Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA174377

Local Sample ID:	blank_media1
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

Select appropriate tab below to view additional metadata details:

Subject:

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

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
blank_media1	SA174377	FL021423	0h	Factor

Collection:

Collection ID:	CO001932
Collection Summary:	Bacteria were grown anaerobically at 37ºC in MAL-M medium. Culture supernatants at 0, 16 and 24h were collected by centrifugation at 16000 g for 20 minutes. Supernatants were filter sterilized at 0.22 µM and stored at -70ºC before analysis.
Sample Type:	Bacterial cells
Storage Conditions:	Described in summary

Treatment:

Treatment ID:	TR001951
Treatment Summary:	The groups differ by bacterial community composition as follows: B, Bacteroidales mix (B. fragilis, B. vulgatus B. ovatus, B. dorei, P. distasonis); E, E. coli mix (E. coli 3_1_53 and E. coli 4_1_47); BE, Bacteroidales-E.coli mix (all seven strains as above); BO, B. ovatus only. Strains were inoculated in equal proportions based on normalized O.D. from overnight input cultures.

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.

Combined analysis:

Analysis ID	AN003018	AN003019
Analysis type	MS	MS
Chromatography type	Normal phase	Reversed phase
Chromatography system	Agilent 1290	Agilent 1200
Column	Phenomenex PFP UPLC (2.1 x 150mm,1.7um)	Agilent Zorbax 300 C18 (250x4.6mm)
MS Type	ESI	ESI
MS instrument type	Triple quadrupole	Triple quadrupole
MS instrument name	Agilent 6495 QQQ	Agilent 6460 QQQ
Ion Mode	NEGATIVE	POSITIVE
Units	µM	µM

Chromatography:

Chromatography ID:	CH002236
Chromatography Summary:	Sugars Analysis
Methods Filename:	methods_summary_ms.docx
Instrument Name:	Agilent 1290
Column Name:	Phenomenex PFP UPLC (2.1 x 150mm,1.7um)
Chromatography Type:	Normal phase

Chromatography ID:	CH002237
Chromatography Summary:	SCFA Analysis
Methods Filename:	methods_summary_ms.docx
Instrument Name:	Agilent 1200
Column Name:	Agilent Zorbax 300 C18 (250x4.6mm)
Chromatography Type:	Reversed phase

MS:

MS ID:	MS002807
Analysis ID:	AN003018
Instrument Name:	Agilent 6495 QQQ
Instrument Type:	Triple quadrupole
MS Type:	ESI
MS Comments:	The raw data was acquired using the Agilent MassHunter® 7.0 software. After data acquisitions, linearly regressed calibration curves of individual compounds were constructed with the analyte-to-internal standard peak area ratios measured from injection of the calibration curves. For those compounds without their isotope-labelling analogues as the internal standards, 13C6-fructose was used a common internal standard. Concentrations of the analytes were calculated by interpolating the calibration curves of individual compounds with their analyte-to-internal standard peak area ratios measured from injection of the sample solutions.
Ion Mode:	NEGATIVE

MS ID:	MS002808
Analysis ID:	AN003019
Instrument Name:	Agilent 6460 QQQ
Instrument Type:	Triple quadrupole
MS Type:	ESI
MS Comments:	A collision energy of 10V was used for multiple reaction monitoring (MRM), and LC-MS/MS data were analysed by Mass Hunter Qualitative Analysis B.06.00 software (Agilent Technologies). The identification and quantification of the SCFAs were carried out based on the retention time and mass fragmentation pattern comparing with standards. Six-point calibration curves made by peak area vs concentration of the pure standards were used to quantify the different SCFA. The linearity of the curves was determined by the coefficient of determination (R2), being higher than 0.99 for all standards. Concentrations of the SCFAs were calculated by interpolating the calibration curves of individual compounds.
Ion Mode:	POSITIVE