{ "METABOLOMICS WORKBENCH":{"STUDY_ID":"ST002317","ANALYSIS_ID":"AN003784","VERSION":"1","CREATED_ON":"October 19, 2022, 8:57 am"}, "PROJECT":{"PROJECT_TITLE":"Mass spectroscopy‑based proteomics and metabolomics analysis of triple‑positive breast cancer cells treated with tamoxifen and/ or trastuzumab","PROJECT_TYPE":"LC-MS/MS","PROJECT_SUMMARY":"HER2-enriched breast cancer with high levels of hormone receptor expression, known as triple positive breast cancer, may represent a new entity with a relatively favourable prognosis against which the combination of chemotherapy, HER-2 inhibition, and endocrine treatment may be considered overtreatment. We explored the effect of the anticancer drugs tamoxifen and trastuzumab, both separately and in combination, on the integrated proteomic and metabolic profile of triple positive breast cancer cells (BT-474). Method We employed ultra-high-performance liquid chromatography-quadrupole time of flight mass spectrometry using a Bruker timsTOF to investigate changes in BT-474 cell line treated with either tamoxifen, trastuzumab or a combination. Differentially abundant metabolites were identified using the Bruker Human Metabolome Database metabolite library and proteins using the Uniprot proteome for Homo sapiens using MetaboScape and MaxQuant, respectively, for identification and quantitation. Results A total of 77 proteins and 85 metabolites were found to significantly differ in abundance in BT-474 treated cells with tamoxifen 5 μM/and or trastuzumab 2.5 μM. Findings suggest that by targeting important cellular signalling pathways which regulate cell growth, apoptosis, proliferation, and chemoresistance, these medicines have a considerable anti-growth effect in BT-474 cells. Pathways enriched for dysregulation include RNA splicing, neutrophil degranulation and activation, cellular redox homeostasis, mitochondrial transmembrane transport, ferroptosis and necroptosis, ABC transporters and central carbon metabolism. Conclusion Our findings in protein and metabolite level research revealed that anti-cancer drug therapy had a significant impact on the key signalling pathways and molecular processes in triple positive BT-474 cell lines.","INSTITUTE":"Sharjah Institute for Medical Research","DEPARTMENT":"Sharjah Institute for Medical Research","LABORATORY":"Biomarker Discovery Group","LAST_NAME":"Facility","FIRST_NAME":"Core","ADDRESS":"M32, SIMR, College of Pharmacy, Health Sciences, University of Sharjah, Sharjah, UAE, Sharjah, 000, United Arab Emirates","EMAIL":"tims-tof@sharjah.ac.ae","PHONE":"+971 6 5057656"}, "STUDY":{"STUDY_TITLE":"Mass spectroscopy‑based proteomics and metabolomics analysis of triple‑positive breast cancer cells treated with tamoxifen","STUDY_SUMMARY":"HER2-enriched breast cancer with high levels of hormone receptor expression, known as triple positive breast cancer, may represent a new entity with a relatively favourable prognosis against which the combination of chemotherapy, HER-2 inhibition, and endocrine treatment may be considered overtreatment. We explored the effect of the anticancer drugs tamoxifen and trastuzumab, both separately and in combination, on the integrated proteomic and metabolic profile of triple positive breast cancer cells (BT-474). Method We employed ultra-high-performance liquid chromatography-quadrupole time of flight mass spectrometry using a Bruker timsTOF to investigate changes in BT-474 cell line treated with either tamoxifen, trastuzumab or a combination. Differentially abundant metabolites were identified using the Bruker Human Metabolome Database metabolite library and proteins using the Uniprot proteome for Homo sapiens using MetaboScape and MaxQuant, respectively, for identification and quantitation. Results A total of 77 proteins and 85 metabolites were found to significantly differ in abundance in BT-474 treated cells with tamoxifen 5 μM/and or trastuzumab 2.5 μM. Findings suggest that by targeting important cellular signalling pathways which regulate cell growth, apoptosis, proliferation, and chemoresistance, these medicines have a considerable anti-growth effect in BT-474 cells. Pathways enriched for dysregulation include RNA splicing, neutrophil degranulation and activation, cellular redox homeostasis, mitochondrial transmembrane transport, ferroptosis and necroptosis, ABC transporters and central carbon metabolism. Conclusion Our findings in protein and metabolite level research revealed that anti-cancer drug therapy had a significant impact on the key signalling pathways and molecular processes in triple positive BT-474 cell lines.","INSTITUTE":"University of Sharjah","DEPARTMENT":"Sharjah Institute for Medical Research","LABORATORY":"Biomarker Discovery Group","LAST_NAME":"Soares","FIRST_NAME":"Nelson","ADDRESS":"M32, SIMR, College of Pharmacy, Health Sciences, University of Sharjah","EMAIL":"nsoares@sharjah.ac.ae","PHONE":"065057656"}, "SUBJECT":{"SUBJECT_TYPE":"Human","SUBJECT_SPECIES":"Homo sapiens","TAXONOMY_ID":"9606"}, "SUBJECT_SAMPLE_FACTORS":[ { "Subject ID":"-", "Sample ID":"1A_93_1_328", "Factors":{"Treatment":"Control"}, "Additional sample data":{"RAW_FILE_NAME":"1A''_93_1_328.d"} }, { "Subject ID":"-", "Sample ID":"1A_93_1_331", "Factors":{"Treatment":"Control"}, "Additional sample data":{"RAW_FILE_NAME":"1A''_93_1_331.d"} }, { "Subject ID":"-", "Sample ID":"1A_92_1_327", "Factors":{"Treatment":"Control"}, "Additional sample data":{"RAW_FILE_NAME":"1A'_92_1_327.d"} }, { "Subject ID":"-", "Sample ID":"1A_92_1_330", "Factors":{"Treatment":"Control"}, "Additional sample data":{"RAW_FILE_NAME":"1A'_92_1_330.d"} }, { "Subject ID":"-", "Sample ID":"1A_91_1_326", "Factors":{"Treatment":"Control"}, "Additional sample data":{"RAW_FILE_NAME":"1A_91_1_326.d"} }, { "Subject ID":"-", "Sample ID":"1A_91_1_329", "Factors":{"Treatment":"Control"}, "Additional sample data":{"RAW_FILE_NAME":"1A_91_1_329.d"} }, { "Subject ID":"-", "Sample ID":"2a_-1_59_1_440", "Factors":{"Treatment":"TAMOXIFEN"}, "Additional sample data":{"RAW_FILE_NAME":"2a''_-1_59_1_440.d"} }, { "Subject ID":"-", "Sample ID":"2a_-2_59_1_441", "Factors":{"Treatment":"TAMOXIFEN"}, "Additional sample data":{"RAW_FILE_NAME":"2a''_-2_59_1_441.d"} }, { "Subject ID":"-", "Sample ID":"2a_-1_58_1_438", "Factors":{"Treatment":"TAMOXIFEN"}, "Additional sample data":{"RAW_FILE_NAME":"2a'_-1_58_1_438.d"} }, { "Subject ID":"-", "Sample ID":"2a_-2_58_1_439", "Factors":{"Treatment":"TAMOXIFEN"}, "Additional sample data":{"RAW_FILE_NAME":"2a'_-2_58_1_439.d"} }, { "Subject ID":"-", "Sample ID":"2a-1_57_1_436", "Factors":{"Treatment":"TAMOXIFEN"}, "Additional sample data":{"RAW_FILE_NAME":"2a-1_57_1_436.d"} }, { "Subject ID":"-", "Sample ID":"2a-2_57_1_437", "Factors":{"Treatment":"TAMOXIFEN"}, "Additional sample data":{"RAW_FILE_NAME":"2a-2_57_1_437.d"} } ], "COLLECTION":{"COLLECTION_SUMMARY":"The BT-474 BC cell line utilized in this study was cultured as monolayers in DMEM medium supplemented with 10% fetal bovine serum and 1% penicillin/streptomycin (Sigma Aldrich, St. Louis, MO, USA). All cultures were incubated at 37 °C in a humidified atmosphere of 5% CO2.","SAMPLE_TYPE":"Breast cancer cells","STORAGE_CONDITIONS":"Described in summary"}, "TREATMENT":{"TREATMENT_SUMMARY":"Triplicate flasks were prepared for each treatment condition for each analysis (metabolomic and proteomic) for a total of 24 flasks. Two million cells were seeded in each 75 cm2 tissue culture flask and incubated for 24 h. The cells were then treated with Tamoxifen (5 μM) and/or Trastuzumab (2.5 μM) for 24 h. These concentrations correspond to the IC50 of these compounds with BT-474 cells, as determined by cytotoxicity assays (data not shown). Control cells were treated with vehicle (dimethyl sulfoxide (DMSO) at 0.5% for 24 h. Following the incubation period, cells were collected by trypsinization and washed twice with phosphate-buffered saline solution (PBS) before re-suspending in 1 mL 1 × PBS for further analysis. Finally, cells were collected as pellets by centrifugation at 1200 rounds per minute (rpm) for 10 min at room temperature. To negate the effect of Circadian rhythms on the response of cells to treatment, cells were kept under the same conditions during the entire incubation period and the cell collection was done concurrently for all samples. In addition, the same number of cells were used for each sample to avoid the effect of variation in cell numbers.","TREATMENT":"Drugs","TREATMENT_COMPOUND":"Tamoxifen and/or Trastuzumab"}, "SAMPLEPREP":{"SAMPLEPREP_SUMMARY":"Sample metabolite extraction A volume of 1 mL of the extraction solvent (methanol+0.1% formic acid) was added to the cell pellets to quench cells. The cells were then vortexed for 2 min to ensure the quantitative extraction of the metabolites and stored on ice for 1 h, during which the samples were vortexed every 15 min. After this, the insoluble cell matrices were collected and transferred to centrifuge tubes, intermittent ultrasonication using the COPLEY sonicator (QSONICA SONICATOR, USA) under 30% amplifier and for 30 s with an ice bath employed throughout the process. Following that, cells debris were then centrifuged (15,000 rpm, 10 min, − 4 °C) and the sample supernatants were collected and transferred to LC vials for drying in the EZ-2 Plus (GeneVac-Ipswich, UK) at 37±1 °C. Dried samples were resuspended with 200 µL (water+0.1% formic acid), and vortexed for 2 min. Finally, the samples were filtered using a hydrophilic Nylon Syringe Filter of 0.45 µm pore size and analyzed by Q-TOF MS","PROCESSING_STORAGE_CONDITIONS":"Described in summary","EXTRACT_STORAGE":"Described in summary"}, "CHROMATOGRAPHY":{"CHROMATOGRAPHY_SUMMARY":"Samples were chromatographically separated by inline reversed-phase chromatography using the Elute HPG 1300 pumps and Elute Autosampler (Bruker, Darmstadt, Germany) with solvent A 0.1% FA in HPLC grade water and solvent B 0.1% FA in ACN. A Hamilton Intensity Solo 2 C18 column (100 mm × 2.1 mm, 1.8 μm beads) was maintained at 35 ℃ (metabolomics analyses).","CHROMATOGRAPHY_TYPE":"Reversed phase LC","INSTRUMENT_NAME":"Bruker Elute HPG 1300","COLUMN_NAME":"Hamilton Intensity Solo 2 C18","FLOW_GRADIENT":"1%B to 99%B in 15 min","FLOW_RATE":"250 uL/min","COLUMN_TEMPERATURE":"35","METHODS_FILENAME":".","SOLVENT_A":"Water (0.1% Formic Acid)","SOLVENT_B":"ACN (0.1% Formic Acid)","METHODS_ID":".","COLUMN_PRESSURE":".","INJECTION_TEMPERATURE":".","INTERNAL_STANDARD":".","INTERNAL_STANDARD_MT":".","RETENTION_INDEX":".","RETENTION_TIME":".","SAMPLE_INJECTION":".","SAMPLING_CONE":".","ANALYTICAL_TIME":".","CAPILLARY_VOLTAGE":".","MIGRATION_TIME":".","OVEN_TEMPERATURE":"35C","PRECONDITIONING":".","RUNNING_BUFFER":".","RUNNING_VOLTAGE":".","SHEATH_LIQUID":".","TIME_PROGRAM":".","TRANSFERLINE_TEMPERATURE":".","WASHING_BUFFER":".","WEAK_WASH_SOLVENT_NAME":".","WEAK_WASH_VOLUME":".","STRONG_WASH_SOLVENT_NAME":".","STRONG_WASH_VOLUME":".","TARGET_SAMPLE_TEMPERATURE":".","SAMPLE_LOOP_SIZE":".","SAMPLE_SYRINGE_SIZE":".","RANDOMIZATION_ORDER":".","CHROMATOGRAPHY_COMMENTS":"."}, "ANALYSIS":{"ANALYSIS_TYPE":"MS"}, "MS":{"INSTRUMENT_NAME":"Bruker timsTOF","INSTRUMENT_TYPE":"QTOF","MS_TYPE":"ESI","ION_MODE":"POSITIVE","MS_COMMENTS":"The MS analysis was performed using a timsTOF (Bruker, Darmstadt, Germany) with Apollo II electrosprayionization (ESI) source. The drying gas was set to flow at 10 L/min and the drying temperature to 220℃ and the nebulizer pressure to 2.2 bar. The capillary voltage was 4500 V and the end plate offset 500 V. For metabolomics 20–1300 m/z. The instrument was operated in auto-MS/MS mode. For metabolomics the collision energy was set to 20 eV, the cycle time to 0.5 s with a relative minimum intensity threshold of 400 counts per thousand and a target intensity of 20,000. Sodium formate was injected as an external calibrant in the first 0.3 min of each LC–MS/MS run."}, "MS_METABOLITE_DATA":{ "Units":"AU", "Data":[{"Metabolite":"L-Arginine","1A_93_1_328":"1678","1A_93_1_331":"2032","1A_92_1_327":"1866","1A_92_1_330":"1960","1A_91_1_326":"2126","1A_91_1_329":"2186","2a_-1_59_1_440":"2364","2a_-2_59_1_441":"2094","2a_-1_58_1_438":"2440","2a_-2_58_1_439":"1524","2a-1_57_1_436":"1810","2a-2_57_1_437":"2042"},{"Metabolite":"L-Proline","1A_93_1_328":"254","1A_93_1_331":"464","1A_92_1_327":"480","1A_92_1_330":"314","1A_91_1_326":"254","1A_91_1_329":"96","2a_-1_59_1_440":"1072","2a_-2_59_1_441":"1566","2a_-1_58_1_438":"700","2a_-2_58_1_439":"694","2a-1_57_1_436":"1108","2a-2_57_1_437":"798"},{"Metabolite":"L-Acetylcarnitine","1A_93_1_328":"0","1A_93_1_331":"0","1A_92_1_327":"0","1A_92_1_330":"0","1A_91_1_326":"0","1A_91_1_329":"0","2a_-1_59_1_440":"16922","2a_-2_59_1_441":"15334","2a_-1_58_1_438":"14726","2a_-2_58_1_439":"15112","2a-1_57_1_436":"21838","2a-2_57_1_437":"21154"},{"Metabolite":"Adenosine 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