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METHOD FOR FABRICATING STABLE-ISOTOPE-LABELED TARGET PEPTIDE FRAGMENT IN MASS SPECTROMETRY

外国特許コード F130007162
整理番号 S2011-0907-N0
掲載日 2013年2月14日
出願国 世界知的所有権機関(WIPO)
国際出願番号 2012JP003965
国際公開番号 WO 2013014853
国際出願日 平成24年6月18日(2012.6.18)
国際公開日 平成25年1月31日(2013.1.31)
優先権データ
  • 特願2011-161351 (2011.7.22) JP
発明の名称 (英語) METHOD FOR FABRICATING STABLE-ISOTOPE-LABELED TARGET PEPTIDE FRAGMENT IN MASS SPECTROMETRY
発明の概要(英語) The present invention addresses the problem of providing a method for fabricating a stable-isotope-labeled target peptide fragment in mass spectrometry, capable of low-cost and simple fabrication. As a solution, a stable-isotope-labeled target peptide fragment in mass spectrometry is fabricated using a method comprising: a step for preparing a stable-isotope-labeled protein by causing a conjugate DNA, which is obtained between tandem-coupled DNA in which two or more DNA has been coupled in tandem for coding two or more species of target peptide fragments and DNA for coding peptide fragments for concentration measurement, to be expressed in a system in which a stable-isotope-labeled amino acid is present; a step for fragmentizing stable-isotope-labeled protein using trypsin to prepare stable-isotope-labeled peptide fragments for concentration measurement and stable-isotope-labeled target peptide fragments; a step for determining the quantity of stable-isotope-labeled peptide fragments for concentration measurement using a liquid chromatography-tandem mass spectrometry device (LC/MS/MS); and a step for calculating the concentration of stable-isotope-labeled target peptide fragments from the quantified value of the stable-isotope-labeled peptide fragments for concentration measurement.
従来技術、競合技術の概要(英語) BACKGROUND ART
In recent years, the progress of the human genome project, the individual difference of the sensitivity to drugs for analyzing the genetic level to be applied to the development of a medicament, pharmacogenomic research has been conducted. An individual difference of the sensitivity to drugs can be diagnosed if it is, without carrying out a trial medication, the appropriate dosage can be from the initial stage of treatment, a so-called personalized for treatment. In particular, in the field of cancer treatment, as compared with the conventional anti-cancer agent of molecular target drugs with fewer side effects have been developed in recent years, as used in clinical practice has been developed (for example, see Patent Document 1). The target agent is a molecular target drugs acting on specific for a protein and therefore, the amount of protein expression in the individual patient by analyzing the profile, which is highly expressed the target protein if it is possible to know, and the appropriate treatment can be in the plan is considered.
However, the current, which are mainly used in clinical inspection method is, for the purpose of detecting a single protein and with, a number of different proteins cannot be detected at the same time. Further, many of the inspection method for this is that the antibodies may be used, such as a specific amount for cross-reactivity it is difficult to perform the inspection (for example, Non-Patent Document 1, reference 2), in addition, different for each protein must be used an antibody from, in order to detect a number of proteins is immense effort and expense. In order to solve such a problem, recently, the development of microarray gene expression analysis method, various types of RNA expression can be determined in one test method for gene expression has been established (for example, see Non-Patent Document 3-6). However, many of the molecular target drug target protein the protein is expressed on the cell membrane, and the amount of protein expression to extremely low correlation with the amount of expression of RNA from, obtained by microarray analysis RNA expression profile, protein expression profiles of the patient need not necessarily be identical to the not considered. From the above, based on the results of microarray analysis, the agent is a molecular target is difficult to individualized therapy, the proteins expressed in practice, can be determined comprehensively establish the inspection method is strongly demanded.
In recent years, mass spectrometry progress been prominently, detection or measurement of a variety of biological material applied in this method. Heretofore, electrospray, ionization mass spectrometry (mass spectrometer; MS) and, prior to the liquid chromatograph mass spectrometer (liquid chromatograph; LC) by connecting a liquid-chromatographic mass spectrometer (LC/MS) - or, 2 are connected in series with a single mass spectrometer (MS/MS) or a tandem mass spectrometer, liquid chromatography tandem mass spectrometer coupled to the front of the liquid chromatography tandem mass spectrometer (LC/MS/MS) - like, have various functions are being developed and the mass spectrometer, the measurement of the biological material, has been widely used to quantify (for example, see Patent Documents 2-4).
Recently, the mass spectrometer using a stable isotope labeling method has been developed, the detection of a biological material has been used to measure. This method, a stable isotope labeled using a protein, a protein in the sample are quantified by mass spectrometry, in the blood serum in the rheumatoid patient diagnostic markers C- (C-reactive protein: CRP) reactive protein and a mammalian tissue in a body fluid sample and the example used for quantitation of amyloid β have been reported. In addition, in Patent Document 5-7, the present inventors, improvement of such a mass spectrometry, LC/MS/MS using a metabolic enzyme membrane proteins and methods for quantifying the absolute expression level of the established all at once. In such a method, the target protein in the target peptide fragment contained in the stable isotope labeled amino acid sequence the same target peptide fragment is used as an internal standard, were digested with trypsin and peptide fragments of the target in the biological sample should be able to quantify the absolute amount, the amount of expression of the target protein that can be obtained. However, for the synthesis of stable isotope labeled peptide fragments, 10 to 1 million yen per type of the cost of a failure from the front, to reduce the cost has been a problem.
  • 出願人(英語)
  • ※2012年7月以前掲載分については米国以外のすべての指定国
  • TOHOKU UNIVERSITY
  • 発明者(英語)
  • OHTSUKI, Sumio
  • TERASAKI, Tetsuya
国際特許分類(IPC)
指定国 National States: AE AG AL AM AO AT AU AZ BA BB BG BH BR BW BY BZ CA CH CL CN CO CR CU CZ DE DK DM DO DZ EC EE EG ES FI GB GD GE GH GM GT HN HR HU ID IL IN IS JP KE KG KM KN KP KR KZ LA LC LK LR LS LT LU LY MA MD ME MG MK MN MW MX MY MZ NA NG NI NO NZ OM PE PG PH PL PT QA RO RS RU RW SC SD SE SG SK SL SM ST SV SY TH TJ TM TN TR TT TZ UA UG US UZ VC VN ZA ZM ZW
ARIPO: BW GH GM KE LR LS MW MZ NA RW SD SL SZ TZ UG ZM ZW
EAPO: AM AZ BY KG KZ RU TJ TM
EPO: AL AT BE BG CH CY CZ DE DK EE ES FI FR GB GR HR HU IE IS IT LT LU LV MC MK MT NL NO PL PT RO RS SE SI SK SM TR
OAPI: BF BJ CF CG CI CM GA GN GQ GW ML MR NE SN TD TG
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