Summary of Study ST001766

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 IDST001766
Study TitleApplication of the redox metabolite detection method for mammalian tissues (part I)
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 LUNA NH2 HILIC 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:SU001843
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
SA164335pooled sample QC 1/3 dilution-
SA164336blank1-
SA164337pooled sample QCc-
SA164338pooled sample Qca-
SA164339pooled sample QC 1/10 dilution-
SA164340pooled sample QC 1/10 dilution a-
SA164341blank2-
SA164342blank3-
SA164343blank4-
SA164344pooled sample QCb-
SA164345kidney2buffer C_Ell
SA164346liver chunk1buffer C_Ell
SA164347liver chunk2buffer C_Ell
SA164348liver chunk3buffer C_Ell
SA164349kidney1buffer C_Ell
SA164350kidney3buffer C_Ell
Showing results 1 to 16 of 16

Collection:

Collection ID:CO001836
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:TR001856
Treatment Summary:Buffer Only

Sample Preparation:

Sampleprep ID:SP001849
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 AN002873
Analysis type MS
Chromatography type HILIC
Chromatography system Thermo Vanquish
Column Phenomenex Luna NH2 (150 x 2mm,3um)
MS Type ESI
MS instrument type Orbitrap
MS instrument name Thermo Q Exactive Orbitrap
Ion Mode UNSPECIFIED
Units ppm

Chromatography:

Chromatography ID:CH002128
Chromatography Summary:LUNA-NH2 chromatography: One  ml of reconstituted sample was injected into a Luna® 3 µm NH2 100 Å, LC Column (150x2 mm, 3 µm particle size; Phenomenex, 00F-4377-B0) 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 5 mM ammonium acetate and 0.2% ammonium hydroxide. Gradient conditions were: 20 min linear gradient from 10% to 90% B; 20-25 min hold at 90% B; 25–26 min from 90% to 10% B; 26–34 min hold at 10% B. The column oven and autosampler tray were held at 30 °C and 4 °C, respectively
Instrument Name:Thermo Vanquish
Column Name:Phenomenex Luna NH2 (150 x 2mm,3um)
Column Temperature:30
Flow Gradient:0 min linear gradient from 10% to 90% B; 20-25 min hold at 90% B; 25-26 min from 90% to 10% B; 26-34 min hold at 10% B.
Solvent A:100% acetonitrile
Solvent B:100% water; 5 mM ammonium acetate; 0.2% ammonium hydroxide
Chromatography Type:HILIC

MS:

MS ID:MS002666
Analysis ID:AN002873
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:HESI
Ion Mode:UNSPECIFIED
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