Analysis of the protein complex associated with 14-3-3 epsilon by a deuterated-leucine labeling quantitative proteomics strategy

By using all unambiguous in vivo deuterated-leucine labeling quantitative proteomic approach, at close to the physiologically relevant level, we systematically profiled multiple proteins interacting with 14-3-3 epsilon, the isoform with least characterized protein interactions in 14-3-3 family in mammalian cells. Among the 19 proteins interacting with 14-3-3 epsilon identified, 6 of them including SKb1Hs, p54nrb, setine/threonine kinase 38, MEP50, 14-3-3 theta and cofilin 2 were the previously unknown interacting partners with 14-3-3 epsilon. The newly identified interactor cofilin 2 was also validated in co-transfection and coimmunoprecipitation. In contrast, with the same stringent criteria only three known partners were identified by conventional tandem affinity purification (TAP) approach. Therefore the 'in-spectra' quantitative marker of deuterated-leucine assisted to precisely identify those genuine interacting partners with minimum requirement of validation using other molecular approaches. (c) 2009 Elsevier B.V. All rights reserved.