Summary of Study ST003910

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	ST003910
Study Title	Molecular fingerprint inference reveals bioactive lipids and microbial metabolites in colitis. Study 3.
Study Type	Study 3: Profiling of bacterial cultures for: Bifidobacterium longum, Clostridium sporogenes, Clostridium innocuum, Escherichia coli BL21(DE3), Streptococcis infantis, Streptococcus parasanguinis.
Study Summary	Untargeted 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 Name	Avila-Pacheco
First Name	Julian
Address	415 Main Street
Email	jravilap@broadinstitute.org
Phone	(617) 714-1729
Submit Date	2025-05-08
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:	SU004045
Subject Type:	Bacteria
Subject Species:	Bifidobacterium longum; Clostridium sporogenes; Clostridium innocuum; Escherichia coli BL21(DE3); Streptococcus infantis; Streptococcus parasanguinis
Taxonomy ID:	216816; 1509; 1522; 469008; 68892; 1318
Genotype Strain:	Bifidobacterium longum, Clostridium sporogenes, Clostridium innocuum, Escherichia coli BL21(DE3), Streptococcis infantis, Streptococcus parasanguinis).

Factors:

Subject type: Bacteria; Subject species: Bifidobacterium longum; Clostridium sporogenes; Clostridium innocuum; Escherichia coli BL21(DE3); Streptococcus infantis; Streptococcus parasanguinis (Factor headings shown in green)

mb_sample_id	local_sample_id	Species	Media	Replicate	Time
SA430140	BL_CHG_0hr_1	Bifidobacterium longum	CHG	R1	0
SA430141	BL_CHG_24hr_1	Bifidobacterium longum	CHG	R1	24
SA430142	BL_CHG_48hr_1	Bifidobacterium longum	CHG	R1	48
SA430143	BL_CHG_6hr_1	Bifidobacterium longum	CHG	R1	6
SA430144	BL_CHG_0hr_2	Bifidobacterium longum	CHG	R2	0
SA430145	BL_CHG_24hr_2	Bifidobacterium longum	CHG	R2	24
SA430146	BL_CHG_48hr_2	Bifidobacterium longum	CHG	R2	48
SA430147	BL_CHG_6hr_2-A	Bifidobacterium longum	CHG	R2	6
SA430148	BL_CHG_0hr_3	Bifidobacterium longum	CHG	R3	0
SA430149	BL_CHG_24hr_3	Bifidobacterium longum	CHG	R3	24
SA430150	BL_CHG_48hr_3	Bifidobacterium longum	CHG	R3	48
SA430151	BL_CHG_6hr_3	Bifidobacterium longum	CHG	R3	6
SA430152	BL_Gifu_0hr_1	Bifidobacterium longum	Gifu	R1	0
SA430153	BL_Gifu_24hr_1	Bifidobacterium longum	Gifu	R1	24
SA430154	BL_Gifu_48hr_1	Bifidobacterium longum	Gifu	R1	48
SA430155	BL_Gifu_6hr_1	Bifidobacterium longum	Gifu	R1	6
SA430156	BL_Gifu_0hr_2	Bifidobacterium longum	Gifu	R2	0
SA430157	BL_Gifu_24hr_2	Bifidobacterium longum	Gifu	R2	24
SA430158	BL_Gifu_48hr_2	Bifidobacterium longum	Gifu	R2	48
SA430159	BL_Gifu_6hr_2	Bifidobacterium longum	Gifu	R2	6
SA430160	BL_Gifu_0hr_3	Bifidobacterium longum	Gifu	R3	0
SA430161	BL_Gifu_24hr_3	Bifidobacterium longum	Gifu	R3	24
SA430162	BL_Gifu_48hr_3	Bifidobacterium longum	Gifu	R3	48
SA430163	BL_Gifu_6hr_3	Bifidobacterium longum	Gifu	R3	6
SA430164	BL_Mega_0hr_1	Bifidobacterium longum	Mega	R1	0
SA430165	BL_Mega_24hr_1	Bifidobacterium longum	Mega	R1	24
SA430166	BL_Mega_48hr_1	Bifidobacterium longum	Mega	R1	48
SA430167	BL_Mega_6hr_1	Bifidobacterium longum	Mega	R1	6
SA430168	BL_Mega_0hr_2	Bifidobacterium longum	Mega	R2	0
SA430169	BL_Mega_24hr_2	Bifidobacterium longum	Mega	R2	24
SA430170	BL_Mega_48hr_2	Bifidobacterium longum	Mega	R2	48
SA430171	BL_Mega_6hr_2	Bifidobacterium longum	Mega	R2	6
SA430172	BL_Mega_0hr_3	Bifidobacterium longum	Mega	R3	0
SA430173	BL_Mega_24hr_3	Bifidobacterium longum	Mega	R3	24
SA430174	BL_Mega_48hr_3	Bifidobacterium longum	Mega	R3	48
SA430175	BL_Mega_6hr_3	Bifidobacterium longum	Mega	R3	6
SA430176	CI_CHG_0hr_1	Clostridium innocuum	CHG	R1	0
SA430177	CI_CHG_24hr_1	Clostridium innocuum	CHG	R1	24
SA430178	CI_CHG_48hr_1	Clostridium innocuum	CHG	R1	48
SA430179	CI_CHG_6hr_1	Clostridium innocuum	CHG	R1	6
SA430180	CI_CHG_0hr_2	Clostridium innocuum	CHG	R2	0
SA430181	CI_CHG_24hr_2	Clostridium innocuum	CHG	R2	24
SA430182	CI_CHG_48hr_2	Clostridium innocuum	CHG	R2	48
SA430183	CI_CHG_6hr_2	Clostridium innocuum	CHG	R2	6
SA430184	CI_CHG_0hr_3	Clostridium innocuum	CHG	R3	0
SA430185	CI_CHG_24hr_3	Clostridium innocuum	CHG	R3	24
SA430186	CI_CHG_48hr_3	Clostridium innocuum	CHG	R3	48
SA430187	CI_CHG_6hr_3	Clostridium innocuum	CHG	R3	6
SA430188	CI_Gifu_0hr_1	Clostridium innocuum	Gifu	R1	0
SA430189	CI_Gifu_24hr_1	Clostridium innocuum	Gifu	R1	24
SA430190	CI_Gifu_48hr_1	Clostridium innocuum	Gifu	R1	48
SA430191	CI_Gifu_6hr_1	Clostridium innocuum	Gifu	R1	6
SA430192	CI_Gifu_0hr_2	Clostridium innocuum	Gifu	R2	0
SA430193	CI_Gifu_24hr_2	Clostridium innocuum	Gifu	R2	24
SA430194	CI_Gifu_48hr_2	Clostridium innocuum	Gifu	R2	48
SA430195	CI_Gifu_6hr_2	Clostridium innocuum	Gifu	R2	6
SA430196	CI_Gifu_0hr_3	Clostridium innocuum	Gifu	R3	0
SA430197	CI_Gifu_24hr_3	Clostridium innocuum	Gifu	R3	24
SA430198	CI_Gifu_48hr_3	Clostridium innocuum	Gifu	R3	48
SA430199	CI_Gifu_6hr_3	Clostridium innocuum	Gifu	R3	6
SA430200	CI_Mega_0hr_1	Clostridium innocuum	Mega	R1	0
SA430201	CI_Mega_24hr_1	Clostridium innocuum	Mega	R1	24
SA430202	CI_Mega_48hr_1	Clostridium innocuum	Mega	R1	48
SA430203	CI_Mega_6hr_1	Clostridium innocuum	Mega	R1	6
SA430204	CI_Mega_0hr_2	Clostridium innocuum	Mega	R2	0
SA430205	CI_Mega_24hr_2	Clostridium innocuum	Mega	R2	24
SA430206	CI_Mega_48hr_2	Clostridium innocuum	Mega	R2	48
SA430207	CI_Mega_6hr_2	Clostridium innocuum	Mega	R2	6
SA430208	CI_Mega_0hr_3	Clostridium innocuum	Mega	R3	0
SA430209	CI_Mega_24hr_3	Clostridium innocuum	Mega	R3	24
SA430210	CI_Mega_48hr_3	Clostridium innocuum	Mega	R3	48
SA430211	CI_Mega_6hr_3	Clostridium innocuum	Mega	R3	6
SA430212	CS_CHG_24_1	Clostridium sporogenes	CHG	R1	24
SA430213	CS_CHG_24_2	Clostridium sporogenes	CHG	R2	24
SA430214	CS_CHG_24_3	Clostridium sporogenes	CHG	R3	24
SA430215	EC_CHG_24_1	Escherichia coli BL21(DE3)	CHG	R1	24
SA430216	EC_CHG_24_2	Escherichia coli BL21(DE3)	CHG	R2	24
SA430217	EC_CHG_24_3	Escherichia coli BL21(DE3)	CHG	R3	24
SA430218	Gifu_0hr_1	NA	Gifu	R1	0
SA430219	Gifu_24hr_1	NA	Gifu	R1	24
SA430220	Gifu_48hr_1	NA	Gifu	R1	48
SA430221	Gifu_6hr_1	NA	Gifu	R1	6
SA430222	Gifu_0hr_2	NA	Gifu	R2	0
SA430223	Gifu_24hr_2	NA	Gifu	R2	24
SA430224	Gifu_48hr_2	NA	Gifu	R2	48
SA430225	Gifu_6hr_2	NA	Gifu	R2	6
SA430226	Gifu_0hr_3	NA	Gifu	R3	0
SA430227	Gifu_24hr_3	NA	Gifu	R3	24
SA430228	Gifu_48hr_3	NA	Gifu	R3	48
SA430229	Gifu_6hr_3	NA	Gifu	R3	6
SA430230	Mega_0hr_1	NA	Mega	R1	0
SA430231	Mega_24hr_1	NA	Mega	R1	24
SA430232	Mega_48hr_1	NA	Mega	R1	48
SA430233	Mega_6hr_1	NA	Mega	R1	6
SA430234	Mega_0hr_2	NA	Mega	R2	0
SA430235	Mega_24hr_2	NA	Mega	R2	24
SA430236	Mega_48hr_2	NA	Mega	R2	48
SA430237	Mega_6hr_2	NA	Mega	R2	6
SA430238	Mega_0hr_3	NA	Mega	R3	0
SA430239	Mega_24hr_3	NA	Mega	R3	24

Showing page 1 of 2 Results: 1 2 Next Showing results 1 to 100 of 185

Collection:

Collection ID:	CO004038
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:	TR004054
Treatment Summary:	NA

Sample Preparation:

Sampleprep ID:	SP004051
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:

SP004051

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:	CH004867
Instrument Name:	Agilent 1290 Infinity II
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:	CH004868
Instrument Name:	Agilent 1290 Infinity II
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:	CH004869
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:	AN006418
Analysis Type:	MS
Chromatography ID:	CH004867
Num Factors:	185
Num Metabolites:	338
Has Mz:	1
Has Rt:	1
Rt Units:	Minutes
Results File:	ST003910_AN006418_Results.txt
Units:	Abudances

Analysis ID:	AN006419
Analysis Type:	MS
Chromatography ID:	CH004868
Num Factors:	185
Num Metabolites:	72
Has Mz:	1
Has Rt:	1
Rt Units:	Minutes
Results File:	ST003910_AN006419_Results.txt
Units:	Abudances

Analysis ID:	AN006420
Analysis Type:	MS
Chromatography ID:	CH004869
Num Factors:	185
Num Metabolites:	86
Has Mz:	1
Has Rt:	1
Rt Units:	Minutes
Results File:	ST003910_AN006420_Results.txt
Units:	Abudances