Preparation and analysis of plant and plastid proteomes by 2DE

Most proteomic analyses require prefractionation and protein purification strategies to achieve maximal proteome coverage, especially in plants in which cells often have a few highly abundant proteins and substances like polyphenols or secondary metabolites that can have significant impact on proteome coverage. Several methods have been developed to reduce cellular complexity and increase protein dynamic range. One approach is the display of the plant cell proteome on a single two-dimensional gel. Other approaches use fractionation strategies to reduce sample complexity to a subset of functionally related proteins or pathway modules. Here we describe a strategy to separate the proteome of a purified cell organelle using two-dimensional gel electrophoresis (2DE). The proteome of plant chloroplasts and nonphotosynthetic plastids was further fractionated by a differential protein solubilization method that is fully compatible with 2DE. The final protein complement of individual fractions comprised approximately 1,000 different protein species that can be fully resolved and visualized in a single 2DE gel.