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.

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Study IDST001683
Study TitleA gut microbe-focused metabolomics pipeline enables mechanistic interrogation of microbiome metabolism.
Study SummaryGut 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
DepartmentMicrobiology & Immunology
LaboratoryJustin Sonnenburg
Last NameHan
First NameShuo
Address299 Campus Drive, Stanford, CA, 94305-5124, USA
Emailshuohan@stanford.edu
Phone-
Submit Date2021-02-06
Publicationsnot published, status to be updated
Raw Data AvailableYes
Raw Data File Type(s)mzML
Analysis Type DetailLC-MS
Release Date2021-05-17
Release Version1
Shuo Han Shuo Han
https://dx.doi.org/10.21228/M8W11P
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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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
SA155124s0194community caecal Bt_Ca_Er_Pd_Et
SA155125s0177community caecal Bt_Ca_Er_Pd_Et
SA155126s0195community caecal Bt_Ca_Er_Pd_Et
SA155127s0187community caecal Bt_Ca_Er_Pd_Et
SA155128s0185community caecal Bt_Ca_Er_Pd_Et
SA155129s0176community caecal Bt_Ca_Er_Pd_Et
SA155130s0178community caecal Bt_Ca_Er_Pd_Et
SA155131s0196community caecal Bt_Ca_Er_Pd_Et
SA155132s0186community caecal Bt_Ca_Er_Pd_Et
SA155133s0201community caecal Cs_Bt_Ca_Er_Pd_Et
SA155134s0219community caecal Cs_Bt_Ca_Er_Pd_Et
SA155135s0220community caecal Cs_Bt_Ca_Er_Pd_Et
SA155136s0218community caecal Cs_Bt_Ca_Er_Pd_Et
SA155137s0213community caecal Cs_Bt_Ca_Er_Pd_Et
SA155138s0202community caecal Cs_Bt_Ca_Er_Pd_Et
SA155139s0212community caecal Cs_Bt_Ca_Er_Pd_Et
SA155140s0200community caecal Cs_Bt_Ca_Er_Pd_Et
SA155141s0214community caecal Cs_Bt_Ca_Er_Pd_Et
SA155142s0162community caecal germ-free
SA155143s0161community caecal germ-free
SA155144s0172community caecal germ-free
SA155145s0163community caecal germ-free
SA155146s0167community caecal germ-free
SA155147s0171community caecal germ-free
SA155148s0170community caecal germ-free
SA155149s0169community caecal germ-free
SA155150s0168community caecal germ-free
SA155151s0054community feces Bt_Ca_Er_Pd_Et
SA155152s0070community feces Bt_Ca_Er_Pd_Et
SA155153s0092community feces Cs_Bt_Ca_Er_Pd_Et
SA155154s0100community feces Cs_Bt_Ca_Er_Pd_Et
SA155155s0082community feces Cs_Bt_Ca_Er_Pd_Et
SA155156s0022community feces germ-free
SA155157s0029community feces germ-free
SA155158s0034community feces germ-free
SA155159s0363community serum Bt_Ca_Er_Pd_Et
SA155160s0368community serum Bt_Ca_Er_Pd_Et
SA155161s0362community serum Bt_Ca_Er_Pd_Et
SA155162s0359community serum Bt_Ca_Er_Pd_Et
SA155163s0355community serum Bt_Ca_Er_Pd_Et
SA155164s0374community serum Bt_Ca_Er_Pd_Et
SA155165s0367community serum Bt_Ca_Er_Pd_Et
SA155166s0366community serum Bt_Ca_Er_Pd_Et
SA155167s0360community serum Cs_Bt_Ca_Er_Pd_Et
SA155168s0357community serum Cs_Bt_Ca_Er_Pd_Et
SA155169s0361community serum Cs_Bt_Ca_Er_Pd_Et
SA155170s0356community serum Cs_Bt_Ca_Er_Pd_Et
SA155171s0373community serum Cs_Bt_Ca_Er_Pd_Et
SA155172s0369community serum Cs_Bt_Ca_Er_Pd_Et
SA155173s0372community serum Cs_Bt_Ca_Er_Pd_Et
SA155174s0365community serum germ-free
SA155175s0364community serum germ-free
SA155176s0358community serum germ-free
SA155177s0371community serum germ-free
SA155178s0370community serum germ-free
SA155179s0286community urine Bt_Ca_Er_Pd_Et
SA155180s0281community urine Bt_Ca_Er_Pd_Et
SA155181s0290community urine Bt_Ca_Er_Pd_Et
SA155182s0295community urine Bt_Ca_Er_Pd_Et
SA155183s0309community urine Bt_Ca_Er_Pd_Et
SA155184s0304community urine Bt_Ca_Er_Pd_Et
SA155185s0314community urine Bt_Ca_Er_Pd_Et
SA155186s0245community urine Cs_Bt_Ca_Er_Pd_Et
SA155187s0250community urine Cs_Bt_Ca_Er_Pd_Et
SA155188s0254community urine Cs_Bt_Ca_Er_Pd_Et
SA155189s0259community urine Cs_Bt_Ca_Er_Pd_Et
SA155190s0277community urine Cs_Bt_Ca_Er_Pd_Et
SA155191s0272community urine Cs_Bt_Ca_Er_Pd_Et
SA155192s0227community urine germ-free
SA155193s0231community urine germ-free
SA155194s0236community urine germ-free
SA155195s1073conventional caecal conventional
SA155196s1072conventional caecal conventional
SA155197s1074conventional caecal conventional
SA155198s1065conventional caecal germ-free
SA155199s1067conventional caecal germ-free
SA155200s1066conventional caecal germ-free
SA155201s1064conventional caecal germ-free
SA155202s1077conventional feces conventional
SA155203s1076conventional feces conventional
SA155204s1075conventional feces conventional
SA155205s1069conventional feces germ-free
SA155206s1068conventional feces germ-free
SA155207s1070conventional feces germ-free
SA155208s1071conventional feces germ-free
SA155209s1061conventional serum conventional
SA155210s1062conventional serum conventional
SA155211s1063conventional serum conventional
SA155212s1060conventional serum germ-free
SA155213s1058conventional serum germ-free
SA155214s1057conventional serum germ-free
SA155215s1059conventional serum germ-free
SA155216s1055conventional urine conventional
SA155217s1054conventional urine conventional
SA155218s1056conventional urine conventional
SA155219s1050conventional urine germ-free
SA155220s1051conventional urine germ-free
SA155221s1052conventional urine germ-free
SA155222s1053conventional urine germ-free
SA155223s1100mono-colonization_2 caecal As
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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.

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.

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 (2.1 mm id x 100 mm, 1.7 µm) Waters Acquity BEH (2.1 mm id x 100 mm, 1.7 µm) 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 (2.1 mm id x 100 mm, 1.7 µm)
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 (2.1 mm id x 100 mm, 1.7 µm)
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)
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
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