Summary of Study ST001765

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 IDST001765
Study TitleOptimization of redox metabolite detection in mammalian cells (part I)
Study SummaryConditions were tested to optimize number of cells and extraction buffer for the detection of redox reactive metabolites from mammalian cells. Four different extraction buffers were compared. Derivatization of glutathione was explored as a condition as well. This is an independent repeat.
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:SU001842
Subject Type:Mammal
Subject Species:Homo sapiens
Taxonomy ID:9606

Factors:

Subject type: Mammal; Subject species: Homo sapiens (Factor headings shown in green)

mb_sample_id local_sample_id Factor
SA164321pooled sample QC f-
SA164322pooled sample QC e-
SA164323pooled sample QC 1/3 dilution-
SA164324pooled sample QC 1/10 dilution-
SA164325blank 2-
SA164326pooled sample QC 1/10 dilution a-
SA164327pooled sample QC d-
SA164328pooled sample QC a-
SA164329blank 3-
SA164330blank 1-
SA164331blank 4-
SA164332pooled sample QC 3x loaded-
SA164333pooled sample QC b-
SA164334pooled sample QC c-
SA164300442_1M_1"buffer A, 1Milion cells"
SA164301442_1M_1.1"buffer A, 1Milion cells"
SA164302442_2M_1"buffer A, 2Milion cells"
SA164303buffer B_1M_2"buffer B, 1Milion cells"
SA164304buffer B_1M_1"buffer B, 1Milion cells"
SA164305buffer B_2M_1"buffer B, 2Milion cells"
SA164306buffer B_2M_2"buffer B, 2Milion cells"
SA164307buffer B_5M_2"buffer B, 5Milion cells"
SA164308buffer B_5M_1"buffer B, 5Milion cells"
SA164309buffer C_1M_2"buffer C, 1Milion cells"
SA164310buffer C_1M_1"buffer C, 1Milion cells"
SA164311buffer C_2M_1"buffer C, 2Milion cells"
SA164312buffer C_2M_2"buffer C, 2Milion cells"
SA164313buffer C_5M_2"buffer C, 5Milion cells"
SA164314buffer C_5M_1"buffer C, 5Milion cells"
SA164315buffer C_Ell_1M_1"buffer C, plus Ellmans, 1Milion cells"
SA164316buffer C_Ell_1M_2"buffer C, plus Ellmans, 1Milion cells"
SA164317buffer C_Ell_2M_1"buffer C, plus Ellmans, 2Milion cells"
SA164318buffer C_Ell_2M_2"buffer C, plus Ellmans, 2Milion cells"
SA164319buffer C_Ell_5M_1"buffer C, plus Ellmans, 5Milion cells"
SA164320buffer C_Ell_5M_2"buffer C, plus Ellmans, 5Milion cells"
Showing results 1 to 35 of 35

Collection:

Collection ID:CO001835
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:K562 Cells

Treatment:

Treatment ID:TR001855
Treatment Summary:K562 Variable Cell count + Buffers: buffer A, 1 Million cells x2, buffer A, 2 Million cells x1, buffer B, 1 Million cells x2, buffer B, 2 Million cells x2, buffer B, 5 Million cells x 2, buffer C, 1 Million cells x 2, buffer C, 2 Million cells x 2, buffer C, 5 Million cells x2, buffer C, plus Ellmans, 1 Million cells x 2, buffer C, plus Ellmans, 2 Million cells x 2, buffer C, plus Ellmans, 5 Million cells x 2,

Sample Preparation:

Sampleprep ID:SP001848
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 AN002872
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:CH002127
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)
Chromatography Type:HILIC

MS:

MS ID:MS002665
Analysis ID:AN002872
Instrument Name:Thermo Q Exactive Orbitrap
Instrument Type:Orbitrap
MS Type:ESI
MS Comments:HESI
Ion Mode:UNSPECIFIED
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