Development of the method for proteome analysis based on a novel chemical derivatization specific to C-termini of proteins

• Project/Area Number: 18310146
• Research Category: Grant-in-Aid for Scientific Research (B)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Living organism molecular science
• Research Institution: Nara Women's University
• Principal Investigator: NAKAZAWA Takashi Nara Women's University, 理学部, 教授 (30175492)
• Co-Investigator(Renkei-kenkyūsha): KUYAMA Hiroki 大阪大学, 蛋白質研究所寄付研究部門, 准教授 (60437332) ; NISHIMURA Osamu 大阪大学, 蛋白質研究所寄付研究部門, 教授 (10374125) ; GASKELL Simonj University of Manchester, Director of the Michael Barber Centre for Mass Spectrometry, Professor [Vice President (Research) of the [Vice President (Research) of the University]
• Project Period (FY): 2006 – 2008
• Project Status: Completed (Fiscal Year 2008)
• Budget Amount: ¥17,880,000 (Direct Cost: ¥15,600,000、Indirect Cost: ¥2,280,000); Fiscal Year 2008: ¥4,290,000 (Direct Cost: ¥3,300,000、Indirect Cost: ¥990,000); Fiscal Year 2007: ¥5,590,000 (Direct Cost: ¥4,300,000、Indirect Cost: ¥1,290,000); Fiscal Year 2006: ¥8,000,000 (Direct Cost: ¥8,000,000)
• Keywords: プロテオミクス / C末端アミノ酸配列解析 / 化学修飾 / MALDI-MS / 質量分析 / オキサゾロン / 翻訳後修飾 / Terminus proteomics / アミノ酸配列解析 / タンパク質 / ペプチド化学 / Terminal Proteomics / タンパク質のC末端 / C末端アミノ酸配列分析 / 活性エステル / C末端標識

Research Abstract

タンパク質のC末端アミノ酸配列解析法の開発のために、本研究ではペプチドの選択的C末端誘導体化の反応条件を最適化した結果、簡単なアミド化において収率を平均60%以上にすることに成功した。この誘導体化と新規N末端標識に基づくC末端ペプチド単離法を組み合わせることで、C末端が翻訳後修飾されたペプチドのマトリックス支援レーザー脱離イオン化質量分析法(MALDI-MS)によるアミノ酸配列解析を可能にした。

Research Products

• [Journal Article] Preparation and conformational analysis of C-glycosyl β^2- and β/β^2-peptides (2009)
  • Author(s): Yoko Inaba
  • Journal Title: Carbohydrate Research 344 (5) Pages: 613-626
  • Peer Reviewed
• [Journal Article] Selective isolation of N-terminal peptides from proteins and their de novo sequencing by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry without regard to unblocking or blocking of N-terminal amino acids (2008)
  • Author(s): Minoru Yamaguchi, Daisuke Nakayama, Keisuke Shima, Hiroki Kuyama, Eiji Ando, Taka-aki Okamura, Norikazu Ueyama, Takashi Nakazawa, Shigemi Norioka, Osamu Nishimura, and Susumu Tsunasawa
  • Journal Title: Rapid Commun. Mass Spectrom 22 Pages: 3313-3319
  • Peer Reviewed
• [Journal Article] Determination of pKa values of individual histidine residues in proteins using mass spectrometry (2008)
  • Author(s): Masaru Miyagi and Takashi Nakazawa
  • Journal Title: (Accelerated article) Anal Chem. 80 Pages: 6481-6487
  • Peer Reviewed
• [Journal Article] Terminal proteomics : N-and C-terminal analyses for high-fidelity identification of proteins using mass spectrometry (2008)
  • Author(s): Takashi Nakazawa, Minoru Yamaguchi, Taka-aki Okamura, Eiji Ando, Osamu Nishimura, and Susumu Tsunasawa
  • Journal Title: Proteomics 8 Pages: 673-685
  • Peer Reviewed
• [Journal Article] Selective isolation of N-terminal peptides from proteins and their de novo sequencing by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry without regard to unblocking or blocking of N-terminal amino acids. (2008)
  • Author(s): Minoru Yamaguchi
  • Journal Title: Rapid Communications in Mass Spectrometry 22 (20) Pages: 3313-3319
  • Peer Reviewed
• [Journal Article] Determination of pK_a values of individual histidine residues in proteins using mass spectrometry (2008)
  • Author(s): Masaru Miyagi
  • Journal Title: Analytical Chemistry. 80(17) Pages: 6481-6487
  • Peer Reviewed
• [Journal Article] Terminal proteomics: N-and C-terminal analyses for high-fidelity identification of proteins using mass spectrometry (2008)
  • Author(s): Nakazawa, Takashi
  • Journal Title: Proteomics 8 Pages: 673-685
  • Peer Reviewed
• [Journal Article] Specific isolation of N-terminal fragments from proteins and their high-fidelity de novo sequencing (2007)
  • Author(s): Minoru Yamaguchi, Takashi Obama, Hiroki Kuyama, Daisuke Nakayama, Eiji Ando, Taka-aki Okamura, Norikazu Ueyama, Takashi Nakazawa, Shigemi Norioka, Osamu Nishimura, and Susumu Tsunasawa
  • Journal Title: Rapid Commun. Mass Spectrom 21 Pages: 3329-3326
  • Peer Reviewed
• [Journal Article] Simultaneous detection of N-terminal fragment ions in a protein mixture using a ruthenium(II) complex (2007)
  • Author(s): Akihiro Ito, Taka-aki Okamura, Hitoshi Yamamoto, Norikazu Ueyama, Minoru Yamaguchi, Hiroki Kuyama, Eiji Ando, Susumu Tsunasawa, Kojiro Ake, Ryoji Masui, Seiki Kuramitsu, Takashi Nakazawa, and Shigemi Norioka
  • Journal Title: Rapid Commu. Mass Spectrom 21 Pages: 2647-2653
  • Peer Reviewed
• [Journal Article] High sequence-coverage detection of proteolytic peptides using a bis(terpyridine)ruthenium(II) complex (2007)
  • Author(s): Akihiro Ito, Taka-aki Okamura, Ken Masui, Maki Kaneko, Ryoji Masui, Kojiro Ake, Seiki Kuramitsu, Minoru Yamaguchi, Hiroki Kuyama, Eiji Ando, Shigemi Norioka, Takashi Nakazawa, Susumu Tsunasawa, Hitoshi Yamamoto, and Norikazu Ueyama
  • Journal Title: Analyst 132 Pages: 358-364
  • NAID: 120004837655
  • Peer Reviewed
• [Journal Article] Tautomerism of histidine 64 associated with proton-transfer in catalysis of carbonic anhydrase (2007)
  • Author(s): Hideto Shimahara, Takuya Yoshida, Yasutaka Shibata, Masato Shimizu, Yoshimasa Kyogoku, Fumio Sakiyama, Takashi Nakazawa, Shin-ichi Tate, Shin-ya Ohki, Takeshi Kato, Hozumi Moriyama, Ken-ichi Kishida, Yasuo Tano, Tadayasu Ohkubo, and Yuji Kobayashi
  • Journal Title: J. Biol. Chem. 282 Pages: 9646-9656
  • Peer Reviewed
• [Journal Article] High sequence-coverage detection of proteolytic peptides using a bis-(terpyridine)ruthenium(II)cnmplex (2007)
  • Author(s): Ito, Akihito
  • Journal Title: Analyst 132 Pages: 358-364
  • Peer Reviewed
• [Journal Article] Simultaneous detection of N-terminal fragment ions in a protein mixture using a ruthenium(II)complex (2007)
  • Author(s): Ito, Akihito
  • Journal Title: Rapid Commun. Mass Spectrom. 21 Pages: 2647-2653
  • Peer Reviewed
• [Journal Article] Specific isolation of N-terminal fragments from proteins and their high-fidelity de novo sequencing (2007)
  • Author(s): Yamaguchi, Minoru
  • Journal Title: Rapid Commun. Mass Spectrom. 21 Pages: 3329-3336
  • Peer Reviewed
• [Journal Article] Tautomerism of histidine 64 associated with proton-transfer in catalysis of carbonic anhydrase (2007)
  • Author(s): Hideto Shimahara
  • Journal Title: Journal of Biological Chemistry 282 Pages: 9646-9656
• [Journal Article] Enhancement of MALDI-MS spectra of Cterminal peptides by the modification of proteins via an active ester generated in situ from an oxazolone (2006)
  • Author(s): Minoru Yamaguchi, Mutsumi Oka, Kimiko Nishida, Mayu Ishida, Ayako Hamazaki, Hiroki Kuyama, Eiji Ando, Taka-aki Okamura, Norikazu Ueyama, Shigemi Norioka, Osamu Nishimura, Susumu Tsunasawa, and Takashi Nakazawa
  • Journal Title: Anal. Chem. 78 Pages: 7861-7869
  • Peer Reviewed
• [Journal Article] Chemistry in proteomics : an interplay between classical methods in chemical modification of proteins and mass spectrometry at the cutting edge (2006)
  • Author(s): Takashi Nakazawa
  • Journal Title: Current Proteomics 3 Pages: 33-54
  • Peer Reviewed
• [Journal Article] Chemistry in Proteomics : An inteiplay between classical methods in chemical modification of proteins and mass spectrometry at the cutting edge (2006)
  • Author(s): Takashi Nakazawa
  • Journal Title: Current Proteomics 3(1) Pages: 33-54
• [Journal Article] Enhancement of MALDI-MS spectra of C-terminal peptides by the modification of proteins via an active ester generated in situ from an oxazolone (2006)
  • Author(s): Minoru Yamaguchi
  • Journal Title: Analytical Chemistry 78(22) Pages: 7861-7869
• [Journal Article] C-Terminal activation of peptides with an active ester via an oxazolone (2006)
  • Author(s): Mutsumi Oka
  • Journal Title: Peptide Science 2006 Pages: 325-326
• [Journal Article] A new approach for detecting C-terminal amidation of proteins and peptides by mass spectrometry in conjunction with chemical derivatization
  • Author(s): Hiroki Kuyama, Chihiro Nakajima, Takashi Nakazawa, Osamu Nishimura, and Susumu Tsunasawa
  • Journal Title: Proteomics, 9 (in press)
  • NAID: 40017076671
  • Peer Reviewed
• [Presentation] Isolation and mass spectrometric sequencing of C-terminal peptides based on exhaustive amidation of proteins (2008)
  • Author(s): Mariko Nakagawa, Minoru Yamaguchi, Hiroki Kuyama, Chihiro Nakajima, Susumu Tsunasawa, Osamu Nishimura, Eiji Ando, and Takashi Nakazawa
  • Organizer: BMB2007(第31回日本分子生物学会・第81回日本生化学会合同大会)
  • Place of Presentation: 神戸ポートアイランド
  • Year and Date: 2008-12-12
• [Presentation] Isolation of C-terminal peptides of proteins by exhaustive amidation followed by proteolytic digestion for sequencing with mass spectrometry (2008)
  • Author(s): Mariko Nakagawa, Minoru Yamaguchi, Hiroki Kuyama, Chihiro Nakajima, Eiji Ando, Susumu Tsunasawa, Osamu Nishimura, and Takashi Nakazawa
  • Organizer: 17^<th> Meeting of Methods in Proteins Structure Analysis
  • Place of Presentation: 北海道大学
  • Year and Date: 2008-08-27
• [Presentation] Isolation of C-terminal peptides of proteins by exhaustive amidation followed by proteolytic digestion for mass spectrometric sequencing (2008)
  • Author(s): Mariko Nakagawa, Minoru Yamaguchi, Hiroki Kuyama, Eiji Ando, Susumu Tsunasawa, Osamu Nishimura, and Takashi Nakazawa
  • Organizer: 56^<th> ASMS Conference on Mass Spectrometry and Allied Topics
  • Place of Presentation: Denver, Colorado, USA
  • Year and Date: 2008-06-02
• [Presentation] Isolation of C-terminal peptides of proteins by exhaustive amidation followed by proteolytic digestion for mass spectrometric sequencing. (2008)
  • Author(s): Takashi Nakazavva
  • Organizer: 56^<th> ASMS Conference on Mass Spectrometry and Allied Tonics
  • Place of Presentation: Denver, Colorado, USA
  • Year and Date: 2008-06-02
• [Presentation] MALDI-MSを用いたタンパク質のC末端ヒドラジド化によるアミノ酸配列解析法 (2007)
  • Author(s): 中島ちひろ, 中川真理子, 水谷文, 辻智恵美, 岡むつみ, 山口実, 綱澤進, 西村紀, 中沢隆
  • Organizer: BMB2007(第30回日本分子生物学会・第80回日本生化学会合同大会)
  • Place of Presentation: パシフィコ横浜
  • Year and Date: 2007-12-12
• [Presentation] C-Terminal Derivatization of proteins via hydrazides for sequencing with MALDI-MS (2007)
  • Author(s): Takashi Nakazawa, Minoru Yamaguchi, Mariko Nakagawa, Mutsumi Oka, Chihiro Nakajima, Hiroki Kuyama, Eiji Ando, Daisuke Nakayama, Taka-aki Okamura, Osamu Nishimura, and Susumu Tsunasawa
  • Organizer: 55^<th> ASMS Conference on Mass Spectrometry and Allied Topics
  • Place of Presentation: Indianapolis, Indiana, USA
  • Year and Date: 2007-06-05
• [Presentation] C-Terminal derivatization of proteins via hydrazides for sequencing with MALDI-MS (2007)
  • Author(s): Nakazawa, Takashi
  • Organizer: 55th ASMS Conference on Mass Spectrometry
  • Place of Presentation: Indianapolis, IN, USA
  • Year and Date: 2007-06-05
• [Presentation] MALDI質量分析のためのC末端ヒドラジド化に基づくペプチドの標識法 (2007)
  • Author(s): 中川真理子,中島ちひろ, 山口実, 岡むつみ, 辻智恵美, 九山浩樹, 安藤英治, 岡村高明, 綱澤進, 西村紀, 中沢隆
  • Organizer: 第7回日本蛋白質科学会年会
  • Place of Presentation: 東北大学
  • Year and Date: 2007-05-24
• [Presentation] C-Terminal activation of peptides with an active ester via an oxazolone (2006)
  • Author(s): Mutsumi Oka, Mariko Yamamoto, Chihiro Nakajima, Chiemi Tsuji, Mariko Nakagawa, Minoru Yamaguchi, Kimiko Nishida, Susumu Tsunasawa and Takashi Nakazawa
  • Organizer: Int Conference of 43^<rd> Japanese Peptide Symposium/4^<th> Peptide Engineering Meeting
  • Place of Presentation: パシフィコ横浜
  • Year and Date: 2006-11-05
• [Presentation] MALDI mass spectrometric analysis of proteins by charge tagging at the C-terminus (2006)
  • Author(s): Takashi Nakazawa, Minoru Yamaguchi, Kimiko Nishida, Mutsumi Oka, Ayako Hamazaki, Hiroki Kuyama, Eiji Ando, Taka-aki Okamura, Norikazu Ueyama, Shigemi Norioka, and Susumu Tsunasawa
  • Organizer: 20^<th> IUBMB International Congress of Biochemistry and Molecular Biology
  • Place of Presentation: 京都国際会議場
  • Year and Date: 2006-06-22
• [Presentation] C-Terminal charge-tagging of proteins for the sequencing with MALDI-MS (2006)
  • Author(s): Takashi Nakazawa, Minoru Yamaguchi, Kimiko Nishida, Mutsumi Oka, Ayako Hamazaki, Hiroki Kuyama, Eiji Ando, Taka-aki Okamura, Norikazu Ueyama, Shigemi Norioka, and Susumu Tsunasawa
  • Organizer: 55^<th> ASMS Conference on Mass Spectrometry and Allied Topics
  • Place of Presentation: Seattle, Washington, USA
  • Year and Date: 2006-05-30
• [Book] 「金属錯体と金属タンパク質の質量分析」生命元素事典(桜井弘編)第4章, 2項〔7〕 (2006)
  • Author(s): 中沢隆、矢野重信
  • Publisher: オーム社
• [Book] 生命元素事典(桜井 弘編)第4章2-7 金属錯体と金属クンパク質の質量分析 (2006)
  • Author(s): 中沢 隆, 矢野重信
  • Publisher: オーム社
• [Remarks]
  • URL: http://www.chem.nara-wu.ac.jp/~nakazawa/
• [Remarks] セミナーでの講演Takashi Nakazawa. "Protein Chemistry and Enzymology as Fundamentals for Proteomics", in Proteomics and Functional Genomics Group Special Seminar, 23^<rd> September 2008, Department of Veterinary Science, University of Liverpool, Liverpool, UK.
• [Patent(Industrial Property Rights)] ペプチドのアミノ酸配列決定法 (2008)
  • Inventor(s): 山口実、安藤英治、乗岡茂巳、上山憲一、岡村高明、中澤隆
  • Industrial Property Rights Holder: 株式会社島津製作所
  • Acquisition Date: 2008-12-19
• [Patent(Industrial Property Rights)] イソチオシアネート基を有する金属錯体、及びそれを用いるタンパク質のアミノ酸配列決定方法 (2008)
  • Inventor(s): 上山憲一、岡村高明、乗岡茂巳、中澤隆、山口実、安藤英治
  • Industrial Property Rights Holder: 株式会社島津製作所
  • Acquisition Date: 2008-11-07
• [Patent(Industrial Property Rights)] ペプチドのアミノ酸配列決定法 (2008)
  • Inventor(s): 山口 実
  • Industrial Property Rights Holder: 島津製作所
  • Acquisition Date: 2008-12-19
• [Patent(Industrial Property Rights)] Method for modifying protein or peptide C-terminal (2007)
  • Inventor(s): Takashi Nakazawa, Minoru Yamaguchi, Hiroki Kuyama, Eiji Ando, Norikazu Ueyama, Taka-aki Okamura, and Shigemi Norioka
  • Industrial Property Rights Holder: Shimadzu Corporation
  • Acquisition Date: 2007-11-13
  • Overseas
• [Patent(Industrial Property Rights)] タンパク質又はペプチドのC末端修飾法、C末端固定化法、及び解析法 (2006)
  • Inventor(s): 中澤隆、岡むつみ、西田希実子、山口 実、九山浩樹、安藤英治、上山憲一、岡村高明、綱澤進
  • Industrial Property Rights Holder: 株式会社 島津製作所、国立大学法人奈良女子大学、国立大学法人大阪大学(JST特許出願支援 : #S2006-0043PTC)
  • Industrial Property Number: 2005-307831
  • Filing Date: 2006-10-20
  • Overseas
• [Patent(Industrial Property Rights)] タンパク質のN末端フラグメントを選択的に回収する方法 (2006)
  • Inventor(s): 乗岡茂巳、上山憲一、岡村高明、中澤隆、山口実、安藤英治、綱澤進
  • Industrial Property Rights Holder: 株式会社島津製作所
  • Acquisition Date: 2006-05-09
• [Patent(Industrial Property Rights)] タンパク質又はペプチドのC末端修飾法、C末端固定化法、及び解析法 (2006)
  • Inventor(s): 中澤 隆ほか8名
  • Industrial Property Rights Holder: 奈良女子大学(4), 島津製作所(4), 大阪大学(2)
  • Industrial Property Number: 2005-307831
  • Filing Date: 2006-10-20