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).ini,.INI, txt
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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Project:

Project ID:PR000075
Project DOI:doi: 10.21228/M86K5H
Project Title:Systems Biology for EnteroPathogens
Project Type:MS analysis
Project Summary:sysbep.org
Institute:Pacific Northwest National Laboratory
Department:Biological Separation and Mass Spectrometry
Last Name:Joshua
First Name:Adkins
Email:Joshua.Adkins@pnnl.gov

Subject:

Subject ID:SU000103
Subject Type:Animal cells
Subject Species:Mus musculus
Taxonomy ID:10090
Genotype Strain:RAW 264.7
Cell Strain Details:RAW 264.7
Cell Primary Immortalized:immortalized
Species Group:Mammal

Factors:

Subject type: Animal cells; Subject species: Mus musculus (Factor headings shown in green)

mb_sample_id local_sample_id Lipopolysaccharide Treatment
SA004283SBEP_Metab_LPSactivated2100 ng/ml
SA004284SBEP_Metab_LPSactivated3100 ng/ml
SA004285SBEP_Metab_LPSactivated1100 ng/ml
SA004286SBEP_Metab_Control3None
SA004287SBEP_Metab_Control2None
SA004288SBEP_Metab_Control1None
Showing results 1 to 6 of 6

Collection:

Collection ID:CO000086
Collection Summary:After stimulation, cells were washed twice with Dulbecco’s PBS, scraped out and harvested into 15-mL centrifuge tubes.
Sample Type:Cell
Collection Method:After stimulation, cells were washed twice with Dulbecco’s PBS, scraped out and harvested into 15-mL centrifuge tubes.
Collection Time:24 hours
Collection Vials:15-mL centrifuge tubes
Tissue Cell Quantity Taken:All

Treatment:

Treatment ID:TR000104
Treatment Summary:Macrophages (RAW 264.7 cells) grown for two day with suplimentation of 100ng | ml lipopolysaccharide | Macrophages (RAW 264.7 cells) grown for two days without suplimentation
Treatment Protocol Comments:Macrophages (RAW 264.7 cells) were seeded at a density of 3.0E6 cells in 150 mm dishes using Dulbecco's Modified Eagle's Medium supplemented with 10% fetal calf serum, then grown for 2 days and stimulated for 24 hours with 100 ng/mL of lipopolysaccharide diluted in fresh medium. A control culture was run in parallel by incubating for the same period of time with fresh medium only. Three biological replicates were performed per condition, and two dishes were used for each replicate. / Macrophages (RAW 264.7 cells) were seeded at a density of 3.0E6 cells in 150 mm dishes using Dulbecco's Modified Eagle's Medium supplemented with 10% fetal calf serum, then grown for 2 days and stimulated for 24 hours with 100 ng/mL of lipopolysaccharide diluted in fresh medium. A control culture was run in parallel by incubating for the same period of time with fresh medium only. Three biological replicates were performed per condition, and two dishes were used for each replicate.
Treatment:Abiotic
Treatment Compound:lipopolysaccharide / fresh medium
Treatment Dose:100 ng/mL /--
Treatment Vehicle:fresh medium
Cell Growth Container:150 mm dishes
Cell Inoc Proc:Macrophages (RAW 264.7 cells) were seeded at a density of 3.0E6 cells in 150 mm dishes
Cell Media:Dulbecco's Modified Eagle's Medium supplemented with 10% fetal calf serum
Cell Harvesting:After stimulation, cells were washed twice with Dulbecco’s PBS, scraped out and harvested into 15-mL centrifuge tubes.

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

Combined analysis:

Analysis ID AN000136
Analysis type MS
Chromatography type GC
Chromatography system Agilent 7890A
Column Agilent HP5-MS (30m × 0.25mm, 0.25 um)
MS Type EI
MS instrument type Single quadrupole
MS instrument name Agilent 5975C
Ion Mode POSITIVE
Units Peak area

Chromatography:

Chromatography ID:CH000096
Chromatography Summary:Agilent 7890A gas chromatograph with a HP-5MS gas chromatography column using Chemstation
Chromatography Comments:Chromatography was carried out on an Agilent 7890A gas chromatograph using the manufacturer's software (Chemstation) and a HP-5MS gas chromatography column (Agilent Technologies, Santa Clara, CA; 30 m x 0.25 mm x 0.25 m film thickness). The sample injection mode was splitless, and 1 L of each sample was injected. The injection port temperature was held at 250 C throughout the analysis. The GC oven was held at 60 C for 1 min after injection, and the temperature was then increased to 325 C by 10 C/min, followed by a 5 min hold at 325 C. The helium gas flow rates for each Experiment were determined by the Agilent Retention Time Locking function based on analysis of deuterated myristic acid and were in the range of 0.450.5 mL/min.
Instrument Name:Agilent 7890A
Column Name:Agilent HP5-MS (30m × 0.25mm, 0.25 um)
Flow Rate:0.45-0.5mL/min
Injection Temperature:250 C
Sample Injection:1 L, Splitless
Analytical Time:37.5 min
Oven Temperature:60 C for 1 min, then increased to 325 C by 10 C/min, followed by a 5 min hold at 325 C
Sample Syringe Size:10 L
Chromatography Type:GC

MS:

MS ID:MS000112
Analysis ID:AN000136
Instrument Name:Agilent 5975C
Instrument Type:Single quadrupole
MS Type:EI
MS Comments:An Agilent GC 7890A coupled with a single quadrupole MSD 5975C (Agilent Technologies, Inc.; Santa Clara, CA, USA) was used, and the samples were blocked and analyzed in random order for each experiment. Data were collected over the mass range 50-550 m/z. A mixture of FAMEs (C8-C28) was analyzed once per day together with the samples for retention index alignment purposes during subsequent data analysis.
Ion Mode:POSITIVE
Scan Range Moverz:50-550 m/z
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