Summary of Study ST001979

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	ST001979
Study Title	Anti-oxidation metabolism measurement in mammalian cells and tissues by quantitative LC/MS method (VI)
Study Summary	Anti-oxidation metabolism measurement in mouse CSF by quantitative LC/MS method.
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	2021-09-15
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:	SU002059
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
SA185499	pooled sample QC 1f	-
SA185500	pooled sample QC 1g	-
SA185501	pooled sample QC 1h	-
SA185502	pooled sample QC 1e	-
SA185503	pooled sample QC 1d	-
SA185504	pooled sample QC 1b	-
SA185505	pooled sample QC 1c	-
SA185506	pooled sample QC 1/3 dilution	-
SA185507	pooled sample QC 1/10 dilution a-2x	-
SA185508	blank 3	-
SA185509	blank 4	-
SA185510	blank 2	-
SA185511	blank 1	-
SA185512	pooled sample QC 1/10 dilution a	-
SA185513	pooled sample QC 1/10 dilution b	-
SA185514	pooled sample QC 1a	-
SA185524	Mouse CSF Ctrl 49	CMV-GFP-1
SA185525	Mouse CSF Ctrl 50	CMV-GFP-2
SA185526	Mouse CSF Ctrl 51	CMV-GFP-3
SA185527	Mouse CSF Ctrl 52	CMV-GFP-4
SA185528	Mouse CSF Ctrl 53	CMV-GFP-5
SA185529	Mouse CSF Ctrl 54	CMV-GFP-6
SA185515	Mouse CSF Ctrl 55	CMV-GFP+MTX-1
SA185516	Mouse CSF Ctrl 56	CMV-GFP+MTX-2
SA185517	Mouse CSF Ctrl 57	CMV-GFP+MTX-3
SA185518	Mouse CSF Ctrl 58	CMV-GFP+MTX-4
SA185519	Mouse CSF Ctrl 59	CMV-GFP+MTX-5
SA185520	Mouse CSF Ctrl 60	CMV-GFP+MTX-6
SA185521	Mouse CSF Ctrl 61	CMV-GFP+MTX-7
SA185522	Mouse CSF Ctrl 62	CMV-GFP+MTX-8
SA185523	Mouse CSF Ctrl 63	CMV-GFP+MTX-9
SA185539	Mouse CSF Ctrl 1	PBS-1
SA185540	Mouse CSF Ctrl 2	PBS-2
SA185541	Mouse CSF Ctrl 3	PBS-3
SA185542	Mouse CSF Ctrl 4	PBS-4
SA185543	Mouse CSF Ctrl 5	PBS-5
SA185544	Mouse CSF Ctrl 6	PBS-6
SA185545	Mouse CSF Ctrl 7	PBS-7
SA185546	Mouse CSF Ctrl 8	PBS-8
SA185547	Mouse CSF Ctrl 9	PBS-9
SA185530	Mouse CSF Ctrl 10	PBS+MTX-1
SA185531	Mouse CSF Ctrl 11	PBS+MTX-2
SA185532	Mouse CSF Ctrl 12	PBS+MTX-3
SA185533	Mouse CSF Ctrl 13	PBS+MTX-4
SA185534	Mouse CSF Ctrl 14	PBS+MTX-5
SA185535	Mouse CSF Ctrl 15	PBS+MTX-6
SA185536	Mouse CSF Ctrl 16	PBS+MTX-7
SA185537	Mouse CSF Ctrl 17	PBS+MTX-8
SA185538	Mouse CSF Ctrl 18	PBS+MTX-9

Showing results 1 to 49 of 49

Showing results 1 to 49 of 49

Collection:

Collection ID:	CO002052
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:	TR002071
Treatment Summary:	Mice were treated with PBS, PBS+ MTX, Null GFP with and without MTX, CAG-GFP with and without MTX, and CMV-GFP with and without MTX. buffers from study design were used for all respective samples. 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:

TR002071

Treatment Summary:

Mice were treated with PBS, PBS+ MTX, Null GFP with and without MTX, CAG-GFP with and without MTX, and CMV-GFP with and without MTX. buffers from study design were used for all respective samples. 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:	SP002065
Sampleprep Summary:	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:

SP002065

Sampleprep Summary:

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	AN003228	AN003229
Analysis type	MS	MS
Chromatography type	HILIC	HILIC
Chromatography system	Thermo Q Exactive Orbitrap	Thermo Q Exactive Orbitrap
Column	EMD Millipore ZIC-HILIC (100 x 2.1mm,3.5um)	EMD Millipore ZIC-HILIC (100 x 2.1mm,3.5um)
MS Type	ESI	ESI
MS instrument type	Orbitrap	Orbitrap
MS instrument name	Thermo Exactive Plus Orbitrap	Thermo Exactive Plus Orbitrap
Ion Mode	UNSPECIFIED	UNSPECIFIED
Units	ppm	ppm

Chromatography:

Chromatography ID:	CH002381
Instrument Name:	Thermo Q Exactive Orbitrap
Column Name:	EMD Millipore ZIC-HILIC (100 x 2.1mm,3.5um)
Chromatography Type:	HILIC

MS:

MS ID:	MS003002
Analysis ID:	AN003228
Instrument Name:	Thermo Exactive Plus Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Xcalibur + Tracefinder
Ion Mode:	UNSPECIFIED

MS ID:	MS003003
Analysis ID:	AN003229
Instrument Name:	Thermo Exactive Plus Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Xcalibur + Tracefinder
Ion Mode:	UNSPECIFIED