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METHOD AND DEVICE FOR BIOMOLECULE ANALYSIS USING RAMAN SPECTROSCOPY

外国特許コード F150008147
整理番号 E095P03WO
掲載日 2015年3月17日
出願国 世界知的所有権機関(WIPO)
国際出願番号 2013JP071844
国際公開番号 WO 2014027652
国際出願日 平成25年8月13日(2013.8.13)
国際公開日 平成26年2月20日(2014.2.20)
優先権データ
  • 特願2012-181140 (2012.8.17) JP
発明の名称 (英語) METHOD AND DEVICE FOR BIOMOLECULE ANALYSIS USING RAMAN SPECTROSCOPY
発明の概要(英語) The present invention provides a device having a sample separation unit, a Raman spectroscopy unit, and a mass spectrometry unit. The present invention further provides: a method for specifying biomolecules, said method combining Raman spectroscopy and mass spectrometry; and a method for identifying the binding site of biomolecules and low-molecular compounds. The present invention also provides a surface-enhanced Raman spectroscopy method exhibiting improved sensitivity.
従来技術、競合技術の概要(英語) BACKGROUND ART
Toxicity and efficacy (such as a drug) is a low molecular weight compound, such as proteins in vivo biological activity on the biological molecule. A low-molecular compound or a target biomolecule to examine the distribution of cells within the living organism, identifying the target biomolecule, by analyzing the specific site on the biological activity is the expression to elucidate the mechanism, the development of effective therapies and therapeutic agents and is the basis of the life thereof is very important in the study.
A target biomolecule within cells in vivo or a method for checking distribution, radioactive compounds, phosphorescent compounds and fluorescent compounds are used in molecular imaging, as well as Raman scattering light of the biomolecules themselves are known. Is in a cell biological or molecular imaging, the kinetics of the drug and the state of the disease which are important for understanding in the art, have been developed in recent years. Is Raman imaging, Raman spectroscopy is used, the laser beam applied to the sample to detect the Raman scattering light, to image the distribution. Is Raman imaging, radioactive compounds, phosphorescent compounds and fluorescent compounds are used for molecular imaging, non-radioactive a small influence on the target molecule for use in a low molecular compound, as is the dynamic state of the cells can be examined easily. In this case, the carbon - carbon triple bond alkynes and the like is used as a label, while minimizing the impact to the target molecule, it is possible to obtain a highly sensitive imaging have been reported (Non-Patent Document 1). Non-Patent Document 1 is, in the nucleic acid analog -2 - ethyl 5 - ' - deoxyuridine (EdU) was taken up by a cell, which has been captured in the nucleus was confirmed by imaging of a Raman microscope has been described by a (non-patent document 1, the first page 6103 in Fig. 2, see Fig. 4). In non-patent document 1, the wave number of the resulting Raman peaks labeled specific Raman image can be obtained. A particular wave number of the images obtained and thus the spatial intensity distribution of the Raman peak.
Low molecular weight compound such as a drug compound and a search for targeted biomolecules, the method for identifying the binding site, a combination of a liquid chromatograph mass spectrometer is used in the LC-MS. LC fractionated sample, the samples in a sequential fractionation exhaustive MS, MS/MS was subjected to analysis such as identifying a target biomolecule, the identification of the binding site. MS in the analysis, a low-molecular compound on the basis of the mass shift from binding of, searching for a target biomolecule. Further, MS/MS analysis, to obtain information such as amino acid sequence of peptide, the binding site can be identified.
LC-MS analysis method such as, in a cell in order to identify the target biomolecule, (1) a low molecular weight compound may be incorporated into the cells, the low molecular compound is allowed to bind to a target biomolecule within a cell, (2) the cell is from a cell lysate, (3) to detect the target biomolecule, (4) by analyzing the target biomolecule, a particular course, or to rupture the cells, (1) cell lysate, (2) a mixture of low molecular compound, binding to a target biomolecule, (3) fractionating a cell lysate of the target biomolecule, (4) analyzed, identified, the series of steps required. In addition, a biological molecule of a specific binding site in the low-molecular compound, the method of identifying, (1) a low molecular compound to bind to the biomolecule, (2) the biomolecule is bound to a low-molecular compound to fragment, (3) the fragment is bound, and (4) binding and fragments, to identify the binding sites is obtained by the processes.
However, not the complex obtained through the sample, using LC-MS an exhaustive search for biological molecules, sequencing and identification of the binding site to perform, requires a great deal of time, and an error is likely to occur. In addition, low molecular weight compound and the manner of the bonds between the biomolecules is unknown, which are based on the mass shift is assumed that the target molecule and is in principle impossible to search for. Instead of the liquid chromatograph and a method for using capillary electrophoresis (CE-MS) is also proposed, in a manner similar to LC-MS, since the cover must be detected, the analysis target is very numerous, complex analysis operation being required for a long time.
Intracellular target molecules to selectively subjected to the analysis of the mass spectrometry method, a low molecular compound and affinity purified using a carrier bound to, the target molecule separation and purification methods have been developed, and widely used. In addition, a functional group reactive with a target biomolecule using after a covalent bond, is introduced into the low-molecular compound in advance as a radioactive, phosphorescent or fluorescent compound can be determined by examining the like, a method for identifying a target biomolecule bound also been used. To specify the site of binding of a target molecule, with regard to a technique, a low molecular weight compound and a fluorophore can be introduced into a method that is widely used. For example, a drug labeled with a specific binding site of the protein, the invention relates to a method of identifying, xanthine as a fluorophore (rhodamine, fluorescein or lodore) dye, cyanine dye, a coumarin dye or complex is used as a labeling agent have been reported (Patent Document 1).
However, low molecular weight compound in the case of using radioactive compounds, the chemical nature of the radioactive isotope and are basically the same for the activity of the target molecule is not affected, the equipment can be used in the radiation management facility is not limited to, further, used in a method to identify the binding sites and increasing the limit of course, that the technique is a simple method. Phosphorescent compound having a large molecular weight fluorescent compounds which bind the target molecule directly to the method, unlike the radioactive compound, is that there is almost no usage restriction, the molecular weight of the fluorophore, as compared to the low-molecular compound becomes larger, the activity or binding characteristics of a low-molecular compound is a problem that may have an influence. Which is a kind of such as anti-cancer (5-FU) neofurtulon 130 molecular weight and on the other hand, in a typical fluorophore Rhodamine 6 g in a molecular weight of 479. 5-FU Rhodamine 6 g in the case where the labeled, anti-fluorescent label or influence the physiological activities of 5-FU may be caused. In addition, that has been extracted from a plant furabagurin Aglaia (flavagline) is an anticancer agent, a cancer cell specific to inhibit cell growth, and the anticancer agent is susceptible to side effects from the elucidation of the mechanism of action in vivo and thus, the drug is labeled with a fluorophore activity might be reduced to less than 1/40 have been reported. Non-Patent Document 2 according to the second right column page 5180, the cell growth suppressing furabagurin 50% is the concentration of IC50 is compared to the 3 nm, 130 nm of the fluorescently labeled furabagurin drops to it. Further, in Non-Patent Document 3, a molecule that binds to the target protein to modify the fluorophore 16F16, and its activity is reported to be erased (Non-Patent Document 3, the first row 901 and the right column pages 13-17).
An improved method of labeling method described above, as the functional group or alkynyl group comprising a first low molecular weight compound bound to the target biomolecule (alkyne), by the click reaction was then introduced into the fluorophore, such as an enzyme and the target biomolecule is decomposed by, a method of fragmenting have been reported (Non-Patent Document 3, page 902 in Fig. 3 the first reference). When this method is used, adverse effects such as disappearance of a target protein is reduced, the operation is complicated, there is a nonspecific binding reaction, a catalyst such as copper is required, as well as a loss of the target molecule by the development of the reaction becomes a problem. Therefore, the amount of sample is not sufficient or the like, may be applied to the practical limit. Of the post-translational modifications within cells with regard to the method of searching for, as an example using the click reaction was taken up by a cell are palmitoyl-lipids, it is then, using the click reaction is modified with a fluorophore, a protein binding to the lipid by a fluorescence analysis a report identifying a (non-patent document 4) and an example, a click reaction of the biotin is introduced into the lipid farneylsation, have been reported for detecting streptavidin (Non-Patent Document 5). However these methods may including the above-described problems associated with the click reaction.
Labeled with a fluorophore or a radioactive material through the search for a target molecule as compared to the method, Raman spectroscopy is, on the basis of the molecular vibrational information, label-free target molecule can be detected. Limitation on the used facilities, or the activity of low-molecular compound does not effect on the binding characteristics, the combination of LC-MS and Raman spectroscopy, to overcome the various problems of the above, can serve as a new detection method. Up to this point, a Raman spectroscopy apparatus and the combination of matrix-assisted laser desorption ionization mass spectrometer, an example of the analysis of the lysozyme have been reported (Patent Document 2, the first column 27, and claim 21 Fig. 31). However, the invention described in Patent Document 2 is an object to be solved, and to increase the sensitivity of Raman spectroscopic analysis, to isolate the sample in order to aggregate state in which the disclosed technique. In addition, in Patent Document 2 is that the mass spectrometer is used, the result confirmed by Raman spectroscopy by an alternative method for re-checking and, low-molecular-weight compound that binds a particular biomolecule, the identification of the binding site for the purpose of the present invention, fundamentally different.
  • 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
  • JAPAN SCIENCE AND TECHNOLOGY AGENCY
  • 発明者(英語)
  • SODEOKA MIKIKO
  • ANDO JUN
  • ASANUMA MIWAKO
  • DODO KOSUKE
  • FUJITA KATSUMASA
国際特許分類(IPC)
指定国 National States: AE AG AL AM AO AT AU AZ BA BB BG BH BN 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 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 PA PE PG PH PL PT QA RO RS RU RW SA 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 KM ML MR NE SN TD TG
参考情報 (研究プロジェクト等) ERATO SODEOKA Live Cell Chemistry AREA
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