Summary of Study ST001975

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	ST001975
Study Title	Anti-oxidative metabolism measurement in mammalian cells and tissues by quantitative LC/MS method (II)
Study Summary	Anti-oxidation metabolism measurement in mouse CSF by quantitative LC/MS method to establish MTX effects on mouse metabolism in mouse controls 0-48hrs in CSF (repeat of 20200124 ChP-MTX-Anti-oxidative-study-test)
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 Available	Yes
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:	SU002055
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
SA185294	pooled sample QCa	-
SA185295	pooled sample QCb	-
SA185296	pooled sample QC1c	-
SA185297	pooled sample QC 1/10 dilution b	-
SA185298	pooled sample QC1a	-
SA185299	pooled sample QC 1/3 dilution a	-
SA185300	pooled sample QC1b	-
SA185301	pooled sample QCd	-
SA185302	pooled sample QCc	-
SA185303	pooled sample QC 1/10 dilution 1a	-
SA185304	pooled sample QC 1/3 dilution	-
SA185305	pooled sample QCe	-
SA185306	pooled sample QC 1/10 dilution a	-
SA185307	blank start	-
SA185308	pooled sample QC 1/10 dilution 1b	-
SA185309	blank end	-
SA185310	blank mid	-
SA185311	mouse HC-R1	female
SA185312	mouse HC-R3	female
SA185313	mouse HC-L2	female
SA185314	mouse HC-L1	female
SA185315	mouse HC-R2	female
SA185316	mouse CSF 19	female 24h
SA185317	mouse CSF 22	female 24h
SA185318	mouse CSF 20	female 24h
SA185319	mouse CSF 21	female 24h
SA185320	mouse CSF 13	female 4h
SA185321	mouse CSF 12	female 4h
SA185322	mouse CSF 14	female 4h
SA185323	mouse CSF 11	female 4h
SA185324	mouse CSF 5	female ctl
SA185325	mouse CSF 4	female ctl
SA185326	mouse CSF 6	female ctl
SA185327	mouse CSF 15	male 24h
SA185328	mouse CSF 18	male 24h
SA185329	mouse CSF 17	male 24h
SA185330	mouse CSF 16	male 24h
SA185331	mouse CSF 25	male 48h
SA185332	mouse CSF 26	male 48h
SA185333	mouse CSF 23	male 48h
SA185334	mouse CSF 24	male 48h
SA185335	mouse CSF 7	male 4h
SA185336	mouse CSF 8	male 4h
SA185337	mouse CSF 10	male 4h
SA185338	mouse CSF 9	male 4h
SA185339	mouse CSF 2	male ctl
SA185340	mouse CSF 1	male ctl
SA185341	mouse CSF 3	male ctl

Showing results 1 to 48 of 48

Showing results 1 to 48 of 48

Collection:

Collection ID:	CO002048
Collection Summary:	All animal care and experimental procedures were approved by the Institutional Animal Care and Use Committees of Boston Children’s Hospital. Mouse strain used was C57BL/6. 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. Hippocampus samples were collected and flash frozen.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:	TR002067
Treatment Summary:	Control CSF + hippocampus samples + buffers from Study Design

Sample Preparation:

Sampleprep ID:	SP002061
Sampleprep Summary:	CSF was collected by puncture of the cisterna magna with a glass capillary [39] and flash-frozen for further analysis. Per condition, 3 µl of precleared CSF were extracted by brief sonication in 200 µl of the indicated extraction buffers. After centrifugation for 10 min 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 to LC-MS micro vials. A small amount of each sample was pooled and serially diluted 3- and 10-fold to be used as quality controls throughout the run of each batch. For hippocampus tissues – chunks were crushed using a hand-held homogenizer (VWR 47747-370) with several pulses while keeping the samples on ice. 300 µl of prechilled extraction buffer was used per 2 mg of tissue. 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:

SP002061

Sampleprep Summary:

CSF was collected by puncture of the cisterna magna with a glass capillary [39] and flash-frozen for further analysis. Per condition, 3 µl of precleared CSF were extracted by brief sonication in 200 µl of the indicated extraction buffers. After centrifugation for 10 min 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 to LC-MS micro vials. A small amount of each sample was pooled and serially diluted 3- and 10-fold to be used as quality controls throughout the run of each batch. For hippocampus tissues – chunks were crushed using a hand-held homogenizer (VWR 47747-370) with several pulses while keeping the samples on ice. 300 µl of prechilled extraction buffer was used per 2 mg of tissue. 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	AN003223
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:	CH002377
Instrument Name:	Thermo Q Exactive Orbitrap
Column Name:	EMD Millipore ZIC-HILIC (100 x 2.1mm,3.5um)
Chromatography Type:	HILIC

MS:

MS ID:	MS002997
Analysis ID:	AN003223
Instrument Name:	Thermo Exactive Plus Orbitrap
Instrument Type:	Orbitrap
MS Type:	ESI
MS Comments:	Xcalibur + Tracefinder
Ion Mode:	UNSPECIFIED