科研之友
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摘要
Proteome-level investigation of the molecular targets in anticancer action of promising pharmaceutical candidates is highly desirable but remains challenging due to the insufficient proteome coverage, limited capacity for biological replicates, and largely unregulated false positive biomarker discovery of current methods. This study described a practical platform strategy to address these challenges, using comparison of drug response proteomic signatures by two promising anti-cancer agents (KX01/KX02) as the model system for method development/optimization. Drug-treated samples were efficiently extracted followed by precipitation/on-pellet-digestion procedure that provides high, reproducible peptide recovery. High-resolution separations were performed on a 75-cm-long heated nano-LC column with a 7-h gradient, with a highly reproducible nano-LC/nanospray configuration. An LTQ Orbitrap hybrid mass spectrometer with a charge overfilling approach to enhance sensitivity was used for detection. Analytical procedures were optimized and well-controlled to achieve high run-to-run reproducibility that permits numerous replicates in one set, and an ion-current-based approach was utilized for quantification. The false positives of biomarker discovery arising from technical variability was controlled based on FBDR measurement by comparing biomarker numbers in each drug-treated group vs. "sham samples", which were analyzed in an order randomly interleaved with the analysis drug-treated samples. More than 1500 unique protein groups were quantified under stringent criteria, and of which about 30% displayed differential expression with FBDR of 0.3-2.1% across groups. Comparison of drug-response proteomic signatures and the subsequent immunoassay. revealed that the action mechanisms of KX01/KX02 are similar but significantly different from vinblastine, which correlates well with clinical and pre-clinical observations. Furthermore, the results strongly supported the hypothesis that KX01/KX02 are dual-action agents (through inhibition of tubulin and Src). Moreover, informative insights into the drug-actions on cell cycle, growth/proliferation, and apoptosis were obtained. This platform technology provides extensive evaluation of drug candidates and facilitates in-depth mechanism studies.
- 出版日期2012-12-21
