Summary of Study ST001683

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 PR001074. The data can be accessed directly via it's Project DOI: 10.21228/M8W11P 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	ST001683
Study Title	A gut microbe-focused metabolomics pipeline enables mechanistic interrogation of microbiome metabolism.
Study Summary	Gut microbes modulate host phenotypes and are associated with numerous health effects in humans, ranging from cancer immunotherapy response to metabolic disease and obesity. However, difficulty in accurate and high-throughput functional analysis of human gut microbes has hindered defining mechanistic connections between individual microbial strains and host phenotypes. One key way the gut microbiome influences host physiology is through the production of small molecules hindered by limited tools calibrated to detect products of anaerobic biochemistry in the gut. Here we construct a microbiome-focused, integrated mass-spectrometry pipeline to accelerate the identification of microbiota-dependent metabolites (MDMs) in diverse sample types. We report the metabolic profiles of 178 gut microbe strains using our library of 833 metabolites. Leveraging this metabolomics resource we establish deviations in the relationships between phylogeny and metabolism, use machine learning to discover novel metabolism in Bacteroides, and employ comparative genomics-based discovery of candidate biochemical pathways. MDMs can be detected in diverse body fluids in gnotobiotic and conventional mice and traced back to corresponding metabolomic profiles of cultured bacteria. Collectively, our microbiome-focused metabolomics pipeline and interactive metabolomics profile explorer are a powerful tool for characterizing microbe and microbe-host interactions.
Institute	Stanford University
Department	Microbiology & Immunology
Laboratory	Justin Sonnenburg
Last Name	Han
First Name	Shuo
Address	299 Campus Drive, Stanford, CA, 94305-5124, USA
Email	shuohan@stanford.edu
Phone	-
Submit Date	2021-02-06
Publications	not published, status to be updated
Raw Data Available	Yes
Raw Data File Type(s)	mzML
Analysis Type Detail	LC-MS
Release Date	2021-05-17
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR001074
Project DOI:	doi: 10.21228/M8W11P
Project Title:	A gut microbe-focused metabolomics pipeline enables mechanistic interrogation of microbiome metabolism
Project Summary:	Gut microbes modulate host phenotypes and are associated with numerous health effects in humans, ranging from cancer immunotherapy response to metabolic disease and obesity. However, difficulty in accurate and high-throughput functional analysis of human gut microbes has hindered defining mechanistic connections between individual microbial strains and host phenotypes. One key way the gut microbiome influences host physiology is through the production of small molecules1–3, yet progress in elucidating this chemical interplay has been hindered by limited tools calibrated to detect products of anaerobic biochemistry in the gut. Here we construct a microbiome-focused, integrated mass-spectrometry pipeline to accelerate the identification of microbiota-dependent metabolites (MDMs) in diverse sample types. We report the metabolic profiles of 178 gut microbe strains using our library of 833 metabolites. Leveraging this metabolomics resource we establish deviations in the relationships between phylogeny and metabolism, use machine learning to discover novel metabolism in Bacteroides, and employ comparative genomics-based discovery of candidate biochemical pathways. MDMs can be detected in diverse body fluids in gnotobiotic and conventional mice and traced back to corresponding metabolomic profiles of cultured bacteria. Collectively, our microbiome-focused metabolomics pipeline and interactive metabolomics profile explorer are a powerful tool for characterizing microbe and microbe-host interactions.
Institute:	Stanford University
Department:	Microbiology and Immunology
Laboratory:	Justin Sonnenburg
Last Name:	Van Treuren
First Name:	Will
Address:	Sherman Fairchild Building, 299 Campus Drive, Stanford CA, 94305
Email:	wdwvt@stanford.edu
Phone:	7209370980
Contributors:	Shuo Han1,, Will Van Treuren1,2,, Curt R. Fischer3, 4, Bryan D. Merrill1,2, Leah Guthrie1, Brian C. DeFelice4, Lalla A. Fall3,5, Dylan Dodd1,5,^, Michael A. Fischbach4,6,^, and Justin L. Sonnenburg1,4,7,^.

Subject:

Subject ID:	SU001760
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Genotype Strain:	Swiss Webster
Age Or Age Range:	10-14 weeks
Gender:	Male

Factors:

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

mb_sample_id	local_sample_id	Experiment	Sample_type	Colonization
SA155124	s0194	community	caecal	Bt_Ca_Er_Pd_Et
SA155125	s0177	community	caecal	Bt_Ca_Er_Pd_Et
SA155126	s0195	community	caecal	Bt_Ca_Er_Pd_Et
SA155127	s0187	community	caecal	Bt_Ca_Er_Pd_Et
SA155128	s0185	community	caecal	Bt_Ca_Er_Pd_Et
SA155129	s0176	community	caecal	Bt_Ca_Er_Pd_Et
SA155130	s0178	community	caecal	Bt_Ca_Er_Pd_Et
SA155131	s0196	community	caecal	Bt_Ca_Er_Pd_Et
SA155132	s0186	community	caecal	Bt_Ca_Er_Pd_Et
SA155133	s0201	community	caecal	Cs_Bt_Ca_Er_Pd_Et
SA155134	s0219	community	caecal	Cs_Bt_Ca_Er_Pd_Et
SA155135	s0220	community	caecal	Cs_Bt_Ca_Er_Pd_Et
SA155136	s0218	community	caecal	Cs_Bt_Ca_Er_Pd_Et
SA155137	s0213	community	caecal	Cs_Bt_Ca_Er_Pd_Et
SA155138	s0202	community	caecal	Cs_Bt_Ca_Er_Pd_Et
SA155139	s0212	community	caecal	Cs_Bt_Ca_Er_Pd_Et
SA155140	s0200	community	caecal	Cs_Bt_Ca_Er_Pd_Et
SA155141	s0214	community	caecal	Cs_Bt_Ca_Er_Pd_Et
SA155142	s0162	community	caecal	germ-free
SA155143	s0161	community	caecal	germ-free
SA155144	s0172	community	caecal	germ-free
SA155145	s0163	community	caecal	germ-free
SA155146	s0167	community	caecal	germ-free
SA155147	s0171	community	caecal	germ-free
SA155148	s0170	community	caecal	germ-free
SA155149	s0169	community	caecal	germ-free
SA155150	s0168	community	caecal	germ-free
SA155151	s0054	community	feces	Bt_Ca_Er_Pd_Et
SA155152	s0070	community	feces	Bt_Ca_Er_Pd_Et
SA155153	s0092	community	feces	Cs_Bt_Ca_Er_Pd_Et
SA155154	s0100	community	feces	Cs_Bt_Ca_Er_Pd_Et
SA155155	s0082	community	feces	Cs_Bt_Ca_Er_Pd_Et
SA155156	s0022	community	feces	germ-free
SA155157	s0029	community	feces	germ-free
SA155158	s0034	community	feces	germ-free
SA155159	s0363	community	serum	Bt_Ca_Er_Pd_Et
SA155160	s0368	community	serum	Bt_Ca_Er_Pd_Et
SA155161	s0362	community	serum	Bt_Ca_Er_Pd_Et
SA155162	s0359	community	serum	Bt_Ca_Er_Pd_Et
SA155163	s0355	community	serum	Bt_Ca_Er_Pd_Et
SA155164	s0374	community	serum	Bt_Ca_Er_Pd_Et
SA155165	s0367	community	serum	Bt_Ca_Er_Pd_Et
SA155166	s0366	community	serum	Bt_Ca_Er_Pd_Et
SA155167	s0360	community	serum	Cs_Bt_Ca_Er_Pd_Et
SA155168	s0357	community	serum	Cs_Bt_Ca_Er_Pd_Et
SA155169	s0361	community	serum	Cs_Bt_Ca_Er_Pd_Et
SA155170	s0356	community	serum	Cs_Bt_Ca_Er_Pd_Et
SA155171	s0373	community	serum	Cs_Bt_Ca_Er_Pd_Et
SA155172	s0369	community	serum	Cs_Bt_Ca_Er_Pd_Et
SA155173	s0372	community	serum	Cs_Bt_Ca_Er_Pd_Et
SA155174	s0365	community	serum	germ-free
SA155175	s0364	community	serum	germ-free
SA155176	s0358	community	serum	germ-free
SA155177	s0371	community	serum	germ-free
SA155178	s0370	community	serum	germ-free
SA155179	s0286	community	urine	Bt_Ca_Er_Pd_Et
SA155180	s0281	community	urine	Bt_Ca_Er_Pd_Et
SA155181	s0290	community	urine	Bt_Ca_Er_Pd_Et
SA155182	s0295	community	urine	Bt_Ca_Er_Pd_Et
SA155183	s0309	community	urine	Bt_Ca_Er_Pd_Et
SA155184	s0304	community	urine	Bt_Ca_Er_Pd_Et
SA155185	s0314	community	urine	Bt_Ca_Er_Pd_Et
SA155186	s0245	community	urine	Cs_Bt_Ca_Er_Pd_Et
SA155187	s0250	community	urine	Cs_Bt_Ca_Er_Pd_Et
SA155188	s0254	community	urine	Cs_Bt_Ca_Er_Pd_Et
SA155189	s0259	community	urine	Cs_Bt_Ca_Er_Pd_Et
SA155190	s0277	community	urine	Cs_Bt_Ca_Er_Pd_Et
SA155191	s0272	community	urine	Cs_Bt_Ca_Er_Pd_Et
SA155192	s0227	community	urine	germ-free
SA155193	s0231	community	urine	germ-free
SA155194	s0236	community	urine	germ-free
SA155195	s1073	conventional	caecal	conventional
SA155196	s1072	conventional	caecal	conventional
SA155197	s1074	conventional	caecal	conventional
SA155198	s1065	conventional	caecal	germ-free
SA155199	s1067	conventional	caecal	germ-free
SA155200	s1066	conventional	caecal	germ-free
SA155201	s1064	conventional	caecal	germ-free
SA155202	s1077	conventional	feces	conventional
SA155203	s1076	conventional	feces	conventional
SA155204	s1075	conventional	feces	conventional
SA155205	s1069	conventional	feces	germ-free
SA155206	s1068	conventional	feces	germ-free
SA155207	s1070	conventional	feces	germ-free
SA155208	s1071	conventional	feces	germ-free
SA155209	s1061	conventional	serum	conventional
SA155210	s1062	conventional	serum	conventional
SA155211	s1063	conventional	serum	conventional
SA155212	s1060	conventional	serum	germ-free
SA155213	s1058	conventional	serum	germ-free
SA155214	s1057	conventional	serum	germ-free
SA155215	s1059	conventional	serum	germ-free
SA155216	s1055	conventional	urine	conventional
SA155217	s1054	conventional	urine	conventional
SA155218	s1056	conventional	urine	conventional
SA155219	s1050	conventional	urine	germ-free
SA155220	s1051	conventional	urine	germ-free
SA155221	s1052	conventional	urine	germ-free
SA155222	s1053	conventional	urine	germ-free
SA155223	s1100	mono-colonization_2	caecal	As

Showing page 1 of 3 Results: 1 2 3 Next Showing results 1 to 100 of 268

Collection:

Collection ID:	CO001753
Collection Summary:	For all experiments, mice were euthanized by CO2 asphyxiation nine days (mono-colonization with Citrobacter portucalensis BEI HM-34 or Anaerostipes sp. BEI HM-220) or four weeks (all other experiments) following colonization, and four sample types (serum, urine, feces, and cecal contents) were harvested from each mouse. Prior to euthanization, urine and feces were collected. Whole blood was collected by cardiac puncture and serum was obtained using microcontainer serum separator tubes from Becton Dickinson following manufacturer’s instructions. The intact cecum was harvested and snap-frozen in liquid nitrogen. A single cecal sample was harvested for mono-association and conventional experiments, and three samples at three different sections of the cecum were harvested for the defined-community experiment. All mouse experiments were conducted under a protocol approved by the Stanford University Institutional Animal Care and Use Committee.
Sample Type:	Feces;Urine;Serum;Cecal contents

Treatment:

Treatment ID:	TR001773
Treatment Summary:	Mouse experiments were performed on gnotobiotic Swiss Webster germ-free mice (males, 10-14 weeks of age, n = 3-8 per group for all experiments) maintained in aseptic isolators, and originally obtained from Taconic Bioscience. For mono-association experiments, mice were colonized with Bacteroides thetaiotaomicron VPI 5482, Clostridium sporogenes ATCC 15579, Citrobacter portucalensis BEI HM-34, or Anaerostipes sp. BEI HM-220904a, by oral gavage (200 uL, ~1 x 107 CFU) and were maintained on a standard chow (LabDiet 5K67). For the defined-community experiment, mice with a six-member community were colonized with a 200 uL mixture consisting of equal volumes from saturated cultures of Bacteroides thetaiotaomicron VPI 5482 (8.7 x 109 CFU), Clostridium sporogenes ATCC 15579 (1.4 x 108 CFU), Edwardsiella tarda ATCC 23685 (3.6 x 1010 CFU), Collinsella aerofaciens ATCC 25986 (1.4 x 109), Eubacterium rectale ATCC 33656 (6.9 x 106 CFU), and Parabacteroides distasonis ATCC 8503 (1.5 x 109 CFU). Mice with a five-member community were colonized with all cultures mixed at the same volumes as described above except for Clostridium sporogenes ATCC 15579, which was not included. Successful colonization and stable community members were determined by 16S amplicon sequencing of the V4 (515f, 806r) region of microbial populations present in the feces and cecal contents from individual mice.

Treatment ID:

TR001773

Treatment Summary:

Mouse experiments were performed on gnotobiotic Swiss Webster germ-free mice (males, 10-14 weeks of age, n = 3-8 per group for all experiments) maintained in aseptic isolators, and originally obtained from Taconic Bioscience. For mono-association experiments, mice were colonized with Bacteroides thetaiotaomicron VPI 5482, Clostridium sporogenes ATCC 15579, Citrobacter portucalensis BEI HM-34, or Anaerostipes sp. BEI HM-220904a, by oral gavage (200 uL, ~1 x 107 CFU) and were maintained on a standard chow (LabDiet 5K67). For the defined-community experiment, mice with a six-member community were colonized with a 200 uL mixture consisting of equal volumes from saturated cultures of Bacteroides thetaiotaomicron VPI 5482 (8.7 x 109 CFU), Clostridium sporogenes ATCC 15579 (1.4 x 108 CFU), Edwardsiella tarda ATCC 23685 (3.6 x 1010 CFU), Collinsella aerofaciens ATCC 25986 (1.4 x 109), Eubacterium rectale ATCC 33656 (6.9 x 106 CFU), and Parabacteroides distasonis ATCC 8503 (1.5 x 109 CFU). Mice with a five-member community were colonized with all cultures mixed at the same volumes as described above except for Clostridium sporogenes ATCC 15579, which was not included. Successful colonization and stable community members were determined by 16S amplicon sequencing of the V4 (515f, 806r) region of microbial populations present in the feces and cecal contents from individual mice.

Sample Preparation:

Sampleprep ID:	SP001766
Sampleprep Summary:	All samples were stored at -80oC until use, and were thawed on ice immediately before extraction. 200 µL of serum and bacterial supernatant samples were used for extraction directly without dilution. Urine samples were diluted 1:20 in LC-MS grade water (Fisher) to reach a final volume of 200 µL prior to extraction. Protein precipitation for bacterial, serum, and urine samples was conducted by adding 1 mL of extraction buffer (see composition below) in 100% methanol (LC-MS grade, Fisher) to 200 µL of each sample in a 2 mL 96-well microplate (Fisher), sealed with a silicone mat (Agilent), and vortexed to mix. For feces and cecal contents, ~ 20 mg of feces or cecal contents were added to ~ 20 mg of acid-washed glass beads (150-212 µm, Sigma) in a 2 mL autoclaved screw top vial. In the same vial, 600 µL of water (LC-MS grade, Fisher) and 600 µL of recovery buffer in 100% methanol (see composition below) were added. Fecal and cecal slurries were homogenized at 4oC using a Mini Beadbeater operating at 3,500 oscillations per minute for 5 minutes. For all sample types, samples were subsequently incubated at room temperature for 5 minutes, followed by centrifugation at 5,000 x g for 10 minutes. Two 440 µL aliquots of the same supernatant were transferred to two separate 2 mL plates and dried under air in a Biotage TurboVap. One of these dried plates was sealed and archived at -80oC. The dried extracts were reconstituted in 200 µL reconstitution buffer (see composition below) in 50% methanol in water (v/v, LC-MS grade, Fisher) by vortexing at max speed for 5 seconds. Reconstituted sample extracts were centrifuged at 2,000 x g for 1 minute, and filtered through a 96-well Durapore PVDF 0.22-µm filter plate (Millipore) into in a 1 mL 96-well plate (Agilent) by centrifugation at 2,000 x g for 10 minutes. Plates were then sealed with 96-well cap mats (Agilent) and stored at -80oC until LC-MS analysis. QC samples were generated by pooling 4 µL from each well of the experiment into a single designated well on the same plate for LC-MS analysis. The extraction buffer consisted of 4-Chloro-phenylalanine (6.8 µM, Carbosynth), Tridecanoic acid (6.8 µM, Sigma), and 2-Flurophenylglycine (3.4 µM, SCBT) in 100% methanol. The reconstitution buffer included the internal standards: Phenylalanine-2,3,4,5,6-d5 (12.5 µM, CIL), Glucose-1,2,3,4,5,6,6-d7 (25 µM, CIL), Methionine-methyl-d3 (12.5 µM, CIL), 4-Hydroxyphenyl-d4-alanine (3.125 µM, CDN), Tryptophan-2,4,5,6,7-d5 (12.5 µM, CDN), Leucine-5,5,5-d3 (12.5 µM, CDN), N-Benzoyl-d5-glycine (6.25 µM, CDN), 4-Bromo-phenylalanine (12.5 µM, Sigma), Progesterone-d9 (3.125 µM, CIL), Di-N-octyl phthalate-3,4,5,6-d4 (12.5 µM, CDN), d19-Decanoic acid (12.5 µM, CDN), d15-Octanoic acid (25 µM, CDN), Indole-2,4,5,6,7-d5-3-acetic acid (12.5 µM, CDN), Carnitine-trimethyl-d9 (3.125 µM, CDN), and d27-Tetradecanoic acid (12.5 µM, CDN), in 50% methanol in water. The final concentration for each internal standard in these buffers was determined by choosing a concentration falling within its linear dynamic range as measured by each analytical method.

Sampleprep ID:

SP001766

Sampleprep Summary:

All samples were stored at -80oC until use, and were thawed on ice immediately before extraction. 200 µL of serum and bacterial supernatant samples were used for extraction directly without dilution. Urine samples were diluted 1:20 in LC-MS grade water (Fisher) to reach a final volume of 200 µL prior to extraction. Protein precipitation for bacterial, serum, and urine samples was conducted by adding 1 mL of extraction buffer (see composition below) in 100% methanol (LC-MS grade, Fisher) to 200 µL of each sample in a 2 mL 96-well microplate (Fisher), sealed with a silicone mat (Agilent), and vortexed to mix. For feces and cecal contents, ~ 20 mg of feces or cecal contents were added to ~ 20 mg of acid-washed glass beads (150-212 µm, Sigma) in a 2 mL autoclaved screw top vial. In the same vial, 600 µL of water (LC-MS grade, Fisher) and 600 µL of recovery buffer in 100% methanol (see composition below) were added. Fecal and cecal slurries were homogenized at 4oC using a Mini Beadbeater operating at 3,500 oscillations per minute for 5 minutes. For all sample types, samples were subsequently incubated at room temperature for 5 minutes, followed by centrifugation at 5,000 x g for 10 minutes. Two 440 µL aliquots of the same supernatant were transferred to two separate 2 mL plates and dried under air in a Biotage TurboVap. One of these dried plates was sealed and archived at -80oC. The dried extracts were reconstituted in 200 µL reconstitution buffer (see composition below) in 50% methanol in water (v/v, LC-MS grade, Fisher) by vortexing at max speed for 5 seconds. Reconstituted sample extracts were centrifuged at 2,000 x g for 1 minute, and filtered through a 96-well Durapore PVDF 0.22-µm filter plate (Millipore) into in a 1 mL 96-well plate (Agilent) by centrifugation at 2,000 x g for 10 minutes. Plates were then sealed with 96-well cap mats (Agilent) and stored at -80oC until LC-MS analysis. QC samples were generated by pooling 4 µL from each well of the experiment into a single designated well on the same plate for LC-MS analysis. The extraction buffer consisted of 4-Chloro-phenylalanine (6.8 µM, Carbosynth), Tridecanoic acid (6.8 µM, Sigma), and 2-Flurophenylglycine (3.4 µM, SCBT) in 100% methanol. The reconstitution buffer included the internal standards: Phenylalanine-2,3,4,5,6-d5 (12.5 µM, CIL), Glucose-1,2,3,4,5,6,6-d7 (25 µM, CIL), Methionine-methyl-d3 (12.5 µM, CIL), 4-Hydroxyphenyl-d4-alanine (3.125 µM, CDN), Tryptophan-2,4,5,6,7-d5 (12.5 µM, CDN), Leucine-5,5,5-d3 (12.5 µM, CDN), N-Benzoyl-d5-glycine (6.25 µM, CDN), 4-Bromo-phenylalanine (12.5 µM, Sigma), Progesterone-d9 (3.125 µM, CIL), Di-N-octyl phthalate-3,4,5,6-d4 (12.5 µM, CDN), d19-Decanoic acid (12.5 µM, CDN), d15-Octanoic acid (25 µM, CDN), Indole-2,4,5,6,7-d5-3-acetic acid (12.5 µM, CDN), Carnitine-trimethyl-d9 (3.125 µM, CDN), and d27-Tetradecanoic acid (12.5 µM, CDN), in 50% methanol in water. The final concentration for each internal standard in these buffers was determined by choosing a concentration falling within its linear dynamic range as measured by each analytical method.

Combined analysis:

Analysis ID	AN002747	AN002748	AN002749
Analysis type	MS	MS	MS
Chromatography type	Reversed phase	Reversed phase	HILIC
Chromatography system	Agilent qTOF 6545	Agilent qTOF 6545	Agilent qTOF 6545
Column	Waters Acquity BEH (100 x 2.1mm,1.7um)	Waters Acquity BEH (100 x 2.1mm,1.7um)	Waters Acquity BEH Amide (150 x 2.1mm,1.7um)
MS Type	ESI	ESI	ESI
MS instrument type	QTOF	QTOF	QTOF
MS instrument name	Agilent qTOF 6545	Agilent qTOF 6545	Agilent qTOF 6545
Ion Mode	POSITIVE	NEGATIVE	POSITIVE
Units	Raw ion count (peak area)	Raw ion count (peak area)	Raw ion count (peak area)

Chromatography:

Chromatography ID:	CH002029
Chromatography Summary:	Published C18 reveres phase methods were implemented with minor modifications. The C18 positive method (ESI+) used mobile phase solvents (LC-MS grade) consisting of 0.1% formic acid (Fisher) in water (A) and 0.1% formic acid in methanol (B). The gradient profile was from 0.5% B to 70% B in 4 minutes, from 70% B to 98% B in 0.5 minutes, and holding at 98% B for 0.9 minute before returning to 0.5% B in 0.2 minutes. The flow rate was 350 µL per minute. The sample injection volume was 5 µL. LC separations were made at 40oC on separate columns fitted with a Vanguard pre-column of the same composition: Waters Acquity BEH 1.7 µm particle size, 2.1 mm id x 100 mm length (C18). Data were collected at a mass range of 70-1000 m/z at an acquisition rate of 2 spectra per second. Specific ion source parameters included Fragmentor (140V), Gas Temp (250oC), Sheath Gas Temp (200oC), and VCap (4000V).
Instrument Name:	Agilent qTOF 6545
Column Name:	Waters Acquity BEH (100 x 2.1mm,1.7um)
Column Temperature:	40
Flow Gradient:	0.5% B to 70% B in 4 minutes, from 70% B to 98% B in 0.5 minutes, and holding at 98% B for 0.9 minute before returning to 0.5% B in 0.2 minutes.
Flow Rate:	350 µL/min
Solvent A:	100% water; 0.1% formic acid
Solvent B:	100% methanol; 0.1% formic acid
Chromatography Type:	Reversed phase

Chromatography ID:	CH002030
Chromatography Summary:	Published C18 reverse phase methods were implemented with minor modifications. The C18 negative method (ESI-) used mobile phase solvents (LC-MS grade) consisting of 6.5 mM ammonium bicarbonate (Sigma) in water at pH 8 (A) and 6.5 mM ammonium bicarbonate in 95:5 v/v methanol:water (B). The gradient profile was from 0.5% B to 70% B in 4 minutes, from 70% B to 98% B in 0.5 minutes, and holding at 98% B for 0.9 minute before returning to 0.5% B in 0.2 minutes. The flow rate was 350 µL per minute. The sample injection volume was 5 µL. LC separations were made at 40oC on separate columns fitted with a Vanguard pre-column of the same composition: Waters Acquity BEH 1.7 µm particle size, 2.1 mm id x 100 mm length. Data were collected at a mass range of 70-1000 m/z at an acquisition rate of 2 spectra per second. Specific ion source parameters included Fragmentor (140V), Gas Temp (250oC), Sheath Gas Temp (200oC), and VCap (4000V).
Instrument Name:	Agilent qTOF 6545
Column Name:	Waters Acquity BEH (100 x 2.1mm,1.7um)
Column Temperature:	40
Flow Gradient:	0.5% B to 70% B in 4 minutes, from 70% B to 98% B in 0.5 minutes, and holding at 98% B for 0.9 minute before returning to 0.5% B in 0.2 minutes.
Flow Rate:	350 µL/min
Solvent A:	100% water; 0.1% formic acid
Solvent B:	100% methanol; 0.1% formic acid
Chromatography Type:	Reversed phase

Chromatography ID:	CH002031
Chromatography Summary:	Published HILIC method was implemented with minor modifications. The HILIC (Hydrophilic Interaction Liquid Chromatography) positive method (ESI+) used mobile phase solvents (LC-MS grade) consisting of 0.125% formic acid and 10 mM ammonium formate (Sigma) in water at pH 3 (A) and 0.125% formic acid in 10 mM ammonium formate in 95:5 v/v acetonitrile:water (B). The gradient profile was held at 100% B for 2 minutes, from 100% B to 70% B in 5 minutes, holding at 70% B for 0.7 minute, from 70% B to 40% B for 1.3 minutes, holding at 40% B for 0.5 minutes, from 40% B to 30% B for 0.75 minutes, before returning to 100% B for 2.5 minutes and holding at 100% B for 4 minutes. The flow rate was 400 µL per minute. The sample injection volume was 3 µL. LC separations were made at 40oC on separate columns fitted with a Vanguard pre-column of the same composition dedicated to each analytical method: Waters Acquity BEH Amide 1.7 µm particle size, 2.1 mm id x 150 mm length. Data were collected at a mass range of 70-1000 m/z at an acquisition rate of 2 spectra per second. Specific ion source parameters included Fragmentor (140V), Gas Temp (250oC), Sheath Gas Temp (200oC), and VCap (4000V).
Instrument Name:	Agilent qTOF 6545
Column Name:	Waters Acquity BEH Amide (150 x 2.1mm,1.7um)
Column Temperature:	40
Flow Gradient:	The gradient profile was held at 100% B for 2 minutes, from 100% B to 70% B in 5 minutes, holding at 70% B for 0.7 minute, from 70% B to 40% B for 1.3 minutes, holding at 40% B for 0.5 minutes, from 40% B to 30% B for 0.75 minutes, before returning to 100% B for 2.5 minutes and holding at 100% B for 4 minutes.
Flow Rate:	400 µL/min
Solvent A:	100% water; 0.125% formic acid; 10 mM ammonium formate, pH 3
Solvent B:	95% acetonitrile/5% water; 0.125% formic acid; 10 mM ammonium formate
Chromatography Type:	HILIC

MS:

MS ID:	MS002544
Analysis ID:	AN002747
Instrument Name:	Agilent qTOF 6545
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	The MS-DIAL software (v. 3.83) was used for analyzing all in vitro and in vivo data on a per-experimental run and per-analytical method basis. QC samples from each experimental run were used for peak alignment. Chemical assignment of molecular features in samples was performed by comparison of recorded RT and m/z information to our reference library constructed from authentic standards. Tolerance windows were set to 0.1 minute RT and 0.01 Da m/z for the C18 methods and 0.2 minute RT and 0.01 Da m/z for the HILIC method. The minimal peak count (height) filter was set to 3000 for all experiments except for select experiments in which the MS exhibited reduced sensitivity. For experiments where detection of internal standards goes beyond the 0.1 (C18 methods) or 0.2 (HILIC method) window, RT correction of the mz-RT reference library was conducted prior to feature annotation in MS-DIAL. The MS-DIAL analysis generated a list of m/z, RT, and ion counts (area under the curve) for high-confidence annotations (matched to the reference library) as well as unknown molecular features. Based on the list of annotations for each experiment, each set of aligned peaks was manually checked using the MS-DIAL graphical user interface. Select metabolite features were removed from this list when: 1) two adjacent but distinct peaks were concurrently assigned to a single molecular feature, 2) odd curvature/shape of the peak led to integration of several “peaks” from separate sections of the same peak, or 3) features were only detected in blank controls. Annotated peaks that passed this inspection were reported in the final output file. After MS-DIAL analysis, data were analyzed with a set of custom bioinformatics pipelines. In short, these pipelines implemented a set of filtration and normalization procedures with the goal of reducing technical variability and controlling for batch effects.
Ion Mode:	POSITIVE

MS ID:	MS002545
Analysis ID:	AN002748
Instrument Name:	Agilent qTOF 6545
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	The MS-DIAL software (v. 3.83) was used for analyzing all in vitro and in vivo data on a per-experimental run and per-analytical method basis. QC samples from each experimental run were used for peak alignment. Chemical assignment of molecular features in samples was performed by comparison of recorded RT and m/z information to our reference library constructed from authentic standards. Tolerance windows were set to 0.1 minute RT and 0.01 Da m/z for the C18 methods and 0.2 minute RT and 0.01 Da m/z for the HILIC method. The minimal peak count (height) filter was set to 3000 for all experiments except for select experiments in which the MS exhibited reduced sensitivity. For experiments where detection of internal standards goes beyond the 0.1 (C18 methods) or 0.2 (HILIC method) window, RT correction of the mz-RT reference library was conducted prior to feature annotation in MS-DIAL. The MS-DIAL analysis generated a list of m/z, RT, and ion counts (area under the curve) for high-confidence annotations (matched to the reference library) as well as unknown molecular features. Based on the list of annotations for each experiment, each set of aligned peaks was manually checked using the MS-DIAL graphical user interface. Select metabolite features were removed from this list when: 1) two adjacent but distinct peaks were concurrently assigned to a single molecular feature, 2) odd curvature/shape of the peak led to integration of several “peaks” from separate sections of the same peak, or 3) features were only detected in blank controls. Annotated peaks that passed this inspection were reported in the final output file. After MS-DIAL analysis, data were analyzed with a set of custom bioinformatics pipelines. In short, these pipelines implemented a set of filtration and normalization procedures with the goal of reducing technical variability and controlling for batch effects.
Ion Mode:	NEGATIVE

MS ID:	MS002546
Analysis ID:	AN002749
Instrument Name:	Agilent qTOF 6545
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	The MS-DIAL software (v. 3.83) was used for analyzing all in vitro and in vivo data on a per-experimental run and per-analytical method basis. QC samples from each experimental run were used for peak alignment. Chemical assignment of molecular features in samples was performed by comparison of recorded RT and m/z information to our reference library constructed from authentic standards. Tolerance windows were set to 0.1 minute RT and 0.01 Da m/z for the C18 methods and 0.2 minute RT and 0.01 Da m/z for the HILIC method. The minimal peak count (height) filter was set to 3000 for all experiments except for select experiments in which the MS exhibited reduced sensitivity. For experiments where detection of internal standards goes beyond the 0.1 (C18 methods) or 0.2 (HILIC method) window, RT correction of the mz-RT reference library was conducted prior to feature annotation in MS-DIAL. The MS-DIAL analysis generated a list of m/z, RT, and ion counts (area under the curve) for high-confidence annotations (matched to the reference library) as well as unknown molecular features. Based on the list of annotations for each experiment, each set of aligned peaks was manually checked using the MS-DIAL graphical user interface. Select metabolite features were removed from this list when: 1) two adjacent but distinct peaks were concurrently assigned to a single molecular feature, 2) odd curvature/shape of the peak led to integration of several “peaks” from separate sections of the same peak, or 3) features were only detected in blank controls. Annotated peaks that passed this inspection were reported in the final output file. After MS-DIAL analysis, data were analyzed with a set of custom bioinformatics pipelines. In short, these pipelines implemented a set of filtration and normalization procedures with the goal of reducing technical variability and controlling for batch effects.
Ion Mode:	POSITIVE