 times diluted
SA226751B2_5xD_3Bacillus megaterium 5 times diluted
SA226752B1_5xD_1Bacillus megaterium 5 times diluted
SA226753B3_5xD_3Bacillus megaterium 5 times diluted
SA226754B2_5xD_2Bacillus megaterium 5 times diluted
SA226755B1_5xD_3Bacillus megaterium 5 times diluted
SA226756B1_5xD_2Bacillus megaterium 5 times diluted
SA226757B2_5xD_1Bacillus megaterium 5 times diluted
SA226758A12_5xC_1Bacillus subtilis 5 times concentrated
SA226759A12_5xC_3Bacillus subtilis 5 times concentrated
SA226760A12_5xC_2Bacillus subtilis 5 times concentrated
SA226761A11_5xC_3Bacillus subtilis 5 times concentrated
SA226762A11_5xC_2Bacillus subtilis 5 times concentrated
SA226763A10_5xC_1Bacillus subtilis 5 times concentrated
SA226764A10_5xC_2Bacillus subtilis 5 times concentrated
SA226765A11_5xC_1Bacillus subtilis 5 times concentrated
SA226766A10_5xC_3Bacillus subtilis 5 times concentrated
SA226767A11_5xD_3Bacillus subtilis 5 times diluted
SA226768A12_5xD_1Bacillus subtilis 5 times diluted
SA226769A12_5xD_3Bacillus subtilis 5 times diluted
SA226770A10_5xD_1Bacillus subtilis 5 times diluted
SA226771A10_5xD_2Bacillus subtilis 5 times diluted
SA226772A12_5xD_2Bacillus subtilis 5 times diluted
SA226773A10_5xD_3Bacillus subtilis 5 times diluted
SA226774A11_5xD_1Bacillus subtilis 5 times diluted
SA226775A11_5xD_2Bacillus subtilis 5 times diluted
SA226776A4_5xC_1Dermabacter sp. 5 times concentrated
SA226777A5_5xC_2Dermabacter sp. 5 times concentrated
SA226778A5_5xC_1Dermabacter sp. 5 times concentrated
SA226779A4_5xC_3Dermabacter sp. 5 times concentrated
SA226780A5_5xC_3Dermabacter sp. 5 times concentrated
SA226781A6_5xC_1Dermabacter sp. 5 times concentrated
SA226782A6_5xC_3Dermabacter sp. 5 times concentrated
SA226783A6_5xC_2Dermabacter sp. 5 times concentrated
SA226784A4_5xC_2Dermabacter sp. 5 times concentrated
SA226785A5_5xD_1Dermabacter sp. 5 times diluted
SA226786A5_5xD_3Dermabacter sp. 5 times diluted
SA226787A6_5xD_1Dermabacter sp. 5 times diluted
SA226788A6_5xD_2Dermabacter sp. 5 times diluted
SA226789A5_5xD_2Dermabacter sp. 5 times diluted
SA226790A4_5xD_3Dermabacter sp. 5 times diluted
SA226791A4_5xD_1Dermabacter sp. 5 times diluted
SA226792A4_5xD_2Dermabacter sp. 5 times diluted
SA226793A6_5xD_3Dermabacter sp. 5 times diluted
SA226794B7_5xC_1Enterococcus faecium 320 5 times concentrated
SA226795B7_5xD_1Enterococcus faecium 320 5 times diluted
SA226796B7_5xC_2Enterococcus faecium 321 5 times concentrated
SA226797B7_5xD_2Enterococcus faecium 321 5 times diluted
SA226798B7_5xC_3Enterococcus faecium 322 5 times concentrated
SA226799B7_5xD_3Enterococcus faecium 322 5 times diluted
SA226800B8_5xC_1Enterococcus faecium 323 5 times concentrated
SA226801B8_5xD_1Enterococcus faecium 323 5 times diluted
SA226802B8_5xC_2Enterococcus faecium 324 5 times concentrated
SA226803B8_5xD_2Enterococcus faecium 324 5 times diluted
SA226804B8_5xC_3Enterococcus faecium 325 5 times concentrated
SA226805B8_5xD_3Enterococcus faecium 325 5 times diluted
SA226806B9_5xC_1Enterococcus faecium 326 5 times concentrated
SA226807B9_5xD_1Enterococcus faecium 326 5 times diluted
SA226808B9_5xC_2Enterococcus faecium 327 5 times concentrated
SA226809B9_5xD_2Enterococcus faecium 327 5 times diluted
SA226810B9_5xC_3Enterococcus faecium 328 5 times concentrated
SA226811B9_5xD_3Enterococcus faecium 328 5 times diluted
SA226812A2_5xC_1Enterococcus faecium 348 5 times concentrated
SA226813A1_5xC_3Enterococcus faecium 348 5 times concentrated
SA226814A1_5xC_2Enterococcus faecium 348 5 times concentrated
SA226815A2_5xC_2Enterococcus faecium 348 5 times concentrated
SA226816A3_5xC_3Enterococcus faecium 348 5 times concentrated
SA226817A2_5xC_3Enterococcus faecium 348 5 times concentrated
SA226818A3_5xC_2Enterococcus faecium 348 5 times concentrated
SA226819A1_5xC_1Enterococcus faecium 348 5 times concentrated
SA226820A3_5xC_1Enterococcus faecium 348 5 times concentrated
SA226821A2_5xD_1Enterococcus faecium 348 5 times diluted
SA226822A1_5xD_3Enterococcus faecium 348 5 times diluted
SA226823A2_5xD_2Enterococcus faecium 348 5 times diluted
SA226824A1_5xD_2Enterococcus faecium 348 5 times diluted
SA226825A3_5xD_2Enterococcus faecium 348 5 times diluted
SA226826A3_5xD_3Enterococcus faecium 348 5 times diluted
SA226827A3_5xD_1Enterococcus faecium 348 5 times diluted
SA226828A2_5xD_3Enterococcus faecium 348 5 times diluted
SA226829A1_5xD_1Enterococcus faecium 348 5 times diluted
SA226830C2_5xC_2Staphylococcus epidermidis 1-1 5 times concentrated
SA226831C1_5xC_3Staphylococcus epidermidis 1-1 5 times concentrated
SA226832C1_5xC_2Staphylococcus epidermidis 1-1 5 times concentrated
SA226833C2_5xC_3Staphylococcus epidermidis 1-1 5 times concentrated
SA226834C3_5xC_1Staphylococcus epidermidis 1-1 5 times concentrated
SA226835C3_5xC_3Staphylococcus epidermidis 1-1 5 times concentrated
SA226836C3_5xC_2Staphylococcus epidermidis 1-1 5 times concentrated
SA226837C1_5xC_1Staphylococcus epidermidis 1-1 5 times concentrated
SA226838C2_5xC_1Staphylococcus epidermidis 1-1 5 times concentrated
SA226839C2_5xD_1Staphylococcus epidermidis 1-1 5 times diluted
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Collection:

Collection ID:CO002387
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:TR002406
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:SP002400
Sampleprep Summary:Full extracts of different bacteria were spiked with NFK-C13 and desalted by Targa C18 cartridges. The desalted extracts were dried by a lyophilizer and reconstituted with water for LC-parallel reaction monitoring (PRM) MS of a panel of indole compounds. The supernatants were analyzed as 5-time diluted and 5-times concentrated, compared to the initial supernatants. The spiked NFK-C13 was 0.5 µM in each sample.
Processing Storage Conditions:On ice
Extract Storage:-80℃

Combined analysis:

Analysis ID AN003770
Chromatography ID CH002789
MS ID MS003513
Analysis type MS
Chromatography type Reversed phase
Chromatography system Thermo Vanquish
Column Waters CORTECS UPLC T3 (150 x 2.1mm,1.6um)
MS Type ESI
MS instrument type Orbitrap
MS instrument name Thermo Orbitrap Exploris 480
Ion Mode POSITIVE
Units peak area

Chromatography:

Chromatography ID:CH002789
Chromatography Summary:Targeted quantitation of select indole compounds in supernatant full extracts was performed on a quadrupole orbitrap mass spectrometer (Exploris 480, Thermo Fisher Scientific) with an H-ESI ionization source in the positive ion mode. The front-end separation used a reversed-phase column (CORTECS UPLC T3, 2.1 x 150 mm, 1.6 μm particle size, 0.52 mL of bed volume, Waters). The autosampler temperature was 4.0 °C, and the column oven temperature was 40.0 °C. The sample injection volume was 10 µL. 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 -5 to 0 minute for the equilibration, 1% from 0 to 1 minute, 90% at 15 minutes, 90% from 15.1 to 19 minutes, and 1% from 19.1 to 20 minutes.
Methods Filename:Chrom.docx
Instrument Name:Thermo Vanquish
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 -5 to 0 minute for the equilibration, 1% from 0 to 1 minute, 90% at 15 minutes, 90% from 15.1 to 19 minutes, and 1% from 19.1 to 20 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:MS003513
Analysis ID:AN003770
Instrument Name:Thermo Orbitrap Exploris 480
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:. Major mass spectrometer parameters were 3500 V for the spray voltage, 50 arb for the sheath gas, 10 arb for the aux gas, 1 arb for the sweep gas, 300 ℃ for the ion transfer tube, and 350 ℃ for the vaporizer temperature. In the full MS mode, the orbitrap resolution was 120,000, with a scan range of 100-1000 m/z. The RF lens was 50%, the AGC target was set to standard, the maximum injection time mode was set to auto, and the microscans were set to 1. The data was collected in the profile format. In the parallel reaction monitoring (PRM) mode, the isolation window was set to 0.7 m/z without isolation offset. The collision energy was set to fix; the normalized collision energy for each compound was defined in Table 1. The other settings remain the same as the DDA mode. The PRM acquisition for each compound was scheduled, and the collision energy was optimized. The orbitrap resolution was 15,000, and the scan range mode was auto. The AGC target was set as standard, the maximum injection time mode was set to auto, the intensity threshold was 1e4, the dynamic exclusion was 6 seconds, and the microscans were set to 1. The data was collected in profile mode. The quantitation was achieved with Skyline. The correlation analysis and PCA were processed in R.
Ion Mode:POSITIVE
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