{
"METABOLOMICS WORKBENCH":{"STUDY_ID":"ST002791","ANALYSIS_ID":"AN004541","VERSION":"1","CREATED_ON":"July 24, 2023, 6:22 am"},

"PROJECT":{"PROJECT_TITLE":"Nucleotide metabolism in pancreatic cancer cells","PROJECT_SUMMARY":"The Experiment analyzes the cancer cell metabolism in two pancreatic cancer cell lines, (i.e. Panc02 and KPC FC 1245) after the knockdown of the protein cytidine deaminase (CDA). Murine Panc02 and KPC FC1245 pancreatic cancer cell lines were genetically engineered using a doxycycline inducible CRISPR/Cas9 platform with a target specific gRNA for CDA and a control non-targeting NT gRNA. CDA knockdown cells show a significant decrease of intracellular uridine levels and the accumulation of intracellular cytidine. Consistent with the decrease in intracellular uridine, sgCda cells show reduced intracellular levels of UMP, UDP and UTP compared to sgNT cells while we see no change in adenine and cytosine nucleotides (i.e., AMP, ADP, ATP and CMP, CDP, CTP, respectively) or in UDP-hexose.","INSTITUTE":"VIB Center for Cancer Biology","LAST_NAME":"Mazzone","FIRST_NAME":"Massimiliano","ADDRESS":"Herestraat 49, box 912, Leuven, Flemish Brabant, 3000, Belgium","EMAIL":"massimiliano.mazzone@kuleuven.be","PHONE":"+32-16-37.32.13"},

"STUDY":{"STUDY_TITLE":"Nucleotide metabolism in pancreatic cancer cells","STUDY_SUMMARY":"The Experiment analyzes the cancer cell metabolism in two pancreatic cancer cell lines, (i.e. Panc02 and KPC FC 1245) after the knockdown of the protein cytidine deaminase (CDA). Murine Panc02 and KPC FC1245 pancreatic cancer cell lines were genetically engineered using a doxycycline inducible CRISPR/Cas9 platform with a target specific gRNA for CDA and a control non-targeting NT gRNA. CDA knockdown cells show a significant decrease of intracellular uridine levels and the accumulation of intracellular cytidine. Consistent with the decrease in intracellular uridine, sgCda cells show reduced intracellular levels of UMP, UDP and UTP compared to sgNT cells while we see no change in adenine and cytosine nucleotides (i.e., AMP, ADP, ATP and CMP, CDP, CTP, respectively) or in UDP-hexose.","INSTITUTE":"VIB-KU Leuven","LAST_NAME":"Mazzone","FIRST_NAME":"Massimiliano","ADDRESS":"Campus Gasthuisberg Herestraat 49, box 912 B-3000 Leuven Belgium","EMAIL":"massimiliano.mazzone@kuleuven.vib.be","PHONE":"+32-16-37.32.13"},

"SUBJECT":{"SUBJECT_TYPE":"Cultured cells","SUBJECT_SPECIES":"Mus musculus","TAXONOMY_ID":"10090","GENDER":"Not applicable"},
"SUBJECT_SAMPLE_FACTORS":[
{
"Subject ID":"-",
"Sample ID":"KPC_NT_1",
"Factors":{"Genotype":"sgNT","Cell Line":"KPC_FC1245"},
"Additional sample data":{"Protein_BCA":"3,852830189","RAW_FILE_NAME":"MCF000545_ST91.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"KPC_NT_2",
"Factors":{"Genotype":"sgNT","Cell Line":"KPC_FC1245"},
"Additional sample data":{"Protein_BCA":"3,725471698","RAW_FILE_NAME":"MCF000545_ST92.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"KPC_NT_3",
"Factors":{"Genotype":"sgNT","Cell Line":"KPC_FC1245"},
"Additional sample data":{"Protein_BCA":"4,10990566","RAW_FILE_NAME":"MCF000545_ST93.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"KPC_NT_4",
"Factors":{"Genotype":"sgNT","Cell Line":"KPC_FC1245"},
"Additional sample data":{"Protein_BCA":"3,421226415","RAW_FILE_NAME":"MCF000545_ST94.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"KPC_CDAKD_1",
"Factors":{"Genotype":"sgCDA","Cell Line":"KPC_FC1245"},
"Additional sample data":{"Protein_BCA":"3,980188679","RAW_FILE_NAME":"MCF000545_ST99.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"KPC_CDAKD_2",
"Factors":{"Genotype":"sgCDA","Cell Line":"KPC_FC1245"},
"Additional sample data":{"Protein_BCA":"4,175943396","RAW_FILE_NAME":"MCF000545_ST100.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"KPC_CDAKD_3",
"Factors":{"Genotype":"sgCDA","Cell Line":"KPC_FC1245"},
"Additional sample data":{"Protein_BCA":"3,664150943","RAW_FILE_NAME":"MCF000545_ST101.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"KPC_CDAKD_4",
"Factors":{"Genotype":"sgCDA","Cell Line":"KPC_FC1245"},
"Additional sample data":{"Protein_BCA":"3,878773585","RAW_FILE_NAME":"MCF000545_ST102.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"Panc02_CDAKD_1",
"Factors":{"Genotype":"sgCDA","Cell Line":"Panc02"},
"Additional sample data":{"Protein_BCA":"531,29","RAW_FILE_NAME":"MCF000215_SR01.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"Panc02_CDAKD_2",
"Factors":{"Genotype":"sgCDA","Cell Line":"Panc02"},
"Additional sample data":{"Protein_BCA":"591,876","RAW_FILE_NAME":"MCF000215_SR02.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"Panc02_CDAKD_3",
"Factors":{"Genotype":"sgCDA","Cell Line":"Panc02"},
"Additional sample data":{"Protein_BCA":"572,783","RAW_FILE_NAME":"MCF000215_SR03.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"Panc02_CDAKD_4",
"Factors":{"Genotype":"sgCDA","Cell Line":"Panc02"},
"Additional sample data":{"Protein_BCA":"588,8695","RAW_FILE_NAME":"MCF000215_SR04.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"Panc02_NT_1",
"Factors":{"Genotype":"sgNT","Cell Line":"Panc02"},
"Additional sample data":{"Protein_BCA":"532,9435","RAW_FILE_NAME":"MCF000215_SR13.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"Panc02_NT_2",
"Factors":{"Genotype":"sgNT","Cell Line":"Panc02"},
"Additional sample data":{"Protein_BCA":"583,156","RAW_FILE_NAME":"MCF000215_SR14.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"Panc02_NT_3",
"Factors":{"Genotype":"sgNT","Cell Line":"Panc02"},
"Additional sample data":{"Protein_BCA":"545,422","RAW_FILE_NAME":"MCF000215_SR15.mzML"}
},
{
"Subject ID":"-",
"Sample ID":"Panc02_NT_4",
"Factors":{"Genotype":"sgNT","Cell Line":"Panc02"},
"Additional sample data":{"Protein_BCA":"579,248","RAW_FILE_NAME":"MCF000215_SR16.mzML"}
}
],
"COLLECTION":{"COLLECTION_SUMMARY":"On day 2, cells were harvested on ice by washing them once in ice-cold saline solution (9 g/l NaCl). Cells were covered with 250 μl of pre-cooled 80% methanol for 2 minutes scraped, transferred to fresh vials, and precipitated overnight at -80°C. Then they were centrifuged at 20.009 g for 15min, 4 degrees C and supernatant transferred to an M/S vial.","SAMPLE_TYPE":"Cultured cells","STORAGE_CONDITIONS":"-80℃"},

"TREATMENT":{"TREATMENT_SUMMARY":"Cancer cells were cultured in DMEM medium supplemented with 10% FBS and 1% Pen/Strep in 6-well plates. On day 1, the cells and empty wells (for background noise control), were washed with PBS, and replenished with DMEM containing 10% FBS, 1% Pen/Strep, 5.5 mM glucose, 2 mM glutamine, 1% Pen/Strep, 0.1 mM cytidine (Sigma-Aldrich), or alternately, 0.1 mM labelled cytidine (13C9, 98% 15N3, 96-98%, Eurisotop) for 48h."},

"SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"The samples were centrifuged at 20.000 x g for 15 min at 4°C, supernatant was transferred to appropriate M/S vial for analysis. The cell pellet was dissolved in 100 μl of 200 mM NaOH (for 20 minutes at 95°C), and the protein concentration was determined by using bicinchoninic acid (BCA) reagent method.","PROCESSING_STORAGE_CONDITIONS":"-80℃","EXTRACT_STORAGE":"-80℃"},

"CHROMATOGRAPHY":{"CHROMATOGRAPHY_SUMMARY":"C18 IP REVERSE PHASE","CHROMATOGRAPHY_TYPE":"Reversed phase","INSTRUMENT_NAME":"Waters Acquity","COLUMN_NAME":"Waters Acquity UPLC HSS T3 (150 x 2.1mm, 1.8um)","SOLVENT_A":"100% water; 10mM tributylamine; 15mM acetic acid","SOLVENT_B":"100% methanol","FLOW_GRADIENT":"The gradient started with 5% solvent B and 95% solvent A and remained at 5% B until 2min post injection. A linear gradient to 37% B was carried out until 7min and increased to 41% until 14min. Between 14 and 26 minutes the gradient increased to 95% of B and remained at 95% B for 4 minutes. At 30 min the gradient returned to 5% B. The chromatography was stopped at 40 min.","FLOW_RATE":"0.25 mL/min","COLUMN_TEMPERATURE":"40"},

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

"MS":{"INSTRUMENT_NAME":"Thermo Q Exactive Orbitrap","INSTRUMENT_TYPE":"Orbitrap","MS_TYPE":"ESI","ION_MODE":"NEGATIVE","MS_COMMENTS":"EI-Maven polly, ThermoFisher Xcalibur, Metabolites were annotated using the inhouse standard metabolite library- elution time and m/z values."},

"MS_METABOLITE_DATA":{
"Units":"AUC",

"Data":[{"Metabolite":"Cytidine","KPC_NT_1":"28223402","KPC_NT_2":"3174356","KPC_NT_3":"13974296","KPC_NT_4":"31187797","KPC_CDAKD_1":"164148316","KPC_CDAKD_2":"61203529","KPC_CDAKD_3":"63084877","KPC_CDAKD_4":"980534","Panc02_CDAKD_1":"1305310162","Panc02_CDAKD_2":"8926774274","Panc02_CDAKD_3":"8648320948","Panc02_CDAKD_4":"1274710013","Panc02_NT_1":"3498402947","Panc02_NT_2":"3955412221","Panc02_NT_3":"2381821314","Panc02_NT_4":"2632888878"},{"Metabolite":"UDP","KPC_NT_1":"108991256","KPC_NT_2":"137762094","KPC_NT_3":"115042954","KPC_NT_4":"132107886","KPC_CDAKD_1":"8567421","KPC_CDAKD_2":"61932077","KPC_CDAKD_3":"66842732","KPC_CDAKD_4":"68226758","Panc02_CDAKD_1":"2193978993","Panc02_CDAKD_2":"1743644098","Panc02_CDAKD_3":"1824406797","Panc02_CDAKD_4":"1850506516","Panc02_NT_1":"2315864644","Panc02_NT_2":"2631132415","Panc02_NT_3":"2437428576","Panc02_NT_4":"287336616"},{"Metabolite":"UMP","KPC_NT_1":"17417638","KPC_NT_2":"2477793","KPC_NT_3":"14165911","KPC_NT_4":"21819336","KPC_CDAKD_1":"10551369","KPC_CDAKD_2":"649234","KPC_CDAKD_3":"7305842","KPC_CDAKD_4":"5885353","Panc02_CDAKD_1":"3135728","Panc02_CDAKD_2":"2601483191","Panc02_CDAKD_3":"2887550602","Panc02_CDAKD_4":"306684733","Panc02_NT_1":"2786814761","Panc02_NT_2":"2982213233","Panc02_NT_3":"2948325387","Panc02_NT_4":"2904765167"},{"Metabolite":"Uridine","KPC_NT_1":"24998308","KPC_NT_2":"21073763","KPC_NT_3":"15980564","KPC_NT_4":"21611477","KPC_CDAKD_1":"137563","KPC_CDAKD_2":"5642838","KPC_CDAKD_3":"5292695","KPC_CDAKD_4":"8223912","Panc02_CDAKD_1":"1657144298","Panc02_CDAKD_2":"1808722227","Panc02_CDAKD_3":"2112223411","Panc02_CDAKD_4":"2924058647","Panc02_NT_1":"6068804178","Panc02_NT_2":"8685912619","Panc02_NT_3":"5299654001","Panc02_NT_4":"6124843779"},{"Metabolite":"UTP","KPC_NT_1":"198402728","KPC_NT_2":"254619974","KPC_NT_3":"279115095","KPC_NT_4":"203107877","KPC_CDAKD_1":"15522391","KPC_CDAKD_2":"156186558","KPC_CDAKD_3":"178140117","KPC_CDAKD_4":"185742304","Panc02_CDAKD_1":"5315675913","Panc02_CDAKD_2":"4080802251","Panc02_CDAKD_3":"4203030526","Panc02_CDAKD_4":"4182507181","Panc02_NT_1":"5437949985","Panc02_NT_2":"6478508159","Panc02_NT_3":"6423958662","Panc02_NT_4":"7361952705"},{"Metabolite":"CDP","KPC_NT_1":"1122614","KPC_NT_2":"1327743","KPC_NT_3":"102451","KPC_NT_4":"1173586","KPC_CDAKD_1":"2345728","KPC_CDAKD_2":"2240025","KPC_CDAKD_3":"2058941","KPC_CDAKD_4":"1981533","Panc02_CDAKD_1":"1279400955","Panc02_CDAKD_2":"1031831161","Panc02_CDAKD_3":"1081047069","Panc02_CDAKD_4":"109651369","Panc02_NT_1":"1067314806","Panc02_NT_2":"116168165","Panc02_NT_3":"1086515814","Panc02_NT_4":"1241615994"},{"Metabolite":"CMP","KPC_NT_1":"502818","KPC_NT_2":"1067148","KPC_NT_3":"540898","KPC_NT_4":"1189809","KPC_CDAKD_1":"1299076","KPC_CDAKD_2":"696441","KPC_CDAKD_3":"586268","KPC_CDAKD_4":"590406","Panc02_CDAKD_1":"1147917069","Panc02_CDAKD_2":"1072233125","Panc02_CDAKD_3":"1133450625","Panc02_CDAKD_4":"1114038746","Panc02_NT_1":"1100599525","Panc02_NT_2":"10944388","Panc02_NT_3":"1052572234","Panc02_NT_4":"1114117923"},{"Metabolite":"CTP","KPC_NT_1":"44755367","KPC_NT_2":"57108646","KPC_NT_3":"60025299","KPC_NT_4":"47160202","KPC_CDAKD_1":"44308309","KPC_CDAKD_2":"45344671","KPC_CDAKD_3":"52586144","KPC_CDAKD_4":"57023085","Panc02_CDAKD_1":"2629652516","Panc02_CDAKD_2":"196619333","Panc02_CDAKD_3":"2040199073","Panc02_CDAKD_4":"2043945541","Panc02_NT_1":"1975033969","Panc02_NT_2":"2215110879","Panc02_NT_3":"2267599573","Panc02_NT_4":"2599611926"},{"Metabolite":"UDP-hexose","KPC_NT_1":"172424796","KPC_NT_2":"199789986","KPC_NT_3":"2655594","KPC_NT_4":"144894659","KPC_CDAKD_1":"80128214","KPC_CDAKD_2":"126526451","KPC_CDAKD_3":"182859265","KPC_CDAKD_4":"135525472","Panc02_CDAKD_1":"4025839579","Panc02_CDAKD_2":"3292239685","Panc02_CDAKD_3":"3577016767","Panc02_CDAKD_4":"3888648917","Panc02_NT_1":"4037093707","Panc02_NT_2":"4521845487","Panc02_NT_3":"4273355015","Panc02_NT_4":"5079009906"},{"Metabolite":"ADP","KPC_NT_1":"4063438688","KPC_NT_2":"4490458710","KPC_NT_3":"3554353544","KPC_NT_4":"4571710669","KPC_CDAKD_1":"4275366446","KPC_CDAKD_2":"3822977105","KPC_CDAKD_3":"3333929188","KPC_CDAKD_4":"3190020937","Panc02_CDAKD_1":"3237275601","Panc02_CDAKD_2":"2572226623","Panc02_CDAKD_3":"2636650174","Panc02_CDAKD_4":"2711604087","Panc02_NT_1":"3175975432","Panc02_NT_2":"3446659929","Panc02_NT_3":"3318696821","Panc02_NT_4":"3797237082"},{"Metabolite":"AMP","KPC_NT_1":"4126056968","KPC_NT_2":"4536124086","KPC_NT_3":"3667779048","KPC_NT_4":"4541566603","KPC_CDAKD_1":"4163800377","KPC_CDAKD_2":"3654802828","KPC_CDAKD_3":"3245063605","KPC_CDAKD_4":"296115205","Panc02_CDAKD_1":"3022757822","Panc02_CDAKD_2":"3019395455","Panc02_CDAKD_3":"2940922713","Panc02_CDAKD_4":"2999762706","Panc02_NT_1":"2823053063","Panc02_NT_2":"2884810581","Panc02_NT_3":"2885486626","Panc02_NT_4":"2543812554"},{"Metabolite":"ATP","KPC_NT_1":"63547267973","KPC_NT_2":"63724959198","KPC_NT_3":"65504286201","KPC_NT_4":"58995852037","KPC_CDAKD_1":"65311628614","KPC_CDAKD_2":"66968652034","KPC_CDAKD_3":"69939579849","KPC_CDAKD_4":"66161080272","Panc02_CDAKD_1":"1035164147","Panc02_CDAKD_2":"7895473992","Panc02_CDAKD_3":"798387881","Panc02_CDAKD_4":"8023780746","Panc02_NT_1":"9416174972","Panc02_NT_2":"1041805351","Panc02_NT_3":"1083269526","Panc02_NT_4":"122973388"}],

"Metabolites":[{"Metabolite":"Cytidine","PubChem ID":"6175","KEGG ID":"C00475"},{"Metabolite":"Uridine","PubChem ID":"6029","KEGG ID":"C00299"},{"Metabolite":"UMP","PubChem ID":"6030","KEGG ID":"C00105"},{"Metabolite":"UDP","PubChem ID":"6031","KEGG ID":"C00015"},{"Metabolite":"UTP","PubChem ID":"6133","KEGG ID":"C00075"},{"Metabolite":"CMP","PubChem ID":"6131","KEGG ID":"C00055"},{"Metabolite":"CDP","PubChem ID":"6132","KEGG ID":"C00112"},{"Metabolite":"CTP","PubChem ID":"6175","KEGG ID":"C00063"},{"Metabolite":"UDP-hexose","PubChem ID":"8629","KEGG ID":"C00029"},{"Metabolite":"AMP","PubChem ID":"6083","KEGG ID":"C00020"},{"Metabolite":"ADP","PubChem ID":"6022","KEGG ID":"C00008"},{"Metabolite":"ATP","PubChem ID":"5957","KEGG ID":"C00002"}]
}

}