公开(公告)号：US07384790B2

公开(公告)日：2008-06-10

申请号：US10489198

申请日：2003-03-24

申请人： Kenji Miyazaki ,  Akira Tsugita ,  Naoyuki Takahashi ,  Takuji Nabetani ,  Toshimasa Yamazaki ,  Kenichi Kamijo

发明人： Kenji Miyazaki ,  Akira Tsugita ,  Naoyuki Takahashi ,  Takuji Nabetani ,  Toshimasa Yamazaki ,  Kenichi Kamijo

IPC分类号： G01N33/68 ,  G01N33/483 ,  G01N27/12

CPC分类号： G01N33/6821 ,  G01N33/6842

摘要： The present invention provides a method for analyzing the C-terminal amino acid sequence of a peptide by applying reaction technique for successively releasing the C-terminal amino acids therefrom, which method can suppress, when releasing the C-terminal amino acids of the peptide in sequence, such as an undesirable side reaction as cleavage of peptide bond in the intermediate position of the peptide, and allows to carry out the chemical treatment thereof under widely applicable conditions. In the method according to the present invention, an alkanoic acid anhydride and a perfluoroalkanoic acid both of vapor phase, which are supplied from a mixture containing an alkanoic acid anhydride with a small amount of a perfluoroalkanoic acid added thereto, are allowed to act on a dry sample of the peptide to be examined in a dry atmosphere at a temperature chosen in a range of 15 to 60° C.; whereby the release of the C-terminal amino acid is resulted from successive formation of a 5-oxazolone structure being followed by cleavage of the 5-oxazolone ring; and then the C-terminal amino acids sequence is identified by analysis based on the decrease in molecular weight in a series of the reaction products obtained.

摘要翻译： 本发明提供了通过应用从其中连续释放C末端氨基酸的反应技术来分析肽的C末端氨基酸序列的方法，当释放肽的C末端氨基酸时，该方法可以抑制 序列，例如作为在肽的中间位置处的肽键的切割的不期望的副反应，并且允许在广泛适用的条件下进行其化学处理。 在根据本发明的方法中，允许从含有链烷酸酐和少量全氟链烷酸的混合物中提供的气相的链烷酸酐和全氟烷酸作用于 在干燥气氛中在15至60℃范围内选择的温度下待检测的肽的干样品; 由此C末端氨基酸的释放是由连续形成5-恶唑酮结构而引起的，随后是5-恶唑酮环的裂解; 然后通过基于所获得的一系列反应产物中分子量的降低的分析来鉴定C-末端氨基酸序列。

公开(公告)号：US20050074832A1

公开(公告)日：2005-04-07

申请号：US10489198

申请日：2003-03-24

申请人： Kenji Miyazaki ,  Akira Tsugita ,  Naoyuki Takahashi ,  Takuji Nabetani ,  Toshimasa Yamazaki ,  Kenichi Kamijo

发明人： Kenji Miyazaki ,  Akira Tsugita ,  Naoyuki Takahashi ,  Takuji Nabetani ,  Toshimasa Yamazaki ,  Kenichi Kamijo

IPC分类号： G01N27/62 ,  G01N33/68 ,  C12Q1/37 ,  G01N33/00

CPC分类号： G01N33/6821 ,  G01N33/6842

摘要： The present invention provides a method for analyzing the C-terminal amino acid sequence of a peptide by applying reaction technique for successively releasing the C-terminal amino acids therefrom, which method can suppress, when releasing the C-terminal amino acids of the peptide in sequence, such a undesirable side reaction as cleavage of peptide bond in the intermediate position of the peptide, and allows to carry out the chemical treatment thereof under widely applicable conditions. In the method according to the present invention, an alkanoic acid anhydride and a perfluoroalkanoic acid both of vapor phase, which are supplied from a mixture containing an alkanoic acid anhydride with a small amount of a perfluoroalkanoic acid added thereto, are allowed to act on a dry sample of the peptide to be examined in a dry atmosphere at a temperature chosen in a range of 15 to 60° C.; whereby the release of the C-terminal amino acid is resulted from successive formation of a 5-oxazolone structure being followed by cleavage of the 5-oxazolone ring; and then the C-terminal amino acids sequence is identified by analysis based on the decrease in molecular weight in a series of the reaction products obtained.

摘要翻译： 本发明提供了通过应用从其中连续释放C末端氨基酸的反应技术来分析肽的C末端氨基酸序列的方法，当释放肽的C末端氨基酸时，该方法可以抑制 序列，这种不利的副反应，如在肽的中间位置处的肽键断裂，并允许在广泛适用的条件下进行化学处理。 在根据本发明的方法中，允许从含有链烷酸酐和少量全氟链烷酸的混合物中提供的气相的链烷酸酐和全氟烷酸作用于 在干燥气氛中在15至60℃范围内选择的温度下待检测的肽的干样品; 由此C末端氨基酸的释放是由连续形成5-恶唑酮结构而引起的，随后是5-恶唑酮环的裂解; 然后通过基于所获得的一系列反应产物中分子量的降低的分析来鉴定C-末端氨基酸序列。

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。

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：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。

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。

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：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）。

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。