2002 Fiscal Year Annual Research Report

創薬を目指すファーマコプロテオミクスの基盤研究

Research Project

Project/Area Number	13877379
Research Institution	Tohoku University
Principal Investigator	後藤順一東北大学, 医学部附属病院, 教授 (80006337)
Co-Investigator(Kenkyū-buntansha)	眞野成康東北大学, 大学院・薬学研究科, 講師 (50323035) 小林典裕神戸薬科大学, 教授 (90205477)
Keywords	プロテオミクス / ファーマコプロテオミクス / ゲノム創薬 / 質量分析法 / ペルオキシレドキシン / アシルアデニレート / イムノアフィニティー抽出 / アフィニティーラベル化
Research Abstract	本格的なポストゲノム時代が到来し、世界の各製薬企業はゲノム情報を活用する新規医薬品開発の方向性を模索している。こうした中、薬物や生理活性物質刺激が誘起する変化を分子レベルで捕らえ、標的蛋白質の機能を解析することにより、新たな創薬標的分子を探索するファーマコプロテオミクス手法の確立に多大な期待が寄せられている。本年度は、リン酸による翻訳後修飾蛋白質解析法を検討するとともに、アフィニティーラベル化法とイムノアフィニティー抽出法を駆使した低分子-蛋白質相互作用解析法の構築を試みた。前年度の結果から、デオキシコール酸刺激によるペルオキシレドキシンIの変動には、リン酸による翻訳後修飾が関与している可能性が示唆された。そこで、2次元ゲル電気泳動マップのディファレンシャルディスプレイを用いるリン酸化蛋白質特定法につき検討した。試料をあらかじめアルカリホスファターゼにより脱リン酸化反応に付した後、2次元ゲル電気泳動法により蛋白質を分離し、マップ化した。次いで、脱リン酸化処理を行わない試料と比較したところ、容易にリン酸化蛋白質を特定できることが判明した。しかも、分離した蛋白質スポットをゲル内にてアルカリホスファターゼ反応に付した後、負イオン検出MALDI-TOFMSにて測定することにより、リン酸化ペプチドを高感度に検出できることが判明した。加えてエキソペプチダーゼを用いるペプチドラダーシークエンス法を用いることにより、そのリン酸化部位の特定も可能であることが明らかとなった。一方、低分子-蛋白質相互作用解析を目的としてアシルアデニレートをアフィニティーラベル化剤として用い、その有用性を吟味したところ、相互作用部位に選択的なラペル化が可能であることが判明した。しかもイムノアフィニティー抽出法を駆使して酵素消化により得たラベル化ペプチドを選択的に抽出し、ラベル化部位の構造解析も可能であることを実証した。

Research Products
(15 results)

All Other

All Publications (15 results)

[Publications] Nariyasu Mano: "Characterization of rat liver bile acid acyl glucuronosyltransferase"Steroids. 67. 257-262 (2002)
[Publications] Takashi Iida: "Potential bile acid metabolites. 24. An efficient synthesis of carboxyl-linked glucosides"Lipids. 37. 101-110 (2002)
[Publications] Norihiro Kobayashi: "A monoclonal antibody-based enzyme-linked immunosorbent assay of glycolithocholic acid sulfate in human urine for liver function test"Steroids. 67. 827-833 (2002)
[Publications] Nariyasu Mano: "Rapid and simple quantitative assay method for diastereomeric flurbiprofen glucuronides in the incubation mixture"Journal of Chromatography B. 776. 125-131 (2002)
[Publications] Norihiro Kobayashi: "Single-chain Fv fragments derived from an anti-11-deoxycortisol antibody : Affinity, specificity, and idiotype analysis"Steroids. 67. 733-742 (2002)
[Publications] Toshiaki Kume: "Isolation of a novel diterpenoid substance with potent neuroprotective activity from fetal calf serum"Proceedings of National Academy of Sciences in USA. 99. 3288-3293 (2002)
[Publications] Takashi Iida: "Potential bile acid metabolites. 25. Synthesis and chemical property of stereoisomeric 3α,7α,16-and 3α,7α,15-trihydroxy-5β-cholan-24-oic acids"Chemical and Pharmaceutical Bulletin. 50. 1335-1340 (2002)
[Publications] Takashi Iida: "Direct capillary gas chromatographic analysis and thermal stability of carboxy-linked glucosides of bile acids"Chromatographia. 56. 489-494 (2002)
[Publications] Nariyasu Mano: "Separation and determination of diastereomeric flurbiprofen acyl glucuronides in human urine by LC/ESI-MS with a simple column-switching technique"Drug Metabolism and Pharmacokinetics. 17. 142-149 (2002)
[Publications] Sookie La: "Enantioseparation of chiral aromatic amino acids by capillary electrophoresis in neutral and charged cyclodextrin selector modes"Electrophoresis. 23. 4123-4131 (2002)
[Publications] Norihiro Kobayashi: "Generation of a novel monoclonal antibody against cortisol-[C-4]-bovine serum albumin conjugate : Application to enzyme-linked immunosorbent assay for urinary and serum cortisol"Analytical Sciences. 18. 1309-1314 (2002)
[Publications] Norihiro Kobayashi: "Idiotype-anti-idiotype-based noncompetitive enzyme-linked immunosorbent assay of ursodeoxycholic acid 7-N-acetylglucosaminides in human urine with subfemtomole range sensitivity"Journal of Immunological Methods. 272. 1-10 (2003)
[Publications] Norihiro Kobayashi: "Monoclonal anti-idiotype antibodies recognizing the variable region of a high-affinity antibody against 11-deoxycortisol. Production, characterization and application to a sensitive noncompetitive immunoassay"Journal of Immunological Methods. 274. 63-75 (2003)
[Publications] Takashi Iida: "^1H and ^<13>C signal assignments of carboxyl-linked glucosides of bile acids"Magnetic Resonance in Chemistry. (in press).
[Publications] Takashi Iida: "Functionalization of unactivated carbons in 3α,6-and 3α,24-dihydroxy-5β-cholan derivatives by dimetyldioxirane"Lipids. (in press).

2002 Fiscal Year Annual Research Report

創薬を目指すファーマコプロテオミクスの基盤研究

Principal Investigator

後藤 順一 東北大学, 医学部附属病院, 教授 (80006337)

Research Products

[Publications] Nariyasu Mano: "Characterization of rat liver bile acid acyl glucuronosyltransferase"Steroids. 67. 257-262 (2002)

[Publications] Takashi Iida: "Potential bile acid metabolites. 24. An efficient synthesis of carboxyl-linked glucosides"Lipids. 37. 101-110 (2002)

[Publications] Norihiro Kobayashi: "A monoclonal antibody-based enzyme-linked immunosorbent assay of glycolithocholic acid sulfate in human urine for liver function test"Steroids. 67. 827-833 (2002)

[Publications] Nariyasu Mano: "Rapid and simple quantitative assay method for diastereomeric flurbiprofen glucuronides in the incubation mixture"Journal of Chromatography B. 776. 125-131 (2002)

[Publications] Norihiro Kobayashi: "Single-chain Fv fragments derived from an anti-11-deoxycortisol antibody : Affinity, specificity, and idiotype analysis"Steroids. 67. 733-742 (2002)

[Publications] Toshiaki Kume: "Isolation of a novel diterpenoid substance with potent neuroprotective activity from fetal calf serum"Proceedings of National Academy of Sciences in USA. 99. 3288-3293 (2002)

[Publications] Takashi Iida: "Potential bile acid metabolites. 25. Synthesis and chemical property of stereoisomeric 3α,7α,16-and 3α,7α,15-trihydroxy-5β-cholan-24-oic acids"Chemical and Pharmaceutical Bulletin. 50. 1335-1340 (2002)

[Publications] Takashi Iida: "Direct capillary gas chromatographic analysis and thermal stability of carboxy-linked glucosides of bile acids"Chromatographia. 56. 489-494 (2002)

[Publications] Nariyasu Mano: "Separation and determination of diastereomeric flurbiprofen acyl glucuronides in human urine by LC/ESI-MS with a simple column-switching technique"Drug Metabolism and Pharmacokinetics. 17. 142-149 (2002)

[Publications] Sookie La: "Enantioseparation of chiral aromatic amino acids by capillary electrophoresis in neutral and charged cyclodextrin selector modes"Electrophoresis. 23. 4123-4131 (2002)

[Publications] Norihiro Kobayashi: "Generation of a novel monoclonal antibody against cortisol-[C-4]-bovine serum albumin conjugate : Application to enzyme-linked immunosorbent assay for urinary and serum cortisol"Analytical Sciences. 18. 1309-1314 (2002)

[Publications] Norihiro Kobayashi: "Idiotype-anti-idiotype-based noncompetitive enzyme-linked immunosorbent assay of ursodeoxycholic acid 7-N-acetylglucosaminides in human urine with subfemtomole range sensitivity"Journal of Immunological Methods. 272. 1-10 (2003)

[Publications] Norihiro Kobayashi: "Monoclonal anti-idiotype antibodies recognizing the variable region of a high-affinity antibody against 11-deoxycortisol. Production, characterization and application to a sensitive noncompetitive immunoassay"Journal of Immunological Methods. 274. 63-75 (2003)

[Publications] Takashi Iida: "^1H and ^<13>C signal assignments of carboxyl-linked glucosides of bile acids"Magnetic Resonance in Chemistry. (in press).

[Publications] Takashi Iida: "Functionalization of unactivated carbons in 3α,6-and 3α,24-dihydroxy-5β-cholan derivatives by dimetyldioxirane"Lipids. (in press).

後藤順一東北大学, 医学部附属病院, 教授 (80006337)