Summary of Study ST002412
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 PR001552. The data can be accessed directly via it's Project DOI: 10.21228/M8342Z 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 | ST002412 |
| Study Title | Metabolic effects of the protein kinase R |
| Study Type | Biomedical research |
| Study Summary | Spleen-derived macrophage from WT or Eif2ak2-/- (gene encoding PKR protein kinase) mice are treated with a synthetic RNA mimetic (polyinosinic:polycytidylic acid) to activate the kinase and metabolites were collected for analysis. The data identified 325 putative metabolites in the cell extracts, with a large number of significant differences between the Eif2ak2- /- and WT sample groups. Metabolite levels are predominantly suppressed in the WT compared to the Eif2ak2-/- cells, with depletion of specific metabolites in amino acid, carbohydrate, lipid and nucleotide pathways, while several amino acid metabolites were significantly elevated in the WT cells compared to the Eif2ak2-/-. The changes appear to delineate a pseudo-starvation response in the WT cells. Phosphate energy metabolism is altered with decreased creatine and phosphocreatine and a compensatory increase in phosphorylated guanidinoacetate in the WT compared to the Eif2ak2- /- cells. There appears to be a constraint in glycolysis in the WT cells, most clearly in the pentose phosphate pathway. |
| Institute | Hudson Institute of Medical Research |
| Department | CIIID |
| Laboratory | Molecular Immunology |
| Last Name | Sadler |
| First Name | Anthony |
| Address | 27-31 Wright st, Clayton, VIC 3168 |
| Anthony.sadler@hudson.org.au | |
| Phone | +61 4 85722722 |
| Submit Date | 2022-12-15 |
| Num Groups | 2 |
| Total Subjects | NA |
| Num Males | NA |
| Num Females | NA |
| Study Comments | KO vs WT |
| Publications | Suppression of the nucleic acid precursor ribose 5-phosphate by RNA-mediated antiviral immunity |
| Raw Data Available | Yes |
| Raw Data File Type(s) | raw(Thermo) |
| Analysis Type Detail | LC-MS |
| Release Date | 2023-01-04 |
| Release Version | 1 |
Select appropriate tab below to view additional metadata details:
Project:
| Project ID: | PR001552 |
| Project DOI: | doi: 10.21228/M8342Z |
| Project Title: | Metabolic effects of the protein kinase R |
| Project Type: | Untargeted LCMS metabolomics |
| Project Summary: | Spleen-derived macrophage from WT or Eif2ak2-/- (gene encoding PKR protein kinase) mice are treated with a synthetic RNA mimetic (polyinosinic:polycytidylic acid) to activate the kinase and metabolites were collected for analysis. The data identified 325 putative metabolites in the cell extracts, with a large number of significant differences between the Eif2ak2- /- and WT sample groups. Metabolite levels are predominantly suppressed in the WT compared to the Eif2ak2-/- cells, with depletion of specific metabolites in amino acid, carbohydrate, lipid and nucleotide pathways, while several amino acid metabolites were significantly elevated in the WT cells compared to the Eif2ak2-/-. The changes appear to delineate a pseudo-starvation response in the WT cells. Phosphate energy metabolism is altered with decreased creatine and phosphocreatine and a compensatory increase in phosphorylated guanidinoacetate in the WT compared to the Eif2ak2- /- cells. There appears to be a constraint in glycolysis in the WT cells, most clearly in the pentose phosphate pathway. |
| Institute: | Hudson Institute of Medical Research |
| Department: | CIIID |
| Laboratory: | Molecular Immunology |
| Last Name: | Sadler |
| First Name: | Anthony |
| Address: | 27-31 Wright St, Clayton, VIC 3168, Australia |
| Email: | Anthony.sadler@hudson.org.au |
| Phone: | +61 4 85722722 |
| Funding Source: | NHMRC grants (1143839, 1043398) and a philanthropic Perpetual Trusties grant (CF07/2408) and a philanthropic Perpetual IMPACT grant. |
| Publications: | Suppression of the nucleic acid precursor ribose 5-phosphate by RNA-mediated antiviral immunity |
| Contributors: | Pushpack Bhattacharjee, Die Wang, Dovile Anderson, Joshua N. Buckler, Eveline de Geus, Feng Alex Yan, Galina Polekhina, Ralf Schittenhelm, Darren J. Creek, Lawrence D. Harris, Anthony J. Sadler |
Subject:
| Subject ID: | SU002501 |
| Subject Type: | Cultured cells |
| Subject Species: | Mus musculus |
| Taxonomy ID: | 10090 |
| Genotype Strain: | Spleen-derived macrophage from WT or Eif2ak2-/- |
| Age Or Age Range: | NA |
| Weight Or Weight Range: | NA |
| Height Or Height Range: | NA |
| Gender: | Not applicable |
Factors:
Subject type: Cultured cells; Subject species: Mus musculus (Factor headings shown in green)
| mb_sample_id | local_sample_id | treatment |
|---|---|---|
| SA241922 | C220181610_Extr_blank_1 | Extraction blank |
| SA241933 | C220181016_Blank_2 | injection blank |
| SA241934 | C220181016_Blank_4 | injection blank |
| SA241935 | C220181016_Blank_3 | injection blank |
| SA241936 | C220181016_Blank_1 | injection blank |
| SA241923 | C220181610_PKR_Null_3 | PKR knockout |
| SA241924 | C220181610_PKR_Null_4 | PKR knockout |
| SA241925 | C220181610_PKR_Null_2 | PKR knockout |
| SA241926 | C220181610_PKR_Null_5 | PKR knockout |
| SA241927 | C220181610_PKR_Null_1 | PKR knockout |
| SA241937 | C220181016_QC_3 | pooled QC |
| SA241938 | C220181016_QC_1 | pooled QC |
| SA241939 | C220181016_QC_2 | pooled QC |
| SA241940 | C220181016_QC_4 | pooled QC |
| SA241928 | C220181610_WT_5 | WT |
| SA241929 | C220181610_WT_3 | WT |
| SA241930 | C220181610_WT_1 | WT |
| SA241931 | C220181610_WT_2 | WT |
| SA241932 | C220181610_WT_4 | WT |
| Showing results 1 to 19 of 19 |
Collection:
| Collection ID: | CO002494 |
| Collection Summary: | Exogenous genes were stably expressed and endogenous gene expression suppressed in murine macrophages by transduction with lentiviral constructs. Gene open reading frames were cloned as NheI-BamHI (New England Biolabs) fragments in the plentiCRISPR v2-Blast (Mohan Babu, Addgene plasmid # 83480; http://n2t.net/addgene:83480; RRID: Addgene_83480). Knockdown of endogenous transcripts was achieved with short hairpin RNA cloned into pLKO.1-puro. Lentiviral particles were packaged using a Lenti-X mix (Takara) by transfection of the plasmid components into HEK293FT cells with Lipofectamine 2000 (Invitrogen). The culture supernatant was collected after 48 hours, centrifuged at 10 000 g, then pipetted onto adherent macrophages. Transduced cells were isolated over 2-3 month by increasing selection (from 1 to 20 ug/mL) with puromycin (Thermo Fisher Scientific) and/or blasticidin (InvivoGen), for pLKO-1-puro-shRNA and plentiCRISPR v2-blast, respectively. Recombinant cells were maintained with antibiotics in Dulbecco’s modified Eagle’s Medium (DMEM, GibCo) supplemented with 10% foetal bovine serum (Bovogen) in a humidified 5% CO 2 incubator at 37 °C. Growth on different carbon sources was tested by plating 6x10 3 cells per well of a 24 well plate with glucose free DMEM (ThermoFisher Scientific) supplemented with 10% FBS (not dialysed), 20 ug/mL puromycin and 20 uM of each sugar. After 72 hours the cells were lifted with trypsin and counted by automation (Countess, Invitrogen). |
| Sample Type: | Macrophages |
Treatment:
| Treatment ID: | TR002513 |
| Treatment Summary: | NA |
Sample Preparation:
| Sampleprep ID: | SP002507 |
| Sampleprep Summary: | Adherent macrophages were lifted with trypsin, suspended in PBS and 2x10 6 cells were collected by centrifugation. The cell pellet was rinsed in 10 mL ice cold 0.9% NaCl in water at 4 °C pelleted, then resuspended in 200 L of 4 °C extraction solvent (a 1:3:1 ratio of chloroform, methanol and water). The sample was subjected to three freeze-thaw cycles then mixed by vortex for 30 minutes at 4 °C and cell debris was removed by centrifugation at 20,000g for 10 minutes at 4 °C. The cleared supernatant was transferred (180 uL) and frozen at -80°C until LCMS analysis. |
| Extract Storage: | -80℃ |
Combined analysis:
| Analysis ID | AN003931 | AN003932 |
|---|---|---|
| Analysis type | MS | MS |
| Chromatography type | HILIC | HILIC |
| Chromatography system | Thermo Dionex Ultimate 3000 RS | Thermo Dionex Ultimate 3000 RS |
| Column | Merck SeQuant ZIC-HILIC (150 x 4.6mm,5um) | Merck SeQuant ZIC-HILIC (150 x 4.6mm,5um) |
| MS Type | ESI | ESI |
| MS instrument type | Orbitrap | Orbitrap |
| MS instrument name | Thermo Q Exactive Orbitrap | Thermo Q Exactive Orbitrap |
| Ion Mode | POSITIVE | NEGATIVE |
| Units | peak height | peak height |
Chromatography:
| Chromatography ID: | CH002910 |
| Chromatography Summary: | pHILIC chromatography at pH9 using Orbitrap high resolution accurate mass MS detection with posneg switching |
| Instrument Name: | Thermo Dionex Ultimate 3000 RS |
| Column Name: | Merck SeQuant ZIC-HILIC (150 x 4.6mm,5um) |
| Column Pressure: | 60 bar at starting conditions |
| Column Temperature: | 25 C |
| Flow Gradient: | 0 min - 80%B, 15 min - 50%B, 18 min - 5%B, 21 min - 5%B, 24 min - 80%B, 32 min - 80%B |
| Flow Rate: | 0.3 ml/min |
| Injection Temperature: | 4 C |
| Internal Standard: | CAPS, CHAPS, PIPES |
| Solvent A: | 100% water; 20 mM ammonium carbonate |
| Solvent B: | 100% acetonitrile |
| Analytical Time: | 32 min |
| Capillary Voltage: | 3.5 kV |
| Oven Temperature: | 25 C |
| Washing Buffer: | syringe wash 50% IPA |
| Sample Loop Size: | 25 uL |
| Sample Syringe Size: | 25 uL |
| Randomization Order: | yes |
| Chromatography Type: | HILIC |
| Chromatography ID: | CH002911 |
| Chromatography Summary: | pHILIC chromatography at pH9 using Orbitrap high resolution accurate mass MS detection with posneg switching |
| Instrument Name: | Thermo Dionex Ultimate 3000 RS |
| Column Name: | Merck SeQuant ZIC-HILIC (150 x 4.6mm,5um) |
| Column Pressure: | 60 bar at starting conditions |
| Column Temperature: | 25 C |
| Flow Gradient: | 0 min - 80%B, 15 min - 50%B, 18 min - 5%B, 21 min - 5%B, 24 min - 80%B, 32 min - 80%B |
| Flow Rate: | 0.3 ml/min |
| Injection Temperature: | 4 C |
| Internal Standard: | CAPS, CHAPS, PIPES |
| Solvent A: | 100% water; 20 mM ammonium carbonate |
| Solvent B: | 100% acetonitrile |
| Analytical Time: | 32 min |
| Capillary Voltage: | 3.5 kV |
| Oven Temperature: | 25 C |
| Washing Buffer: | syringe wash 50% IPA |
| Sample Loop Size: | 25 uL |
| Sample Syringe Size: | 25 uL |
| Randomization Order: | yes |
| Chromatography Type: | HILIC |
MS:
| MS ID: | MS003669 |
| Analysis ID: | AN003931 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | .raw files containing both pos and neg data (produced by using polarity switching) were converted to a readable format .mzxml, these files were combined and features aligned. Compiled data matrix was imported to IDEOM for metabolite ID and filtering. |
| Ion Mode: | POSITIVE |
| Capillary Voltage: | 4 kV |
| Collision Gas: | NA |
| Ion Source Temperature: | 120 C |
| Mass Accuracy: | 1-2 ppm |
| Acquisition Parameters File: | Metabolomics_pHILIC_Parkville_v1.pdf |
| Analysis Protocol File: | PQMS3-MBPF-WIN-0501_analysis.pdf |
| MS ID: | MS003670 |
| Analysis ID: | AN003932 |
| Instrument Name: | Thermo Q Exactive Orbitrap |
| Instrument Type: | Orbitrap |
| MS Type: | ESI |
| MS Comments: | .raw files containing both pos and neg data (produced by using polarity switching) were converted to a readable format .mzxml, these files were combined and features aligned. Compiled data matrix was imported to IDEOM for metabolite ID and filtering. |
| Ion Mode: | NEGATIVE |
| Capillary Voltage: | 3.5 kV |
| Collision Gas: | NA |
| Ion Source Temperature: | 120 C |
| Mass Accuracy: | 1-2 ppm |
| Acquisition Parameters File: | Metabolomics_pHILIC_Parkville_v1.pdf |
| Analysis Protocol File: | PQMS3-MBPF-WIN-0501_analysis.pdf |
