Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA091411

Local Sample ID:	12 200 L3
Subject ID:	SU001326
Subject Type:	Invertebrate
Subject Species:	Brugia malayi
Taxonomy ID:	6279
Species Group:	Invertebrates

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Subject:

Subject ID:	SU001326
Subject Type:	Invertebrate
Subject Species:	Brugia malayi
Taxonomy ID:	6279
Species Group:	Invertebrates

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
12 200 L3	SA091411	FL013171	L3	Group

Collection:

Collection ID:	CO001320
Collection Summary:	All parasites were obtained from FR3 (Filariasis Research Reagent Resource Center; BEI Resources, Manassas, VA, USA) where they were isolated and separated by sex from infected gerbils (Meriones unguiculatus) or mosquitoes (Aedes aegypti). Worms were flash-frozen and shipped to the New York Blood Center for processing. Stages used for metabolomics analysis included L3 larvae from mosquitoes, adult male and female worms at 120dpi, and microfilaria. The number of worms per sample were 20 adult female worms, 40 adult males, 2X106 microfilariae, and 200 L3 larvae per biological replicate. Samples were washed in 1x PBS and run in triplicate. Adult male and female worms were picked individually from PBS and each biological was weighed. The microfilaria and L3 samples were spun down, the PBS pipetted off, and weighed directly into a metabolomics 2mL screw cap vial with total amounts ranging from 1.3 mg (adult males) to 15.8 mg (microfilaria). Metabolites were extracted and the data analyzed as described in the Supplementary Information.
Sample Type:	Worms

Treatment:

Treatment ID:	TR001341
Treatment Summary:	All parasites were obtained from FR3 (Filariasis Research Reagent Resource Center; BEI Resources, Manassas, VA, USA) where they were isolated and separated by sex from infected gerbils (Meriones unguiculatus) or mosquitoes (Aedes aegypti). Worms were flash-frozen and shipped to the New York Blood Center for processing. Stages used for metabolomics analysis included L3 larvae from mosquitoes, adult male and female worms at 120dpi, and microfilaria. The number of worms per sample were 20 adult female worms, 40 adult males, 2X106 microfilariae, and 200 L3 larvae per biological replicate. Samples were washed in 1x PBS and run in triplicate. Adult male and female worms were picked individually from PBS and each biological was weighed. The microfilaria and L3 samples were spun down, the PBS pipetted off, and weighed directly into a metabolomics 2mL screw cap vial with total amounts ranging from 1.3 mg (adult males) to 15.8 mg (microfilaria). Metabolites were extracted and the data analyzed as described in the Supplementary Information.

Treatment ID:

TR001341

Treatment Summary:

All parasites were obtained from FR3 (Filariasis Research Reagent Resource Center; BEI Resources, Manassas, VA, USA) where they were isolated and separated by sex from infected gerbils (Meriones unguiculatus) or mosquitoes (Aedes aegypti). Worms were flash-frozen and shipped to the New York Blood Center for processing. Stages used for metabolomics analysis included L3 larvae from mosquitoes, adult male and female worms at 120dpi, and microfilaria. The number of worms per sample were 20 adult female worms, 40 adult males, 2X106 microfilariae, and 200 L3 larvae per biological replicate. Samples were washed in 1x PBS and run in triplicate. Adult male and female worms were picked individually from PBS and each biological was weighed. The microfilaria and L3 samples were spun down, the PBS pipetted off, and weighed directly into a metabolomics 2mL screw cap vial with total amounts ranging from 1.3 mg (adult males) to 15.8 mg (microfilaria). Metabolites were extracted and the data analyzed as described in the Supplementary Information.

Sample Preparation:

Sampleprep ID:	SP001334
Sampleprep Summary:	Metabolite extraction – The mass of the weighed worm samples was used to scale the metabolite extraction to a ratio of 16.5 mg / 1 mL extraction solvent. Freezing 80% acetonitrile was added directly to each vial containing the samples, along with zirconium disruption beads (0.5 mm, RPI) and homogenized for 3 min at 4°C in a BeadBlasterTM with a 30 sec on, 30 sec off pattern. The resulting lysate was centrifuged at 21,000 x g for 3 min, and 90% of the supernatant volume was transferred to a 1.5 mL microfuge tube for speed vacuum concentration, no heating. The dry extracts were resolublized in a volume of LCMS grade water 1/10th of that used for the homogenization step, sonicated in a water bath for 3 min, and transferred to a glass insert for analysis.

Sampleprep ID:

SP001334

Sampleprep Summary:

Metabolite extraction – The mass of the weighed worm samples was used to scale the metabolite extraction to a ratio of 16.5 mg / 1 mL extraction solvent. Freezing 80% acetonitrile was added directly to each vial containing the samples, along with zirconium disruption beads (0.5 mm, RPI) and homogenized for 3 min at 4°C in a BeadBlasterTM with a 30 sec on, 30 sec off pattern. The resulting lysate was centrifuged at 21,000 x g for 3 min, and 90% of the supernatant volume was transferred to a 1.5 mL microfuge tube for speed vacuum concentration, no heating. The dry extracts were resolublized in a volume of LCMS grade water 1/10th of that used for the homogenization step, sonicated in a water bath for 3 min, and transferred to a glass insert for analysis.

Combined analysis:

Analysis ID	AN002087	AN002088
Analysis type	MS	MS
Chromatography type	HILIC	Reversed phase
Chromatography system	Thermo Dionex Ultimate 3000 RS	Thermo Dionex Ultimate 3000 RS
Column	SeQuant ZIC-pHILIC (150 x 2.1mm,5um)	Waters Acquity BEH Phenyl (150 x 2.1mm,1.7um)
MS Type	ESI	ESI
MS instrument type	Orbitrap	Orbitrap
MS instrument name	Thermo Q Exactive HF hybrid Orbitrap	Thermo Q Exactive HF hybrid Orbitrap
Ion Mode	UNSPECIFIED	UNSPECIFIED
Units

Chromatography:

Chromatography ID:	CH001524
Instrument Name:	Thermo Dionex Ultimate 3000 RS
Column Name:	SeQuant ZIC-pHILIC (150 x 2.1mm,5um)
Chromatography Type:	HILIC

Chromatography ID:	CH001525
Instrument Name:	Thermo Dionex Ultimate 3000 RS
Column Name:	Waters Acquity BEH Phenyl (150 x 2.1mm,1.7um)
Chromatography Type:	Reversed phase

MS:

MS ID:	MS001938
Analysis ID:	AN002087
Instrument Name:	Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Positive/Negative Polarity Switching
Ion Mode:	UNSPECIFIED

MS ID:	MS001939
Analysis ID:	AN002088
Instrument Name:	Thermo Q Exactive HF hybrid Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Positive/Negative Polarity Switching
Ion Mode:	UNSPECIFIED