Summary of Study ST003132

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 PR001945. The data can be accessed directly via it's Project DOI: 10.21228/M89M7J 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	ST003132
Study Title	Untargeted Metabolomics on Mouse colonic mucosa
Study Type	Mouse
Study Summary	While there is strong evidence for interactions between the microbiota-gut-brain axis and host physiology in the context of chronic stress, limited research has investigated the role of the microbiome in host response to acute stress. Determining the underlying mechanisms by which stress-induced microbiota changes may provoke functional changes in the gut and brain is critical for developing future therapeutics to alleviate the adverse consequences of traumatic stress. Here, we aimed to identify a biological signature of gut metabolites that are significantly altered following exposure to acute restraint stress. Adult male C57Bl/6 conventional, germ-free and colonized germ-free mice underwent a 15-minute restraint stress exposure. Caecal contents were collected from naïve mice and stressed mice, either immediately or 45 minutes following stress. Colonic mucosa underwent untargeted metabolomics analysis.
Institute	University College Cork
Department	Psychiatry
Laboratory	Microbiota-Gut-Brain Axis Group
Last Name	Clarke
First Name	Gerard
Address	Gaol Walk, Cork
Email	G.Clarke@ucc.ie
Phone	+353-21-4901408
Submit Date	2024-03-19
Num Groups	9
Total Subjects	63
Num Males	63
Analysis Type Detail	Other
Release Date	2024-03-22
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR001945
Project DOI:	doi: 10.21228/M89M7J
Project Title:	The microbiota drives diurnal rhythms in tryptophan metabolism in the stressed gut
Project Type:	Untargeted metabolomics by Metabolon
Project Summary:	Adult male C57Bl/6 conventional, germ-free and colonized germ-free mice underwent a 15-minute restraint stress exposure. Caecal contents and colonic mucosal scrapings were collected from naïve mice and stressed mice, either immediately or 45 minutes following stress. Caecal contents and colonic mucosal scrapings underwent untargeted metabolomics analysis.
Institute:	University College Cork
Department:	Psychiatry
Laboratory:	Microbiota-Gut-Brain Axis Group
Last Name:	Clarke
First Name:	Gerard
Address:	Gaol Walk, Cork
Email:	G.Clarke@ucc.ie
Phone:	+353-21-4901408
Funding Source:	The research was conducted in the APC Microbiome Ireland which is funded by Science Foundation Ireland (SFI/12/RC/2273). This project in part is a collaborative agreement (FA9550-17-1-0016) funded by European Office of Aerospace Research and Development, Air Force Office of Scientific Research and 711 Human Performance Wing, Air Force Research Laboratory.

Subject:

Subject ID:	SU003249
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Genotype Strain:	C57Bl/6J
Age Or Age Range:	8-10 weeks
Weight Or Weight Range:	20-29g
Gender:	Male
Animal Animal Supplier:	Taconic
Animal Housing:	Gnotobiotic isolators/conventional housing
Animal Light Cycle:	12/12
Animal Feed:	RM1A (P) and RM3A (P) from Special Diet Services

Factors:

Subject type: Mammal; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id	local_sample_id	Sample source	Factor
SA339239	ColGF8.5	Colonic mucosa	ColGF_0min
SA339240	ColGF8.6	Colonic mucosa	ColGF_0min
SA339241	ColGF8.4	Colonic mucosa	ColGF_0min
SA339242	ColGF8.2	Colonic mucosa	ColGF_0min
SA339243	ColGF8.1	Colonic mucosa	ColGF_0min
SA339244	ColGF8.7	Colonic mucosa	ColGF_0min
SA339245	ColGF8.3	Colonic mucosa	ColGF_0min
SA339246	ColGF9.6	Colonic mucosa	ColGF_45min
SA339247	ColGF9.7	Colonic mucosa	ColGF_45min
SA339248	ColGF9.5	Colonic mucosa	ColGF_45min
SA339249	ColGF9.4	Colonic mucosa	ColGF_45min
SA339250	ColGF9.3	Colonic mucosa	ColGF_45min
SA339251	ColGF9.1	Colonic mucosa	ColGF_45min
SA339252	ColGF9.2	Colonic mucosa	ColGF_45min
SA339253	ColGF7.3	Colonic mucosa	ColGF_Pre
SA339254	ColGF7.1	Colonic mucosa	ColGF_Pre
SA339255	ColGF7.4	Colonic mucosa	ColGF_Pre
SA339256	ColGF7.2	Colonic mucosa	ColGF_Pre
SA339257	ColGF7.6	Colonic mucosa	ColGF_Pre
SA339258	ColGF7.5	Colonic mucosa	ColGF_Pre
SA339259	ColGF7.7	Colonic mucosa	ColGF_Pre
SA339260	Conv5.3	Colonic mucosa	Conv_0min
SA339261	Conv5.2	Colonic mucosa	Conv_0min
SA339262	Conv5.5	Colonic mucosa	Conv_0min
SA339263	Conv5.7	Colonic mucosa	Conv_0min
SA339264	Conv5.1	Colonic mucosa	Conv_0min
SA339265	Conv5.6	Colonic mucosa	Conv_0min
SA339266	Conv5.4	Colonic mucosa	Conv_0min
SA339267	Conv6.3	Colonic mucosa	Conv_45min
SA339268	Conv6.2	Colonic mucosa	Conv_45min
SA339269	Conv6.4	Colonic mucosa	Conv_45min
SA339270	Conv6.6	Colonic mucosa	Conv_45min
SA339271	Conv6.7	Colonic mucosa	Conv_45min
SA339272	Conv6.1	Colonic mucosa	Conv_45min
SA339273	Conv6.5	Colonic mucosa	Conv_45min
SA339274	Conv4.3	Colonic mucosa	Conv_Pre
SA339275	Conv4.1	Colonic mucosa	Conv_Pre
SA339276	Conv4.7	Colonic mucosa	Conv_Pre
SA339277	Conv4.6	Colonic mucosa	Conv_Pre
SA339278	Conv4.2	Colonic mucosa	Conv_Pre
SA339279	Conv4.4	Colonic mucosa	Conv_Pre
SA339280	Conv4.5	Colonic mucosa	Conv_Pre
SA339281	GF2.4	Colonic mucosa	GF_0min
SA339282	GF2.5	Colonic mucosa	GF_0min
SA339283	GF2.6	Colonic mucosa	GF_0min
SA339284	GF2.3	Colonic mucosa	GF_0min
SA339285	GF2.7	Colonic mucosa	GF_0min
SA339286	GF2.2	Colonic mucosa	GF_0min
SA339287	GF2.1	Colonic mucosa	GF_0min
SA339288	GF3.5	Colonic mucosa	GF_45min
SA339289	GF3.7	Colonic mucosa	GF_45min
SA339290	GF3.1	Colonic mucosa	GF_45min
SA339291	GF3.4	Colonic mucosa	GF_45min
SA339292	GF3.6	Colonic mucosa	GF_45min
SA339293	GF3.3	Colonic mucosa	GF_45min
SA339294	GF3.2	Colonic mucosa	GF_45min
SA339295	GF1.3	Colonic mucosa	GF_Pre
SA339296	GF1.2	Colonic mucosa	GF_Pre
SA339297	GF1.4	Colonic mucosa	GF_Pre
SA339298	GF1.1	Colonic mucosa	GF_Pre
SA339299	GF1.7	Colonic mucosa	GF_Pre
SA339300	GF1.6	Colonic mucosa	GF_Pre
SA339301	GF1.5	Colonic mucosa	GF_Pre

Showing results 1 to 63 of 63

Showing results 1 to 63 of 63

Collection:

Collection ID:	CO003242
Collection Summary:	Upon entering the cull room, mice were immediately decapitated, trunk blood collected, and tissues harvested. Caecal contents and intestinal samples were manually dissected and stored in PCR-grade microfuge tubes at -80°C until analyses.
Sample Type:	Colon
Storage Conditions:	-80?

Treatment:

Treatment ID:	TR003258
Treatment Summary:	All animal work carried was approved by the Animal Experimentation Ethics Committee of University College Cork and Health Products Regulatory Authority (HPRA) before beginning this study. All experimentation was carried out in accordance with European Directive 2010/63/EU and was approved by both the Animal Experimentation Ethics Committee of University College Cork (Project Authorization AE19130/P160) and United States Air Force Surgeon General's Office of Research Oversight and Compliance. C57/BL6 mice breeding pairs were acquired from Taconic Biosciences, and F1-generation male and female offspring were used in all experiments. Germ-free, ex-germ-free, and conventional mice were housed 2-4 mice/cage under a 12-hour light/dark cycle and maintained on ad libitum autoclaved water and autoclaved, pelleted diet (Special Diet Services). Housing conditions for germ-free, conventional, and colonized germ-free adhered to the same environmental conditions of temperature (21 ± 1°C) and humidity (55%-60%). Germ-free mice were housed in gnotobiotic flexible-film isolators. Colonized germ-free mice were born and maintained as germ-free mice in gnotobiotic flexible-film isolators until postnatal day 21 when they were removed from the isolators and, for the remaining duration of this study, re-located to the standard animal facility and housed in wire-top cages that contained used-bedding from age- and sex-matched conventional mice. The acute restraint stress procedure was performed using a clean perforated polypropylene screw-cap 50 mL conical tubes. Cages were randomly assigned to either non-stress or stress groups. Each mouse that underwent stress was placed into the 50 mL tube and restrained for 15 minutes.

Treatment ID:

TR003258

Treatment Summary:

All animal work carried was approved by the Animal Experimentation Ethics Committee of University College Cork and Health Products Regulatory Authority (HPRA) before beginning this study. All experimentation was carried out in accordance with European Directive 2010/63/EU and was approved by both the Animal Experimentation Ethics Committee of University College Cork (Project Authorization AE19130/P160) and United States Air Force Surgeon General's Office of Research Oversight and Compliance. C57/BL6 mice breeding pairs were acquired from Taconic Biosciences, and F1-generation male and female offspring were used in all experiments. Germ-free, ex-germ-free, and conventional mice were housed 2-4 mice/cage under a 12-hour light/dark cycle and maintained on ad libitum autoclaved water and autoclaved, pelleted diet (Special Diet Services). Housing conditions for germ-free, conventional, and colonized germ-free adhered to the same environmental conditions of temperature (21 ± 1°C) and humidity (55%-60%). Germ-free mice were housed in gnotobiotic flexible-film isolators. Colonized germ-free mice were born and maintained as germ-free mice in gnotobiotic flexible-film isolators until postnatal day 21 when they were removed from the isolators and, for the remaining duration of this study, re-located to the standard animal facility and housed in wire-top cages that contained used-bedding from age- and sex-matched conventional mice. The acute restraint stress procedure was performed using a clean perforated polypropylene screw-cap 50 mL conical tubes. Cages were randomly assigned to either non-stress or stress groups. Each mouse that underwent stress was placed into the 50 mL tube and restrained for 15 minutes.

Sample Preparation:

Sampleprep ID:	SP003256
Sampleprep Summary:	Samples were prepared using the automated MicroLab STAR® system from Hamilton Company. Several recovery standards were added prior to the first step in the extraction process for QC purposes. To remove protein, dissociate small molecules bound to protein or trapped in the precipitated protein matrix, and to recover chemically diverse metabolites, proteins were precipitated with methanol under vigorous shaking for 2 min (Glen Mills GenoGrinder 2000) followed by centrifugation. The resulting extract was divided into five fractions: two for analysis by two separate reverse phase (RP)/UPLC-MS/MS methods with positive ion mode electrospray ionization (ESI), one for analysis by RP/UPLC-MS/MS with negative ion mode ESI, one for analysis by HILIC/UPLC-MS/MS with negative ion mode ESI, and one sample was reserved for backup. Samples were placed briefly on a TurboVap® (Zymark) to remove the organic solvent. The sample extracts were stored overnight under nitrogen before preparation for analysis.

Sampleprep ID:

SP003256

Sampleprep Summary:

Samples were prepared using the automated MicroLab STAR® system from Hamilton Company. Several recovery standards were added prior to the first step in the extraction process for QC purposes. To remove protein, dissociate small molecules bound to protein or trapped in the precipitated protein matrix, and to recover chemically diverse metabolites, proteins were precipitated with methanol under vigorous shaking for 2 min (Glen Mills GenoGrinder 2000) followed by centrifugation. The resulting extract was divided into five fractions: two for analysis by two separate reverse phase (RP)/UPLC-MS/MS methods with positive ion mode electrospray ionization (ESI), one for analysis by RP/UPLC-MS/MS with negative ion mode ESI, one for analysis by HILIC/UPLC-MS/MS with negative ion mode ESI, and one sample was reserved for backup. Samples were placed briefly on a TurboVap® (Zymark) to remove the organic solvent. The sample extracts were stored overnight under nitrogen before preparation for analysis.

Combined analysis:

Analysis ID	AN005139	AN005140	AN005141	AN005142
Analysis type	MS	MS	MS	MS
Chromatography type	Reversed phase	Reversed phase	Reversed phase	HILIC
Chromatography system	Waters Acquity	Waters Acquity	Waters Acquity	Waters Acquity
Column	Waters Acquity BEH C18 (100 x 2.1mm, 1.7um)	Waters Acquity BEH C18 (100 x 2.1mm, 1.7um)	Waters Acquity BEH C18 (100 x 2.1mm, 1.7um)	Waters Acquity BEH Amide (150 x 2.1mm, 1.7um)
MS Type	Other	Other	Other	Other
MS instrument type	Orbitrap	Orbitrap	Orbitrap	Orbitrap
MS instrument name	Thermo Q Exactive Orbitrap	Thermo Q Exactive Orbitrap	Thermo Q Exactive Orbitrap	Thermo Q Exactive Orbitrap
Ion Mode	POSITIVE	POSITIVE	NEGATIVE	NEGATIVE
Units	Raw Peak Area	Raw Peak Area	Raw Peak Area	Raw Peak Area

Chromatography:

Chromatography ID:	CH003889
Chromatography Summary:	Low pH polar (LC/MS Pos early)
Instrument Name:	Waters Acquity
Column Name:	Waters Acquity BEH C18 (100 x 2.1mm, 1.7um)
Column Temperature:	40-50
Flow Gradient:	Linear gradient from 5% B to 80% B over 3.35 minutes
Flow Rate:	0.35 mL/min
Solvent A:	100% water; 0.1% formic acid; 0.05% PFPA, pH ~2.5
Solvent B:	100% methanol; 0.1% formic acid; 0.05% PFPA, pH ~2.5
Chromatography Type:	Reversed phase

Chromatography ID:	CH003890
Chromatography Summary:	Low pH Lipophilic (LC/MS Pos late)
Instrument Name:	Waters Acquity
Column Name:	Waters Acquity BEH C18 (100 x 2.1mm, 1.7um)
Column Temperature:	40-50
Flow Gradient:	Linear gradient from 40% B to 99.5% B over 1.0 minute, hold 99.5% B for 2.4 minutes.
Flow Rate:	0.60 mL/min
Solvent A:	100% water; 0.1% formic acid; 0.05% PFPA, pH ~2.5
Solvent B:	50% methanol/50% acetonitrile; 0.1% formic acid; 0.05% PFPA, pH ~2.5
Chromatography Type:	Reversed phase

Chromatography ID:	CH003891
Chromatography Summary:	High pH (LC/MS Neg)
Instrument Name:	Waters Acquity
Column Name:	Waters Acquity BEH C18 (100 x 2.1mm, 1.7um)
Column Temperature:	40-50
Flow Gradient:	Linear gradient from 0.5 to 70% B over 4.0 minutes, then rapid gradient to 99% B in 0.5 minutes.
Flow Rate:	0.35 mL/min
Solvent A:	100% water; 6.5 mM ammonium bicarbonate, pH 8
Solvent B:	95% methanol/5% water; 6.5 mM ammonium bicarbonate
Chromatography Type:	Reversed phase

Chromatography ID:	CH003892
Chromatography Summary:	HILIC (LC/MS Polar Neg)
Instrument Name:	Waters Acquity
Column Name:	Waters Acquity BEH Amide (150 x 2.1mm, 1.7um)
Column Temperature:	40-50
Flow Gradient:	Linear gradient from 5% B to 50% B in 3.5 minutes, then linear gradient from 50% B to 95% B in 2 minutes.
Flow Rate:	0.50 mL/min
Solvent A:	15% water/5% methanol/80% acetonitrile; 10 mM ammonium formate, (effective pH 10.16 with NH4OH)
Solvent B:	50% water/50% acetonitrile; 10 mM ammonium formate, (effective pH 10.60 with NH4OH)
Chromatography Type:	HILIC

MS:

MS ID:	MS004875
Analysis ID:	AN005139
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	Other
MS Comments:	Metabolon (LC/MS Pos early)
Ion Mode:	POSITIVE

MS ID:	MS004876
Analysis ID:	AN005140
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	Other
MS Comments:	Metabolon (LC/MS Pos late)
Ion Mode:	POSITIVE

MS ID:	MS004877
Analysis ID:	AN005141
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	Other
MS Comments:	Metabolon (LC/MS Neg)
Ion Mode:	NEGATIVE

MS ID:	MS004878
Analysis ID:	AN005142
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Orbitrap
MS Type:	Other
MS Comments:	Metabolon (LC/MS Polar)
Ion Mode:	NEGATIVE