Summary of Study ST003911

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 PR002363. The data can be accessed directly via it's Project DOI: 10.21228/M8653M 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.

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Study IDST003911
Study TitleMolecular fingerprint inference reveals bioactive lipids and microbial metabolites in colitis. Study 4
Study TypeStudy 4: Profiling of bacterial cultures for: Eggerthella lenta, Fusobacterium nucleatum
Study SummaryUntargeted metabolomics provides a sensitive readout of small molecules in biofluids , though considerable effort is still required to assign compound annotations to yet unidentified features . Here, we developed MS1-Tools, a machine learning model trained on LC-MS1 profiles of verified metabolites, to infer molecular fingerprints for unknown features. In stool samples, MS1-Tools achieved test accuracy of >74% (correct within top 3 candidates), drastically reducing the number of reference standards to be tested. Predicted identities were validated with MS/MS and standards, revealing diverse markers and drivers of inflammation, including amino-acid conjugations with primary metabolites, and platelet-activating factor analogs that we show to bind the PAF receptor. MS1-Tools enabled integration with bacterial culturomics to uncover production of N-lactoyl-phenylalanine (Lac-Phe) – an appetite-regulating metabolite – by inflammation-associated Lactobacillales and Bifidobacteriales. Structural protein search and bacterial genetics enabled validation of multiple bacterial Lac-Phe-producing dipeptidases, demonstrating broad applicability of MS1-Tools to resolve inflammation-associated metabolites and their origins.
Institute
Broad Institute of MIT and Harvard
Last NameAvila-Pacheco
First NameJulian
Address415 Main Street
Emailjravilap@broadinstitute.org
Phone(617) 714-1729
Submit Date2025-05-08
Raw Data AvailableYes
Raw Data File Type(s)mzML, raw(Thermo)
Analysis Type DetailLC-MS
Release Date2025-08-04
Release Version1
Julian Avila-Pacheco Julian Avila-Pacheco
https://dx.doi.org/10.21228/M8653M
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

Select appropriate tab below to view additional metadata details:

Project:

Project ID:PR002363
Project DOI:doi: 10.21228/M8653M
Project Title:Molecular fingerprint inference reveals bioactive lipids and microbial metabolites in colitis
Project Summary:Untargeted metabolomics provides a sensitive readout of small molecules in biofluids, but requires targeted approaches to resolve ~90% of features for which tandem mass spectra (MS/MS) are not collected. By training on a subset of verified metabolites and their profiles in LC-MS, we derive a probabilistic model to predict molecular fingerprints in human stool and blood samples. These predictions, which do not utilize MS/MS, were accurate for >44% (correct top ranked candidate) or >75% (correct within top 3) of test metabolites, drastically reducing the number of reference standards that would need to be to be tested. These predictions revealed markers and drivers of inflammation, including amino acid derivatives and lysophospholipids with herein demonstrated platelet-activating factor receptor (PAF-R) activity. Integration with bacterial culturomics facilitates tracking the source of inflammation-associated metabolites to their origins in the gut microbiome.
Institute:Broad Institute of MIT and Harvard
Last Name:Avila-Pacheco
First Name:Julian
Address:415 Main Street
Email:jravilap@broadinstitute.org
Phone:+1 (617) 714-1729

Subject:

Subject ID:SU004046
Subject Type:Bacteria
Subject Species:Eggerthella lenta; Fusobacterium nucleatum
Taxonomy ID:84112; 851
Genotype Strain:Eggerthella lenta, Fusobacterium nucleatum

Factors:

Subject type: Bacteria; Subject species: Eggerthella lenta; Fusobacterium nucleatum (Factor headings shown in green)

mb_sample_id local_sample_id Species Media Timepoint Replicate
SA430328EL_BHI-R_24hr_1Eggerthella lenta BHI-R 24 R1
SA430329EL_BHI-R_24hr_2Eggerthella lenta BHI-R 24 R2
SA430330EL_BHI-R_24hr_3Eggerthella lenta BHI-R 24 R3
SA430331EL_BHI-R_48hr_1Eggerthella lenta BHI-R 48 R1
SA430332EL_BHI-R_48hr_2Eggerthella lenta BHI-R 48 R2
SA430333EL_BHI-R_48hr_3Eggerthella lenta BHI-R 48 R3
SA430334EL_BHI-R_6hr_1Eggerthella lenta BHI-R 6 R1
SA430335EL_BHI-R_6hr_2Eggerthella lenta BHI-R 6 R2
SA430336EL_BHI-R_6hr_3Eggerthella lenta BHI-R 6 R3
SA430325EL_BHI-R_0hr_1Eggerthella lenta BHI-R - R1
SA430326EL_BHI-R_0hr_2Eggerthella lenta BHI-R - R2
SA430327EL_BHI-R_0hr_3Eggerthella lenta BHI-R - R3
SA430340EL_CHG-R_24hr_1Eggerthella lenta CHG-R 24 R1
SA430341EL_CHG-R_24hr_2Eggerthella lenta CHG-R 24 R2
SA430342EL_CHG-R_24hr_3Eggerthella lenta CHG-R 24 R3
SA430343EL_CHG-R_48hr_1Eggerthella lenta CHG-R 48 R1
SA430344EL_CHG-R_48hr_2Eggerthella lenta CHG-R 48 R2
SA430345EL_CHG-R_48hr_3Eggerthella lenta CHG-R 48 R3
SA430346EL_CHG-R_6hr_1Eggerthella lenta CHG-R 6 R1
SA430347EL_CHG-R_6hr_2Eggerthella lenta CHG-R 6 R2
SA430348EL_CHG-R_6hr_3Eggerthella lenta CHG-R 6 R3
SA430337EL_CHG-R_0hr_1Eggerthella lenta CHG-R - R1
SA430338EL_CHG-R_0hr_2Eggerthella lenta CHG-R - R2
SA430339EL_CHG-R_0hr_3Eggerthella lenta CHG-R - R3
SA430352EL_Gifu-R_24hr_1Eggerthella lenta Gifu-R 24 R1
SA430353EL_Gifu-R_24hr_2Eggerthella lenta Gifu-R 24 R2
SA430354EL_Gifu-R_24hr_3Eggerthella lenta Gifu-R 24 R3
SA430355EL_Gifu-R_48hr_1Eggerthella lenta Gifu-R 48 R1
SA430356EL_Gifu-R_48hr_2Eggerthella lenta Gifu-R 48 R2
SA430357EL_Gifu-R_48hr_3Eggerthella lenta Gifu-R 48 R3
SA430358EL_Gifu-R_6hr_1Eggerthella lenta Gifu-R 6 R1
SA430359EL_Gifu-R_6hr_2Eggerthella lenta Gifu-R 6 R2
SA430360EL_Gifu-R_6hr_3Eggerthella lenta Gifu-R 6 R3
SA430349EL_Gifu-R_0hr_1Eggerthella lenta Gifu-R - R1
SA430350EL_Gifu-R_0hr_2Eggerthella lenta Gifu-R - R2
SA430351EL_Gifu-R_0hr_3Eggerthella lenta Gifu-R - R3
SA430364EL_Mega-R_24hr_1Eggerthella lenta Mega-R 24 R1
SA430365EL_Mega-R_24hr_2Eggerthella lenta Mega-R 24 R2
SA430366EL_Mega-R_24hr_3Eggerthella lenta Mega-R 24 R3
SA430367EL_Mega-R_48hr_1Eggerthella lenta Mega-R 48 R1
SA430368EL_Mega-R_48hr_2Eggerthella lenta Mega-R 48 R2
SA430369EL_Mega-R_48hr_3Eggerthella lenta Mega-R 48 R3
SA430370EL_Mega-R_6hr_1Eggerthella lenta Mega-R 6 R1
SA430371EL_Mega-R_6hr_2Eggerthella lenta Mega-R 6 R2
SA430372EL_Mega-R_6hr_3Eggerthella lenta Mega-R 6 R3
SA430361EL_Mega-R_0hr_1Eggerthella lenta Mega-R - R1
SA430362EL_Mega-R_0hr_2Eggerthella lenta Mega-R - R2
SA430363EL_Mega-R_0hr_3Eggerthella lenta Mega-R - R3
SA430376FB_CHG_24hr_1Fusobacterium nucleatum CHG 24 R1
SA430377FB_CHG_24hr_2Fusobacterium nucleatum CHG 24 R2
SA430378FB_CHG_24hr_3Fusobacterium nucleatum CHG 24 R3
SA430379FB_CHG_48hr_1Fusobacterium nucleatum CHG 48 R1
SA430380FB_CHG_48hr_2Fusobacterium nucleatum CHG 48 R2
SA430381FB_CHG_48hr_3Fusobacterium nucleatum CHG 48 R3
SA430382FB_CHG_6hr_1Fusobacterium nucleatum CHG 6 R1
SA430383FB_CHG_6hr_2Fusobacterium nucleatum CHG 6 R2
SA430384FB_CHG_6hr_3Fusobacterium nucleatum CHG 6 R3
SA430373FB_CHG_0hr_1Fusobacterium nucleatum CHG - R1
SA430374FB_CHG_0hr_2Fusobacterium nucleatum CHG - R2
SA430375FB_CHG_0hr_3Fusobacterium nucleatum CHG - R3
SA430388FB_Gifu_24hr_1Fusobacterium nucleatum Gifu 24 R1
SA430389FB_Gifu_24hr_2Fusobacterium nucleatum Gifu 24 R2
SA430390FB_Gifu_24hr_3Fusobacterium nucleatum Gifu 24 R3
SA430391FB_Gifu_48hr_1Fusobacterium nucleatum Gifu 48 R1
SA430392FB_Gifu_48hr_2Fusobacterium nucleatum Gifu 48 R2
SA430393FB_Gifu_48hr_3Fusobacterium nucleatum Gifu 48 R3
SA430394FB_Gifu_6hr_1Fusobacterium nucleatum Gifu 6 R1
SA430395FB_Gifu_6hr_2Fusobacterium nucleatum Gifu 6 R2
SA430396FB_Gifu_6hr_3Fusobacterium nucleatum Gifu 6 R3
SA430385FB_Gifu_0hr_1Fusobacterium nucleatum Gifu - R1
SA430386FB_Gifu_0hr_2Fusobacterium nucleatum Gifu - R2
SA430387FB_Gifu_0hr_3Fusobacterium nucleatum Gifu - R3
SA430400FB_Mega_24hr_1Fusobacterium nucleatum Mega 24 R1
SA430401FB_Mega_24hr_2Fusobacterium nucleatum Mega 24 R2
SA430402FB_Mega_24hr_3Fusobacterium nucleatum Mega 24 R3
SA430403FB_Mega_48hr_1Fusobacterium nucleatum Mega 48 R1
SA430404FB_Mega_48hr_2Fusobacterium nucleatum Mega 48 R2
SA430405FB_Mega_48hr_3Fusobacterium nucleatum Mega 48 R3
SA430406FB_Mega_6hr_1Fusobacterium nucleatum Mega 6 R1
SA430407FB_Mega_6hr_2Fusobacterium nucleatum Mega 6 R2
SA430408FB_Mega_6hr_3Fusobacterium nucleatum Mega 6 R3
SA430397FB_Mega_0hr_1Fusobacterium nucleatum Mega - R1
SA430398FB_Mega_0hr_2Fusobacterium nucleatum Mega - R2
SA430399FB_Mega_0hr_3Fusobacterium nucleatum Mega - R3
SA430412BHI-R_24hr_1Unspent media BHI-R 24 R1
SA430413BHI-R_24hr_2Unspent media BHI-R 24 R2
SA430414BHI-R_24hr_3Unspent media BHI-R 24 R3
SA430415BHI-R_48hr_1Unspent media BHI-R 48 R1
SA430416BHI-R_48hr_2Unspent media BHI-R 48 R2
SA430417BHI-R_48hr_3Unspent media BHI-R 48 R3
SA430418BHI-R_6hr_1Unspent media BHI-R 6 R1
SA430419BHI-R_6hr_2Unspent media BHI-R 6 R2
SA430420BHI-R_6hr_3Unspent media BHI-R 6 R3
SA430409BHI-R_0hr_1Unspent media BHI-R - R1
SA430410BHI-R_0hr_2Unspent media BHI-R - R2
SA430411BHI-R_0hr_3Unspent media BHI-R - R3
SA430424CHG-R_24hr_1Unspent media CHG-R 24 R1
SA430425CHG-R_24hr_2Unspent media CHG-R 24 R2
SA430426CHG-R_24hr_3Unspent media CHG-R 24 R3
SA430427CHG-R_48hr_1Unspent media CHG-R 48 R1
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Collection:

Collection ID:CO004039
Collection Summary:All strains were cultured anaerobically at 37°C in different media types according to the experiment being performed. The media compositions used in this study include BHI-R, CHG-R, Gifu-R, and Mega-R, each formulated with specific components to support microbial growth. BHI-R was prepared by dissolving 18.5 g of Brain Heart Infusion (BHI) and 5 g of L-arginine in 500 mL of Milli-Q water, followed by sterilization via autoclaving (10 lbs, 115°C, 15 minutes). CHG-R was based on CHG media, which contained 18.5 g of BHI, 0.5 g each of D-(+)-cellobiose, D-(+)-maltose monohydrate, and D-(-)-fructose, and 0.25 g of cysteine in 500 mL of Milli-Q water. After filter sterilization, additional supplements, including 5 mL each of Vitamin K + Hematin (1%), trace mineral supplement, and vitamin supplement, were added in an anaerobic chamber. Gifu-R was derived from commercially available Gifu Anaerobic Media (GAM) (Himedia, Cat # M1801), prepared by suspending 59 g of GAM in 1000 mL of purified/distilled water and autoclaving under the same conditions as BHI-R. Mega-R was formulated with a complex mixture of components, including 5 g of Trypticase Peptone (BBL), 2.5 g each of Yeast Extract (Bacto) and Meat Extract, and multiple carbohydrate sources such as 1 g of D-(+)-glucose and 0.5 g each of D-(+)-cellobiose, D-(+)-maltose monohydrate, and D-(-)-fructose, all dissolved in 500 mL of Milli-Q water. Additional components included 50 mL of 1 M potassium phosphate buffer (pH 7.2), 20 mL of TYG salts solution, 1 mL of 25% Tween 80, and various supplements such as SCFA, CaCl₂, FeSO₄, and resazurin. Like CHG-R, Mega-R was supplemented with 5 mL each of Vitamin K + Hematin (1%), trace mineral supplement, and vitamin supplement. For CHG-R, Gifu-R, and Mega-R, an additional 5 g of L-arginine was incorporated into 500 mL of each respective medium to create their corresponding arginine-enriched formulations. All cultures were grown in an anaerobic chamber (Coy Laboratory Products) with an atmosphere of 5% CO2, 5% H2, and 90% N2 at 37°C. The isolates were streaked onto CHG plates and incubated for 48 hours. Once colonies were visible, a single colony from each plate was picked and processed for 16S rRNA Sanger sequencing using primer sequences 27F: AGAGTTTGATCMTGGCTCAG and 1492R: GGTTACCTTGTTACGACTT. Once the identity of the bacterial strains was confirmed, a single colony from each strain was inoculated in triplicates in 6 mL of the respective media types (CHG, Gifu, Mega) and incubated overnight. The overnight cultures were inoculated into fresh media and normalized to the OD600 of 0.05. At each time point after inoculation (6h, 24h, 48 h), 200 μL of the bacterial culture were collected.
Sample Type:Bacterial cells

Treatment:

Treatment ID:TR004055
Treatment Summary:NA

Sample Preparation:

Sampleprep ID:SP004052
Sampleprep Summary:LC–MS samples were prepared from bacterial cultures for each profiling method as follows: - HILIC-pos: Bacterial cultures (10 μL) were extracted with the addition of nine volumes of 74.9:24.9:0.2 v/v/v acetonitrile/methanol/formic acid containing stable isotope-labeled internal standards (valine-d8, Isotec; and phenylalanine-d8, Cambridge Isotope Laboratories). The samples were centrifuged (10 min, 9,000g, 4°C), and the supernatants (10 μL) injected directly onto column. - C8-pos: Bacterial cultures (10 μL) using 190 μL isopropanol containing 1-dodecanoyl-2-tridecanoyl-sn-glycero-3-phosphocholine as an internal standard (Avanti Polar Lipids; Alabaster, AL). After centrifugation (10 min, 9,000g, ambient temperature), supernatants (2 μL) were injected directly onto column. - C18-neg: Bacterial cultures (30 μL) were extracted using 90 μl methanol containing 15R-15-methyl Prostaglandin A2,15R-15-methyl Prostaglandin F2α, 15S-15-methyl Prostaglandin D2, 15S-15-methyl Prostaglandin E1, and 15S-15-methyl Prostaglandin E2 as internal standards (Cayman Chemical Co.) and centrifuged (10 min, 9,000g, 4°C). The supernatants (10 μL) were injected onto column. - HILIC-neg: Bacterial cultures (30 μL) were extracted with the addition of four volumes of 80% methanol containing inosine-15N4, thymine-d4 and glycocholate-d4 internal standards (Cambridge Isotope Laboratories). The samples were centrifuged (10 min, 9,000g, 4°C) and the supernatants 10 μL) were injected directly onto column.

Chromatography:

Chromatography ID:CH004870
Instrument Name:Agilent 1290 Infinity II
Column Name:Waters Atlantis HILIC (150 x 2mm, 3um)
Column Temperature:30°C
Flow Gradient:Isocratically with 5% mobile phase A for 1 minute followed by a linear gradient to 40% mobile phase B over 10 minutes
Flow Rate:250 µL/min
Solvent A:100% Water; 10 mM Ammonium formate; 0.1% Formic acid
Solvent B:100% Acetonitrile; 0.1% Formic acid
Chromatography Type:HILIC
  
Chromatography ID:CH004871
Instrument Name:Agilent 1290 Infinity II
Column Name:Waters Acquity BEH C8 (100 x 2.1mm, 1.7um)
Column Temperature:40°C
Flow Gradient:The column was eluted at a flow rate of 450 µL/min isocratically for 1 minute at 80% mobile phase A, followed by a linear gradient to 80% mobile-phase B over 2 minutes, a linear gradient to 100% mobile phase B over 7 minutes, and then 3 minutes at 100% mobile-phase B.
Flow Rate:450 µL/min
Solvent A:95% Water/5% Methanol; 10 mM Ammonium acetate; 0.1% Acetic acid
Solvent B:100% Methanol; 0.1% Acetic acid
Chromatography Type:Reversed phase
  
Chromatography ID:CH004872
Instrument Name:Shimadzu Nexera X2
Column Name:Waters ACQUITY UPLC BEH C18 (150 x 1.7mm,2.1um)
Column Temperature:45°C
Flow Gradient:The column was eluted isocratically at a flow rate of 450 µL/min with 20% mobile phase A for 3 minutes followed by a linear gradient to 100% mobile phase B over 12 minutes.
Flow Rate:450 µL/min
Solvent A:100% Water; 0.01% Formic acid
Solvent B:100% Acetonitrile; 0.01% Acetic acid
Chromatography Type:Reversed phase

Analysis:

Analysis ID:AN006421
Analysis Type:MS
Chromatography ID:CH004870
Num Factors:168
Num Metabolites:338
Has Mz:1
Has Rt:1
Rt Units:Minutes
Results File:ST003911_AN006421_Results.txt
Units:Abudances
  
Analysis ID:AN006422
Analysis Type:MS
Chromatography ID:CH004871
Num Factors:168
Num Metabolites:72
Has Mz:1
Has Rt:1
Rt Units:Minutes
Results File:ST003911_AN006422_Results.txt
Units:Abudances
  
Analysis ID:AN006423
Analysis Type:MS
Chromatography ID:CH004872
Num Factors:168
Num Metabolites:83
Has Mz:1
Has Rt:1
Rt Units:Minutes
Results File:ST003911_AN006423_Results.txt
Units:Abudances
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