title: 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.
  
  
  CONCLUSIONS
  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.
authors.affiliation: UMR 5280, Institut des sciences analytiques, Université de Lyon, Lyon 1, 69622 Villeurbanne cedex, France. Electronic address: jordane.biarc@univ-lyon1.fr.
authors.forename: Jordane
authors.initials: J
authors.lastname: Biarc
authors.affiliation: CNRS UMR5286, INSERM U1052, Centre de Recherche en Cancérologie de Lyon, Université de Lyon, Centre Léon Bérard, F-69008 Lyon, France; Laboratoire de Biochimie, Hôpitaux de Saint-Etienne, Université de Saint-Etienne, France.
authors.forename: Philippe
authors.initials: P
authors.lastname: Gonzalo
authors.affiliation: CNRS UMR5286, INSERM U1052, Centre de Recherche en Cancérologie de Lyon, Université de Lyon, Centre Léon Bérard, F-69008 Lyon, France.
authors.forename: Ivan
authors.initials: I
authors.lastname: Mikaelian
authors.affiliation: CNRS UMR5286, INSERM U1052, Centre de Recherche en Cancérologie de Lyon, Université de Lyon, Centre Léon Bérard, F-69008 Lyon, France.
authors.forename: Laurent
authors.initials: L
authors.lastname: Fattet
authors.affiliation: CNRS UMR5286, INSERM U1052, Centre de Recherche en Cancérologie de Lyon, Université de Lyon, Centre Léon Bérard, F-69008 Lyon, France.
authors.forename: Mathieu
authors.initials: M
authors.lastname: Deygas
authors.affiliation: CNRS UMR5286, INSERM U1052, Centre de Recherche en Cancérologie de Lyon, Université de Lyon, Centre Léon Bérard, F-69008 Lyon, France.
authors.forename: Germain
authors.initials: G
authors.lastname: Gillet
authors.affiliation: UMR 5280, Institut des sciences analytiques, Université de Lyon, Lyon 1, 69622 Villeurbanne cedex, France.
authors.forename: Jérôme
authors.initials: J
authors.lastname: Lemoine
authors.affiliation: CNRS UMR5286, INSERM U1052, Centre de Recherche en Cancérologie de Lyon, Université de Lyon, Centre Léon Bérard, F-69008 Lyon, France.
authors.forename: Ruth
authors.initials: R
authors.lastname: Rimokh
mesh_terms: D001940:Breast / Q000378:metabolism*; D002460:Cell Line; D002853:Chromatography, Liquid / Q000379:methods*; D004847:Epithelial Cells / Q000378:metabolism*; D058750:Epithelial-Mesenchymal Transition / Q000502:physiology*; D005260:Female; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods*; D010449:Peptide Mapping / Q000379:methods; D013194:Staining and Labeling; D016212:Transforming Growth Factor beta / Q000737:chemistry / Q000378:metabolism*; D018631:ras Proteins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D016212:Transforming Growth Factor beta; D018631:ras Proteins
keywords: Cell signaling; Epithelial–mesenchymal transition signature; Label-free quantification; Proteomics; Single reaction monitoring