Summary of Study ST001772
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.
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 | ST001772 |
Study Title | Optimization of redox metabolite detection in mammalian cells (part II) |
Study Summary | Conditions 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 glutathion was explored as a condition as well. |
Institute | Boston Children's Hospital, Harvard Medical School |
Department | Pathology |
Laboratory | Naama Kanarek |
Last Name | Petrova |
First Name | Boryana |
Address | 300 Longwood Av, Boston, MA, 2115, USA |
boryana.petrova@childrens.harvard.edu | |
Phone | 6173557433 |
Submit Date | 2021-04-22 |
Raw Data Available | Yes |
Raw Data File Type(s) | raw(Thermo) |
Analysis Type Detail | LC-MS |
Release Date | 2021-05-17 |
Release Version | 1 |
Select appropriate tab below to view additional metadata details:
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: | SU001849 |
Subject Type: | Mammal |
Subject Species: | Homo sapiens |
Taxonomy ID: | 9606 |
Species Group: | Mammals |
Factors:
Subject type: Mammal; Subject species: Homo sapiens (Factor headings shown in green)
mb_sample_id | local_sample_id | Factor |
---|---|---|
SA164462 | pooled sample QC 1/10 dilution a | - |
SA164463 | pooled sample QC 1/3 dilution | - |
SA164464 | pooled sample QC5 | - |
SA164465 | pooled sample QC2 | - |
SA164466 | pooled sample QC3 | - |
SA164467 | pooled sample QC1 | - |
SA164468 | blank1 | - |
SA164469 | blank3 | - |
SA164470 | blank2 | - |
SA164471 | blank4 | - |
SA164472 | blank5 | - |
SA164473 | pooled sample QC 1/10 dilution b | - |
SA164474 | pooled sample QC4 | - |
SA164475 | 1M K562 cell buffer A_5_1 | buffer A |
SA164476 | 1M K562 cell buffer A_1_1 | buffer A |
SA164477 | 1M K562 cell buffer A_1_2 | buffer A |
SA164478 | 1M K562 cell buffer A_2_1 | buffer A |
SA164479 | 1M K562 cell buffer A_2_2 | buffer A |
SA164480 | 1M K562 cell buffer A_5_2 | buffer A |
SA164481 | 1M K562 cell buffer B_1_2a | buffer B |
SA164482 | 1M K562 cell buffer B_1_2 | buffer B |
SA164483 | 1M K562 cell buffer B_2_1 | buffer B |
SA164484 | 1M K562 cell buffer B_2_2 | buffer B |
SA164485 | 1M K562 cell buffer B_5_1 | buffer B |
SA164486 | 1M K562 cell buffer B_5_2 | buffer B |
SA164487 | 1M K562 cell buffer C_1_2 | buffer C buffer |
SA164488 | 1M K562 cell buffer C_5_1 | buffer C buffer |
SA164489 | 1M K562 cell buffer C_2_2 | buffer C buffer |
SA164490 | 1M K562 cell buffer C_2_1 | buffer C buffer |
SA164491 | 1M K562 cell buffer C_1_1 | buffer C buffer |
SA164492 | 1M K562 cell buffer C_5_2 | buffer C buffer |
SA164493 | 1M K562 cell buffer C_Ell_1_1 | buffer C_Ell |
SA164494 | 1M K562 cell buffer C_Ell_5_2 | buffer C_Ell |
SA164495 | 1M K562 cell buffer C_Ell_2_2 | buffer C_Ell |
SA164496 | 1M K562 cell buffer C_Ell_2_1 | buffer C_Ell |
SA164497 | 1M K562 cell buffer C_Ell_1_2 | buffer C_Ell |
SA164498 | 1M K562 cell buffer C_Ell_5_1 | buffer C_Ell |
Showing results 1 to 37 of 37 |
Collection:
Collection ID: | CO001842 |
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: | TR001862 |
Treatment Summary: | 1M K562 cells + Buffers from Study Design |
Sample Preparation:
Sampleprep ID: | SP001855 |
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 | AN002879 |
---|---|
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: | CH002134 |
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: | MS002672 |
Analysis ID: | AN002879 |
Instrument Name: | Thermo Q Exactive Orbitrap |
Instrument Type: | Orbitrap |
MS Type: | ESI |
MS Comments: | HESI |
Ion Mode: | UNSPECIFIED |