Summary of Study ST003799

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.

Study ID	ST003799
Study Title	Molecular fingerprint inference reveals bioactive lipids and microbial metabolites in colitis. Study 2.
Study Type	Bacterial cell cultures
Study 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	(617) 714-1729
Submit Date	2025-03-04
Raw Data Available	Yes
Raw Data File Type(s)	mzML, raw(Thermo)
Analysis Type Detail	LC-MS
Release Date	2025-08-04
Release Version	1

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:	SU003933
Subject Type:	Bacteria
Subject Species:	Dorea longicatena
Taxonomy ID:	88431
Genotype Strain:	Flavonifractor plautii, Dorea longicatena, Turicibacter sanguinis, Clostridium bolteae, Clostridium symbiosum, Clostridium innocuum, Clostridium clostridioforme, Streptococcus parasanguinis

Factors:

Subject type: Bacteria; Subject species: Dorea longicatena (Factor headings shown in green)

mb_sample_id	local_sample_id	Species	Media	Replicate
SA416010	CB-R1_CHG_24h	Clostridium bolteae	CHG	R1
SA416011	CB-R1_CHG_6h	Clostridium bolteae	CHG	R1
SA416012	CB-R1_CHG_48h	Clostridium bolteae	CHG	R1
SA416013	CB-R2_CHG_48h	Clostridium bolteae	CHG	R2
SA416014	CB-R2_CHG_24h	Clostridium bolteae	CHG	R2
SA416015	CB-R2_CHG_6h	Clostridium bolteae	CHG	R2
SA416016	CB-R3_CHG_24h	Clostridium bolteae	CHG	R3
SA416017	CB-R3_CHG_48h	Clostridium bolteae	CHG	R3
SA416018	CB-R3_CHG_6h	Clostridium bolteae	CHG	R3
SA416019	CB-R1_Gifu_6h	Clostridium bolteae	Gifu	R1
SA416020	CB-R1_Gifu_24h	Clostridium bolteae	Gifu	R1
SA416021	CB-R1_Gifu_48h	Clostridium bolteae	Gifu	R1
SA416022	CB-R2_Gifu_48h	Clostridium bolteae	Gifu	R2
SA416023	CB-R2_Gifu_6h	Clostridium bolteae	Gifu	R2
SA416024	CB-R2_Gifu_24h	Clostridium bolteae	Gifu	R2
SA416025	CB-R3_Gifu_6h	Clostridium bolteae	Gifu	R3
SA416026	CB-R3_Gifu_24h	Clostridium bolteae	Gifu	R3
SA416027	CB-R3_Gifu_48h	Clostridium bolteae	Gifu	R3
SA416028	CB-R1_Mega_6h	Clostridium bolteae	Mega	R1
SA416029	CB-R1_Mega_24h	Clostridium bolteae	Mega	R1
SA416030	CB-R1_Mega_48h	Clostridium bolteae	Mega	R1
SA416031	CB-R2_Mega_6h	Clostridium bolteae	Mega	R2
SA416032	CB-R2_Mega_48h	Clostridium bolteae	Mega	R2
SA416033	CB-R2_Mega_24h	Clostridium bolteae	Mega	R2
SA416034	CB-R3_Mega_6h	Clostridium bolteae	Mega	R3
SA416035	CB-R3_Mega_24h	Clostridium bolteae	Mega	R3
SA416036	CB-R3_Mega_48h	Clostridium bolteae	Mega	R3
SA416037	CC-R1_CHG_24h	Clostridium clostridioforme	CHG	R1
SA416038	CC-R1_CHG_48h	Clostridium clostridioforme	CHG	R1
SA416039	CC-R1_CHG_6h	Clostridium clostridioforme	CHG	R1
SA416040	CC-R2_CHG_24h	Clostridium clostridioforme	CHG	R2
SA416041	CC-R2_CHG_48h	Clostridium clostridioforme	CHG	R2
SA416042	CC-R2_CHG_6h	Clostridium clostridioforme	CHG	R2
SA416043	CC-R3_CHG_24h	Clostridium clostridioforme	CHG	R3
SA416044	CC-R3_CHG_48h	Clostridium clostridioforme	CHG	R3
SA416045	CC-R3_CHG_6h	Clostridium clostridioforme	CHG	R3
SA416046	CC-R1_Gifu_6h	Clostridium clostridioforme	Gifu	R1
SA416047	CC-R1_Gifu_48h	Clostridium clostridioforme	Gifu	R1
SA416048	CC-R1_Gifu_24h	Clostridium clostridioforme	Gifu	R1
SA416049	CC-R2_Gifu_6h	Clostridium clostridioforme	Gifu	R2
SA416050	CC-R2_Gifu_24h	Clostridium clostridioforme	Gifu	R2
SA416051	CC-R2_Gifu_48h	Clostridium clostridioforme	Gifu	R2
SA416052	CC-R3_Gifu_48h	Clostridium clostridioforme	Gifu	R3
SA416053	CC-R3_Gifu_24h	Clostridium clostridioforme	Gifu	R3
SA416054	CC-R3_Gifu_6h	Clostridium clostridioforme	Gifu	R3
SA416055	CC-R1_Mega_24h	Clostridium clostridioforme	Mega	R1
SA416056	CC-R1_Mega_6h	Clostridium clostridioforme	Mega	R1
SA416057	CC-R1_Mega_48h	Clostridium clostridioforme	Mega	R1
SA416058	CC-R2_Mega_24h	Clostridium clostridioforme	Mega	R2
SA416059	CC-R2_Mega_6h	Clostridium clostridioforme	Mega	R2
SA416060	CC-R2_Mega_48h	Clostridium clostridioforme	Mega	R2
SA416061	CC-R3_Mega_48h	Clostridium clostridioforme	Mega	R3
SA416062	CC-R3_Mega_24h	Clostridium clostridioforme	Mega	R3
SA416063	CC-R3_Mega_6h	Clostridium clostridioforme	Mega	R3
SA416064	CI-R1_CHG_24h	Clostridium innocuum	CHG	R1
SA416065	CI-R1_CHG_6h	Clostridium innocuum	CHG	R1
SA416066	CI-R1_CHG_48h	Clostridium innocuum	CHG	R1
SA416067	CI-R2_CHG_6h	Clostridium innocuum	CHG	R2
SA416068	CI-R2_CHG_24h	Clostridium innocuum	CHG	R2
SA416069	CI-R2_CHG_48h	Clostridium innocuum	CHG	R2
SA416070	CI-R3_CHG_6h	Clostridium innocuum	CHG	R3
SA416071	CI-R3_CHG_48h	Clostridium innocuum	CHG	R3
SA416072	CI-R3_CHG_24h	Clostridium innocuum	CHG	R3
SA416073	CI-R1_Mega_24h	Clostridium innocuum	Mega	R1
SA416074	CI-R1_Mega_48h	Clostridium innocuum	Mega	R1
SA416075	CI-R1_Mega_6h	Clostridium innocuum	Mega	R1
SA416076	CI-R2_Mega_24h	Clostridium innocuum	Mega	R2
SA416077	CI-R2_Mega_48h	Clostridium innocuum	Mega	R2
SA416078	CI-R2_Mega_6h	Clostridium innocuum	Mega	R2
SA416079	CI-R3_Mega_48h	Clostridium innocuum	Mega	R3
SA416080	CI-R3_Mega_24h	Clostridium innocuum	Mega	R3
SA416081	CI-R3_Mega_6h	Clostridium innocuum	Mega	R3
SA416082	CS-R1_CHG_24h	Clostridium symbiosum	CHG	R1
SA416083	CS-R1_CHG_6h	Clostridium symbiosum	CHG	R1
SA416084	CS-R1_CHG_48h	Clostridium symbiosum	CHG	R1
SA416085	CS-R2_CHG_24h	Clostridium symbiosum	CHG	R2
SA416086	CS-R2_CHG_6h	Clostridium symbiosum	CHG	R2
SA416087	CS-R2_CHG_48h	Clostridium symbiosum	CHG	R2
SA416088	CS-R3_CHG_24h	Clostridium symbiosum	CHG	R3
SA416089	CS-R3_CHG_6h	Clostridium symbiosum	CHG	R3
SA416090	CS-R3_CHG_48h	Clostridium symbiosum	CHG	R3
SA416091	CS-R1_Gifu_6h	Clostridium symbiosum	Gifu	R1
SA416092	CS-R1_Gifu_24h	Clostridium symbiosum	Gifu	R1
SA416093	CS-R1_Gifu_48h	Clostridium symbiosum	Gifu	R1
SA416094	CS-R2_Gifu_24h	Clostridium symbiosum	Gifu	R2
SA416095	CS-R2_Gifu_6h	Clostridium symbiosum	Gifu	R2
SA416096	CS-R2_Gifu_48h	Clostridium symbiosum	Gifu	R2
SA416097	CS-R3_Gifu_6h	Clostridium symbiosum	Gifu	R3
SA416098	CS-R3_Gifu_24h	Clostridium symbiosum	Gifu	R3
SA416099	CS-R3_Gifu_48h	Clostridium symbiosum	Gifu	R3
SA416100	CS-R1_Mega_24h	Clostridium symbiosum	Mega	R1
SA416101	CS-R1_Mega_48h	Clostridium symbiosum	Mega	R1
SA416102	CS-R1_Mega_6h	Clostridium symbiosum	Mega	R1
SA416103	CS-R2_Mega_48h	Clostridium symbiosum	Mega	R2
SA416104	CS-R2_Mega_6h	Clostridium symbiosum	Mega	R2
SA416105	CS-R2_Mega_24h	Clostridium symbiosum	Mega	R2
SA416106	CS-R3_Mega_6h	Clostridium symbiosum	Mega	R3
SA416107	CS-R3_Mega_48h	Clostridium symbiosum	Mega	R3
SA416108	CS-R3_Mega_24h	Clostridium symbiosum	Mega	R3
SA416109	DL-R1_CHG_6h	Dorea longicatena	CHG	R1

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Collection:

Collection ID:	CO003926
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:	TR003942
Treatment Summary:	NA

Sample Preparation:

Sampleprep ID:	SP003939
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.

Sampleprep ID:

SP003939

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:	CH004735
Instrument Name:	Shimadzu Nexera X2
Column Name:	Waters Atlantis HILIC (150 x 2 mm, 3 µm)
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:	CH004736
Instrument Name:	Shimadzu Nexera X2
Column Name:	Waters Acquity BEH C8 (100 x 2.1 mm, 1.7 µm)
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:	CH004737
Instrument Name:	Shimadzu Nexera X2
Column Name:	Phenomenex Luna NH2 (150 x 2.1 mm, 3 µm)
Column Temperature:	30°C
Flow Gradient:	The column was eluted with initial conditions of 10% mobile phase A and 90% mobile phase B followed by a 10 min linear gradient to 100% mobile phase A.
Flow Rate:	400 µL/min
Solvent A:	100% water; 20 mM ammonium acetate; 20 mM ammonium hydroxide
Solvent B:	75% acetonitrile/25% methanol; 10 mM ammonium hydroxide
Chromatography Type:	HILIC

Chromatography ID:	CH004738
Instrument Name:	Shimadzu Nexera X2
Column Name:	Waters ACQUITY UPLC BEH C18 (150 x 1.7 mm,2.1 µm)
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:	AN006244
Analysis Type:	MS
Chromatography ID:	CH004735
Num Factors:	73
Num Metabolites:	199
Has Mz:	1
Has Rt:	1
Rt Units:	Minutes
Results File:	ST003799_AN006244_Results.txt
Units:	Abudances

Analysis ID:	AN006245
Analysis Type:	MS
Chromatography ID:	CH004736
Num Factors:	73
Num Metabolites:	36
Has Mz:	1
Has Rt:	1
Rt Units:	Minutes
Results File:	ST003799_AN006245_Results.txt
Units:	Abudances

Analysis ID:	AN006246
Analysis Type:	MS
Chromatography ID:	CH004737
Num Factors:	73
Num Metabolites:	91
Has Mz:	1
Has Rt:	1
Rt Units:	Minutes
Results File:	ST003799_AN006246_Results.txt
Units:	A

Analysis ID:	AN006247
Analysis Type:	MS
Chromatography ID:	CH004738
Num Factors:	73
Num Metabolites:	59
Has Mz:	1
Has Rt:	1
Rt Units:	Minutes
Results File:	ST003799_AN006247_Results.txt
Units:	Abudances