Construction of a high-performance system to collect protein information by the combination of non-denaturing two-dimensional electrophoresis and mass spectrometry
| 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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Report
(3 results)
Research Products
(38 results)
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[Journal Article] Regulation of IL-27p28 gene by lipopolysaccharide in dendritic DC2.4 cells2006
Author(s)
Kamakura, M. Morisawa, K, Komi, H. Tomatani, A, Saito, F. Konishi, Y. Jin, Y. Manabe, T. Kuroda, M. Imai, S. Mizuguchi, H. Taniguchi, T
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Journal Title
Biochem. Biophys. Res. Commun 349
Pages: 1372-1377
Description
「研究成果報告書概要(欧文)」より
Related Report
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