Profiling Cell Lines Nuclear Sub-proteome
Proteins are very dynamic within the cell and their localization and trafficking between subcellular compartments are critical for their correct function. Indeed, the abnormal localization of a protein might lead to the pathogenesis of several diseases. The association of cell fractionation methods and mass spectrometry based proteomic methods allow both the localization and quantification of proteins in different sub-compartments. Here we present a detailed protocol for enrichment, identification, and quantitation of the nuclear proteome in cell lines combining nuclear subproteome enrichment by differential centrifugation and high-throughput proteomics.
Key wordsNuclear fractionation Subcellular proteomics Protein localization Cell Line Mass spectrometry
This research was supported by FAPESP (Young Scientist Grant—Proc.No. 2011/0947-1), CNPq, Center for Cell Based Thereapy—CTC-CEPID (Proc.FAPESP 2013/08135-2) and CISBi-NAP. A.G.M. C.S.P, M.L.G., C.H.T., and D.A. received fellowships from FAPESP Proc. No., 2014/16839-2, 2012/09682-4, 2013/08755-0, 2013/07675-3, and 2012/02518-4, respectively. A.P receives a PNPD fellowship from CAPES. V.M.F. receives a fellowship from CNPq, Proc.No. (308561/2014-7). We thank Profs. Emanuel Carrilho and Daniel Cardoso for allowing our data collection with the LTQ-Orbitrap Velos at the Analytical Central – Chemistry Institute of São Carlos—University of São Paulo.
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