Proteomic Analysis of Barley Cell Nuclei at Different Phases of Cell Cycle

Hundreds of proteins are involved in maintaining organization of eukaryotic cell nuclei, in the processes of nuclear and cell division, as well as gene expression. However, except for histones and a few other nuclear proteins, only a fraction of these proteins is known in plants. The plant nuclear proteome is yet to be explored. Attempts to characterize proteins of sub-cellular components may be compromised by the alternation of their biochemical composition during their isolation and subsequent protein purification. Typically, a series of fractionation steps is needed to obtain purified nuclei. These approaches are time consuming, laborious and the isolated fractions are often contaminated by cytoplasmic proteins. We have developed an alternative and efficient method for plant nuclei isolation, which does not affect their protein content and which involves only a single step. The nuclei are purified by flow cytometric sorting, which allows discrimination between G1, S and G2 phase nuclei, and minimizes the risk of contamination by non-nuclear proteins. Our preliminary results indicate that flow sorting coupled with protein/peptide separation and mass spectrometry permits a comprehensive characterization of the proteome of plant cell nuclei in various phases of cell division cycle.