Summary of Study ST003085

This data is available at the NIH Common Fund's National Metabolomics Data Repository (NMDR) website, the Metabolomics Workbench, https://www.metabolomicsworkbench.org, where it has been assigned Project ID PR001918. The data can be accessed directly via it's Project DOI: 10.21228/M8ST6K This work is supported by NIH grant, U2C- DK119886.

See: https://www.metabolomicsworkbench.org/about/howtocite.php

This study contains a large results data set and is not available in the mwTab file. It is only available for download via FTP as data file(s) here.

Study ID	ST003085
Study Title	Metabolome changes in embryonic CSF (Part 7)
Study Summary	Untargeted MS analysis of embryonic and maternal CSF. Metabolome changes in embryonic and maternal CSF and serum at E14.5, Saline VS PolyI:C injected into mother, collection at 48 hours.
Institute	Boston Children's Hospital, Harvard Medical School
Laboratory	Kanarek Lab
Last Name	Petrova
First Name	Boryana
Address	Enders 1116.2 300 Longwood Ave
Email	Boryana.Petrova@childrens.harvard.edu
Phone	6179197352
Submit Date	2024-01-16
Raw Data Available	Yes
Raw Data File Type(s)	raw(Thermo)
Analysis Type Detail	LC-MS
Release Date	2024-07-29
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR001918
Project DOI:	doi: 10.21228/M8ST6K
Project Title:	Metabolomics of Mouse Embryonic CSF Following Maternal Immune Activation
Project Summary:	The embryonic cerebrospinal fluid (eCSF) is critical for the developing central nervous system (CNS), from neurogenesis to lifelong cognitive functions. Changes in eCSF composition due to inflammation can impact brain function. We recently identified an abnormal cytokine signature in eCSF following maternal immune activation (MIA), a mouse model of autism spectrum disorder (ASD). We hypothesized that MIA leads to other alterations in eCSF composition and employed untargeted metabolomics to profile changes in the eCSF metabolome in mice after inducing MIA with polyI:C. We report these data here as a resource, including a comprehensive MS1 and MS2 reference dataset, and present additional datasets comparing two mouse strains (CD-1 and C57Bl/6) and two developmental time points (E12.5 and E14.5). Targeted metabolomics further validated changes in eCSF upon MIA. We show a significant elevation of glucocorticoids and kynurenine pathway related metabolites. Both pathways are relevant for suppressing inflammation or could be informative as disease biomarkers. Our resource should inform future mechanistic studies regarding the etiology of MIA neuropathology and roles and contributions of eCSF metabolites to brain development.
Institute:	Boston Childrens Hospital
Last Name:	Petrova
First Name:	Boryana
Address:	300 Longwood Av, Boston, MA, 2115, USA
Email:	boryana.petrova@childrens.harvard.edu
Phone:	6173557433

Subject:

Subject ID:	SU003200
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090

Factors:

Subject type: Mammal; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id	local_sample_id	Sample source	Developmental Timepoint	Maternal Treatment (injection)	Collection Timepoint
SA332891	8	embryonic CSF	E14.5	PolyIC	48 Hrs
SA332892	9	embryonic CSF	E14.5	PolyIC	48 Hrs
SA332893	11	embryonic CSF	E14.5	PolyIC	48 Hrs
SA332894	10	embryonic CSF	E14.5	PolyIC	48 Hrs
SA332895	7	embryonic CSF	E14.5	saline	48 Hrs
SA332896	6	embryonic CSF	E14.5	saline	48 Hrs
SA332897	5	embryonic CSF	E14.5	saline	48 Hrs
SA332898	4	embryonic CSF	E14.5	saline	48 Hrs
SA332899	2	mock	-	-	-
SA332900	3	mock	-	-	-
SA332901	1	mock	-	-	-

Showing results 1 to 11 of 11

Showing results 1 to 11 of 11

Collection:

Collection ID:	CO003193
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, eCSF, was collected from the cisterna magna of the pup embryos and mother, 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.
Sample Type:	Cerebrospinal fluid

Treatment:

Treatment ID:	TR003209
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 12.5-14.5. Samples were collected either 3hrs or 48hrs post injection.

Sample Preparation:

Sampleprep ID:	SP003206
Sampleprep Summary:	Per condition, 5uL of maternal serum, CSF, were extracted by brief sonication in 200/100μl (serum/csf) 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 followed the same protocol, except that an additional 20-minute centrifugation was administered, and homogenization for 5 minutes. Final LCMS water reconstitution values for tissue type were 50uL (maternal and embryonic serum), 15uL (maternal CSF and eCSF),. 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:

SP003206

Sampleprep Summary:

Per condition, 5uL of maternal serum, CSF, were extracted by brief sonication in 200/100μl (serum/csf) 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 followed the same protocol, except that an additional 20-minute centrifugation was administered, and homogenization for 5 minutes. Final LCMS water reconstitution values for tissue type were 50uL (maternal and embryonic serum), 15uL (maternal CSF and eCSF),. 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	AN005044	AN005045
Analysis type	MS	MS
Chromatography type	HILIC	HILIC
Chromatography system	Thermo Vanquish	Thermo Vanquish
Column	SeQuant ZIC- pHILIC (150 x 2.1mm, 5um)	SeQuant ZIC- pHILIC (150 x 2.1mm, 5um)
MS Type	ESI	ESI
MS instrument type	Orbitrap	Orbitrap
MS instrument name	Thermo Q Exactive Plus Orbitrap	Thermo Q Exactive Plus Orbitrap
Ion Mode	POSITIVE	NEGATIVE
Units	a.u.	a.u.

Chromatography:

Chromatography ID:	CH003813
Chromatography Summary:	ZIC-pHILIC
Instrument Name:	Thermo Vanquish
Column Name:	SeQuant ZIC- pHILIC (150 x 2.1mm, 5um)
Column Temperature:	25
Flow Gradient:	0-20 min: linear gradient from 20% to 80% B; 20–20.5 min: from 80% to 20% B; 20.5–28 min: hold at 20% B.
Flow Rate:	0.150mL/minute
Solvent A:	100% acetonitrile
Solvent B:	100% water; 20mM ammonium carbonate, 0.1% ammonium hydroxide
Chromatography Type:	HILIC

MS:

MS ID:	MS004782
Analysis ID:	AN005044
Instrument Name:	Thermo Q Exactive Plus Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	HESI, See uploaded BCH_Kanarek_Lab_protocol.pdf for metabolite metadata "Tags" key and explanation
Ion Mode:	POSITIVE

MS ID:	MS004783
Analysis ID:	AN005045
Instrument Name:	Thermo Q Exactive Plus Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	HESI, See uploaded BCH_Kanarek_Lab_protocol.pdf for metabolite metadata "Tags" key and explanation
Ion Mode:	NEGATIVE