Combination of a discovery LC-MS/MS analysis and a label-free quantification for the characterization of an epithelial-mesenchymal transition signature.

Abstract Disease phenotype reorganizations are the consequences of signaling pathway perturbations and protein abundance modulations. Characterizing the protein signature of a biological event allows the identification of new candidate biomarkers, new targets for treatments and selective patient therapy. The combination of discovery LC–MS/MS analyses and targeted mass spectrometry using selected reaction monitoring (SRM) mode has emerged as a powerful technology for biomarker identification and quantification owing to faster development time and multiplexing capability. The epithelial–mesenchymal transition (EMT) is a process that controls local invasion and metastasis generation by stimulating changes in adhesion and migration of cells but also in metabolic pathways. In this study, the non-transformed human breast epithelial cell line MCF10A, treated by TGFβ or overexpressing mutant K-Ras v12 , two EMT inducers frequently involved in cancer progression, was used to characterize protein abundance changes during an EMT event. The LC–MS/MS analysis and label-free quantification revealed that TGFβ and K-Ras v12 induce a similar pattern of protein regulation and that besides the expected cytoskeletal changes, a strong increase in the anabolism and energy production machinery was observed. Biological Significance To our knowledge, this is the first proteomic analysis combining a label-free quantification with an SRM validation of proteins regulated by TGFβ and K-Rasv12. This study reveals new insights in the characterization of the changes occurring during an epithelial–mesenchymal transition (EMT) event. Notably, a strong increase in the anabolism and energy production machinery was observed upon both EMT inducers.