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Mass spectrometric analysis of proteins using stable isotopes of pyrylium derivates as labels 新技術説明会

外国特許コード F110002364
掲載日 2011年1月13日
出願国 アメリカ合衆国
出願番号 45223308
公報番号 20100190261
公報番号 8071392
出願日 平成20年6月19日(2008.6.19)
公報発行日 平成22年7月29日(2010.7.29)
公報発行日 平成23年12月6日(2011.12.6)
国際出願番号 JP2008061243
国際公開番号 WO2008156139
国際出願日 平成20年6月19日(2008.6.19)
国際公開日 平成20年12月24日(2008.12.24)
優先権データ
  • 特願2007-164249 (2007.6.21) JP
  • 2008WO-JP61243 (2008.6.19) WO
発明の名称 (英語) Mass spectrometric analysis of proteins using stable isotopes of pyrylium derivates as labels 新技術説明会
発明の概要(英語) (US8071392)
The present invention provides a protein analysis method using a combination of two or more kinds of stable isotopes of a compound represented by the formula (I): wherein R1, R2 and R3 are the same or different and each is hydrogen, halogen or alkyl, or a salt thereof, as a labeling compound to produce a difference in the mass of the same kind of protein contained in each sample.
特許請求の範囲(英語) [claim1]
1. A method for determining the quantitative ratio of a target protein contained in two or more kinds of protein-containing samples using a mass spectrometer, which method comprises: (i) providing two or more kinds of protein-containing samples to be analyzed;
(ii) preparing an internal standard sample by preparing starting samples with the same total protein content from all the corresponding protein-containing samples to be analyzed, and mixing equal amounts of the starting samples to obtain the internal standard sample;
(iii) labeling the target protein in each of the protein-containing samples to be analyzed and the internal standard sample with a combination of two or more kinds of stable isotopes of a compound represented by the formula (I):
wherein R1, R2 and R3 are the same or different and each is hydrogen, halogen or alkyl, or a salt thereof having a different mass number due to isotope, thereby producing a difference in the mass of the target protein contained in each of the protein-containing samples and the internal standard sample;
(iv) mixing the respective protein-containing samples and the internal standard sample;
(v) (a) separating the target protein in the mixture obtained in step (iv) from other proteins and cleaving the target protein with a restriction enzyme at a particular amino acid site to give a sample containing isotope-labeled peptides derived from the target protein, or (b) cleaving the target protein in the mixture obtained in step (iv) with a restriction enzyme at a particular amino acid site to give a sample containing isotope-labeled peptides derived from the target protein; and
(vi) measuring an MS spectrum of the isotope-labeled peptides derived from the target protein determining an MS spectral intensity of each of the same kind of peptide derived from the target protein having a mass difference due to isotope labeling, determining the ratios of the MS spectral intensity derived from the protein-containing samples to the MS spectral intensity derived from the internal standard sample, determining the quantitative ratio based on the intensity ratios, and correcting the quantitative ratio by removing the overlap with the isotope peak of the peptide due to naturally occurring isotope.
[claim2]
2. The method of claim 1, wherein the compound of the formula (I) is 2,4,6-trimethyl pyrylium.
[claim3]
3. The method of claim 1, wherein the mass difference between stable isotopes contained in said combination is two or more.
[claim4]
4. The method of claim 1, wherein the mass difference between stable isotopes contained in said combination is four or more.
[claim5]
5. The method of claim 1, wherein the salt of the compound represented by the formula (I) is 2,4,6-trimethyl pyrylium tetrafluoroborate salt, and wherein the mass difference between stable isotopes contained in said combination is 4 or 8.
[claim6]
6. The method of claim 1, wherein the two or more kinds of protein-containing samples comprise (i) samples derived from affected and normal region of tissues, or (ii) samples derived from a certain kind of cell culture in various developmental stages.
[claim7]
7. The method of claim 1, wherein the isotope is 13C isotope.
  • 発明者/出願人(英語)
  • MATSUKAWA SHIGERU
  • FUKUI UNIVERSITY
  • TAIYO NIPPON SANSO
国際特許分類(IPC)
米国特許分類/主・副
  • G01N033/68A12
  • S01N458/15
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