Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA071664

Local Sample ID:	PCC 7002_Method 1_Rep2
Subject ID:	SU001099
Subject Type:	Bacteria
Subject Species:	Synechococcus
Taxonomy ID:	1129

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Subject:

Subject ID:	SU001099
Subject Type:	Bacteria
Subject Species:	Synechococcus
Taxonomy ID:	1129

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
PCC 7002_Method 1_Rep2	SA071664	FL011231	Method 1	Extraction Method Used

Collection:

Collection ID:	CO001093
Collection Summary:	20 mL Cyanobacterial cells grown at exponential phase were filtered and extracted with the extraction buffers suggested.
Sample Type:	Bacterial cells

Treatment:

Treatment ID:	TR001113
Treatment Summary:	Various cells were extracted using the extraction solutions described as Method 1, Method 2A and Method 2B

Sample Preparation:

Sampleprep ID:	SP001106
Sampleprep Summary:	The cell suspension was filtered through a 0.8 µm nylon membrane and the filtered cells was quickly placed in 1.6 ml cold methanol (100%) or methanol-water mixture (80:20 v/v). The mixture was incubated at -80 °C for 1 hour following which the cell-solvent mixture was removed and placed in a 15 mL centrifuge tube. The cells sticking to the filter were then scraped off with chloroform (2.4 mL) and mixed with the cell-solvent mixture. The mixture was vortexed for 20 minutes under cold conditions (vortexed for 30s and kept on ice for 1 minute and the cycle repeated). Two mL of MilliQ water or NH4OH solution in water (at concentrations of 0.2 M, 0.4 M) was added into the mixture to separate the phases. The mixture was vortexed for 10 minutes, centrifuged for 15 minutes at 3200g at 4 °C to obtain two separate phases and the top aqueous-rich layer was collected (approximately 3 mL), evaporated to dryness and stored at -80 °C until further analysis. The stored pellets were reconstituted in 100 μL of 50/50 methanol-water mixture and filtered before injecting on LC/MS.

Sampleprep ID:

SP001106

Sampleprep Summary:

The cell suspension was filtered through a 0.8 µm nylon membrane and the filtered cells was quickly placed in 1.6 ml cold methanol (100%) or methanol-water mixture (80:20 v/v). The mixture was incubated at -80 °C for 1 hour following which the cell-solvent mixture was removed and placed in a 15 mL centrifuge tube. The cells sticking to the filter were then scraped off with chloroform (2.4 mL) and mixed with the cell-solvent mixture. The mixture was vortexed for 20 minutes under cold conditions (vortexed for 30s and kept on ice for 1 minute and the cycle repeated). Two mL of MilliQ water or NH4OH solution in water (at concentrations of 0.2 M, 0.4 M) was added into the mixture to separate the phases. The mixture was vortexed for 10 minutes, centrifuged for 15 minutes at 3200g at 4 °C to obtain two separate phases and the top aqueous-rich layer was collected (approximately 3 mL), evaporated to dryness and stored at -80 °C until further analysis. The stored pellets were reconstituted in 100 μL of 50/50 methanol-water mixture and filtered before injecting on LC/MS.

Combined analysis:

Analysis ID	AN001726
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Shimadzu 20AD
Column	Phenomenex Synergi Hydro RP (100 x 2mm,2.5um)
MS Type	ESI
MS instrument type	Triple TOF
MS instrument name	ABI Sciex 5600+ TripleTOF
Ion Mode	NEGATIVE
Units	Peak Area

Chromatography:

Chromatography ID:	CH001220
Instrument Name:	Shimadzu 20AD
Column Name:	Phenomenex Synergi Hydro RP (100 x 2mm,2.5um)
Flow Rate:	300 microliter/min
Solvent A:	100% water; 10 mM tributylamine
Solvent B:	100% methanol
Chromatography Type:	Reversed phase

MS:

MS ID:	MS001596
Analysis ID:	AN001726
Instrument Name:	ABI Sciex 5600+ TripleTOF
Instrument Type:	Triple TOF
MS Type:	ESI
Ion Mode:	NEGATIVE