Analysis of phosphorylation signal using a Phos-tag quencher

• Project/Area Number: 26460036
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Multi-year Fund
• Section: 一般
• Research Field: Physical pharmacy
• Research Institution: Hiroshima University
• Principal Investigator: Koike Tohru 広島大学, 医歯薬保健学研究院(薬), 教授 (90186586)
• Project Period (FY): 2014-04-01 – 2017-03-31
• Project Status: Completed (Fiscal Year 2016)
• Budget Amount: ¥4,940,000 (Direct Cost: ¥3,800,000、Indirect Cost: ¥1,140,000); Fiscal Year 2016: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000); Fiscal Year 2015: ¥1,560,000 (Direct Cost: ¥1,200,000、Indirect Cost: ¥360,000); Fiscal Year 2014: ¥1,820,000 (Direct Cost: ¥1,400,000、Indirect Cost: ¥420,000)
• Keywords: プロテオミクス / 蛍光分析 / キナーゼ / フォスファターゼ / Phos-tag / リン酸化分子 / リン酸化プロテオミクス / 消光性分子 / フォスタグ / リン酸化反応 / 亜鉛錯体 / リン酸化 / 亜鉛化合物

Outline of Final Research Achievements

By utilizing an original analytical method using Phos-tag derivatives, we have developed convenient and reliable protocols for real-time analysis of phosphorylation status of kinase or phosphatase substrates. Since the analytical volume of μM-level sample is between μL and mL, the general fluorometric apparatus can be used in a standard chemical laboratory. We believe that our Phos-tag technology will result in great progress in phophophroteomics.

Research Products

• [Journal Article] Specific glutamic acid residues in targeted proteins induce exaggerated retardations in Phos-tag SDS-PAGE migration (2017)
  • Author(s): E.Kinoshita, E. Kinoshita-Kikuta, K. Karata, T. Kawano, A. Nishiyama, M. Yamato, and T. Koike
  • Journal Title: Electrophoresis Volume: 38 Issue: 8 Pages: 1139-1146
  • DOI: 10.1002/elps.201600520
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] A Phos-tag SDS-PAGE method that effectively uses phosphoproteomic data for profiling the phosphorylation dynamics of MEK1. (2016)
  • Author(s): Kinoshita E, Kinoshita-Kikuta E, Kubota Y, Takekawa M and Koike T.
  • Journal Title: Proteomics Volume: 16 Issue: 13 Pages: 1825-1836
  • DOI: 10.1002/pmic.201500494
  • Peer Reviewed / Open Access
• [Journal Article] Expression and phosphorylation state analysis of intracellular protein kinases using Multi- PK antibody and Phos- tag SDS- PAGE. (2015)
  • Author(s): Sugiyama, Y., Katayama, S., Kameshita, I., Morisawa, K., Higuchi, T., Todaka, H., Eiji Kinoshita, Kinoshita- Kikuta, E., Koike, T., Taniguchi, T., Sakamoto, S.
  • Journal Title: MethodsX Volume: 2 Pages: 469-474
  • DOI: 10.1016/j.mex.2015.11.007
  • Peer Reviewed / Open Access / Int'l Joint Research
• [Journal Article] A Phos-tag-based fluorescence quenching system for activity assay and inhibitor screening for alkaline phosphatase (2014)
  • Author(s): Kinoshita-Kikuta, E., Kurosaki, H., Kunisada, N., Eiji Kinoshita, and Koike, T.
  • Journal Title: Am. J. Anal. Chem. Volume: 5 Issue: 12 Pages: 796-804
  • DOI: 10.4236/ajac.2014.512088
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Journal Article] Identification of two phosphorylated species of β-catenin involved in the ubiquitin-proteasome pathway by using two-dimensional Phos-tag affinity electrophoresis (2014)
  • Author(s): E. Kinoshita-Kikuta, E. Kinoshita, and T. Koike
  • Journal Title: Journal of Electrophoresis Volume: 58 Issue: 1 Pages: 1-4
  • DOI: 10.2198/jelectroph.58.1
  • NAID: 130004713130
  • ISSN: 1349-9394, 1349-9408
  • Peer Reviewed / Open Access / Acknowledgement Compliant
• [Presentation] 大腸菌ハイブリッドセンサーキナーゼにおけるリン酸基転移反応様式の検証 (2016)
  • Author(s): 木下英司，木下恵美子，江口陽子，小池 透
  • Organizer: 日本生化学会第89回大会
  • Place of Presentation: 仙台市
  • Year and Date: 2016-09-25
• [Presentation] Phos-tag SDS-PAGE methodology that effectively uses phosphoproteomic data for profiling the phosphorylation dynamics of MEK1 (2016)
  • Author(s): Eiji Kinoshita, Emiko Kinoshita-Kikuta, and Tohru Koike
  • Organizer: HUPO2016 台湾大会
  • Place of Presentation: Taipei (Taiwan)
  • Year and Date: 2016-09-18
  • Int'l Joint Research
• [Presentation] フォスタグ技術を用いたヒスチジンキナーゼにおける自己リン酸化ダイナミクスのプロファイリング (2016)
  • Author(s): 木下英司，木下恵美子，小池 透
  • Organizer: 日本プロテオーム学会2016年大会
  • Place of Presentation: 東京都
  • Year and Date: 2016-07-28
• [Presentation] Development of Thio-tag magnetic bead for rapid and selective separation of thiol-containing molecules (2015)
  • Author(s): Maho Kawaguchi, Norinao Koretake, Emiko Kinoshita-Kikuta, Eiji Kinoshita, and Tohru Koike
  • Organizer: 環太平洋国際化学会議 PACIFICHEM 2015
  • Place of Presentation: 米国 ホノルル市
  • Year and Date: 2015-12-15
  • Int'l Joint Research
• [Presentation] リン酸基捕捉能を持つダーククエンチャーを用いた蛍光分析法 (2015)
  • Author(s): 國貞夏実，木下恵美子，木下英司，小池 透
  • Organizer: 第66回日本電気泳動学会総会
  • Place of Presentation: 東京都
  • Year and Date: 2015-09-04
• [Book] Kinomics: Approaches and Applications (2015)
  • Author(s): E. Kinoshita-Kikuta, E. Kinoshita, and T. Koike
  • Total Pages: 15
  • Publisher: Wiley-VCH Verlag GmbH & Co. KGaA
• [Book] Protein Modifications in Pathogenic Dysregulation of Signaling (2015)
  • Author(s): E. Kinoshita-Kikuta, E. Kinoshita, and T. Koike
  • Total Pages: 15
  • Publisher: Springer Japan