Summary of Study ST001768

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 IDST001768
Study TitleApplication of the redox metabolite detection method for mammalian tissues (part III)
Study SummaryThis study was aimed at optimizing redox metabolites detection from mammalian tissues. Three different chromatographic conditions were compared as well as three different extraction conditions. This study was run on ZIC-pHILIC chromatography.
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 Date2021-04-22
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:SU001845
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 Factor
SA164367pooled sample QCc-
SA164368blank1-
SA164369pooled sample QC 1/3 dilution-
SA164370pooled sample Qca-
SA164371pooled sample QC 1/10 dilution a-
SA164372blank2-
SA164373pooled sample QC 1/10 dilution b-
SA164374pooled sample QCb-
SA164375kidney3buffer C_Ell
SA164376liver chunk1buffer C_Ell
SA164377liver chunk2buffer C_Ell
SA164378liver chunk3buffer C_Ell
SA164379kidney1buffer C_Ell
SA164380kidney2buffer C_Ell
Showing results 1 to 14 of 14

Collection:

Collection ID:CO001838
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:Liver/Kidney

Treatment:

Treatment ID:TR001858
Treatment Summary:Buffer only

Sample Preparation:

Sampleprep ID:SP001851
Sampleprep Summary:Metabolites were quenched as quickly as possible while working with the samples at low temperatures. Cells were handled at 4ºC or on dry ice, extraction buffer was pre-chilled at -20ºC. Samples were analyzed by LC-MS on the same day of extraction (if impractical, best alternative is to store dried samples at -80ºC). Unless indicated otherwise, 1 million cells or about 2 mg of tissue was extracted per condition and a minimum of three replicates per condition was used in each experiment. K562 cells that are non-adherent, were collected by brief centrifugation at 4ºC using a table-top centrifuge (4,500 rpm, 1.5 min) and washed briefly in 0.9% NaCl (high grade salt and LC-MS-grade water Fisher Scientific W6500 or Sigma Aldrich 1.15333). 300 µl of prechilled extraction buffer were added per sample. For 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. 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. Samples were vortexed briefly (10 sec) and sonicated for 3 min in a 4ºC water bath. Samples were then centrifuged for 10 min, 4ºC, at maximum speed on a benchtop centrifuge (Eppendorf) and the cleared supernatant was transferred to a new tube. Samples were dried using a nitrogen dryer while on ice, and special attention was given to minimize the time of drying and to not let samples idle in the dryer (Reacti-Vap™ Evaporator, Thermo Fisher Scientific, TS-18826) once the drying process was completed. Needles were continuously adjusted to the surface of the liquid as the samples evaporated to expedite the drying process. Samples were reconstituted in 30 µl LC-MS-grade water by brief sonication in a 4ºC water bath. Extracted metabolites were spun for 2 min at maximum speed on a bench-top centrifuge and cleared supernatant was transferred to LC-MS micro vials (National Scientific, C5000-45B). 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.

Combined analysis:

Analysis ID AN002875
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:CH002130
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:MS002668
Analysis ID:AN002875
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:HESI
Ion Mode:UNSPECIFIED
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