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1. 发明申请

US20100167326A1 METHOD FOR CLEAVAGE OF PEPTIDIC BOND AT C TERMINAL OF PEPTIDE AND A METHOD FOR DETERMINATION OF C TERMINAL AMINO ACID SEQUENCE OF PEPTIDE 审中-公开
标题翻译：肽末端肽结合蛋白的方法和确定肽末端氨基酸序列的方法
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。

2. 发明申请

US20110053196A1 METHOD FOR MODIFYING A PEPTIDE AND A METHOD FOR IDENTIFYING A PEPTIDE 审中-公开
标题翻译：用于修饰肽的方法和鉴定肽的方法
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。

3. 发明授权

US08119411B2 Method for analyzing C-terminal amino acid sequence of peptide using mass spectrometry 失效
标题翻译：使用质谱分析肽的C-末端氨基酸序列的方法
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。

4. 发明申请

US20090140136A1 Method Of Analyzing Protein 审中-公开
标题翻译：分析蛋白质的方法
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）。

5. 发明申请

US20060030052A1 Method of analyzing c-terminal amino acid sequence of peptide 失效
标题翻译：分析肽末端氨基酸序列的方法
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。

6. 发明授权

US07651859B2 Method of analyzing c-terminal amino acid sequence of peptide 失效
标题翻译：分析肽末端氨基酸序列的方法
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。

7. 发明申请

US20080213911A1 Method of Analyzing C-Terminal Amino Acid Sequence of Peptide 失效
标题翻译：分析C端氨基酸序列的方法
公开(公告)号：US20080213911A1
公开(公告)日：2008-09-04
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：通过使用链烷酸酐顺序地降解分析物肽（S101）。将具有缺失一个或多个C末端氨基酸的肽的原始肽和一系列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，基于通过质谱法获得的分子量测定来自C末端的原始肽的氨基酸序列（S104）。

8. 发明申请

US20060177887A1 Method of analyzing a protein 审中-公开
标题翻译：分析蛋白质的方法
公开(公告)号：US20060177887A1
公开(公告)日：2006-08-10
申请号：US11322320
申请日：2006-01-03
申请人： Kenji Miyazaki , Hiroaki Torii , Kenichi Kamijo , Akira Tsugita
发明人： Kenji Miyazaki , Hiroaki Torii , Kenichi Kamijo , Akira Tsugita
IPC分类号： C12Q1/37 , G06F19/00 , G01N33/00
CPC分类号： G01N33/6842 , G01N30/6095 , G01N30/7233 , G01N33/6821 , G01N33/6848
摘要： Provided a method of acquiring information on internal sequence which is applicable to a trace amount of protein and has a high reliability. A protein to be analyzed is limitedly cleaved at a specified position of amino acid to prepare a mixture of a plurality of peptide fragments; one or more peptide fragments are isolated and purified from the peptide fragment mixture; C-terminal amino acids of the isolated and purified peptide fragment are successively released by chemical reaction to prepare a mixture containing a series of resulting products; mass spectrometry is applied to both the reaction products of the successive truncation and unreacted peptide fragment not subjected to the successive truncation reaction; then results of the mass spectrometry is analyzed to acquire chemical structure information of the target protein.
摘要翻译：提供一种获取适用于微量蛋白质并具有高可靠性的内部序列信息的方法。要分析的蛋白质在氨基酸的指定位置被有限地切割以制备多个肽片段的混合物; 从肽片段混合物中分离和纯化一个或多个肽片段; 分离和纯化的肽片段的C-末端氨基酸通过化学反应连续释放，制备含有一系列产物的混合物; 对连续截断的反应产物和未进行连续截短反应的未反应的肽片段进行质谱分析; 然后分析质谱结果，获得目标蛋白质的化学结构信息。

9. 发明授权

US07670841B2 Method of analyzing C-terminal amino acid sequence of peptide 失效
标题翻译：分析肽的C-末端氨基酸序列的方法
公开(公告)号：US07670841B2
公开(公告)日：2010-03-02
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。

10. 发明申请

US20060057731A1 Method for analyzing c-terminal amono acid sequence of peptide 失效
标题翻译：分析肽末端氨基酸序列的方法
公开(公告)号：US20060057731A1
公开(公告)日：2006-03-16
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。

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