公开(公告)号：US5439650A

公开(公告)日：1995-08-08

申请号：US970941

申请日：1992-11-03

申请人： Akira Tsugita ,  Masaharu Kamo ,  Toyoaki Uchida ,  Ikuo Nanno ,  Yasuhiro Nomoto ,  Seitaro Takahashi

发明人： Akira Tsugita ,  Masaharu Kamo ,  Toyoaki Uchida ,  Ikuo Nanno ,  Yasuhiro Nomoto ,  Seitaro Takahashi

IPC分类号： G01N33/50 ,  B01J19/00 ,  C07D277/20 ,  C07D277/40 ,  C40B40/10 ,  C40B60/14 ,  G01N1/28 ,  B01S8/20 ,  G05F7/00

CPC分类号： B01J19/0053 ,  B01J19/0046 ,  G01N1/28 ,  B01J2219/00286 ,  B01J2219/0059 ,  B01J2219/00725 ,  C40B40/10 ,  C40B60/14 ,  Y10S436/807

摘要： An reaction vessel utilizes a sample carrier composed of a magnetic core and a surface coating effective to support sample of protein or peptide. The sample carrier is floated magnetically and positioned within a reaction chamber. Edman reagent is applied to the sample to effect amino acid sequence analysis of protein or peptide from amino-terminal. By such construction, reaction efficiency is increased to produce sequentially thiazolinon amino-acid derivatives to thereby increase number of identified remaining amino acids, thereby enabling microanalysis of sample.

摘要翻译： 反应容器使用由磁芯和表面涂层组成的样品载体，其有效地支持蛋白质或肽的样品。 样品载体磁性浮动并定位在反应室内。 将Edman试剂应用于样品，以从氨基末端进行蛋白质或肽的氨基酸序列分析。 通过这种结构，提高反应效率以依次产生噻唑啉氨基酸衍生物，从而增加鉴定的残留氨基酸的数量，由此可以进行样品的微量分析。

公开(公告)号：US5538896A

公开(公告)日：1996-07-23

申请号：US211339

申请日：1994-05-09

申请人： Akira Tsugita ,  Masaharu Kamo ,  Mitsuru Sano

发明人： Akira Tsugita ,  Masaharu Kamo ,  Mitsuru Sano

IPC分类号： G01N33/68 ,  G01N33/00

CPC分类号： G01N33/6824

摘要： To easily detect an amino acid derivative with high sensitivity in carrying out amino acid sequence analysis of a protein from an amino terminal, 2-anilino-5-thiazolinone amino acid derivative is reacted with a volatile primary amine represented by the general formula X-NH.sub.2, wherein X denotes a hydrocarbon containing a halogen to form a phenylthiocarbamyl amino acid derivative, and the phenylthiocarbamyl amino acid derivative is detected by using a gas chromatograph provided with an ECD. Accordingly, amino acid sequencing may be performed with high sensitivity without the need to use harmful radioisotope labeled or fluorescent amino compounds.

摘要翻译： PCT No.PCT / JP93 / 00744 Sec。 371日期1994年5月9日 102（e）日期1994年5月9日PCT提交1993年6月2日PCT公布。 公开号WO94 / 02854 日期1994年2月3日为了容易地从氨基末端进行氨基酸序列分析蛋白质的高灵敏度检测氨基酸衍生物，将2-苯胺基-5-噻唑啉酮氨基酸衍生物与由 通式X-NH 2，其中X表示含有卤素以形成苯基硫代氨基甲酰基氨基酸衍生物的烃，苯基硫代氨基甲酰基氨基酸衍生物通过使用具有ECD的气相色谱仪进行检测。 因此，可以高灵敏度地进行氨基酸测序，而不需要使用有害的放射性同位素标记或荧光氨基化合物。

公开(公告)号：US6046053A

公开(公告)日：2000-04-04

申请号：US862625

申请日：1997-05-23

申请人： Akira Tsugita ,  Keiji Takamoto ,  Tatsuaki Ataka ,  Toshihiko Sakuhara ,  Toyoaki Uchida

发明人： Akira Tsugita ,  Keiji Takamoto ,  Tatsuaki Ataka ,  Toshihiko Sakuhara ,  Toyoaki Uchida

IPC分类号： G01N31/00 ,  C07K1/00 ,  G01N33/68 ,  G01N33/50 ,  G01N33/00

CPC分类号： G01N33/6821

摘要： A process is disclosed for sequencing proteins or peptides from the C-terminal end. The process comprises the steps of reacting the peptide or protein with an alkyl acid anhydride to convert the carboxy-terminal thereof into oxazolone, liberating the C-terminal amino acid by reaction with acid and alcohol or with ester, and identifying the liberated amino acid or amino acid derivative.

摘要翻译： 公开了从C末端测序蛋白质或肽的方法。 该方法包括以下步骤：使肽或蛋白质与烷基酸酐反应，将其羧基末端转化成恶唑酮，通过与酸和醇或酯反应释放C端氨基酸，并鉴定释放的氨基酸或 氨基酸衍生物。

公开(公告)号：US5521097A

公开(公告)日：1996-05-28

申请号：US931931

申请日：1992-08-18

申请人： Toyoaki Uchida ,  Akira Tsugita ,  Keiji Takamoto ,  Kazuo Satake

发明人： Toyoaki Uchida ,  Akira Tsugita ,  Keiji Takamoto ,  Kazuo Satake

IPC分类号： C07K1/12 ,  G01N33/68 ,  G01N33/00

CPC分类号： G01N33/6821 ,  C07K1/128 ,  Y10T436/24

摘要： A protein or peptide is treated by a vapor containing an organic acid represented by the following general formula CF.sub.3 --(CF.sub.2)n--COOH (n is zero or more integer). The resulting reaction mixture is processed by a mass spectrometer to obtain a mass spectrum to measure a mass of respective chemical species contained in the reaction mixture. Alternatively, the reaction mixture is processed by an amino acid analyzer to determine an amino acid sequence of the protein or peptide from the carboxy-terminal. According to another method, the protein or peptide is treated by an anhydride of the organic acid represented by the following general formula CF.sub.3 --(CF.sub.2)n--COOH (n is zero or more integer). The resulting reaction mixture is processed by a mass spectrometer to obtain a mass spectrum to measure a mass of respective chemical species contained in the reaction mixture to determine an amino acid sequence of the protein or peptide from the carboxy-terminal.

摘要翻译： 通过含有由以下通式CF 3 - （CF 2）n -COOH表示的有机酸的蒸气处理蛋白质或肽（n为0以上整数）。 通过质谱仪处理得到的反应混合物，得到质谱以测量反应混合物中各化学物质的质量。 或者，通过氨基酸分析仪处理反应混合物以从羧基端确定蛋白质或肽的氨基酸序列。 根据另一种方法，蛋白质或肽由下列通式CF 3 - （CF 2）n -COOH表示的有机酸的酸酐（n为0以上整数）处理。 通过质谱仪处理所得反应混合物以获得质谱以测量反应混合物中所含的各化学物质的质量，以确定羧基末端的蛋白质或肽的氨基酸序列。

公开(公告)号：US5962642A

公开(公告)日：1999-10-05

申请号：US854187

申请日：1997-05-09

申请人： Akira Tsugita ,  Keiji Takamoto ,  Tatsuaki Ataka ,  Toshihiko Sakuhara ,  Toyoaki Uchida

发明人： Akira Tsugita ,  Keiji Takamoto ,  Tatsuaki Ataka ,  Toshihiko Sakuhara ,  Toyoaki Uchida

IPC分类号： C07K7/06 ,  G01N33/68 ,  C07K1/00 ,  C07K99/00

CPC分类号： G01N33/6842 ,  C07K7/06 ,  G01N33/6824

摘要： A protein or peptide which has an amino-terminus serine or threonine which has an acetylated alpha-amino group is allowed to react with an acid, and then allowed to react with an isothiocyanate under acidic conditions to thereby obtain a thiocarbamyl compound. Then the compound is to be analyzed using Edman degradation. Analysis can be performed with fewer operation steps and without using enzymes.

摘要翻译： 具有具有乙酰化α-氨基的氨基末端丝氨酸或苏氨酸的蛋白质或肽与酸反应，然后在酸性条件下与异硫氰酸酯反应，从而得到硫代氨基甲酰基化合物。 然后使用Edman降解分析化合物。 可以用较少的操作步骤进行分析，而不使用酶。

公开(公告)号：US5051369A

公开(公告)日：1991-09-24

申请号：US227184

申请日：1988-08-02

申请人： Akira Tsugita ,  Toyoaki Uchida

发明人： Akira Tsugita ,  Toyoaki Uchida

IPC分类号： C07D311/86 ,  G01N33/52 ,  G01N33/68

CPC分类号： G01N33/6824 ,  G01N33/52 ,  Y10T436/141111

摘要： A method for detecting amino acid derivatives in which 2-anilino-5-thiazolinone derivatives of amino acids are reacted with an amino compound of fluorescein or rhodamine derivative amino compounds to form phenylthiocarbamyl amino acids derivatives. The phenylithiocarbamyl amino acids derivatives are detected with a high level of sensitivity by a fluorescence spectrophotometer.

摘要翻译： 用于检测其中氨基酸的2-苯胺基-5-噻唑啉酮衍生物与荧光素或罗丹明衍生物氨基化合物的氨基化合物反应以形成苯基硫代氨基甲酰基氨基酸衍生物的氨基酸衍生物的方法。 通过荧光分光光度计以高灵敏度检测苯硫基氨基甲酰氨基酸衍生物。

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：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：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：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）。