| Project/Area Number |
15201043
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| Research Category |
Grant-in-Aid for Scientific Research (A)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Applied genomics
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| Research Institution | Osaka University |
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Principal Investigator |
OKAMURA Taka-aki (2004-2006) Osaka University, Graduate School of Science, Associate Professor, 大学院理学研究科, 講師 (90252569)
乗岡 茂巳 (2003) 大阪大学, 大学院・生命機能研究科, 教授 (70198638)
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| Co-Investigator(Kenkyū-buntansha) |
UEYAMA Norikazu Osaka University, Graduate School of Science, Researcher, 大学院理学研究科, 特任研究員 (80093376)
NAKAZAWA Takashi Nara Women's University, Department of Chemistry, Associate Professor, 理学部, 助教授 (30175492)
TSUNASAWA Susumu Shimadzu Corporation, Life Science Laboratory, Senior R & D Manager, 分析計測事業部ライフサイエンス研究所, 部長(研究職) (30029962)
KUYAMA Hiroki Osaka University, Institure for Protein Research, Associate Professor, 蛋白質研究所, 寄附研究部門助教授 (60437332)
望月 正雄 大阪大学, 大学院・生命機能研究科, 特任助手(常勤) (20379085)
小田 和明 北海道医療大学, 薬学部, 教授 (80094829)
岡村 高明 大阪大学, 大学院・理学研究科, 助手 (90252569)
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| Project Period (FY) |
2003 – 2006
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| Project Status |
Completed (Fiscal Year 2006)
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| Budget Amount *help |
¥50,830,000 (Direct Cost: ¥39,100,000、Indirect Cost: ¥11,730,000)
Fiscal Year 2006: ¥7,930,000 (Direct Cost: ¥6,100,000、Indirect Cost: ¥1,830,000)
Fiscal Year 2005: ¥9,880,000 (Direct Cost: ¥7,600,000、Indirect Cost: ¥2,280,000)
Fiscal Year 2004: ¥9,880,000 (Direct Cost: ¥7,600,000、Indirect Cost: ¥2,280,000)
Fiscal Year 2003: ¥23,140,000 (Direct Cost: ¥17,800,000、Indirect Cost: ¥5,340,000)
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| Keywords | Proteome / Metal Complex / Protein / Mass Analysis / Amino Acid Sequencing / N-terminal Peptide / C-terminal Peptide / Labeling Reagent / N端ペプチド / C端ペプチド / ビオチン / プロテオミクス / プロテオーム解析 / 質量分析計 / de novo sequencing / オキサゾロン |
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| Research Abstract |
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 peptide
2. Development of N-terminal labeling reagent having ruthenium compound and analytical method of protein complexes using this reagent
3. Selective C-terminal modification of proteins and peptides and new analytical methods for proteome
4. Rapid and efficient MALDI-TOF MS peak detection of 2-nitrobenzenesulfenyl-labeled peptides using the combination of HPLC and an automatic spotting apparatus
The 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.
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