#METABOLOMICS WORKBENCH MaMa_20230716_065245 DATATRACK_ID:4162 STUDY_ID:ST002791 ANALYSIS_ID:AN004541 PROJECT_ID:PR001739 VERSION 1 CREATED_ON July 24, 2023, 6:22 am #PROJECT PR:PROJECT_TITLE Nucleotide metabolism in pancreatic cancer cells PR:PROJECT_SUMMARY The Experiment analyzes the cancer cell metabolism in two pancreatic cancer cell PR:PROJECT_SUMMARY lines, (i.e. Panc02 and KPC FC 1245) after the knockdown of the protein cytidine PR:PROJECT_SUMMARY deaminase (CDA). Murine Panc02 and KPC FC1245 pancreatic cancer cell lines were PR:PROJECT_SUMMARY genetically engineered using a doxycycline inducible CRISPR/Cas9 platform with a PR:PROJECT_SUMMARY target specific gRNA for CDA and a control non-targeting NT gRNA. CDA knockdown PR:PROJECT_SUMMARY cells show a significant decrease of intracellular uridine levels and the PR:PROJECT_SUMMARY accumulation of intracellular cytidine. Consistent with the decrease in PR:PROJECT_SUMMARY intracellular uridine, sgCda cells show reduced intracellular levels of UMP, UDP PR:PROJECT_SUMMARY and UTP compared to sgNT cells while we see no change in adenine and cytosine PR:PROJECT_SUMMARY nucleotides (i.e., AMP, ADP, ATP and CMP, CDP, CTP, respectively) or in PR:PROJECT_SUMMARY UDP-hexose. PR:INSTITUTE VIB Center for Cancer Biology PR:LAST_NAME Mazzone PR:FIRST_NAME Massimiliano PR:ADDRESS Herestraat 49, box 912, Leuven, Flemish Brabant, 3000, Belgium PR:EMAIL massimiliano.mazzone@kuleuven.be PR:PHONE +32-16-37.32.13 #STUDY ST:STUDY_TITLE Nucleotide metabolism in pancreatic cancer cells ST:STUDY_SUMMARY The Experiment analyzes the cancer cell metabolism in two pancreatic cancer cell ST:STUDY_SUMMARY lines, (i.e. Panc02 and KPC FC 1245) after the knockdown of the protein cytidine ST:STUDY_SUMMARY deaminase (CDA). Murine Panc02 and KPC FC1245 pancreatic cancer cell lines were ST:STUDY_SUMMARY genetically engineered using a doxycycline inducible CRISPR/Cas9 platform with a ST:STUDY_SUMMARY target specific gRNA for CDA and a control non-targeting NT gRNA. CDA knockdown ST:STUDY_SUMMARY cells show a significant decrease of intracellular uridine levels and the ST:STUDY_SUMMARY accumulation of intracellular cytidine. Consistent with the decrease in ST:STUDY_SUMMARY intracellular uridine, sgCda cells show reduced intracellular levels of UMP, UDP ST:STUDY_SUMMARY and UTP compared to sgNT cells while we see no change in adenine and cytosine ST:STUDY_SUMMARY nucleotides (i.e., AMP, ADP, ATP and CMP, CDP, CTP, respectively) or in ST:STUDY_SUMMARY UDP-hexose. ST:INSTITUTE VIB-KU Leuven ST:LAST_NAME Mazzone ST:FIRST_NAME Massimiliano ST:ADDRESS Campus Gasthuisberg Herestraat 49, box 912 B-3000 Leuven Belgium ST:EMAIL massimiliano.mazzone@kuleuven.vib.be ST:PHONE +32-16-37.32.13 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Mus musculus SU:TAXONOMY_ID 10090 SU:GENDER Not applicable #SUBJECT_SAMPLE_FACTORS: SUBJECT(optional)[tab]SAMPLE[tab]FACTORS(NAME:VALUE pairs separated by |)[tab]Raw file names and additional sample data SUBJECT_SAMPLE_FACTORS - KPC_NT_1 Genotype:sgNT | Cell Line:KPC_FC1245 Protein_BCA=3,852830189; RAW_FILE_NAME=MCF000545_ST91.mzML SUBJECT_SAMPLE_FACTORS - KPC_NT_2 Genotype:sgNT | Cell Line:KPC_FC1245 Protein_BCA=3,725471698; RAW_FILE_NAME=MCF000545_ST92.mzML SUBJECT_SAMPLE_FACTORS - KPC_NT_3 Genotype:sgNT | Cell Line:KPC_FC1245 Protein_BCA=4,10990566; RAW_FILE_NAME=MCF000545_ST93.mzML SUBJECT_SAMPLE_FACTORS - KPC_NT_4 Genotype:sgNT | Cell Line:KPC_FC1245 Protein_BCA=3,421226415; RAW_FILE_NAME=MCF000545_ST94.mzML SUBJECT_SAMPLE_FACTORS - KPC_CDAKD_1 Genotype:sgCDA | Cell Line:KPC_FC1245 Protein_BCA=3,980188679; RAW_FILE_NAME=MCF000545_ST99.mzML SUBJECT_SAMPLE_FACTORS - KPC_CDAKD_2 Genotype:sgCDA | Cell Line:KPC_FC1245 Protein_BCA=4,175943396; RAW_FILE_NAME=MCF000545_ST100.mzML SUBJECT_SAMPLE_FACTORS - KPC_CDAKD_3 Genotype:sgCDA | Cell Line:KPC_FC1245 Protein_BCA=3,664150943; RAW_FILE_NAME=MCF000545_ST101.mzML SUBJECT_SAMPLE_FACTORS - KPC_CDAKD_4 Genotype:sgCDA | Cell Line:KPC_FC1245 Protein_BCA=3,878773585; RAW_FILE_NAME=MCF000545_ST102.mzML SUBJECT_SAMPLE_FACTORS - Panc02_CDAKD_1 Genotype:sgCDA | Cell Line:Panc02 Protein_BCA=531,29; RAW_FILE_NAME=MCF000215_SR01.mzML SUBJECT_SAMPLE_FACTORS - Panc02_CDAKD_2 Genotype:sgCDA | Cell Line:Panc02 Protein_BCA=591,876; RAW_FILE_NAME=MCF000215_SR02.mzML SUBJECT_SAMPLE_FACTORS - Panc02_CDAKD_3 Genotype:sgCDA | Cell Line:Panc02 Protein_BCA=572,783; RAW_FILE_NAME=MCF000215_SR03.mzML SUBJECT_SAMPLE_FACTORS - Panc02_CDAKD_4 Genotype:sgCDA | Cell Line:Panc02 Protein_BCA=588,8695; RAW_FILE_NAME=MCF000215_SR04.mzML SUBJECT_SAMPLE_FACTORS - Panc02_NT_1 Genotype:sgNT | Cell Line:Panc02 Protein_BCA=532,9435; RAW_FILE_NAME=MCF000215_SR13.mzML SUBJECT_SAMPLE_FACTORS - Panc02_NT_2 Genotype:sgNT | Cell Line:Panc02 Protein_BCA=583,156; RAW_FILE_NAME=MCF000215_SR14.mzML SUBJECT_SAMPLE_FACTORS - Panc02_NT_3 Genotype:sgNT | Cell Line:Panc02 Protein_BCA=545,422; RAW_FILE_NAME=MCF000215_SR15.mzML SUBJECT_SAMPLE_FACTORS - Panc02_NT_4 Genotype:sgNT | Cell Line:Panc02 Protein_BCA=579,248; RAW_FILE_NAME=MCF000215_SR16.mzML #COLLECTION CO:COLLECTION_SUMMARY On day 2, cells were harvested on ice by washing them once in ice-cold saline CO:COLLECTION_SUMMARY solution (9 g/l NaCl). Cells were covered with 250 μl of pre-cooled 80% CO:COLLECTION_SUMMARY methanol for 2 minutes scraped, transferred to fresh vials, and precipitated CO:COLLECTION_SUMMARY overnight at -80°C. Then they were centrifuged at 20.009 g for 15min, 4 degrees CO:COLLECTION_SUMMARY C and supernatant transferred to an M/S vial. CO:SAMPLE_TYPE Cultured cells CO:STORAGE_CONDITIONS -80℃ #TREATMENT TR:TREATMENT_SUMMARY Cancer cells were cultured in DMEM medium supplemented with 10% FBS and 1% TR:TREATMENT_SUMMARY Pen/Strep in 6-well plates. On day 1, the cells and empty wells (for background TR:TREATMENT_SUMMARY noise control), were washed with PBS, and replenished with DMEM containing 10% TR:TREATMENT_SUMMARY FBS, 1% Pen/Strep, 5.5 mM glucose, 2 mM glutamine, 1% Pen/Strep, 0.1 mM cytidine TR:TREATMENT_SUMMARY (Sigma-Aldrich), or alternately, 0.1 mM labelled cytidine (13C9, 98% 15N3, TR:TREATMENT_SUMMARY 96-98%, Eurisotop) for 48h. #SAMPLEPREP SP:SAMPLEPREP_SUMMARY The samples were centrifuged at 20.000 x g for 15 min at 4°C, supernatant was SP:SAMPLEPREP_SUMMARY transferred to appropriate M/S vial for analysis. The cell pellet was dissolved SP:SAMPLEPREP_SUMMARY in 100 μl of 200 mM NaOH (for 20 minutes at 95°C), and the protein SP:SAMPLEPREP_SUMMARY concentration was determined by using bicinchoninic acid (BCA) reagent method. SP:PROCESSING_STORAGE_CONDITIONS -80℃ SP:EXTRACT_STORAGE -80℃ #CHROMATOGRAPHY CH:CHROMATOGRAPHY_SUMMARY C18 IP REVERSE PHASE CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Waters Acquity CH:COLUMN_NAME Waters Acquity UPLC HSS T3 (150 x 2.1mm, 1.8um) CH:SOLVENT_A 100% water; 10mM tributylamine; 15mM acetic acid CH:SOLVENT_B 100% methanol CH:FLOW_GRADIENT The gradient started with 5% solvent B and 95% solvent A and remained at 5% B CH:FLOW_GRADIENT until 2min post injection. A linear gradient to 37% B was carried out until 7min CH:FLOW_GRADIENT and increased to 41% until 14min. Between 14 and 26 minutes the gradient CH:FLOW_GRADIENT increased to 95% of B and remained at 95% B for 4 minutes. At 30 min the CH:FLOW_GRADIENT gradient returned to 5% B. The chromatography was stopped at 40 min. CH:FLOW_RATE 0.25 mL/min CH:COLUMN_TEMPERATURE 40 #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Thermo Q Exactive Orbitrap MS:INSTRUMENT_TYPE Orbitrap MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS EI-Maven polly, ThermoFisher Xcalibur, Metabolites were annotated using the MS:MS_COMMENTS inhouse standard metabolite library- elution time and m/z values. #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS AUC MS_METABOLITE_DATA_START Samples KPC_NT_1 KPC_NT_2 KPC_NT_3 KPC_NT_4 KPC_CDAKD_1 KPC_CDAKD_2 KPC_CDAKD_3 KPC_CDAKD_4 Panc02_CDAKD_1 Panc02_CDAKD_2 Panc02_CDAKD_3 Panc02_CDAKD_4 Panc02_NT_1 Panc02_NT_2 Panc02_NT_3 Panc02_NT_4 Factors Genotype:sgNT | Cell Line:KPC_FC1245 Genotype:sgNT | Cell Line:KPC_FC1245 Genotype:sgNT | Cell Line:KPC_FC1245 Genotype:sgNT | Cell Line:KPC_FC1245 Genotype:sgCDA | Cell Line:KPC_FC1245 Genotype:sgCDA | Cell Line:KPC_FC1245 Genotype:sgCDA | Cell Line:KPC_FC1245 Genotype:sgCDA | Cell Line:KPC_FC1245 Genotype:sgCDA | Cell Line:Panc02 Genotype:sgCDA | Cell Line:Panc02 Genotype:sgCDA | Cell Line:Panc02 Genotype:sgCDA | Cell Line:Panc02 Genotype:sgNT | Cell Line:Panc02 Genotype:sgNT | Cell Line:Panc02 Genotype:sgNT | Cell Line:Panc02 Genotype:sgNT | Cell Line:Panc02 Cytidine 28223402 3174356 13974296 31187797 164148316 61203529 63084877 980534 1305310162 8926774274 8648320948 1274710013 3498402947 3955412221 2381821314 2632888878 UDP 108991256 137762094 115042954 132107886 8567421 61932077 66842732 68226758 2193978993 1743644098 1824406797 1850506516 2315864644 2631132415 2437428576 287336616 UMP 17417638 2477793 14165911 21819336 10551369 649234 7305842 5885353 3135728 2601483191 2887550602 306684733 2786814761 2982213233 2948325387 2904765167 Uridine 24998308 21073763 15980564 21611477 137563 5642838 5292695 8223912 1657144298 1808722227 2112223411 2924058647 6068804178 8685912619 5299654001 6124843779 UTP 198402728 254619974 279115095 203107877 15522391 156186558 178140117 185742304 5315675913 4080802251 4203030526 4182507181 5437949985 6478508159 6423958662 7361952705 CDP 1122614 1327743 102451 1173586 2345728 2240025 2058941 1981533 1279400955 1031831161 1081047069 109651369 1067314806 116168165 1086515814 1241615994 CMP 502818 1067148 540898 1189809 1299076 696441 586268 590406 1147917069 1072233125 1133450625 1114038746 1100599525 10944388 1052572234 1114117923 CTP 44755367 57108646 60025299 47160202 44308309 45344671 52586144 57023085 2629652516 196619333 2040199073 2043945541 1975033969 2215110879 2267599573 2599611926 UDP-hexose 172424796 199789986 2655594 144894659 80128214 126526451 182859265 135525472 4025839579 3292239685 3577016767 3888648917 4037093707 4521845487 4273355015 5079009906 ADP 4063438688 4490458710 3554353544 4571710669 4275366446 3822977105 3333929188 3190020937 3237275601 2572226623 2636650174 2711604087 3175975432 3446659929 3318696821 3797237082 AMP 4126056968 4536124086 3667779048 4541566603 4163800377 3654802828 3245063605 296115205 3022757822 3019395455 2940922713 2999762706 2823053063 2884810581 2885486626 2543812554 ATP 63547267973 63724959198 65504286201 58995852037 65311628614 66968652034 69939579849 66161080272 1035164147 7895473992 798387881 8023780746 9416174972 1041805351 1083269526 122973388 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name PubChem ID KEGG ID Cytidine 6175 C00475 Uridine 6029 C00299 UMP 6030 C00105 UDP 6031 C00015 UTP 6133 C00075 CMP 6131 C00055 CDP 6132 C00112 CTP 6175 C00063 UDP-hexose 8629 C00029 AMP 6083 C00020 ADP 6022 C00008 ATP 5957 C00002 METABOLITES_END #END