#METABOLOMICS WORKBENCH Lu_Group_20220804_044709_mwtab.txt DATATRACK_ID:3383 STUDY_ID:ST002255 ANALYSIS_ID:AN003683 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 POSITIVE 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 ATP 965854.7853 1296434.751 1054809.166 1064943.207 1228774.88 1264282.634 1558328.42 1593004.036 1396503.757 1768318.266 2-Deoxyadenosine 157536.5908 202723.3092 185656.401 212623.3324 290384.8798 39010.27947 92929.54801 241028.2863 38397.31973 43601.53982 Adenosine 15204304.68 17745613.81 19444757.21 18913790.43 22441609.57 26851121.92 51796125.58 50987615.66 23633976.04 45809333.58 Inosine-5'-monophosphate 42240.90721 31336.06478 46220.57104 52479.22626 31379.5499 87331.86139 20521.30772 34347.36703 98812.03453 51929.34235 Adenosine 5'-monophosphate 925046.673 602696.5537 1136123.896 1248403.548 838107.0076 2445745.932 454843.3328 939240.6193 2470928.672 1327749.876 3-Isobutyl-1-methylxanthine 12155.85391 10408.87398 4785.848249 3858.890906 10935.46387 26046.85136 67689.66287 653751.1696 35636460.61 42421.6857 cAMP 40779.30582 38244.52239 37406.53384 36464.79899 34374.77574 54284.32504 66328.07868 98566.59017 75036.21044 57956.56341 Inosine 854544.1352 1011851.579 1101557.131 1072419.45 1305676.27 1654004.589 2925016.382 3018941.226 1427315.07 2646929.464 MS_METABOLITE_DATA_END #METABOLITES METABOLITES_START metabolite_name m/z RT ATP 508.01 1.94 2-Deoxyadenosine 252.1 3.03 Adenosine 268.25 2.7 Inosine-5'-monophosphate 349.06 1.32 Adenosine 5'-monophosphate 348.07 1.32 3-Isobutyl-1-methylxanthine 223.25 9.08 cAMP 330.22 3.13 Inosine 269.09 2.7 METABOLITES_END #END