Summary of Study ST001761

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 PR001127. The data can be accessed directly via it's Project DOI: 10.21228/M81D6M This work is supported by NIH grant, U2C- DK119886.

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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.

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Study IDST001761
Study TitleApplication of the redox metabolite detection method for mouse biofluids
Study SummaryThis study was aimed at optimizing redox metabolites detection from mammalian biofluids. Three different extraction conditions were compared, including derivatization of glutathione. This study was with mouse cerebrospinal fluid.
Institute
Boston Children's Hospital, Harvard Medical School
DepartmentPathology
LaboratoryNaama Kanarek
Last NamePetrova
First NameBoryana
Address300 Longwood Av, Boston, MA, 2115, USA
Emailboryana.petrova@childrens.harvard.edu
Phone6173557433
Submit Date2021-04-21
Raw Data AvailableYes
Raw Data File Type(s)raw(Thermo)
Analysis Type DetailLC-MS
Release Date2021-05-17
Release Version1
Boryana Petrova Boryana Petrova
https://dx.doi.org/10.21228/M81D6M
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Project:

Project ID:PR001127
Project DOI:doi: 10.21228/M81D6M
Project Title:Redox metabolism measurement in mammalian cells and tissues by quantitative LC/MS method (part I)
Project Summary:This study aimed to optimize the detection of several key redox-reactive metabolites from mammalian cells and tissues. We explored three different chromatographic methods and optimized sample preparation, extraction buffer and conditions as well as mass spectrometry detection parameters. The established method was tested and validated using biologically relevant ROS-inducing conditions. This study can be a valuable resource for the metabolomics community.
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:SU001838
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 EXTRACTION BUFFER
SA164189pooled sample QC 1/3 dilution-
SA164190pooled sample QC-
SA164191pooled sample QC d-
SA164192pooled sample QC 1/10 dilution a-
SA164193blank-
SA164194blank1-
SA164195pooled sample QC b-
SA164196pooled sample QC c-
SA164197pooled sample QC 1/10 dilution-
SA164198mouse_CSF_buffer B_6buffer B
SA164199mouse_CSF_buffer B_1buffer B
SA164200mouse_CSF_buffer B_2buffer B
SA164201mouse_CSF_buffer B_3buffer B
SA164202mouse_CSF_buffer B_4buffer B
SA164203mouse_CSF_buffer B_5buffer B
SA164204mouse_CSF_buffer C_6buffer C
SA164205mouse_CSF_buffer C_4buffer C
SA164206mouse_CSF_buffer C_3buffer C
SA164207mouse_CSF_buffer C_2buffer C
SA164208mouse_CSF_buffer C_1buffer C
SA164209mouse_CSF_buffer C_5buffer C
SA164210mouse_CSF_buffer C_Ell_1buffer C_Ellmans
SA164211mouse_CSF_buffer C_Ell_5buffer C_Ellmans
SA164212mouse_CSF_buffer C_Ell_6buffer C_Ellmans
SA164213mouse_CSF_buffer C_Ell_3buffer C_Ellmans
SA164214mouse_CSF_buffer C_Ell_2buffer C_Ellmans
SA164215mouse_CSF_buffer C_Ell_4buffer C_Ellmans
Showing results 1 to 27 of 27

Collection:

Collection ID:CO001831
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. Pure CSF samples were collected from the cisterna magna [39]. Blood samples were collected from the retromandibular vein. The 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. K562 cells used in this manuscript were authenticated by short tandem repeat analysis and tested negative for mycoplasma. Cells were cultured in RPMI (Genesee Scientific) up to a density of 2 Million cells per ml. For redox chemical treatment experiments, cells were seeded at 1 Million cells per ml cell density in 6-well plates and drugs were added for 4h at the following concentrations: methotrexate: 5 µM; oligomycin: 80 µg/ml; H2O2: 1 mM; diamide: 0.5 mM; DMSO, which served as control: 0.6 µl per 1 mL of cell culture media (equivalent to volume used for oligomycin).
Sample Type:CSF

Treatment:

Treatment ID:TR001851
Treatment Summary:Tissue and cells were extracted using either: 1. buffer A 40:40:20 of acetonitrile:methanol:water, supplemented with 0.1M formic acid and isotopically-labeled internal standards (17 amino acids and reduced glutathione, Cambridge Isotope Laboratories, MSK-A2-1.2 and CNLM-6245-10). 2. Extraction buffer “B”: 80% LC/MS-grade methanol, 20% 25 mM Ammonium Acetate and 2.5 mM Na-Ascorbate prepared in LC/MS water and supplemented with isotopically labeled internal standards (17 amino acids and isotopically labelled reduced glutathione, Cambridge Isotope Laboratories, MSK-A2-1.2 and CNLM-6245-10). or 3. Extraction buffer “C” and “C + Ellman’s”: Solution 1: 100% LC-MS Methanol Solution 2: 25mM Ammonium Acetate and 2.5mM Na-Ascorbate in LC-MS water supplemented with isotopically labelled reduced glutathione and isotopically labeled internal standards (17 amino acids and reduced glutathione, Cambridge Isotope Laboratories, MSK-A2-1.2 and CNLM-6245-10). Ellman’s reagent (5,5′-Dithiobis(2-nitrobenzoic acid),D8130, Sigma Aldrich): 20 mM in “Solution 2”. Final composition is 4:1 solution 1:solution 2.

Sample Preparation:

Sampleprep ID:SP001844
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. Extraction buffer “A”: 40:40:20 of acetonitrile:methanol:water, supplemented with 0.1M formic acid and isotopically-labeled internal standards (17 amino acids and reduced glutathione, Cambridge Isotope Laboratories, MSK-A2-1.2 and CNLM-6245-10). Extraction buffer “B”: 80% LC-MS-grade methanol, 20% 25 mM Ammonium Acetate and 2.5 mM Na-Ascorbate prepared in LC-MS water and supplemented with isotopically labeled internal standards (17 amino acids and isotopically labelled reduced glutathione, Cambridge Isotope Laboratories, MSK-A2-1.2 and CNLM-6245-10). Extraction buffer “C” and “C + Ellman’s”: Solution 1: 100% LC-MS Methanol Solution 2: 25mM Ammonium Acetate and 2.5mM Na-Ascorbate in LC-MS water supplemented with isotopically labelled reduced glutathione and isotopically labeled internal standards (17 amino acids and reduced glutathione, Cambridge Isotope Laboratories, MSK-A2-1.2 and CNLM-6245-10). Ellman’s reagent (5,5′-Dithiobis(2-nitrobenzoic acid),D8130, Sigma Aldrich): 20 mM in “Solution 2”. Final composition is 80% solution 1 and 20% solution 2.

Combined analysis:

Analysis ID AN002868
Analysis type MS
Chromatography type HILIC
Chromatography system Thermo Vanquish
Column EMD Millipore ZIC HILIC (150 x 2.1mm,5um)
MS Type ESI
MS instrument type Orbitrap
MS instrument name Thermo Q Exactive Orbitrap
Ion Mode UNSPECIFIED
Units ppm

Chromatography:

Chromatography ID:CH002123
Chromatography Summary:ZIC-pHILIC chromatography: One ml of reconstituted sample was injected into a ZIC-pHILIC 150 × 2.1 mm (5 µm particle size) column (EMD Millipore) operated on a Vanquish™ Flex UHPLC Systems (Thermo Fisher Scientific, San Jose, CA). 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.
Instrument Name:Thermo Vanquish
Column Name:EMD Millipore ZIC HILIC (150 x 2.1mm,5um)
Column Temperature:25
Flow Gradient:linear gradient from 20% to 80% B; 20-20.5 min: from 80% to 20% B; 20.5-28 min: hold at 20% B.
Solvent A:100% acetonitrile
Solvent B:100% water; 20 mM ammonium carbonate; 0.1% ammonium hydroxide
Chromatography Type:HILIC

MS:

MS ID:MS002661
Analysis ID:AN002868
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:HESI
Ion Mode:UNSPECIFIED
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