Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA174398

Local Sample ID:	BE5_16h
Subject ID:	SU001939
Subject Type:	Bacteria
Subject Species:	Bacteroides spp. and Escherichia spp. (mixed communities)
Taxonomy ID:	B. fragilis 3_1_12; B. vulgatus 3_1_40A; B. ovatus 3_8_47; B. dorei 5_1_36; P. distasonis 2_1_33B; E. coli 3_1_53; E. coli 4_1_47

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Sample Preparation:

Sampleprep ID:	SP001945
Sampleprep Summary:	Sugars Analysis Low molecular weight sugars and NAc-sugar amines were quantified by LC-MRM/MS at The Metabolomics Innovation Centre (TMIC) commercial service (University of Victoria , Genome BC Proteomics Centre), according to previously published UPLC-MRM/MS methods (Han et al 2016, Electrophoresis), with necessary modifications. In brief, a mixed stock solution of 13 low-MW sugars and 4 N-acetyl sugar amines was prepared with the use of their standard substances in 80% methanol at 500µM for each compound. This solution was then serially diluted with the same solvent to prepare calibration solutions in a concentration range of 0.002 to 125µM. For chemical derivatization, 20µL of each medium sample or each calibration solution was mixed in turn with 20µL of an internal standard solution containing 13C6-glucose, 13C6-mannose, 13C6-fructose and 13C5-ribose in water, 40µL of 200-mM 3-nitrophenylhydrazine hydrochloride solution in 60% methanol and 40µL of 200-mM EDC.HCl solution prepared in a mixed solvent of methanol/water/pyridine (60:40:5, v/v/v). The mixture was allowed to react at 50ºC for 90 min. SCFA Analysis Quantification of short-chain fatty acids was performed in-house according to a method developed by Han et al., with minor modifications(Han et al 2015, Analytica Chimica Acta). Briefly, 500µL of supernatant were mixed with 500µL of 50 % acetonitrile, then the mixture was vortexed for 5 minutes and centrifuged at 7000 x g for 5 minutes. 50 µL of the organic phase were derivatized adding 20µL of 200mM 3NPH in 50 % acetonitrile and 20 µL 120 mM EDC solution of 6 % pyridine in 50 % acetonitrile. The mixture was left under agitation at 40ºC for 30 minutes. After this time reaction was stopped adding 100 µL of 0.1 % formic acid in 90 % acetonitrile.

Sampleprep ID:

SP001945

Sampleprep Summary:

Sugars Analysis Low molecular weight sugars and NAc-sugar amines were quantified by LC-MRM/MS at The Metabolomics Innovation Centre (TMIC) commercial service (University of Victoria , Genome BC Proteomics Centre), according to previously published UPLC-MRM/MS methods (Han et al 2016, Electrophoresis), with necessary modifications. In brief, a mixed stock solution of 13 low-MW sugars and 4 N-acetyl sugar amines was prepared with the use of their standard substances in 80% methanol at 500µM for each compound. This solution was then serially diluted with the same solvent to prepare calibration solutions in a concentration range of 0.002 to 125µM. For chemical derivatization, 20µL of each medium sample or each calibration solution was mixed in turn with 20µL of an internal standard solution containing 13C6-glucose, 13C6-mannose, 13C6-fructose and 13C5-ribose in water, 40µL of 200-mM 3-nitrophenylhydrazine hydrochloride solution in 60% methanol and 40µL of 200-mM EDC.HCl solution prepared in a mixed solvent of methanol/water/pyridine (60:40:5, v/v/v). The mixture was allowed to react at 50ºC for 90 min. SCFA Analysis Quantification of short-chain fatty acids was performed in-house according to a method developed by Han et al., with minor modifications(Han et al 2015, Analytica Chimica Acta). Briefly, 500µL of supernatant were mixed with 500µL of 50 % acetonitrile, then the mixture was vortexed for 5 minutes and centrifuged at 7000 x g for 5 minutes. 50 µL of the organic phase were derivatized adding 20µL of 200mM 3NPH in 50 % acetonitrile and 20 µL 120 mM EDC solution of 6 % pyridine in 50 % acetonitrile. The mixture was left under agitation at 40ºC for 30 minutes. After this time reaction was stopped adding 100 µL of 0.1 % formic acid in 90 % acetonitrile.