Summary of Study ST002465

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 PR001468. The data can be accessed directly via it's Project DOI: 10.21228/M8ZT5R 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	ST002465
Study Title	Map of microbially induced metabolic changes across diverse body sites in mice - Mouse Data
Study Summary	Tissue samples from contents along the intestinal tract and systemic sites in mice that did not have any bacteria (germ free) or colonized with a simplified microbiota or more complex microbiota.
Institute	University of Calgary
Department	Physiology and Pharmacology
Laboratory	McCoy
Last Name	Brown
First Name	Kirsty
Address	3330 Hospital Dr NW
Email	kirsty.brown12@gmail.com; kathy.mccoy@ucalgary.ca
Phone	2508692232
Submit Date	2023-02-04
Total Subjects	72
Num Males	36
Num Females	36
Raw Data Available	Yes
Raw Data File Type(s)	mzXML
Analysis Type Detail	LC-MS
Release Date	2023-02-05
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR001468
Project DOI:	doi: 10.21228/M8ZT5R
Project Title:	Microbiota alter the metabolome in an age and sex dependent manner in mice
Project Summary:	Tissue samples from contents along the intestinal tract and systemic sites in mice that did not have any bacteria (germ free) or colonized with a simplified microbiota or more complex microbiota.
Institute:	University of Calgary
Department:	Physiology and Pharmacology
Laboratory:	McCoy
Last Name:	BROWN
First Name:	KIRSTY
Address:	2500 University Dr NW, CALGARY, AB, T2N 1N4, Canada
Email:	kirsty.brown1@ucalgary.ca; Kathy.mccoy@ucalgary.ca
Phone:	2508692232
Funding Source:	Canadian Institute of Health Research, Natural Science and Engineering Research Council of Canada, Alberta Innovates Health Solutions
Publications:	Microbiota alter the metabolome in an age and sex dependent manner in mice
Contributors:	Kirsty Brown, Carolyn A. Thomson, Ryan Groves, Vina Fan, Ian A. Lewis, Kathy D. McCoy

Subject:

Subject ID:	SU002555
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Age Or Age Range:	3-12 weeks
Animal Animal Supplier:	In house breeding
Animal Housing:	Germ-free and gnotobiotic isolators for germ and gnotobiotic mice. Individually ventilated cages for specific pathogen free mice
Animal Light Cycle:	12 hours
Animal Feed:	LabDiet (Cat# 5010; Laboratory Autoclavable Rodent Diet)

Factors:

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

mb_sample_id	local_sample_id	Site	Microbiota	Age(weeks)	Sex
SA246529	cec_gf_12_F_1	Cecum_content	gf	12	F
SA246530	cec_gf_12_F_4	Cecum_content	gf	12	F
SA246531	cec_gf_12_F_3	Cecum_content	gf	12	F
SA246532	cec_gf_12_F_2	Cecum_content	gf	12	F
SA246533	cec_gf_12_M_4	Cecum_content	gf	12	M
SA246534	cec_gf_12_M_1	Cecum_content	gf	12	M
SA246535	cec_gf_12_M_3	Cecum_content	gf	12	M
SA246536	cec_gf_12_M_2	Cecum_content	gf	12	M
SA246537	cec_gf_3_F_4	Cecum_content	gf	3	F
SA246538	cec_gf_3_F_1	Cecum_content	gf	3	F
SA246539	cec_gf_3_F_3	Cecum_content	gf	3	F
SA246540	cec_gf_3_F_2	Cecum_content	gf	3	F
SA246541	cec_gf_3_M_4	Cecum_content	gf	3	M
SA246542	cec_gf_3_M_3	Cecum_content	gf	3	M
SA246543	cec_gf_3_M_2	Cecum_content	gf	3	M
SA246544	cec_gf_3_M_1	Cecum_content	gf	3	M
SA246545	cec_gf_8_F_4	Cecum_content	gf	8	F
SA246546	cec_gf_8_F_3	Cecum_content	gf	8	F
SA246547	cec_gf_8_F_2	Cecum_content	gf	8	F
SA246548	cec_gf_8_F_1	Cecum_content	gf	8	F
SA246549	cec_gf_8_M_4	Cecum_content	gf	8	M
SA246550	cec_gf_8_M_3	Cecum_content	gf	8	M
SA246551	cec_gf_8_M_1	Cecum_content	gf	8	M
SA246552	cec_gf_8_M_2	Cecum_content	gf	8	M
SA246553	cec_sdm_12_F_4	Cecum_content	sdm	12	F
SA246554	cec_sdm_12_F_1	Cecum_content	sdm	12	F
SA246555	cec_sdm_12_F_3	Cecum_content	sdm	12	F
SA246556	cec_sdm_12_F_2	Cecum_content	sdm	12	F
SA246557	cec_sdm_12_M_4	Cecum_content	sdm	12	M
SA246558	cec_sdm_12_M_1	Cecum_content	sdm	12	M
SA246559	cec_sdm_12_M_3	Cecum_content	sdm	12	M
SA246560	cec_sdm_12_M_2	Cecum_content	sdm	12	M
SA246561	cec_sdm_3_F_4	Cecum_content	sdm	3	F
SA246562	cec_sdm_3_F_3	Cecum_content	sdm	3	F
SA246563	cec_sdm_3_F_2	Cecum_content	sdm	3	F
SA246564	cec_sdm_3_F_1	Cecum_content	sdm	3	F
SA246565	cec_sdm_3_M_4	Cecum_content	sdm	3	M
SA246566	cec_sdm_3_M_3	Cecum_content	sdm	3	M
SA246567	cec_sdm_3_M_2	Cecum_content	sdm	3	M
SA246568	cec_sdm_3_M_1	Cecum_content	sdm	3	M
SA246569	cec_sdm_8_F_4	Cecum_content	sdm	8	F
SA246570	cec_sdm_8_F_3	Cecum_content	sdm	8	F
SA246571	cec_sdm_8_F_1	Cecum_content	sdm	8	F
SA246572	cec_sdm_8_F_2	Cecum_content	sdm	8	F
SA246573	cec_sdm_8_M_3	Cecum_content	sdm	8	M
SA246574	cec_sdm_8_M_4	Cecum_content	sdm	8	M
SA246575	cec_sdm_8_M_2	Cecum_content	sdm	8	M
SA246576	cec_sdm_8_M_1	Cecum_content	sdm	8	M
SA246577	cec_spf_12_F_3	Cecum_content	spf	12	F
SA246578	cec_spf_12_F_4	Cecum_content	spf	12	F
SA246579	cec_spf_12_F_2	Cecum_content	spf	12	F
SA246580	cec_spf_12_F_1	Cecum_content	spf	12	F
SA246581	cec_spf_12_M_4	Cecum_content	spf	12	M
SA246582	cec_spf_12_M_3	Cecum_content	spf	12	M
SA246583	cec_spf_12_M_2	Cecum_content	spf	12	M
SA246584	cec_spf_3_F_4	Cecum_content	spf	3	F
SA246585	cec_spf_3_F_3	Cecum_content	spf	3	F
SA246586	cec_spf_3_F_2	Cecum_content	spf	3	F
SA246587	cec_spf_3_F_1	Cecum_content	spf	3	F
SA246588	cec_spf_3_M_4	Cecum_content	spf	3	M
SA246589	cec_spf_3_M_3	Cecum_content	spf	3	M
SA246590	cec_spf_3_M_1	Cecum_content	spf	3	M
SA246591	cec_spf_3_M_2	Cecum_content	spf	3	M
SA246592	cec_spf_8_F_4	Cecum_content	spf	8	F
SA246593	cec_spf_8_F_1	Cecum_content	spf	8	F
SA246594	cec_spf_8_F_3	Cecum_content	spf	8	F
SA246595	cec_spf_8_F_2	Cecum_content	spf	8	F
SA246596	cec_spf_8_M_4	Cecum_content	spf	8	M
SA246597	cec_spf_8_M_1	Cecum_content	spf	8	M
SA246598	cec_spf_8_M_3	Cecum_content	spf	8	M
SA246599	cec_spf_8_M_2	Cecum_content	spf	8	M
SA246600	col_gf_12_F_4	Colon_content	gf	12	F
SA246601	col_gf_12_F_3	Colon_content	gf	12	F
SA246602	col_gf_12_F_2	Colon_content	gf	12	F
SA246603	col_gf_12_F_1	Colon_content	gf	12	F
SA246604	col_gf_12_M_4	Colon_content	gf	12	M
SA246605	col_gf_12_M_2	Colon_content	gf	12	M
SA246606	col_gf_12_M_3	Colon_content	gf	12	M
SA246607	col_gf_12_M_1	Colon_content	gf	12	M
SA246608	col_gf_3_F_4	Colon_content	gf	3	F
SA246609	col_gf_3_F_3	Colon_content	gf	3	F
SA246610	col_gf_3_F_1	Colon_content	gf	3	F
SA246611	col_gf_3_F_2	Colon_content	gf	3	F
SA246612	col_gf_3_M_4	Colon_content	gf	3	M
SA246613	col_gf_3_M_1	Colon_content	gf	3	M
SA246614	col_gf_3_M_3	Colon_content	gf	3	M
SA246615	col_gf_3_M_2	Colon_content	gf	3	M
SA246616	col_gf_8_F_4	Colon_content	gf	8	F
SA246617	col_gf_8_F_1	Colon_content	gf	8	F
SA246618	col_gf_8_F_3	Colon_content	gf	8	F
SA246619	col_gf_8_F_2	Colon_content	gf	8	F
SA246620	col_gf_8_M_4	Colon_content	gf	8	M
SA246621	col_gf_8_M_3	Colon_content	gf	8	M
SA246622	col_gf_8_M_2	Colon_content	gf	8	M
SA246623	col_gf_8_M_1	Colon_content	gf	8	M
SA246624	col_sdm_12_F_3	Colon_content	sdm	12	F
SA246625	col_sdm_12_F_1	Colon_content	sdm	12	F
SA246626	col_sdm_12_F_2	Colon_content	sdm	12	F
SA246627	col_sdm_12_M_1	Colon_content	sdm	12	M
SA246628	col_sdm_12_M_4	Colon_content	sdm	12	M

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Collection:

Collection ID:	CO002548
Collection Summary:	Mice were anaesthetized with isoflurane and blood was collected by retro-orbital bleed to serum separation collection tubes (BD, NJ, USA). Urine was collected from the mouse either before it was euthanized, immediately following euthanization or directly from the bladder following euthanization. Mice were euthanized by cervical dislocation. 1.5mL of ice-cold sterile PBS was injected into the peritoneal cavity, massaged for 20 seconds then removed and collected. The spleen and a segment of left lobe of the liver was collected. The intestine was excised and content from the entire length of the jejunum, ileum, cecum, and colon were collected. For the purposes of these experiments, the first 10cm of the small intestine was considered duodenum, and the remaining length was split in half with the proximal segment considered jejunum and the distal segment considered ileum. Following collection, blood was centrifuged (10,000 xg, 10 minutes, 4°C) and serum transferred into a 1.5mL Eppendorf tube. All collected samples were placed in liquid nitrogen immediately after collection and stored at -80°C until processing. For processing liver tissue, spleen, and intestinal content, ~50mg of tissue or content was added to a pre-weighed 2mL safe-lock tube containing a steel bead (3mm, Qiagen, Hilden, Germany). Tubes were reweighed and 5X v/w of ice-cold 50% methanol was added. Samples were homogenized (2 minutes, 30Hz), beads were removed, and samples stored at -20°C for 1 hour. Samples were centrifuged (max. speed, 15 minutes, 4֯C), supernatant was recovered and combined 1:4 with 50% methanol to obtain a final dilution of D20. For serum and urine, sample was combined 1:1 with 100% methanol and stored and -20°C for 1 hour. Samples were centrifuged (max. speed, 15 minutes, 4°C), supernatant was recovered and combined 1:25 with 50% methanol to obtain a final dilution of D50. Samples were centrifuged (max. speed, 15 minutes, 4°C) and supernatants were recovered and stored at -80°C until further processing.
Sample Type:	intestine content / liver / spleen / urine / peritoneal fluid / serum
Storage Conditions:	Described in summary

Treatment:

Treatment ID:	TR002567
Treatment Summary:	GF and OMM12-colonized gnotobiotic mice were housed in flexible-film isolators within the International Microbiome Centre (IMC) and SPF mice were maintained in the Mouse Barrier Unit (MBU) at the University of Calgary. GF mice were routinely tested for the absence of bacteria by aerobic and anaerobic culture, gram staining, and vital dye (DNA-dye Sytox green) staining of caecal contents and all mice were routinely screened for the presence of pathogens. For SPF colonization by co-housing, 8-week-old GF animals were exported, transferred into sterile individually ventilated cages and transported from the IMC to the MBU where a colonizer SPF mouse was transferred into each cage. For OMM12 colonization by co-housing, 8-week-old GF animals were transferred into isolators containing OMM12-colonized mice and an OMM12-colonized mouse was added to each cage. All animals were fed identical autoclaved diets and maintained with 12-hour light-dark cycle.

Treatment ID:

TR002567

Treatment Summary:

GF and OMM12-colonized gnotobiotic mice were housed in flexible-film isolators within the International Microbiome Centre (IMC) and SPF mice were maintained in the Mouse Barrier Unit (MBU) at the University of Calgary. GF mice were routinely tested for the absence of bacteria by aerobic and anaerobic culture, gram staining, and vital dye (DNA-dye Sytox green) staining of caecal contents and all mice were routinely screened for the presence of pathogens. For SPF colonization by co-housing, 8-week-old GF animals were exported, transferred into sterile individually ventilated cages and transported from the IMC to the MBU where a colonizer SPF mouse was transferred into each cage. For OMM12 colonization by co-housing, 8-week-old GF animals were transferred into isolators containing OMM12-colonized mice and an OMM12-colonized mouse was added to each cage. All animals were fed identical autoclaved diets and maintained with 12-hour light-dark cycle.

Sample Preparation:

Sampleprep ID:	SP002561
Sampleprep Summary:	Mice were anaesthetized with isoflurane and blood was collected by retro-orbital bleed to serum separation collection tubes (BD, NJ, USA). Urine was collected from the mouse either before it was euthanized, immediately following euthanization or directly from the bladder following euthanization. Mice were euthanized by cervical dislocation. 1.5mL of ice-cold sterile PBS was injected into the peritoneal cavity, massaged for 20 seconds then removed and collected. The spleen and a segment of left lobe of the liver was collected. The intestine was excised and content from the entire length of the jejunum, ileum, cecum, and colon were collected. For the purposes of these experiments, the first 10cm of the small intestine was considered duodenum, and the remaining length was split in half with the proximal segment considered jejunum and the distal segment considered ileum. Following collection, blood was centrifuged (10,000 xg, 10 minutes, 4°C) and serum transferred into a 1.5mL Eppendorf tube. All collected samples were placed in liquid nitrogen immediately after collection and stored at -80°C until processing.

Sampleprep ID:

SP002561

Sampleprep Summary:

Mice were anaesthetized with isoflurane and blood was collected by retro-orbital bleed to serum separation collection tubes (BD, NJ, USA). Urine was collected from the mouse either before it was euthanized, immediately following euthanization or directly from the bladder following euthanization. Mice were euthanized by cervical dislocation. 1.5mL of ice-cold sterile PBS was injected into the peritoneal cavity, massaged for 20 seconds then removed and collected. The spleen and a segment of left lobe of the liver was collected. The intestine was excised and content from the entire length of the jejunum, ileum, cecum, and colon were collected. For the purposes of these experiments, the first 10cm of the small intestine was considered duodenum, and the remaining length was split in half with the proximal segment considered jejunum and the distal segment considered ileum. Following collection, blood was centrifuged (10,000 xg, 10 minutes, 4°C) and serum transferred into a 1.5mL Eppendorf tube. All collected samples were placed in liquid nitrogen immediately after collection and stored at -80°C until processing.

Combined analysis:

Analysis ID	AN004021
Analysis type	MS
Chromatography type	HILIC
Chromatography system	Thermo Vanquish
Column	Thermo Syncronis HILIC (100 x 2.1mm, 1.7um)
MS Type	ESI
MS instrument type	Orbitrap
MS instrument name	Thermo Q Exactive HF hybrid Orbitrap
Ion Mode	NEGATIVE
Units	AUC

Chromatography:

Chromatography ID:	CH002971
Chromatography Summary:	All metabolomics data were collected at the Calgary Metabolomics Research Facility (CMRF). Briefly, samples were centrifuged (20,817 xg, 15 minutes, 4°C), then 200µL was transferred to a deep-well 96-well plate (Thermo FisherF) for LC-MS analysis. Data were collected on a Q Exactive™ HF Hybrid Quadrupole-Orbitrap™ Mass Spectrometer (Thermo-Fisher) coupled to a Vanquish™ UHPLC System (Thermo-Fisher). Metabolites were chromatographically separated on Syncronis HILIC UHPLC column (2.1mm x 100mm x 1.7um, Thermo-Fisher) at the flow rate of 600uL/min using a binary solvent system (solvent A, 20mM ammonium formate pH 3.0 in MS grade H20 and solvent B, MS grade acetonitrile with 0.1% formic acid (%v/v)) and the following gradient: 0-2 mins, 100 %B; 2-7 mins, 100-80 %B; 7-10 mins, 80-5 %B; 10-12 mins, 5% B; 12-13 mins, 5-100 %B; 13-15 mins, 100 %B. The samples injection volume was 2uL. MassM data were acquired in MS1, negative full scan mode at a resolution of 240,000 scanning from 50-750m/z.
Instrument Name:	Thermo Vanquish
Column Name:	Thermo Syncronis HILIC (100 x 2.1mm, 1.7um)
Column Temperature:	30C
Flow Gradient:	2 stage linear gradient
Flow Rate:	600uL/min
Sample Injection:	2uL
Solvent A:	100% water; 20mM ammonium formate (pH3)
Solvent B:	100% acetonitrile; 0.1% formic acid
Analytical Time:	15 minutes
Washing Buffer:	10% MeOH in H2O + 0.1% formic acid
Target Sample Temperature:	4
Chromatography Type:	HILIC

MS:

MS ID:	MS003768
Analysis ID:	AN004021
Instrument Name:	Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Metabolomics data files were processed with ms-mint (version 0.1.8.3; mint.resistancedb.org) Python package for targeted metabolomics and verified using El-Maven software package. For targeted analysis, metabolites were identified by matching observed m/z signals and chromatographic retention times to those observed from commercial metabolite standards library (MSMLSTM Sigma-Aldrich) containing 639 standards, 397 of which were detectable using our LC-MS and sample preparation methods and 140 of which were observed a detectable levels in the biological samples.
Ion Mode:	NEGATIVE