Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA247741

Local Sample ID:	None_Azathioprine_R4
Subject ID:	SU002563
Subject Type:	Bacteria
Subject Species:	Veillonella parvula
Taxonomy ID:	29466

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Subject:

Subject ID:	SU002563
Subject Type:	Bacteria
Subject Species:	Veillonella parvula
Taxonomy ID:	29466

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
None_Azathioprine_R4	SA247741	FL031147	None	Strain
None_Azathioprine_R4	SA247741	FL031147	Azathioprine	Drug

Collection:

Collection ID:	CO002556
Collection Summary:	All strain experiments were done in the Xavier lab. Strains and growth conditions: we utilized the following strains of Veillonella for growth and metabolic analysis; Veillonella parvula SKV38 [DR071], Veillonella parvula SKV38 xdh::cat* [DR214], Veillonella parvula SKV38 pucD::cat* [DR213]. All strains were first streaked on an agar plate with SK media (composition: yeast extract 10 gL-1, casitone 10 gL-1, NaCl 2 gL-1, K2HPO4 0.4 gL-1) supplemented with 50 mM lactate and 40 mM KNO3 (SKLN medium) and antibiotics if required. From this agar plate, a single colony was selected and inoculated in 5 mL SKLN media and grown for 24 hours. Next, overnight cells from this inoculum were grown using a 1/50 inoculum of the overnight culture, on either SK, SK + 50 mM lactate (SKL), SK + 40 mM nitrate (SKN), and SKLN. Supernatants were collected at the mid-exponential phase (OD600~0.4) and collected for metabolomic analyses (HILIC-pos metabolite profiling method described above). For analysis of the metabolism of the IBD drugs, cells were prepared as described above, and mid-exponential phase cells were collected, washed twice with sterile SK, and resuspended in 100 µl of SK. A volume of 6 ml of SKN was then inoculated using 90 µl of the bacterial suspension. Those 6 ml were split in three parts: a) 1.5 ml SK, b) 1.5 ml SK + 20 µM 6-mercaptopurine, and c) 1.5 ml SK + 20 µM 6-azathioprine. Additionally, sterile SK was prepared in an identical fashion to serve as a control for the experiment. Cells were incubated in a plate reader (4 wells/treatment) with low shaking and 37ºC for about ~5 h, and when the OD600 reached 0.4 cells were collected. For collection, 700 µl of the suspensions were centrifuged at 21,000 g for 2 min, and 400 µl of the supernatant harvested and stored at -20 ºC for analysis. Preparation of thiopurine drugs: 20 mM solutions were prepared using 4.25 mg of 6-mercaptopurine (Sigma 852678) or 6.9 mg of azathioprine (Sigma A4638) dissolved in 1 ml of DMSO. The drugs were prepared and used the same day.
Sample Type:	Culture Media

Treatment:

Treatment ID:	TR002575
Treatment Summary:	A volume of 6 ml of SKN was then inoculated using 90 µl of the bacterial suspension. Those 6 ml were split in three parts: a) 1.5 ml SK, b) 1.5 ml SK + 20 µM 6-mercaptopurine, and c) 1.5 ml SK + 20 µM 6-azathioprine. Preparation of thiopurine drugs: 20 mM solutions were prepared using 4.25 mg of 6-mercaptopurine (Sigma 852678) or 6.9 mg of azathioprine (Sigma A4638) dissolved in 1 ml of DMSO. The drugs were prepared and used the same day.

Treatment ID:

TR002575

Treatment Summary:

A volume of 6 ml of SKN was then inoculated using 90 µl of the bacterial suspension. Those 6 ml were split in three parts: a) 1.5 ml SK, b) 1.5 ml SK + 20 µM 6-mercaptopurine, and c) 1.5 ml SK + 20 µM 6-azathioprine. Preparation of thiopurine drugs: 20 mM solutions were prepared using 4.25 mg of 6-mercaptopurine (Sigma 852678) or 6.9 mg of azathioprine (Sigma A4638) dissolved in 1 ml of DMSO. The drugs were prepared and used the same day.

Sample Preparation:

Sampleprep ID:	SP002569
Sampleprep Summary:	Bacterial supernatant (media) metabolites were profiled using the HILIC-pos and HILIC-neg methods in order to estimate purines and thiopurines metabolism. Media samples were prepared as follows: mid-exponential Veillonella cultures (OD600 = 0.3-0.4) were harvested by centrifugation at 20,000g at 4°C for 1 minute, supernatants (spent media) were aliquoted and stored at -80°C until metabolite profiling was conducted. For the HILIC-pos method, media samples (10 µL) were extracted using 90 µL HILIC extraction solution (74.9:24.9:0.2 v/v/v acetonitrile/methanol/formic acid) with internal standards (valine-d8, Sigma-Aldrich; St. Louis, MO; and phenylalanine-d8) and extracts were cleared by centrifugation (10 min, 9,000 x g, Room Temperature). For media profiled in the HILIC-neg mode, 30 µL of media and metabolites extracted using 80% methanol containing inosine-15N4, thymine-d4 and glycocholate-d4 internal standards (Cambridge Isotope Laboratories; Andover, MA). Extracts were cleared by centrifugation (10 min, 9,000 x g, 4C) prior to analysis.

Sampleprep ID:

SP002569

Sampleprep Summary:

Bacterial supernatant (media) metabolites were profiled using the HILIC-pos and HILIC-neg methods in order to estimate purines and thiopurines metabolism. Media samples were prepared as follows: mid-exponential Veillonella cultures (OD600 = 0.3-0.4) were harvested by centrifugation at 20,000g at 4°C for 1 minute, supernatants (spent media) were aliquoted and stored at -80°C until metabolite profiling was conducted. For the HILIC-pos method, media samples (10 µL) were extracted using 90 µL HILIC extraction solution (74.9:24.9:0.2 v/v/v acetonitrile/methanol/formic acid) with internal standards (valine-d8, Sigma-Aldrich; St. Louis, MO; and phenylalanine-d8) and extracts were cleared by centrifugation (10 min, 9,000 x g, Room Temperature). For media profiled in the HILIC-neg mode, 30 µL of media and metabolites extracted using 80% methanol containing inosine-15N4, thymine-d4 and glycocholate-d4 internal standards (Cambridge Isotope Laboratories; Andover, MA). Extracts were cleared by centrifugation (10 min, 9,000 x g, 4C) prior to analysis.

Combined analysis:

Analysis ID	AN004039	AN004040
Analysis type	MS	MS
Chromatography type	HILIC	HILIC
Chromatography system	Shimadzu Nexera X2	Shimadzu Nexera X2
Column	Waters Atlantis HILIC (150 x 2 mm, 3 μm)	Phenomenex Luna NH2 (150 x 2.1mm,3um)
MS Type	ESI	ESI
MS instrument type	Orbitrap	Orbitrap
MS instrument name	Thermo Q Exactive Plus Orbitrap	Thermo Q Exactive Plus Orbitrap
Ion Mode	POSITIVE	NEGATIVE
Units	Abundance	Abundance

Chromatography:

Chromatography ID:	CH002987
Instrument Name:	Shimadzu Nexera X2
Column Name:	Waters Atlantis HILIC (150 x 2 mm, 3 μm)
Column Temperature:	30C
Flow Gradient:	Isocratically with 5% mobile phase A for 1 minute followed by a linear gradient to 40% mobile phase B over 10 minutes
Flow Rate:	250 µL/min
Solvent A:	100% water; 10 mM ammonium formate; 0.1% formic acid
Solvent B:	100% acetonitrile; 0.1% formic acid
Chromatography Type:	HILIC

Chromatography ID:	CH002988
Instrument Name:	Shimadzu Nexera X2
Column Name:	Phenomenex Luna NH2 (150 x 2.1mm,3um)
Column Temperature:	30C
Flow Gradient:	The column was eluted with initial conditions of 10% mobile phase A and 90% mobile phase B followed by a 10 min linear gradient to 100% mobile phase A.
Flow Rate:	400 µL/min
Solvent A:	100% water; 20 mM ammonium acetate; 20 mM ammonium hydroxide
Solvent B:	75% acetonitrile/25% methanol; 10 mM ammonium hydroxide
Chromatography Type:	HILIC

MS:

MS ID:	MS003786
Analysis ID:	AN004039
Instrument Name:	Thermo Q Exactive Plus Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Raw data were processed using TraceFinder 3.3 software (Thermo Fisher Scientific; Waltham, MA) and Progenesis QI (Nonlinear Dynamics; Newcastle upon Tyne, UK). Metabolite identities were confirmed using authentic reference standards or reference samples.
Ion Mode:	POSITIVE

MS ID:	MS003787
Analysis ID:	AN004040
Instrument Name:	Thermo Q Exactive Plus Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Raw data were processed using TraceFinder 3.3 software (Thermo Fisher Scientific; Waltham, MA) and Progenesis QI (Nonlinear Dynamics; Newcastle upon Tyne, UK). Metabolite identities were confirmed using authentic reference standards or reference samples.
Ion Mode:	NEGATIVE