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MB Sample ID: SA307155

Local Sample ID:Neg_10'
Subject ID:SU002943
Subject Type:Bacteria
Subject Species:Bacteroides thetaiotaomicron
Taxonomy ID:8188
Subject Comments:Fecal derived communities and isolates, supernatant was assayed

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Subject:

Subject ID:SU002943
Subject Type:Bacteria
Subject Species:Bacteroides thetaiotaomicron
Taxonomy ID:8188
Subject Comments:Fecal derived communities and isolates, supernatant was assayed

Factors:

Local Sample IDMB Sample IDFactor Level IDLevel ValueFactor Name
Neg_10'SA307155FL036968bacterial communityGenotype
Neg_10'SA307155FL036968BHI spent by Enterococcus faecalisTreatment

Collection:

Collection ID:CO002936
Collection Summary:Isolates were obtained via plating of in vitro communities –, derived from culturing fecal samples from humanized mice –, on agar plates made with various complex media and frozen as glycerol stocks, as previously described (https://doi.org/https://doi.org/10.1016/j.chom.2021.12.008, https://www.biorxiv.org/content/10.1101/2023.01.13.523996v1). Frozen stocks were streaked onto BHI-blood agar plates (5% defibrinated horse blood in 1.5% w/v agar). Resulting colonies were inoculated into 3 mL of Brain Heart Infusion (BHI) (BD #2237500) or modified Gifu Anaerobic Medium (mGAM) (HyServe #05433) in test tubes. All culturing werewas performed at 37 °C without shaking in an anaerobic chamber (Coy). To minimize potential physiological changes from freeze-thaw cycles and changes in growth medium, cultures were diluted 1:200 every 48 h for 3 passages before growth or metabolomics measurements. After the first passage, subsequent passages were performed in 96-well polystyrene plates (Greiner Bio-One #655161) filled with 200 μL of growth medium.
Sample Type:Bacterial cells

Treatment:

Treatment ID:TR002952
Treatment Summary:Many combinations of bacterial isolates were assayed. details can be found in the publicly available preprint here: https://www.biorxiv.org/content/10.1101/2022.05.30.494065v1.abstract

Sample Preparation:

Sampleprep ID:SP002949
Sampleprep Summary:Spent media were collected as described above and immediately stored at -80 °C. Samples were thawed only once, immediately before LC-MS/MS. Thawed samples were kept on ice, each sample was homogenized by pipetting prior to dispensing. Two 20-µL aliquots of supernatant were removed from each sample well and dispensed into two shallow 96-well polypropylene plates, maintained on ice. Additionally, 5 µL were removed from each sample and combined into a homogenous pool; this pool was dispensed in 20-µL aliquots and prepared in parallel with samples. These pooled samples were used for in-run quality control, injected at predefined intervals over the course of analysis to ensure consistent instrument performance over time. Samples were analyzed using two complementary chromatography methods: reversed phase (C18) and hydrophilic interaction chromatography (HILIC). All samples were analyzed by positive and negative mode electrospray ionization (ESI+, ESI-). Sample analysis order was randomized to minimize potential bias in data acquisition. Procedural blanks were prepared by extracting 20 µL of water in place of bacterial supernatant. Procedural blanks were inserted throughout the run as additional quality control.

Combined analysis:

Analysis ID AN004629 AN004630
Analysis type MS MS
Chromatography type Reversed phase Reversed phase
Chromatography system Thermo Vanquish Thermo Vanquish
Column Agilent Zorbax SB-C18 (100 x 3.0 mm, 1.8 um) Agilent Zorbax SB-C18 (100 x 3.0 mm, 1.8 um)
MS Type ESI ESI
MS instrument type Orbitrap Orbitrap
MS instrument name Thermo Q Exactive HF hybrid Orbitrap Thermo Q Exactive HF hybrid Orbitrap
Ion Mode POSITIVE NEGATIVE
Units counts, height counts, height

Chromatography:

Chromatography ID:CH003483
Chromatography Summary:Bacterial supernatant were analyzed via reversed phase (C18) coupled to a Thermo Q-Exactive HF high resolution mass spectrometer. Prepared samples were injected onto an Agilent Zorbax SB-C18 column (100 mm length × 3 mm id; 1.8 μm particle size) with an additional Phenomenex KrudKatcher pre-column (2 μm depth x 0.004in ID) maintained at 40°C coupled to an Thermo Vanquish UPLC. The mobile phases were prepared with 0.1% formic acid in either 100% LC-MS grade water for mobile phase (A), 100% water or mobile phase (B), 100% acetonitrile. Gradient elution was performed as follows 3% (B) maintained 0–0.43 min to 97% (B) at 9 min, maintained until 11 min, returning to initial conditions at 11.5 min and equilibrating until the end of the run at 14 min. Flow rate is maintained at 0.4 mL/min. Each sample was analyzed in both positive and negative ionization modes (ESI+, ESI-) via subsequent injections. Full MS-ddMS2 data was collected, an inclusion list was used to prioritize MS2 selection of metabolites from our in-house ‘local’ library, when additional scan bandwidth was available MS2 was collected in a data-dependent manner. Mass range was 60-900 mz, resolution was 60k (MS1) and 15k (MS2), centroid data was collected, loop count was 4, isolation window was 1.5 Da. Metabolomics data was processed using MS-DIAL v4.60 (https://www.nature.com/articles/s41587-020-0531-2) and queried against a combination of our in-house MS2 library and MassBank of North America, the largest freely available spectral repository (https://doi.org/10.1002/mas.21535). Features were excluded from analysis if peak height was not at least 5-fold greater in one or more samples compared to the procedural blank average.
Instrument Name:Thermo Vanquish
Column Name:Agilent Zorbax SB-C18 (100 x 3.0 mm, 1.8 um)
Column Temperature:40C
Flow Gradient:Gradient elution was performed from 100% (B) at 0–2 min to 70% (B) at 7.7 min, 40% (B) at 9.5 min, 30% (B) at 10.25 min, 100% (B) at 12.75 min, isocratic until 16.75 min with a column flow ofGradient elution was performed as follows 3% (B) maintained 0–0.43 min to 97% (B) at 9 min, maintained until 11 min, returning to initial conditions at 11.5 min and equilibrating until the end of the run at 14 min.
Flow Rate:0.4 mL/min.
Solvent A:Water + 0.1% formic acid
Solvent B:Acetonitrile + 0.1% formic acid
Chromatography Type:Reversed phase

MS:

MS ID:MS004375
Analysis ID:AN004629
Instrument Name:Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Full MS-ddMS2 data was collected, an inclusion list was used to prioritize MS2 selection of metabolites from our in-house ‘local’ library, when additional scan bandwidth was available MS2 was collected in a data-dependent manner. Mass range was 60-900 mz, resolution was 60k (MS1) and 15k (MS2), centroid data was collected, loop count was 4, isolation window was 1.5 Da. Metabolomics data was processed using MS-DIAL v4.60. Features were excluded from analysis if peak height was not at least 5-fold greater in one or more samples compared to the procedural blank average.
Ion Mode:POSITIVE
  
MS ID:MS004376
Analysis ID:AN004630
Instrument Name:Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:Full MS-ddMS2 data was collected, an inclusion list was used to prioritize MS2 selection of metabolites from our in-house ‘local’ library, when additional scan bandwidth was available MS2 was collected in a data-dependent manner. Mass range was 60-900 mz, resolution was 60k (MS1) and 15k (MS2), centroid data was collected, loop count was 4, isolation window was 1.5 Da. Metabolomics data was processed using MS-DIAL v4.60. Features were excluded from analysis if peak height was not at least 5-fold greater in one or more samples compared to the procedural blank average.
Ion Mode:NEGATIVE
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