{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST002809","ANALYSIS_ID":"AN004569","VERSION":"1","CREATED_ON":"August 7, 2023, 1:13 pm"},

"PROJECT":{"PROJECT_TITLE":"Role of cilia in mitochondrial function","PROJECT_SUMMARY":"Autosomal dominant polycystic kidney disease (ADPKD), the most common potentially lethal genetic disease in humans and the fourth leading cause of kidney disease, exhibits features of both a ciliary and metabolic disorder. Our previous research revealed that cells overexpressing Exoc5 with elongated cilia demonstrate enhanced recovery from oxidative stress. To investigate the connection between primary cilia and metabolism, we conducted an unbiased metabolomics screen. Global metabolic profiling was performed on canine MDCK cells (Control, Exoc5 ciliary targeting sequence mutation (CTS-mut), Exoc5 knockdown (KD), Exoc5 overexpression (OE)) and murine cells (Ift88 knockout (KO), Ift88 rescue). Knockdown (KD) or ciliary targeting sequence mutation (CTS-mut) in Exoc5, a central exocyst component, resulted in cilia loss. Similarly, Ift88 knockout (KO) resulted in cilia loss. For each experimental group, we cultivated six independent replicates of Exoc5 OE, KD, CTS-mut, and control MDCK cells, as well as six independent replicates of murine Ift88 KO and rescue cells. Cell pellets were obtained from the cultures, and we analyzed the global metabolic profiles for all 36 cell pellets. The most significant findings from the metabolomics screen indicated defects in tryptophan metabolism. This discovery suggests a potential link between primary cilia function and tryptophan-related metabolic pathways. Further exploration of these findings may shed light on the underlying mechanisms and implications for ADPKD pathogenesis and metabolic disturbances.","INSTITUTE":"Medical University of South Carolina","DEPARTMENT":"Medicine","LAST_NAME":"Lipschutz","FIRST_NAME":"Josh","ADDRESS":"96 Jonathan Lucas St, Charleston, SC 29425","EMAIL":"lipschut@musc.edu","PHONE":"843-792-7659"},

"STUDY":{"STUDY_TITLE":"Role of cilia in mitochondrial function","STUDY_TYPE":"cultured cells","STUDY_SUMMARY":"Autosomal dominant polycystic kidney disease (ADPKD), the most common potentially lethal genetic disease in humans and the fourth leading cause of kidney disease, exhibits features of both a ciliary and metabolic disorder. Our previous research revealed that cells overexpressing Exoc5 with elongated cilia demonstrate enhanced recovery from oxidative stress. To investigate the connection between primary cilia and metabolism, we conducted an unbiased metabolomics screen. Global metabolic profiling was performed on canine MDCK cells (Control, Exoc5 ciliary targeting sequence mutation (CTS-mut), Exoc5 knockdown (KD), Exoc5 overexpression (OE)) and murine cells (Ift88 knockout (KO), Ift88 rescue). Knockdown (KD) or ciliary targeting sequence mutation (CTS-mut) in Exoc5, a central exocyst component, resulted in cilia loss. Similarly, Ift88 knockout (KO) resulted in cilia loss. For each experimental group, we cultivated six independent replicates of Exoc5 OE, KD, CTS-mut, and control MDCK cells, as well as six independent replicates of murine Ift88 KO and rescue cells. Cell pellets were obtained from the cultures, and we analyzed the global metabolic profiles for all 36 cell pellets. The most significant findings from the metabolomics screen indicated defects in tryptophan metabolism. This discovery suggests a potential link between primary cilia function and tryptophan-related metabolic pathways. Further exploration of these findings may shed light on the underlying mechanisms and implications for ADPKD pathogenesis and metabolic disturbances.","INSTITUTE":"Medical University of South Carolina","DEPARTMENT":"Medicine","LAST_NAME":"Lipschutz","FIRST_NAME":"Josh","ADDRESS":"96 Jonathan Lucas St, Charleston, SC 29425","EMAIL":"lipschut@musc.edu","PHONE":"8437927659"},

"SUBJECT":{"SUBJECT_TYPE":"Mammal","SUBJECT_SPECIES":"Mouse/canine","TAXONOMY_ID":"9606"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"MUSC-00277",
"Factors":{"Genotype":"EXOC5_KO"},
"Additional sample data":{"Description":"Exoc5 ciliary targeting sequence point mutant MDCK cells"}
},
{
"Subject ID":"-",
"Sample ID":"MUSC-00278",
"Factors":{"Genotype":"EXOC5_KO"},
"Additional sample data":{"Description":"Exoc5 ciliary targeting sequence point mutant MDCK cells"}
},
{
"Subject ID":"-",
"Sample ID":"MUSC-00279",
"Factors":{"Genotype":"EXOC5_KO"},
"Additional sample data":{"Description":"Exoc5 ciliary targeting sequence point mutant MDCK cells"}
},
{
"Subject ID":"-",
"Sample ID":"MUSC-00280",
"Factors":{"Genotype":"EXOC5_KO"},
"Additional sample data":{"Description":"Exoc5 ciliary targeting sequence point mutant MDCK cells"}
},
{
"Subject ID":"-",
"Sample ID":"MUSC-00281",
"Factors":{"Genotype":"EXOC5_KO"},
"Additional sample data":{"Description":"Exoc5 ciliary targeting sequence point mutant MDCK cells"}
},
{
"Subject ID":"-",
"Sample ID":"MUSC-00282",
"Factors":{"Genotype":"EXOC5_KO"},
"Additional sample data":{"Description":"Exoc5 ciliary targeting sequence point mutant MDCK cells"}
},
{
"Subject ID":"-",
"Sample ID":"MUSC-00283",
"Factors":{"Genotype":"EXOC5_KD"},
"Additional sample data":{"Description":"Exoc5 knockdown MDCK cells"}
},
{
"Subject ID":"-",
"Sample ID":"MUSC-00284",
"Factors":{"Genotype":"EXOC5_KD"},
"Additional sample data":{"Description":"Exoc5 knockdown MDCK cells"}
},
{
"Subject ID":"-",
"Sample ID":"MUSC-00285",
"Factors":{"Genotype":"EXOC5_KD"},
"Additional sample data":{"Description":"Exoc5 knockdown MDCK cells"}
},
{
"Subject ID":"-",
"Sample ID":"MUSC-00286",
"Factors":{"Genotype":"EXOC5_KD"},
"Additional sample data":{"Description":"Exoc5 knockdown MDCK cells"}
},
{
"Subject ID":"-",
"Sample ID":"MUSC-00287",
"Factors":{"Genotype":"EXOC5_KD"},
"Additional sample data":{"Description":"Exoc5 knockdown MDCK cells"}
},
{
"Subject ID":"-",
"Sample ID":"MUSC-00288",
"Factors":{"Genotype":"EXOC5_KD"},
"Additional sample data":{"Description":"Exoc5 knockdown MDCK cells"}
},
{
"Subject ID":"-",
"Sample ID":"MUSC-00289",
"Factors":{"Genotype":"EXOC5_OE"},
"Additional sample data":{"Description":"Exoc5 overexpressing MDCK cells"}
},
{
"Subject ID":"-",
"Sample ID":"MUSC-00290",
"Factors":{"Genotype":"EXOC5_OE"},
"Additional sample data":{"Description":"Exoc5 overexpressing MDCK cells"}
},
{
"Subject ID":"-",
"Sample ID":"MUSC-00291",
"Factors":{"Genotype":"EXOC5_OE"},
"Additional sample data":{"Description":"Exoc5 overexpressing MDCK cells"}
},
{
"Subject ID":"-",
"Sample ID":"MUSC-00292",
"Factors":{"Genotype":"EXOC5_OE"},
"Additional sample data":{"Description":"Exoc5 overexpressing MDCK cells"}
},
{
"Subject ID":"-",
"Sample ID":"MUSC-00293",
"Factors":{"Genotype":"EXOC5_OE"},
"Additional sample data":{"Description":"Exoc5 overexpressing MDCK cells"}
},
{
"Subject ID":"-",
"Sample ID":"MUSC-00294",
"Factors":{"Genotype":"EXOC5_OE"},
"Additional sample data":{"Description":"Exoc5 overexpressing MDCK cells"}
},
{
"Subject ID":"-",
"Sample ID":"MUSC-00295",
"Factors":{"Genotype":"WT"},
"Additional sample data":{"Description":"MDCK wild-type control cells"}
},
{
"Subject ID":"-",
"Sample ID":"MUSC-00296",
"Factors":{"Genotype":"WT"},
"Additional sample data":{"Description":"MDCK wild-type control cells"}
},
{
"Subject ID":"-",
"Sample ID":"MUSC-00297",
"Factors":{"Genotype":"WT"},
"Additional sample data":{"Description":"MDCK wild-type control cells"}
},
{
"Subject ID":"-",
"Sample ID":"MUSC-00298",
"Factors":{"Genotype":"WT"},
"Additional sample data":{"Description":"MDCK wild-type control cells"}
},
{
"Subject ID":"-",
"Sample ID":"MUSC-00299",
"Factors":{"Genotype":"WT"},
"Additional sample data":{"Description":"MDCK wild-type control cells"}
},
{
"Subject ID":"-",
"Sample ID":"MUSC-00300",
"Factors":{"Genotype":"WT"},
"Additional sample data":{"Description":"MDCK wild-type control cells"}
},
{
"Subject ID":"-",
"Sample ID":"MUSC-00301",
"Factors":{"Genotype":"IFT88_KO"},
"Additional sample data":{"Description":"Intraflagellar transport protein 88 (Ift88) null mouse cells"}
},
{
"Subject ID":"-",
"Sample ID":"MUSC-00302",
"Factors":{"Genotype":"IFT88_KO"},
"Additional sample data":{"Description":"Intraflagellar transport protein 88 (Ift88) null mouse cells"}
},
{
"Subject ID":"-",
"Sample ID":"MUSC-00303",
"Factors":{"Genotype":"IFT88_KO"},
"Additional sample data":{"Description":"Intraflagellar transport protein 88 (Ift88) null mouse cells"}
},
{
"Subject ID":"-",
"Sample ID":"MUSC-00304",
"Factors":{"Genotype":"IFT88_KO"},
"Additional sample data":{"Description":"Intraflagellar transport protein 88 (Ift88) null mouse cells"}
},
{
"Subject ID":"-",
"Sample ID":"MUSC-00305",
"Factors":{"Genotype":"IFT88_KO"},
"Additional sample data":{"Description":"Intraflagellar transport protein 88 (Ift88) null mouse cells"}
},
{
"Subject ID":"-",
"Sample ID":"MUSC-00306",
"Factors":{"Genotype":"IFT88_KO"},
"Additional sample data":{"Description":"Intraflagellar transport protein 88 (Ift88) null mouse cells"}
},
{
"Subject ID":"-",
"Sample ID":"MUSC-00307",
"Factors":{"Genotype":"IFT88_KO_WT"},
"Additional sample data":{"Description":"Ift88 rescue mouse cells"}
},
{
"Subject ID":"-",
"Sample ID":"MUSC-00308",
"Factors":{"Genotype":"IFT88_KO_WT"},
"Additional sample data":{"Description":"Ift88 rescue mouse cells"}
},
{
"Subject ID":"-",
"Sample ID":"MUSC-00309",
"Factors":{"Genotype":"IFT88_KO_WT"},
"Additional sample data":{"Description":"Ift88 rescue mouse cells"}
},
{
"Subject ID":"-",
"Sample ID":"MUSC-00310",
"Factors":{"Genotype":"IFT88_KO_WT"},
"Additional sample data":{"Description":"Ift88 rescue mouse cells"}
},
{
"Subject ID":"-",
"Sample ID":"MUSC-00311",
"Factors":{"Genotype":"IFT88_KO_WT"},
"Additional sample data":{"Description":"Ift88 rescue mouse cells"}
},
{
"Subject ID":"-",
"Sample ID":"MUSC-00312",
"Factors":{"Genotype":"IFT88_KO_WT"},
"Additional sample data":{"Description":"Ift88 rescue mouse cells"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"Six independent replicates of Exoc5 OE, KD, CTS-mut, and control MDCK cells were grown in cell culture in 15 cm dishes. The cells were then washed in PBS, scraped, and centrifuged to a pellet and snap frozen in liquid nitrogen. Similarly six independent replicates of murine Ift88 KO and rescue cells were grown in cell culture, washed with PBS, scraped, centrifuged to a pellet and snap frozen in liquid nitrogen.","SAMPLE_TYPE":"Cultured kidney cells"},

"TREATMENT":{"TREATMENT_SUMMARY":"No treatment"},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Samples were thawed on ice prior to extraction. Samples were prepared using the automated MicroLab STAR® system from Hamilton Company. Several recovery standards were added prior to the first step in the extraction process for QC purposes. In order to dissociate small molecules bound to or trapped in proteins, lysate was precipitated with methanol under vigorous shaking for 2 min (Glen Mills GenoGrinder 2000) followed by centrifugation. The resulting extract was divided into multiple fractions: two for analysis by two separate reverse phase (RP)/UPLC-MS/MS methods with positive ion mode electrospray ionization (ESI), one for analysis by RP/UPLC-MS/MS with negative ion mode ESI, one for analysis by HILIC/UPLC-MS/MS with negative ion mode ESI, and remaining fractions reserved for backup. Samples were dried under warm nitrogen to remove the organic solvent. The sample extracts were stored sealed at -80oC if not analyzed immediately."},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_TYPE":"HILIC","INSTRUMENT_NAME":"Waters Acquity","COLUMN_NAME":"Waters Acquity BEH Amide (150 x 2.1mm, 1.7um)","SOLVENT_A":"10 mM ammonium formate in 15% water/ 5% methanol/ 80% acetonitrile (effective pH 10.16 with NH4OH)","SOLVENT_B":"10 mM ammonium formate in 50% water/ 50% acetonitrile (effective pH 10.60 with NH4OH)","FLOW_GRADIENT":"Linear gradient from 5% B to 50% B in 3.5 minutes, then linear gradient from 50% B to 95% B in 2 minutes minutes.","FLOW_RATE":"0.50 mL/min","COLUMN_TEMPERATURE":"65"},

"ANALYSIS":{"ANALYSIS_TYPE":"MS"},

"MS":{"INSTRUMENT_NAME":"Thermo Q Exactive Orbitrap","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","ION_MODE":"NEGATIVE","MS_COMMENTS":"Proprietary analytical software for integration and peak picking (Metabolon). Normalized raw area counts for each sample are normalized by Bradford protein concentration. E.ach metabolite is then rescaled to set the median equal to 1. Lastly, missing values are imputed with the minimum."},

"MS_METABOLITE_DATA":{
"Units":"Normalized/scaled raw area counts",

"Data":[{"Metabolite":"1,2-dioleoyl-GPG (18:1/18:1)","MUSC-00277":"","MUSC-00278":"","MUSC-00279":"","MUSC-00280":"","MUSC-00281":"","MUSC-00282":"","MUSC-00283":"","MUSC-00284":"","MUSC-00285":"","MUSC-00286":"","MUSC-00287":"","MUSC-00288":"","MUSC-00289":"","MUSC-00290":"","MUSC-00291":"","MUSC-00292":"","MUSC-00293":"","MUSC-00294":"","MUSC-00295":"","MUSC-00296":"","MUSC-00297":"","MUSC-00298":"","MUSC-00299":"","MUSC-00300":"","MUSC-00301":"0.5964","MUSC-00302":"0.4724","MUSC-00303":"0.5492","MUSC-00304":"0.5031","MUSC-00305":"0.4494","MUSC-00306":"0.6117","MUSC-00307":"1.4924","MUSC-00308":"1.5026","MUSC-00309":"1.5756","MUSC-00310":"1.3883","MUSC-00311":"1.7092","MUSC-00312":"1.7671"},{"Metabolite":"1,2-dioleoyl-GPI (18:1/18:1)","MUSC-00277":"","MUSC-00278":"","MUSC-00279":"","MUSC-00280":"","MUSC-00281":"","MUSC-00282":"","MUSC-00283":"","MUSC-00284":"","MUSC-00285":"","MUSC-00286":"","MUSC-00287":"","MUSC-00288":"","MUSC-00289":"","MUSC-00290":"","MUSC-00291":"","MUSC-00292":"","MUSC-00293":"","MUSC-00294":"","MUSC-00295":"","MUSC-00296":"","MUSC-00297":"","MUSC-00298":"","MUSC-00299":"","MUSC-00300":"","MUSC-00301":"0.9031","MUSC-00302":"0.7514","MUSC-00303":"0.8236","MUSC-00304":"0.73","MUSC-00305":"0.8116","MUSC-00306":"0.9977","MUSC-00307":"1.0087","MUSC-00308":"1.0038","MUSC-00309":"1.0023","MUSC-00310":"1.0232","MUSC-00311":"1.2841","MUSC-00312":"1.4921"},{"Metabolite":"1-oleoyl-GPC (18:1)","MUSC-00277":"0.9182","MUSC-00278":"0.8464","MUSC-00279":"0.6544","MUSC-00280":"0.6099","MUSC-00281":"0.584","MUSC-00282":"0.8764","MUSC-00283":"0.8552","MUSC-00284":"0.9434","MUSC-00285":"0.8103","MUSC-00286":"0.8595","MUSC-00287":"1","MUSC-00288":"0.8824","MUSC-00289":"1.5841","MUSC-00290":"1.7934","MUSC-00291":"1.2937","MUSC-00292":"1.553","MUSC-00293":"1.7524","MUSC-00294":"1.6998","MUSC-00295":"1.1017","MUSC-00296":"1.5977","MUSC-00297":"1.6289","MUSC-00298":"1.7865","MUSC-00299":"0.584","MUSC-00300":"1.2548","MUSC-00301":"1.2191","MUSC-00302":"0.8928","MUSC-00303":"1.0557","MUSC-00304":"0.9853","MUSC-00305":"0.8642","MUSC-00306":"1.299","MUSC-00307":"1.0147","MUSC-00308":"0.4025","MUSC-00309":"0.9242","MUSC-00310":"0.748","MUSC-00311":"1.2198","MUSC-00312":"1.2354"},{"Metabolite":"1-oleoyl-GPE (18:1)","MUSC-00277":"1","MUSC-00278":"0.9933","MUSC-00279":"0.7882","MUSC-00280":"0.8162","MUSC-00281":"0.7595","MUSC-00282":"0.9562","MUSC-00283":"0.8699","MUSC-00284":"0.9181","MUSC-00285":"0.8371","MUSC-00286":"0.8335","MUSC-00287":"1.0003","MUSC-00288":"0.8833","MUSC-00289":"1.0287","MUSC-00290":"1.1481","MUSC-00291":"0.8562","MUSC-00292":"1","MUSC-00293":"1.1546","MUSC-00294":"1.0913","MUSC-00295":"1.1451","MUSC-00296":"1.5116","MUSC-00297":"1.4848","MUSC-00298":"1.6209","MUSC-00299":"1.0645","MUSC-00300":"1.2225","MUSC-00301":"2.2092","MUSC-00302":"1.314","MUSC-00303":"0.5769","MUSC-00304":"1.0758","MUSC-00305":"0.7207","MUSC-00306":"1.2944","MUSC-00307":"0.3008","MUSC-00308":"0.9242","MUSC-00309":"1.8423","MUSC-00310":"0.8534","MUSC-00311":"2.1765","MUSC-00312":"0.8652"},{"Metabolite":"1-oleoyl-GPI (18:1)","MUSC-00277":"0.657","MUSC-00278":"0.4279","MUSC-00279":"0.4726","MUSC-00280":"0.3759","MUSC-00281":"0.4543","MUSC-00282":"0.6042","MUSC-00283":"1.0029","MUSC-00284":"0.8486","MUSC-00285":"1.8242","MUSC-00286":"0.9455","MUSC-00287":"1.2885","MUSC-00288":"0.7977","MUSC-00289":"1.8044","MUSC-00290":"1.5134","MUSC-00291":"1.7689","MUSC-00292":"1.7344","MUSC-00293":"1.6039","MUSC-00294":"1.5665","MUSC-00295":"0.8996","MUSC-00296":"0.9971","MUSC-00297":"1.3632","MUSC-00298":"1.544","MUSC-00299":"1.0087","MUSC-00300":"0.9247","MUSC-00301":"2.2713","MUSC-00302":"0.9455","MUSC-00303":"0.4609","MUSC-00304":"0.788","MUSC-00305":"0.4813","MUSC-00306":"0.9109","MUSC-00307":"0.322","MUSC-00308":"1.7436","MUSC-00309":"2.1335","MUSC-00310":"1.0545","MUSC-00311":"2.5097","MUSC-00312":"1.1412"},{"Metabolite":"1-palmitoleoyl-GPC* 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}

}