Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA255648

Local Sample ID:	Toxoplasma infected-1
Subject ID:	SU002645
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090

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Subject:

Subject ID:	SU002645
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
Toxoplasma infected-1	SA255648	FL032733	Infected	Factor

Collection:

Collection ID:	CO002638
Collection Summary:	At 7 weeks post-infection, isoflurane-anesthetized mice were retro-orbitally bled and sera was flash frozen and sent to the National Institute of Health (NIH) West Coast Metabolomics Center (UC Davis) for untargeted mass spectrometry analysisusing the primary metabolism assay (ALEXCIS GCTOF-MS) or the complex lipids (CSH-QTOF MS) assay. Detected meatbolites were identified based on retention time and mass spectra from MassBank of North America, curated by the NIH West Coast Metabolomics Center , and reported as raw peak heights. The raw peak heights from each analytical platform were normalized to the average peak heights of the identified metabolites in uninfected group. The resulting data were analyzed for fold-change and multiple unpaired t-test and visualized using volcano plots to identify the differential expression of metabolites in response to T. gondii-induced cachexia
Sample Type:	Blood (serum)

Treatment:

Treatment ID:	TR002657
Treatment Summary:	To generate cysts for infection, 8-10 week female CBA/J mice were infected with 10 Me49 bradyzoite cysts by intraperitoneal injection. 4–8 weeks following infection, mice were euthanized by CO2 inhalation and cysts were harvest from brains homogenate passed through a 70 μm filter. Homegenate was washed 3 times in PBS, stained with dolichos biflorus agglutinin conjugated to FITC (Vector labs) at a 1:500 dilution. The number of cysts were determined by counting FITCpositive cysts at 20x magnification using an EVOS FL imaging system (Thermo Fisher). For experimental infections 10–14-week-old male C57BL/6 mice were infected with 10 Me49 bradyzoite cysts by intraperitoneal infection resuspended in 200 Μl PBS per mouse using a 5G 5/8” tuberculin syringe. Prior to infection, mice were cross-housed on dirty, wood chip bedding for two weeks to normalize commensal microbiota and limit the effect of eating corn husk bedding on dietary metabolites. At experimental endpoints, mice were fasted for 4 hours and isoflurane anaesthetized to isolate sera via retro-orbital bleed and/or euthanized by CO2 asphyxiation to harvest tissues for weighing and histological analysis.

Treatment ID:

TR002657

Treatment Summary:

To generate cysts for infection, 8-10 week female CBA/J mice were infected with 10 Me49 bradyzoite cysts by intraperitoneal injection. 4–8 weeks following infection, mice were euthanized by CO2 inhalation and cysts were harvest from brains homogenate passed through a 70 μm filter. Homegenate was washed 3 times in PBS, stained with dolichos biflorus agglutinin conjugated to FITC (Vector labs) at a 1:500 dilution. The number of cysts were determined by counting FITCpositive cysts at 20x magnification using an EVOS FL imaging system (Thermo Fisher). For experimental infections 10–14-week-old male C57BL/6 mice were infected with 10 Me49 bradyzoite cysts by intraperitoneal infection resuspended in 200 Μl PBS per mouse using a 5G 5/8” tuberculin syringe. Prior to infection, mice were cross-housed on dirty, wood chip bedding for two weeks to normalize commensal microbiota and limit the effect of eating corn husk bedding on dietary metabolites. At experimental endpoints, mice were fasted for 4 hours and isoflurane anaesthetized to isolate sera via retro-orbital bleed and/or euthanized by CO2 asphyxiation to harvest tissues for weighing and histological analysis.

Sample Preparation:

Sampleprep ID:	SP002651
Sampleprep Summary:	At 7 weeks post-infection, isoflurane-anesthetized mice were retro-orbitally bled and sera was flash frozen and sent to the National Institute of Health (NIH) West Coast Metabolomics Center (UC Davis) for untargeted mass spectrometry analysisusing the primary metabolism assay (ALEXCIS GCTOF-MS) or the complex lipids (CSH-QTOF MS) assay. Detected meatbolites were identified based on retention time and mass spectra from MassBank of North America, curated by the NIH West Coast Metabolomics Center , and reported as raw peak heights (Table S1 and S3). The raw peak heights from each analytical platform were normalized to the average peak heights of the identified metabolites in uninfected group. The resulting data were analyzed for fold-change and multiple unpaired t-test and visualized using volcano plots to identify the differential expression of metabolites in response to T. gondii-induced cachexia

Sampleprep ID:

SP002651

Sampleprep Summary:

At 7 weeks post-infection, isoflurane-anesthetized mice were retro-orbitally bled and sera was flash frozen and sent to the National Institute of Health (NIH) West Coast Metabolomics Center (UC Davis) for untargeted mass spectrometry analysisusing the primary metabolism assay (ALEXCIS GCTOF-MS) or the complex lipids (CSH-QTOF MS) assay. Detected meatbolites were identified based on retention time and mass spectra from MassBank of North America, curated by the NIH West Coast Metabolomics Center , and reported as raw peak heights (Table S1 and S3). The raw peak heights from each analytical platform were normalized to the average peak heights of the identified metabolites in uninfected group. The resulting data were analyzed for fold-change and multiple unpaired t-test and visualized using volcano plots to identify the differential expression of metabolites in response to T. gondii-induced cachexia

Combined analysis:

Analysis ID	AN004192
Analysis type	MS
Chromatography type	Reversed phase
Chromatography system	Agilent 1290 Infinity
Column	Waters ACQUITY UPLC CSH C18 (100 x 2.1mm,1.7um)
MS Type	ESI
MS instrument type	QTOF
MS instrument name	Agilent 6530 QTOF
Ion Mode	POSITIVE
Units	reads

Chromatography:

Chromatography ID:	CH003106
Instrument Name:	Agilent 1290 Infinity
Column Name:	Waters ACQUITY UPLC CSH C18 (100 x 2.1mm,1.7um)
Column Temperature:	65
Flow Gradient:	15%-99%
Flow Rate:	0.6 ml/min
Solvent A:	60% water/40% acetonitrile; 5 mM ammonium formate; 0.1% formic acid
Solvent B:	90% isopropanol/10% methanol; 5 mM ammonium formate; 0.1% formic acid
Chromatography Type:	Reversed phase

MS:

MS ID:	MS003939
Analysis ID:	AN004192
Instrument Name:	Agilent 6530 QTOF
Instrument Type:	QTOF
MS Type:	ESI
MS Comments:	Samples extracted using Matyash extraction procedure which includes MTBE, MeOH, and H2O. The organic (upper) phase was dried down and submitted for resuspension and injection onto the LC while the aqueous (bottom) phase was dried down and submitted to derivatization for GC. They are resuspended with 110 uL of a solution of 9:1 methanol: toluene and 50 ng/mL CUDA. This is then shaken for 20 seconds, sonicated for 5 minutes at room temperature, and then centrifuged for 2 minutes at 16100 rcf. The samples are then aliquoted into three parts. 33 uL are aliquoted into a vial with a 50 uL glass insert for positive and negative mode lipidomics. The last part is aliquoted into an eppendorf tube to be used as a pool. The samples are then loaded up on an Agilent 1290 Infinity LC stack. The positive and negative mode were run on an Agilent 6530 with a scan range of m/z 120-1200 Da with an acquisition speed of 2 spectra/s. Between 0.5 and 2 uL were injected onto a Waters ACQUITY UPLC CSH C18 1.7um 2.1x100mm Column with VanGuard PreColumn 2.1x5 mm . The gradient used is 0 min 15% (B), 0–2 min 30% (B), 2–2.5 min 48% (B), 2.5–11 min 82% (B), 11–11.5 min 99% (B), 11.5–12 min 99% (B), 12–12.1 min 15% (B), 12.1–15 min 15% (B) with a flow rate of 0.6 mL/min. The mass resolution for the Agilent 6530 is 10,000 for ESI (+).
Ion Mode:	POSITIVE