公开(公告)号：US09481710B2

公开(公告)日：2016-11-01

申请号：US13202474

申请日：2010-01-27

申请人： Sumio Ohtsuki ,  Junichi Kamiie ,  Tetsuya Terasaki

发明人： Sumio Ohtsuki ,  Junichi Kamiie ,  Tetsuya Terasaki

IPC分类号： C12Q1/37 ,  A61K38/04 ,  C07K5/00 ,  C07K7/00 ,  C07K16/00 ,  C07K17/00 ,  C07K7/06 ,  C07K7/08 ,  G01N33/68 ,  H01J49/00

CPC分类号： C07K7/06 ,  C07K7/08 ,  G01N33/6848 ,  H01J49/00

摘要： Disclosed is an evaluation peptide for evaluating the efficiency of a pretreatment in the quantification of a protein using a mass spectrometer, having high reliability and high general versatility. Also disclosed is an artificial standard protein comprising the evaluation peptide. Further disclosed is a method for quantifying a protein utilizing the artificial standard protein. Specifically disclosed is a method for selecting a peptide which consists of an amino acid sequence not agreeing with that in a naturally occurring protein and a variant thereof and capable of being detected by mass spectrometry and which has an amino acid that can be recognized by a protein-digesting enzyme, and using the peptide as an evaluation peptide for use in the quantification of a protein by a mass spectrometer.

摘要翻译： 公开了一种用于评估在使用质谱仪定量蛋白质中的预处理效率的评估肽，其具有高可靠性和高通用性。 还公开了包含评价肽的人造标准蛋白质。 另外公开了利用人造标准蛋白质定量蛋白质的方法。 具体公开的是选择由与天然存在的蛋白质及其变体不同的氨基酸序列组成的能够通过质谱法检测并且具有可被蛋白质识别的氨基酸的肽的方法 并且使用肽作为用于通过质谱仪定量蛋白质的评估肽。

公开(公告)号：US20120021446A1

公开(公告)日：2012-01-26

申请号：US13202474

申请日：2010-01-27

申请人： Sumio Ohtsuki ,  Junichi Kamiie ,  Tetsuya Terasaki

发明人： Sumio Ohtsuki ,  Junichi Kamiie ,  Tetsuya Terasaki

IPC分类号： C12Q1/37 ,  C07K7/08 ,  H01J49/26 ,  C07K7/06

CPC分类号： C07K7/06 ,  C07K7/08 ,  G01N33/6848 ,  H01J49/00

摘要： Disclosed is an evaluation peptide for evaluating the efficiency of a pretreatment in the quantification of a protein using a mass spectrometer, having high reliability and high general versatility. Also disclosed is an artificial standard protein comprising the evaluation peptide. Further disclosed is a method for quantifying a protein utilizing the artificial standard protein. Specifically disclosed is a method for selecting a peptide which consists of an amino acid sequence not agreeing with that in a naturally occurring protein and a variant thereof and capable of being detected by mass spectrometry and which has an amino acid that can be recognized by a protein-digesting enzyme, and using the peptide as an evaluation peptide for use in the quantification of a protein by a mass spectrometer.

摘要翻译： 公开了一种用于评估在使用质谱仪定量蛋白质中的预处理效率的评估肽，其具有高可靠性和高通用性。 还公开了包含评价肽的人造标准蛋白质。 另外公开了利用人造标准蛋白质定量蛋白质的方法。 具体公开的是选择由与天然存在的蛋白质及其变体不同的氨基酸序列组成的能够通过质谱法检测并且具有可被蛋白质识别的氨基酸的肽的方法 并且使用肽作为用于通过质谱仪定量蛋白质的评估肽。

公开(公告)号：US20110177491A1

公开(公告)日：2011-07-21

申请号：US13120705

申请日：2009-09-28

申请人： Junichi Kamiie ,  Sumio Ohtsuki ,  Tetsuya Terasaki

发明人： Junichi Kamiie ,  Sumio Ohtsuki ,  Tetsuya Terasaki

IPC分类号： C12Q1/00 ,  C12N9/00

CPC分类号： G01N33/6848 ,  C12Q1/37

摘要： There are provided a peptide consisting of an amino acid sequence for simultaneously quantifying absolute amounts of metabolizing enzyme proteins in a biological sample at high sensitivity and a method for using the same. A peptide which can be detected at high sensitivity with a mass spectrometer that enables highly sensitive simultaneous quantification of metabolizing enzymes, intracellular proteins, is selected, and the amino acid sequence thereof is identified. Using a stable-isotope-labeled peptide having the same amino acid sequence as the amino acid sequence of this peptide to be quantified, a mass spectrometry by LC-MS/MS is performed at predetermined concentration levels of the stable-isotope-labeled peptide to create a calibration curve. The stable-isotope-labeled peptide is added to a peptide fragment obtained by fragmenting metabolizing enzyme proteins to be quantified in a sample with trypsin, amass spectrometry by LC-MS/MS is performed to calculate amass spectrum area ratio of the metabolizing enzyme protein peptides to be quantified and the stable-isotope-labeled peptide, and a quantitative value is obtained from the area ratio using the calibration curve.

摘要翻译： 提供了由高灵敏度同时量化生物样品中代谢酶蛋白质的绝对量的氨基酸序列组成的肽及其使用方法。 选择能够以高灵敏度检测的肽，其能够通过使能高度敏感地同时定量代谢酶，细胞内蛋白质的质谱仪来检测，并鉴定其氨基酸序列。 使用具有与待定量的该肽的氨基酸序列相同的氨基酸序列的稳定同位素标记的肽，通过LC-MS / MS的质谱法以稳定同位素标记的肽的预定浓度水平进行， 创建一个校准曲线。 将稳定同位素标记的肽加入到通过用胰蛋白酶在样品中定量的代谢酶蛋白质片段获得的肽片段中，通过LC-MS / MS进行氨卡分析，计算代谢酶蛋白质肽的氨基酸谱面积比 待测量和稳定同位素标记的肽，并使用校准曲线从面积比值获得定量值。

公开(公告)号：US09163276B2

公开(公告)日：2015-10-20

申请号：US14233592

申请日：2012-06-18

申请人： Sumio Ohtsuki ,  Tetsuya Terasaki

发明人： Sumio Ohtsuki ,  Tetsuya Terasaki

IPC分类号： C12Q1/37 ,  A61K38/00 ,  C07K2/00 ,  C07K4/00 ,  C07K5/00 ,  C07K7/00 ,  G01N33/68 ,  G01N30/72 ,  G01N30/88

CPC分类号： C12Q1/37 ,  G01N30/7233 ,  G01N33/6848 ,  G01N2030/8827 ,  G01N2458/15

摘要： An object of the present invention is to provide a method for producing a stable isotope-labeled target peptide fragment in mass spectrometry, which achieves inexpensive and convenient production. As a solution to attain the object, the stable isotope-labeled target peptide fragment in mass spectrometry is produced using a method comprising the steps of: expressing a DNA conjugate in a system having a stable isotope-labeled amino acid to thereby prepare a stable isotope-labeled protein, wherein the DNA conjugate comprises: a tandemly linked DNA in which two or more DNAs encoding one or more types of target peptide fragments are linked in tandem; and a DNA encoding a peptide fragment for concentration measurement; subjecting the stable isotope-labeled protein to fragmentation treatment with trypsin to prepare a stable isotope-labeled peptide fragment for concentration measurement and stable isotope-labeled target peptide fragments; quantifying the stable isotope-labeled peptide fragment for concentration measurement using a liquid chromatograph-tandem mass spectrometer (LC/MS/MS); and calculating the concentration of the stable isotope-labeled target peptide fragment of each type from the quantification value of the stable isotope-labeled peptide fragment for concentration measurement.

摘要翻译： 本发明的目的是提供一种在质谱法中制备稳定的同位素标记的靶肽片段的方法，其实现了便宜且方便的生产。 作为实现该目标的解决方案，使用包括以下步骤的方法产生质谱中的稳定同位素标记的靶肽片段：在具有稳定的同位素标记的氨基酸的系统中表达DNA缀合物，从而制备稳定的同位素 标记的蛋白质，其中所述DNA缀合物包含：串联连接的DNA，其中编码一种或多种类型的靶肽片段的两个或多个DNA串联连接; 和编码用于浓度测量的肽片段的DNA; 用胰蛋白酶对稳定同位素标记的蛋白质进行片段化处理，制备用于浓度测定和稳定同位素标记的靶肽片段的稳定同位素标记的肽片段; 使用液相色谱 - 串联质谱仪（LC / MS / MS）定量稳定同位素标记的肽片段进行浓度测量; 并从稳定同位素标记的肽片段的定量值中计算每种类型的稳定同位素标记的靶肽片段的浓度，用于浓度测定。

公开(公告)号：US20140255966A1

公开(公告)日：2014-09-11

申请号：US14233592

申请日：2012-06-18

申请人： Sumio Ohtsuki ,  Tetsuya Terasaki

发明人： Sumio Ohtsuki ,  Tetsuya Terasaki

IPC分类号： C12Q1/37

CPC分类号： C12Q1/37 ,  G01N30/7233 ,  G01N33/6848 ,  G01N2030/8827 ,  G01N2458/15

摘要： An object of the present invention is to provide a method for producing a stable isotope-labeled target peptide fragment in mass spectrometry, which achieves inexpensive and convenient production. As a solution to attain the object, the stable isotope-labeled target peptide fragment in mass spectrometry is produced using a method comprising the steps of: expressing a DNA conjugate in a system having a stable isotope-labeled amino acid to thereby prepare a stable isotope-labeled protein, wherein the DNA conjugate comprises: a tandemly linked DNA in which two or more DNAs encoding one or more types of target peptide fragments are linked in tandem; and a DNA encoding a peptide fragment for concentration measurement; subjecting the stable isotope-labeled protein to fragmentation treatment with trypsin to prepare a stable isotope-labeled peptide fragment for concentration measurement and stable isotope-labeled target peptide fragments; quantifying the stable isotope-labeled peptide fragment for concentration measurement using a liquid chromatograph-tandem mass spectrometer (LC/MS/MS); and calculating the concentration of the stable isotope-labeled target peptide fragment of each type from the quantification value of the stable isotope-labeled peptide fragment for concentration measurement.

摘要翻译： 本发明的目的是提供一种在质谱法中制备稳定的同位素标记的靶肽片段的方法，其实现了便宜且方便的生产。 作为实现该目标的解决方案，使用包括以下步骤的方法产生质谱中的稳定同位素标记的靶肽片段：在具有稳定的同位素标记的氨基酸的系统中表达DNA缀合物，从而制备稳定的同位素 标记的蛋白质，其中所述DNA缀合物包含：串联连接的DNA，其中编码一种或多种类型的靶肽片段的两个或多个DNA串联连接; 和编码用于浓度测量的肽片段的DNA; 用胰蛋白酶对稳定同位素标记的蛋白质进行片段化处理，制备用于浓度测定和稳定同位素标记的靶肽片段的稳定同位素标记的肽片段; 使用液相色谱 - 串联质谱仪（LC / MS / MS）定量稳定同位素标记的肽片段进行浓度测量; 并从稳定同位素标记的肽片段的定量值中计算每种类型的稳定同位素标记的靶肽片段的浓度，用于浓度测定。