Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA174903

Local Sample ID:	bdivrou_mero_1_met_2
Subject ID:	SU001955
Subject Type:	Other organism
Subject Species:	Babesia divergens
Taxonomy ID:	32595
Genotype Strain:	Rouen 1986

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Subject:

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

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
bdivrou_mero_1_met_2	SA174903	FL021497	Wild-type	Genotype

Collection:

Collection ID:	CO001948
Collection Summary:	Merozoites were isolated from B. divergens infected erythrocite (iRBC) cultures at 40% parasitemia. The content from iRBC culture flasks was transferred to Falcon tubes and spinned at 600 x g and 4 ºC for 5 min. Then, supernatants were filtered once using 5 μm filters and twice using 1.2 μm filters (Versapor membranes) with the help of a 1 mL syringe. Subsequently, the resulting volume was spinned on a Falcon tube at 2000 x g and 4 ºC for 2 min. The supernatants were discarded, and merozoite-containing pellets were placed on ice.
Sample Type:	merozoites isolated from RBC

Treatment:

Treatment ID:	TR001967
Treatment Summary:	Since this was a qualitative study aiming to characterize specific metabolites from the Babesia divergens merozoites, only one sample group was included and no treatment was performed

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.

Combined analysis:

Analysis ID	AN003081	AN003082
Analysis type	MS	MS
Chromatography type	Reversed phase	Reversed phase
Chromatography system	Agilent 1290 Infinity II	Agilent 1290 Infinity II
Column	Agilent Zorbax RRHD SB-C18 (100 x 2.1mm,1.8um)	Agilent Zorbax RRHD SB-C18 (100 x 2.1mm,1.8um)
MS Type	ESI	ESI
MS instrument type	Triple quadrupole	Triple quadrupole
MS instrument name	Agilent 6460 QQQ	Agilent 6460 QQQ
Ion Mode	NEGATIVE	NEGATIVE
Units

Chromatography:

Chromatography ID:	CH002253
Chromatography Summary:	The chromatographic method consists on a subtle adaptation of the Agilent dMRM method, which uses tributylamine as an ion pairing reagent (for more information, see https://www.agilent.com/cs/library/technicaloverviews/public/5991-6482EN.pdf)
Instrument Name:	Agilent 1290 Infinity II
Column Name:	Agilent Zorbax RRHD SB-C18 (100 x 2.1mm,1.8um)
Chromatography Type:	Reversed phase

MS:

MS ID:	MS002827
Analysis ID:	AN003081
Instrument Name:	Agilent 6460 QQQ
Instrument Type:	Triple quadrupole
MS Type:	ESI
MS Comments:	Method 1: Data was acquired in MRM mode. To achieve a higher signal from low-abundance metabolites, transitions corresponding to metabolites of interests were separated in two MS methods (see method 2). Transitions utilized in this study are subtle modifications of the transition dataset contained in the Agilent dMRM database and method (see https://www.agilent.com/cs/library/technicaloverviews/public/5991-6482EN.pdf). Feature assignment was performed by matching transitions and RT with these contained in the Agilent dMRM Database and Method, and further confirmed by addition of authentic MS-grade standards. The software workflow included user visualization in Agilent MassHunter Workstation Software Qualitative Analysis (version B.09.00), and further compound integration using Agilent MassHunter Workstation Software Quantitative Analysis (version B.09.00). Only metabolites with signal-to-noise ratio higher than 3 (limit of detection) were reported.
Ion Mode:	NEGATIVE

MS ID:	MS002828
Analysis ID:	AN003082
Instrument Name:	Agilent 6460 QQQ
Instrument Type:	Triple quadrupole
MS Type:	ESI
MS Comments:	Method 1: Data was acquired in MRM mode. To achieve a higher signal from low-abundance metabolites, transitions corresponding to metabolites of interests were separated in two MS methods (see method 2). Transitions utilized in this study are subtle modifications of the transition dataset contained in the Agilent dMRM database and method (see https://www.agilent.com/cs/library/technicaloverviews/public/5991-6482EN.pdf). Feature assignment was performed by matching transitions and RT with these contained in the Agilent dMRM Database and Method, and further confirmed by addition of authentic MS-grade standards. The software workflow included user visualization in Agilent MassHunter Workstation Software Qualitative Analysis (version B.09.00), and further compound integration using Agilent MassHunter Workstation Software Quantitative Analysis (version B.09.00). Only metabolites with signal-to-noise ratio higher than 3 (limit of detection) were reported.
Ion Mode:	NEGATIVE