Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA155232

Local Sample ID:	s1164
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

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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.