Integrated data-independent acquisition and thermal proteome profiling for proteomic characterization of lamotrigine-treated MCF-7 cells

: Lamotrigine, a clinically approved anticonvulsant, has recently gained interest for its potential anticancer effects. However, the molecular mechanisms underlying its activity in breast cancer remain poorly understood. Here, an integrated mass spectrometry-based proteomic strategy combining data-independent acquisition (DIA) and thermal proteome profiling (TPP) was applied to investigate the cellular effects and potential targets of lamotrigine in the human breast cancer cell line MCF-7. The DIA-based quantitative approach identified 6622 proteins, of which 142 were significantly modulated following lamotrigine exposure. Differentially expressed proteins were mainly associated with mitochondrial energy metabolism, chromatin organization, and protein translation. TPP experiments identified significant thermal stability alterations in ABRAXAS2, MT-CYB, TMEM97, and MTA2 upon lamotrigine treatment. Notably, both DIA and TPP analyses highlighted mitochondrial alterations, with consistent involvement of MT-CYB, suggesting mitochondrial dysfunction as a potential central mechanism of lamotrigine's action. Overall, these findings characterize proteome-level alterations induced by lamotrigine and support its further evaluation in breast cancer.