Plant Membrane Proteomics - using isolated membranes and proteins to compare and quantify subproteomes

Sammanfattning: Membrane proteins are particularly important to characterize, since they are involved in cellular processes of utmost importance in control and regulation of cells, such as transport across membranes, signal transduction and photosynthesis. The word "proteome" means all proteins expressed by a e.g. a cell or a tissue, at a certain time point and "proteomics" is the large scale study of these proteins. This thesis concerns mass spectrometry based quantitative proteomics of isolated plant membranes and proteins to gain more insights into the subproteomes. An important prerequisite for the investigations of plant plasma membrane (PM) subproteomes is the isolation of pure membranes, which we obtained by using aqueous two-phase partitioning (paper I). By using highly pure membranes, we were able to identify and quantify tissue specific isoforms in both poplar (Paper II) and Arabidopsis (Paper III). A large number of proteins, unique to the wood-forming xylem PM of poplar, were identified. We applied a quantitative approach using metabolic labeling and mass spectrometric determination of 14N/15N-ratios to determine the distribution differences for e.g. PM aquaporin isofoms, between leaf and root tissue in Arabidopsis (Paper III). Focus was then shifted to the Arabidopsis subproteome and the chloroplast-localized small heat shock protein, Hsp21 (Paper IV). This protein is plays a crucial role for plant survival during periods of stress, by interacting with and preventing stress-sensitive proteins from unfolding. Using our metabolic labeling approach and 14N/15N-ratios, we could detect and quantify the translocation of Hsp21 from the soluble stromal phase to the thylakoid membrane in heat-stressed plants, which was quite unique for Hsp21 compared to other thylakoid membrane proteins (Paper V). Quantitative proteomics using metabolic labeling and 14N/15N-ratios has proven to be a robust and useful tool to investigate differences in plant subproteomes and to investigate the location, abundance and behavior of membrane proteins both in vivo and in vitro.