Development of Highly Sensitive, Accurate, and Quantitative Analytical System for Proteome

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As a post-genome project, proteome analysis is one of the most important problems. Generally, proteome analyses identify precursor proteins on genes by determining internal sequences of the target protein referred to genome data base. However, these methods can not determine accurately mature proteins working in important biological systems. In this project, the following results were obtained.1. Establishment of a new method for isolation and characterization of N-terminal peptide2. Development of N-terminal labeling reagent having ruthenium compound and analytical method of protein complexes using this reagent3. Selective C-terminal modification of proteins and peptides and new analytical methods for proteome4. Rapid and efficient MALDI-TOF MS peak detection of 2-nitrobenzenesulfenyl-labeled peptides using the combination of HPLC and an automatic spotting apparatusThe first is selective N-terminal labeling by a reagent having biotin and disulfide moieties following isolation using avidin-biotin technique. The disulfide bond was oxidatively cleaved to release N-terminal peptide, which was analyzed by mass spectroscopy to give amino acid sequences. The second is selective labeling of proteins by ruthenium complex. After enzymatic digestion, the labeled N-terminal fragment was exclusively detected without requiring any separation. Using this reagent to a mixture of proteins afforded simultaneous detection of all the N-termini of constituent proteins. The next one is selective C-terminal labeling via oxazolone. intermediate, which enabled successful determination of C-terminal amino acid sequences. These original works in this project resulted in many reports, domestic and overseas patents, and commercialized products.