Metabolomics Workbench : NIH Data Repository

MB Sample ID: SA219096

Local Sample ID:	11
Subject ID:	SU002372
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090

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Subject:

Subject ID:	SU002372
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090

Factors:

Local Sample ID	MB Sample ID	Factor Level ID	Level Value	Factor Name
11	SA219096	FL026530	CSF3 saline	Factor

Collection:

Collection ID:	CO002365
Collection Summary:	All animal studies were performed under the protocol approved by the Institutional Animal Care and Use Committee (IACUC) of Boston Children’s Hospital (BCH). All mice were housed in a 12h light-dark cycle with ad libitum access to food and water. CSF was collected from the cisterna magna and centrifuged at 5,000g for 10 min. at 4 °C to remove any tissue debris. CSF samples were kept on ice and analyzed the same day as collection. Blood samples were coagulated and centrifuged. Liver and kidney were collected and flash frozen. Tissue chunks were cut on a glass plate while kept chilled on top of dry ice.
Collection Protocol Comments:	Liver, Serum included
Sample Type:	CSF

Treatment:

Treatment ID:	TR002384
Treatment Summary:	For polyI:C experiments: Pregnant female mice were injected intraperitoneally (route of administration) with a single dose of 20 mg/kg polyI:C (sigma aldrich) and controls will receive an injection of 0.9% saline embryonic day 12.5. Samples were collected either 3hrs or 48hrs post injection.

Sample Preparation:

Sampleprep ID:	SP002378
Sampleprep Summary:	Per condition, 5uL of maternal serum, CSF, were extracted by brief sonication in 200 μl 80% methanol, supplemented with isotopically labeled internal standards (17 amino acids and reduced glutathione, Cambridge Isotope Laboratories, MSK-A2-1.2 and CNLM-6245-10). 10uL Embryonic CSF (eCSF) was extracted per condition using the previously described buffer quantity, and the flash frozen liver chunks were extracted in 300uL of the previously described buffer. After centrifugation for 10min. at maximum speed on a benchtop centrifuge (Eppendorf) the cleared supernatant from CSF and eCSF was dried using a nitrogen dryer. Samples were reconstituted in LCMS water by brief sonication. Extracted metabolites were spun again and cleared supernatant was transferred in LC-MS microvials. Serum and liver chunks followed the same protocol, except that an additional 20-minute centrifugation was administered, and homogenization for 5 minutes was administered to liver chunks. Final LCMS water reconstitution values for tissue type were 50uL (maternal serum), 15uL (maternal CSF and eCSF), and 100 uL (liver chunks). A small amount of each sample was pooled and serially diluted 3 and 10-fold to be used as quality controls throughout the batch run. Two microliters (equivalent to 0.2 ul of CSF) of reconstituted sample were injected into a ZIC-pHILIC 150 × 2.1 mm (5 µm particle size) column (EMD Millipore) operated on a Dionex UltiMate 3000 UPLC system (Thermo Fisher Scientific). Chromatographic separation was achieved using the following conditions: buffer A was acetonitrile; buffer B was 20 mM ammonium carbonate, 0.1% ammonium hydroxide. Gradient conditions were: linear gradient from 20% to 80% B; 20–20.5 min: from 80% to 20% B; 20.5–28 min: hold at 20% B. The column oven and autosampler tray were held at 25 °C and 4 °C, respectively. The MS data acquisition was on a QExactive benchtop orbitrap mass spectrometer equipped with an Ion Max source and a HESI II probe and was performed in a range of m/z= 70–1000, with the resolution set at 70,000, the AGC target at 1x106, and the maximum injection time (Max IT) at 20 msec. For tSIM scans, the resolution was set at 70,000, the AGC target was 1x105, and the max IT was 100 msec. Relative quantitation of polar metabolites was performed with TraceFinder 4.1 (Thermo Fisher Scientific) using a 5 ppm mass tolerance and referencing an in-house library of chemical standards. Pooled samples and fractional dilutions were prepared as quality controls and only those metabolites were taken for further analysis, for which the correlation between the dilution factor and the peak area was >0.95 (high confidence metabolites).

Sampleprep ID:

SP002378

Sampleprep Summary:

Per condition, 5uL of maternal serum, CSF, were extracted by brief sonication in 200 μl 80% methanol, supplemented with isotopically labeled internal standards (17 amino acids and reduced glutathione, Cambridge Isotope Laboratories, MSK-A2-1.2 and CNLM-6245-10). 10uL Embryonic CSF (eCSF) was extracted per condition using the previously described buffer quantity, and the flash frozen liver chunks were extracted in 300uL of the previously described buffer. After centrifugation for 10min. at maximum speed on a benchtop centrifuge (Eppendorf) the cleared supernatant from CSF and eCSF was dried using a nitrogen dryer. Samples were reconstituted in LCMS water by brief sonication. Extracted metabolites were spun again and cleared supernatant was transferred in LC-MS microvials. Serum and liver chunks followed the same protocol, except that an additional 20-minute centrifugation was administered, and homogenization for 5 minutes was administered to liver chunks. Final LCMS water reconstitution values for tissue type were 50uL (maternal serum), 15uL (maternal CSF and eCSF), and 100 uL (liver chunks). A small amount of each sample was pooled and serially diluted 3 and 10-fold to be used as quality controls throughout the batch run. Two microliters (equivalent to 0.2 ul of CSF) of reconstituted sample were injected into a ZIC-pHILIC 150 × 2.1 mm (5 µm particle size) column (EMD Millipore) operated on a Dionex UltiMate 3000 UPLC system (Thermo Fisher Scientific). Chromatographic separation was achieved using the following conditions: buffer A was acetonitrile; buffer B was 20 mM ammonium carbonate, 0.1% ammonium hydroxide. Gradient conditions were: linear gradient from 20% to 80% B; 20–20.5 min: from 80% to 20% B; 20.5–28 min: hold at 20% B. The column oven and autosampler tray were held at 25 °C and 4 °C, respectively. The MS data acquisition was on a QExactive benchtop orbitrap mass spectrometer equipped with an Ion Max source and a HESI II probe and was performed in a range of m/z= 70–1000, with the resolution set at 70,000, the AGC target at 1x106, and the maximum injection time (Max IT) at 20 msec. For tSIM scans, the resolution was set at 70,000, the AGC target was 1x105, and the max IT was 100 msec. Relative quantitation of polar metabolites was performed with TraceFinder 4.1 (Thermo Fisher Scientific) using a 5 ppm mass tolerance and referencing an in-house library of chemical standards. Pooled samples and fractional dilutions were prepared as quality controls and only those metabolites were taken for further analysis, for which the correlation between the dilution factor and the peak area was >0.95 (high confidence metabolites).

Combined analysis:

Analysis ID	AN003738
Analysis type	MS
Chromatography type	HILIC
Chromatography system	Thermo Vanquish
Column	SeQuant ZIC-pHILIC (150 x 2.1mm,5um)
MS Type	ESI
MS instrument type	Orbitrap
MS instrument name	Thermo Q Exactive Plus Orbitrap
Ion Mode	UNSPECIFIED
Units	ppm

Chromatography:

Chromatography ID:	CH002769
Chromatography Summary:	ZIC-pHILIC
Instrument Name:	Thermo Vanquish
Column Name:	SeQuant ZIC-pHILIC (150 x 2.1mm,5um)
Chromatography Type:	HILIC

MS:

MS ID:	MS003486
Analysis ID:	AN003738
Instrument Name:	Thermo Q Exactive Plus Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	HESI
Ion Mode:	UNSPECIFIED