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MB Sample ID: SA176980

Local Sample ID:	81_P-FW-Cr-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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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.