Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA247703

Local Sample ID:	LN_C2
Subject ID:	SU002562
Subject Type:	Bacteria
Subject Species:	Veillonella parvula
Taxonomy ID:	29466

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Subject:

Subject ID:	SU002562
Subject Type:	Bacteria
Subject Species:	Veillonella parvula
Taxonomy ID:	29466

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
LN_C2	SA247703	FL031136	Cell	Matrix
LN_C2	SA247703	FL031136	Experiment	Sample Type
LN_C2	SA247703	FL031136	Lactate+Nitrate	Tretment

Collection:

Collection ID:	CO002555
Collection Summary:	The strain Veillonella parvula SKV38 xdh::cat*. Briefly, V. parvula was grown on SK agar (composition: yeast extract 10 gL-1, casitone 10 gL-1, NaCl 2 gL-1, K2HPO4 0.4 gL-1) supplemented with either DL-lactate (50 mM lactate), potassium nitrate (40 mM KNO3), or both, incubated under anaerobic conditions at 37ºC overnight and then inoculated into the respective liquid media in biological triplicates. Cells and supernatants were harvested at mid-exponential phase (OD600=0.3), after centrifugation at 10,000g for 5 min. Both cell pellets and resulting supernatants were stored at -80C until processing for metabolite extraction and metabolomic analysis. A titration of cell pools and media pools was conducted (10,20,40,60,80,100%) and used to filter the data based on the correlation of the abundance of detected features with
Sample Type:	Bacterial cells

Treatment:

Treatment ID:	TR002574
Treatment Summary:	The strain was 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 5mL 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.

Treatment ID:

TR002574

Treatment Summary:

The strain was 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 5mL 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.

Sample Preparation:

Sampleprep ID:	SP002568
Sampleprep Summary:	Bacterial metabolites were profiled using the HILIC-pos and C8-pos methods for mapping Veillonella metabolites present in human stool. Bacterial 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, while cell pellets were resuspended in ice-cold PBS. Cells were then spun twice at 20,000g for 1 minute and all PBS supernatant removed and discarded. Cell pellet weights were estimated and all the samples harvested were stored at -80°C until metabolite profiling was conducted. For cells profiled in the C8-pos, cell pellets were resuspended in 20 µL of H2O and metabolites extracted using 380 µL of isopropanol containing 1,2-didodecanoyl-sn-glycero-3-phosphocholine (Avanti Polar Lipids; Alabaster, AL), 10 ul of media was extracted using 190 µL of isopropanol containing internal standards. Extracts were incubated at room temperature in the dark before centrifugation (10 min, 9,000 x g, Room Temperature). For cells profiled in the HILIC-pos mode, cell pellets were resuspended in 20 µL of H2O and metabolites extracted using180 µL HILIC extraction solution with internal standards (valine-d8, Sigma-Aldrich; St. Louis, MO; and phenylalanine-d8, Cambridge Isotope Laboratories; Andover, MA), while 10 µL of media was extracted with 90 µL of extraction solution. Extracts were cleared by centrifugation (10 min, 9,000 x g, 4C) prior to analysis.

Sampleprep ID:

SP002568

Sampleprep Summary:

Bacterial metabolites were profiled using the HILIC-pos and C8-pos methods for mapping Veillonella metabolites present in human stool. Bacterial 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, while cell pellets were resuspended in ice-cold PBS. Cells were then spun twice at 20,000g for 1 minute and all PBS supernatant removed and discarded. Cell pellet weights were estimated and all the samples harvested were stored at -80°C until metabolite profiling was conducted. For cells profiled in the C8-pos, cell pellets were resuspended in 20 µL of H2O and metabolites extracted using 380 µL of isopropanol containing 1,2-didodecanoyl-sn-glycero-3-phosphocholine (Avanti Polar Lipids; Alabaster, AL), 10 ul of media was extracted using 190 µL of isopropanol containing internal standards. Extracts were incubated at room temperature in the dark before centrifugation (10 min, 9,000 x g, Room Temperature). For cells profiled in the HILIC-pos mode, cell pellets were resuspended in 20 µL of H2O and metabolites extracted using180 µL HILIC extraction solution with internal standards (valine-d8, Sigma-Aldrich; St. Louis, MO; and phenylalanine-d8, Cambridge Isotope Laboratories; Andover, MA), while 10 µL of media was extracted with 90 µL of extraction solution. Extracts were cleared by centrifugation (10 min, 9,000 x g, 4C) prior to analysis.

Combined analysis:

Analysis ID	AN004037	AN004038
Analysis type	MS	MS
Chromatography type	HILIC	Reversed phase
Chromatography system	Shimadzu Nexera X2	Shimadzu Nexera X2
Column	Waters Atlantis HILIC (150 x 2 mm, 3 μm)	Waters Acquity BEH C8 (100 x 2.1mm, 1.7um)
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	POSITIVE
Units	Abundance	Abundance

Chromatography:

Chromatography ID:	CH002985
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:	CH002986
Instrument Name:	Shimadzu Nexera X2
Column Name:	Waters Acquity BEH C8 (100 x 2.1mm, 1.7um)
Column Temperature:	40C
Flow Gradient:	The column was eluted at a flow rate of 450 µL/min isocratically for 1 minute at 80% mobile phase A, followed by a linear gradient to 80% mobile-phase B over 2 minutes, a linear gradient to 100% mobile phase B over 7 minutes, and then 3 minutes at 100% mobile-phase B.
Flow Rate:	450 µL/min
Solvent A:	95% water/5% methanol; 10 mM ammonium acetate; 0.1% acetic acid
Solvent B:	100% methanol; 0.1% acetic acid
Chromatography Type:	Reversed phase

MS:

MS ID:	MS003784
Analysis ID:	AN004037
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:	MS003785
Analysis ID:	AN004038
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