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2007 Fiscal Year Final Research Report Summary

Construction of a high-performance system to collect protein information by the combination of non-denaturing two-dimensional electrophoresis and mass spectrometry

Research Project

Project/Area Number 18550076
Research Category

Grant-in-Aid for Scientific Research (C)

Allocation TypeSingle-year Grants
Section一般
Research Field Analytical chemistry
Research InstitutionEhime University

Principal Investigator

MANABE Takashi  Ehime University, Faculty of Science, Professor (60094281)

Co-Investigator(Kenkyū-buntansha) SHIMAZAKI Yoji  Ehime University, Faculty of Science, Ass. Professor (80284389)
Project Period (FY) 2006 – 2007
Keywordsprotein interaction / protein complex / non-denaturing / two-dimensional electrophoresis / mass spectrometry / micro 2-DE / plasma protein / peptide mass fumernrintine
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.

  • Research Products

    (30 results)

All 2008 2007 2006 Other

All Journal Article (16 results) (of which Peer Reviewed: 8 results) Presentation (13 results) Remarks (1 results)

  • [Journal Article] Noncovalent interactions in human plasma proteins analyzed by the comparisons of non-denaturing and denaturing micro 2-DE patterns after polypeptide assignment using MALDI-MS and PMF2008

    • Author(s)
      真鍋 敬
    • Journal Title

      Electrophoresis 29

      Pages: 2672-2688

    • Description
      「研究成果報告書概要(和文)」より
    • Peer Reviewed
  • [Journal Article] Noncovalent interactions in human plasma proteins analyzed by the comparisons of non-denaturing and denaturing micro 2-DE patterns after polypeptide assignment using MALDI-MS and PMF2008

    • Author(s)
      Manabe, T., Jin, Y
    • Journal Title

      Electrophoresis 29(in press)

    • Description
      「研究成果報告書概要(欧文)」より
  • [Journal Article] Alkaline extraction of human plasma proteins from nondenaturing micro-2-D gels for protein/polypeptide mass measurement and peptide mass fingerprinting using MALDI-TOF MS2007

    • Author(s)
      金 亜
    • Journal Title

      Electrophoresis 28

      Pages: 449-459

    • Description
      「研究成果報告書概要(和文)」より
    • Peer Reviewed
  • [Journal Article] Assignment of human plasma polypeptides on a nondenaturing 2-D gelusing MALDI-MS and PMF and comparisons with the results of intact protein mapping2007

    • Author(s)
      真鍋 敬
    • Journal Title

      Electrophoresis 28

      Pages: 843-863

    • Description
      「研究成果報告書概要(和文)」より
    • Peer Reviewed
  • [Journal Article] Analysis of protein/polypeptide interactions in human plasma using non denaturing micro 2-DE followed by 3-D SDS-PAGE and MS.2007

    • Author(s)
      真鍋 敬
    • Journal Title

      Electrophoresis 28

      Pages: 2065-2079

    • Description
      「研究成果報告書概要(和文)」より
    • Peer Reviewed
  • [Journal Article] キャピラリー電気泳動法による生体試料中の過塩素酸塩の定量2007

    • Author(s)
      加藤 徳雄
    • Journal Title

      分析化学 56

      Pages: 675-678

    • Description
      「研究成果報告書概要(和文)」より
    • Peer Reviewed
  • [Journal Article] Cleavage of fibrinogen alpha chains during isoelectric focusing of human plasma under non-denaturing conditions analyzed by micro two-dimen sional gel electrophoresis and matrix-assisted laser desorption/ionization mass spectrometry2007

    • Author(s)
      真鍋 敬
    • Journal Title

      J. Electrop. 51

      Pages: 27-34

    • Description
      「研究成果報告書概要(和文)」より
    • Peer Reviewed
  • [Journal Article] Alkaline extraction of human plasma proteins from nondenaturing micro-2-D gels for protein/polypeptide mass measurement and peptide mass fingerprinting using MALDI-TOF MS2007

    • Author(s)
      Jin, Y., Manabe, T
    • Journal Title

      Electrophoresis 28

      Pages: 449-459

    • Description
      「研究成果報告書概要(欧文)」より
  • [Journal Article] Assignment of human plasma polypeptides on a nondenaturing 2-D gel using MALDI-MS and PMF and comparisons with the results of intact protein mapping2007

    • Author(s)
      Manabe, T., Jin, Y
    • Journal Title

      Electrophoresis 28

      Pages: 843-863

    • Description
      「研究成果報告書概要(欧文)」より
  • [Journal Article] Analysis of protein/polypeptide interactions in human plasma using nondenaturing micro 2-DE followed by 3-D SDS-PAGE and MS2007

    • Author(s)
      Manabe, T., Jin, Y
    • Journal Title

      Electrophoresis 28

      Pages: 2065-2079

    • Description
      「研究成果報告書概要(欧文)」より
  • [Journal Article] Determination of perchlorate in biological samples by capillary electrophoresis.(in Japanese)2007

    • Author(s)
      Kato, N., Morimoto, S., Manabe, T
    • Journal Title

      Bunseki Kagaku 56

      Pages: 675-678

    • Description
      「研究成果報告書概要(欧文)」より
  • [Journal Article] Cleavage of fibrinogen alpha chains during isoelectric focusing of human plasma under non-denaturing conditions analyzed by micro two-dimensional gel electrophoresis and matrix-assisted laser desorption/ionization mass spectrometry2007

    • Author(s)
      Manabe, T., Jin, Y., Yamaguchi, N., Sugiyama, T., Ikari, K
    • Journal Title

      J. Electrop 51

      Pages: 27-34

    • Description
      「研究成果報告書概要(欧文)」より
  • [Journal Article] Regulation of IL-27p28 gene by lipopolysaccharide in dendritic DC2.4 cells2006

    • Author(s)
      鎌倉 真紀
    • Journal Title

      Biochem. Biophys. Res. Commun. 349

      Pages: 1372-1377

    • Description
      「研究成果報告書概要(和文)」より
    • Peer Reviewed
  • [Journal Article] Analysis of hydrolytic activity of phospholipase Calfa from porcine retina on retinyl ester and phosphatidyl choline using non-denaturing two-dimensional electrophoresis and mass spectrometry2006

    • Author(s)
      島崎 洋次
    • Journal Title

      J. Chromatogr. B. 843

      Pages: 42-46

    • Description
      「研究成果報告書概要(和文)」より
    • Peer Reviewed
  • [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
    • Journal Title

      Biochem. Biophys. Res. Commun 349

      Pages: 1372-1377

    • Description
      「研究成果報告書概要(欧文)」より
  • [Journal Article] Analysis of hydrolytic activity of phospholipase Calfa from porcine retina on retinyl ester and phosphatidyl choline using non-denaturing two-dimensional electrophoresis and mass spectrometry2006

    • Author(s)
      Shimazaki, Y., Kishi, IL, Mori, M., Yasuda, C., Manabe, T
    • Journal Title

      J. Chromatogr B 843

      Pages: 42-46

    • Description
      「研究成果報告書概要(欧文)」より
  • [Presentation] ヒト血漿タンパク質の細管(内径1.4mm)等電点電気泳動におけるアガロースゲルとポリアクリルアミドゲルの性能比較2007

    • Author(s)
      真鍋 敬
    • Organizer
      第27回キャピラリー電気泳動シンポジウム
    • Place of Presentation
      静岡県浜松市
    • Year and Date
      2007-11-15
    • Description
      「研究成果報告書概要(和文)」より
  • [Presentation] アガロースゲルを等電点電気泳動に用いた非変性2次元電気泳動によるヒト血漿中の高分子量タンパク質の解析2007

    • Author(s)
      真鍋 敬
    • Organizer
      第27回キャピラリー電気泳動シンポジウム
    • Place of Presentation
      静岡県浜松市クリエート浜松
    • Year and Date
      2007-11-15
    • Description
      「研究成果報告書概要(和文)」より
  • [Presentation] 非変性条件2次元ゲル電気泳動と質量分析によるヒト血漿タンパク質相互作用の解析2007

    • Author(s)
      真鍋 敬
    • Organizer
      第58回日本電気泳動学会総会
    • Place of Presentation
      山口県宇部市宇部全日空ホテル
    • Year and Date
      2007-11-08
    • Description
      「研究成果報告書概要(和文)」より
  • [Presentation] 非変性2次元電気泳動と質量分析を用いたヒト血漿タンパク質相互作用の解析法の検討2007

    • Author(s)
      真鍋 敬
    • Organizer
      日本分析化学会第56年会
    • Place of Presentation
      徳島県徳島市徳島大学工学部
    • Year and Date
      2007-09-20
    • Description
      「研究成果報告書概要(和文)」より
  • [Presentation] 非変性2次元電気泳動と質量分析によるヒト血漿タンパク質相互作用の解析2007

    • Author(s)
      真鍋 敬
    • Organizer
      日本ヒトプロテオーム機第5回大会
    • Place of Presentation
      東京都江東区日本科学未来館
    • Year and Date
      2007-07-30
    • Description
      「研究成果報告書概要(和文)」より
  • [Presentation] Assignment of human plasma polypeptides and polypeptide interactions on a non-denaturing micro 2-D gel using MALDI-MS and peptide mass fingerprinting2006

    • Author(s)
      Manabe, T
    • Organizer
      APCE2006
    • Place of Presentation
      Kyoto
    • Year and Date
      20061100
    • Description
      「研究成果報告書概要(欧文)」より
  • [Presentation] Assignment of human plasma polypeptides on a non-denaturing two-dimensional gel using MALDI-MS and peptide mass fingerprinting, comparisons with the results of intact protein mapping2006

    • Author(s)
      Manabe, T
    • Organizer
      7th Siena Meeting From Genome to Proteome
    • Place of Presentation
      Siena (Itary)
    • Year and Date
      20060900
    • Description
      「研究成果報告書概要(欧文)」より
  • [Presentation] Direct targeting of human plasma for matrix-assisted laser desorption/iionization and analysis of plasma proteins by time of flight mass spectrometry2006

    • Author(s)
      Manabe, T
    • Organizer
      7th Siena Meeting From Genome to Proteome
    • Place of Presentation
      Siena (Itary)
    • Year and Date
      20060900
    • Description
      「研究成果報告書概要(欧文)」より
  • [Presentation] Alkaline extraction of human plasma proteins from a non-denaturing micro two-dimensional gel for protein/polypeptide mass measurement and peptide mass fingerprinting using MALDI-TOF MS2006

    • Author(s)
      Manabe, T
    • Organizer
      7th Siena Meeting From Genome to Proteome
    • Place of Presentation
      Siena (Itary)
    • Year and Date
      20060900
    • Description
      「研究成果報告書概要(欧文)」より
  • [Presentation] Assignment of human plasma polypeptides and polypeptide interactions on a non-denmaturing micro 2-D gel using MALDI-MS and peptide mass fingerprinting2006

    • Author(s)
      真鍋 敬
    • Organizer
      APCE2006
    • Place of Presentation
      京都市西京区京都大学桂キャンパス
    • Year and Date
      2006-11-14
    • Description
      「研究成果報告書概要(和文)」より
  • [Presentation] Assignment of human plasma polypeptides on a non-denaturing two-dimensional gel using MALDI-MS and peptide mass fingerprinting, compari sons with the results of intact protein mapping.2006

    • Author(s)
      真鍋 敬
    • Organizer
      7th Siena Meeting From Genome to Proteome
    • Place of Presentation
      シエナ(イタリア)
    • Year and Date
      2006-09-05
    • Description
      「研究成果報告書概要(和文)」より
  • [Presentation] Direct targeting of human plasma for matrix-assisted laser desorption/ii onization and analysis of plasma proteins by time of flight mass spectr ometry.2006

    • Author(s)
      真鍋 敬
    • Organizer
      7th Siena Meeting From Genome to Proteome
    • Place of Presentation
      シエナ(イタリア)
    • Year and Date
      2006-09-05
    • Description
      「研究成果報告書概要(和文)」より
  • [Presentation] Alkaline extraction of human plasma proteins from a non-denaturing micro two-dimensional gel for protein/polypeptide mass measurement and peptide mass fingerprinting using MALDI-TOF MS.2006

    • Author(s)
      真鍋 敬
    • Organizer
      7th Siena Meeting From Genome to Proteome
    • Place of Presentation
      シエナ(イタリア)
    • Year and Date
      2006-09-05
    • Description
      「研究成果報告書概要(和文)」より
  • [Remarks] 「研究成果報告書概要(和文)」より

    • URL

      http://www.ehime-u.ac.jp/~manabet/home02.htm

URL: 

Published: 2010-02-04  

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