公开(公告)号：US20110053196A1

公开(公告)日：2011-03-03

申请号：US12278711

申请日：2007-02-08

申请人： Kenji Miyazaki ,  Yo Tabuse ,  Hirotaka Minagawa ,  Emiko Saito ,  Ken'ichi Kamijo ,  Akira Tsugita ,  Kozue Tsugita

发明人： Kenji Miyazaki ,  Yo Tabuse ,  Hirotaka Minagawa ,  Emiko Saito ,  Ken'ichi Kamijo ,  Akira Tsugita ,  Kozue Tsugita

IPC分类号： C12Q1/37 ,  C07K1/113

CPC分类号： G01N33/6803 ,  C07K1/107

摘要： The present invention is to provide a method for easily and specifically modifying specific amino acid residue(s) constituting a peptide and to provide a methodology of improving the accuracy of identification of the peptide using a new information of the peptide obtained from the number of modified amino acid residue by said specific modification method as mentioned. The method for modifying a peptide according to the present invention is characterized:A method for modifying a peptide, wherein the peptide as supported in a substrate and an aqueous solution of perhalogenated carboxylic acid containing an alcohol is reacted to selectively esterify a glutamic acid residue of said peptide.The method for identifying a peptide according to the present invention is characterized:A method for identifying a peptide comprising the steps of: reacting the peptide as supported in a substrate and an aqueous solution of perhalogenated carboxylic acid containing an alcohol to selectively esterify glutamic acid residue of said peptide; immersing said substrate in a protease solution to obtain a peptide fragment originated from said peptide; measuring a molecular weight of said peptide fragment; and determining said peptide based on said molecular weight.

摘要翻译： 本发明提供一种容易且特异性地修饰构成肽的特定氨基酸残基的方法，并且提供使用从修饰的数量获得的肽的新信息来提高肽的鉴定准确度的方法 通过所述具体的改性方法制备氨基酸残基。 本发明的肽的修饰方法的特征在于：对肽进行修饰的方法，其中负载在底物中的肽和含有醇的全卤代羧酸的水溶液进行反应以选择性地酯化谷氨酸残基 所述肽。 本发明鉴定肽的方法的特征在于：一种鉴定肽的方法，包括以下步骤：将载体载于底物中的肽与含有醇的全卤代羧酸水溶液反应以选择性酯化谷氨酸残基 的所述肽; 将所述底物浸入蛋白酶溶液中以获得源自所述肽的肽片段; 测量所述肽片段的分子量; 并基于所述分子量测定所述肽。

公开(公告)号：US20100167326A1

公开(公告)日：2010-07-01

申请号：US12278878

申请日：2007-02-08

申请人： Kenji Miyazaki ,  Ken'ichi Kamijo ,  Akira Tsugita ,  Kozue Tsugita

发明人： Kenji Miyazaki ,  Ken'ichi Kamijo ,  Akira Tsugita ,  Kozue Tsugita

IPC分类号： C12Q1/37 ,  C07K1/107 ,  G01N33/00

CPC分类号： C07K1/107 ,  C07K1/128 ,  G01N33/6821 ,  G01N33/6848

摘要： The present invention is to provide a method for cleavage of peptidic bond at C terminal of peptide and a method for determination of C terminal amino acid sequence of peptide without the decrease of the sensitivity and with preventing the subsidiary reaction. The method for cleavage of peptidic bond at C terminal of peptide according to the present invention is characterized as follows: A method for cleavage of peptidic bond at C terminal of peptide comprising the steps of: reacting a peptide with a solution of perhalogenated carboxylic acid containing alcohol to esterify glutamic acid residue of said peptide; and reacting said peptide with alkanoic acid anhydride to obtain C terminal-deleted peptide in which the amino acid residue of said peptide at C terminal is sequentially deleted. The method for determination of C terminal amino acid sequence of peptide according to the present invention is characterized as follows: A method for determination of C terminal amino acid sequence of peptide comprising the steps of: reacting a peptide with a solution of perhalogenated carboxylic acid containing alcohol to esterify glutamic acid residue of said peptide; reacting said peptide with alkanoic acid anhydride to obtain C terminal-deleted peptide in which the amino acid residue of said peptide at C terminal is sequentially deleted; measuring molecular weight of said C terminal-deleted peptide; and grasping said peptide based on said molecular weight.

摘要翻译： 本发明提供肽的C末端肽键的切割方法以及肽的C末端氨基酸序列的测定方法，而不会降低灵敏度并防止辅助反应。 根据本发明的肽在C末端切割肽键的方法的特征如下：肽的C末端切割肽键的方法，其包括以下步骤：使肽与含有全卤代羧酸的溶液反应 醇以酯化所述肽的谷氨酸残基; 并使所述肽与链烷酸酐反应，得到C末端缺失的肽，其中C末端的所述肽的氨基酸残基被顺序删除。 根据本发明的肽的C末端氨基酸序列的测定方法的特征如下：肽的C末端氨基酸序列的测定方法，包括以下步骤：使肽与含有卤素的全卤代羧酸的溶液反应 醇以酯化所述肽的谷氨酸残基; 使所述肽与链烷酸酐反应，得到C末端缺失的肽，其中所述肽在C末端的氨基酸残基被顺序删除; 测量所述C末端缺失肽的分子量; 并基于所述分子量掌握所述肽。

公开(公告)号：US08207301B2

公开(公告)日：2012-06-26

申请号：US12452216

申请日：2008-06-16

申请人： Hirotaka Minagawa ,  Kenji Miyazaki ,  Yo Tabuse ,  Kenichi Kamijo ,  Shuichi Kaneko

发明人： Hirotaka Minagawa ,  Kenji Miyazaki ,  Yo Tabuse ,  Kenichi Kamijo ,  Shuichi Kaneko

IPC分类号： C07K14/435

CPC分类号： C07K14/4748 ,  G01N33/57438

摘要： Provided are: a method of assessing hepatocellular carcinoma by using a protein with a different phosphorylated state in hepatocellular carcinoma cells compared with non-hepatocellular carcinoma cells; and a hepatocellular carcinoma protein marker for detecting hepatocellular carcinoma formed of the protein. The hepatocellular carcinoma protein marker for detecting hepatocellular carcinoma includes tumor rejection antigen gp96 formed of the amino acid represented by SEQ ID NO: 1, and is measured for its phosphorylated state to detect the presence or absence of hepatocellular carcinoma.

摘要翻译： 提供：与非肝细胞癌细胞相比，通过使用肝细胞癌细胞中具有不同磷酸化状态的蛋白质来评估肝细胞癌的方法; 和用于检测由蛋白质形成的肝细胞癌的肝细胞癌蛋白标记物。 用于检测肝细胞癌的肝细胞癌蛋白标志物包括由SEQ ID NO：1表示的氨基酸形成的肿瘤排斥抗原gp96，并测量其磷酸化状态以检测肝细胞癌的存在或不存在。

公开(公告)号：US20100248256A1

公开(公告)日：2010-09-30

申请号：US12452216

申请日：2008-06-16

申请人： Hirotaka Minagawa ,  Kenji Miyazaki ,  Yo Tabuse ,  Kenichi Kamijo ,  Shuichi Kaneko

发明人： Hirotaka Minagawa ,  Kenji Miyazaki ,  Yo Tabuse ,  Kenichi Kamijo ,  Shuichi Kaneko

IPC分类号： G01N33/53 ,  C07K14/435

CPC分类号： C07K14/4748 ,  G01N33/57438

摘要： Provided are: a method of assessing hepatocellular carcinoma by using a protein with a different phosphorylated state in hepatocellular carcinoma cells compared with non-hepatocellular carcinoma cells; and a hepatocellular carcinoma protein marker for detecting hepatocellular carcinoma formed of the protein. The hepatocellular carcinoma protein marker for detecting hepatocellular carcinoma includes tumor rejection antigen gp96 formed of the amino acid represented by SEQ ID NO: 1, and is measured for its phosphorylated state to detect the presence or absence of hepatocellular carcinoma.

摘要翻译： 提供：与非肝细胞癌细胞相比，通过使用肝细胞癌细胞中具有不同磷酸化状态的蛋白质来评估肝细胞癌的方法; 和用于检测由蛋白质形成的肝细胞癌的肝细胞癌蛋白标记物。 用于检测肝细胞癌的肝细胞癌蛋白标志物包括由SEQ ID NO：1表示的氨基酸形成的肿瘤排斥抗原gp96，并测量其磷酸化状态以检测肝细胞癌的存在或不存在。

公开(公告)号：US20120225440A1

公开(公告)日：2012-09-06

申请号：US13471629

申请日：2012-05-15

申请人： Hirotaka Minagawa ,  Kenji Miyazaki ,  Yo Tabuse ,  Kenichi Kamijo ,  Shuichi Kaneko

发明人： Hirotaka Minagawa ,  Kenji Miyazaki ,  Yo Tabuse ,  Kenichi Kamijo ,  Shuichi Kaneko

IPC分类号： G01N33/574 ,  G01N27/62 ,  G01N21/64

CPC分类号： C07K14/4748 ,  G01N33/57438

摘要： A method of detecting hepatocellular carcinoma includes using an isolated protein including an amino acid sequence represented by SEQ ID NO: 1.

摘要翻译： 检测肝细胞癌的方法包括使用包含SEQ ID NO：1所示氨基酸序列的分离蛋白。

公开(公告)号：US20070026456A1

公开(公告)日：2007-02-01

申请号：US10570205

申请日：2004-09-02

申请人： Kenichi Kamijo ,  Hisao Kawaura ,  Toru Sano ,  Yo Tabuse ,  Hirotaka Minagawa ,  Kenji Miyazaki

发明人： Kenichi Kamijo ,  Hisao Kawaura ,  Toru Sano ,  Yo Tabuse ,  Hirotaka Minagawa ,  Kenji Miyazaki

IPC分类号： G01N33/53

CPC分类号： G01N33/6803

摘要： A biomolecule is analyzed with high accuracy. A biomolecule is analyzed with high likelihood. A biomolecule as an analysis subject is modified with a marker binding or adsorbing to only its specific portion (S101). Next, the biomolecule modified with the marker is developed on a base plate (S102). The arrangement of the biomolecule on the base plate is detected using a marker (S103). Then, scanning is performed along the shape of the biomolecule present on the detected position (S104). The biomolecule is analyzed based on an information relating to the shape or arrangement of the biomolecule obtained by scanning or an information relating to the arrangement of the marker on the biomolecule (S105).

摘要翻译： 以高精度分析生物分子。 分析生物分子的可能性很高。 作为分析对象的生物分子用仅结合或吸附到其特定部分的标记进行修饰（S 101）。 接下来，在基板上显影用标记物修饰的生物分子（S102）。 使用标记检测基板上的生物分子的排列（S103）。 然后，沿检测位置上存在的生物分子的形状进行扫描（S104）。 基于与通过扫描获得的生物分子的形状或排列相关的信息或与生物分子上的标记的排列有关的信息来分析生物分子（S105）。

公开(公告)号：US08119411B2

公开(公告)日：2012-02-21

申请号：US12973158

申请日：2010-12-20

申请人： Kenji Miyazaki ,  Akira Tsugita ,  Kenichi Kamijo ,  Hiroaki Torii

发明人： Kenji Miyazaki ,  Akira Tsugita ,  Kenichi Kamijo ,  Hiroaki Torii

IPC分类号： G01N33/00

CPC分类号： G01N33/6821 ,  G01N33/6842 ,  G01N33/6851

摘要： The present invention provides a method for analyzing the C-terminal amino acid sequence of a peptide by using a reaction for successively releasing the C-terminal amino acids of the peptide, which method can suppress, when successively releasing the C-terminal amino acids of a peptide of long amino acid length, such a undesirable side reaction as cleavage of peptide bond in the intermediate position of the peptide and can carry out the chemical treatment thereof under widely applicable conditions; In the method, a dry sample of a peptide with long amino acid length is beforehand subjected to an N-acylation treatment; by using a reaction reagent where an alkanoic acid anhydride is combined with a small amount of a perfluoroalkanoic acid, successive release of C-terminal amino acids is conducted under mild conditions; a hydrolysis treatment is applied; then, selective fragmentization at site of arginine residue is performed by digestion by trypsin; thereafter, decreases in molecular weight are measured for the C-terminal side fragments derived from a series of reaction products with use of a MALDI-TOF-MS apparatus; thereby, the C-terminal amino acid sequence of the peptide sample is identified.

摘要翻译： 本发明提供了通过使用连续释放肽的C末端氨基酸的反应来分析肽的C末端氨基酸序列的方法，当连续释放C末端氨基酸的C末端氨基酸时，该方法可以抑制 长氨基酸长度的肽，这种不利的副反应，如在肽的中间位置处的肽键断裂，并且可以在广泛适用的条件下进行化学处理; 在该方法中，将具有长氨基酸长度的肽的干样品预先进行N-酰化处理; 通过使用其中链烷酸酐与少量全氟链烷酸组合的反应试剂，在温和条件下连续释放C-末端氨基酸; 应用水解处理; 然后通过胰蛋白酶消化进行精氨酸残基位点的选择性碎裂; 此后，使用MALDI-TOF-MS装置测量衍生自一系列反应产物的C-末端侧片段的分子量的降低; 鉴定出肽样品的C-末端氨基酸序列。

公开(公告)号：US20090140136A1

公开(公告)日：2009-06-04

申请号：US11547516

申请日：2005-02-08

申请人： Hiroaki Torii ,  Kenji Miyazaki ,  Kenichi Kamijo ,  Akira Tsugita

发明人： Hiroaki Torii ,  Kenji Miyazaki ,  Kenichi Kamijo ,  Akira Tsugita

IPC分类号： B01D59/44

CPC分类号： G01N33/6848 ,  G01N33/68

摘要： The primary structure or the modification state of a protein is analyzed in detail. First, an analyte protein is subjected to PMF analysis (S101), and the gene of the protein is identified. Unidentified peaks not corresponding to the peaks of hypothetical peptide fragments are extracted, by comparing the hypothetical mass spectrum with the mass spectrum obtained by mass spectrometry of a sample protein (S102). Then, the unidentified peaks obtained are analyzed, and thus, the structure or properties of the protein, the presence and the kind of modification of amino acid residues, amino acid substitution, generation of mutants, and terminal cleavage are analyzed (S103).

摘要翻译： 详细分析蛋白质的一级结构或修饰状态。 首先，对分析物蛋白进行PMF分析（S101），鉴定出蛋白质的基因。 通过将假想质谱与通过样品蛋白质谱质谱获得的质谱进行比较，提取不符合假想肽片段的峰的未鉴定的峰（S102）。 然后，分析所得到的未鉴定的峰，分析蛋白质的结构或性质，氨基酸残基的修饰，氨基酸取代，突变体的产生和末端切割的存在和种类（S103）。

公开(公告)号：US20060030052A1

公开(公告)日：2006-02-09

申请号：US10536824

申请日：2003-11-28

申请人： Kenji Miyazaki ,  Akira Tsugita ,  Kenichi Kamijo ,  Takuji Nabetani

发明人： Kenji Miyazaki ,  Akira Tsugita ,  Kenichi Kamijo ,  Takuji Nabetani

IPC分类号： G01N33/00

CPC分类号： G01N33/6842 ,  G01N33/6821

摘要： The present invention provides, as a method of analyzing the C-terminal amino acid sequence of a peptide with use of reaction technique for successively releasing the C-terminal amino acids, in which undesirable side reactions, such as cleavage of a peptide bond at the middle of the peptide, can be prevented and chemical treatments therein can be carried out under widely applicable conditions in the course of successive release of the C-terminal amino acids from a peptide, such a method comprising steps of dehydrating the gel on which a target peptide that has been separated by gel electrophoresis is held in the bound state; immersing it in a mixture solution of an alkanoic acid anhydride added with a small amount of a perfluoroalkanoic acid in a dipolar aprotic solvent to re-swell the gel carrier, forming a 5-oxazolone structure, at a temperature chosen in the range of from 30° C. to 80° C., followed by the cleavage of the 5-oxazolone ring to release the C-terminal amino acids, and then specifying the C-terminal amino acid sequence of the peptide based on the measured decrease in the molecular weight of a series of reaction products resulting therefrom.

摘要翻译： 本发明提供了使用用于连续释放C-末端氨基酸的反应技术分析肽的C-末端氨基酸序列的方法，其中不期望的副反应，例如在 肽的中间，可以在广泛适用的条件下在肽的C末端氨基酸连续释放的过程中进行其中的化学处理，这种方法包括使凝胶脱水，其中将目标 通过凝胶电泳分离的肽保持在结合状态; 将其加入到加入少量全氟链烷酸的链烷酸与偶极非质子传递溶剂的混合溶液中，使溶胶载体重新溶胀，形成5-恶唑酮结构，温度范围为30℃ ℃至80℃，然后切割5-恶唑酮环以释放C末端氨基酸，然后根据测定的分子量减少指定肽的C-末端氨基酸序列 的一系列由此产生的反应产物。

公开(公告)号：US07651859B2

公开(公告)日：2010-01-26

申请号：US10538305

申请日：2003-12-04

申请人： Kenji Miyazaki ,  Akira Tsugita ,  Kenichi Kamijo ,  Takuji Nabetani

发明人： Kenji Miyazaki ,  Akira Tsugita ,  Kenichi Kamijo ,  Takuji Nabetani

IPC分类号： G01N33/00

CPC分类号： G01N33/6821 ,  G01N33/6842

摘要： The present invention provides, as for a method for analyzing the C-terminal amino acid sequence of a peptide by using a reaction for successively releasing the C-terminal amino acids of the peptide, which method can suppress, when successively releasing the C-terminal amino acids of a peptide of long amino acid length, such a undesirable side reaction as cleavage of peptide bond in the intermediate position of the peptide and can carry out the chemical treatment thereof under widely applicable conditions, a following method wherein a dry sample of a peptide with long amino acid length is beforehand subjected to an N-acylation treatment; by using a reaction reagent where an alkanoic acid anhydride is combined with a small amount of a perfluoroalkanoic acid, successive release of C-terminal amino acids is conducted under mild conditions; a hydrolysis treatment is applied; then, selective fragmentization at site of arginine residue is performed by digestion by trypsin; thereafter, decreases in molecular weight are measured for the C-terminal side fragments derived from a series of reaction products by analysis in negative mode of a MALDI-TOF-MS apparatus; thereby, the C-terminal amino acid sequence of the peptide sample is identified.

摘要翻译： 本发明提供了通过使用连续释放肽的C末端氨基酸的反应来分析肽的C末端氨基酸序列的方法，该方法可以在连续释放C端 具有长氨基酸长度的肽的氨基酸，这种不利的副反应，如在肽的中间位置处的肽键断裂，并且可以在广泛适用的条件下进行化学处理，以下方法，其中将 具有长氨基酸长度的肽预先进行N-酰化处理; 通过使用其中链烷酸酐与少量全氟链烷酸组合的反应试剂，在温和条件下连续释放C-末端氨基酸; 应用水解处理; 然后通过胰蛋白酶消化进行精氨酸残基位点的选择性碎裂; 此后，通过MALDI-TOF-MS装置的负模式的分析，测量衍生自一系列反应产物的C末端侧片段的分子量的降低; 鉴定出肽样品的C-末端氨基酸序列。