公开(公告)号：US07579165B2

公开(公告)日：2009-08-25

申请号：US10962575

申请日：2004-10-13

申请人： Shigemi Norioka ,  Minoru Yamaguchi ,  Hiroki Kuyama ,  Takashi Obama ,  Eiji Ando ,  Takashi Nakazawa ,  Norikazu Ueyama ,  Taka-aki Okamura

发明人： Shigemi Norioka ,  Minoru Yamaguchi ,  Hiroki Kuyama ,  Takashi Obama ,  Eiji Ando ,  Takashi Nakazawa ,  Norikazu Ueyama ,  Taka-aki Okamura

IPC分类号： C07K1/13 ,  C07K1/14 ,  C07K14/00 ,  C12P21/06 ,  C12Q1/00 ,  C12Q1/34 ,  C12Q1/37 ,  G01N33/53 ,  G01N33/38 ,  G01N33/483

CPC分类号： C12N9/2462 ,  C07K1/1077 ,  C07K1/128 ,  C07K14/78

摘要： The present invention provides a method for effectively introducing sulfonic acid groups into the N-terminus of a protein or a peptide. The method comprises a modification step to react a N-terminus in a protein or peptide with a compound A which includes disulfide group and a cleavage step to cleave a disulfide bond of the disulfide group to convert into a sulfonic acid group. The present invention also provides a method of analyzing proteins or peptides easily and effectively on mass spectrometry and an intermediate that can be used to effectively derivatize proteins or peptides as sulfonic acid derivatives.

摘要翻译： 本发明提供了将磺酸基团有效地引入蛋白质或肽的N-末端的方法。 该方法包括使蛋白质或肽中的N末端与包含二硫键的化合物A和切割二硫键的二硫键以切割成磺酸基团的切割步骤进行反应的修饰步骤。 本发明还提供了一种在质谱分析中容易且有效地分析蛋白质或肽的方法，以及可用于有效地将蛋白质或肽衍生为磺酸衍生物的中间体。

公开(公告)号：US20050084927A1

公开(公告)日：2005-04-21

申请号：US10962575

申请日：2004-10-13

申请人： Shigemi Norioka ,  Minoru Yamaguchi ,  Hiroki Kuyama ,  Takashi Obama ,  Eiji Ando ,  Takashi Nakazawa ,  Norikazu Ueyama ,  Taka-aki Okamura

发明人： Shigemi Norioka ,  Minoru Yamaguchi ,  Hiroki Kuyama ,  Takashi Obama ,  Eiji Ando ,  Takashi Nakazawa ,  Norikazu Ueyama ,  Taka-aki Okamura

IPC分类号： C07K1/107 ,  C07K1/12 ,  C07K14/78 ,  C12N9/36 ,  C12P21/06 ,  C07K14/00

CPC分类号： C12N9/2462 ,  C07K1/1077 ,  C07K1/128 ,  C07K14/78

摘要： The present invention provides a method for effectively introducing sulfonic acid groups into the N-terminus of a protein or a peptide; a method capable of analyzing proteins or peptides easily and effectively on mass spectrometry; an intermediate that can be used to effectively derivatize proteins or peptides as sulfonic acid derivatives. A method for derivatizing a protein or peptide to a sulfonic acid derivative, comprising steps of: modification step to react a N-terminus in a protein or peptide with a compound A which includes disulfide group, to obtain a protein or peptide modified with the compound A at the N-terminus; and cleavage step to cleave a disulfide bond of the disulfide group to convert into a sulfonic acid group, thereby converting the modified protein or peptide into a sulfonic acid derivative. A method for analyzing the amino acid sequence of a protein or peptide, wherein the sulfonic acid derivative of a protein or a peptide obtained by the above method is subjected to mass spectrometry. A protein or peptide modified with a disulfide group-containing group at the N-terminus.

摘要翻译： 本发明提供了有效地将磺酸基引入蛋白质或肽的N末端的方法; 能够在质谱上容易且有效地分析蛋白质或肽的方法; 可用于有效地将蛋白质或肽衍生为磺酸衍生物的中间体。 一种将蛋白质或肽衍生为磺酸衍生物的方法，包括以下步骤：使蛋白质或肽中的N末端与包含二硫键的化合物A反应的修饰步骤，以获得用化合物修饰的蛋白质或肽 A在N端; 以及切割二硫键的二硫键转化为磺酸基的切割步骤，从而将修饰的蛋白质或肽转化为磺酸衍生物。 分析蛋白质或肽的氨基酸序列的方法，其中通过上述方法获得的蛋白质或肽的磺酸衍生物进行质谱分析。 在N末端用含二硫基的基团修饰的蛋白质或肽。

公开(公告)号：US07294707B2

公开(公告)日：2007-11-13

申请号：US10958298

申请日：2004-10-06

申请人： Takashi Nakazawa ,  Minoru Yamaguchi ,  Hiroki Kuyama ,  Eiji Ando ,  Norikazu Ueyama ,  Taka-aki Okamura ,  Shigemi Norioka

发明人： Takashi Nakazawa ,  Minoru Yamaguchi ,  Hiroki Kuyama ,  Eiji Ando ,  Norikazu Ueyama ,  Taka-aki Okamura ,  Shigemi Norioka

IPC分类号： C07K1/00

CPC分类号： C07K1/003

摘要： A simple and low-cost method of selectively modifying the C-terminal of a protein or peptide is provided. A method of modifying the C-terminal of a protein or peptide comprises forming an intramolecular oxazolone ring at the C-terminal of the protein or peptide that requires C-terminal modification, and then performing a ring-opening of the oxazolone ring to produce a protein or peptide with a modified C-terminal. Preferred forms include a method in which by reacting the oxazolone ring with a compound containing a nucleophilic group to effect an oxazolone ring-opening, a protein or peptide is produced in which the C-terminal is modified with the compound containing the nucleophilic group, as well as a method in which by reacting the oxazolone ring with an active esterifying agent to effect a ring-opening, the oxazolone is converted to an active ester, which by subsequent reaction with a compound containing a nucleophilic group, produces a protein or peptide in which the C-terminal has been modified with the compound containing the nucleophilic group.

摘要翻译： 提供了选择性修饰蛋白质或肽的C-末端的简单且低成本的方法。 修饰蛋白质或肽的C末端的方法包括在需要C末端修饰的蛋白质或肽的C-末端形成分子内的恶唑酮环，然后进行恶唑酮环的开环以产生 具有修饰C末端的蛋白质或肽。 优选的形式包括通过使恶唑酮环与含有亲核基团的化合物反应以进行恶唑酮开环的方法，制备其中C-末端用含有亲核基团的化合物修饰的蛋白质或肽作为 以及通过使恶唑酮环与活性酯化剂反应以开环的方法，将恶唑酮转化成活性酯，其随后与含有亲核基团的化合物反应，产生蛋白质或肽 其中C-末端已经与含有亲核基团的化合物一起被修饰。

公开(公告)号：US20050085622A1

公开(公告)日：2005-04-21

申请号：US10958298

申请日：2004-10-06

申请人： Takashi Nakazawa ,  Minoru Yamaguchi ,  Hiroki Kuyama ,  Eiji Ando ,  Norikazu Ueyama ,  Taka-aki Okamura ,  Shigemi Norioka

发明人： Takashi Nakazawa ,  Minoru Yamaguchi ,  Hiroki Kuyama ,  Eiji Ando ,  Norikazu Ueyama ,  Taka-aki Okamura ,  Shigemi Norioka

IPC分类号： C07K1/113 ,  C07K1/00 ,  C07K1/08 ,  C07K14/00

CPC分类号： C07K1/003

摘要： A simple and low-cost method of selectively modifying the C-terminal of a protein or peptide is provided. A method of modifying the C-terminal of a protein or peptide comprises forming an intramolecular oxazolone ring at the C-terminal of the protein or peptide that requires C-terminal modification, and then performing a ring-opening of the oxazolone ring to produce a protein or peptide with a modified C-terminal. Preferred forms include a method in which by reacting the oxazolone ring with a compound containing a nucleophilic group to effect an oxazolone ring-opening, a protein or peptide is produced in which the C-terminal is modified with the compound containing the nucleophilic group, as well as a method in which by reacting the oxazolone ring with an active esterifying agent to effect a ring-opening, the oxazolone is converted to an active ester, which by subsequent reaction with a compound containing a nucleophilic group, produces a protein or peptide in which the C-terminal has been modified with the compound containing the nucleophilic group.

摘要翻译： 提供了选择性修饰蛋白质或肽的C-末端的简单且低成本的方法。 修饰蛋白质或肽的C末端的方法包括在需要C末端修饰的蛋白质或肽的C-末端形成分子内的恶唑酮环，然后进行恶唑酮环的开环以产生 具有修饰C末端的蛋白质或肽。 优选的形式包括通过使恶唑酮环与含有亲核基团的化合物反应以进行恶唑酮开环的方法，制备其中C-末端用含有亲核基团的化合物修饰的蛋白质或肽作为 以及通过使恶唑酮环与活性酯化剂反应以开环的方法，将恶唑酮转化成活性酯，其随后与含有亲核基团的化合物反应，产生蛋白质或肽 其中C-末端已经与含有亲核基团的化合物一起被修饰。

公开(公告)号：US07041472B2

公开(公告)日：2006-05-09

申请号：US10739111

申请日：2003-12-19

申请人： Shigemi Norioka ,  Norikazu Ueyama ,  Taka-aki Okamura ,  Takashi Nakazawa ,  Minoru Yamaguchi ,  Eiji Ando

发明人： Shigemi Norioka ,  Norikazu Ueyama ,  Taka-aki Okamura ,  Takashi Nakazawa ,  Minoru Yamaguchi ,  Eiji Ando

IPC分类号： C12P21/06

CPC分类号： C12P21/06

摘要： The present invention provides a method for selectively collecting the N-terminal peptide fragments of a protein of interest whether or not the protein of interest is modified on the N-terminus. A method for selectively collecting the N-terminal peptide fragment of a protein, comprising: a protection step (1) of protecting side chain-amino groups of amino acid residues containing side chain-amino groups of a protein of interest to obtain a protected protein protected on the side chain-amino groups; a fragmentation step (2) of cleaving the protected protein into one N-terminal peptide fragment (a) containing the N-terminus of the peptide of interest and one or more of peptide fragments (b) other than the N-terminal peptide fragment (a); and a separation step (3) of separating the N-terminal peptide fragment (a) from the other peptide fragments (b) by selectively eluting the N-terminal peptide fragment (a) based on the difference in their reactivity or affinity to substrate, wherein the selective elution is achieved either by allowing the other peptide fragments (b) to bind to the substrate while allowing the N-terminal peptide fragment (a) to elute, or by allowing the N-terminal peptide fragment (a) to bind to the substrate while allowing the other peptide fragments (b) to elute and subsequently eluting the bound N-terminal peptide fragment (a).

摘要翻译： 本发明提供了一种用于选择性收集目的蛋白质的N-末端肽片段的方法，无论目的蛋白质是否在N末端被修饰。 一种用于选择性收集蛋白质的N-末端肽片段的方法，包括：保护步骤（1）保护含有目的蛋白质的侧链 - 氨基的氨基酸残基的侧链氨基以获得受保护的蛋白质 保护侧链氨基; 将保护的蛋白质切割成包含目标肽的N末端的一个N-末端肽片段（a）和除了N-末端肽片段之外的一个或多个肽片段（b）的片段化步骤（2） 一个）; 以及通过基于它们对底物的反应性或亲和力的差异来选择性地洗脱N末端肽片段（a），从而将N-末端肽片段（a）与其它肽片段（b）分离的分离步骤（3） 其中所述选择性洗脱通过使其它肽片段（b）与底物结合同时允许N-末端肽片段（a）洗脱，或通过使N-末端肽片段（a）与 同时允许其他肽片段（b）洗脱并随后洗脱结合的N-末端肽片段（a）。

公开(公告)号：US20090325227A1

公开(公告)日：2009-12-31

申请号：US12090747

申请日：2006-10-23

申请人： Takashi Nakazawa ,  Mutsumi Oka ,  Kimiko Nishida ,  Minoru Yamaguchi ,  Hiroki Kuyama ,  Eiji Ando ,  Norikazu Ueyama ,  Takaaki Okamura ,  Susumu Tsunasawa

发明人： Takashi Nakazawa ,  Mutsumi Oka ,  Kimiko Nishida ,  Minoru Yamaguchi ,  Hiroki Kuyama ,  Eiji Ando ,  Norikazu Ueyama ,  Takaaki Okamura ,  Susumu Tsunasawa

IPC分类号： C12P21/00 ,  C07K1/107 ,  G01N33/68

CPC分类号： C07K17/00 ,  C07K1/1072 ,  C07K1/113

摘要： The present invention provides a method for inexpensively, easily and efficiently modifying the C-terminus of a protein or peptide; a method for easily and reliably isolating a C-terminal peptide fragment of a protein or peptide; and a method for rapidly, accurately and reliably determining an amino acid sequence of a protein or peptide by using a mass spectroscope. A method comprising the step of adding a formylation reagent and a catalyst to a protein or peptide to convert a carboxyl group into an aldehyde group. A method comprising the step of reacting a nucleophilic reagent with the aldehyde group to modify the C-terminus of the protein or peptide. A method comprising the step of reacting a support having a nucleophilic group to immobilize the protein or peptide. A method comprising the step of fragmenting the immobilized protein or peptide, washing the support, and isolating the C-terminal peptide fragment from the support. A method comprising the step of subjecting the isolated C-terminal peptide fragment to mass spectrometry and determining an amino acid sequence.

摘要翻译： 本发明提供了一种廉价，容易且有效地修饰蛋白质或肽的C末端的方法; 容易且可靠地分离蛋白质或肽的C末端肽片段的方法; 以及通过使用质谱仪快速，准确，可靠地测定蛋白质或肽的氨基酸序列的方法。 一种方法，其包括将甲酰化试剂和催化剂加入到蛋白质或肽中以将羧基转化为醛基的步骤。 一种包括使亲核试剂与醛基反应以修饰蛋白质或肽的C末端的步骤的方法。 一种包括使具有亲核基团的载体反应以固定蛋白质或肽的方法。 一种方法，其包括将固定的蛋白质或肽片段化，洗涤载体和从载体中分离C末端肽片段的步骤。 一种方法，包括使分离的C-末端肽片段进行质谱分析和测定氨基酸序列的步骤。

公开(公告)号：US20090142851A1

公开(公告)日：2009-06-04

申请号：US12325115

申请日：2008-11-28

申请人： Keisuke Shima ,  Minoru Yamaguchi ,  Hiroki Kuyama ,  Eiji Ando ,  Osamu Nishimura ,  Susumu Tsunasawa ,  Kazuhiro Sonomura

发明人： Keisuke Shima ,  Minoru Yamaguchi ,  Hiroki Kuyama ,  Eiji Ando ,  Osamu Nishimura ,  Susumu Tsunasawa ,  Kazuhiro Sonomura

IPC分类号： G01N33/68 ,  C07K1/14 ,  C12P21/06

CPC分类号： C07K1/1072 ,  C07K1/128 ,  G01N33/6821

摘要： The present invention provides a method for specifically recovering a C-terminal peptide fragment, and a method for easily determining the sequence of a C-terminal peptide fragment, which is difficult to be determined by a conventional method, with the use of a mass spectrometer, in particular a method capable of de novo sequencing of a C-terminal peptide fragment. A method for selectively recovering a C-terminal peptide of a protein, comprising the steps of: in a cleavage product of a protein containing a C-terminal peptide fragment (A) having an α-amino group but not having an ε-amino group and the other peptide fragments (B) having an α-amino group and an ε-amino group, selectively modifying the α-amino groups to obtain a C-terminal peptide fragment modified (A′) and the other peptide fragments modified (B′); and separating the C-terminal peptide fragment modified (A′) from the modified cleavage product by allowing a carrier to hold the other peptide fragments modified (B′) via the ε-amino group. A method for determining the amino acid sequence of a C-terminal peptide of a protein, comprising the steps of: selectively recovering a C-terminal peptide of a protein by the above method; and determining the amino acid sequence by subjecting a recovered C-terminal peptide fragment to mass spectrometry measurement.

摘要翻译： 本发明提供了特异性回收C末端肽片段的方法，以及通过使用质谱仪容易地测定难以用常规方法测定的C末端肽片段的序列的方法 ，特别是能够重新测序C-末端肽片段的方法。 一种选择性回收蛋白质C末端肽的方法，包括以下步骤：在含有具有α-氨基但不具有ε-氨基的C-末端肽片段（A）的蛋白质的切割产物中 和具有α-氨基和ε-氨基的其它肽片段（B），选择性地修饰α-氨基以获得修饰的C末端肽片段（A'）和修饰的其它肽片段（B' ）; 以及通过允许载体通过ε-氨基保持修饰的其它肽片段（B'），从修饰的切割产物中分离修饰的C末端肽片段（A'）。 一种确定蛋白质C-末端肽的氨基酸序列的方法，包括以下步骤：通过上述方法选择性地回收蛋白质的C-末端肽; 并通过对回收的C-末端肽片段进行质谱测量来测定氨基酸序列。

公开(公告)号：US08068200B2

公开(公告)日：2011-11-29

申请号：US11311675

申请日：2005-12-19

申请人： Minoru Yamaguchi ,  Yasushi Nakajima ,  Ryota Mizusako

发明人： Minoru Yamaguchi ,  Yasushi Nakajima ,  Ryota Mizusako

IPC分类号： G02F1/1337 ,  G02F1/1343

CPC分类号： G02F1/133711 ,  G02F1/133707 ,  G02F1/1343 ,  G02F1/1393 ,  G02F2001/133742 ,  G02F2001/13712 ,  G02F2201/122

摘要： A liquid crystal display device has an opposing substrate on which an opposing electrode is formed, a TFT substrate on which pixel electrodes arranged in matrix, thin film transistors connected to the pixel electrodes respectively, and gate lines and data lines for the thin film transistors are formed, vertical alignment films formed on the opposing inner surfaces of these substrates, and a liquid crystal layer disposed between the vertical alignment films and having negative dielectric anisotropy. Each pixel electrode has a slit formed for separating each pixel into a plurality of sub-pixels by partially eliminating the pixel electrode with a connecting portion left at which adjoining electrode portions of each pixel electrode is connected with each other. The width W1 of the pixel electrode that runs in a direction in which the slit is formed and width W2 of the connecting portion have a ratio W2/W1 of 0.13 or lower.

摘要翻译： 液晶显示装置具有形成有对置电极的对置基板，分别配置有像素电极的TFT基板，与像素电极分别连接的薄膜晶体管，以及用于薄膜晶体管的栅极线和数据线 形成在这些基板的相对的内表面上的垂直取向膜，以及设置在垂直取向膜之间并具有负介电各向异性的液晶层。 每个像素电极具有形成的狭缝，用于通过部分地去除像素电极以将每个像素分离成多个子像素，其中每个像素电极的相邻电极部分彼此连接留下连接部分。 沿形成狭缝的方向延伸的像素电极的宽度W1和连接部的宽度W2的比值W2 / W1为0.13以下。

公开(公告)号：US20080074599A1

公开(公告)日：2008-03-27

申请号：US11862037

申请日：2007-09-26

申请人： Minoru Yamaguchi ,  Hiromitsu Ishii

发明人： Minoru Yamaguchi ,  Hiromitsu Ishii

IPC分类号： G02F1/1337

CPC分类号： G02F1/136213 ,  G02F1/133707 ,  G02F1/1393 ,  G02F2001/134345

摘要： A liquid crystal display device provides, on the internal side of one substrate of a pair of substrates a plurality of pixel electrodes, a plurality of TFTs, a plurality of scan lines and signal lines, and a plurality of convex sections each formed on the internal side of the other substrate so as to be located substantially on a center part of each of the plurality of predetermined regions provided in each of the plurality of pixels. Further, the liquid crystal display device provides an opposing electrode on the internal side of the one substrate. Then, the liquid crystal display device provides vertical alignment films on the respective internal sides of the pair of substrates, and a liquid crystal layer having negative dielectric anisotropy and inserted in the gap between the pair of substrates.

摘要翻译： 液晶显示装置在一对基板的一个基板的内侧设置多个像素电极，多个TFT，多条扫描线和信号线，以及多个凸部，每个形成在内部 以便基本上位于设置在多个像素中的每一个中的多个预定区域中的每一个的中心部分上。 此外，液晶显示装置在一个基板的内侧提供相对电极。 然后，液晶显示装置在一对基板的各个内侧上提供垂直取向膜，并且在一对基板之间的间隙中插入具有负介电各向异性的液晶层。

公开(公告)号：US20060066791A1

公开(公告)日：2006-03-30

申请号：US11238027

申请日：2005-09-28

申请人： Ryota Mizusako ,  Minoru Yamaguchi ,  Yasushi Nakajima

发明人： Ryota Mizusako ,  Minoru Yamaguchi ,  Yasushi Nakajima

IPC分类号： G02F1/1337

CPC分类号： G02F1/133707 ,  G02F1/133371 ,  G02F2001/133742

摘要： A liquid crystal display device comprises a TFT substrate where TFTs are formed, an opposing substrate (CF substrate), a liquid crystal with a negative dielectric anisotropy filled between both substrates, a pixel electrode, and an auxiliary electrode formed around the pixel electrode. The pixel electrode has a recess portion formed by etching a gate insulation film. The liquid crystal molecules are aligned tilted toward the center of a pixel according to the shape of the recess portion.

摘要翻译： 液晶显示装置包括形成TFT的TFT基板，相对基板（CF基板），在两基板之间填充有负介电各向异性的液晶，像素电极和形成在像素电极周围的辅助电极。 像素电极具有通过蚀刻栅极绝缘膜形成的凹部。 根据凹部的形状，液晶分子朝向像素的中心倾斜。