公开(公告)号：US20110183428A1

公开(公告)日：2011-07-28

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： G01N33/48

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：US07879616B2

公开(公告)日：2011-02-01

申请号：US10540814

申请日：2003-12-25

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：US07588944B2

公开(公告)日：2009-09-15

申请号：US10589495

申请日：2004-08-24

申请人： Kenji Miyazaki ,  Akira Tsugita ,  Kenichi Kamijo

发明人： Kenji Miyazaki ,  Akira Tsugita ,  Kenichi Kamijo

IPC分类号： G01N33/00

CPC分类号： G01N33/6842 ,  G01N33/6821 ,  Y10T436/24

摘要： An analyte peptide is selectively degraded sequentially by using an alkanoic anhydride (S101). The original peptide and a series of degradation reaction products having peptide in which one or more C-terminal-sided amino acids are deleted, are subjected to a certain posttreatment (S102). The molecular weight of the reaction products is measured by mass spectrometry (S103). And, the amino acid sequence of the original peptide from C-terminal is determined, based on the molecular weight obtained by mass spectrometry (S104).

摘要翻译： 分析物肽通过使用链烷酸酐依次选择性降解（S101）。 将原来的肽和一系列具有缺失一个或多个C末端氨基酸的肽的降解反应产物进行一定的后处理（S102）。 通过质谱法测定反应产物的分子量（S103）。 并且，基于通过质谱法得到的分子量，测定来自C-末端的原始肽的氨基酸序列（S104）。

公开(公告)号：US20070148720A1

公开(公告)日：2007-06-28

申请号：US10583600

申请日：2004-12-17

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： C12Q1/37 ,  G01N33/00

CPC分类号： G01N27/62

摘要： The present invention provides an approach for identifying with high accuracy, a known protein or a variant of the known protein derived from the same genomic gene as in a target protein to be analyzed, based on a mass spectrometric result of a plurality of peptide fragments obtained from site-specific enzymatic digestion of the target protein to be analyzed by referring to nucleotide sequences of genes encoding known proteins on a database and to deduced full-length amino acid sequences thereof. In the approach of the present invention, a candidate known protein of identification is specified with high accuracy by such a process comprising steps of comparing actually measured molecular weight values of the peptide fragments derived from the target protein to be analyzed, which are obtained with the use of peptide fragmentation by the site-specific proteolytic treatment, with predicted molecular weight values of peptide fragments predicted from the deduced full-length amino acid sequences of the known proteins and making comparison in terms of the numbers of matching fragments, the consecutiveness of amino acid sequences of matching fragments of the known protein, and the prediction of variation in a mismatching fragment.

摘要翻译： 本发明提供了一种基于获得的多个肽片段的质谱结果，高精度鉴定来自与要分析的靶蛋白相同的基因组基因的已知蛋白质或已知蛋白质的变体的方法 通过参考在数据库上编码已知蛋白质的基因的核苷酸序列并推断其全长氨基酸序列来分析待分析的靶蛋白的位点特异性酶促消化。 在本发明的方法中，通过这样的方法以高精度指定候选的已知的鉴定蛋白质，该方法包括以下步骤：比较衍生自待分析的靶蛋白的肽片段的实测值的分子量值， 通过位点特异性蛋白水解处理使用肽断裂，从已知蛋白质的推导的全长氨基酸序列预测的肽片段的预测分子量值，并根据匹配片段的数量进行比较，氨基酸的连续性 已知蛋白质的匹配片段的酸序列，以及错配片段变异的预测。

公开(公告)号：US20060134720A1

公开(公告)日：2006-06-22

申请号：US10540814

申请日：2003-12-25

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： C12Q1/37

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：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。