Improved proteomic analysis of nuclear proteins, as exemplified by the comparison of two myeloid cell lines nuclear proteomes.
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 proteins lies in their strong interaction with nuclear acids. Several experimental procedures have been tested to increase, in the nuclear extract, the ratio of nuclear proteins compared 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 protein expression 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 proteins but 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.
Laboratoire de Chimie et Biologie des Métaux, Université Joseph Fourier-Grenoble, iRTSV/LCBM, CEA Grenoble, 17 avenue des Martyrs, 38054 Grenoble cedex 9, France. cecile.lelong@ujf-grenoble.fr
Cécile
C
Lelong
Mireille
M
Chevallet
Hélène
H
Diemer
Sylvie
S
Luche
Alain
A
Van Dorsselaer
Thierry
T
Rabilloud
D000818:Animals; D045744:Cell Line, Tumor; D002467:Cell Nucleus / Q000378:metabolism*; D003713:Dendritic Cells / Q000166:cytology / Q000378:metabolism*; D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods / Q000592:standards; D008264:Macrophages / Q000166:cytology / Q000378:metabolism*; D013058:Mass Spectrometry / Q000379:methods / Q000592:standards; D051379:Mice; D009687:Nuclear Proteins / Q000378:metabolism*; D011499:Protein Processing, Post-Translational / Q000502:physiology; D020543:Proteome / Q000378:metabolism*; D040901:Proteomics / Q000379:methods* / Q000592:standards; D012984:Software
D009687:Nuclear Proteins; D020543:Proteome