Summary of Study ST003088
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 PR001918. The data can be accessed directly via it's Project DOI: 10.21228/M8ST6K 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 | ST003088 |
Study Title | Metabolome changes in embryonic CSF (Part 10) |
Study Summary | Untargeted MS analysis of maternal liver, CSF, serum, and embryonic CSF and liver, 3h post saline or PolyI:C administrated to mother. |
Institute | Boston Children's Hospital, Harvard Medical School |
Laboratory | Kanarek Lab |
Last Name | Petrova |
First Name | Boryana |
Address | Enders 1116.2 300 Longwood Ave |
Boryana.Petrova@childrens.harvard.edu | |
Phone | 6179197352 |
Submit Date | 2024-01-18 |
Raw Data Available | Yes |
Raw Data File Type(s) | raw(Thermo) |
Analysis Type Detail | LC-MS |
Release Date | 2024-07-29 |
Release Version | 1 |
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Sample Preparation:
Sampleprep ID: | SP003209 |
Sampleprep Summary: | Per condition, 5uL of maternal serum, CSF, were extracted by brief sonication in 200/100μl (serum/csf) 80% methanol, supplemented with isotopically labeled internal standards (17 amino acids and reduced glutathione, Cambridge Isotope Laboratories, MSK-A2-1.2 and CNLM-6245-10). 10uL Embryonic CSF (eCSF) was extracted per condition using the previously described buffer quantity, and the flash frozen liver chunks were extracted in 300uL of the previously described buffer. After centrifugation for 10min. at maximum speed on a benchtop centrifuge (Eppendorf) the cleared supernatant from CSF and eCSF was dried using a nitrogen dryer. Samples were reconstituted in LCMS water by brief sonication. Extracted metabolites were spun again and cleared supernatant was transferred in LC-MS microvials. Serum followed the same protocol, except that an additional 20-minute centrifugation was administered, and homogenization for 5 minutes. Final LCMS water reconstitution values for tissue type were 50uL (maternal and embryonic serum), 15uL (maternal CSF and eCSF),. A small amount of each sample was pooled and serially diluted 3 and 10-fold to be used as quality controls throughout the batch run. Two microliters (equivalent to 0.2 ul of CSF) of reconstituted sample were injected into a ZIC-pHILIC 150 × 2.1 mm (5 µm particle size) column (EMD Millipore) operated on a Dionex UltiMate 3000 UPLC system (Thermo Fisher Scientific). 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. The MS data acquisition was on a QExactive benchtop orbitrap mass spectrometer equipped with an Ion Max source and a HESI II probe and was performed in a range of m/z= 70–1000, with the resolution set at 70,000, the AGC target at 1x106, and the maximum injection time (Max IT) at 20 msec. For tSIM scans, the resolution was set at 70,000, the AGC target was 1x105, and the max IT was 100 msec. Relative quantitation of polar metabolites was performed with TraceFinder 4.1 (Thermo Fisher Scientific) using a 5 ppm mass tolerance and referencing an in-house library of chemical standards. Pooled samples and fractional dilutions were prepared as quality controls and only those metabolites were taken for further analysis, for which the correlation between the dilution factor and the peak area was >0.95 (high confidence metabolites). |