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：US07901942B2

公开(公告)日：2011-03-08

申请号：US12093133

申请日：2006-10-27

申请人： Junichi Kamiie ,  Sumio Otsuki ,  Tetsuya Terasaki

发明人： Junichi Kamiie ,  Sumio Otsuki ,  Tetsuya Terasaki

IPC分类号： G01N33/00

CPC分类号： G01N30/88 ,  G01N30/7266 ,  G01N30/8679 ,  G01N33/6848 ,  G01N2030/8813 ,  G01N2030/8818 ,  G01N2030/8827 ,  G01N2030/8831 ,  G01N2333/705 ,  H01J49/00

摘要： A method is provided for quantifying a plasma membrane protein present by using a stable-isotope labeled peptide as a probe by mass spectrometry in a simple, quick and accurate manner. A plasma membrane protein is fragmented to prepare an oligopeptide fragment, identified by LC/MS/MS. A subject peptide for quantification is selected if the peptide is obtained by fragmenting with a protease, the peptide is specific to a target molecule, and if the peptide has a high total score value based on selective criteria for hydrophobic amino acids content, sequence conditions, number of amino acid residues, specific amino acid sequence conditions, etc. According to these criteria, a subject peptide fragment that can be ionized by ESI method is selected. By using the subject peptide for and a stable-isotope labeled peptide, the plasma membrane protein is quantified accurately by mass spectrometry.

摘要翻译： 提供了一种通过以简单，快速和准确的方式通过质谱法使用稳定同位素标记肽作为探针来定量存在的质膜蛋白质的方法。 将质膜蛋白质片段化以制备通过LC / MS / MS鉴定的寡肽片段。 如果通过用蛋白酶片段化获得肽，则该肽对靶分子是特异性的，并且如果该肽基于疏水性氨基酸含量，序列条件的选择性标准具有高的总分值，则选择用于定量的主题肽， 氨基酸残基数，特定氨基酸序列条件等。根据这些标准，选择可通过ESI方法离子化的主题肽片段。 通过使用目标肽和稳定同位素标记的肽，通过质谱法精确定量质膜蛋白质。

公开(公告)号：US20100029005A1

公开(公告)日：2010-02-04

申请号：US12093133

申请日：2006-10-27

申请人： Junichi Kamiie ,  Sumio Otsuki ,  Tetauya Terasaki

发明人： Junichi Kamiie ,  Sumio Otsuki ,  Tetauya Terasaki

IPC分类号： G01N33/68

CPC分类号： G01N30/88 ,  G01N30/7266 ,  G01N30/8679 ,  G01N33/6848 ,  G01N2030/8813 ,  G01N2030/8818 ,  G01N2030/8827 ,  G01N2030/8831 ,  G01N2333/705 ,  H01J49/00

摘要： The present invention is to provide a method for quantifying a plasma membrane protein present in a cell membrane, by using a stable-isotope labeled peptide as a probe by mass spectrometry, in a simple, quickly and accurate manner. A plasma membrane protein is fragmented to prepare an oligopeptide fragment, identified by LC-MSMS, and by setting as essential criteria that the peptide is obtained by fragmentizing with a protease, and the peptide is specific to a target molecule, selective criteria with score are set for hydrophobic amino acids content, sequence conditions, number of amino acid residues, specific amino acid sequence conditions, etc. According to criterion for selecting a subject peptide for quantification for selecting preferentially a peptide with high score, a peptide fragment that can be ionized by ESI method is selected, and by using the subject peptide for quantification and a stable-isotope labeled peptide, the plasma membrane protein is quantified accurately by mass spectrometry.

摘要翻译： 本发明提供一种以简单，快速且准确的方式通过质谱法使用稳定同位素标记肽作为探针来定量细胞膜中存在的质膜蛋白质的方法。 将质膜蛋白质片段化以制备通过LC-MSMS鉴定的寡肽片段，并且通过设定通过用蛋白酶片段化获得肽的基本标准，并且肽对靶分子是特异性的，得分的选择性标准 设定疏水性氨基酸含量，序列条件，氨基酸残基数，特定氨基酸序列条件等。根据选择目标肽进行定量以优先选择具有高分数的肽的标准，可以离子化的肽片段 通过ESI方法，通过使用目标肽进行定量和稳定同位素标记的肽，通过质谱法精确定量质膜蛋白质。