{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST002412","ANALYSIS_ID":"AN003932","VERSION":"1","CREATED_ON":"December 19, 2022, 8:20 am"},

"PROJECT":{"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","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"},

"STUDY":{"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","DEPARTMENT":"CIIID","LABORATORY":"Molecular Immunology","LAST_NAME":"Sadler","FIRST_NAME":"Anthony","ADDRESS":"27-31 Wright st, Clayton, VIC 3168","EMAIL":"Anthony.sadler@hudson.org.au","PHONE":"+61 4 85722722","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"},

"SUBJECT":{"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"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"C220181016_Blank_1",
"Factors":{"treatment":"injection blank"},
"Additional sample data":{"RAW_FILE_NAME":"C220181016_Blank_1"}
},
{
"Subject ID":"-",
"Sample ID":"C220181016_Blank_2",
"Factors":{"treatment":"injection blank"},
"Additional sample data":{"RAW_FILE_NAME":"C220181016_Blank_2"}
},
{
"Subject ID":"-",
"Sample ID":"C220181016_Blank_3",
"Factors":{"treatment":"injection blank"},
"Additional sample data":{"RAW_FILE_NAME":"C220181016_Blank_3"}
},
{
"Subject ID":"-",
"Sample ID":"C220181016_Blank_4",
"Factors":{"treatment":"injection blank"},
"Additional sample data":{"RAW_FILE_NAME":"C220181016_Blank_4"}
},
{
"Subject ID":"-",
"Sample ID":"C220181610_Extr_blank_1",
"Factors":{"treatment":"Extraction blank"},
"Additional sample data":{"RAW_FILE_NAME":"C220181610_Extr_blank_1"}
},
{
"Subject ID":"-",
"Sample ID":"C220181610_PKR_Null_1",
"Factors":{"treatment":"PKR knockout"},
"Additional sample data":{"RAW_FILE_NAME":"C220181610_PKR_Null_1"}
},
{
"Subject ID":"-",
"Sample ID":"C220181610_PKR_Null_2",
"Factors":{"treatment":"PKR knockout"},
"Additional sample data":{"RAW_FILE_NAME":"C220181610_PKR_Null_2"}
},
{
"Subject ID":"-",
"Sample ID":"C220181610_PKR_Null_3",
"Factors":{"treatment":"PKR knockout"},
"Additional sample data":{"RAW_FILE_NAME":"C220181610_PKR_Null_3"}
},
{
"Subject ID":"-",
"Sample ID":"C220181610_PKR_Null_4",
"Factors":{"treatment":"PKR knockout"},
"Additional sample data":{"RAW_FILE_NAME":"C220181610_PKR_Null_4"}
},
{
"Subject ID":"-",
"Sample ID":"C220181610_PKR_Null_5",
"Factors":{"treatment":"PKR knockout"},
"Additional sample data":{"RAW_FILE_NAME":"C220181610_PKR_Null_5"}
},
{
"Subject ID":"-",
"Sample ID":"C220181016_QC_1",
"Factors":{"treatment":"pooled QC"},
"Additional sample data":{"RAW_FILE_NAME":"C220181016_QC_1"}
},
{
"Subject ID":"-",
"Sample ID":"C220181016_QC_2",
"Factors":{"treatment":"pooled QC"},
"Additional sample data":{"RAW_FILE_NAME":"C220181016_QC_2"}
},
{
"Subject ID":"-",
"Sample ID":"C220181016_QC_3",
"Factors":{"treatment":"pooled QC"},
"Additional sample data":{"RAW_FILE_NAME":"C220181016_QC_3"}
},
{
"Subject ID":"-",
"Sample ID":"C220181016_QC_4",
"Factors":{"treatment":"pooled QC"},
"Additional sample data":{"RAW_FILE_NAME":"C220181016_QC_4"}
},
{
"Subject ID":"-",
"Sample ID":"C220181610_WT_1",
"Factors":{"treatment":"WT"},
"Additional sample data":{"RAW_FILE_NAME":"C220181610_WT_1"}
},
{
"Subject ID":"-",
"Sample ID":"C220181610_WT_2",
"Factors":{"treatment":"WT"},
"Additional sample data":{"RAW_FILE_NAME":"C220181610_WT_2"}
},
{
"Subject ID":"-",
"Sample ID":"C220181610_WT_3",
"Factors":{"treatment":"WT"},
"Additional sample data":{"RAW_FILE_NAME":"C220181610_WT_3"}
},
{
"Subject ID":"-",
"Sample ID":"C220181610_WT_4",
"Factors":{"treatment":"WT"},
"Additional sample data":{"RAW_FILE_NAME":"C220181610_WT_4"}
},
{
"Subject ID":"-",
"Sample ID":"C220181610_WT_5",
"Factors":{"treatment":"WT"},
"Additional sample data":{"RAW_FILE_NAME":"C220181610_WT_5"}
}
],
"COLLECTION":{"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_SUMMARY":"NA"},

"SAMPLEPREP":{"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℃"},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_SUMMARY":"pHILIC chromatography at pH9 using Orbitrap high resolution accurate mass MS detection with posneg switching","CHROMATOGRAPHY_TYPE":"HILIC","INSTRUMENT_NAME":"Thermo Dionex Ultimate 3000 RS","COLUMN_NAME":"Merck SeQuant ZIC-HILIC (150 x 4.6mm,5um)","SOLVENT_A":"100% water; 20 mM ammonium carbonate","SOLVENT_B":"100% acetonitrile","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","COLUMN_TEMPERATURE":"25 C","COLUMN_PRESSURE":"60 bar at starting conditions","INJECTION_TEMPERATURE":"4 C","INTERNAL_STANDARD":"CAPS, CHAPS, PIPES","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"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS","LABORATORY_NAME":"Monash Proteomics and Metabolomics Facility","OPERATOR_NAME":"Dovile Anderson","DETECTOR_TYPE":"Full scan","SOFTWARE_VERSION":"XCalibur","ACQUISITION_DATE":"17/10/2018","ANALYSIS_PROTOCOL_FILE":"PQMS3-MBPF-WIN-0501_analysis.pdf","ACQUISITION_PARAMETERS_FILE":"Metabolomics_pHILIC_Parkville_v1.pdf","DATA_FORMAT":".RAW"},

"MS":{"INSTRUMENT_NAME":"Thermo Q Exactive Orbitrap","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","ION_MODE":"NEGATIVE","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.","CAPILLARY_VOLTAGE":"3.5 kV","COLLISION_GAS":"NA","ION_SOURCE_TEMPERATURE":"120 C","MASS_ACCURACY":"1-2 ppm"},

"MS_METABOLITE_DATA":{
"Units":"peak height",

"Data":[{"Metabolite":"(R)-2-Hydroxyglutarate","C220181016_Blank_1":"58580","C220181016_Blank_2":"35405","C220181016_Blank_3":"40585","C220181016_Blank_4":"35505","C220181610_Extr_blank_1":"55080","C220181610_PKR_Null_1":"2826787","C220181610_PKR_Null_2":"3198475","C220181610_PKR_Null_3":"4566972","C220181610_PKR_Null_4":"1864725","C220181610_PKR_Null_5":"2661820","C220181016_QC_1":"3936661","C220181016_QC_2":"4024397","C220181016_QC_3":"3031512","C220181016_QC_4":"3740026","C220181610_WT_1":"847565","C220181610_WT_2":"844402","C220181610_WT_3":"810489","C220181610_WT_4":"337327","C220181610_WT_5":"882146"},{"Metabolite":"(S)-2-Acetolactate","C220181016_Blank_1":"25320","C220181016_Blank_2":"23838","C220181016_Blank_3":"24867","C220181016_Blank_4":"25764","C220181610_Extr_blank_1":"25150","C220181610_PKR_Null_1":"70090","C220181610_PKR_Null_2":"67466","C220181610_PKR_Null_3":"113034","C220181610_PKR_Null_4":"77595","C220181610_PKR_Null_5":"147843","C220181016_QC_1":"68871","C220181016_QC_2":"61672","C220181016_QC_3":"56394","C220181016_QC_4":"78246","C220181610_WT_1":"37300","C220181610_WT_2":"35163","C220181610_WT_3":"48321","C220181610_WT_4":"43350","C220181610_WT_5":"45677"},{"Metabolite":"(S)-Lactate","C220181016_Blank_1":"29786","C220181016_Blank_2":"32220","C220181016_Blank_3":"28630","C220181016_Blank_4":"0","C220181610_Extr_blank_1":"114603","C220181610_PKR_Null_1":"432117","C220181610_PKR_Null_2":"537857","C220181610_PKR_Null_3":"883588","C220181610_PKR_Null_4":"225594","C220181610_PKR_Null_5":"418525","C220181016_QC_1":"327398","C220181016_QC_2":"235518","C220181016_QC_3":"211407","C220181016_QC_4":"251059","C220181610_WT_1":"266941","C220181610_WT_2":"208485","C220181610_WT_3":"380240","C220181610_WT_4":"49534","C220181610_WT_5":"293204"},{"Metabolite":"(S)-Malate","C220181016_Blank_1":"88080","C220181016_Blank_2":"82166","C220181016_Blank_3":"83297","C220181016_Blank_4":"73815","C220181610_Extr_blank_1":"94800","C220181610_PKR_Null_1":"45904124","C220181610_PKR_Null_2":"41073364","C220181610_PKR_Null_3":"38346972","C220181610_PKR_Null_4":"24781594","C220181610_PKR_Null_5":"25842358","C220181016_QC_1":"26495118","C220181016_QC_2":"25987750","C220181016_QC_3":"20471230","C220181016_QC_4":"26104038","C220181610_WT_1":"26319662","C220181610_WT_2":"32468070","C220181610_WT_3":"16424074","C220181610_WT_4":"10735779","C220181610_WT_5":"17848764"},{"Metabolite":"[FA (24:0)] 15Z-tetracosenoic acid","C220181016_Blank_1":"0","C220181016_Blank_2":"0","C220181016_Blank_3":"0","C220181016_Blank_4":"0","C220181610_Extr_blank_1":"0","C220181610_PKR_Null_1":"886359","C220181610_PKR_Null_2":"1033543","C220181610_PKR_Null_3":"1108225","C220181610_PKR_Null_4":"905945","C220181610_PKR_Null_5":"901761","C220181016_QC_1":"2050945","C220181016_QC_2":"828927","C220181016_QC_3":"675546","C220181016_QC_4":"956331","C220181610_WT_1":"807347","C220181610_WT_2":"648879","C220181610_WT_3":"671811","C220181610_WT_4":"529580","C220181610_WT_5":"754343"},{"Metabolite":"[FA trihydroxy(4:0)] 2,3,4-trihydroxy-butanoic acid","C220181016_Blank_1":"34220","C220181016_Blank_2":"39982","C220181016_Blank_3":"31497","C220181016_Blank_4":"32561","C220181610_Extr_blank_1":"34454","C220181610_PKR_Null_1":"2736035","C220181610_PKR_Null_2":"4282617","C220181610_PKR_Null_3":"6088661","C220181610_PKR_Null_4":"1658601","C220181610_PKR_Null_5":"2562410","C220181016_QC_1":"2369027","C220181016_QC_2":"3513825","C220181016_QC_3":"2583939","C220181016_QC_4":"2490110","C220181610_WT_1":"2009431","C220181610_WT_2":"1872431","C220181610_WT_3":"3033398","C220181610_WT_4":"450403","C220181610_WT_5":"1558490"},{"Metabolite":"[PE(16:1)] 1-(1Z-hexadecenyl)-sn-glycero-3-phosphoethanolamine","C220181016_Blank_1":"0","C220181016_Blank_2":"0","C220181016_Blank_3":"0","C220181016_Blank_4":"0","C220181610_Extr_blank_1":"0","C220181610_PKR_Null_1":"351990","C220181610_PKR_Null_2":"298007","C220181610_PKR_Null_3":"387896","C220181610_PKR_Null_4":"283090","C220181610_PKR_Null_5":"460003","C220181016_QC_1":"0","C220181016_QC_2":"764706","C220181016_QC_3":"678093","C220181016_QC_4":"841764","C220181610_WT_1":"654218","C220181610_WT_2":"708067","C220181610_WT_3":"717308","C220181610_WT_4":"431236","C220181610_WT_5":"315780"},{"Metabolite":"[PR] Farnesyl pyrophosphate","C220181016_Blank_1":"0","C220181016_Blank_2":"0","C220181016_Blank_3":"0","C220181016_Blank_4":"0","C220181610_Extr_blank_1":"0","C220181610_PKR_Null_1":"0","C220181610_PKR_Null_2":"51077","C220181610_PKR_Null_3":"91727","C220181610_PKR_Null_4":"0","C220181610_PKR_Null_5":"0","C220181016_QC_1":"176562","C220181016_QC_2":"107422","C220181016_QC_3":"71400","C220181016_QC_4":"99371","C220181610_WT_1":"0","C220181610_WT_2":"0","C220181610_WT_3":"57177","C220181610_WT_4":"0","C220181610_WT_5":"0"},{"Metabolite":"[SP (16:0)] N-(hexadecanoyl)-sphing-4-enine","C220181016_Blank_1":"0","C220181016_Blank_2":"0","C220181016_Blank_3":"0","C220181016_Blank_4":"0","C220181610_Extr_blank_1":"0","C220181610_PKR_Null_1":"1096665","C220181610_PKR_Null_2":"1137684","C220181610_PKR_Null_3":"1070332","C220181610_PKR_Null_4":"1391512","C220181610_PKR_Null_5":"656678","C220181016_QC_1":"1159254","C220181016_QC_2":"1084501","C220181016_QC_3":"910673","C220181016_QC_4":"1115715","C220181610_WT_1":"1052228","C220181610_WT_2":"1201063","C220181610_WT_3":"1257003","C220181610_WT_4":"582852","C220181610_WT_5":"906379"},{"Metabolite":"[SP (24:0)] N-(tetracosanoyl)-sphing-4-enine-1-phosphate","C220181016_Blank_1":"0","C220181016_Blank_2":"0","C220181016_Blank_3":"0","C220181016_Blank_4":"0","C220181610_Extr_blank_1":"0","C220181610_PKR_Null_1":"675363","C220181610_PKR_Null_2":"553070","C220181610_PKR_Null_3":"430255","C220181610_PKR_Null_4":"651208","C220181610_PKR_Null_5":"686276","C220181016_QC_1":"0","C220181016_QC_2":"883477","C220181016_QC_3":"625614","C220181016_QC_4":"963372","C220181610_WT_1":"605176","C220181610_WT_2":"668029","C220181610_WT_3":"433689","C220181610_WT_4":"772514","C220181610_WT_5":"794074"},{"Metabolite":"[SP] Sphinganine-1-phosphate","C220181016_Blank_1":"0","C220181016_Blank_2":"0","C220181016_Blank_3":"0","C220181016_Blank_4":"0","C220181610_Extr_blank_1":"0","C220181610_PKR_Null_1":"91161","C220181610_PKR_Null_2":"265357","C220181610_PKR_Null_3":"450144","C220181610_PKR_Null_4":"128089","C220181610_PKR_Null_5":"241060","C220181016_QC_1":"20833","C220181016_QC_2":"84659","C220181016_QC_3":"106368","C220181016_QC_4":"127870","C220181610_WT_1":"0","C220181610_WT_2":"0","C220181610_WT_3":"0","C220181610_WT_4":"0","C220181610_WT_5":"0"},{"Metabolite":"[ST (12:0) homo-seco-cholestatrien-tetrol","C220181016_Blank_1":"324564","C220181016_Blank_2":"111043","C220181016_Blank_3":"259925","C220181016_Blank_4":"191755","C220181610_Extr_blank_1":"1803368","C220181610_PKR_Null_1":"1044865","C220181610_PKR_Null_2":"1285148","C220181610_PKR_Null_3":"1283964","C220181610_PKR_Null_4":"1613173","C220181610_PKR_Null_5":"1591464","C220181016_QC_1":"483055","C220181016_QC_2":"1477702","C220181016_QC_3":"1713907","C220181016_QC_4":"1738808","C220181610_WT_1":"1234672","C220181610_WT_2":"1417165","C220181610_WT_3":"1377385","C220181610_WT_4":"1833910","C220181610_WT_5":"1493148"},{"Metabolite":"[ST (12:0) 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}