#METABOLOMICS WORKBENCH Lu_Group_20220804_044709_mwtab.txt DATATRACK_ID:3383 STUDY_ID:ST002255 ANALYSIS_ID:AN003684 PROJECT_ID:PR001346 VERSION 1 CREATED_ON August 11, 2022, 8:44 am #PROJECT PR:PROJECT_TITLE Functional metabolic molecule were identified as novel therapeutic targets to PR:PROJECT_TITLE facilitate gemcitabine treatment against pancreatic cancer PR:PROJECT_TYPE Targeted MS quantitative analysis PR:PROJECT_SUMMARY Characteristics of pancreatic cancer metabolomics with gemcitabine treatment PR:INSTITUTE Shanghai Center for Systems Biomedicine, Shanghai Jiaotong University PR:DEPARTMENT Shanghai Center for Systems Biomedicine PR:LABORATORY Lu Group PR:LAST_NAME Lu PR:FIRST_NAME Haitao PR:ADDRESS 800 Dongchuan RD. Minhang District, Shanghai, Shanghai, 200240, China PR:EMAIL jingjing2018@sjtu.edu.cn PR:PHONE 18818211315 #STUDY ST:STUDY_TITLE Functional metabolic molecules were identified as novel therapeutic targets to ST:STUDY_TITLE facilitate gemcitabine treatment against pancreatic cancer (Cells metabolomics ST:STUDY_TITLE with ATP) ST:STUDY_SUMMARY With the development of frontier technologies in system biology, traditional ST:STUDY_SUMMARY omics-drove phenotypic studies are insufficient to decipher the diseases. ST:STUDY_SUMMARY Therefore, for a thorough understanding of the molecular mechanisms of diseases ST:STUDY_SUMMARY to investigate novel drug targets, traditional phenotypic studies must be broken ST:STUDY_SUMMARY through to the functional exploration of molecules. Meanwhile, the intuitive ST:STUDY_SUMMARY role of small molecule compounds (metabolites) in pathogenesis, precision ST:STUDY_SUMMARY diagnosis and therapy are gradually recognized compared to macromolecules such ST:STUDY_SUMMARY as DNA, RNA and proteins. Therefore, we pioneeringly proposed Spatial Temporal ST:STUDY_SUMMARY Operative Real Metabolomics (STORM) strategy that established a relationship ST:STUDY_SUMMARY between metabolic phenotypes and functions to accurately character abnormal ST:STUDY_SUMMARY metabolisms and further identify operative functional molecules as novel ST:STUDY_SUMMARY therapeutic targets. Here, given the difficulty of pancreatic cancer (PC) ST:STUDY_SUMMARY treatment and the high resistance of clinical drugs, we were committed to ST:STUDY_SUMMARY explore new targets and drugs of pancreatic cancer from a small molecular ST:STUDY_SUMMARY functional perspective via STORM strategy. Fortunately, based on targeted ST:STUDY_SUMMARY metabolomics, we found that gemcitabine, one of the most effective clinical ST:STUDY_SUMMARY anti-PC drugs, served as a dual modulator that promote the accumulation of ST:STUDY_SUMMARY functional metabolic molecules in purine metabolism to activate down-streamed ST:STUDY_SUMMARY kinases. And the quantitative consequences of related enzymes annotated the ST:STUDY_SUMMARY unique molecular mechanisms of purine metabolism regulations by gemcitabine. ST:STUDY_SUMMARY Collectively, we broadened the cognitions of gemcitabine in tumor inhibition, ST:STUDY_SUMMARY providing potential strategies for treating PC with small molecules ST:STUDY_SUMMARY modification. Even more importantly, with the integration of multiple frontier ST:STUDY_SUMMARY technologies, the STORM strategy has proven to be well adapted to the phenotypic ST:STUDY_SUMMARY era of functional molecules devoted to innovate molecule mechanism annotation ST:STUDY_SUMMARY and therapeutic discovery. ST:INSTITUTE Shanghai Center for Systems Biomedicine, Shanghai Jiaotong University ST:DEPARTMENT Shanghai Center for Systems Biomedicine ST:LABORATORY Lu Group ST:LAST_NAME Lu ST:FIRST_NAME Haitao ST:ADDRESS 800 Dongchuan RD. Minhang District, Shanghai, Shanghai, 200240, China ST:EMAIL jingjing2018@sjtu.edu.cn ST:PHONE 18818211315 #SUBJECT SU:SUBJECT_TYPE Cultured cells SU:SUBJECT_SPECIES Homo sapiens SU:TAXONOMY_ID 9606 #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 - ASPC-CATP-1 Treatment:25µl DMSO and 40µM ATP for 72h RAW_FILE_NAME=ASPC-CATP-1 SUBJECT_SAMPLE_FACTORS - ASPC-CATP-2 Treatment:25µl DMSO and 40µM ATP for 72h RAW_FILE_NAME=ASPC-CATP-2 SUBJECT_SAMPLE_FACTORS - ASPC-CATP-3 Treatment:25µl DMSO and 40µM ATP for 72h RAW_FILE_NAME=ASPC-CATP-3 SUBJECT_SAMPLE_FACTORS - ASPC-CATP-4 Treatment:25µl DMSO and 40µM ATP for 72h RAW_FILE_NAME=ASPC-CATP-4 SUBJECT_SAMPLE_FACTORS - ASPC-CATP-5 Treatment:25µl DMSO and 40µM ATP for 72h RAW_FILE_NAME=ASPC-CATP-5 SUBJECT_SAMPLE_FACTORS - ASPC-GATP-1-r002 Treatment:50µM gemcitabine and 40µM ATP for 72h RAW_FILE_NAME=ASPC-GATP-1-r002 SUBJECT_SAMPLE_FACTORS - ASPC-GATP-2 Treatment:50µM gemcitabine and 40µM ATP for 72h RAW_FILE_NAME=ASPC-GATP-2 SUBJECT_SAMPLE_FACTORS - ASPC-GATP-3 Treatment:50µM gemcitabine and 40µM ATP for 72h RAW_FILE_NAME=ASPC-GATP-3 SUBJECT_SAMPLE_FACTORS - ASPC-GATP-4 Treatment:50µM gemcitabine and 40µM ATP for 72h RAW_FILE_NAME=ASPC-GATP-4 SUBJECT_SAMPLE_FACTORS - ASPC-GATP-5 Treatment:50µM gemcitabine and 40µM ATP for 72h RAW_FILE_NAME=ASPC-GATP-5 #COLLECTION CO:COLLECTION_SUMMARY After 72 hours of drug treatment, the cells were washed twice with ice PBS and CO:COLLECTION_SUMMARY scraped in 80% ice methanol CO:SAMPLE_TYPE Tumor cells #TREATMENT TR:TREATMENT_SUMMARY Cells were evenly divided into 3 groups of 6 discs each. 24h after cell TR:TREATMENT_SUMMARY inoculation, gemcitabine administration group(GATP) was treated with gemcitabine TR:TREATMENT_SUMMARY with final concentration of 50μM for 72h and ATP with a final concentration of TR:TREATMENT_SUMMARY 40μM, Meanwhile, the control group (CATP) was given the same volume of DMSO TR:TREATMENT_SUMMARY with 40μM ATP. One plate of cells was taken from each group for counting, and TR:TREATMENT_SUMMARY the rest were collected for metabolite extraction #SAMPLEPREP SP:SAMPLEPREP_SUMMARY The cells were cultured as described above and fixed with 1 ml of 80% ice-cold SP:SAMPLEPREP_SUMMARY menthol after being washed twice with ice-cold PBS. The cells were scraped from SP:SAMPLEPREP_SUMMARY the plates, and 0.5-mm beads were added to process the cells by grinding and SP:SAMPLEPREP_SUMMARY shaking. The supernatants were collected after centrifugation and deproteinized SP:SAMPLEPREP_SUMMARY by mixing with 800μL acetonitrile on ice. Then, the supernatants collected and SP:SAMPLEPREP_SUMMARY spun down under nitrogen at room temperature. The samples were resuspended in SP:SAMPLEPREP_SUMMARY 100 μL of distilled H2O, and 5 μL was used for LC-TQ-MS-based metabolome SP:SAMPLEPREP_SUMMARY assay. #CHROMATOGRAPHY CH:CHROMATOGRAPHY_TYPE Reversed phase CH:INSTRUMENT_NAME Agilent 1290 Infinity CH:COLUMN_NAME Waters Acquity HSS T3 column (100 mm×2.1, 1.8 μm) #ANALYSIS AN:ANALYSIS_TYPE MS #MS MS:INSTRUMENT_NAME Agilent 6495 QQQ MS:INSTRUMENT_TYPE Triple quadrupole MS:MS_TYPE ESI MS:ION_MODE NEGATIVE MS:MS_COMMENTS Agilent MassHunter Workstation Data Acquisition Agilent MassHunter #MS_METABOLITE_DATA MS_METABOLITE_DATA:UNITS counts MS_METABOLITE_DATA_START Samples ASPC-CATP-1 ASPC-CATP-2 ASPC-CATP-3 ASPC-CATP-4 ASPC-CATP-5 ASPC-GATP-1-r002 ASPC-GATP-2 ASPC-GATP-3 ASPC-GATP-4 ASPC-GATP-5 Factors Treatment:25µl DMSO and 40µM ATP for 72h Treatment:25µl DMSO and 40µM ATP for 72h Treatment:25µl DMSO and 40µM ATP for 72h Treatment:25µl DMSO and 40µM ATP for 72h Treatment:25µl DMSO and 40µM ATP for 72h Treatment:50µM gemcitabine and 40µM ATP for 72h Treatment:50µM gemcitabine and 40µM ATP for 72h Treatment:50µM gemcitabine and 40µM ATP for 72h Treatment:50µM gemcitabine and 40µM ATP for 72h Treatment:50µM gemcitabine and 40µM ATP for 72h 2'-Deoxyinosine 3779.453655 3376.698667 3058.968688 2872.935811 2805.735082 1996.521413 2766.506833 2817.770542 1388.694785 2617.347688 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name m/z RT 2'-Deoxyinosine 251.1 3.79 METABOLITES_END #END