Enhancing the practicability of the method for proteome analysis based on a new isolation scheme of C-terminal peptides.

• Project/Area Number: 21510225
• Research Category: Grant-in-Aid for Scientific Research (C)
• Allocation Type: Single-year Grants
• Section: 一般
• Research Field: Living organism molecular science
• Research Institution: Nara Women's University
• Principal Investigator: NAKAZAWA Takashi 奈良女子大学, 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, 特任准教授 (60437332)
• Project Period (FY): 2009 – 2011
• Project Status: Completed (Fiscal Year 2011)
• Budget Amount: ¥4,680,000 (Direct Cost: ¥3,600,000、Indirect Cost: ¥1,080,000); Fiscal Year 2011: ¥1,040,000 (Direct Cost: ¥800,000、Indirect Cost: ¥240,000); Fiscal Year 2010: ¥1,300,000 (Direct Cost: ¥1,000,000、Indirect Cost: ¥300,000); Fiscal Year 2009: ¥2,340,000 (Direct Cost: ¥1,800,000、Indirect Cost: ¥540,000)
• Keywords: タンパク質 / C末端ペプチド / プロテオミクス / アミノ酸配列解析 / 質量分析 / 化学修飾 / 安定同位体標識 / 強塩基性陰イオン交換体 / ペプチドの分離 / イオン交換クロマトグラフィー / 文化財科学

Research Abstract

We have established a method to identify proteins by MALDI mass spectrometry of the C-terminal peptide obtained from a protein in which all the carboxyl groups were protected by exhaustive amidation. The optimized protocol to concentrate or isolate the C-terminal peptide from the proteolytic digest of the fully amidated protein includes the use of a strong anion-exchange HPLC column to remove anionic peptides.

Research Products

• [Journal Article] Polymorphism of Collagen Triple Helix Revealed by (19)F NMR of Mode 1 Peptide [Pro-4(R)-Hydroxyprolyl-Gly](3)-[Pro-4(R)-Fluoroprolyl-Gly]-[Pro-4(R)-Hydroxyprolyl-Gly](3) (2012)
  • Author(s): Kawahara K, Nemoto N, Motooka D, Nishi Y, Doi M, Uchiyama S, Nakazawa T, Nishiuchi Y, Yoshida T, Ohkubo T, Kobayashi Y
  • Journal Title: J Phys Chem B. Volume: 116 Issue: 23 Pages: 6908-6915
  • DOI: 10.1021/jp212631q
  • Peer Reviewed
• [Journal Article] Enzymatic conversion of arginine to citrulline for improving the fragmentation of N^α-tris(2, 4, 6-trimethoxyphenyl) phosphonium acetylated peptides by tandem mass spectrometry (2011)
  • Author(s): Hiroki Kuyama, Chihiro Nakajima, Takashi, Nakazawa, Osamu Nishimura
  • Journal Title: Anal. Methods Volume: 3 Issue: 12 Pages: 2829-2835
  • DOI: 10.1039/c1ay05177f
  • Peer Reviewed
• [Journal Article] Specific racemization of heavy-chain cysteine-220 in the hinge region of immunoglobulin gamma 1 as a possible cause of degradation during storage (2011)
  • Author(s): Masato Amano, Jun Hasegawa, Naoki Kobayashi, Naoyuki Kishi, Takashi Nakazawa, Susumu Uchiyama, Kiichi Fukui.
  • Journal Title: Anal. Chem Volume: 83 Issue: 10 Pages: 3857-3864
  • DOI: 10.1021/ac200321v
  • Peer Reviewed
• [Journal Article] A method for N-terminal de novo sequencing of N^α-blocked proteins by mass spectrometry (2011)
  • Author(s): Chihiro Nakajima, Hiroki Kuyama, Takashi Nakazawa, Osamu Nishimura, and Susumu Tsunasawa
  • Journal Title: Analyst Volume: 136 Issue: 1 Pages: 113-119
  • DOI: 10.1039/c0an00384k
  • NAID: 10028237483
  • Peer Reviewed
• [Journal Article] Enzymatic conversion of arginine to citrulline for improving the fragmentation of N-tris(2,4,6-trimethoxyphenyl)phosphonium acetylaled peptides by tandem mass spectrometry (2011)
  • Author(s): Hiroki Kuyama
  • Journal Title: Anal.Methods Volume: 3 Pages: 2829-2835
  • Peer Reviewed
• [Journal Article] The triple helical structure and stability of collagen model peptide with 4(S)-hydroxyprolyl-pro-gly units (2011)
  • Author(s): Motooka D, Kawahara K, Nakamura S, Doi M, Nishi Y, Nishiuchi Y, Kee Kang Y, Nakazawa T, Uchiyama S, Yoshida T, Ohkubo T, Kobayashi Y
  • Journal Title: Biopolymers Volume: 98 Issue: 2 Pages: 111-121
  • DOI: 10.1002/bip.21730
  • Peer Reviewed
• [Journal Article] A method for N^α-terminal de novo sequencing of N-blocked proteins by mass spectrometry (2011)
  • Author(s): Chihiro Nakajima
  • Journal Title: Analyst Volume: 136 Pages: 113-119
  • Peer Reviewed
• [Journal Article] Specific racemization of heavy-chain cysteine-220 in the hinge region of immunoglobulin gamma 1 as a possible cause of degradation during storage (2011)
  • Author(s): Masato Amano
  • Journal Title: Anal.Chem. Volume: 83(in press)
  • Peer Reviewed
• [Journal Article] Conversion of arginine to ornithine for improving the fragmentation pattern of peptides labeled with the N-terminal tris(2, 4, 6-trimethoxyphenyl) phosphonium group in tandem mass spectrometry (2010)
  • Author(s): Hiroki Kuyama, Chihiro Nakajima, Takashi, Nakazawa, Osamu Nishimura
  • Journal Title: Anal. Methods Volume: 2 Issue: 11 Pages: 1792-1797
  • DOI: 10.1039/c0ay00439a
  • NAID: 10028237480
  • Peer Reviewed
• [Journal Article] Conversion of arginine to omithine for improving the fragmentation pattern of peptides labeled with the N-terminal tris(2,4,6-trimethoxyphenyl)phosphonium group in tandem mass spectrometry (2010)
  • Author(s): Hiroki Kuyama
  • Journal Title: Anal.Methods Volume: 2 Pages: 1792-1797
  • Peer Reviewed
• [Journal Article] Structure and reaction mechanism of human nicotinamide phosphoribosyl-transferase (2010)
  • Author(s): Ryo Takahashi
  • Journal Title: J.Biochem. 147 Pages: 95-107
  • Peer Reviewed
• [Journal Article] A new approach for detecting C-terminal amidation of proteins and peptides by mass spectrometry in conjunction with chemical derivatization (2009)
  • Author(s): Hiroki Kuyama, Chihiro Nakajima, Takashi Nakazawa, Osamu Nishimura, Susumu Tsunasawa
  • Journal Title: Proteomics Volume: 9 Issue: 16 Pages: 4063-4070
  • DOI: 10.1002/pmic.200900267
  • NAID: 40017076671
  • Peer Reviewed
• [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 Pages: 613-624
  • Peer Reviewed
• [Journal Article] A new approach for detecting C-terminal amidation of proteins and pep-tides by mass spectrometry in conjunction with chemical derivatization (2009)
  • Author(s): Hiroki Kuyama
  • Journal Title: Proteomics 9 Pages: 4063-4070
  • Peer Reviewed
• [Journal Article] C-Terminal sequencing of proteins by MALDI mass spectrometry through the specific derivatization of theα-carboxyl group with 3-aminopropyltris(2, 4, 6-trimethoxyphenyl) phosphonium bromide
  • Author(s): Chihiro Nakajima, Hiroki Kuyama, Takashi Nakazawa, Osamu Nishimura
  • Journal Title: Anal. Bioanal. Chem Volume: (in press)
  • Peer Reviewed
• [Presentation] 質量分析による牽牛子塚古墳出土夾紵棺断片中の絹の確認 (2011)
  • Author(s): 宮路淳子, 河原一樹, 六車美保, 松尾良樹, 岡田文男, 中沢隆
  • Organizer: 第28回日本文化財科学会
  • Place of Presentation: 筑波大学
  • Year and Date: 2011-06-11
• [Presentation] 質量分析による牽牛子塚古墳出上夾紵棺断片中の絹の確認 (2011)
  • Author(s): 中沢隆
  • Organizer: 第28回日本文化財科学会
  • Place of Presentation: 筑波大学
  • Year and Date: 2011-06-11
• [Presentation] Identification of animal species by the MALDI-MS of collagen in animal glues used in archaeological materials (2010)
  • Author(s): Takashi Nakazawa, Yuzo Yamazaki, Atsuko Miyaji, Yoshiki Matsuo
  • Organizer: 58^<th> ASMS Conference on Mass Spectrometry and Allied Topics
  • Place of Presentation: Salt Lake City, Utah, USA
  • Year and Date: 2010-05-27
• [Presentation] Isolation of C-Terminal Peptides by Strong Anion-Exchanger from Proteolytic Digests of Fully Amidated Proteins for Mass Spectrometric Sequencing (2009)
  • Author(s): Mariko Nakagawa, Hiroki Kuyama, Minoru Yamaguchi, Eiji Ando, Osamu Nishimura, Susumu Tsunasawa, Takashi Nakazawa
  • Organizer: 57^<th> ASMS Conference on Mass Spectrometry and Allied Topics
  • Place of Presentation: Philadelphia, PA, USA
  • Year and Date: 2009-06-02
• [Remarks]
  • URL: http://www.nara-wu.ac.jp/proteome/aprp/index.html
• [Remarks]
  • URL: http://www.nara-wu.ac.jp/proteome/aprp/index.html
• [Patent(Industrial Property Rights)] タンパク質又はペプチドのC末端を修飾する方法 (2010)
  • Inventor(s): 中澤隆,山口実、九山浩樹、安藤英治、上山憲一、岡村高明、乗岡茂巳
  • Industrial Property Rights Holder: (株)島津製作所
  • Acquisition Date: 2010-07-30
• [Patent(Industrial Property Rights)] 生体試料の構造解析法 (2009)
  • Inventor(s): 中澤隆,安藤英治,山口実
  • Industrial Property Rights Holder: (株)島津製作所
  • Acquisition Date: 2009-11-20
• [Patent(Industrial Property Rights)] 生体試料の構造解析法 (2009)
  • Inventor(s): 中澤隆
  • Industrial Property Rights Holder: (株)島津製作所
  • Acquisition Date: 2009-11-20