Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA204503

Local Sample ID:	WT-0-1P
Subject ID:	SU002215
Subject Type:	Bacteria
Subject Species:	Escherichia coli
Taxonomy ID:	562

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Subject:

Subject ID:	SU002215
Subject Type:	Bacteria
Subject Species:	Escherichia coli
Taxonomy ID:	562

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
WT-0-1P	SA204503	FL025113	standard growth conditions	Treatment

Collection:

Collection ID:	CO002208
Collection Summary:	After 18h of culture, the sample pellet was isolated. The bacterial pellets harvested from 50 mL of broth culture were mixed with 1.2 mL 80% ice-cold methanol (added to internal standard 0.001mg/ml 4-chloro-DL-phenylalanine), then vortexed for 30 s, and placed on dry ice for 30 min. The samples were centrifuged at 18000 × g for 15 min at 4 °C. The frozen samples were ground with beads and the homogenates were centrifuged at 18000 × g for 15 min at 4 °C. The supernatant was mixed with 800μL ice-cold acetonitrile, and then left to stand for 20 minutes in an ice bath. After centrifugation at 18000 × g 4℃ for 15 min, the supernatant was removed and filtered through 0.22μm membrane.
Sample Type:	Bacterial cells

Treatment:

Treatment ID:	TR002227
Treatment Summary:	M63 medium (1.36% monopotassium phosphate, 0.2% ammonium sulfate, 0.024% magnesium sulfate, 0.001% calcium chloride, and 0.0015% nicotinic acid) was used to form MG1655 and UTI89 mutants. In addition, add ferric chloride solution to the medium to prepare 10μM iron M63 medium, we cultured the wild UTI89 strain in the presence of 10μM iron. The E. coli strain was incubated in LB-agar plate for 12 hours, one colony was isolated to LB broth for further 4 hours incubation, then diluted the solution into M63 medium at a ratio of 1:100 and the cultures were incubated for another18 h at 37°C, 200rpm to culture E. coli.

Treatment ID:

TR002227

Treatment Summary:

M63 medium (1.36% monopotassium phosphate, 0.2% ammonium sulfate, 0.024% magnesium sulfate, 0.001% calcium chloride, and 0.0015% nicotinic acid) was used to form MG1655 and UTI89 mutants. In addition, add ferric chloride solution to the medium to prepare 10μM iron M63 medium, we cultured the wild UTI89 strain in the presence of 10μM iron. The E. coli strain was incubated in LB-agar plate for 12 hours, one colony was isolated to LB broth for further 4 hours incubation, then diluted the solution into M63 medium at a ratio of 1:100 and the cultures were incubated for another18 h at 37°C, 200rpm to culture E. coli.

Sample Preparation:

Sampleprep ID:	SP002221
Sampleprep Summary:	The bacterial pellets harvested from 50 mL of broth culture were mixed with 1.2 mL 80% ice-cold methanol (added to internal standard 0.001mg/ml 4-chloro-DL-phenylalanine), then vortexed for 30 s, and placed on dry ice for 30 min. The samples were centrifuged at 18000 × g for 15 min at 4 °C. The frozen samples were ground with beads and the homogenates were centrifuged at 18000 × g for 15 min at 4 °C. The supernatant was mixed with 800μL ice-cold acetonitrile, and then left to stand for 20 minutes in an ice bath. After centrifugation at 18000 × g 4℃ for 15 min, the supernatant was removed and filtered through 0.22μm membrane. For LC/MS based metabolomics analysis, the dried samples were dissolved in 200μL water and 5μL aliquots were analyzed.

Sampleprep ID:

SP002221

Sampleprep Summary:

The bacterial pellets harvested from 50 mL of broth culture were mixed with 1.2 mL 80% ice-cold methanol (added to internal standard 0.001mg/ml 4-chloro-DL-phenylalanine), then vortexed for 30 s, and placed on dry ice for 30 min. The samples were centrifuged at 18000 × g for 15 min at 4 °C. The frozen samples were ground with beads and the homogenates were centrifuged at 18000 × g for 15 min at 4 °C. The supernatant was mixed with 800μL ice-cold acetonitrile, and then left to stand for 20 minutes in an ice bath. After centrifugation at 18000 × g 4℃ for 15 min, the supernatant was removed and filtered through 0.22μm membrane. For LC/MS based metabolomics analysis, the dried samples were dissolved in 200μL water and 5μL aliquots were analyzed.

Combined analysis:

Analysis ID	AN003483	AN003484
Analysis type	MS	MS
Chromatography type	Reversed phase	Reversed phase
Chromatography system	Agilent 1290 Infinity	Agilent 1290 Infinity
Column	Waters Acquity BEH HSS T3 (100 x 2.1mm,1.8um)	Waters Acquity BEH HSS T3 (100 x 2.1mm,1.8um)
MS Type	ESI	ESI
MS instrument type	Triple quadrupole	Triple quadrupole
MS instrument name	Agilent 6495 QQQ	Agilent 6495 QQQ
Ion Mode	POSITIVE	NEGATIVE
Units	peak area	peak area

Chromatography:

Chromatography ID:	CH002572
Instrument Name:	Agilent 1290 Infinity
Column Name:	Waters Acquity BEH HSS T3 (100 x 2.1mm,1.8um)
Chromatography Type:	Reversed phase

MS:

MS ID:	MS003244
Analysis ID:	AN003483
Instrument Name:	Agilent 6495 QQQ
Instrument Type:	Triple quadrupole
MS Type:	ESI
MS Comments:	Agilent MassHunter Workstation Data Acquisition Agilent MassHunter QualitativeAnalysis B.07.00 Agilent MassHunter Quantitative Analysis (for QQQ)
Ion Mode:	POSITIVE

MS ID:	MS003245
Analysis ID:	AN003484
Instrument Name:	Agilent 6495 QQQ
Instrument Type:	Triple quadrupole
MS Type:	ESI
MS Comments:	Agilent MassHunter Workstation Data Acquisition Agilent MassHunter QualitativeAnalysis B.07.00 Agilent MassHunter Quantitative Analysis (for QQQ)
Ion Mode:	NEGATIVE