Summary of Study ST001976

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 PR001255. The data can be accessed directly via it's Project DOI: 10.21228/M8GT41 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	ST001976
Study Title	Anti-oxidation metabolism measurement in mammalian cells and tissues by quantitative LC/MS method (III)
Study Summary	Anti-oxidative metabolism measurement in mouse CSF by quantitative LC/MS method of mouse CSF at 24H of MTX treatment, for either control GFP or SOD3-overexpressing ChP mice.
Institute	Boston Children's Hospital, Harvard Medical School
Last Name	Petrova
First Name	Boryana
Address	300 Longwood Ave
Email	boryana.petrova@childrens.harvard.edu
Phone	6173557433
Submit Date	2021-09-14
Raw Data File Type(s)	raw(Thermo)
Analysis Type Detail	LC-MS
Release Date	2022-08-29
Release Version	1

Select appropriate tab below to view additional metadata details:

Project:

Project ID:	PR001255
Project DOI:	doi: 10.21228/M8GT41
Project Title:	Gene-therapy enhances CSF’s anti-oxidative activity to mitigate chemotherapy side effects
Project Type:	Anti-oxidative Metabolism Measurement in CSF of MTX-treated mice under gene therapy by quantitative LC/MS method
Project Summary:	In order to test the protective activity of SOD3 levels on the redox state of the CSF of MTX-treated mice, targeted metabolomics on CSF was employed at different timepoints after treatment, on mice of both sexes, at various levels of impacted hSOD3 expression.
Institute:	Boston Childrens Hospital
Department:	Pathology
Laboratory:	Naama Kanarek
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:	SU002056
Subject Type:	Mammal
Subject Species:	Mus musculus
Taxonomy ID:	10090
Gender:	Male and female

Factors:

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

mb_sample_id	local_sample_id	Factor
SA185342	pooled sample QCa	-
SA185343	pooled sample QC 1/10 dilution b	-
SA185344	pooled sample QC 1/10 dilution a	-
SA185345	pooled sample QCb	-
SA185346	pooled sample QCc	-
SA185347	pooled sample QC 1/3 dilution	-
SA185348	pooled sample QCf	-
SA185349	pooled sample QCd	-
SA185350	blank end	-
SA185351	pooled sample QCg	-
SA185352	blank start 3	-
SA185353	blank start 2	-
SA185354	blank start 1	-
SA185355	blank start 4	-
SA185356	mouse CSF 1	24h GFP 1
SA185357	mouse CSF 2	24h GFP 2
SA185358	mouse CSF 3	24h GFP 3
SA185359	mouse CSF 4	24h GFP 4
SA185360	mouse CSF 5	24h GFP 5
SA185361	mouse CSF 6	24h GFP 6
SA185362	mouse CSF 7	24h GFP 7
SA185363	mouse CSF 8	24h MTX 1
SA185364	mouse CSF 9	24h MTX 2
SA185365	mouse CSF 10	24h MTX 3
SA185366	mouse CSF 11	24h MTX 4
SA185367	mouse CSF 12	24h MTX 5
SA185368	mouse CSF 13	24h MTX 6
SA185369	mouse CSF 14	24h MTX 7
SA185377	mouse CSF 15	24h SOD3-1
SA185378	mouse CSF 16	24h SOD3-2
SA185379	mouse CSF 17	24h SOD3-3
SA185380	mouse CSF 18	24h SOD3-4
SA185381	mouse CSF 19	24h SOD3-5
SA185370	mouse CSF 20	24h SOD3+MTX-1
SA185371	mouse CSF 21	24h SOD3+MTX-2
SA185372	mouse CSF 22	24h SOD3+MTX-3
SA185373	mouse CSF 23	24h SOD3+MTX-4
SA185374	mouse CSF 24	24h SOD3+MTX-5
SA185375	mouse CSF 25	24h SOD3+MTX-6
SA185376	mouse CSF 26	24h SOD3+MTX-7

Showing results 1 to 40 of 40

Showing results 1 to 40 of 40

Collection:

Collection ID:	CO002049
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). CD-1 mice (timed pregnant and 6-8 weeks) and Sprague Dawley rats (6-8 weeks) were purchased (Charles River Laboratories). Male and female animals were used equally for all studies. All animals 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 10,000g for 10 min. at 4 °C to remove any tissue debris (38). CSF samples were flash frozen and stored at −80°C until use.
Sample Type:	CSF

Treatment:

Treatment ID:	TR002068
Treatment Summary:	Control AAV-GFP, MTX, AAV-SOD3 + buffers from Study Design. The pAAV-IRES-hrGFP vector was purchased from Agilent Technologies (240075) and pcDNA3.1-myc HisA (-)/ human SOD3 (myc-his tag at C-terminal) (36). Virus production and purification were performed by the Viral Core at BCH according to standard procedures (37). CD-1 mice (6-8 weeks) or Sprague Dawley rats (8 weeks) were given a single intravenous injection of 75 mg/kg MTX (Fisher Scientific, AAJ66364MD) dissolved in 0.9 % NaCl (Mountainside Medical, 0409-4888-10). MTX was freshly prepared for each experiment. MTX in mouse serum or CSF was assessed by MTX ELISA according to the manufacturer's instructions (Enzo Life Sciences, 142-0001).

Treatment ID:

TR002068

Treatment Summary:

Control AAV-GFP, MTX, AAV-SOD3 + buffers from Study Design. The pAAV-IRES-hrGFP vector was purchased from Agilent Technologies (240075) and pcDNA3.1-myc HisA (-)/ human SOD3 (myc-his tag at C-terminal) (36). Virus production and purification were performed by the Viral Core at BCH according to standard procedures (37). CD-1 mice (6-8 weeks) or Sprague Dawley rats (8 weeks) were given a single intravenous injection of 75 mg/kg MTX (Fisher Scientific, AAJ66364MD) dissolved in 0.9 % NaCl (Mountainside Medical, 0409-4888-10). MTX was freshly prepared for each experiment. MTX in mouse serum or CSF was assessed by MTX ELISA according to the manufacturer's instructions (Enzo Life Sciences, 142-0001).

Sample Preparation:

Sampleprep ID:	SP002062
Sampleprep Summary:	CSF was collected from the cisterna magna and centrifuged at 10,000g for 10 min. at 4 °C to remove any tissue debris (38). CSF samples were flash frozen and stored at −80°C until use. For characterization by mass spectrometry, CSF was acquired 4h, 24h, and 48h following a single 75 mg/kg MTX injection from 6-8 mice and flash frozen for further analysis. Per condition, 3 μl of CSF were extracted by brief sonication in 240 μl 100% methanol, supplemented with isotopically labeled internal standards (17 amino acids and reduced glutathione, Cambridge Isotope Laboratories, MSK-A2-1.2 and CNLM-6245-10) and 60 μl 20 mM Ellman’s reagent in water (Sigma-Aldrich, D8130). After centrifugation for 10min. at maximum speed on a benchtop centrifuge (Eppendorf) the cleared supernatant was dried using a nitrogen dryer and reconstituted in 30 µl water by brief sonication. Extracted metabolites were spun again and cleared supernatant was transferred in LC-MS microvials. 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). Normalization for biological material amounts was based on the total integrated peak area values of high-confidence metabolites within an experimental batch after normalizing to the averaged factor from all mean-centered chromatographic peak areas of isotopically labeled amino acids internal standards (Cambridge Isotope Laboratories). The data were Log transformed and Pareto scaled for MetaboAnalyst-based statistical or pathway analysis (41). We profiled 200 metabolites, 85 of which were detected in CSF and passed our quality control protocol.

Sampleprep ID:

SP002062

Sampleprep Summary:

CSF was collected from the cisterna magna and centrifuged at 10,000g for 10 min. at 4 °C to remove any tissue debris (38). CSF samples were flash frozen and stored at −80°C until use. For characterization by mass spectrometry, CSF was acquired 4h, 24h, and 48h following a single 75 mg/kg MTX injection from 6-8 mice and flash frozen for further analysis. Per condition, 3 μl of CSF were extracted by brief sonication in 240 μl 100% methanol, supplemented with isotopically labeled internal standards (17 amino acids and reduced glutathione, Cambridge Isotope Laboratories, MSK-A2-1.2 and CNLM-6245-10) and 60 μl 20 mM Ellman’s reagent in water (Sigma-Aldrich, D8130). After centrifugation for 10min. at maximum speed on a benchtop centrifuge (Eppendorf) the cleared supernatant was dried using a nitrogen dryer and reconstituted in 30 µl water by brief sonication. Extracted metabolites were spun again and cleared supernatant was transferred in LC-MS microvials. 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). Normalization for biological material amounts was based on the total integrated peak area values of high-confidence metabolites within an experimental batch after normalizing to the averaged factor from all mean-centered chromatographic peak areas of isotopically labeled amino acids internal standards (Cambridge Isotope Laboratories). The data were Log transformed and Pareto scaled for MetaboAnalyst-based statistical or pathway analysis (41). We profiled 200 metabolites, 85 of which were detected in CSF and passed our quality control protocol.

Combined analysis:

Analysis ID	AN003224
Analysis type	MS
Chromatography type	HILIC
Chromatography system	Thermo Q Exactive Orbitrap
Column	EMD Millipore ZIC-HILIC (100 x 2.1mm,3.5um)
MS Type	ESI
MS instrument type	Orbitrap
MS instrument name	Thermo Exactive Plus Orbitrap
Ion Mode	UNSPECIFIED
Units	ppm

Chromatography:

Chromatography ID:	CH002378
Instrument Name:	Thermo Q Exactive Orbitrap
Column Name:	EMD Millipore ZIC-HILIC (100 x 2.1mm,3.5um)
Chromatography Type:	HILIC

MS:

MS ID:	MS002998
Analysis ID:	AN003224
Instrument Name:	Thermo Exactive Plus Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Xcalibur + Tracefinder
Ion Mode:	UNSPECIFIED