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MB Sample ID: SA176940

Local Sample ID:	86_E-FW-DF-Inc_1
Subject ID:	SU001990
Subject Type:	Algae
Subject Species:	Chlamydomonas reinhardtii;Desmodesmus sp.;Phaeodactylum tricornutum;Microchloropsis salina
Taxonomy ID:	3055;91202;2950;2511165

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Subject:

Subject ID:	SU001990
Subject Type:	Algae
Subject Species:	Chlamydomonas reinhardtii;Desmodesmus sp.;Phaeodactylum tricornutum;Microchloropsis salina
Taxonomy ID:	3055;91202;2950;2511165

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
86_E-FW-DF-Inc_1	SA176940	FL021941	E - exometabolome	Type
86_E-FW-DF-Inc_1	SA176940	FL021941	FW - freshwater	Medium
86_E-FW-DF-Inc_1	SA176940	FL021941	DF - Desmodesmus sp.	Algal strain or abiotic control condition
86_E-FW-DF-Inc_1	SA176940	FL021941	Inc - incubated	Incubation

Collection:

Collection ID:	CO001983
Collection Summary:	Axenic algal were grown in glass 125 mL Erlenmeyer flasks that had previously been baked in a muffle furnace at 550°C for two hours to remove trace organics that could contaminate metabolomics analyses. Each flask contained 50 mL of the appropriate medium (defined freshwater or saltwater medium) and was capped with a foam stopper and covered with aluminum foil to avoid contamination during the experiment. Flasks were incubated with shaking at 90 rpm in a light incubator, with a 12-hour day/night light cycle. Daytime illumination was 3500 lux, and the incubator temperature was maintained at 22°C. Algal inocula were prepared by first growing algal stock cultures under experimental conditions (flasks, media, incubation) for one week to acclimate cultures to those conditions. Experimental flasks were inoculated by transferring 2 mL of inoculum culture to flasks with the appropriate medium. Cultures were harvested for metabolomic analysis from five replicate flasks for each alga after 8 days of incubation. Spent medium and cell pellets were separated by centrifugation. Half of the culture was transferred to a 50 mL falcon tube and centrifuged at 5000 g for eight minutes at 4°C. The supernatant was carefully transferred to a clean Falcon tube, and the remainder of the culture was added to the tube containing the pellet and the centrifugation procedure was repeated. After separation by centrifugation, the supernatant was filtered through a 0.45µm pore size syringe filter to remove residual algal cells. Filtered spent medium and cell pellets were frozen on dry ice and stored at -80°C until extraction.
Sample Type:	Algae
Storage Conditions:	-80℃

Treatment:

Treatment ID:	TR002002
Treatment Summary:	Treatments consisted of 4 different axenic algal strains growth separately for comparison: Chlamydomonas reinhardtii, Desmodesmus sp., Phaeodactylum tricornutum, and Microchloropsis salina. C. reinhartdii was grown in defined freshwater mediunm. Desmodesmus was grown separately in defined freshwater and saltwater medium. P. tricornutum and M. salina were grown separately in defined saltwater medium. Separate axenic controls were conducted for the defined freshwater and saltwater media. Metabolite extraction controls were also prepared using the same extraction protocol.

Treatment ID:

TR002002

Treatment Summary:

Treatments consisted of 4 different axenic algal strains growth separately for comparison: Chlamydomonas reinhardtii, Desmodesmus sp., Phaeodactylum tricornutum, and Microchloropsis salina. C. reinhartdii was grown in defined freshwater mediunm. Desmodesmus was grown separately in defined freshwater and saltwater medium. P. tricornutum and M. salina were grown separately in defined saltwater medium. Separate axenic controls were conducted for the defined freshwater and saltwater media. Metabolite extraction controls were also prepared using the same extraction protocol.

Sample Preparation:

Sampleprep ID:	SP001996
Sampleprep Summary:	In order to disrupt cells for metabolite extraction, cell pellets were resuspended in 1 mL sterile water, frozen at -80°C, and lyophilized. Lyophilized pellets were disrupted using a sterilized steel ball and vortexing three times for five seconds. Disrupted pellets were then resuspended in 50 mL sterile water and filtered and frozen in the same manner as spent medium. Thus, the spent medium and cell pellet samples corresponded to the same original sample volume. Samples for LC-MS/MS metabolomics analysis were extracted by solid phase extraction using Waters Sep-Pak C18 3 mL cartridges. Because chlorophyll and biomass content were similar across samples, samples were normalized to the same volume (30 mL). Cartridges were first preconditioned with 3 mL methanol, and the rinsed with 6 mL ultrapure water. Metabolites were extracted by passing 30 mL of sample through the cartridge. Columns with extracted metabolites were rinsed with 6 mL ultrapure water. Metabolites were eluted with 1 mL methanol into a 1.5 mL polypropylene tube and the dried in a vacuum centrifuge with a refrigerated vapor trap. Dried metabolite extracts resuspended in 150 µL methanol with matrix control internal standards, vortexed for 5 to 10 seconds, and sonicated in a water bath sonicator with ice for ten minutes. Samples were centrifuged at 10,000 g for five minutes at 4°C to pellet insoluble salts and proteins. Supernatant was transferred to a 0.2 µm pore size centrifuge filter device, and centrifuged at 5,000 g for five minutes at 4°C. Filtrate was transferred to an autosampler vial for LC-MS/MS analysis.

Sampleprep ID:

SP001996

Sampleprep Summary:

In order to disrupt cells for metabolite extraction, cell pellets were resuspended in 1 mL sterile water, frozen at -80°C, and lyophilized. Lyophilized pellets were disrupted using a sterilized steel ball and vortexing three times for five seconds. Disrupted pellets were then resuspended in 50 mL sterile water and filtered and frozen in the same manner as spent medium. Thus, the spent medium and cell pellet samples corresponded to the same original sample volume. Samples for LC-MS/MS metabolomics analysis were extracted by solid phase extraction using Waters Sep-Pak C18 3 mL cartridges. Because chlorophyll and biomass content were similar across samples, samples were normalized to the same volume (30 mL). Cartridges were first preconditioned with 3 mL methanol, and the rinsed with 6 mL ultrapure water. Metabolites were extracted by passing 30 mL of sample through the cartridge. Columns with extracted metabolites were rinsed with 6 mL ultrapure water. Metabolites were eluted with 1 mL methanol into a 1.5 mL polypropylene tube and the dried in a vacuum centrifuge with a refrigerated vapor trap. Dried metabolite extracts resuspended in 150 µL methanol with matrix control internal standards, vortexed for 5 to 10 seconds, and sonicated in a water bath sonicator with ice for ten minutes. Samples were centrifuged at 10,000 g for five minutes at 4°C to pellet insoluble salts and proteins. Supernatant was transferred to a 0.2 µm pore size centrifuge filter device, and centrifuged at 5,000 g for five minutes at 4°C. Filtrate was transferred to an autosampler vial for LC-MS/MS analysis.

Combined analysis:

Analysis ID	AN003109	AN003110
Analysis type	MS	MS
Chromatography type	Reversed phase	Reversed phase
Chromatography system	Agilent 1290	Agilent 1290
Column	Agilent Sorbax Eclipse Plus C18 RRHD (50 x 2.1 mm, 1.8 um)	Agilent Sorbax Eclipse Plus C18 RRHD (50 x 2.1 mm, 1.8 um)
MS Type	ESI	ESI
MS instrument type	Single quadrupole	Single quadrupole
MS instrument name	Thermo Q Exactive Orbitrap	Thermo Q Exactive Orbitrap
Ion Mode	NEGATIVE	POSITIVE
Units	peak height	peak height

Chromatography:

Chromatography ID:	CH002296
Instrument Name:	Agilent 1290
Column Name:	Agilent Sorbax Eclipse Plus C18 RRHD (50 x 2.1 mm, 1.8 um)
Chromatography Type:	Reversed phase

MS:

MS ID:	MS002891
Analysis ID:	AN003109
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Single quadrupole
MS Type:	ESI
MS Comments:	Feature detection conducted using MZmine 2
Ion Mode:	NEGATIVE

MS ID:	MS002892
Analysis ID:	AN003110
Instrument Name:	Thermo Q Exactive Orbitrap
Instrument Type:	Single quadrupole
MS Type:	ESI
MS Comments:	Feature detection conducted using MZmine 2
Ion Mode:	POSITIVE