Improved proteomic analysis of nuclear proteins, as exemplified by the comparison of two myeloid cell lines nuclear proteomes
2012
Abstract One of the challenges of the proteomic analysis by 2D-gel is to visualize the low abundance proteins, particularly those localized in the organelles. An additional problem with
nuclear proteinslies in their strong interaction with nuclear acids. Several experimental procedures have been tested to increase, in the nuclear extract, the ratio of
nuclear proteinscompared to contaminant proteins, and also to obtain reproducible conditions compatible with 2D-
gel electrophoresis. The NaCl procedure has been chosen. To test the interest of this procedure, the
nuclear proteinexpression profiles of macrophages and dendritic cells have been compared with a proteomic approach by 2D-
gel electrophoresis. Delta2D software and mass spectrometry analyses have allowed pointing out some proteins of interest. We have chosen some of them, involved in transcriptional regulation and/or chromatin structure for further validations. The immunoblotting experiments have shown that most of the observed changes are due to post-translational modifications, thereby exemplifying the interest of the 2D gel approach. Finally, this approach allowed us to reach not only high abundance
nuclear proteinsbut also lower abundance proteins, such as the HP1 proteins and reinforces the interest of using 2DE-gel in proteomics because of its ability to visualize intact proteins with their modifications.
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