Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA174902

Local Sample ID:	bdivrou_mero_2_met_2
Subject ID:	SU001955
Subject Type:	Other organism
Subject Species:	Babesia divergens
Taxonomy ID:	32595
Genotype Strain:	Rouen 1986

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Sample Preparation:

Sampleprep ID:	SP001961
Sampleprep Summary:	Metabolite extraction and quenching: four volumes of cold methanol were added to one volume isolated B. divergens merozoite pellets placed on ice and quickly mixed using a high speed vortex. Then, samples were placed in an ice bath for 10 min, Subsequently, samples were transferred to liquid nitrogen for 10 min and thawed in an ice bath for 10 min (this freeze-thaw cycle was repeated two times). Samples were then centrifuged at 5725 x g and 4 ºC for 5 min. Supernatants containing the metabolite extracts were transferred to new tubes, while the remaining pellets were re-extracted twice by adding 400 μL of cold methanol and undergoing the liquid nitrogen freeze-thaw cycles described above. Supernatants obtained for each biological replicate were combined and filtered through 0.22 μm nylon syringe filters and stored at -80 ºC until use. Prior to LC-QqQ/MS analysis, supernatants underwent LC-QqQ/MS-specific sample preparation. First supernatants were thawed on ice and vortex mixed for 2 min. On ice, 300 µL of each supernatant were transferred to LC/MS vials and evaporated using a vacuum concentrator at <10 bar for 2h. The dried extracts were then reconstituted in 60 µL of Milli-Q water with the help of an ultrasonic bath for 5 min. Then, samples were subjected to LC-QqQ/MS analysis.

Sampleprep ID:

SP001961

Sampleprep Summary:

Metabolite extraction and quenching: four volumes of cold methanol were added to one volume isolated B. divergens merozoite pellets placed on ice and quickly mixed using a high speed vortex. Then, samples were placed in an ice bath for 10 min, Subsequently, samples were transferred to liquid nitrogen for 10 min and thawed in an ice bath for 10 min (this freeze-thaw cycle was repeated two times). Samples were then centrifuged at 5725 x g and 4 ºC for 5 min. Supernatants containing the metabolite extracts were transferred to new tubes, while the remaining pellets were re-extracted twice by adding 400 μL of cold methanol and undergoing the liquid nitrogen freeze-thaw cycles described above. Supernatants obtained for each biological replicate were combined and filtered through 0.22 μm nylon syringe filters and stored at -80 ºC until use. Prior to LC-QqQ/MS analysis, supernatants underwent LC-QqQ/MS-specific sample preparation. First supernatants were thawed on ice and vortex mixed for 2 min. On ice, 300 µL of each supernatant were transferred to LC/MS vials and evaporated using a vacuum concentrator at <10 bar for 2h. The dried extracts were then reconstituted in 60 µL of Milli-Q water with the help of an ultrasonic bath for 5 min. Then, samples were subjected to LC-QqQ/MS analysis.