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Development of Analytical Procedures for the Phosphoproteome Research

Research Project

Project/Area Number 15390013
Research Category

Grant-in-Aid for Scientific Research (B)

Allocation TypeSingle-year Grants
Section一般
Research Field Physical pharmacy
Research InstitutionHiroshima University

Principal Investigator

KOIKE Tohru  Hiroshima University, Graduate School of Biomedical Sciences, Professor, 大学院医歯薬学総合研究科, 教授 (90186586)

Co-Investigator(Kenkyū-buntansha) KINOSHITA Eiji  Hiroshima University, Graduate School of Biomedical Sciences, Associate Professor, 大学院医歯薬学総合研究科, 助教授 (80304418)
Project Period (FY) 2003 – 2006
Project Status Completed (Fiscal Year 2006)
Budget Amount *help
¥12,000,000 (Direct Cost: ¥12,000,000)
Fiscal Year 2006: ¥2,200,000 (Direct Cost: ¥2,200,000)
Fiscal Year 2005: ¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 2004: ¥2,100,000 (Direct Cost: ¥2,100,000)
Fiscal Year 2003: ¥5,600,000 (Direct Cost: ¥5,600,000)
KeywordsPhosphoproteomics / Postgenome Research / Metal Complex / Western Analysis / Affinity Electrophoresis / Kinase Profiling / 亜鉛化合物 / ゲル蛍光染色 / アフィニティークロマトグラフィー / SPR分析
Research Abstract

Protein phosphorylation is essential for the regulatory events of biological processes, such as signal transduction, apoptosis, proliferation, differentiation, and metabolism, in all living cells. The balance of the kinase and phosphatase reactions controls the phosphorylation status of a certain protein. Perturbation of the balance triggers severe pathologies, such as cancer and inflammation. Methods for the determination of the phosphorylation status of a protein are thus very important with respect to the evaluation of the basis for understanding the molecular origins of diseases and for drug design. Recently, we reported that a dinuclear metal complex of 1,3-bis[bis(pyridin-2-ylmethyl) amino]propan-2-olate acts as a phosphate-binding tag molecule, Phos-tag, in an aqueous solution. This finding has contributed to the development of phosphate-selective MS analysis, phosphate-affinity chromatography, phosphate-selective Western analysis, and phosphate-affinity electrophoresis. For example, the phosphate-affinity electrophoresis, Phos-tag SDS-PAGE offers the following significant advantages: i) Radioactive and chemical labels are avoided.ii) The time-course quantitative ratio of the phosphorylated and nonphosphorylated proteins can be determined.iii) The phosphate-binding specificity is independent of the amino acid sequence context. iv) A downstream procedure, such as Western blotting analysis, is applicable. v) The procedure is almost identical to that of the general SDS-PAGE system. We believe that phosphoproteomics would progress greatly by combining our Phos-tag technology and existing methods using high-quality antibodies and convenient mass spectrometers.

Report

(5 results)
  • 2006 Annual Research Report   Final Research Report Summary
  • 2005 Annual Research Report
  • 2004 Annual Research Report
  • 2003 Annual Research Report
  • Research Products

    (32 results)

All 2007 2006 2005 2004 2003 Other

All Journal Article (29 results) Book (1 results) Patent(Industrial Property Rights) (1 results) Publications (1 results)

  • [Journal Article] Separation of a phosphorylated histidine protein using phosphate affinity polyacrylamide gel electrophoresis2007

    • Author(s)
      Seiji Yamada
    • Journal Title

      Analytical Biochemistry 360

      Pages: 160-162

    • NAID

      120000878053

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Label-free kinase profiling using phosphate-affinity polyacrylamidegel electrophresis2007

    • Author(s)
      Emiko Kinoshita-Kikuta
    • Journal Title

      Molecular & Cellular Proteomics 6

      Pages: 356-366

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Separation of a phosphorylated histidine protein using phosphate affinity polyacrylamide gel electrophoresis2007

    • Author(s)
      S.Yamada, H.Nakamura, E.Kinoshita, E.Kinoshita-Kikuta, T.Koike, Y.Shiro
    • Journal Title

      Analytical Biochemistry 360

      Pages: 160-162

    • NAID

      120000878053

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Label-free kinase profiling using phosphate-affinity polyacrylamide gel electrophresis2007

    • Author(s)
      E.Kinoshita-Kikuta, Y.Aoki, E.Kinoshita, T.Koike
    • Journal Title

      Molecular & Cellular Proteomics 6

      Pages: 356-366

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Phosphate-binding tag : A new tool to visualize phosphorylated proteins2006

    • Author(s)
      Eiji Kinoshita
    • Journal Title

      Molecular & Cellular Proteomics 5

      Pages: 749-757

    • NAID

      120000878114

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2006 Annual Research Report 2006 Final Research Report Summary
  • [Journal Article] Enrichment of phosphorylated proteins from cell lysate using phosphate-affinity chromatography at physiological pH2006

    • Author(s)
      Emiko Kinoshita-Kikuta
    • Journal Title

      Proteomics 6

      Pages: 5088-5095

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2006 Annual Research Report 2006 Final Research Report Summary
  • [Journal Article] Phosphate-binding tag : A new tool to visualize phosphorylated proteins2006

    • Author(s)
      E.Kinoshita, E.Kinoshita-Kikuta, K.Takiyama, T.Koike
    • Journal Title

      Molecular & Cellular Proteomics 5

      Pages: 749-757

    • NAID

      120000878114

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Enrichment of phosphorylated proteins from cell lysate using phosphate-affinity chromatography at physiological pH2006

    • Author(s)
      E.Kinoshita-Kikuta, E.Kinoshita, A.Yamada, M.Endo, T.Koike
    • Journal Title

      Proteomics 6

      Pages: 5088-5095

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Detection and Quantification of On-Chip Phosphorylated Peptides by Surface Plasmon Resonance Imaging Techniques Using a Phosphate Capture Molecule2005

    • Author(s)
      Kazuki Inamori
    • Journal Title

      Analytical Chemistry 77

      Pages: 3979-3985

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2006 Final Research Report Summary 2005 Annual Research Report
  • [Journal Article] A heteroduplex-preferential T_m depressor for the specificity-enhanced DNA polymerase chain reaction2005

    • Author(s)
      Eiji Kinoshita
    • Journal Title

      Analytical Biochemistry 337

      Pages: 154-160

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Novel immobilized zinc(II) affinity chromatography for Phosphopeptides and phosphorylated proteins2005

    • Author(s)
      Eiji Kinoshita
    • Journal Title

      Journal of Separation Science 28

      Pages: 155-162

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] An Alkoxide-bridged Dinuclear Zinc(II) Hexaazacryptate : a Novel Phosphate Capture Molecule in Aqueous Solution2005

    • Author(s)
      Eiji Kinoshita
    • Journal Title

      Bulletin of the Chemical Society of Japan 78

      Pages: 125-131

    • NAID

      10014271953

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Reliable and Cost-effective Screening of Inherited Heterozygosity by Using Zn(II)-Cyclen Polyacrylamide Gel Electrophoresis2005

    • Author(s)
      Eiji Kinoshita
    • Journal Title

      Clinical Chemistry 51

      Pages: 2195-2198

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Detection and Quantification of On-Chip Phosphorylated Peptides by Surface Plasmon Resonance Imaging Techniques Using a Phosphate Capture Molecule2005

    • Author(s)
      K.Inamori, M.Kyo, Y.Nishiya, Y.Inoue, T.Sonoda, E.Kinoshita, T.Koike, Y.Katayama
    • Journal Title

      Analytical Chemistry 77

      Pages: 3979-3985

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Novel immobilized zinc(II) affinity chromatography for phosphopeptides and phosphorylated proteins2005

    • Author(s)
      E.Kinoshita, A.Yamada, H.Takeda, E.Kinoshita-Kikuta, T.Koike
    • Journal Title

      Journal of Separation Science 28

      Pages: 155-162

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] An Alkoxide-bridged Dinuclear Zinc(II) Hexaazacryptate : a Novel Phosphate Capture Molecule in Aqueous Solution2005

    • Author(s)
      E.Kinoshita, K.Ishikawa, E.Kinoshita-Kikuta, K.Ohtani, M.Shiro, T.Koike
    • Journal Title

      Bulletin of the Chemical Society of Japan 78

      Pages: 125-131

    • NAID

      10014271953

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] An Alkoxide-bridged Dinuclear Zinc(II) Hexaazacryptate: a Novel Phosphate Capture Molecule in Aqueous Solution2005

    • Author(s)
      Eiji Kinosita
    • Journal Title

      Bulletin of the Chemical Society Japan 78

      Pages: 125-131

    • NAID

      10014271953

    • Related Report
      2005 Annual Research Report
  • [Journal Article] Novel immobilized zinc(II) affinity chromatography for phosphopeptides and phosphorylated proteins2005

    • Author(s)
      Eiji Kinoshita
    • Journal Title

      Journal of Separation Science 28

      Pages: 155-162

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Recognition of phosphate monoester dianion by an alkoxide-bridged dinuclear zinc(II) complex2004

    • Author(s)
      Eiji Kinoshita
    • Journal Title

      Dalton Transactions

      Pages: 1189-1193

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Inhibition of NADPH-dependent enzyme activities using the dinuclear zinc chelate. Phos-tag, and reactivation depending on phosphate ion concentrations2004

    • Author(s)
      Yoshiaki Nishiya
    • Journal Title

      Journal of Analytical Bio-Science 27

      Pages: 224-228

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Quantitative analysis of lysophosphatidic acid by time-of-flight mass spectrometry using a phosphate capture molecule2004

    • Author(s)
      Tamotsu Tanaka
    • Journal Title

      Journal of Lipid Research 45

      Pages: 2145-2150

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Production of 1,2-Didocosahexaenoyl Phosphatidylcholine by Bonito Muscle Lysophosphatidylcholine/Transacylase2004

    • Author(s)
      Kaoru Hirano
    • Journal Title

      Journal of Biochemistry 136

      Pages: 477-483

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Recognition of phosphate monoester dianion by an alkoxide-bridged dinuclear zinc(II) complex2004

    • Author(s)
      E.Kinoshita, M.Takahashi, H.Takeda, M.Shiro, T.Koike
    • Journal Title

      Dalton Transactions

      Pages: 1189-1193

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Quantitative analysis of lysophosphatidic acid by time-of-flight mass spectrometry using a phosphate capture molecule2004

    • Author(s)
      T.Tanaka, H.Tsutsui, K.Hirano, T.Koike, A.Tokumura, K.Satouchi
    • Journal Title

      Journal of Lipid Research 45

      Pages: 2145-2150

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Recognition of phosphate monoester dianion by an alkoxide-bridged dinuclear zinc(II) complex"2004

    • Author(s)
      Eiji Kinoshita
    • Journal Title

      Dalton Transactions

      Pages: 1189-1193

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Quantitative analysis of lysophosphatidic acid by time-of-flight mass spectrometry using a phosphate-capture molecule2004

    • Author(s)
      Tamotsu Tanaka
    • Journal Title

      Journal of Lipid Research 45

      Pages: 2145-2150

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Production of 1,2-didocosahexaenoyl phosphatidylcholine by bonito muscle lysophosphatidylcholine/transacylase2004

    • Author(s)
      Kaoru Hirano
    • Journal Title

      Journal of Biochemistry 136

      Pages: 447-483

    • Related Report
      2004 Annual Research Report
  • [Journal Article] Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of phosphorylated compounds using a novel phosphate capture molecule2003

    • Author(s)
      Hironori Takeda
    • Journal Title

      Rapid Communication of Mass Spectrometry 17

      Pages: 2075-2081

    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Journal Article] Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of phosphorylated compounds using a novel phosphate capture molecule2003

    • Author(s)
      H.Takeda, A.Kawasaki, M.Takahashi, A.Yamada, T.Koike
    • Journal Title

      Rapid Communication of Mass Spectrometry 17

      Pages: 2075-2081

    • Description
      「研究成果報告書概要(欧文)」より
    • Related Report
      2006 Final Research Report Summary
  • [Book] 実験医学2006

    • Author(s)
      木下英司, 木下恵美子, 小池 透
    • Total Pages
      6
    • Publisher
      株式会社 羊土社
    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2006 Annual Research Report 2006 Final Research Report Summary
  • [Patent(Industrial Property Rights)] リン酸基を有する物質の染色方法2005

    • Inventor(s)
      小池 透
    • Industrial Property Rights Holder
      広島大学
    • Industrial Property Number
      2005-230583
    • Filing Date
      2005-08-09
    • Description
      「研究成果報告書概要(和文)」より
    • Related Report
      2006 Final Research Report Summary 2005 Annual Research Report
  • [Publications] Hironori Takeda: "Matrix-assisted laser desorption/ionization time-of-flight mass spectrometry of phosphorylated compounds by using a novel phosphate capture molecule"Rapid Communications in Mass Spectrometry. 17. 2075-2081 (2003)

    • Related Report
      2003 Annual Research Report

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Published: 2003-04-01   Modified: 2016-04-21  

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