Summary of Study ST000084

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 PR000075. The data can be accessed directly via it's Project DOI: 10.21228/M86K5H This work is supported by NIH grant, U2C- DK119886.

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Study IDST000084
Study TitleModel-driven multi-omic data analysis elucidates metabolic immunomodulators of macrophage activation
Study Typegrowth condition, timecourse
Study SummaryMacrophages are central players in immune response, manifesting divergent phenotypes to control inflammation and innate immunity through release of cytokines and other signaling factors. Recently, the focus on metabolism has been reemphasized as critical signaling and regulatory pathways of human pathophysiology, ranging from cancer to aging, often converge on metabolic responses. Here, we used genome-scale modeling and multi-omics (transcriptomics, proteomics, and metabolomics) analysis to assess metabolic features that are critical for macrophage activation. A genome-scale metabolic network for the RAW 264.7 cell line was constructed to determine metabolic modulators of activation. Metabolites well-known to be associated with immunoactivation (glucose and arginine) and immunosuppression (tryptophan and vitamin D3) were among the most critical effectors. Intracellular metabolic mechanisms were assessed, identifying a suppressive role for de-novo nucleotide synthesis. Finally, underlying metabolic mechanisms of macrophage activation were identified by analyzing multi-omic data obtained from LPS-stimulated RAW cells in the context of our flux-based predictions. This study demonstrates that the role of metabolism in regulating activation may be greater than previously anticipated and elucidates underlying connections between activation and metabolic effectors. This submission corresponds to the metabolomics data from this study.
Institute
Pacific Northwest National Laboratory
DepartmentBiological Separation and Mass Spectrometry
Last NameMetz
First NameThomas
Emailthomas.metz@pnnl.gov
Submit Date2014-06-25
Num Groups2
Total Subjects12
Raw Data AvailableYes
Raw Data File Type(s)cdf, d
Uploaded File Size102 M
Analysis Type DetailGC-MS
Release Date2014-08-06
Release Version1
Thomas Metz Thomas Metz
https://dx.doi.org/10.21228/M86K5H
ftp://www.metabolomicsworkbench.org/Studies/ application/zip

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Sample Preparation:

Sampleprep ID:SP000099
Sampleprep Summary:Suspentions softly centrifuged, buffer removed, amonium bicarbonate added, metabolites extraxted with chloroform/methanol (2:1, v/v), vortexed, centrifuged, aqueous layer dried in vacuum concentrator, derivatization with methoxyamine in pyridine, N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA), and 1% trimethylchlorosilane (TMCS)
Sampleprep Protocol Comments:Cell suspensions were softly centrifuged (230 × g for 5 min) and as much buffer as possible was removed. Then, 170 µL of 150 mM ammonium bicarbonate was added to the cell pellet and the cell suspensions were transferred to 2 mL micro-centrifuge tubes for extraction. Subsequently, the water soluble metabolites were extracted with four volumes of chilled (-20°C) chloroform/methanol (2:1, v/v). After vortexing, the samples were centrifuged (12,000 × g for 5 min) and the upper (aqueous) layers containing water-soluble metabolites were transferred into glass vials, followed by drying in a vacuum concentrator. For the derivatization, 20 µL of methoxyamine in pyridine (30 mg/mL) were added to each sample, followed by incubation at 37°C with shaking for 90 min to protect carbonyl groups. Next, 80 µL of N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) with 1% trimethylchlorosilane (TMCS) were added to each vial, followed by incubation at 37°C with shaking for 30 min to derivatize hydroxyl and amine groups. The samples were then allowed to cool to room temperature.
Processing Method:Homogenization
Extraction Method:The water soluble metabolites were extracted with four volumes of chilled (-20°C) chloroform/methanol (2:1, v/v). After vortexing, the samples were centrifuged (12,000 × g for 5 min) and the upper (aqueous) layers containing water-soluble metabolites were transferred into glass vials, followed by drying in a vacuum concentrator.
Extract Concentration Dilution:chilled (-20°C) chloroform/methanol (2:1, v/v)
Extract Enrichment:Vacuum Concentrator
Sample Resuspension:20 µL of methoxyamine in pyridine (30 mg/mL)
Sample Derivatization:20 µL of methoxyamine in pyridine (30 mg/mL), 80 µL of N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) with 1% trimethylchlorosilane (TMCS),
Cell Type:Macrophage
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