| Project/Area Number |
18550076
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| Research Category |
Grant-in-Aid for Scientific Research (C)
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| Allocation Type | Single-year Grants |
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| Section | 一般 |
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| Research Field |
Analytical chemistry
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| Research Institution | Ehime University |
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Principal Investigator |
MANABE Takashi Ehime University, Faculty of Science, Professor (60094281)
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| Co-Investigator(Kenkyū-buntansha) |
SHIMAZAKI Yoji Ehime University, Faculty of Science, Ass. Professor (80284389)
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| Project Period (FY) |
2006 – 2007
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| Project Status |
Completed (Fiscal Year 2007)
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| Budget Amount *help |
¥4,060,000 (Direct Cost: ¥3,700,000、Indirect Cost: ¥360,000)
Fiscal Year 2007: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000)
Fiscal Year 2006: ¥2,500,000 (Direct Cost: ¥2,500,000)
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| Keywords | protein interaction / protein complex / non-denaturing / two-dimensional electrophoresis / mass spectrometry / micro 2-DE / plasma protein / peptide mass fumernrintine / ヒト血漿 / 非変性条件 / 2次元ゲル電気泳動 / マイクロ / MALDI-TOF-MS / マイクロゲル / MALDI-TOF MS |
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| Research Abstract |
A high-performance system to collect information on intact proteins and protein complexes was constructed combining non-denaturing micro two-dimensional gel electrophoresis (we named this type of 2-DE as Type-I micro 2-DE) and mass spectrometric assignment of the proteins, using human plasma as a model of complex protein mixtures. Information on intact proteins and protein complexes was firstly collected by assigning polypeptides in all the stained spots on the Type-I 2-DE gel by matrix-assisted laser desorption-ioninzation mass spectrometry (MALDI MS) and peptide mass fingerprinting. Secondly, using the same conditions of non-denaturing isoelectric focusing as the non-denaturing 2-DE, second dimension electrophoresis was run in the presence of sodium dodecyl sulfate (SDS) (we named this type of 2-DE as Type-II micro 2-DE) and again the polypeptides in all the stained spots were assigned by mass spectrometry. The comparisons between the results of polypeptide assignment provided information on non-covalent interactions between protein subunits and/or between different proteins, which should be crucial to exert the specific functions of the proteins or protein complexes. Thirdly, we developed a method to extract proteins from each stained spot on the Type-I micro 2-DE gel and analyze them using SDS-gel electrophoresis (third-dimension electrophoresis).The analysis of the stained bands on each lane of the SDS gel revealed the constituent polypeptides included in the stained spot, thus providing detailed information on the protein-protein interactions. Combining the results obtained by these three methods, together with the high-efficiency of the micro 2-DE system, we could collect information on proteins and protein complexes in human plasma.
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