Mass spectrometry based Cellular Thermal Shift Assay (MS-CETSA) enables proteome-wide analysis of drug-induced changes in protein thermal stability in intact cells. This project aimed to perform preliminary work for establishing a CETSA-based proteomic platform to compare cellular molecular responses upon exposure to multiple anticancer drugs.
A panel of 24 anticancer compounds with diverse mechanisms of action was selected, including drugs targeting cell growth signaling pathways, DNA damage response, cell-cycle regulation, apoptosis, epigenetic regulation, and cellular stress. MV4-11 acute myeloid leukemia (AML) cells were used as the model system. Before MS-CETSA analysis, treatment time and dose were optimized for each compound using IncuCyte-based live-cell imaging. Relative Growth Index and Cell Viability were evaluated together to select conditions that induced early drug responses while maintaining good cell viability.
Tandem Mass Tag (TMT) of 32 channels were used for the high-throughput MS-CETSA quantitation. A benchmark experiment was also performed to assess the reliability of combining Deuterium-labelled and non-Deuterium-labelled TMT channels. The results indicate that comparisons made within the same labelling type were more stable, whereas comparisons across Deuterium-labelled and non-Deuterium-labelled channels showed increased quantitative variation. Comparison with the published Venetoclax-induced response in Kasumi-1 AML cells revealed overlapping hits, which suggests that Venetoclax treatment induces a reproducible apoptosis-associated CETSA signature in AML cells.
Overall, this project established optimized treatment conditions for 24 compounds and provided a basis for future large-scale MS-CETSA analysis of anticancer drug mechanisms.