Summary of Study ST002306
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 | ST002306 |
| Study Title | Metabolomics profiling of full extracts of bacterial culture supernatants. |
| Study Type | MS quantitative analysis |
| Study Summary | Separate LC-MS runs of AhR-active and inactive 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 |
| 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 |
Select appropriate tab below to view additional metadata details:
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: | SU002392 |
| 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 |
|---|---|---|---|
| SA226722 | MSR_3 | Bacillus megaterium | Active |
| SA226723 | MSR_2 | Bacillus megaterium | Active |
| SA226724 | MSR_1 | Bacillus megaterium | Active |
| SA226725 | SAM_2 | Enterococcus faecium | Inactive |
| SA226726 | SAM_3 | Enterococcus faecium | Inactive |
| SA226727 | SAM_1 | Enterococcus faecium | Inactive |
| Showing results 1 to 6 of 6 |
Collection:
| Collection ID: | CO002385 |
| Collection Summary: | Bacteria were cultured in deep well plates. Bacteria supernatants were filtered through a 0.22-micron filter to prepare cell-free culture supernatants. The supernatants were processed by solid phase extraction and dried for LC-MS. |
| Collection Protocol Filename: | Full_sample_collection.docx |
| Sample Type: | Bacterial cells |
| Storage Conditions: | -80℃ |
Treatment:
| Treatment ID: | TR002404 |
| Treatment Summary: | The supernatants were processed by solid phase extraction and dried for LC-MS. |
Sample Preparation:
| Sampleprep ID: | SP002398 |
| Sampleprep Summary: | The supernatants were processed by solid phase extraction and dried for LC-MS. |
| Processing Storage Conditions: | On ice |
| Extract Storage: | -80℃ |
Combined analysis:
| Analysis ID | AN003768 |
|---|---|
| Analysis type | MS |
| Chromatography type | Reversed phase |
| Chromatography system | Waters Acquity 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: | CH002787 |
| Chromatography Summary: | 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. |
| Methods Filename: | Full_chromatography method.docx |
| Instrument Name: | Waters Acquity H-Class |
| Column Name: | Waters CORTECS UPLC T3 (150 x 2.1mm,1.6um) |
| Column Pressure: | 8000 psi |
| Column Temperature: | 40 |
| Flow Gradient: | 90 |
| Flow Rate: | 0.3 mL |
| Solvent A: | 100% water; 0.1% formic acid |
| Solvent B: | 100% acetonitrile; 0.1% formic acid |
| Chromatography Type: | Reversed phase |
MS:
| MS ID: | MS003511 |
| Analysis ID: | AN003768 |
| Instrument Name: | Waters Synapt-G2-Si |
| Instrument Type: | QTOF |
| MS Type: | ESI |
| MS Comments: | TOF MS modes 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 |
