Comprehensive analysis of protein interactions for determination of protein function in rice

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Protein chip has been one of the most promising tools for identification of expressed proteins and study of protein-protein interaction in proteome analysis. We developed a novel protein chip plate, which is made of diamond-like carbon-coated stainless steel (DLC), and modified with N-hydroxysuccinimide ester on the surface of plate. Proteins separated by SDS gel electrophoresis or two-dimensional electrophoresis (2-DE) were successfully electroblotted and immobilized covalently onto the DLC plate to produce easily and rapidly a high-density protein chip. High blotting efficiency (25-70 %) of proteins from the gels onto the DLC plate was obtained by improvement of electrophoresis and electroblotting techniques. Using the DLC plate, we established the techniques for identification of proteins immobilized on the DLC plate, for detection of proteins or peptides interacted with proteins immobilized on the plate using matrix-assisted laser desorption ionization/time-of-flight mass spectrometry, and also we developed the technique for identification of post-translational modification of proteins on the plate. In this study, we applied these techniques to identification of a number of proteins and N-linked glycoproteins of the rice calli and soybean seeds, which were separated by 2-DE. The developed techniques have a great potential to make possible the high-throughput proteome analysis in plants.