| During the last few years genomics and proteomics have made a huge development. For many applications 2D gel electrophoresis followed by mass spectrometry for protein analysis is sufficient. However, 2D gel electrophoresis has also a number of shortcomings. First, the amount of protein that can be applied onto a 2D gel is very limited and hence low-abundant proteins cannot be detected. Secondly, protein complexes are disrupted and cannot be analysed by this technique. Thirdly, proteins separated by 2D gel electrophoresis usually are biochemically inactive. These shortcomings already slow down the development of proteomics. In the proposed research we will therefore not only use 2D gel electrophoresis, but also use column chromatography for protein purification. In our approach we will start with the separation of proteins using column chromatographic techniques under highly defined conditions. For example, proteins can be separated by ion exchange chromatography and subsequently the fractions obtained are separated by hydrophobic interaction chromatography. For this chromatography alternative column materials can be used and the conditions for an optimal separation of the proteins will be established. Use will be made of a highly automatic system. The protein fractions obtained, usually between 400 and 1000, will be analyzed for defined biochemical properties. This analysis will be done with robotic methods. The active fractions will be subjected to another round(s) of chromatography and analysis for the biochemical properties. The active fractions will be analyzed for their protein composition and MALDI-TOF mass spectrometry will reveal the identity of the proteins of interest. Proteins will be identified and characterized that are relevant for current research in the Institute of Molecular Plant Sciences, which has a major focus on signal transduction processes and regulatory circuits. We ask for the following equipment: - Pharmacia Explorer FPLC system with multi-sample injection, fraction collector compatible with robotic systems for analysis of fractions and various columns. - Q-Biogene Fastprep cell homogenizer. - Complete 2D gel electrophoresis system for analysis and purification of proteins. Investment in this equipment at IMP will help the institute to maintain its strength and competitiveness in the future. We will closely collaborate with Dr. R. van der Heijden (LACDR, Leiden) who has state-of-the-art equipment for mass spectroscopy. This collaboration will lead to a very strong center for protein analysis. |
