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

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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.