新規なプロテインチップと質量分析による電気泳動で分離されたタンパク質間相互作用の分析

抄録

Since proteins interact with other proteins to perform their particular cellular task, analysis of the protein-protein interaction is important to determine the function of proteins. Protein-protein interaction has been analyzed by several methods such as yeast two-hybrid system, immunoaffinity purification technique and protein chip. Among them, protein chip has been considered the most promising tools for high-throughput analysis of protein-protein interaction at protein level. In the conventional technique, proteins expressed by DNA (RNA) or natural proteins have been purified and immobilized on the chemically modified glass plate or membrane to produce the protein chip. However, purification of the proteins has been laborious and time-consuming, and the purification of a number of proteins has been often impossible. This is a limiting factor to perform the high-throughput analysis of protein-protein interaction. We developed a technique for high-throughput analysis of the interaction using a novel protein chip. In this technique, proteins are separated by gel electrophoresis, and electroblotted onto the diamond-like carbon coated (DLC) stainless steel plate, of which surface is modified with N-hydroxysuccinimide ester, to produce a high-density of protein chip. Proteins in the gels can be immobilized covalently on the DLC plate with high blotting efficiency (30-70 %). Proteins extracted from the cells were probed with the proteins on the DLC plate, and the interacted proteins were detected by matrix assisted laser desorption ionization (MALDI)-time-of-flight mass spectrometry using the plate as a MALDI sample target. This technique has a great potential of high-throughput analysis of proteins interacted with thousands of proteins separated by two-dimensional gel electrophoresis.