WO2014027652A1 - ラマン分光法を用いた生体分子の解析方法及び装置 - Google Patents
ラマン分光法を用いた生体分子の解析方法及び装置 Download PDFInfo
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- G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
- G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
- G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures
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- C12N9/14—Hydrolases (3)
- C12N9/48—Hydrolases (3) acting on peptide bonds (3.4)
- C12N9/50—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25)
- C12N9/64—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue
- C12N9/6421—Proteinases, e.g. Endopeptidases (3.4.21-3.4.25) derived from animal tissue from mammals
- C12N9/6472—Cysteine endopeptidases (3.4.22)
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- C12Y—ENZYMES
- C12Y304/00—Hydrolases acting on peptide bonds, i.e. peptidases (3.4)
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- C12Y304/22001—Cathepsin B (3.4.22.1)
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/65—Raman scattering
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- G01N21/62—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light
- G01N21/63—Systems in which the material investigated is excited whereby it emits light or causes a change in wavelength of the incident light optically excited
- G01N21/65—Raman scattering
- G01N21/658—Raman scattering enhancement Raman, e.g. surface plasmons
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- G01N27/416—Systems
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- G01N27/44721—Arrangements for investigating the separated zones, e.g. localising zones by optical means
- G01N27/44726—Arrangements for investigating the separated zones, e.g. localising zones by optical means using specific dyes, markers or binding molecules
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- G01N30/02—Column chromatography
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- G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph
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- G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
- G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
- G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
- G01N33/585—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with a particulate label, e.g. coloured latex
- G01N33/587—Nanoparticles
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- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J49/00—Particle spectrometers or separator tubes
- H01J49/004—Combinations of spectrometers, tandem spectrometers, e.g. MS/MS, MSn
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- G01N2570/00—Omics, e.g. proteomics, glycomics or lipidomics; Methods of analysis focusing on the entire complement of classes of biological molecules or subsets thereof, i.e. focusing on proteomes, glycomes or lipidomes
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- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/74—Optical detectors
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- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/78—Detectors specially adapted therefor using more than one detector
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- H—ELECTRICITY
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- H01J49/00—Particle spectrometers or separator tubes
- H01J49/02—Details
- H01J49/04—Arrangements for introducing or extracting samples to be analysed, e.g. vacuum locks; Arrangements for external adjustment of electron- or ion-optical components
- H01J49/0409—Sample holders or containers
- H01J49/0418—Sample holders or containers for laser desorption, e.g. matrix-assisted laser desorption/ionisation [MALDI] plates or surface enhanced laser desorption/ionisation [SELDI] plates
Definitions
- the present invention relates to an analysis method and apparatus for identifying a biomolecule that binds to a low molecular compound, in particular, an intracellular or extracellular biomolecule, or identifying a binding site between a biomolecule and a low molecular compound. More specifically, the present invention relates to an apparatus including a sample separation unit, a Raman spectroscopic unit, and a mass spectrometric unit, a method for identifying a biomolecule that combines Raman spectroscopic analysis and mass spectrometry, and binding between a biomolecule and a low molecular compound. The present invention relates to a method for identifying a site. The present invention also relates to surface enhanced Raman spectroscopy.
- a low molecular weight compound (drug, etc.) having toxicity or medicinal effect acts on a biomolecule such as a protein in a living body and exhibits biological activity. It is an effective treatment to investigate the distribution of target biomolecules for low molecular weight compounds in vivo or in cells, identify the target biomolecule, analyze the specific site of action, and elucidate the mechanism of biological activity. It is extremely important in the development of methods and therapeutics and the life research that forms the basis for them.
- molecular imaging using a radioactive compound, a phosphorescent compound or a fluorescent compound, and Raman imaging for detecting scattered light of the biomolecule itself are known.
- Molecular imaging in a living body or in a cell is an important technique for understanding disease states, drug dynamics, and the like, and has been rapidly developed in recent years.
- Raman imaging Raman spectroscopy is used to detect the Raman scattered light of the laser irradiated on the sample and image the distribution.
- Raman imaging uses a low-molecular compound that is non-radioactive and has little influence on the target molecule.
- Non-patent Document 1 describes that 5-ethyl-2′-deoxyuridine (EdU), which is a nucleic acid analog, was taken into cells and confirmed that this was taken into cell nuclei by imaging with a Raman microscope. (See Non-Patent Document 1, page 6103, FIG. 2 and FIG. 4).
- EdU 5-ethyl-2′-deoxyuridine
- a Raman image is obtained at a wave number at which a Raman peak peculiar to a label is obtained. Therefore, the obtained image is a spatial intensity distribution of a Raman peak of a specific wave number.
- LC-MS combining a liquid chromatograph and a mass spectrometer is used as a method for searching for low molecular compounds such as drugs and biomolecules targeted by the compound and identifying the binding site.
- the sample is fractionated by LC, and the fractionated sample is sequentially and comprehensively subjected to MS and MS / MS analysis to identify the target biomolecule and identify the binding site.
- MS analysis a target biomolecule is searched based on a mass shift derived from the binding of a low-molecular compound.
- the binding site can be identified by acquiring information such as the amino acid sequence of the peptide by MS / MS analysis.
- a low molecular compound is taken into the cell and the low molecular compound is bound to a target biomolecule in the cell.
- Crush cells (3) detect target biomolecules from cell lysate, (4) analyze and identify target biomolecules, or (1) crush cells, (2) cell lysates A series of processes of mixing a low molecular weight compound and binding with a target biomolecule, (3) fractionating a cell lysate, and (4) analyzing and identifying the target biomolecule are required.
- a method for selectively purifying intracellular target molecules for analysis such as mass spectrometry has been developed and widely used by affinity purification using a carrier bound with a low molecular weight compound to separate and purify target molecules.
- the bound target biomolecule can be determined by examining the radioactive, phosphorescent or fluorescent compound previously introduced into the low molecular weight compound.
- An identifying method is also used.
- a technique for identifying and identifying a binding site of a target molecule a method of observing by introducing a fluorophore into a low molecular compound is widely used.
- Patent Document 1 a method for identifying and identifying a binding site between a labeled drug and a protein, a method using a xanthine dye (rhodamine, fluorescein or rhodol), a cyanine dye, a coumarin dye or a complex dye as a fluorophore has been reported.
- a xanthine dye rhodamine, fluorescein or rhodol
- cyanine dye a coumarin dye or a complex dye as a fluorophore
- radioactive compounds when radioactive compounds are used as low-molecular compounds, the chemical properties of the radioisotopes are basically the same and do not affect the activity of the target molecule, but the facilities that can be used are limited to radiation management facilities. Furthermore, it is difficult to say that it is a simple method because the method of identifying the binding site is greatly limited in usage. Unlike the radioactive compounds, the method of directly binding a phosphorescent compound or a fluorescent compound with a large molecular weight to the target molecule has almost no restrictions on use, but the molecular weight of the fluorophore is lower than that of a low molecular weight compound. There is a problem that it can affect the activity or binding properties of molecular compounds.
- fluorouracil a kind of anticancer agent
- Rhodamine 6G a typical fluorophore
- 5-FU fluorouracil
- Rhodamine 6G a typical fluorophore
- 5-FU the physiological activity of the anticancer agent 5-FU by fluorescence labeling may be affected.
- the anticancer drug flavagline extracted from a plant called Aglaia inhibits cell growth specifically in cancer cells, and anticancer drugs are unlikely to cause side effects, so elucidation of the mechanism of action in vivo is required.
- labeling with a fluorophore reduces the drug activity to 1/40 or less.
- Non-Patent Document 3 reports that the activity disappears when the molecule 16F16 that binds to the target protein is modified with a fluorophore (Non-Patent Document 3, page 901, right column, lines 13-17). .
- Non-patent document 4 For specifying a protein that binds to the lipid and a report example in which a biotin tag is introduced into farnesyl lipid by a click reaction and detected with streptavidin.
- these methods also include the above-mentioned problems associated with the click reaction.
- Raman spectroscopy can detect a target molecule without labeling based on molecular vibration information. Since there are no restrictions on the facilities used and there is no effect on the activity and binding properties of low-molecular compounds, the combination of Raman spectroscopy and LC-MS can be a new detection method that overcomes the various problems described above. So far, an example of analyzing lysozyme by combining a Raman spectroscope and a matrix-assisted laser desorption / ionization mass spectrometer has been reported (Patent Document 2, column 27, FIG. 31 and claim 21).
- Patent Document 2 the problem to be solved by the invention described in Patent Document 2 is to increase the sensitivity of Raman spectroscopic analysis.
- a technique for aggregating the sample in an isolated state is disclosed.
- the reason why the mass spectrometer is used in Patent Document 2 is to reconfirm the result confirmed by Raman spectroscopy by another method.
- the identification of the biomolecule that binds to the low molecular weight compound and the binding site This is fundamentally different from the present invention for identification purposes.
- Identifying a target molecule that binds to a biomolecule in a wide variety of biomolecules contained in a living cell and identifying the binding site is an extremely important technology for developing effective therapeutic agents for various diseases.
- a practical and simple method and an analysis apparatus therefor are not known.
- a molecule containing a radioisotope a molecule having a phosphorescent compound or a fluorescent compound bound thereto is used as a labeling agent
- the activity (binding ability) of the target molecule may be reduced or eliminated by introducing a fluorophore having a large molecular weight.
- molecules to which a phosphorescent compound or a fluorescent compound is bound have various problems such that they can be firmly bound to the column non-specifically in chromatography and can be difficult to isolate and recover.
- an object of the present invention is to provide a method and apparatus for identifying a target molecule that binds to a biomolecule by a practical and simple method and identifying the binding site.
- Another object of the present invention is to provide a surface enhanced Raman spectroscopy (SERS) method with enhanced sensitivity.
- SERS surface enhanced Raman spectroscopy
- the present inventors used a fractionated sample for Raman spectroscopic analysis, and then subjected to mass spectrometry for biomolecules that bind to low molecular compounds.
- the present invention has been completed by discovering that it can be specified and that a binding site between a low molecular weight compound and a biomolecule can be specified.
- the present inventors have found that the sensitivity of SERS can be enhanced by using an aggregation accelerator. That is, the present invention is as follows.
- An apparatus for identifying a biomolecule that binds to a low molecular compound or for identifying a binding site between a low molecular compound and a biomolecule comprising a sample separation unit, a Raman spectroscopic unit, and a mass spectrometric unit
- sample separation unit is a liquid chromatograph or a capillary electrophoresis apparatus.
- liquid chromatograph is any one type of high performance liquid chromatograph selected from the group consisting of normal phase, reverse phase, molecular sieve, and ion exchange chromatograph.
- the Raman spectroscopic unit is a linear or non-linear Raman spectroscopic device having a laser unit for irradiating a Raman excitation laser beam and a spectrum analyzing unit for spectrally analyzing Raman scattered light. The device described.
- the mass spectrometer includes a mass spectrometer that uses a matrix-assisted laser desorption ionization method, an electrospray ionization method, or an atmospheric pressure chemical ionization method as an ionization method .
- the low molecular weight compound has an alkynyl group, nitrile group, diazonio group, isocyanate ester group, isonitrile group, ketene group, carbodiimide group, thiocyanate ester group, azide having a scattering spectrum in the silent region of the Raman spectrum in the molecule.
- the apparatus according to any one of [1] to [6], comprising at least one substituent selected from the group consisting of a group, a diazo group, an alkynediyl group, and deuterium.
- biomolecule is at least one biomolecule selected from the group consisting of proteins, peptides, nucleic acids, sugars and lipids.
- the plate according to [9] or [10] which is made of metal, glass, quartz, calcium fluoride, or magnesium fluoride.
- a method for identifying a binding site between a biomolecule and a low molecular weight compound comprising the following steps: (1) subjecting the fractionated biomolecule fragment bound to the low molecular weight compound to Raman spectroscopic analysis; and (2) subjecting all or part of the fraction subjected to Raman spectroscopic analysis to mass spectrometry. Including Detects a fraction having a Raman peak derived from a low molecular weight compound bound to a biomolecule fragment by Raman spectroscopic analysis, obtains a mass analysis result of the fraction having a low molecular weight compound-derived Raman peak, and obtains the mass information of the biomolecule.
- biomolecule is fragmented by an enzyme selected from the group consisting of a proteolytic enzyme, a peptide degrading enzyme, a nucleolytic enzyme, a glycolytic enzyme, and a lipolytic enzyme, or by chemical degradation.
- an enzyme selected from the group consisting of a proteolytic enzyme, a peptide degrading enzyme, a nucleolytic enzyme, a glycolytic enzyme, and a lipolytic enzyme, or by chemical degradation.
- a screening method for identifying a biomolecule that binds to a low molecular weight compound comprising the following steps: (1) subjecting a fraction containing a biomolecule bound to a low molecular compound to Raman spectroscopic analysis, and (2) subjecting all or a part of the fraction subjected to Raman spectroscopic analysis to mass spectrometry, Including Detects a fraction having a Raman peak derived from a low molecular compound by Raman spectroscopic analysis, obtains a mass analysis result of a fraction having a Raman peak derived from a low molecular compound, and collates this with the mass information of the biomolecule. And a method for identifying a biomolecule that binds to the low molecular weight compound.
- a sample containing a biomolecule bound to the low molecular weight compound is obtained by (A) incorporating a low molecular weight compound into a cell, binding it to the intracellular biomolecule, and disrupting the cell, or (B) [17] The method according to [17], wherein the method is prepared by disrupting cells, adding a low molecular compound to the cell disruption solution, and binding the cells with biomolecules in the cells.
- the low molecular weight compound has an alkynyl group, nitrile group, diazonio group, isocyanate group, isonitrile group, ketene group, carbodiimide group, thiocyanate group, azide having a scattering spectrum in the silent region of the Raman spectrum in the molecule.
- biomolecule is at least one biomolecule selected from the group consisting of proteins, peptides, nucleic acids, sugars and lipids.
- the fractionated fraction is a droplet as it is or a droplet mixed with a solvent, the droplet is arranged on a plate having a cleaned surface, and included in the droplet
- Organic acid is trifluoroacetic acid, difluoroacetic acid, monofluoroacetic acid, trifluoromethanesulfonic acid, difluoromethanesulfonic acid, 3,3,3-trifluoropropionic acid, trichloroacetic acid, dichloroacetic acid, monochloroacetic acid, trichloromethane
- the method according to [28] which is selected from the group consisting of sulfonic acid, dichloromethanesulfonic acid, 3,3,3-trichloropropionic acid, formic acid, acetic acid, propionic acid, methanesulfonic acid, and combinations thereof.
- a low molecular weight compound that binds to a biomolecule has an alkynyl group, nitrile group, diazonio group, isocyanate group, isonitrile group, ketene group, carbodiimide group, thiocyanate having a scattering spectrum in the silent region of the Raman spectrum.
- a surface enhanced Raman spectroscopic analysis method comprising:
- the target molecule is a biomolecule, a fragment of a biomolecule, a biomolecule bonded to a low molecular compound having a Raman peak in the silent region, or a biomolecule fragment bonded to a low molecular compound having a Raman peak in the silent region. [31] or [32].
- biomolecule is at least one biomolecule selected from the group consisting of proteins, peptides, nucleic acids, sugars and lipids.
- Organic acid is trifluoroacetic acid, difluoroacetic acid, monofluoroacetic acid, trifluoromethanesulfonic acid, difluoromethanesulfonic acid, 3,3,3-trifluoropropionic acid, trichloroacetic acid, dichloroacetic acid, monochloroacetic acid, trichloromethane Any one of [31] to [34] selected from the group consisting of sulfonic acid, dichloromethanesulfonic acid, 3,3,3-trichloropropionic acid, formic acid, acetic acid, propionic acid, methanesulfonic acid, and combinations thereof Method.
- a low molecular weight compound that binds to a biomolecule has an alkynyl group, nitrile group, diazonio group, isocyanate group, isonitrile group, ketene group, carbodiimide group, thiocyanate having a scattering spectrum in the silent region of the Raman spectrum.
- the droplets of the solution containing aggregates are arranged on a plate having a cleaned surface, and the solvent contained in the droplets is evaporated.
- An analysis method comprising subjecting all or part of the solution or fraction subjected to the surface enhanced Raman spectroscopy (SERS) analysis method according to any one of [31] to [38] to mass spectrometry.
- SERS surface enhanced Raman spectroscopy
- the liquid chromatography-Raman spectroscopic-mass spectrometry (LC-R-MS) or capillary electrophoresis-Raman spectroscopic-mass spectrometric (CE-R-MS) apparatus is a novel biomolecular analysis that has not been known so far.
- This is a device that can be used for conventional liquid chromatography-mass spectrometry (LC-MS) and capillary electrophoresis-mass spectrometry (CE-MS) devices that comprehensively search for biomolecules, determine sequencing and identify binding sites, etc. Compared to this, it is an excellent analysis device that can shorten the processing time and has high accuracy.
- the method according to the present invention when used, complementary information regarding a measurement target can be acquired by Raman spectroscopic analysis and mass spectrometry, and a target biomolecule can be identified more quickly and reliably.
- the SERS method using the aggregation accelerator of the present invention has an increased measurement sensitivity and an improved detection limit. Further, in the SERS method using the aggregation accelerator of the present invention, the correlation between the amount of the sample to be measured and the SERS signal intensity is improved, and variation in measurement is suppressed.
- the Raman spectroscopic unit according to the present invention can perform non-destructive and non-contact measurement using a Raman spectroscopic device without modifying the sample.
- a low molecular weight compound can be selectively detected with high sensitivity by a Raman label having a characteristic Raman peak.
- a Raman label having a characteristic Raman peak since there are almost no compounds or deuterium with triple bonds such as alkyne molecules in living organisms, when alkyne or deuterium is used as a Raman label, low molecular weight compounds from complex mixtures such as cell lysates are used.
- the target biomolecule bound to can be identified. The same applies to other types of Raman labels having a Raman peak in the silent region.
- the Raman spectroscopic analysis according to the present invention When the Raman spectroscopic analysis according to the present invention is performed, not only low molecular compounds but also molecular vibration information derived from biomolecules can be obtained. Therefore, there is an advantage that the coexistence of the low molecular compounds and the biomolecules can be confirmed.
- the conventional fluorescence labeling method confirms the presence or absence of a low-molecular compound from the fluorescence intensity of a single channel, whereas the Raman spectroscopy according to the present invention provides multi-dimensional vibrational spectroscopy information, and thus has multiple scattering peaks. From the strength, it is possible to confirm the presence or absence of both low molecular weight compounds and biomolecules. Furthermore, in the case of peptides and the like, information on the backbone structure and side chains can also be obtained from the shape of the spectrum.
- the method according to the present invention uses a low-molecular compound as it is or can suppress the molecular weight of a tag added to an analysis target compound, so that it is low in comparison with the conventional fluorescent labeling method using a large fluorophore.
- the target biomolecule can be specifically identified / detected and identified without changing the biochemical characteristics of the molecular compound. That is, when the Raman label of the present invention is used, the artifact due to modification is small compared to the fluorophore.
- a mass spectrum of a protein or peptide can be obtained by a mass spectrometer, and a binding site between a low molecular compound and a target biomolecule can be identified based on the mass spectrum.
- the amino acid sequence of a protein or peptide can also be determined by mass spectrometry using MS / MS analysis.
- protein post-translational modifications can be analyzed.
- Non-Patent Document 2 The conventional method of combining an alkyne tag with a click reaction (Non-Patent Document 2, etc.) has a problem that there is a loss of the target compound related to the click reaction operation and a non-specific reaction occurs.
- the analysis target compound is used as it is as a low molecular compound in Raman spectroscopy, or the analysis target compound added with an alkyne tag is used as a low molecular compound, and this is used as it is in Raman spectroscopy. For this reason, the problems involved in the conventional method are solved.
- C shows the peak of alkyne obtained from the Raman spectrum of each fraction (FIG. 9-3G).
- D shows the mass spectrum of fraction number 4, and E shows the mass spectrum of fraction number 12.
- F represents the intensities of fractions 1-16 at 1211.5 m / z and 1229/6 m / z. It is a Raman spectrum of each fraction corresponding to FIG. 9-1C. It is the figure which compared the online Raman measurement and the offline Raman measurement. A is an online Raman detection method, and B is an offline Raman detection method. It is a figure which shows the apparatus which concerns on 1 aspect of this invention. It is a figure shown about the effect of SERS.
- the Raman intensity of RAT8-AOMK increased by more than 10 3 when silver nanoparticles with a diameter of 40 nm were used. It is a figure which shows the sample spotted with the multi spot metal substrate. Spot the fraction sample as shown in the schematic diagram in the upper center.
- the upper right is a 384 multi-spot metal substrate.
- the lower right shows a photograph in which the peptides in the solution aggregate as the droplets on the substrate dries. It is a figure which shows the example of the plate which can be used for the apparatus which concerns on this invention.
- the upper left is a microscope substrate fixing plate.
- the lower left is the Raman microscope sample stage.
- FIG. 6 is a view showing a Raman spectrum of fraction numbers 35 to 94.
- fractions 57-66 a characteristic Raman peak was observed around 2106 cm -1 . It is a figure which shows the mass spectrometry result about the spot after Raman spectroscopy. From the spot where the Raman peak was obtained, a peptide bound with a low molecular weight compound having a Raman label was detected.
- A is fraction number 62
- B is 60
- C is 57.
- A shows experimental values and calculated values corresponding to fraction number 62.
- B shows the experimental value and calculated value corresponding to fraction number 60.
- C shows the experimental value and calculated value corresponding to fraction number 57.
- a comparison of the Raman spectrum of RAT8-AOMK itself and the spectrum of fraction numbers 35-75 is shown in A.
- C shows the Raman peak intensities at 1609 cm -1 and 2107 cm -1 .
- the dotted line is the intensity of 1609 cm ⁇ 1 derived from the phenyl ring, and the solid line is the intensity of 2107 cm ⁇ 1 derived from the alkyne.
- the lower part of FIG. 24 shows structures and molecular weights before and after the binding of RAT8-AOMK to cathepsin B. It is a figure which shows the Raman spectrum of RAT8-AOMK.
- Peptide A-2 (no cleavage error, + RAT8-AOMK), m / z 2435.0067. It is a figure which shows the MALDI mass spectrum of fraction number 58-62 (peptide B-1). The scale was fixed at 2E 7 .
- Peptide B-1 (1 cleavage mistake, + RAT8-AOMK + CAM), m / z 2890.2559. It is a figure which shows the MALDI mass spectrum of fraction number 60-65 (peptide A-1). The scale was fixed at 2E 7 .
- Peptide A-1 (no cleavage error, + RAT8-AOMK + CAM), m / z 2492.0282.
- FIG. 30 summarizes the results of FIGS.
- the ion count number of the target peptide of fraction number 50-69 is shown.
- the horizontal axis is the fraction number, and the vertical axis is the ion count number.
- the excitation wavelength was 532 nm.
- the SERS spectrum of RAT8-AOMK when using gold nanoparticles is shown.
- the excitation wavelength was 660 nm.
- the upper spectrum is with gold nanoparticles and the lower spectrum is without gold nanoparticles.
- the result of SERS measurement by mixing a dispersion of silver nanoparticles with an aqueous solution of alkyne-labeled / unlabeled peptide is shown.
- the upper spectrum is that of an alkyne labeled peptide and the lower spectrum is that of a normal peptide.
- the result of comparing the case of introducing a fluorophore with a click reaction and the case of Raman spectroscopy is shown.
- the UV chromatogram (1) at the bottom of the figure is the case of Raman spectroscopy of the present invention
- the UV chromatogram (2) is the case where a fluorophore is introduced by a click reaction. In the case of the click reaction, there was a 57.5-74.2% sample reduction.
- the black circle is a Raman chromatogram
- the white circle is a UV romanogram with a fluorophore introduced by a click reaction.
- An apparatus includes a sample separation unit, a Raman spectroscopic unit, and a mass spectrometric unit.
- the sample separation unit, the Raman spectroscopic unit, and the mass spectrometry unit are connected in this order.
- An example of the sample separation unit is shown in FIG. 1, an example of the Raman spectroscopic unit is shown in FIG. 2, and an example of the mass analysis unit is shown in FIG. Each of these will be described.
- the sample separation unit according to the present invention can separate various molecules in a sample from each other.
- Specific examples of the sample separation unit include, but are not limited to, a liquid chromatograph and a capillary electrophoresis apparatus.
- the sample separation unit can be an isoelectric focusing device.
- a sample refers to a sample that may contain a compound to be measured.
- An example of a sample separation unit according to the present invention is shown in FIG. First, the sample is fed from the sample injection unit 1 to the fractionation unit 3 through the liquid feeding line 2. Next, the fractionation unit 3 performs fractionation.
- the fractionation unit 3 may include various chromatography columns, liquid chromatography columns, and electrophoresis capillaries, but is not limited thereto.
- the detection unit 5 can be, for example, an ultraviolet (UV) light detector.
- the detection means in the detection unit 5 is preferably a nondestructive inspection. In FIG. 1, it is indicated by a right-pointing arrow that the separated fraction is further connected to the next Raman spectroscopic unit.
- the sample fractionated by the sample separation unit can be detected by Raman spectroscopic analysis, and therefore the detection unit 5 in FIG. 1 can be omitted. That is, FIG. 1 is merely an example, and the detection unit 5 is not an essential component for the sample separation unit.
- a liquid chromatograph refers to a chromatograph that uses a liquid as the mobile phase.
- substances contained in a mobile phase are eluted or eluted from columns packed with a solid phase carrier at different rates based on the difference in the degree of interaction with the solid phase carrier.
- a specific substance contained in the mobile phase is separated from other substances using the difference in elution rate.
- the principle of liquid chromatographic separation may be any, and examples include distribution, adsorption, molecular exclusion, molecular sieving, ion exchange, and the like.
- the chromatograph may be normal phase or reverse phase.
- the liquid chromatograph is high performance liquid chromatography (HPLC) using a liquid pressurized to a high pressure as the mobile phase.
- HPLC high performance liquid chromatography
- any solvent that dissolves the solute may be used as the mobile phase, such as water, aqueous solution, aqueous solution containing salts, organic solvent, methanol, ethanol, isopropanol, n-propanol, etc.
- the basic compound contained in the aqueous solution may be adsorbed by the residual silanol present in the column and the chromatogram peak may tail.
- separation may be performed by adding an acid such as trifluoroacetic acid. As described below, such an acid can be removed from the sample used in the next step by removing the solvent used in the separation operation after liquid chromatography.
- the Raman measurement can be performed regardless of the presence or absence of a solvent.
- the solvent used before the Raman measurement For example, it is desirable to use a low boiling polar solvent as the main component of the solvent of the mobile phase of the chromatogram. This is because such a low boiling point polar solvent can be easily removed by evaporation.
- the low boiling point polar solvent is a solvent having a low boiling point and polarity, and examples thereof include acetonitrile, methanol, dichloromethane, trichloromethane and the like.
- a low boiling polar solvent is preferred as the mobile phase used in the liquid chromatograph.
- a concentration gradient can be brought about in the separation solution, thereby increasing the resolution of the sample.
- the solvent used for the liquid chromatograph can be removed from the sample used for the Raman measurement.
- a person skilled in the art can appropriately set conditions such as a separation solvent and a concentration gradient to be applied according to the sample to be separated.
- Capillary electrophoresis refers to a method in which electrophoresis is performed in a sufficiently thin capillary tube to separate substances contained in a sample.
- a capillary electrophoresis apparatus typically includes a capillary tube and a voltage application unit.
- one of the capillaries is a sample injection part, and the other is a sample elution part. For example, referring to the sample separation unit in FIG.
- This capillary electrophoresis includes capillary zone electrophoresis (CZE), micelle conduction chromatography (MEKC), capillary gel electrophoresis (CGE), capillary isoelectric focusing (cIEF), and the like.
- CZE capillary zone electrophoresis
- MEKC micelle conduction chromatography
- CGE capillary gel electrophoresis
- cIEF capillary isoelectric focusing
- a person skilled in the art can appropriately set the operating conditions such as the solvent used for capillary electrophoresis, the type of capillary, and the applied voltage.
- the fraction fractionated by capillary electrophoresis can be subjected to the next step in a dry state by evaporating the solvent as in the liquid chromatograph.
- the Raman spectroscopic unit referred to in this specification is a linear or non-linear Raman spectroscopic device including a laser unit that emits Raman excitation laser light and a spectral analysis unit that performs spectral analysis of Raman scattered light.
- FIG. 2 shows a micro Raman analysis apparatus as an example of the Raman spectroscopic unit according to the present invention.
- a sample 9 is placed on the sample stage 10 and analyzed by a Raman microscope.
- Laser light for Raman excitation is irradiated from the laser unit 6, and this is reflected by the difleic filter 7 and irradiated to the sample 9 focused by the objective lens 8.
- the Raman scattered light is dispersed by the spectroscope 11 and detected by a detection mechanism such as a charge coupled device (CCD) in the detector 12.
- the spectrum analysis unit includes a spectroscope 11, a detection unit 12, and an arbitrary electronic calculation unit for acquiring the Raman scattering spectrum of FIG.
- the collected Raman scattered light is indicated by an upward arrow entering the spectrometer 11.
- An entrance slit or a lens can be appropriately used for condensing light.
- the Raman spectroscopic unit according to the present invention is not limited to a microscopic Raman analyzing apparatus, but includes all known Raman spectroscopic analyzing apparatuses such as a dispersion type laser Raman spectroscopic apparatus and an FT-Raman spectroscopic analyzing apparatus.
- the spectrum analysis unit included in the Raman spectroscopic unit according to the present invention may include a device that detects Raman scattered light with an interferometer instead of the spectroscope.
- a filter having a limited transmission wavelength band may be used for the Raman scattered light detection unit, and the scattered light transmitted through the filter may be directly detected by a detection mechanism such as a CCD without using a spectroscope or the like.
- a detection mechanism such as a CCD
- a wavelength tunable filter is used, a Raman spectrum can be acquired by scanning the transmission wavelength band.
- the structure which acquires a Raman spectrum by using the wavelength variable laser light source for the laser for Raman excitation, and scanning the wavelength of excitation laser light may be sufficient. Whether it is a spectroscope, interferometer, filter, or scanning of excitation laser light, it is possible to detect the intensity of a specific Raman peak or information on the Raman spectrum by detecting Raman scattered light in any configuration.
- the sample is placed on the sample stage as shown in FIG. 2, it is also possible to send the sample through a liquid feed line and perform so-called “on-line” measurement.
- a person skilled in the art can appropriately analyze the presence / absence of the target molecule in the sample from the obtained Raman spectrum pattern (profile). Analysis can also be performed manually or with the aid of a computer.
- the laser for Raman excitation a semiconductor laser, a diode-excited solid (DPSS) laser, a gas laser, a liquid laser, or the like can be used, but is not limited thereto.
- Raman spectroscopic analysis is a well-known technique in this technical field. For example, the principle is explained in Raman spectroscopy (published by the Japan Spectroscopic Society, Measurement Method Series 17) edited by Hiroo Hamaguchi and Atsuko Hirakawa. Yes. Briefly, Raman spectroscopy uses the so-called “Raman effect”, in which light with a wavelength different from the wavelength of incident light in scattered light is generated when light such as laser light is incident on a chemical substance. This is a spectroscopic analysis method. The difference between the frequency of Raman scattered light and the frequency of incident light is called Raman shift.
- Raman shift is specific to the structure of the molecule, knowledge about the molecular structure can be obtained by measuring the Raman shift.
- the Raman spectrum of a molecule whose chemical structure has been elucidated is measured in advance to obtain a profile, and the Raman spectrum pattern of the sample is compared with the profile to determine whether the molecule exists in a sample. This can be detected. Detecting means confirming the presence of a certain compound in a certain sample.
- Raman spectroscopy has the advantage of being a non-destructive analytical method. Linear Raman spectroscopy is also called Raman scattering spectroscopy, also known as spontaneous Raman scattering spectroscopy, having an intensity proportional to the incident light intensity.
- Nonlinear Raman spectroscopy is Raman spectroscopy based on higher-order nonlinear optical effects, and means Raman scattering spectroscopy having an intensity proportional to the second or higher order of the intensity of incident light.
- Raman spectroscopy include nonlinear Raman spectroscopy such as stimulated Raman scattering, hyper-Raman scattering, and coherent / anti-Stokes Raman scattering.
- An example of a Raman spectrum is shown in FIG. As shown in FIG. 4, the molecular vibration of paclitaxel itself is detected as a peak.
- Raman spectroscopy in this example is described in J. Ling et al., Applied Optic, 41, (28), 6006 (2002). Measurements were made using a Renishaw Model 2000 Raman spectroscopy system, Ti: sapphire laser. The sample was a powder, and measurement was performed with a 20 ⁇ lens and 30 seconds exposure.
- the present invention provides Raman spectroscopy using surface enhanced Raman spectroscopy (SERS).
- SERS surface enhanced Raman spectroscopy
- the surface enhanced Raman spectroscopy (SERS) of the present invention can be used in an apparatus or method according to the present invention.
- Raman spectroscopy since scattered light is weak, a long-term measurement is generally required. However, when SERS is used, a Raman signal can be enhanced and a rapid measurement can be performed.
- SERS is known as one of the Raman spectroscopic methods, and performs Raman spectroscopic analysis using a metal particle colloid or a substrate containing a metal.
- the surface plasmon of the metal is excited by the laser, and as a result, the electromagnetic field surrounding the metal increases and the Raman signal generated in proportion to the electromagnetic field is enhanced. Furthermore, a chemical interaction including the transfer of electrons occurs between a molecule near the metal surface and the metal, thereby enhancing the Raman signal.
- the action of either or both of the electromagnetic and chemical enhancement mechanisms described above greatly enhances the measured Raman signal.
- the metal used for SERS include, but are not limited to, iron, cobalt, nickel, tin, indium, germanium, copper, silver, gold, platinum, palladium, aluminum, titanium, ruthenium, and the like.
- the metal can be a metal nanoparticle, a metal nanostructure or a metal nanostructure.
- FIG. 12 shows that the intensity of the Raman peak of RAT8-AOMK increased by 10 3 or more when silver nanoparticles having a diameter of 40 nm were used. The exposure time was 10 seconds.
- FIG. 34 shows a SERS spectrum when gold nanoparticles are used. The lower spectrum of FIG.
- the SERS effect of the present invention is not limited to silver nanoparticles, and it is considered that the Raman signal is enhanced when metal nanoparticles such as gold nanoparticles are used in general.
- the SERS sample is spotted on a substrate cleaned with a commercially available silver nanoparticle dispersed aqueous solution and dried, and then the sample is overlaid on it, or the sample solution is mixed with the silver nanoparticle dispersed aqueous solution, or the mixed sample is spotted. It can be prepared by post-drying.
- the substrate a substrate obtained by applying a silver nanoparticle-dispersed aqueous solution to the entire surface of the substrate by a mechanical coating method such as a spin coating method can be used.
- the diameter of the metal nanoparticles is not particularly limited and is preferably small.
- the diameter (diameter) of the particle is a length equal to the diameter of a sphere having the same volume as the particle.
- the particle having a diameter of 40 nm means that a value obtained by averaging the diameters obtained as described above of a large number of particles is 40 nm.
- metal nanostructures such as nanorods, nanowires, nanocubes, nanoprisms, and shell structures, and it is preferable that the sizes thereof are also small.
- the size of the metal nanostructure refers to the length of the structure in the longitudinal direction.
- the size of the metal nanostructure being 40 nm means that the average size of various metal nanostructures is 40 nm.
- the size of the metal particle, metal nanostructure or metal nanostructure is preferably equal to or less than the mean free path of electrons that vibrate in the metal by light.
- the diameter of the particle In the case of a metal particle, the diameter of the particle In the case of a nanostructure or a metal nanostructure, the length of the nanostructure is 200 nm or less, more preferably 100 nm or less, and still more preferably 50 nm or less.
- Examples of the aggregation accelerator (organic acid) used in the present invention include halogenated organic acids containing fluorine or chlorine atoms in the molecule, such as trifluoroacetic acid, difluoroacetic acid, monofluoroacetic acid, trifluoromethanesulfonic acid, difluoromethanesulfonic acid or Fluorine-containing organic acids such as 3,3,3-trifluoropropionic acid and chlorine-containing organic acids such as trichloroacetic acid, dichloroacetic acid, monochloroacetic acid, trichloromethanesulfonic acid, dichloromethanesulfonic acid or 3,3,3-trichloropropionic acid Hydrocarbon organic acids such as formic acid, acetic acid, methanesulfonic acid, propionic acid can be used.
- halogenated organic acids containing fluorine or chlorine atoms in the molecule such as trifluoroacetic acid, difluoroacetic acid, monofluor
- trifluoroacetic acid trifluoroacetic acid, difluoroacetic acid, trifluoromethanesulfonic acid, difluoromethanesulfonic acid, 3, 3,3-trifluoropropionic acid, formic acid, acetic acid, propionic acid or Tan sulfonic acid is preferred.
- Organic acids containing bromine or iodine atoms in the molecule are more decomposable than fluorine-containing or chlorine-containing compounds, and have little effect on SERS due to the reaction between decomposition products and alkynyl groups, etc. Rather, it is not preferable because it is considered to have an inhibitory effect.
- the addition amount of the aggregation accelerator (organic acid) of the present invention is not limited as long as uniform aggregate formation is promoted, for example, 0.001 to 10 mol%, 0.01 to 1 mol%, preferably 0.05 to 0.5. mol%.
- a person skilled in the art prepares a plurality of solutions containing various organic acids together with a target molecule or a plurality of solutions containing various concentrations of organic acid together with a target molecule, and these are mixed with metal nanoparticles, and the solution is liquidated.
- the droplets By forming the droplets on the plate and observing the bright field image with a microscope, the formation of aggregates and the uniformity thereof can be easily confirmed. Thereby, the organic acid which can be used for this invention can be confirmed. Also, an appropriate amount of organic acid can be determined.
- Such screening can be performed at high throughput, for example using automated equipment, with only routine work by preparing a large number of spots on the plate.
- the aggregation promoter of the present invention can be used as follows.
- an aqueous solution containing the aggregation promoter of the present invention and a target molecule is mixed with metal nanoparticles, and then the target molecule-metal nanoparticle complex is aggregated.
- an aqueous solution containing the aggregation promoter of the present invention but not the target molecule is mixed with the metal nanoparticles, and then the metal nanoparticles are aggregated.
- the target molecule is added, and the target molecule and the aggregated metal nanoparticles are allowed to interact with each other.
- the inventors of the present invention have confirmed that the aggregation promoter of the present invention not only increases the detection limit of SERS measurement but also increases the correlation between the injection amount of a sample that emits a SERS signal and the SERS signal intensity. That is, the aggregation promoter of the present invention has an effect of stabilizing SERS measurement.
- the action of the aggregation accelerator of the present invention is not desired to be bound by a specific logic, but is considered to be due to the following mechanism.
- a target molecule-metal nanoparticle complex is formed, and the metal nanoparticles are aggregated while involving the target molecule. It is done.
- the formation of aggregates is observed even when the aggregation accelerator (organic acid) of the present invention is not used, but the distribution of aggregates is uneven and the SERS measurement results vary. It is mentioned that it occurred.
- the aggregation accelerator (organic acid) of the present invention when used, uniformly distributed aggregates were formed and the SERS effect was enhanced (see Example 12).
- the target molecule is a peptide
- the peptide if the peptide is present in an excessive amount, this exceeds the aggregating action of the aggregation promoter (organic acid) of the present invention, and the formation of aggregates was not observed.
- the aggregation promoter of the present invention may be used for any sample as long as SERS measurement can be performed. That is, the aggregation promoter of the present invention can be used for a sample that may contain a biomolecule, a sample separated by liquid chromatography or capillary electrophoresis, and other SERS measurements. Any sample can be used. That is, the SERS measurement using the aggregation accelerator of the present invention is performed as described in 1. above.
- the present invention is not limited to the case of using the above-mentioned apparatus, and can be used for any surface enhanced Raman spectroscopy (SERS) method.
- the target molecule to be analyzed needs to emit a SERS signal.
- the target molecule preferably aggregates when mixed together with the metal nanoparticles, or interacts with the metal nanoparticles pre-aggregated.
- the target molecule is, for example, a biomolecule that emits a SERS signal, a fragment of a biomolecule that emits a SERS signal, a biomolecule that binds to a low molecular compound that emits a SERS signal, or a biomolecule that binds to a low molecular compound that emits a SERS signal. It can be a fragment.
- the target molecule can be contained in a fraction fractionated in advance by the sample separation unit of 1.1 above, for example, by liquid chromatography or capillary electrophoresis.
- RAT8-AOMK is explained in 2.2.4 below.
- the Raman spectroscopic method according to the present invention can perform so-called “on-line” analysis in which measurement is performed while a sample from a sample separation unit is fed.
- the Raman spectroscopy according to the present invention can perform so-called “off-line” analysis in which a sample from a sample separation unit is sent to a plate, spotted on a plate, and the spot is measured.
- FIG. 10 compares the characteristics of the online Raman detection method (A) and the offline Raman detection method (B). The left (A) in FIG.
- the measurement 10 is on-line detection in which the liquid feeding is directly measured by Raman
- the right (B) is off-line detection in which the liquid feeding is once spotted on the plate and then this is measured by Raman.
- the measurement may be performed offline.
- the measurement sensitivity of on-line Raman spectroscopy is in units of mM, but if off-line, the sensitivity is in units of ⁇ M (several pmol for peptides).
- the solvent of the spot on the plate is dried and evaporated to eliminate the problem of background light. Can be avoided.
- FIG. 11 illustrates one aspect of the apparatus according to the present invention.
- the mixed sample is introduced into the sample separation unit, and the sample separation unit uses the fraction by the liquid chromatograph, and the obtained fraction is spotted on the plate.
- the measurement sensitivity of Raman spectroscopic analysis can be improved by drying each spot on the plate and aggregating the sample. Measurement sensitivity is improved by about three orders of magnitude or more compared to the case where drying and aggregation are not performed (dissolved state). Further, since the Raman spectroscopic analysis is performed off-line, the measurement can be performed without being affected by the background light of the solvent used in the liquid chromatograph and without being restricted by the liquid feeding speed. After the Raman spectroscopic analysis, a part or all of the spots showing a Raman peak are subjected to mass spectrometry (MS).
- MS mass spectrometry
- Silent region When the Raman spectroscopic analysis is performed without fractionating the cell lysate, a region where a peak is detected and a region where a peak is not detected or hardly detected appear.
- a region where a Raman peak is not detected or hardly detected when the cell lysate is subjected to Raman spectroscopic analysis is referred to as a “silent region”.
- Raman peak of protein is mainly observed at about 800 ⁇ 1800 cm -1 and 2800 ⁇ 3000 cm -1, it is hardly detected in the 1800 ⁇ 2800 cm -1. These Raman peaks are all assigned to specific amino acid residues.
- tryptophan-derived peaks are near 1011 cm -1 and 1554 cm -1
- amide-derived peaks are around 1250 cm -1 and 1660 cm -1
- CH 2 -derived peaks are around 1430 cm -1
- CH 3 The derived peak appears in the vicinity of 2933 cm ⁇ 1 (see FIG. 6).
- the silent region as used herein can be 1800-2800 cm ⁇ 1 .
- Raman spectroscopic analysis is 500 cm -1 or higher, 700 cm -1 or higher, 1000 cm -1 or higher, 1200 cm -1 or higher, 1400 cm -1 or higher, 1600 cm -1 or higher, or 1800 cm -1 or higher, 3000 cm. less than -1, less than 2900 cm -1, less than 2800 cm -1, can be performed 2700 cm less than -1, or in the region of less than 2600 cm -1.
- the silent region is basically the same regardless of the biomaterial used.
- Mass Spectrometer is an apparatus that ionizes molecules contained in a sample by an appropriate ionization method and measures a mass spectrum of the molecules.
- FIG. 3 shows an example of a mass spectrometer according to the present invention.
- a mass spectrometer including the sample unit, the separation unit, and the analysis unit constitutes the mass analysis unit according to the present invention.
- the sample portion first, the sample 14 is placed on the sample stage 13. Next, the sample is ionized by an appropriate ionization means, and the sample is caused to fly by electrostatic force.
- the laser part 15 is illustrated as an ionization means.
- the ions accelerated by the accelerating electrode 16 are separated in accordance with the mass-to-charge ratio by an electric or magnetic action in the separation unit, and then detected by the ion detector 17 in the analysis unit, whereby the mass spectrum. Can be obtained.
- the ion detector 17 is preferably connected to a signal processor 18, and the obtained signal is preferably processed by an electronic computer.
- the ion detector 17, the signal processing unit 18, and an arbitrary computer are collectively referred to as an “analysis unit” of the mass analyzer.
- the mass spectrum obtained by processing the signal in the analysis unit usually represents the mass-to-charge ratio (m / z) as the horizontal axis and the detection intensity as the vertical axis.
- Examples of ionization methods for mass spectrometry include matrix-assisted laser desorption ionization (MALDI), electrospray ionization (ESI), atmospheric pressure chemical ionization (ACPI), electron ionization (EI), and chemical ionization (CI). ) Law.
- MALDI matrix-assisted laser desorption ionization
- ESI electrospray ionization
- ACPI atmospheric pressure chemical ionization
- EI electron ionization
- CI chemical ionization
- a person skilled in the art can appropriately change and adapt the configuration of the mass spectrometer according to these ionization means.
- a sample is mixed in a matrix such as an aromatic organic compound to produce a crystal, and ionization is performed by irradiating this with a laser.
- the matrix to be used is not particularly limited, but ⁇ -cyano-4-hydroxycinnamic acid (CHCA), sinapinic acid (SA), trans-4-hydroxy-3-methoxycinnamic acid (ferulic acid), 3 -Hydroxypicolinic acid (HPA), 1,8-dihydroxy-9,10-dihydroanthracen-9-one (disranol), 2,5-dihydroxybenzoic acid (DHB) and the like.
- CHCA ⁇ -cyano-4-hydroxycinnamic acid
- SA sinapinic acid
- ferulic acid trans-4-hydroxy-3-methoxycinnamic acid
- HPA 3 -Hydroxypicolinic acid
- 1,8-dihydroxy-9,10-dihydroanthracen-9-one diisranol
- 2,5-dihydroxybenzoic acid DHB
- the separation unit used for mass spectrometry separates the ionized sample.
- Examples of the type of separation unit include a time-of-flight type, a magnetic field deflection type, a quadrupole type, an ion trap type, and a Fourier transform type.
- TOF time-of-flight
- an ionized sample is accelerated in a pulse manner to detect a time difference until it reaches a detector. The mass can be calculated from this time difference.
- the acceleration electrode 16 shown in FIG. 3 is arranged only in a part of the space in which ions fly, so that acceleration is performed in a pulse manner.
- the mass spectrometry may be MS / MS analysis.
- MS / MS analysis mass spectrometry is performed in tandem. In this method, only a specific ion is taken out in the first separation section, cleaved, and the resulting fragment ions are analyzed in the second separation section. Fragment ion analysis can be performed on a single device or on two different devices. For example, when a peptide is obtained by digesting a protein with a protease and MS / MS analysis is performed on the peptide fragment, peaks in which the peptide is sequentially fragmented are detected, and the amino acid sequence of the peptide is determined from the mass information of those peaks. can do.
- MS / MS analysis is a technique well known in the art. See, for example, A. K. Shukla et. Al., J. Mass Spectrom. 35, 1069 (2000).
- the apparatus according to the present invention includes a liquid chromatograph, a Raman spectroscope, and a mass spectrometer (LC-R-MS).
- LC-R-MS mass spectrometer
- An example is shown in FIG.
- the mixed sample is fractionated by liquid chromatography (LC), and the fraction containing low molecular weight compounds (drugs) is narrowed down by Raman spectroscopy.
- a fraction showing a Raman peak can be analyzed with a mass spectrometer (MS) to identify a biomolecule that binds to a low molecular compound.
- MS mass spectrometer
- the apparatus according to the present invention comprises a capillary electrophoresis apparatus, a Raman spectrometer and a mass spectrometer (CE-R-MS).
- CE-R-MS mass spectrometer
- the basic principle of the present invention is the same as in the LC-R-MS except that the sample is separated by a capillary electrophoresis apparatus (CE).
- CE capillary electrophoresis apparatus
- liquid phase isoelectric focusing or the like can be used as the sample separation unit, and the same applies to the case where the sample separation unit is based on other separation means.
- the sample separation unit, the Raman spectroscopic unit, and the mass spectrometry unit are connected in this order.
- Linking means that the devices of the apparatus are connected so that the sample can be delivered.
- the sample separation unit, the Raman spectroscopic unit, and the mass spectrometric unit are connected in this order.
- the sample separated by the sample separation unit is introduced into the Raman spectroscopic unit, and then the Raman spectroscopic analysis is performed.
- Means that the apparatus is configured to deliver samples in the order of introduction into the mass spectrometer. Delivery of the sample from the sample separating unit to the Raman spectroscopic unit and from the Raman spectroscopic unit to the mass spectrometric unit can be performed manually or by an automated apparatus.
- the sample can be transferred continuously by a liquid feed line, or can be intermittently performed by spotting the sample once on a plate or the like, or by separating the sample into individual fractions by a fraction collector.
- the sample separation unit, the Raman spectroscopic unit, and the mass spectrometric unit may be physically independent devices, and the sample (fraction) separated from the sample separation unit may be manually or automated by the Raman spectroscopic unit.
- the apparatus or method according to the present invention is a system in which the sample (fraction) introduced and introduced from the Raman spectroscopic section into the mass spectrometric section is manually or by an automated apparatus.
- the apparatus according to the present invention may be an apparatus in which a sample separation unit, a Raman spectroscopic unit, and a mass analysis unit are integrated. The apparatus according to the present invention having such a configuration can overcome the problems included in the prior art.
- the present invention provides a method for identifying a biomolecule using the apparatus according to the present invention and a method for identifying a binding site between a biomolecule and a low molecular compound.
- a biomolecule or a fragment thereof bound with a low molecular weight compound that can be identified by Raman spectroscopy is fractionated by a sample separation unit without requiring complicated pretreatment or the like.
- Each of the fractions (droplets, etc.) fractionated can be arranged on a plate, dried and aggregated biomolecules or fragments thereof can be directly measured by the Raman spectroscopic unit. Thereafter, all the fractions or a part of the fractions specified to have a Raman peak can be directly analyzed by the mass spectrometer without any special treatment.
- biomolecule refers to a protein, peptide, nucleic acid, sugar, or lipid that exists extracellularly or intracellularly.
- the biomolecules referred to in this specification may be derived from any living body or organism, such as viruses, prokaryotes, eukaryotes, fungi, plants, higher plants, animals, insects, higher animals, mammals. They may be derived from animals, rodents such as mice and rats, primates such as monkeys and chimpanzees, humans, or cultured cells or tissues thereof.
- a nucleic acid encompassed by a biomolecule refers to a single-stranded or double-stranded nucleic acid comprising at least 10, preferably 50, 300, 500, or 1000 or more bases, preferably a specific low It interacts with molecular compounds.
- the nucleic acid may be DNA or RNA.
- RNA includes tRNA, ribosomal RNA, and ribozymes.
- the nucleic acid can include a promoter region, an enhancer region, a silencer region, and a terminator region.
- the saccharide contained in the biomolecule referred to in the present specification includes a polysaccharide, and the polysaccharide preferably interacts with a specific low molecular weight compound.
- sugars include proteoglycans or proteoglycan analogs such as hyaluronic acid, chitin, heparan sulfate, keratan sulfate, dermatan sulfate, sialic acid, and chondroitin sulfate.
- the lipid contained in the biomolecule referred to in this specification includes lipids contained in the organisms exemplified above, and preferably interacts with a specific low molecular weight compound.
- lipids examples include phospholipids such as sphingophospholipids and glycerophospholipids, glycolipids such as sphingoglycolipids and glyceroglycolipids, and complex lipids that form extracellular or cell membranes such as lipoprotein lipids, sulfolipids, or galactolipids. Can be mentioned.
- the low molecular weight compound according to the present invention refers to a compound having a low molecular weight and capable of binding to or binding to a specific biomolecule.
- the low molecular weight compound according to the present invention has a low molecular weight compared to a biomolecule.
- the biomolecule viewed from the low molecular weight compound may be called a target.
- Low molecular compounds or compounds that form the basis of low molecular compounds (also referred to as compounds to be analyzed) include drugs, drug candidate compounds, physiologically active substances, metabolites, vitamins, hormones, ligands that bind to specific receptor proteins, Also included are protein agonists, protein antagonists, compounds that bind to proteins through protein post-translational modification mechanisms, and the like. These include naturally occurring compounds and analogs (analogues) that are similar in chemical structure to them.
- the low molecular compound according to the present invention may be any compound as long as it gives a scattering peak that can be distinguished from a biomolecule in Raman spectroscopy, or a scattering peak that can be distinguished from a biomolecule by Raman labeling.
- Raman peak of low molecular weight compounds A compound that has a characteristic Raman peak or a Raman peak that can be distinguished from biomolecules that coexist in cells or in a mixture, especially biomolecules that are targets of low molecular weight compounds. If present, this can be used in the present invention as it is as a low molecular compound. Since such a low molecular weight compound can be detected by Raman spectroscopy as it is, there is an advantage that there is no need for modification with a fluorophore or the like. In addition, a region where a Raman peak derived from a compound is observed in the range of 500 to 1800 cm ⁇ 1 is sometimes referred to as a “fingerprint region”.
- a substituent that has an extremely small influence on the binding to a biomolecule even when introduced into the compound is introduced. It can be used as a low molecular compound.
- a substituent is also called a Raman label.
- the substituent or the Raman label has a scattering spectrum in a silent region. As described above, the silent region is a wave number region in which little or no signal is observed in a Raman spectrum derived from a biomolecule.
- the substituent or Raman label has a relatively strong Raman scattered light, and shows a characteristic peak in a wave number region different from a Raman peak derived from a biomolecule when detecting a target low molecular compound.
- the alkynyl group an ethynyl group (CH ⁇ C-), propargyl (CH ⁇ CCH 2 -, also referred to as a 2-propynyl group), but-3-yn-1-yl group (HC ⁇ CCH 2 CH 2 -) , But-2-yn-1-yl group (CH 3 —C ⁇ CCH 2 —) and the like. Any of these can be the Raman label of the present invention.
- the low molecular weight compound according to the present invention preferably has an alkynyl group, a nitrile group, or deuterium.
- the Raman label may be introduced directly into the compound of interest or may be attached to the compound of interest through a suitable spacer molecule.
- the alkynyl group may be introduced directly or as an alkynyl group (ethynylphenyl group) bonded to a phenyl group.
- the spacer molecule is a phenyl group. Therefore, a compound that binds to a specific biomolecule but does not have a characteristic Raman peak or does not have a Raman peak that can be distinguished from a biomolecule is Raman-labeled by introducing the above-described substituent, and the compound according to the present invention.
- Spacer molecules include methylene (-CH 2- ), ethylene (-CH 2 CH 2- ), propane-1,3-diyl (-CH 2 CH 2 CH 2- ), phenylene (-C 6 H 4 -), an oxyethylene group (-OCH 2 CH 2 -), oxypropylene group (-OCH 2 CH (CH 3) -) but the like are not limited to.
- Acyloxymethyl ketone (AOMK) is known as a cysteine protease inhibitor. The principle is that AOMK modifies the cysteine residue at the active center of the enzyme protein and loses its activity as a protease (see FIG. 24).
- a person skilled in the art determines that if a Raman label is introduced at a site away from the methyl ketone group in the AOMK compound, it does not affect the cysteine residue modification reaction so much, and the position for introducing the Raman label is determined. be able to.
- a “Raman-labeled compound library” in which a Raman label is introduced comprehensively at any position in the compound is prepared, and the Raman-labeled compound library has a predetermined biomolecule binding activity, for example, protein inhibitory activity.
- examples of the low molecular compound according to the present invention include drugs, drug candidate compounds, antibiotics, and agricultural chemicals that bind to such proteins or peptides.
- bioactive substances such as metabolites, vitamins such as coenzymes, hormones, ligands that bind to specific receptor proteins, protein agonists, protein antagonists, and compounds that bind to proteins through protein post-translational modification mechanisms.
- Such low molecular weight compounds are preferably Raman labeled or have characteristic Raman spectra that are distinguishable from other biomolecules.
- the compound N-Boc-AOMK that binds to the protein cathepsin B introduces an alkyne group that is a kind of Raman label with 4-nitrophenyl-4-ethynylbenzyl carbonate, and the low molecular compound RAT8- Can be AOMK.
- examples of the low molecular weight compound according to the present invention include intercalating drugs for double-stranded nucleic acids such as proflavine and actinomycin D, netropsin, destamycin, etc. Group-bonded drugs, and DNA-cleaving drugs such as calichemicin.
- Such low molecular weight compounds are preferably Raman labeled or have characteristic Raman spectra that are distinguishable from other biomolecules.
- examples of the low molecular weight compound include low molecular weight antibiotics exhibiting lectin-like activity such as pradomycin A, B, C, D, E, FA-1, FA-2, and benanomycin A. It is done.
- Such low molecular weight compounds are preferably Raman-labeled or have a characteristic Raman spectrum that can be distinguished from other biomolecules.
- the substances according to the present invention that bind to such sugars include C-type lectins such as R-type lectin, calnexin, calreticulin, selectin, collectin, galectin, legumes.
- C-type lectins such as R-type lectin, calnexin, calreticulin, selectin, collectin, galectin, legumes.
- examples include lectins composed of type I lectins such as lectins, L-type lectins, P-type lectins, annexins and siglecs, and sugar chain specific antibodies.
- Such substances are also included in the low molecular weight compounds referred to in the present invention because they bind to biomolecules.
- a specific antibody that binds to a sugar can introduce an alkyne group by incorporating an amino acid that has been genetically engineered to be alkyne-modified into a protein.
- the low molecular weight compound according to the present invention includes a polyether antibiotic such as monensin, lasalocid, salinomycin, an anesthetic such as isoflurane, sevofuran, desflurane, vitamin, etc.
- a polyether antibiotic such as monensin, lasalocid, salinomycin
- an anesthetic such as isoflurane, sevofuran, desflurane, vitamin, etc.
- fat-soluble vitamins such as A (retinoid), vitamin D, vitamin E, and vitamin K.
- Such low molecular weight compounds are preferably Raman labeled or have characteristic Raman spectra that are distinguishable from other biomolecules.
- the low molecular compound according to the present invention is a Raman-labeled amino acid.
- the low molecular weight compound according to the present invention is a low molecular weight peptide having a Raman-labeled amino acid.
- FIG. 6 shows an example of an alkyne-labeled peptide and its Raman spectrum.
- the Raman spectroscopic unit according to the present invention can clearly identify both peptides.
- Focusing on a low molecular weight peptide having physiological activity one or more amino acids among them are Raman-labeled, or a peptide in which a Raman-labeled amino acid is substituted is prepared.
- this Raman labeled low molecular weight peptide is used, the biomolecule that binds to the peptide can be identified by the apparatus or method according to the present invention.
- the binding site between the peptide and the biomolecule can be identified. That is, the target of a low molecular weight peptide (for example, peptide hormone) having physiological activity can be searched, and its action site can be identified.
- Alkylated peptides can be prepared by solid phase synthesis (Fmoc) methods. Explaining in the above example, if the amino acid sequence added next to lysine (K) is propargylglycine (X) by solid-phase synthesis and the amino acid sequence is added in order from the C-terminal to TPCQPWQE, the resulting alkyne is obtained. Peptides can be obtained.
- a peptide may be synthesized by a solid-phase synthesis method, and any side chain functional group in the peptide may be alkyned.
- the bond includes a covalent bond, a coordinate bond, and an interaction.
- a covalent bond refers to a chemical bond formed by a plurality of atoms sharing each other's electrons.
- a coordinate bond refers to a chemical bond in which electrons are provided only from one of the atoms involved in the bond.
- the interaction refers to an action based on an intermolecular force acting between two molecules, and includes an ion-ion interaction, an action caused by a hydrogen bond, a dipole interaction, a hydrophobic interaction, and a combination thereof.
- Method for identifying the binding site between a biomolecule and a low molecular weight compound "Identifying" the binding site between a biomolecule and a low molecular weight compound is where the low molecular weight compound is bound or interacts with the biomolecule. It means to decide. When the apparatus according to the present invention is used, a binding site between a biomolecule and a low molecular compound can be identified.
- the method for identifying a binding site between a biomolecule and a low molecular compound includes the following steps. (1) The fractionated biomolecule fragment bound to the low molecular weight compound is subjected to Raman spectroscopic analysis, and (2) all or a part of the fraction subjected to Raman spectroscopic analysis is subjected to mass spectrometry.
- a fraction having a Raman peak derived from a low molecular weight compound bound to a biomolecule fragment is detected by Raman spectroscopic analysis, and a mass spectrometry result of the fraction having a Raman peak derived from a low molecular weight compound is obtained.
- the binding site of the low molecular compound in the biomolecule is identified by collating with the mass information of the molecule.
- a low molecular compound and a biomolecule are bound, and then the biomolecule bound to the low molecular compound can be fragmented and fractionated. Such a fraction can be used in the above step (1).
- a biomolecule fragment refers to a unit obtained by cleaving a biomolecule, which is a polymer compound, at one or more sites to obtain a unit having a lower molecular weight.
- a biomolecule when the biomolecule is a protein, it can be subjected to protease treatment to obtain a fragment (peptide) in which the peptide bond is cleaved.
- protease include, but are not limited to, serine protease, aspartic protease, metalloprotease, cysteine protease and the like.
- Biomolecules can also be chemically decomposed with cyanogen bromide, N-bromosuccinimide, hydroxylamine, and the like.
- the biomolecule is a peptide.
- the biomolecule contains a triglyceride lipid, it can be subjected to treatment with a lipolytic enzyme such as lipase to obtain a degraded fragment (fatty acid).
- a lipolytic enzyme such as lipase
- examples of the lipase include, but are not limited to, triacylglyceride lipase, phospholipase, lipoprotein lipase, esterase and the like. The same applies to other types of biomolecules.
- enzymes that degrade sugar can be used, such as ⁇ -amylase, ⁇ -amylase, glucoamylase, isoamylase, pullulanase, maltotriohydrolase, ⁇ -glucosidase, cyclodextrin, glucanotransferase, Examples include, but are not limited to, amyloglucosidase, dextranase, ⁇ -galactosidase, sialidase, cellulase, ⁇ -mannosidase, ⁇ -mannosidase and the like.
- a nucleic acid fragment can be obtained by treating with a nucleic acid degrading enzyme such as a restriction enzyme that specifically cleaves double-stranded DNA or a single-stranded RNA cleaving enzyme ribonuclease.
- a nucleic acid degrading enzyme such as a restriction enzyme that specifically cleaves double-stranded DNA or a single-stranded RNA cleaving enzyme ribonuclease.
- Fragmentation refers to the degradation of biomolecules into lower molecular weight fragments by appropriate degrading enzymes or physical or chemical treatment. Fragmentation can be performed by enzyme treatment or chemical treatment as described above. A person skilled in the art can appropriately select the enzyme or compound to be used and determine the treatment conditions.
- FIG. 3 An example of an apparatus for identifying a binding site between a biomolecule and a low molecular compound is shown in FIG.
- HPLC is connected to a UV detector, which is connected to a spotter, which is then connected to a Raman spectroscopic unit, and then this is connected to a mass spectrometric unit.
- This apparatus can be used in a method for identifying a binding site between a biomolecule and a low molecular compound according to the present invention. This method will be described with reference to FIG. 18.
- a biomolecule eg, protein
- the obtained fragment (peptide) mixture is fractionated by HPLC in the sample separation section, and detected by a UV detector to obtain a UV chromatogram.
- each fraction is spotted on a MALDI plate.
- the arrayed peptide array is subjected to Raman spectroscopy and mapped according to Raman intensity. By subjecting the fraction showing a Raman signal to MALDI-MS and performing mass spectrometry, it is possible to identify a peptide to which a low molecular compound is bound, thereby identifying a binding site between the low molecular compound and the biomolecule.
- RAT8-AOMK is an alkyne labeled cathepsin B inhibitor.
- FIG. 19-1 schematically illustrates the procedure of bond analysis. As shown in the upper part of FIG. 19-1, RAT8-AOMK is first bound to cathepsin B and then fragmented (digested) by protease treatment.
- screening can be performed with the Raman peak of the low-molecular compound itself.
- screening can be performed with the Raman peak of the low molecular compound itself, and it can be detected which fraction contains the low molecular compound.
- FIG. 18 and the present specification and drawings the configuration in which the sample separation unit has the UV detector is illustrated for convenience.
- the UV detector of the sample separation unit is not essential for the apparatus or method according to the present invention, and the low molecular weight The compound can be detected by the Raman spectroscopic unit.
- mass analysis results are obtained, and this is “verified” with biomolecule mass information to bind the low molecular compound in the biomolecule.
- To identify the site in the example of protein, whether or not the mass spectrum result of the obtained peptide fragment matches the calculated mass of the region corresponding to a part of the protein from which the peptide is derived. By judging, it refers to identifying the binding site where the low molecular weight compound is bound in the protein.
- a person skilled in the art can provide information on biomolecules such as proteins, peptides, nucleic acids, lipids and sugars as needed, including DDBJ / NIG, EMBL / EBI, GenBank / NCBI, NIAS DNA Bank, PIR, SWISS-PROT & TrEMBL , GenPept, PRF, Japan Glycoscience Integrated Database (JCGGDB), LipidBank, and other suitable known databases. Further, whether or not the amino acid sequences match can be determined using software such as Mascot (Matrix Science Inc.). Furthermore, the bound amino acid of a low molecular compound can also be confirmed by MS / MS analysis.
- the screening method for identifying a biomolecule that binds to the low molecular weight compound according to the present invention includes the following steps. (1) A fraction containing a biomolecule bound to a low molecular compound was subjected to Raman spectroscopic analysis, a fraction containing the biomolecule bound to the low molecular compound was detected, and (2) subjected to Raman spectroscopic analysis. Subject all or part of the fraction to mass spectrometry.
- the fraction having a Raman peak derived from a low molecular compound is detected by Raman spectroscopy, and the mass analysis result of the fraction having a Raman peak derived from the low molecular compound is obtained. Collation is performed to identify biomolecules that bind to the low molecular weight compound.
- a low molecular compound is first added to a mixture containing a target biomolecule to bind the low molecular compound and the biomolecule, and then this can be fractionated by a sample separation means. . Such separated fractions can be used in step (1).
- Biomolecules The screening method and binding site identification method according to the present invention can be used for various biomolecules including proteins, peptides, nucleic acids, sugars, and lipids.
- the screening method according to the present invention can be used for protein screening. For example, in the case of screening an organism or virus whose base sequence of the entire genome has been decoded to determine which protein in a certain organism activity binds and acts with a low molecular weight compound having a certain drug activity, A mass analysis result of a protein that binds to the low molecular weight compound is obtained. Further, when the protein that binds to the low molecular weight compound is decomposed by protease treatment and fragmented into peptides, and mass analysis is performed, mass information of the peptide fragments can be obtained. Furthermore, the amino acid sequence of a peptide can be determined by performing MS / MS analysis.
- the obtained amino acid sequence can be collated with the sequence information of any protein encoded in the entire genome sequence that has been decoded, and the protein that binds to the low molecular weight compound can be identified.
- the binding site can also be identified by using the above-mentioned “3. Identification method of binding site between biomolecule and low molecular compound”. The same applies to other biomolecules such as nucleic acids, sugars or lipids, and when there is comprehensive mass information about various nucleic acids, sugars or lipids contained in a sample, the above method according to the present invention is carried out.
- Mass spectrometry results of nucleic acids, sugars or lipids that bind to the low molecular compounds are obtained, and this can be collated with the comprehensive mass information to identify those that bind to the low molecular compounds. Hereinafter, these will be described.
- nucleic acids The method according to the invention can also be used for nucleic acids. For example, when there is information on various nucleic acid molecules of a certain cell, when it is desired to determine which nucleic acid molecule binds to a low molecular compound, the nucleic acid molecule that binds to the low molecular compound is performed by carrying out the method according to the present invention. Mass information can be obtained. Further, by performing MS / MS analysis, a mass spectrum of nucleic acid sequentially decomposed can be obtained, and this can be collated with the aforementioned mass information to identify which nucleic acid molecule the low molecular weight compound binds to. In addition, after specifying the bound nucleic acid, the binding site can be determined by carrying out the method according to the present invention.
- the method according to the invention can also be used for sugars.
- the method according to the present invention is performed, and Mass information of the capsular polysaccharide that binds to the molecular compound can be obtained. It is also possible to obtain a mass spectrum of polysaccharides sequentially decomposed by performing MS / MS analysis, which can be collated with the aforementioned mass information to identify which capsular polysaccharide the low molecular weight compound binds to. .
- the method according to the present invention can be performed to determine the binding site.
- the method according to the invention can also be used for lipids.
- the method according to the present invention when there is comprehensive information on the molecules constituting the lipid bilayer membrane of a cell, when it is desired to determine which lipid molecule a low molecular weight compound binds to, the method according to the present invention is performed, A mass spectrum of a lipid molecule that binds to a low molecular compound can be obtained. By comparing this with comprehensive information on the lipid, it is possible to identify which lipid molecule the low-molecular compound binds to. In addition, after identifying the bound lipid molecule, the method according to the present invention can be performed to determine the binding site.
- a system using RAT8-AOMK as a low molecular compound and protein cathepsin B as a biomolecule is described as a representative example.
- the biomolecules to which the apparatus and method according to the present invention are applied are not limited to proteins in principle.
- the silent region is basically the same for other components of cells, such as nucleic acids, sugars, and lipids.
- the Raman spectrum of the HeLa cell is shown in FIG. The Raman peak is not particularly observed in the region of 1800 to 2800 cm ⁇ 1 , and it can be seen that the silent region is the same range in the cell lysate. See also Non-Patent Document 1.
- the apparatus and method according to the present invention can be applied to biomolecules such as nucleic acids, sugars, and lipids, when a group having a peak in a silent region such as alkyne is used as a Raman label.
- a solution containing biomolecules bound to low molecular weight compounds causes cells to take in low molecular weight compounds, bind them to intracellular biomolecules, and disrupt the cells. Can be prepared.
- a solution containing a biomolecule bound to a low molecular compound can also be prepared by crushing cells, then adding the low molecular compound to the cell lysate and allowing it to bind to intracellular biomolecules.
- the sample used in the method according to the present invention may be a fraction obtained by liquid chromatography using a low-boiling polar solvent and water as a separation solvent.
- the low boiling polar solvent is as described in 1.1.1.
- the fraction to be analyzed by Raman spectroscopy is a droplet as it is or a droplet mixed with a solvent. It is possible to prepare a spot for Raman spectroscopic analysis by arranging on a simple plate and evaporating the solvent contained in the droplet. Since the spots are prepared by evaporation of the solvent and the Raman spectroscopic analysis is performed with a Raman microscope, the plate preferably has a cleaned surface. The surface of the plate being cleaned means that there are no liquids, solid contaminants, inorganic or organic impurities, fingerprints, dust, cloudiness, scratches, etc. on the surface that would interfere with Raman spectroscopy.
- Cleaning can be performed by washing the plate surface with water, an aqueous detergent containing a surfactant, or an organic solvent, and then drying the plate.
- the plate used preferably has a water repellent surface. More preferably, the water-repellent surface of the plate is cleaned.
- Water repellent means to repel water
- the water repellent surface of a plate refers to the surface of the plate that repels water.
- the water repellent surface can be realized, for example, by a process of applying a water repellent having a surface tension remarkably smaller than that of water, such as a fluorine water repellent or a silicone water repellent, to the plate.
- the plate having a water repellent surface may be a plate made of metal, glass, quartz, calcium fluoride or magnesium fluoride, and preferably has little or no influence on the results of Raman spectroscopy and mass spectrometry.
- the arrangement of the droplets on the plate can be performed using a micropipette. This operation can be performed manually or by automated equipment. Examples of the plate include, but are not limited to, a 96-well plate and a 384-well plate that are widely used in the art.
- FIG. 13 shows an example of spotting the fraction sample on the plate. On the left side of FIG. 13, a solution fractionated by HPLC is fed and spotted on a plate. The order of spotting is only an example. As shown in the lower right of FIG. 13, as the droplets on the substrate dries, the peptides in the solution aggregate in a ring shape. When this ring portion is analyzed with a Raman microscope, a Raman spectrum can be obtained efficiently and with high sensitivity.
- Raman spectroscopic analysis and mass spectrometry may be performed using commercially available plates, but it is also possible to prepare and use a microscope substrate fixing plate suitable for the sample stage of the Raman microscope.
- . 14 and 15-1 show examples of plates that can be used in the apparatus according to the present invention.
- the microscope substrate fixing plate is shown in the upper left of FIG.
- the photograph is a multi-spot metal substrate viewed from the back.
- the lower left of FIG. 14 shows the sample stage of the Raman microscope.
- the right side of FIG. 14 shows a state where a microscope substrate fixing plate is mounted on the sample stage. In this state, Raman spectroscopic analysis is performed. Detecting a spot having the target Raman peak is also called Raman screening.
- the fraction of the sample subjected to the Raman spectroscopic analysis is then subjected to mass spectrometry. Therefore, the plate used for the Raman spectroscopic analysis can be directly analyzed by the mass spectrometer. It is preferable. Therefore, the present inventors have developed a plate that can smoothly perform Raman spectroscopy and mass spectrometry of a spotted sample. Examples thereof are shown in FIGS. When this plate is used, the sample screened by the Raman spectroscopic unit can be directly analyzed by the mass spectrometric unit. In FIG. 16, it was confirmed that a quartz substrate advantageous for Raman measurement can be used as it is for MALDI-MS measurement. In the upper left of FIG.
- the left spot is alkyne peptide 1 and the right spot is peptide 1.
- the left arrow points to the alkyne peptide spot
- the right arrow points to the peptide 1 spot.
- the upper right of FIG. 16 shows the Raman spectra of alkyne peptide 1 and peptide 1, and both samples can be identified by the presence or absence of a Raman peak at 2123 cm ⁇ 1 .
- the lower right of FIG. 16 is the MALDI-MS measurement result, and the mass difference between the alkyne peptide 1 and the unlabeled peptide 1 can be confirmed from the spectrum, which is in good agreement with the calculated value. This result corresponds to FIGS.
- FIG. 17 shows a multi-spot substrate whose entire surface is made of quartz.
- the substrate is treated with hydrochloric acid, sulfuric acid, nitric acid or the like, acid removed by washing with water, washed with a low boiling point solvent such as acetone, and dried.
- the substrate needs to be clean on the surface, but is preferably water-repellent so that biological materials can aggregate at high density, such as dimethyldichlorosilane, trimethylchlorosilane, etc. in which no signal is observed in the silent region of the Raman spectrum.
- the substrate can be produced by performing a water repellent treatment with a silicone water repellent or a fluorine water repellent. As shown in the lower left of FIG. 17, the substrate is fixed to the base with a magnet. This plate can be used in the method and apparatus according to the present invention.
- the sample is directly used for the Raman spectroscopic unit.
- Specific biomolecules or fragments can be easily detected or separated and identified.
- the Raman spectroscopic unit is connected to the mass spectrometric unit, the fraction specified by the Raman spectroscopic analysis can be analyzed as it is by a subsequent mass spectrometer, thereby enabling sequencing of biomolecules and low molecular weight compounds.
- the binding site can be easily identified. Sequencing refers to, for example, determination of an amino acid sequence when the biomolecule is a protein or peptide, and determination of the sequence of sugars constituting the polysaccharide when the biomolecule is a polysaccharide.
- the apparatus or method according to the present invention can also be used for analysis of post-translational modification of proteins.
- a protein when a protein is translated and then modified at a specific site with a lipid such as a farnesyl group or a palmitoyl group (for example, a cysteine residue in the case of a palmitoyl group), the lipid is Raman-labeled in the present invention.
- a low molecular weight compound is used and used in the apparatus or method according to the present invention, a protein that binds to a Raman-labeled lipid can be identified, and the binding site thereof can also be identified.
- the Raman-labeled lipid may be incorporated into the cell and bound to the target protein, or may be bound to the target protein in addition to the cell lysate.
- the lipid is modified with a fluorophore, there is a problem that the cellular mechanism of protein post-translational modification does not recognize the fluorescently modified lipid and cannot perform subsequent analysis.
- Non-patent Document 4 lipid is incorporated into cells and then modified with a fluorophore using a click reaction
- Non-patent Document 5 a report example for specifying a protein that binds to the lipid by fluorescence analysis or click reaction to lipid
- a biotin tag is introduced with and detected with streptavidin
- these methods have problems of complicated handling, nonspecific reaction, and loss of target protein due to reaction operation.
- a Raman-labeled lipid that has little or no influence on the cellular mechanism of protein post-translational modification is used, and a biomolecule that binds to the Raman-labeled lipid is identified. And its binding site can be identified.
- the post-translational modification of the protein is a sugar modification.
- the apparatus and method according to the present invention comprehensively search for biomolecules to identify those that bind to low molecular compounds, or bind biomolecules to low molecular compounds.
- Site identification can be performed.
- a low molecular weight compound can be selectively detected with high sensitivity by a Raman label having a Raman peak of the compound itself or a characteristic Raman peak such as an alkynyl group.
- the low molecular weight compound can be used as it is, or the molecular weight of the tag added to the analysis target compound can be kept low, and compared with the conventional fluorescent labeling method using a large fluorophore.
- the target biomolecule can be specifically identified / detected and identified without changing the biochemical properties of the compound.
- Raman spectroscopic analysis according to the present invention not only low molecular compounds but also molecular vibration information derived from biomolecules can be obtained, so there is an advantage not found in the prior art that the coexistence of low molecular compounds and biomolecules can be confirmed. is there.
- Non-patent Document 2 Non-patent Document 2
- the conventional method of combining an alkyne tag with a click reaction has a problem that there is a loss of a target substance related to the click reaction operation and a non-specific reaction occurs.
- the alkyne tag itself can be analyzed by Raman spectroscopy in the method according to the present invention, problems such as loss of the target substance and non-specific reactions that were included in the conventional method are solved.
- the SERS method using the aggregation accelerator of the present invention is characterized in that the measurement sensitivity is increased and the detection limit is improved.
- the distribution of aggregates is made uniform, so the correlation between the amount of sample to be measured and the SERS signal is improved, and variations in measurement are suppressed.
- the long-time measurement which is a weak point of Raman measurement, can be shortened.
- Trifluoroacetic acid (TFA) for sample separation in liquid chromatography is from Wako Pure Chemical Industries, Ltd., distilled water containing 0.1% formic acid, and acetonitrile (MeCN) containing 0.1% formic acid (FA) are from Kanto. Obtained from Chemical Co., Ltd. Note that acetonitrile and distilled water for semi-micro HPLC were obtained from Nacalai Tesque.
- ⁇ Raman spectroscopy> An example of the measurement conditions is as follows. Wavelength: 532 nm, laser intensity: 30 mW, exposure time: 30 seconds, objective magnification: 40 times, numerical aperture: 0.75, in dry air, irradiation: spot irradiation.
- the laser is focused on a ring area (dried, agglomerated and powdered) where the peptide is highly concentrated. The spectra were acquired five times for each spot and averaged to obtain one spectrum.
- MALDI mass spectra were acquired using LTQ Orbitrap XL (Thermo Fisher Scientific) equipped with a MALDI ion source. Samples were mixed with ⁇ -cyano-4-hydroxycinnamic acid (CHCA) or 2,5-dihydroxybenzoic acid (DHB) (Bruker). MALDI mass spectra were acquired by FT mode (resolution 30,000 or 60,000). These spectra were acquired manually. The parameters are as follows: Scan range: m / z 800-4000, Laser energy ( ⁇ J): 2-4 (for CHCA) or 6-8 (for DHB).
- Nano LC-MS and MS / MS were acquired using LTQ Orbitrap XL (Thermo Fisher Scientific) equipped with an ESI ion source.
- a nano HPLC system (Ultimate 3000, DIONEX), a trap column (ZORBAX 300SB C18 (inside diameter 0.3 ⁇ 5 mm), Agilent) and a tip column (NTCC-360, inside diameter 0.075 mm, Nikyo Technos) were used.
- Mobile phase A was distilled water containing 0.1% formic acid and 4% acetonitrile, and mobile phase B was acetonitrile containing 0.1% formic acid.
- ESI mass spectra were acquired in FT mode (resolution 60,000) and MS / MS spectra were acquired in ion trap mode.
- Database search for identifying proteins or modified peptides was performed by database MS / MS ion search (MatrixScience mascot search engine) using a peptide sequencing program (Protein Discoverer, Thermo Thermo Fisher Scientific).
- Example 1 Raman spectroscopic analysis of a low molecular weight compound having a Raman label
- a low molecular weight peptide having an amino acid sequence of EQWPQCPTXK SEQ ID NO: 4
- a peptide in which X is isoleucine and a peptide in which X is propargylglycine were synthesized.
- the former is called peptide 1
- the latter is called alkyne peptide 1.
- Peptide 1 was synthesized by the solid phase synthesis (Fmoc) method.
- alkyne peptide 1 was also synthesized by solid phase synthesis (both peptides were synthesized at the RIKEN Brain Science Institute).
- a commercial product was used as propargylglycine.
- FIGS. 8-1 and 8-2 The results obtained by fractionating alkyne peptide 1 with a liquid chromatograph and then subjecting it to Raman spectroscopic analysis are shown in FIGS. 8-1 and 8-2.
- Liquid chromatography and Raman spectroscopic analysis were performed using the methods and conditions described in [Experimental methods] above.
- the sample was fractionated according to the retention time by liquid chromatography.
- the result of measuring this with a UV detector is A in FIG.
- FIG. 8B which is an enlarged view of this result, a UV peak was observed at 28 to 29 minutes, and peptides were detected.
- D in Fig. 8-2 shows Raman spectra of fraction numbers 1 to 17.
- a Raman peak characteristic of alkyne was observed in fractions 7 to 8.
- FIG. 7 shows the mass analysis result of the sample subjected to Raman spectroscopic analysis.
- a peak of alkyne peptide 1 was detected in the vicinity of m / z 1211, and a peak of peptide 1 was detected in the vicinity of m / z 1229.
- FIG. 9 shows the results of separating the alkyne-labeled peptide and the unlabeled peptide from the mixture using the method according to the present invention.
- the sample was fractionated according to the retention time by liquid chromatography. The result of measuring this with a UV detector is shown in FIG. 9-1A.
- FIG. 9-1B which is an enlargement of this result, UV (280 nm) peaks were observed at 31 minutes and 33.2 minutes.
- the results of Raman spectroscopy of these fractions are shown in FIG. 9-3G. A Raman peak was observed in fractions 3 and 4, and no significant peak was observed in fractions 12 and 13.
- RAT8-AOMK low molecular weight compound RAT8-AOMK (S) -3- (2-((((4-ethynylbenzyl) oxy) carbonyl) amino) -3-phenylpropanamido) -2-oxopropyl 2 , 6-Dimethylbenzoate) (hereinafter referred to as RAT8-AOMK) was prepared by the following procedure.
- the reaction was quenched with water saturated NaHCO 3 and ethyl acetate, extracted twice with ethyl acetate and dried over magnesium sulfate.
- the organic layer was concentrated under reduced pressure, and then purified on a silica gel column using ethyl acetate / hexane (2/1) solvent to obtain colorless and amorphous bromomethyl ketone (1.55 g, 57%).
- Raman spectroscopy was performed using a Raman microspectroscope (Nanophoton, Raman-11).
- a laser with a wavelength of 532 nm was used as the laser light source.
- the intensity of the laser was 30 mW on the sample surface after passing through the objective lens, and the exposure time was 30 seconds.
- An objective lens with a magnification of 40 times and a numerical aperture of 0.75 was used. Point illumination was selected as the laser illumination pattern.
- Raman spectra in the wavenumber range of 710-3100 cm -1 were acquired.
- FIGS. The results of Raman spectroscopic analysis are shown in FIGS.
- an alkyne-derived Raman peak was observed in the vicinity of 2100 cm ⁇ 1 as shown in the upper right of FIG.
- Raman spectra at various sample concentrations are shown.
- an alkyne-derived Raman peak was observed around 2109 cm ⁇ 1 as shown in the upper right of FIG.
- concentrations is shown in the lower stage of FIG. 26, the Raman peak derived from protein other than the peak derived from alkyne was confirmed simultaneously.
- Example 5 Liquid Chromatography ⁇ Nano LC-Probot>
- the sample lot FL-S10 prepared by the above method was lyophilized and dissolved in 26 ⁇ l of water. 25 ⁇ l of 26 ⁇ l of the sample was injected into a nano LC (NanoFrontier nLC, Hitachi) equipped with a UV detector (MU701, GL science) and fractionated. The flow rate was 250 nL / min. Fractions were spotted on a MALDI plate (ITOP plate, Thermo) using a fraction collector (Probot, Dionnex) at a spotting rate of 20 seconds / spot. The result of UV chromatogram is shown in FIG. FIG. 20C shows the order in which the fractions are spotted. UV absorption was observed in fraction numbers 35-75 as shown in FIG. 20A. About this range, the Raman spectroscopic analysis and the mass spectrometry were performed in the following procedures next.
- Example 6 MALDI mass spectral analysis of concentrated RAT8-AOMK-labeled cathepsin B spotted
- FIG. 27 shows bright field images of fraction numbers 1 to 94 spotted on the MALDI plate. In the spot, the solvent was evaporated by a drying process, and the sample was concentrated.
- the sample lot FL-S10 containing this concentrated RAT8-AOMK-labeled cathepsin B was measured by Raman spectroscopy. Since alkyne signals were detected from multiple spots, mass spectra were manually acquired from the same target plate using a MALDI-Orbitrap instrument.
- FIG. 20A shows the relationship between the retention time in the UV chromatogram and the fraction number. Below that, a Raman spectrum plotting the peak intensity of alkyne (2107 cm ⁇ 1 ) is shown correspondingly (FIG. 20B). An alkyne signal was obtained for fractions 57-66, indicating the presence of a RAT8-AOMK labeled peptide.
- Raman spectroscopy was performed using a Raman microspectroscope (Nanophoton, Raman-11). A laser with a wavelength of 532 nm was used as the laser light source. The intensity of the laser was 30 mW on the sample surface after passing through the objective lens, and the exposure time was 30 seconds. An objective lens with a magnification of 40 times and a numerical aperture of 0.75 was used. Point illumination was selected as the laser illumination pattern. Raman spectra in the wavenumber range of 710-3100 cm -1 were acquired. Silver nanoparticles were not used.
- FIG. 21 shows the Raman spectrum obtained from fraction numbers 35-94.
- the Raman spectrum is acquired 5 times for each spot, and the averaged Raman spectrum is shown.
- the alkyne peak intensity of fraction numbers 35-75 is shown in FIG. 20C.
- Fig. 27 shows bright field images of 94 sample spots on the MALDI plate.
- the Raman measurement was performed with the Raman microscope focused on the agglomerated part. Fraction number 35 coincides with the starting point where the UV peak intensity begins to increase. After Raman measurement, MALDI mass spectrometry was performed for each spot according to the following procedure.
- FIG. 28 is a mass spectrum of fraction number 56-60.
- the theoretical m / z value of peptide A-2 (DQGSCGSCWAFGAVEAISDR + RAT8-AOMK) is shown at the bottom of FIG.
- the region corresponding to the theoretical spectrum of peptide A-2 (bottom row) is shown in parentheses at the top. The strongest peak was detected in fraction number 57.
- FIG. 29 is a mass spectrum of fraction numbers 58-62.
- the theoretical m / z value of peptide B-1 (EIRDQGSCGSCWAFGAVEAISDR + carbamidomethyl + RAT8-AOMK) is shown at the bottom of FIG.
- EIRDQGSCGSCWAFGAVEAISDR + carbamidomethyl + RAT8-AOMK is shown at the bottom of FIG.
- FIG. 30 is a mass spectrum of fraction numbers 60-65.
- the theoretical m / z value of peptide A-1 (DQGSCGSCWAFGAVEAISDR + carbamidomethyl + RAT8-AOMK) is shown at the bottom of FIG.
- FIG. 31 shows a superposition of these results.
- Peptide A-2 was found most frequently in fraction number 57, peptide B-1 in fraction number 60, and peptide A-1 in fraction number 62 (fraction number is the number of wells on the MALDI plate). Number).
- FIG. 22 shows the MALDI mass spectrum obtained for the spots detected by Raman screening.
- the left alkyne peak intensity map of FIG. 22-1 corresponds to the peak intensity profile of FIG.
- the peak of the AOMK-labeled peptide was observed (A, B, C in FIG. 22-1).
- FIG. 22-2 the experimental results are compared with the calculated values. From the fraction number that showed a Raman peak, a mass spectrum that closely matched the calculated mass of the peptide fragment bound with the low molecular compound RAT8-AOMK was obtained.
- the mass spectrum of fraction number 62 showed a spectrum having a peak at m / z 2492 and the like, which was assigned to peptide A-1 (DQGSCGSCWAFGAVEAISDR + carbamidomethyl + RAT8-AOMK).
- the calculated mass to charge ratio (m / z) of C 109 H 150 N 28 O 36 S 2 is 2492.0282 Da.
- the mass spectrum of fraction number 60 was assigned to peptide B-1 (EIRDQGSCGSCWAFGAVEAISDR + carbamidomethyl + RAT8-AOMK).
- the calculated m / z of C 126 H 180 N 34 O 41 S 2 is 2890.2559 Da.
- the mass spectrum of fraction 57 was assigned to peptide A-2 (DQGSCGSCWAFGAVEAISDR + RAT8-AOMK).
- the calculated m / z of C 107 H 147 N 27 O 35 S 2 is 2435.0067.
- Mass spectrometry was performed on a spot where no alkyne signal was observed (fraction number 50), and an unlabeled peptide fragment was confirmed (peptide NGPVEGAFSVYSDFLLYK, SEQ ID NO: 5, 2004.98 Da). This indicates that MALDI mass spectral analysis can be performed on unlabeled peptides as well as labeled peptides.
- the spots where AOMK-labeled peptide and unlabeled peptide were observed by MALDI mass spectrometry are roughly shown in FIG. Only peptide was detected in fraction number 50. In the vicinity of fraction number 57-66, an AOMK-labeled peptide was detected.
- the intact RAT8-AOMK (neat) has two unique Raman peaks (bottom of FIG. 23A). One peak is 2106 cm ⁇ 1 , which is attributed to the alkyne vibration. Another peak is vibration due to the phenyl ring at 1610 cm ⁇ 1 .
- the RAT8-AOMK (neat) as it is and the Raman spectrum of fraction numbers 35-75 are shown correspondingly. In fraction No. 57-66, a peak was observed at 1609 cm ⁇ 1 .
- CHCA In MALDI-Orbitrap analysis, CHCA or DHB was used as a matrix.
- CHCA is suitable for automatic analysis because it is easy to form a uniform co-crystal and a spectrum can be obtained with high sensitivity.
- co-crystals of DHB are not uniform and are needle-like, but in many cases, the peptide coverage (inclusion range) of the protein is increased. Therefore, it is important to select a matrix according to the properties of the analysis sample and the purpose of measurement. Below, the theoretical mass of each peptide is described. The above experimental results agree well with these theoretical values.
- Example 7 MALDI-MS / MS spectral analysis of spotted concentrated RAT8-AOMK-labeled cathepsin B
- MS / MS analysis was performed using a MALDI-Orbitrap apparatus on the fraction that was measured by spectroscopy and the alkyne signal was detected.
- FIG. 33 shows an MS / MS spectrum obtained from the fraction in which peptide A-1 (DQGSCGSCWAFGAVEAISDR + carbamidomethyl + RAT8-AOMK) was detected.
- FIG. 14 The lower left of FIG. 14 shows the sample stage of the Raman microscope.
- the right side of FIG. 14 shows a state where a microscope substrate fixing plate is mounted on the sample stage. In this state, Raman screening (Raman spectroscopic analysis) is performed.
- a metal plate for MALDI manufactured by Thermo was used after confirming the cleaning of the surface.
- a quartz substrate is advantageous for Raman measurement.
- synthetic quartz Starbar Japan, ⁇ 25 mm ⁇ 0.17 mm
- This can be used as it is for MALDI-MS measurement.
- a quartz substrate advantageous for Raman measurement is shown on the left of FIG.
- the results of spotting peptides and alkyne peptides on this substrate and performing Raman spectroscopic analysis are shown in the upper right of FIG.
- a Raman peak specific to alkyne was observed at 2123 cm ⁇ 1 , whereas in the peptide, no Raman peak was observed in this region.
- FIG. 16 shows the result of using this quartz substrate for MALDI-MS measurement as it is.
- An alkyne peptide peak was detected in the vicinity of m / z 1211, and a peptide peak was detected in the vicinity of m / z 1229.
- the materials used are a peptide whose amino acid sequence is EQWPQCPTXK (SEQ ID NO: 4), a peptide where X is isoleucine, and an alkyne peptide where X is propargylglycine.
- the Raman spectrum of FIG. 6 corresponds to the upper right of FIG. 16, and the mass spectrum of FIG. 7 corresponds to the lower right of FIG.
- FIG. 17 shows a multi-spot substrate made entirely of quartz.
- the quartz substrate was fixed to the pedestal with a magnet as shown in the lower part of FIG.
- Example 9 SERS Measurement Using Gold Nanoparticles
- a gold nanoparticle dispersion liquid (EMGC50, BBI) having a diameter of 50 nm was dropped on a glass substrate and dried.
- 1 ⁇ l of 6 mM RAT8-AOMK dissolved in DMSO was dropped onto the dried gold nanoparticle aggregate.
- 1 ⁇ l of 6 mM RAT8-AOMK dissolved in DMSO was dropped on a glass substrate without gold nanoparticles.
- Raman measurement was performed on each droplet.
- a Raman microspectroscope (Nanophoton, Raman-11) was used.
- a laser having a wavelength of 660 nm was used as the laser light source.
- Line illumination was selected as the laser illumination pattern.
- the intensity of the laser was 3.5 mW on the sample surface after passing through the objective lens, and the exposure time was 10 seconds.
- As the objective lens an objective lens having a numerical aperture of 0.75 and a magnification of 40 times was used. 400 points of Raman spectra obtained along the line were averaged to obtain a spectrum for each droplet. Raman spectra in the wave number range of 1250-2400 cm -1 were acquired. The results are shown in FIG. The enhancement of Raman signal was confirmed by the use of gold nanoparticles.
- Example 10 SERS measurement when mixed with silver nanoparticle dispersion 15 ⁇ l of silver nanoparticle dispersion (EMSC50, BBI) having a diameter of 40 nm and 15 ⁇ l of water in which 10 pmol of alkyne peptide 1 was dissolved were mixed. Injected into one compartment of a glass bottom well (EzView 384 well glass bottom assay plate, AGC technoglass). Similarly, a solution prepared by dissolving 10 pmol of silver nanoparticles and unlabeled peptide 1 was also injected into different compartments of the same well plate. The well plate was covered with tape and stored in a refrigerator (4 ° C.) for 1 day, and then Raman measurement was performed.
- EMC50 silver nanoparticle dispersion
- Raman spectroscopy a Raman microspectroscope (Nanophoton, Raman-11) was used. A laser with a wavelength of 532 nm was used as the laser light source. Line illumination was selected as the laser illumination pattern. The intensity of the laser was 240 mW on the sample surface after passing through the objective lens, the exposure time was 1 second / line, and 25 lines were measured per sample. As the objective lens, an objective lens having a numerical aperture of 0.75 and a magnification of 40 times was used. By averaging 10,000 Raman spectra of 1 line 400 points ⁇ 25 lines obtained along the line, Raman spectra in the wave number range of 710 to 3100 cm ⁇ 1 were obtained for each solution. The results are shown in FIG.
- Example 11 and comparative example Comparison sample preparation when using the Raman spectroscopy of the present invention and when introducing a fluorophore via the conventional click reaction Cathepsin B (10 ⁇ g, CALBIOCHEM, catalog number 219362) was added to 100 ⁇ l Bogyo buffer (50 It was dissolved in mM acetic acid (pH 5.6), 5 mM MgCl2, 2 mM DTT). This was left to stand at room temperature for 15 minutes and mixed with 20 mM RAT8-AOMK in 1.0 ⁇ l DMSO. After incubating the mixture at 37 ° C. for 3 hours, the protein was incubated on ice for 3 hours and precipitated by TCA precipitation.
- Cathepsin B 10 ⁇ g, CALBIOCHEM, catalog number 219362
- Centrifugation was performed at 20000 G for 20 minutes to obtain a precipitate. After removing the supernatant, 1 ml of acetone was added, and centrifugation was performed at 20000 G for 15 minutes. Centrifugation and acetone treatment were repeated three times. After removing acetone for 30 seconds under vacuum, the precipitate was dissolved in 10 ⁇ l of denaturing buffer (7M GuHCl, 1M Tris-HCl (pH 8.5)).
- Click-iT protein reaction buffer kit (C10276, Invitrogen) was used for the click reaction. 100 ⁇ l of 40 ⁇ M Alexa Fluor 488 azide was added. 50 ⁇ l of water was added and vortexed for 5 seconds. 10 ⁇ l of CuSO 4 (component B) was added and vortexed for 5 seconds. 10 ⁇ l of Click-iT reaction buffer additive 1 solution was added and vortexed for 5 seconds. This was left for 3 minutes. 20 ⁇ l of Click-iT reaction buffer additive 2 solution was added and vortexed for 5 seconds.
- UV Chromatogram Acquisition 100 ⁇ l of the peptide mixture prepared as described above was lyophilized and dissolved in 50 ⁇ l of water. 50 ⁇ l solution was injected into the nano-LC system (nanoFrontier, Hitachi).
- the experimental conditions for Raman spectroscopy without click reaction were 250 nl / min flow rate, 20 s / spot (Probot, Dionnex), 384 well glass bottom plate (EzView 384 well glass bottom assay plate, AGC technoglass For fluorescence analysis with click reaction, a flow rate of 250 nl / min, 20 s / spot (Probot, Dionnex) and 384-well water-repellent MALDI plates (ITOP, Thermo) It was a measurement.
- UV chromatograms were acquired using a UV detector (MU701, GL science) at 215 nm. The gradients were 0 min 5%, 60 min 80%, 60.01 min 95%, 75 min 95%, 75.01 min 0%, 90 min 0%. The total planned fraction was 192.
- the retention time from 20 to 85 minutes is shown.
- the chart of the retention time of 41 to 57.5 minutes was expanded to the lower part of FIG.
- the click reaction is used as a comparative example (2), a sample loss of 57.5 to 74.2% is observed as compared with the case (1) of the present invention that does not use the click reaction. It was done.
- Raman Chromatogram Raman measurement was performed after the droplets on the 384 well glass bottom plate were dried.
- a Raman microspectrophotometer (Nanophoton Co., Ltd., Raman-11) was used.
- a laser with a wavelength of 532 nm was used as the laser light source.
- Point illumination was selected as the laser illumination pattern.
- the intensity of the laser was 180 mW on the sample surface after passing through the objective lens, and the exposure time was 30 seconds.
- An objective lens having a numerical aperture of 0.75 and a magnification of 40 times was used. Spectra were acquired at different positions on the peptide aggregate 5 times per sample and averaged. The same measurement was performed for all 192 wells.
- Fluorescence measurement was performed after the droplets on the 384-well water-repellent MALDI plate were dried. Fluorescence measurement was performed using a fluorescence imager (Pharos FX, Biorad). The excitation wavelength was selected to be 488 nm. A resolution of 50 ⁇ m was selected. In the obtained fluorescence image, the maximum value of the fluorescence intensity at each spot position was calculated at 192 points, and an intensity profile was created as a fluorescence chromatogram.
- FIG. 37 shows the difference between the Raman chromatogram and the fluorescence chromatogram profile.
- the holding time of 40 to 60 minutes is shown enlarged.
- the fluorescence signal was seen at 47.5-51.0 minutes, whereas in the Raman chromatogram, the Raman signal was seen in a narrower range of 49.5-52.0 minutes.
- many non-specific signals were observed before and after these three peaks.
- Example 12 SERS measurement of TFA-added and TFA-free alkyne-labeled peptide 15 ⁇ l of alkyne-labeled peptide (alkyne peptide 1) containing 15 ⁇ l of 40 nm silver nanoparticles (EMSC40, manufactured by British Biocell International) at a predetermined concentration.
- EQWPQCPTXK; X propargylglycine) /0.3% TFA aqueous solution and left at 4 ° C. for 1 day. SERS measurement was performed using this sample.
- the SERS measurement was automatically performed using a Raman microspectroscope (Nanophoton Corporation, Raman-11) using a 532 nm excitation laser.
- the laser output after the objective lens was 240 mW and the exposure time was 1 to 3 seconds.
- An objective lens with a magnification of 40 times and a numerical aperture of 0.75 was used.
- Line illumination was selected as the laser illumination pattern.
- the alkyne intensity was obtained from a wave number of 1958 cm- 1 .
- the sensitivity of detection of alkyne for SERS in the TFA-added system was 100 fmol (femtomole) on a peptide basis.
- the TFA-free system For the TFA-free system, a sample was prepared in the same manner as the TFA-added system except that TFA was not added in the sample preparation, and SERS measurement was performed. As a result, the detection sensitivity of SERS was 3 pmol (picomoles) on a peptide basis. However, in the TFA-free system, the injection volume of alkyne peptide 1 did not necessarily correlate with the SERS intensity.
- the detection sensitivity of alkyne is about 30 times higher and the SERS intensity is about 4 to 5 times higher than the detection sensitivity of the TFA-free system, and the operability of SERS measurement is greatly improved.
- the injection volume of alkyne peptide 1 and the SERS intensity correlated well in the dynamic range of 100 to fmol to 100 pmol, and the measurement system was stabilized. This is presumably because the aggregates were uniformly distributed by the aggregation accelerator (organic acid) of the present invention.
- Example 13 SERS measurement of TFA-added RAT8-AOMK-labeled cathepsin B
- a labeled sample of cathepsin B with RAT8-AOMK was prepared in the same manner as in Example 3 and digested with trypsin. According to the procedure shown below, a sample containing RAT8-AOMK-labeled cathepsin B fragment was fractionated into TFA-added wells using nano LC-UV-probot, mixed with silver nanoparticles, aggregated, and SERS measurement was performed .
- Sample fractionation was performed by freeze-drying the prepared 100 ⁇ l peptide mixture, dissolving it in a 50 ⁇ l solution, and injecting all the samples into Nano LC-UV-Probot.
- Peptide separation with nano LC-UV-Probot was performed under the following conditions: Flow rate: 250 nl / min Fractionation: 384 well glass bottom plate (EzView 384 well glass bottom assay plate, AGC technoglass) Top, 20 seconds per spot UV chromatogram: 215 nm Concentration gradient: 0 minutes 5%, 60 minutes 80% / 60.01 minutes 95%, 75 minutes 95% / 75.01 minutes 0%, 90 minutes 0% Fractionation: 20 seconds per well The sample was fractionated into a glass bottom well plate containing 25 ⁇ l of 0.1% TFA aqueous solution in advance. The fractionated samples were each fractioned at 15 ⁇ l for SERS and 10 ⁇ l for mass spectrometry. 15 ⁇ l of 40 nm silver nanoparticles were added to the SERS sample, and the sample was left at 4 ° C. for 1 day for SERS measurement.
- a Raman microspectroscope (Nanophoton Corporation, Raman-11) using a 532 nm excitation laser with line illumination was used, and automatic measurement was performed using HTS software.
- the laser output after the objective lens was 130 mW, and the exposure time was 1 to 3 seconds.
- An objective lens with a magnification of 40 times and a numerical aperture of 0.75 was used.
- Line illumination was selected as the laser illumination pattern.
- the alkyne intensity was obtained from a wave number between 1981 and 1900 cm- 1 .
- the SERS measurement time was significantly shortened to 38 minutes / 192 wells because the addition of TFA made the distribution of peptide and silver nanoparticle aggregates uniform and the laser focus setting operation easier.
- the apparatus and method according to the present invention can identify a biomolecule that binds to a low molecular compound, and can identify a binding site between the low molecular compound and the biomolecule. Therefore, by using the apparatus and method according to the present invention, it is possible to search for a protein that is a target of a drug in the field of drug discovery, and to identify a drug binding site in the protein. Furthermore, the present invention allows analysis of protein post-translational modifications in the field of biology. In addition, all or part of the amino acid sequence of the protein or peptide identified using the apparatus or method according to the present invention can be determined by MS / MS analysis. Further, the present invention enables highly sensitive SERS measurement.
- Sample injection unit 2 Liquid feeding line 3: Fractionation unit 4: Liquid feeding line 5: Detection unit 6: Laser unit 7: Mirror 8: Objective lens 9: Sample 10: Sample stage 11: Spectrometer 12: Detection unit 13: Sample stage 14: Sample 15: Laser unit 16: Accelerating electrode 17: Detection unit 18: Signal processing unit
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Abstract
Description
(1)分画された、低分子化合物と結合した生体分子の断片をラマン分光分析に供すること、及び
(2)ラマン分光分析に供した画分の全部又は一部を質量分析に供すること、
を含み、
ラマン分光分析により生体分子断片と結合した低分子化合物由来のラマンピークを有する画分を検出し、低分子化合物由来ラマンピークを有する画分の質量分析結果を取得し、これを生体分子の質量情報と照合して、生体分子内の前記低分子化合物の結合部位を同定する方法。
(1)低分子化合物と結合した生体分子を含む画分をラマン分光分析に供すること、及び
(2)ラマン分光分析に供した画分の全部又は一部を質量分析に供すること、
を含み、
ラマン分光分析により低分子化合物由来のラマンピークを有する画分を検出し、低分子化合物に由来するラマンピークを有する画分の質量分析結果を取得し、これを生体分子の質量情報と照合して、前記低分子化合物に結合する生体分子を特定する方法。
(2)前記凝集体について表面増強ラマン分光(SERS)分析を行う工程、
を含む表面増強ラマン分光分析方法。
(2)前記凝集体に、目的分子を含む溶液を添加する工程、
(3)工程(2)により得られた金属ナノ粒子又は金属ナノ構造と目的分子との複合体について表面増強ラマン分光(SERS)分析を行う工程、
を含む表面増強ラマン分光分析方法。
本発明に係る装置は、試料分離部、ラマン分光部及び質量分析部を有する。試料分離部、ラマン分光部及び質量分析部はこの順序で連結されている。試料分離部の例を図1に、ラマン分光部の例を図2に、質量分析部の例を図3に示す。これらについてそれぞれ説明する。
本発明に係る試料分離部は、試料中の様々な分子を互いに分離することができるものである。試料分離部の具体例としては、限定するものではないが、液体クロマトグラフ及びキャピラリー電気泳動装置等が挙げられる。また試料分離部を等電点電気泳動装置とすることもできる。試料とは、測定対象の化合物を含む可能性のあるサンプルをいう。本発明に係る試料分離部の一例を図1に示す。まず試料注入部1から分画部3へと送液ライン2により試料が送液される。次いで分画部3にて分画が行われる。分画部3は、各種クロマトグラフィーカラム、液体クロマトグラフィーカラムや電気泳動用キャピラリーを有しうるがこれらに限られない。次いで分画された画分は送液ライン4中を送液されて検出部5に送られる。検出部5は、例えば紫外(UV)光検出器であり得る。検出部5での検出手段は非破壊検査であることが好ましい。図1では分離された画分はさらに次のラマン分光部へと連結されていることを右向きの矢印で示してある。本発明に係る装置では、試料分離部で分画された試料を、ラマン分光分析により検出することも可能であるため、図1における検出部5は省略することも可能である。すなわち図1は単なる例示であり、検出部5は試料分離部に必須の構成要素ではない。
液体クロマトグラフとは、移動相として液体を使用するクロマトグラフをいう。液体クロマトグラフにおいて、移動相中に含まれる物質は、固相担体との相互作用の程度の違いに基づき異なる速度で固相担体が充填されたカラムから溶離又は溶出する。この溶離速度の違いを利用して移動相中に含まれる特定の物質を他の物質から分離する。液体クロマトグラフ分離の原理はどのようなものであってもよく、例えば分配、吸着、分子排斥、分子ふるい、イオン交換等が挙げられる。またクロマトグラフは順相であっても逆相であってもよい。好ましくは、液体クロマトグラフは、移動相として高圧に加圧した液体を用いる高速液体クロマトグラフィー(HPLC)である。液体クロマトグラフにおいては移動相として、溶質を溶解するものであればどのような溶媒を用いてもよく、水、水溶液、塩類を含む水溶液、有機溶媒、メタノール、エタノールやイソプロパノール,n-プロパノール等のアルコール、アセトニトリル、ジクロロメタン、トリクロロメタン、酢酸、トリフルオロ酢酸、トリクロロ酢酸、アセトン、シクロヘキサノン、メチルエチルケトン、酢酸エチル、炭酸ジメチル、炭酸ジエチル、イソオクタン、n-ヘキサン、n-ヘプタン、ジエチルエ-テル、シクロヘキサン、トルエン、テトラヒドロフラン、ベンゼン、ジオキサン、ジメチルホルムアミド、ジメチルスルホキシド等及びこれらの適当な組合せが挙げられる。
キャピラリー電気泳動法とは、電気泳動を十分に細い毛細管の中で行い、試料中に含まれる物質を分離する方法をいう。毛細管を用いることで対流の発生を抑制でき、通常の電気泳動と比較して物質の分離能を高めることができる。キャピラリー電気泳動装置は、典型的には毛細管及び電圧印加部を有する。通常は毛細管の一方が試料注入部であり、他方は試料溶出部である。例えば図1の試料分離部で説明すると、試料は試料注入部1から注入され、分画部3のキャピラリーにて分離され、検出部5に送液される。試料溶出部は適宜、検出部及び/又はフラクションコレクターに連結されていてもよい。このキャピラリー電気泳動には、キャピラリーゾーン電気泳動(CZE)、ミセル導電クロマトグラフィー(MEKC)、キャピラリーゲル電気泳動(CGE)やキャピラリー等電点電気泳動(cIEF)等も含まれる。当業者であれば、キャピラリー電気泳動に用いる溶媒やキャピラリーの種類、印加電圧などの運転条件を適当に設定することができる。キャピラリー電気泳動により分画した画分は、上記液体クロマトグラフと同様に、溶媒を蒸発させ、乾燥状態で次の工程に供することができる。
本明細書にいうラマン分光部とは、ラマン励起用レーザー光を照射するレーザー部とラマン散乱光をスペクトル解析するスペクトル解析部を含む線形又は非線形ラマン分光装置である。図2に本発明に係るラマン分光部の一例として顕微ラマン分析装置を示す。試料台10の上に試料9を配置し、これをラマン顕微装置で分析する。レーザー部6からラマン励起用レーザー光が照射され、これがダイフロイックフィルター7で反射されて対物レンズ8により焦点を合わせた試料9に照射される。ラマン散乱光は分光器11により分光され、検出部12において電荷結合素子(CCD)のような検出機構により検出される。スペクトル解析部は、図2のラマン散乱スペクトルを取得するための分光器11、検出部12及び任意の電子計算部を有する。図2では集光されるラマン散乱光を分光器11に入る上向きの矢印で示してある。集光には適宜入射スリットやレンズが使用され得る。図2はあくまで一例であり、本発明に係るラマン分光部は顕微ラマン分析装置に限らず、分散型レーザーラマン分光分析装置、FT-ラマン分光分析装置等の公知のあらゆるラマン分光分析装置を包含する。例えば本発明に係るラマン分光部に含まれるスペクトル解析部は、ラマン散乱光を分光器の代わりに干渉計で検出する機器を備えていてもよい。また、ラマン散乱光の検出部に透過波長帯を限定したフィルターを使用し、フィルターを透過してきた散乱光を、分光器等を介さず、直接CCDのような検出機構により検出する構成でもよい。この場合、波長可変フィルターを用いれば、透過波長帯を走査することでラマンスペクトルの取得も可能となる。さらに、ラマン励起用レーザーに波長可変レーザー光源を用い、励起レーザー光の波長を走査することで、ラマンスペクトルを取得する構成でもよい。分光器であれ干渉計であれ、フィルターであれ、また励起レーザー光の走査であれ、いずれの構成でもラマン散乱光を検出することで、対象試料における特定のラマンピークの強度や、ラマンスペクトルの情報を得ることができる。また、図2に示されるように試料を試料台に載せる構成とは別に、試料を送液ラインで送液し、いわゆる「オンライン」で測定を行うこともできる。当業者であれば、得られたラマンスペクトルのパターン(プロファイル)から、適宜、試料中の目的分子の存在/不在を解析することができる。解析は手作業で又は電子計算機の補助により行うこともできる。ラマン励起用レーザーとしては、半導体レーザー、ダイオード励起固体(DPSS)レーザー、ガスレーザー、液体レーザー等を用いることができるが、これらに限定されない。
ラマン分光分析は当技術分野において周知の技術であり、例えば浜口宏夫、平川暁子 編 ラマン分光法(日本分光学会出版、測定法シリーズ17)にその原理が解説されている。簡単に説明すると、ラマン分光法は、化学物質にレーザー光などの光を入射したときに、散乱光の中の入射光の波長とは異なる波長の光が発生する、いわゆる「ラマン効果」を利用して行う分光分析法である。ラマン散乱光の振動数と入射光の振動数の差をラマンシフトという。ラマンシフトは分子の構造に特有であるため、ラマンシフトを測定することで分子構造に関する知見が得られる。また、化学構造が解明されている分子のラマンスペクトルを予め測定してプロファイルを取得しておき、ある試料中に当該分子が存在するか否かをその試料のラマンスペクトルパターンと前記プロファイルとを比較することにより検出することができる。検出するとは、ある試料中にある化合物が存在することを確認することをいう。ラマン分光法には非破壊分析法であるという利点がある。線形ラマン分光とは、入射光強度に比例する強度をもつラマン散乱分光、別名自発ラマン散乱分光ともいう。非線形ラマン分光とは、高次の非線形光学効果によるラマン分光で、入射光の強度の2次以上に比例する強度をもつラマン散乱分光をいう。ラマン分光法としては、誘導ラマン散乱、ハイパーラマン散乱、コヒーレント・反ストークスラマン散乱などの非線形ラマン分光法が挙げられる。ラマンスペクトルの一例を図4に示す。図4に示されているように、パクリタキセル自身の分子振動がピークとして検出される。この例でのラマン分光分析はJ.Ling et al., Applied Optic, 41, (28), 6006(2002).に記載されている。Renishaw Model 2000ラマン分光システム、Ti:サファイアレーザーを用いて測定した。試料は粉体であり、20倍レンズ、 30秒露光にて測定を行った。
本発明は、ある実施形態において、表面増強ラマン分光(SERS)を用いたラマン分光分析法を提供する。ある実施形態において本発明の表面増強ラマン分光(SERS)は本発明に係る装置又は方法に用いることができる。ラマン分光法では、散乱光が弱いため一般的に長時間の測定が必要とされることがあるが、SERSを用いるとラマンシグナルを増強でき、迅速な測定が可能となる。SERSはラマン分光法の1つとして知られており、金属粒子コロイド又は金属を含む基材を用いてラマン分光分析を行う。このとき、金属の表面プラズモンがレーザーにより励起され、その結果、金属を取り囲む電磁場が増大し、電磁場に比例して生じるラマンシグナルを増強する。さらに、金属表面近傍の分子と金属間には、電子の授受を含む化学的な相互作用を生じ、ラマンシグナルを増強する。上記の電磁気学的・化学的増強メカニズムのいずれか、又は両方が作用することにより、測定されるラマンシグナルが大幅に増強される。SERSに用いる金属としては、鉄、コバルト、ニッケル、スズ、インジウム、ゲルマニウム、銅、銀、金、プラチナ、パラジウム、アルミニウム、チタン、ルテニウム等が挙げられるが、これらに限られない。金属は金属ナノ粒子、金属ナノ構造又は金属ナノ構造物とすることができる。また、試料に金属膜を被覆する処理を行うこともできる。この被覆処理は独立に行うこともでき、又は前記被覆処理を行う処理室を本発明に係るラマン分光部の一部とすることもできる。SERSの効果の一例を図12に示す。図12には直径40 nmの銀ナノ粒子を用いるとRAT8-AOMKのラマンピークの強度が103以上増大したことが示されている。露光時間は10秒であった。図12左において下のスペクトルが銀粒子を使用しなかったものであり、上のスペクトルが銀粒子を使用したときに得られたものである。図12中央も同様である。別の例として図34に金ナノ粒子を用いた場合のSERSスペクトルを示す。図34の下のスペクトルが金ナノ粒子無し、上のスペクトルが金ナノ粒子を使用した結果である。このように本発明のSERS効果は銀ナノ粒子に限られず、金ナノ粒子等、金属ナノ粒子全般を用いた場合にラマンシグナルが増強すると考えられる。SERS試料は、市販の銀ナノ粒子分散水溶液を清浄化した基板にスポッティング後乾燥し、その上から試料を重層するか、試料溶液に銀ナノ粒子分散水溶液を混合するか、又は混合した試料をスポッティング後乾燥することにより調製できる。さらに、基板としては、スピンコート法等機械的塗布法で銀ナノ粒子分散水溶液を基板一面に塗布、乾燥したものを用いることができる。また、金属ナノ粒子の径は、特に制限なく、小さい方が好ましい。ここで、粒子の直径(径)とは、当該粒子と同じ容積を有する球体の直径に等しい長さとする。また、直径40 nmの粒子とは多数の粒子の前記のようにして得られる直径を平均した値が40 nmであることをいう。金属ナノ構造には、ナノロッド、ナノワイヤ、ナノキューブ、ナノプリズム、シェル構造等様々な形状があり、それらの大きさも同様に小さい方が好ましい。金属ナノ構造の大きさは、当該構造の長手方向の長さをいうものとする。また金属ナノ構造のサイズが40 nmであるとは、種々の金属ナノ構造の大きさの平均が40 nmであることをいう。金属粒子、金属ナノ構造又は金属ナノ構造物のサイズは光によって金属中で振動する電子の平均自由行程以下であることが好ましく、具体的には金属粒子の場合には当該粒子の直径が、金属ナノ構造又は金属ナノ構造物の場合には当該ナノ構造の長さが200 nm以下であり、より好ましくは100 nm以下、さらに好ましくは50 nm以下である。
SERS測定をする際に、金属ナノ粒子又は金属ナノ構造と生体分子及び低分子化合物に結合した生体分子との均一な凝集体の形成を促進させるために有機酸を添加することができる。有機酸添加に起因して、均一に分布した凝集体が形成されると、SERS効果が高まるとともに、ラマン分光測定に用いるレーザーの焦点設定操作が容易となり測定時間を大幅に短縮することができる。そのため、自動測定に極めて有利となる。本明細書では、このようなSERS効果を高めるために添加する酸のことを便宜上、凝集促進剤、又は添加剤と呼ぶことがある。
本発明の凝集促進剤はSERS測定を行うことが可能で有れば、どのような試料に対して使用してもよい。すなわち、本発明の凝集促進剤は、生体分子を含み得る試料や、液体クロマトグラフ又はキャピラリー電気泳動により分離された試料に対して使用できるのはもちろんのこと、SERS測定を行うことのできる他のあらゆる試料についても使用できる。つまり本発明の凝集促進剤を用いたSERS測定は、上記1.の装置を用いる場合に限定されず、あらゆる表面増強ラマン分光(SERS)法に用いることができる。ただし、分析する目的分子はSERSシグナルを発するものである必要がある。また、目的分子は好ましくは金属ナノ粒子と一緒に混合すると凝集するか、又は予め凝集させた金属ナノ粒子と相互作用するものである。この場合、目的分子は例えばSERSシグナルを発する生体分子、SERSシグナルを発する生体分子の断片、SERSシグナルを発する低分子化合物と結合した生体分子、又はSERSシグナルを発する低分子化合物と結合した生体分子の断片であり得る。目的分子は、予め上記1.1の試料分離部により、例えば液体クロマトグラフィーやキャピラリー電気泳動により分画された画分に含まれ得る。
本発明に係るラマン分光法は、試料分離部からの試料を送液しながら測定を行う、いわゆる「オンライン」分析が可能である。また、本発明に係るラマン分光法は、試料分離部からの試料を送液後、プレートにスポッティングし、そのスポットについて測定を行う、いわゆる「オフライン」分析も可能である。図10にオンラインラマン検出法(A)とオフラインラマン検出法(B)の特徴を比較して示す。図10の左(A)が送液をそのままラマン計測するオンライン検出であり、右(B)が送液を一旦プレート上にスポッティングし、次いでこれをラマン計測するオフライン検出である。例えばオンラインで測定を行った場合にラマンピークの強度が十分でないときは、測定をオフラインで行ってもよい。一般にオンラインでのラマン分光法の測定感度はmM単位であるが、オフラインであれば感度はμM単位(ペプチドで数pmol)である。また、測定をオンラインで行う場合には溶媒の背景光が混入するという問題があるのに対して、オフラインであれば、プレート上のスポットの溶媒を乾燥させて蒸発させることで背景光の問題を回避できる。オンライン測定の場合は、プレート上へのスポッティングの必要がなく、装置の構成が簡便ですむという利点がある。当業者であれば、ラマンスペクトルの測定をオンラインで行うのが好ましいか、オフラインで行うのが好ましいか、試料濃度や測定条件に応じて適宜判断することができる。また、これに応じて装置の構成を適宜変更することができる。
細胞破砕液を分画せずにそのままラマン分光分析すると、ピークが検出される領域と検出されない又はほとんど検出されない領域が現れる。細胞破砕液をラマン分光分析したときに、ラマンピークが検出されない又はほとんど検出されない領域のことを本明細書において「サイレント領域」という。例えばタンパク質のラマンピークは主に800~1800 cm-1及び2800~3000 cm-1付近に見られ、1800~2800 cm-1にはほとんど検出されない。これらのラマンピークはいずれも特定のアミノ酸残基に帰属されている。例えばトリプトファン由来のピークは1011 cm-1 及び1554 cm-1付近に、アミド由来のピークは1250 cm-1及び1660 cm-1付近に、CH2由来のピークは1430 cm-1付近に、CH3由来のピークは2933 cm-1付近に現れる(図6参照)。しかしながら、1800~2800 cm-1という波長領域では、生体分子由来のラマンピークはほとんど観察されない。したがって、本明細書でいうサイレント領域とは、1800~2800 cm-1でありうる。また、ラマン分光分析は、500 cm-1以上、700 cm-1以上、1000 cm-1以上、1200 cm-1 以上、1400 cm-1以上1600 cm-1以上又は1800 cm-1以上、3000 cm-1未満、2900 cm-1未満、2800 cm-1 未満、2700 cm-1 未満、又は2600 cm-1未満の領域で行うことができる。サイレント領域は用いる生体材料によらず、基本的に同一である。
質量分析部とは、適当なイオン化法により試料中に含まれる分子をイオン化し、当該分子の質量スペクトルを測定する装置をいう。図3に本発明に係る質量分析部の一例を示す。試料部、分離部、分析部を合わせた質量分析計が本発明に係る質量分析部を構成する。試料部ではまず試料ステージ13に試料14を載せる。次に試料を適当なイオン化手段によりイオン化し、静電力によって装置内を飛行させる。図3ではイオン化手段としてレーザー部15を例示してある。加速電極16により加速させて飛行するイオンを分離部において電気的又は磁気的な作用等により質量電荷比に応じて分離し、その後それを分析部におけるイオン検出器17で検出することで、質量スペクトルを得ることができる。イオン検出器17は好ましくは信号処理部18に接続されており、得られた信号は好ましくは電子計算機により処理される。本明細書では、イオン検出器17、信号処理部18及び任意の電子計算機を合わせて質量分析器の「分析部」という。分析部で信号を処理して得られる質量スペクトルは通常、質量電荷比(m/z)を横軸、検出強度を縦軸として表す。質量分析のためのイオン化方式としては、例えばマトリックス支援レーザー脱離イオン化(MALDI)法、エレクトロスプレーイオン化(ESI)法、大気圧化学イオン化(ACPI)法、電子イオン化(EI)法、化学イオン化(CI)法等が挙げられる。当業者であれば、これらのイオン化手段に応じて適宜、質量分析装置の構成を変更し、適合化することができる。一例としてMALDI法では、芳香族有機化合物などのマトリックス中に試料を混合して結晶を作製し、これにレーザーを照射することでイオン化を行う。用いるマトリックスとしては、特に限定するものではないが、α-シアノ-4-ヒドロキシ桂皮酸(CHCA)、シナピン酸(SA)、trans-4-ヒドロキシ-3-メトキシケイ皮酸(フェルラ酸)、3-ヒドロキシピコリン酸(HPA)、1,8-ジヒドロキシ-9,10-ジヒドロアントラセン-9-オン(ジスラノール)及び2,5-ジヒドロキシ安息香酸(DHB)等が挙げられる。MALDI法はタンパク質などの高分子化合物であっても分子を破壊することなく安定してイオン化することができるという利点がある。
質量分析に用いる分離部ではイオン化された試料を分離する。分離部の種類としては、飛行時間型、磁場偏向型、四重極型、イオントラップ型及びフーリエ変換型等が挙げられる。飛行時間型(TOF)の分離部を備えた質量分析では、イオン化した試料をパルス的に加速し、検出器に到達するまでの時間差を検出する。この時間差から質量を算出することができる。この場合、図3に示す加速電極16はイオンが飛行する空間の一部のみに配置されることでパルス的に加速が行われ、イオンが飛行する空間の大部分では電場・磁場はかけられていない。これらの分離部は上記イオン化法のいずれと組み合わせてもよいが、特にMALDIとTOFの組合せが好ましい。このような構成をMALDI-TOF型質量分析器と呼ぶことがある。
本発明のある実施形態において、質量分析は、MS/MS分析とすることができる。MS/MS分析では質量分析をタンデムに行う。この方法では第一の分離部で特定のイオンだけを取り出し、これを解裂させ、生じたフラグメントイオンを第二の分離部で分析する。フラグメントイオンの分析は単一の装置でも行うことができ、又は異なる2つの装置で行ってもよい。例えばタンパク質をプロテアーゼで消化してペプチドを取得し、当該ペプチド断片についてMS/MS分析を行うと、ペプチドが順次フラグメント化されたピークが検出され、それらのピークの質量情報からペプチドのアミノ酸配列を決定することができる。MS/MS分析は当技術分野で周知の手法である。例えば、A. K. Shukla et. al., J. Mass Spectrom. 35, 1069 (2000)等を参照されたい。
ある実施形態において、本発明に係る装置は、液体クロマトグラフ、ラマン分光装置及び質量分析計(LC-R-MS)を有する。その一例を図5に示す。混合試料を液体クロマトグラフ(LC)により分画し、ラマン分光法により低分子化合物(薬剤)が存在する画分を絞り込む。さらにラマンピークを示した画分を質量分析計(MS)で分析し、低分子化合物と結合する生体分子を特定することができる。別の実施形態において、本発明に係る装置は、キャピラリー電気泳動装置、ラマン分光装置及び質量分析計(CE-R-MS)を有する。この場合も試料の分離をキャピラリー電気泳動装置(CE)により行う他はLC-R-MSの場合と同様であり、本発明の基本的な原理は同じである。これらの他、試料分離部として液相等電点電気泳動等を使用することもでき、試料分離部が他の分離手段による場合についても同様である。
本発明は、本発明に係る装置を用いて生体分子を特定する方法、及び生体分子と低分子化合物との結合部位を同定する方法を提供する。
本発明に係る装置又は方法は生体分子を分析するものである。本明細書でいう「生体分子」とは、細胞外又は細胞内に存在する、タンパク質、ペプチド、核酸、糖又は脂質をいう。本明細書でいう生体分子はどのような生体又は生物に由来するものであってもよく、例えばウイルス、原核生物、真核生物、真菌類、植物、高等植物、動物、昆虫、高等動物、哺乳動物、マウスやラット等のげっ歯類、サルやチンパンジー等の霊長類、ヒト、又はこれらの培養細胞や培養組織由来であってもよい。本明細書でいう生体分子に包含されるタンパク質及びペプチドは、天然及び/又は合成アミノ酸がペプチド結合により結合している高分子化合物をいう。本明細書でいう生体分子に包含される核酸は、少なくとも10、好ましくは50、300、500、又は1000以上の塩基を含む、一本鎖又は二本鎖の核酸をいい、好ましくは特定の低分子化合物と相互作用するものである。核酸はDNAであってもよく、又はRNAであってもよい。RNAにはtRNAやリボソームRNA、リボザイムも含まれる。核酸はプロモーター領域、エンハンサー領域、サイレンサー領域、ターミネーター領域を含むものであり得る。これらは好ましくは特定の転写調節因子、転写開始因子などと結合するものである。本明細書でいう生体分子に含まれる糖は、多糖類を包含し、この多糖類は好ましくは特定の低分子化合物と相互作用するものである。糖の例としては、ヒアルロン酸、キチン、ヘパラン硫酸、ケラタン硫酸、デルマタン硫酸、シアル酸、コンドロイチン硫酸等のプロテオグリカン或いはプロテオグリカン類縁体が挙げられる。本明細書でいう生体分子に含まれる脂質は、上記に例示した生物に含まれる脂質を包含し、好ましくは特定の低分子化合物と相互作用するものである。脂質の例としては、スフィンゴリン脂質、グリセロリン脂質等のリン脂質、スフィンゴ糖脂質、グリセロ糖脂質等の糖脂質、リポタンパク脂質、スルホ脂質又はガラクト脂質等の細胞外或いは細胞膜を形成する複合脂質が挙げられる。
本発明に係る低分子化合物とは、分子量が低く、特定の生体分子に結合する化合物又は結合する可能性がある候補化合物をいう。ある実施形態において本発明に係る低分子化合物は生体分子と比較して分子量が低い。ある低分子化合物が特定の生体分子に結合する場合、低分子化合物からみた生体分子のことを標的と呼ぶことがある。低分子化合物、又は低分子化合物の基となる化合物(分析対象化合物ともいう)には、薬剤、薬剤候補化合物、生理活性物質、代謝物質、ビタミン、ホルモン、特定の受容体タンパク質に結合するリガンド、タンパク質のアゴニスト、タンパク質のアンタゴニスト、タンパク質翻訳後修飾機構によりタンパク質に結合する化合物等も含まれる。これらには、天然に存在する化合物及びそれらと化学構造が類似するアナログ(類似体)が含まれる。本発明に係る低分子化合物は、ラマン分光において、生体分子と識別可能な散乱ピークを与えるもの、またはラマン標識により生体分子と識別可能な散乱ピークを与えるものであれば、いかなる化合物でもよい。
特徴的なラマンピークや、細胞中や混合物中に共存する生体分子、特に低分子化合物の標的となる生体分子と識別できるようなラマンピークを有する化合物であれば、これをそのまま低分子化合物として本発明に使用することができる。このような低分子化合物は、そのままラマン分光により検出できるため、蛍光団等による修飾の必要がないという利点がある。また、500~1800 cm-1の範囲で、化合物に由来するラマンピークが認められる領域を「フィンガープリント(指紋)領域」ということがある。
また、特徴的なラマンピークを有しない化合物については、当該化合物に導入しても生体分子との結合に対する影響がきわめて小さい置換基を導入し、これを本発明に係る低分子化合物として使用することができる。このような置換基をラマン標識ともいい、好ましくは前記置換基又はラマン標識はサイレント領域に散乱スペクトルを有するものである。サイレント領域は上記に説明したとおり、生体分子由来のラマンスペクトルにおいてシグナルがほとんど又は全く観察されない波数領域である。前記置換基又はラマン標識はラマン散乱光が比較的強く、標的低分子化合物を検出する際に生体分子由来のラマンピークと異なる波数域に特徴的なピークを示すため、標的である低分子化合物を高感度かつ選択的に検出するのに好都合である。これらは蛍光団等で修飾する必要がなくそのままラマン分光法で検出できる。サイレント領域に散乱スペクトルを有する置換基としては、アルキニル基、ニトリル基(-C≡N)、重水素(C-D、C-D2、C-D3)、ジアゾニオ基(-N+≡N)、イソシアン酸エステル基(-N=C=O)、イソニトリル基(-N+≡C-)、ケテン基(>C=C=O)、カルボジイミド基(-N=C=N-)、チオシアン酸エステル基(-N=C=S)、アジド基(-N=N+=N-)、ジアゾ基(>C+=N-=N)、アルキンジイル基、エチニレン基(-C≡C-)、1,3-ブタジイニレン(-C≡CC≡C-)等を含む化合物が挙げられるがこれらに限定されない(浜口宏夫、平川暁子 編 ラマン分光法(日本分光学会 測定法シリーズ17)も参照されたい)。アルキニル基としては、エチニル基(CH≡C-)、プロパルギル基(CH≡CCH2-、2-プロピニル基ともいう)、ブタ-3-イン-1-イル基(HC≡CCH2CH2-)、ブタ-2-イン-1-イル基(CH3-C≡CCH2-)などが挙げられるがこれらに限定されない。これらはいずれも本発明のラマン標識となり得る。本発明に係る低分子化合物は、好ましくはアルキニル基、ニトリル基、又は重水素を有する。
ラマン標識は目的の化合物に直接導入してもよく、又は適当なスペーサー分子を介して目的の化合物に結合させることもできる。例えば目的の化合物にアルキニル基を導入したい場合、アルキニル基を直接導入してもよく、又はフェニル基と結合したアルキニル基(ethynylphenyl基)として導入することもできる。この場合、スペーサー分子はフェニル基である。したがって特定の生体分子に結合するが特徴的なラマンピークを有しない又は生体分子と識別できるようなラマンピークを有しない化合物は、上記置換基を導入することによりラマン標識して、本発明に係る装置又は方法に使用することができる。当業者であれば、適当なスペーサー分子を用いて化合物をラマン標識することができる。スペーサー分子としては、メチレン基(-CH2-)、エチレン基(-CH2CH2-)、プロパン-1,3-ジイル基(-CH2CH2CH2-)、フェニレン基(-C6H4-)、オキシエチレン基(-OCH2CH2-)、オキシプロピレン基(-OCH2CH(CH3)-)等が挙げられるがこれらに限られない。
当業者であれば、化合物の構造に鑑み、どの位置にどのようなラマン標識を導入すればよいか、適宜選択することができる。また、有機合成の分野における通常の技術を有する当業者であれば、適宜、ラマン標識された化合物を合成し、本発明に係る低分子化合物とすることができる。具体例を用いて説明すると、当業者であれば、有機合成の分野における通常の技術を用いてカテプシンBの阻害剤であるアシルオキシメチルケトン(AOMK)をアルキンでラマン標識し、AOMK誘導体(以下、RAT8-AOMKともいう)を合成することができる。このRAT8-AOMKはカテプシンBに結合し、その酵素活性を阻害する。RAT8-AOMKのカテプシンB阻害活性はIC50=0.3 μMである。RAT8-AOMK合成は、N-Boc-AOMK(IC50=0.05 μM)を4-ニトロフェニル-4-エチニルベンジルカルボネートと反応させることにより得ることができる。アシルオキシメチルケトン(AOMK)はシステインプロテアーゼ阻害剤として知られている。その原理は、AOMKにより酵素タンパク質の活性中心のシステイン残基が修飾され、プロテアーゼとしての活性が失われる、というものである(図24参照)。
生体分子がタンパク質又はペプチドである場合、本発明に係る低分子化合物としては、そのようなタンパク質又はペプチドに結合する、薬剤、薬剤候補化合物、抗生物質、農薬等の生理活性物質、代謝物質、補酵素等のビタミン、ホルモン、特定の受容体タンパク質に結合するリガンド、タンパク質のアゴニスト、タンパク質のアンタゴニスト、タンパク質翻訳後修飾機構によりタンパク質に結合する化合物等が挙げられる。このような低分子化合物は、好ましくはラマン標識されているか、又は他の生体分子と識別可能な特徴的なラマンスペクトルを有する。一例として、タンパク質カテプシンBに結合する化合物N-Boc-AOMKは、4-ニトロフェニル-4-エチニルベンジルカルボネートによりラマン標識の一種であるアルキン基を導入し、本発明に係る低分子化合物RAT8-AOMKとすることができる。
本明細書において低分子化合物と生体分子とが「結合した」という場合、結合には、共有結合、配位結合及び相互作用が含まれ、低分子化合物による生体分子中の特定の部位への結合をいう。共有結合とは、複数の原子が互いの電子を共有することによる化学結合をいう。配位結合とは、結合に関与する原子の一方からのみ電子が提供される化学結合をいう。相互作用とは、2つの分子の間に働く分子間力に基づく作用をいい、イオン間相互作用、水素結合による作用、双極子相互作用、疎水性相互作用、及びこれらの組合せが挙げられる。
生体分子と低分子化合物との結合部位を「同定する」とは、低分子化合物が生体分子のどこに結合しているか、又はどこと相互作用するか、を決定することをいう。本発明に係る装置を使用すると生体分子と低分子化合物との結合部位を同定することができる。
(1)分画された、低分子化合物と結合した生体分子断片をラマン分光分析に供すること、及び
(2)ラマン分光分析に供した画分の全部又は一部を質量分析に供すること。
本明細書において生体分子の断片とは、高分子化合物である生体分子の結合を1以上の箇所で切断し、より分子量の低い単位にしたものをいう。例えば生体分子がタンパク質である場合、これをプロテアーゼ処理に供してペプチド結合が切断された断片(ペプチド)を得ることができる。プロテアーゼとしては、セリンプロテアーゼ、アスパラギン酸プロテアーゼ、メタロプロテアーゼ、システインプロテアーゼ等が挙げられるがこれらに限定されない。また、臭化シアン、N-ブロモサクシンイミド、ヒドロキシルアミン等により生体分子を化学的に分解することもできる。生体分子がペプチドである場合についても同様である。生体分子がトリグリセリド脂質を含む場合は、これをリパーゼ等の脂質分解酵素での処理に供して分解された断片(脂肪酸)を得ることができる。リパーゼとしてはトリアシルグリセリドリパーゼ、ホスホリパーゼ、リポタンパクリパーゼ、エステラーゼ等が挙げられるがこれらに限定されない。他種の生体分子についても同様である。生体分子が糖であれば、糖を分解する酵素が使用可能であり、α-アミラーゼ、β-アミラーゼ、グルコアミラーゼ、イソアミラーゼ、プルラナーゼ、マルトトリオヒドロラーゼ、α-グルコシダーゼ、シクロデキストリン、グルカノトランスフェラーゼ、アミログルコシダーゼ、デキストラナーゼ、β‐ガラクトシダーゼ、シアリダーゼ、セルラーゼ、α―マンノシダーゼ、β―マンノシダーゼ等が挙げられるが、これらに限定されない。生体分子が核酸であれば、二重鎖DNAを特異的に切断する制限酵素等のデオキシリボヌクレアーゼ、一重鎖RNA切断酵素のリボヌクレアーゼ等の核酸分解酵素で処理し、核酸断片を得ることができる。断片化とは、適当な分解酵素や物理的又は化学的処理により生体分子をより分子量の低い断片に分解することをいう。断片化は上記のような酵素処理や化学的処理により行うことができる。当業者であれば適宜、用いる酵素や化合物等を選択し、処理条件を決定することができる。
生体分子と低分子化合物との結合部位を同定するための装置の例を図18に示す。装置の構成としては、HPLCがUV検出器に連結され、これがスポッターに連結され、次にこれがラマン分光部に連結され、次いでこれが質量分析部に連結されている。この装置は、本発明に係る生体分子と低分子化合物との結合部位を同定する方法に使用することができる。この方法について図18を参照しながら説明すると、まず生体分子(例えばタンパク質)を低分子化合物と結合させる。次いでこれを適当なプロテアーゼ処理により断片化(消化)する。得られた断片(ペプチド)の混合物を試料分離部のHPLCで分画し、UV検出器で検出してUVクロマトグラムを得る。次にそれぞれの画分をMALDI用プレート上にスポッティングする。配列されたペプチドアレイをラマン分光分析に供し、ラマン強度にしたがってマッピングする。ラマンシグナルを示した画分をMALDI-MSに供して質量分析を行うことで、低分子化合物が結合したペプチドが特定でき、これにより低分子化合物と生体分子との結合部位が同定できる。
結合部位の同定について具体例を用いて説明する。本発明者らは3.の方法を用いて生体分子カテプシンBと低分子化合物RAT8-AOMKの結合部位を解析した。RAT8-AOMKはアルキン標識されたカテプシンB阻害剤である。図19-1上段では結合解析の手順を模式化した。図19-1上段に示すように、まずRAT8-AOMKをカテプシンBに結合させ、次いでこれをプロテアーゼ処理により断片化(消化)する。HPLC分画を経て、プレートに画分をスポッティングし、スポットを乾燥させた後、ラマン分光分析を行う。図19-2の左にアルキン強度の分布が模式的に示されており、強度の高い画分を質量分析に供する。質量分析により種々のペプチドの質量スペクトルが得られ、これを解析することで、どのペプチド断片にRAT8-AOMKが結合しているか特定でき、これによりRAT8-AOMKがカテプシンBに結合している部位を同定できる。
本明細書において質量分析結果を取得し、これを生体分子の質量情報と「照合」して、生体分子内の前記低分子化合物の結合部位を同定する、とは、タンパク質の例で説明すると、取得されたペプチド断片の質量スペクトル結果が、当該ペプチドが由来するタンパク質の一部に相当する領域の計算上の質量と一致するか否か判断することにより、低分子化合物がタンパク質中のどこに結合しているか結合部位を同定することをいう。当業者であれば、必要に応じてタンパク質、ペプチド、核酸、脂質や糖のような生体分子に関する情報をDDBJ / NIG、EMBL / EBI、GenBank / NCBI、NIAS DNA Bank、PIR、SWISS-PROT & TrEMBL、GenPept、PRF、日本糖鎖科学統合データベース(JCGGDB)、LipidBank等の適当な公知のデータベースから取得することができる。また、アミノ酸配列が一致しているか否かはMascot(Matrix Science Inc.)等のソフトウエアを用いて決定することができる。さらに、MS/MS解析により、低分子化合物の結合アミノ酸を確認することもできる。
本発明に係る装置を使用すると、低分子化合物と結合する生体分子を特定することができる。本発明に係る低分子化合物と結合する生体分子を特定するスクリーニング方法は、以下の工程を含む。
(1)低分子化合物と結合した生体分子を含む画分をラマン分光分析に供し、前記低分子化合物と結合した生体分子を含む画分を検出すること、及び
(2)ラマン分光分析に供した画分の全部又は一部を質量分析に供すること。
本発明に係るスクリーニング方法及び結合部位同定方法は、タンパク質、ペプチド、核酸、糖、脂質を含む種々の生体分子に用いることができる。
本発明に係るスクリーニング方法はタンパク質のスクリーニングに使用することができる。例えば全ゲノムの塩基配列が解読されている生物又はウイルスについて、ある薬剤活性を有する低分子化合物が、当該生物又はウイルス中のどのタンパク質と結合し作用するかスクリーニングする場合、上記方法を実施すると、前記低分子化合物と結合するタンパク質の質量分析結果が得られる。また、前記低分子化合物と結合するタンパク質を、プロテアーゼ処理により分解し、ペプチドに断片化した後質量分析を行うと、当該ペプチド断片の質量情報が得られる。さらに、MS/MS分析にかけることで、ペプチドのアミノ酸配列が決定できる。得られたアミノ酸配列を、解読されている全ゲノム配列中にコードされているあらゆるタンパク質の配列情報と照合し、前記低分子化合物と結合するタンパク質を特定することができる。ペプチドについても同様である。さらに、上記「3.生体分子と低分子化合物との結合部位の同定方法」を用いると、その結合部位を同定することもできる。他の生体分子、例えば核酸、糖又は脂質についても同様であり、ある試料中に含まれる種々の核酸、糖又は脂質について網羅的な質量情報がある場合に本発明に係る上記方法を実施すると、低分子化合物と結合する核酸、糖又は脂質の質量分析結果が得られ、これを前記網羅的な質量情報と照合して、低分子化合物と結合するものを特定することができる。以下、これらについて説明する。
本発明に係る方法は核酸に使用することもできる。例えばある細胞の種々の核酸分子の情報がある場合において、低分子化合物がどの核酸分子と結合するか決定したいときは、本発明に係る方法を実施して、前記低分子化合物と結合する核酸分子の質量情報を得ることができる。またMS/MS分析を行うことによって核酸を順次分解したものの質量スペクトルを得ることもでき、これを前述の質量情報と照合して前記低分子化合物がどの核酸分子と結合するか特定できる。また、結合した核酸を特定した後、本発明に係る方法を実施してその結合部位を決定することもできる。
本発明に係る方法は糖に使用することもできる。例えばある病原菌の複数の莢膜多糖類の構造が解明されている場合において、低分子化合物がどの莢膜多糖類と結合するか決定したいときは、本発明に係る方法を実施して、前記低分子化合物と結合する莢膜多糖類の質量情報を得ることができる。またMS/MS分析を行うことによって多糖類を順次分解したものの質量スペクトルを得ることもでき、これを前述の質量情報と照合して前記低分子化合物がどの莢膜多糖類と結合するか特定できる。また、結合した莢膜多糖類を特定した後、本発明に係る方法を実施してその結合部位を決定することもできる。
本発明に係る方法は脂質にも使用可能である。例として、細胞の脂質二重膜を構成する分子に関する網羅的な情報がある場合に、低分子化合物がどの脂質分子と結合するか決定したいときは、本発明に係る方法を実施して、前記低分子化合物と結合する脂質分子の質量スペクトルを得ることができる。これを前記脂質に関する網羅的な情報と照合して、前記低分子化合物がどの脂質分子と結合するか特定できる。また、結合した脂質分子を特定した後、本発明に係る方法を実施して、その結合部位を決定することもできる。
低分子化合物と結合した生体分子を含む溶液は、細胞に低分子化合物を取り込ませ、細胞内の生体分子と結合させ、該細胞を破砕することにより調製することができる。また、低分子化合物と結合した生体分子を含む溶液は、細胞を破砕し、次いで細胞破砕液に低分子化合物を加え、細胞内の生体分子と結合させることにより調製することもできる。
本発明に係る方法に用いる試料は、分離溶媒として低沸点極性溶媒及び水を用いる液体クロマトグラフにより分画したものであってもよい。低沸点極性溶媒は1.1.1に記載したとおりである。
本発明に係る方法に関し、ラマン分光分析しようとする画分は、そのままの液滴とし又は溶媒と混合した液滴とし、前記液滴を適当なプレート上に配列すること、及び前記液滴に含まれる溶媒を蒸発させることによりラマン分光分析に供するスポットを調製することができる。溶媒の蒸発によりスポットを調製すること、及びラマン分光分析をラマン顕微鏡にて行うことから、プレートは清浄化された面を有することが好ましい。プレートの面が清浄化されているとは、ラマン分光分析の支障となるような液体、固体汚染物、無機、有機不純物、指紋、ゴミ、曇り、キズなどが面の上にないことをいう。清浄化は、水、界面活性剤を含む水性洗浄剤、又は有機溶媒によりプレート表面を洗浄し、その後プレートを乾燥させることにより行うことができる。また、用いるプレートは好ましくは、撥水面を有する。プレートの撥水面が清浄化されていればさらに好ましい。撥水とは水をはじくことをいい、プレートの撥水面とは、プレートの水をはじく面をいう。撥水面は、例えばプレートにフッ素系撥水剤やシリコーン系撥水剤のような水より著しく表面張力の小さい撥水剤を塗布する処理により実現することができる。撥水面を有するプレートは金属、ガラス、石英、フッ化カルシウム又はフッ化マグネシウム製のプレートであってもよく、好ましくはラマン分光分析及び質量分析の結果に影響をほとんど又は全く及ぼさないものである。液滴のプレート上への配列は、マイクロピペットを用いて行うことができる。この操作は手動で又は自動化された装置により行うことができる。プレートとしては当技術分野で広く用いられている96ウェルプレートや384ウェルプレート等が挙げられるが、これらに限られない。プレート上への分画試料のスポッティングの例を図13に示す。図13左では、HPLCで分画した溶液を送液し、これをプレート上にスポッティングする。スポッティングの順番はあくまで一例である。図13右下に示すように、基板上の液滴が乾燥するに伴い、溶液中のペプチドが環状に凝集する。このリング部分をラマン顕微鏡で分析すると効率よく、高感度でラマンスペクトルを得ることができる。
ラマン分光分析及び質量分析は、市販のプレートを用いて行ってもよいが、ラマン顕微鏡の試料ステージに適合する顕微鏡用基板固定プレートを作製して用いることもできる。図14及び図15-1に本発明に係る装置に使用可能なプレートの例を示す。図14の左上には顕微鏡用基板固定プレートを示す。写真はマルチスポット金属基板を背面からみたものである。図14の左下はラマン顕微鏡の試料台を示す。図14の右は試料台に顕微鏡用基板固定プレートを装着した状態である。この状態でラマン分光分析を行う。目的のラマンピークを有するスポットを検出することをラマンスクリーニングともいう。
本発明において、試料を試料分離部の液体クロマトグラフ又はキャピラリー電気泳動装置により分画後、その試料を直接ラマン分光部に用いることが従来の技術にない特徴であり、これにより、特定の生体分子或いは断片を容易に検出又は分離し特定することができる。さらに、ラマン分光部は質量分析部と連結されているため、ラマン分光分析により特定された画分をそのまま後続する質量分析計により解析することができ、これにより生体分子の配列決定や低分子化合物との結合部位の同定が簡便にできる。配列決定とは、例えば生体分子がタンパク質又はペプチドの場合はアミノ酸配列の決定をいい、生体分子が多糖類の場合は多糖類を構成する糖の配列の決定をいう。
以上をまとめると、本発明に係る装置及び方法は、網羅的に生体分子を探索して低分子化合物と結合するものを特定し、又は生体分子と低分子化合物との結合部位の同定を行うことができる。本発明は化合物自身のラマンピーク、又はアルキニル基等特徴的なラマンピークを有するラマン標識により、低分子化合物を選択的に高感度で検出することができる。
液体クロマトグラフィーでの試料分離のためのトリフルオロ酢酸(TFA)は和光純薬工業株式会社から、0.1 %ギ酸を含有する蒸留水、及び0.1 %ギ酸(FA)を含有するアセトニトリル(MeCN)は関東化学株式会社から入手した。なお、セミマイクロHPLCのためのアセトニトリル及び蒸留水については、ナカライテスク社から入手した。
<液体クロマトグラフィー>
液体クロマトグラフィーの一例は次のとおりである。試料を用意し、UV検出器を備えたHPLC (Ultimate3000、 DIONEX)に注入し、フラクションコレクター(Probot、 DIONEX)で分画した。流速は50 μl/分とした。必要に応じて、分離溶媒に0.1 % TFAを含む蒸留水―アセトニトリル混合溶媒を用い、アセトニトリル濃度勾配をかけた。
測定条件の一例は次のとおりである。波長:532 nm、 レーザー強度: 30 mW、 露光時間: 30秒、対物倍率: 40倍、開口数:0.75、乾燥空気中、 照射: 点照射。レーザーの焦点は、ペプチドが高度に濃縮されているリング領域(乾燥・凝集して粉体の状態)に合わせる。各スポットについて5回反復してスペクトルを取得し、平均化して1つのスペクトルを得た。
MALDI質量スペクトルは、MALDIイオン源を備えたLTQ Orbitrap XL (Thermo Fisher Scientific社)を用いて取得した。サンプルは、α-シアノ-4-ヒドロキシ桂皮酸(CHCA)又は2,5-ジヒドロキシ安息香酸(DHB) (Bruker社)と混合した。MALDI質量スペクトルはFTモードにより取得した(分解能30,000又は60,000)。こうしたスペクトルは手動で取得した。パラメーターは次のとおりである。スキャン範囲: m/z 800-4000、 レーザーエネルギー(μJ): (CHCAについては)2-4又は(DHBについては)6-8。
本発明に係るLC-R-MSと比較するために、LC-MSを次の手順で行う。ナノLC-MS及びMS/MSは、ESIイオン源を備えたLTQ Orbitrap XL(Thermo Fisher Scientific)を用いて取得した。ナノHPLCシステム(Ultimate 3000, DIONEX), トラップカラム(ZORBAX 300SB C18 (内径0.3 × 5 mm), Agilent)及びtip column (NTCC-360、内径0.075 mm、日京テクノス)を用いた。移動相Aは、0.1%ギ酸、4%アセトニトリルを含む蒸留水であり、移動相Bは0.1%ギ酸を含むアセトニトリルであった。サンプルを0.1% TFA又は適当な濃度の n-デシル-β-D-グルコピラノシド (DG) (MP Biomedicals)で希釈し、200 nL/分の流速で移動相Bが0-80%/30分のグラジエント法を用いて溶出させた。ESI質量スペクトルはFTモードで取得し(分解能60,000)、MS/MSスペクトルはイオントラップモードで取得した。
ラマン標識を有する低分子化合物のラマン分光分析
アミノ酸配列がEQWPQCPTXK(配列番号4)である低分子量ペプチドにおいて、XがイソロイシンであるペプチドとXがプロパルギルグリシンであるペプチドを合成した。以下、本実施例において前者をペプチド1、後者をアルキンペプチド1と呼ぶ。ペプチド1は、固相合成(Fmoc)法により合成した。同様にアルキンペプチド1も固相合成法により合成した(いずれのペプチドも(独)理化学研究所脳科学総合研究センターにおいて合成)。プロパルギルグリシンは市販品を用いた。これらの構造を図6の上段に示す。
(S)-3-(2-((((4-エチニルベンジル)オキシ)カルボニル)アミノ)-3-フェニルプロパンアミド)-2-オキソプロピル2,6-ジメチルベンゾエート)(以下、RAT8-AOMKという)は、次の手順により調製した。
メチルエチル2-(2-((tert-ブトキシカルボニル)アミノ)-3-フェニルプロパンアミド)アセテート (2.0 g, 5.7 mmol)のTHF(29 ml)及びメタノール(29 ml)溶液に19 mlの10%水酸化ナトリウムを加え、10℃で10分間撹拌した。反応後7.5% 塩酸で中和し、ジクロロメタンで6回抽出した。減圧下に溶媒除去後THF (27 ml)溶液とし、N-メチルモルホリン(970 μl, 8.8 mmol)及びクロロギ酸イソブチル(1.05 ml, 8.1 mmol)を加え、10℃、30分間撹拌後、ジアゾメタン/ジエチルエーテルを加えた。3時間以上室温にて撹拌し、33% HBrの酢酸 (10.5 ml) 及び水 (10.5 ml)溶液を滴下し、0 ℃、10 分間撹拌した。反応を水飽和のNaHCO3及び酢酸エチルで停止し、酢酸エチルで2回抽出し、硫酸マグネシウムで乾燥した。有機層を減圧下で濃縮後、酢酸エチル/ヘキサン(2/1)溶媒を用いてシリカゲルカラムで精製し、無色、非結晶状のブロモメチルケトン(1.55 g、 57%)を得た。
1H-NMR (400 MHz, CDCl3) δ: 7.31-7.19 (6H, m), 7.04 (2H, d, J = 7.7 Hz), 6.83 (1H, brs), 5.11 (1H, d, J = 7.8 Hz), 4.88 (2H, s), 4.47 (1H, brs), 4.21 (2H, m), 3.10 (2H, m), 2.37 (6H, s), 1.39 (9H, s)
13C-NMR (100 MHz, CDCl3) δ: 198.6, 171.8, 168.9, 155.4, 136.4, 135.5, 132.1, 129.8, 129.2, 128.6, 127.7, 127.0, 80.3, 66.6,
55.6, 46.6, 38.2, 28.2, 19.8
MS (ESI) m/z値: 491 [(M+Na)+]
HRMS (ESI) 計算値 C26H32N2O6Na: 491.2153(実測値: 491.2165)。
N-Boc-AOMK (83 mg、177 μmol)のジクロロメタン (0.75 ml)溶液にトリフルオロ酢酸を加え30分間撹拌した。溶媒を減圧除去し、THF(0.74 ml)溶液として4-ニトロフェニル-4-エチニルベンジルカルボネート(35 mg、118 μmol)、 N,N-ジイソプロピルエチルアミン(160 μl、1.6 mmol)及び4-ジメチルアミノピリジン(14 mg、0.12 mmol)を加えて室温で2時間撹拌した。反応後、水及び酢酸エチルを加え、水層と有機層を分離し、水層を2回酢酸エチルで抽出し、硫酸マグネシウムで乾燥した。有機層を減圧下で濃縮後、シリカゲルカラムで精製した。続いてゲルろ過し、無色、非晶質のRAT8-AOMK (34.7 mg、 56 %)を得た。
1H-NMR (400 MHz, CDCl3) δ: 7.43 (2H, d, J = 8.3 Hz), 7.28-7.16 (8H,m), 7.04 (2H, d, J = 7.8 Hz), 6.75 (1H, brs), 5.50 (1H, brd, J = 7.2 Hz), 5.06 (1H, d, J = 12.7 Hz), 5.00 (1H, d, J = 12.7 Hz), 4.86(2H, s), 4.53 (1H, m), 4.17 (2H, m), 3.09 (2H, m), 3.09 (1H, s), 2.36 (6H, s)
13C-NMR (100 MHz, CDCl3) δ: 198.6, 171.3,169.0, 155.8, 136.8, 136.1, 135.6, 132.2, 132.1, 129.9, 129.2, 128.7, 127.7, 127.7, 127.1, 121.9, 83.2, 77.6, 66.6, 66.5, 56.0, 46.5,38.4, 19.9
MS (ESI) m/z値: 549 [(M+Na)+]
HRMS (ESI) 計算値C31H30N2O6Na: 549.1996(実測値: 549.2012)。
RAT8-AOMK標識されたカテプシンBを含む試料ロットFL-S10は次の手順で調製した。カテプシンB (6 μg, 約200 pmol, CALBIOCHEM カタログ番号219362)を300 μlの標識バッファー(50 mM酢酸(pH 5.6), 5 mM MgCl2, 2 mM ジチオトレイトール(DTT))中に溶解させた。これを室温で15分静置した後、3.0μlのジメチルスルホキシド(DMSO)に溶解した2 mM RAT8-AOMKを3 μl加えた。この混合物を37℃で3時間インキュベートした後、タンパク質(カテプシンB)をTCA沈殿法により沈殿させた。得られた沈殿を20 μlの変性バッファー(7 M グアニジン塩酸塩(GuHCl)), 1M Tris-HCl (pH 8.5))に溶解させ、37℃で1時間インキュベートした。DTT及びヨードアセトアミド(IAA)による還元とアルキル化の後、1.5 μlのトリプシン(100 ng/μl)を試料に添加し、37℃で数時間インキュベートした。ロットFL-S10を下記で、最終サンプルとよぶことがある。最終サンプルの9/10をスポッティング実験に使用した。
調製したRAT8-AOMK試料そのもの、及びRAT8-AOMK標識カテプシンBを含む試料そのもののラマンスペクトルを測定した。
<ナノLC-Probot>
上記の方法で調製した試料ロットFL-S10を凍結乾燥させ、26 μlの水に溶解させた。試料26 μl中の25 μlをUV検出器 (MU701、 GL science)を備えたナノLC(NanoFrontier nLC、 Hitachi社製)に注入し分画した。流速は250 nL/分とした。画分はフラクションコレクター(Probot、 Dionnex)を用いて20 秒/スポットというスポッティング速度でMALDI用プレート(ITOP plate, Thermo)にスポッティングした。UVクロマトグラムの結果を図20のAに示す。図20のCは画分をスポッティングした順番を示す。図20Aに示されているとおり画分番号35~75にUV吸収が観察された。この範囲について、次にラマン分光分析及び質量分析を以下の手順で行った。
濃縮RAT8-AOMK標識カテプシンB(ロットFL-S10)を含む溶液をナノLC-UV-probotによりMALDI用プレートにスポッティングした(250 nl/分、20 秒/ウェル、約200 pmol)。図27にMALDI用プレートにスポッティングした画分番号1~94の明視野像を示す。スポットでは乾燥工程により溶媒を蒸発させ、試料を濃縮した。この濃縮されたRAT8-AOMK標識カテプシンBを含む試料ロットFL-S10をラマン分光法により測定した。複数のスポットからアルキンシグナルが検出されたので、MALDI-Orbitrap装置を用いて同じ標的プレートから質量スペクトルを手動で取得した。
ラマン分光分析は、実施例5に記載のように行った。結果は図21及び23に示す。
マトリクス: DHB
質量範囲: m/z800-4,000
質量スペクトル取得モード: フーリエ変換(FT)、分解能30,000、レーザーエネルギー 5~8μJ
<LCによる試料の分離及びUVでの検出>
ナノLC-UV-probotを用いてRAT8-AOMK標識カテプシンBの断片を含む試料を分画した。ナノLCに注入した試料のUVクロマトグラム結果を図20Aに示す。便宜上、保持時間30分~60分の領域を拡大して示す。UV検出は214 nmにて行った。使用した溶媒は水-アセトニトリルであった。この実験ではアセトニトリル濃度(勾配)を60分で5%から80%へと線形に増大させた。カテプシンBの出発量は200 pmol (3 μg)であった。最終サンプル(ロットFL-S10)の9/10をナノLCに注入した。これらの画分をMALDI用プレートにスポッティングした。LCでの溶出時間が30分である時点から開始して、20秒間隔で画分を回収し、これらの画分をスポッティングした。合計の画分数は94であった。図20AではUVクロマトグラムでの保持時間と画分番号の関係を示す。その下にアルキン(2107 cm-1)のピーク強度をプロットしたラマンスペクトルを対応して示す(図20B)。画分番号57-66についてアルキンシグナルが得られ、RAT8-AOMK標識されたペプチドの存在が示された。
ラマン分光はラマン顕微分光装置(ナノフォトン株式会社、Raman-11)を用いて行った。レーザー光源は波長532 nmのレーザーを用いた。レーザーの強度は、対物レンズ透過後の試料面で30 mWであり、露光時間は30秒であった。倍率40倍, 開口数0.75の対物レンズを用いた。レーザーの照明パターンはポイント照明を選択した。710~3100 cm-1の波数範囲のラマンスペクトルを取得した。銀ナノ粒子は使用しなかった。
画分番号56-60 [57]: ペプチドA-2、DQGSCGSCWAFGAVEAISDR (+RAT8-AOMK)
画分番号59-67 [60]: ペプチドB-1、EIRDQGSCGSCWAFGAVEAISDR (+RAT8-AOMK, +カルバミドメチル)
画分番号61-69 [62]: ペプチドA-1、DQGSCGSCWAFGAVEAISDR (+RAT8-AOMK, +カルバミドメチル)
([]内の番号は最も強いイオンピークが観察された画分である)。
ペプチドA, 切断ミス:なし
DQGSCGSCWAFGAVEAISDR; C85 H127N25 O31 S2
*モノアイソトピック質量2057.857 Da, 平均質量2059.197 Da
(* モノアイソトピック質量は対象分子を構成する各元素の主同位体のみに基づく質量をいう。)
ペプチドB, 切断ミス:1ヶ所
EIRDQGSCGSCWAFGAVEAISDR; C102 H157N31 O36 S2
モノアイソトピック質量2456.085 Da, 平均質量2457.654 Da
<各修飾による増加>
カルバミドメチル(Cys)
モノアイソトピック質量57.021464 Da, 平均質量57.0513 Da
組成H3 C2 N O
RAT8-AOMK (Cys)
モノアイソトピック質量376.142307 Da, 平均質量376.4052 Da
組成C22 H20 N2O4
<計算されたm/z>
ペプチドA系列
ペプチドA-1
DQGSCGSCWAFGAVEAISDR ; C109 H150 N28 O36 S2
カルバミドメチル(Cys); RAT8-AOMK (Cys)
モノアイソトピックm/z 2492.0282
ペプチドA-2
DQGSCGSCWAFGAVEAISDR ; C107 H147 N27 O35 S2
RAT8-AOMK (Cys)
モノアイソトピックm/z 2435.0067
ペプチドB系列
ペプチドB-1
EIRDQGSCGSCWAFGAVEAISDR; C126 H180N34 O41 S2
カルバミドメチル (Cys); RAT8-AOMK (Cys)
モノアイソトピックm/z 2890.2559。
実施例6で示した濃縮RAT8-AOMK標識カテプシンBと同様の実験条件でMALDI用プレートにスポッティングした試料をラマン分光法により測定し、アルキンシグナルが検出された画分に対して、MALDI-Orbitrap装置を用いてMS/MS分析を行った。図33は、ペプチドA-1 (DQGSCGSCWAFGAVEAISDR +カルバミドメチル+RAT8-AOMK)が検出された画分から得られたMS/MSスペクトルを示す。得られたフラグメントイオンを解析したところ、ペプチドのC末端から数えて12番目トリプトファン残基のN末端で開裂したC末端側フラグメントイオン (y12)と、13番目システイン残基のN末端側で開裂したC末端側フラグメントイオン(y13)の間に、RAT8-AOMKの質量に相当する376.1 Daとシステイン残基に相当する103.0 Daの和から、脱水による18.0 Daを引いた質量数の差が見られることが分かった。一方、C末端から数えて15番目グリシン残基のN末端で開裂したC末端側フラグメントイオン(y15)と、16番目システイン残基のN末端側で開裂したC末端側フラグメントイオン(y16)の間に、カルバミドメチルの質量に相当する57.0 Daとシステインに相当する103.0 Daの和に相当する質量数の差が見られることが分かった。上記の結果から、ペプチド DQGSCGSCWAFGAVEAISDRに含まれる2つのシステイン残基のうち、C末端から数えて13番目のシステイン残基が、RAT8-AOMKによる修飾を受けていることが決定された。
<MALDI MS/MS分析>
マトリクス: CHCA
質量範囲: m/z200-3,000
質量スペクトル取得モード:フーリエ変換(FT)、分解能15,000、レーザーエネルギー 5~8 μJ
MS/MS方式: HCD (higher energy collision dissociation)
<MS/MS分析に供するペプチド>
ペプチドA-1
[実施例8] 金属基板及び石英基板
本発明者らは、スポットされた試料のラマン分光及び質量分析をスムーズに行うプレートを開発した。図14にスポットされた試料のラマン分光及び質量分析をスムーズに行うプレートを示す。図14の左上には顕微鏡用基板固定プレートを示す。写真はマルチスポット金属基板を背面からみたものである。図14の左下はラマン顕微鏡の試料台を示す。図14の右は試料台に顕微鏡用基板固定プレートを装着した状態である。この状態でラマンスクリーニング(ラマン分光分析)を行う。マルチスポット金属基板は、MALDI用金属プレート(Thermo社製)を、表面の清浄化を確認し用いた。
直径50 nmの金ナノ粒子分散液(EMGC50, BBI)をガラス基板に滴下し、乾燥させた。乾燥した金ナノ粒子凝集体の上に、DMSOに溶解した6mM RAT8-AOMKを1 μl滴下した。比較のために、金ナノ粒子のないガラス基板上にも、同様にDMSOに溶解した6mM RAT8-AOMKを1 μl滴下した。それぞれの液滴に対して、ラマン測定を行った。ラマン分光は、ラマン顕微分光装置(ナノフォトン株式会社, Raman-11)を用いた。レーザー光源は波長660nmのレーザーを用いた。レーザーの照明パターンはライン照明を選択した。レーザーの強度は、対物レンズ透過後の試料面で3.5 mWであり、露光時間は10秒であった。対物レンズには開口数0.75、倍率40倍の対物レンズを用いた。ラインに沿って得られた400点のラマンスペクトルを平均して、それぞれの液滴に対するスペクトルとした。1250~2400cm-1の波数範囲のラマンスペクトルを取得した。結果を図34に示す。金ナノ粒子の使用によりラマンシグナルの増強が確認された。
直径40 nmの銀ナノ粒子分散液(EMSC50, BBI) 15 μlと、アルキンペプチド1を10 pmol溶解した水15 μlとを混合し、ガラスボトムウェル(EzView 384ウェル ガラス底アッセイプレート、AGCテクノグラス)の内の1区画に注入した。同様に、銀ナノ粒子と非標識のペプチド1を10pmol溶解した溶液も、同一ウェルプレートの異なる区画に注入した。ウェルプレートにテープで蓋をして、冷蔵庫(4℃)で1日保存した後、ラマン測定を行った。ラマン分光は、ラマン顕微分光装置(ナノフォトン株式会社, Raman-11)を用いた。レーザー光源は波長532nmのレーザーを用いた。レーザーの照明パターンはライン照明を選択した。レーザーの強度は、対物レンズ透過後の試料面で240 mWであり、露光時間は1秒/ラインで、1サンプルにつき25ライン測定した。対物レンズには開口数0.75、倍率40倍の対物レンズを用いた。ラインに沿って得られた1 line 400点× 25 lineの10000個のラマンスペクトルを平均して、それぞれの溶液に対して、710~3100cm-1の波数範囲のラマンスペクトルを取得した。結果を図35に示す。
本発明のラマン分光法を用いた場合と、従来法のクリック反応を介して蛍光団を導入する場合の比較
試料調製
カテプシンB (10 μg, CALBIOCHEM, カタログ番号219362)を100 μlのBogyoバッファー(50 mM酢酸(pH5.6)、5 mM MgCl2、2 mM DTT)に溶解させた。これを室温で15分静置し、1.0 μlのDMSO中20 mM RAT8-AOMKと混合した。この混合物を37℃で3時間インキュベート後、該タンパク質を氷上で3時間インキュベートしてTCA沈降により沈降させた。沈殿物を取得するために、20000 G、20分の遠心分離を行った。上清を除いた後、1 mlのアセトンを加え、遠心分離を20000 Gにて15分行った。遠心分離とアセトン処理は3回反復して行った。真空下で30秒にわたりアセトンを除去した後、該沈殿物を10μlの変性バッファー(7M GuHCl, 1M Tris-HCl (pH 8.5))に溶解させた。
上記のように調製した100 μlのペプチド混合物を凍結乾燥させ、50 μlの水に溶解させた。50 μl溶液をナノ-LCシステム(nanoFrontier, Hitachi)に注入した。実験条件は、クリック反応無しでのラマン分光分析については、250 nl/分の流速、20 s/spot (Probot, Dionnex)で、384ウェルガラス底プレート (EzView 384 ウェルガラス底アッセイプレート、AGCテクノグラス)での測定というものであり、クリック反応有りでの蛍光分析については、250 nl/分の流速、20 s/spot (Probot, Dionnex)で、384ウェル撥水性MALDIプレート(ITOP, Thermo)での測定というものであった。1画分当たり1.5 μlの水をプロボット(probot)のサイドポートから加えることで、ガラスウェルプレート上の水滴が安定して分配されるよう補助した。UVクロマトグラムはUV検出器(MU701, GL science)を215 nmにて使用して取得した。勾配は、0分 5 %、60分 80 %、60.01分 95 %、75分 95 %、75.01分 0 %、90分 0 %というものであった。合計画分数は192であった。図中には20~85 分での保持時間を示した。図36上段に示すUVクロマトグラムにおけるピーク高を比較するために、41~57.5 分の保持時間のチャートを図36下段に拡大した。図36の下段に示されるように、比較例としてクリック反応を用いた場合(2)は、クリック反応を用いない本発明の場合(1)と比較して、57.5~74.2 %のサンプル喪失が観察された。
384ウェルガラス底プレート上の液滴が乾燥するのを待って、ラマン測定を行った。ラマン分光はラマン顕微分光装置(ナノフォトン株式会社, Raman-11)を用いた。レーザー光源は波長532 nmのレーザーを用いた。レーザーの照明パターンはポイント照明を選択した。レーザーの強度は、対物レンズ透過後の試料面で180 mWであり、露光時間は30秒であった。対物レンズには開口数0.75, 倍率40倍の対物レンズを用いた。1サンプルにつき5回、ペプチド凝集体上の異なる位置でスペクトルを取得し、その平均をとった。192ウェル全てについて同様の測定をおこなった。710~3100 cm-1の波数範囲のラマンスペクトルを取得した。得られたスペクトルに対して移動平均によるスムージングをかけた。アルキン由来のラマンピークのピークトップ2108 cm-1から、ピーク底部の2091 cm-1の値を引き算して、各ウェルにおけるアルキンのラマン強度を算出し、ラマンクロマトグラムとして強度プロファイルを作成した。
384ウェル撥水性 MALDIプレート上の液滴が乾燥するのを待って、蛍光測定を行った。蛍光測定は、蛍光イメージャー(Pharos FX, Biorad)を用いて行った。励起波長は488 nmを選択した。分解能は50 μmを選択した。得られた蛍光画像において、各スポット位置における蛍光強度の最大値を192点算出し、蛍光クロマトグラムとして強度プロファイルを作成した。
15 μlの40 nm銀ナノ粒子(EMSC40、ブリティシュ バイオセル インターナショナル製)を所定濃度にした15 μlのアルキン標識ペプチド(アルキンペプチド1; EQWPQCPTXK; X=プロパギルグリシン)/0.3 %TFA水溶液に加え、4℃で1日放置した。この試料を用いてSERS測定を行った。
実施例3と基本的に同様の操作でRAT8-AOMKによるカテプシンBの標識試料を調製し、トリプシン消化した。以下に示す手順に従って、ナノLC-UV-probotを用いてRAT8-AOMK標識カテプシンBの断片を含む試料をTFA添加したウェルに分画した後、銀ナノ粒子と混合、凝集しSERS測定を行った。
流速: 250 nl/min
分画: 384ウェルガラスガラス底プレート (EzView 384 ウェルガラス底アッセイプレート、AGCテクノグラス)
上、スポットあたり20秒
UVクロマトグラム: 215 nm
濃度勾配: 0分 5 %、60分 80 %/60.01分 95 %、75分 95 %/ 75.01分 0 %、90分 0 %
分画: ウェルあたり20秒
予め25 μlの0.1 %TFA水溶液を含むガラスボトムウェルプレートに試料を分画した。分画された試料は、それぞれSERS用に15 μl、質量分析用に10 μl分別した。SERS用試料には15 μlの40 nm銀ナノ粒子を加え、4℃で1日放置後SERS測定を行った。
2:送液ライン
3:分画部
4:送液ライン
5:検出部
6:レーザー部
7:ミラー
8:対物レンズ
9:試料
10:試料台
11:分光器
12:検出部
13:試料ステージ
14:試料
15:レーザー部
16:加速電極
17:検出部
18:信号処理部
配列番号2:ペプチドA-2
配列番号3:ペプチドB-1
配列番号4:ペプチド1
配列番号5:非標識のペプチド断片
本明細書で引用した全ての刊行物、特許および特許出願をそのまま参考として本明細書にとり入れるものとする。
Claims (39)
- 低分子化合物と結合する生体分子を特定するため、又は、低分子化合物と生体分子との結合部位を同定するための装置であって、試料分離部、ラマン分光部及び質量分析部を有し、該試料分離部、ラマン分光部及び質量分析部がこの順序で連結されている前記装置。
- 試料分離部が液体クロマトグラフ又はキャピラリー電気泳動装置である、請求項1記載の装置。
- 液体クロマトグラフが、順相、逆相、分子ふるい、及びイオン交換クロマトグラフからなる群より選択されるいずれか1種の高速液体クロマトグラフである、請求項2記載の装置。
- ラマン分光部が、ラマン励起用レーザー光を照射するレーザー部及びラマン散乱光をスペクトル解析するスペクトル解析部を有する線形又は非線形ラマン分光装置である、請求項1~3のいずれか1項記載の装置。
- 質量分析部が、イオン化方式としてマトリックス支援レーザー脱離イオン化法、エレクトロスプレーイオン化法又は大気圧化学イオン化法を用いる質量分析計を含む、請求項1~4のいずれか1項記載の装置。
- 低分子化合物が、生体分子と識別可能なラマンピークを与える、請求項1~5のいずれか1項記載の装置。
- 低分子化合物が、分子内にラマンスペクトルのサイレント領域に散乱スペクトルを有するアルキニル基、ニトリル基、ジアゾニオ基、イソシアン酸エステル基、イソニトリル基、ケテン基、カルボジイミド基、チオシアン酸エステル基、アジド基、ジアゾ基、アルキンジイル基及び重水素からなる群より選択される少なくとも1種の置換基を含む、請求項1~6のいずれか1項記載の装置。
- 生体分子が、タンパク質、ペプチド、核酸、糖及び脂質からなる群より選択される少なくとも1種の生体分子である、請求項1~7のいずれか1項記載の装置。
- 請求項1に記載の装置に用いるための、清浄化された面を有するプレート。
- 清浄化された面が撥水面を含む、請求項9に記載のプレート。
- 金属、ガラス、石英、フッ化カルシウム、又はフッ化マグネシウム製である、請求項9または10記載のプレート。
- 生体分子と低分子化合物との結合部位を同定する方法であって、以下の工程、
(1)分画された、低分子化合物と結合した生体分子の断片をラマン分光分析に供すること、及び
(2)ラマン分光分析に供した画分の全部又は一部を質量分析に供すること、
を含み、
ラマン分光分析により生体分子断片と結合した低分子化合物由来のラマンピークを有する画分を検出し、低分子化合物由来ラマンピークを有する画分の質量分析結果を取得し、これを生体分子の質量情報と照合して、生体分子内の前記低分子化合物の結合部位を同定する前記方法。 - 低分子化合物と結合した生体分子を断片化し、分画することにより、前記分画された、低分子化合物と結合した生体分子断片を用意する、請求項12記載の方法。
- 前記低分子化合物と結合した生体分子が、無細胞条件下で低分子化合物と生体分子とを混合することにより得られたものである、請求項12又は13記載の方法。
- タンパク質分解酵素、ペプチド分解酵素、核酸分解酵素、糖分解酵素及び脂質分解酵素からなる群より選択される酵素又は化学的分解により生体分子を断片化する、請求項13記載の方法。
- 低分子化合物と結合する生体分子を特定するスクリーニング方法であって、以下の工程、
(1)低分子化合物と結合した生体分子を含む画分をラマン分光分析に供すること、及び
(2)ラマン分光分析に供した画分の全部又は一部を質量分析に供すること、
を含み、
ラマン分光分析により低分子化合物由来のラマンピークを有する画分を検出し、低分子化合物に由来するラマンピークを有する画分の質量分析結果を取得し、これを生体分子の質量情報と照合して、前記低分子化合物に結合する生体分子を特定する前記方法。 - 低分子化合物と結合した生体分子を含む試料を分画して、前記低分子化合物と結合した生体分子を含む画分を用意する、請求項16記載の方法。
- 前記低分子化合物と結合した生体分子を含む試料が、(A)細胞に低分子化合物を取り込ませ、細胞内の生体分子と結合させ、該細胞を破砕することにより、又は(B)細胞を破砕し、細胞破砕液に低分子化合物を加え、細胞内の生体分子と結合させることにより、調製されたものである、請求項17記載の方法。
- 低分子化合物が、生体分子と識別可能なラマンピークを与える、請求項12~18のいずれか1項記載の方法。
- 低分子化合物が、分子内にラマンスペクトルのサイレント領域に散乱スペクトルを有するアルキニル基、ニトリル基、ジアゾニオ基、イソシアン酸エステル基、イソニトリル基、ケテン基、カルボジイミド基、チオシアン酸エステル基、アジド基、ジアゾ基、アルキンジイル基及び重水素からなる群より選択される少なくとも1種の置換基を含む、請求項12~19のいずれか1項記載の方法。
- 生体分子が、タンパク質、ペプチド、核酸、糖及び脂質からなる群より選択される少なくとも1種の生体分子である、請求項12~20のいずれか1項記載の方法。
- 分画を液体クロマトグラフ又はキャピラリー電気泳動により行う、請求項13又は17記載の方法。
- 分画された前記画分を、そのままの液滴とし又は溶媒と混合した液滴とし、前記液滴を清浄化された面を有するプレート上に配列すること、及び前記液滴に含まれる溶媒を蒸発させることによりラマン分光分析に供するスポットを調製することを含む、請求項12~22のいずれか1項記載の方法。
- プレートの前記清浄化された面が撥水面を含む、請求項23に記載の方法。
- プレートが金属、ガラス、石英、フッ化カルシウム、又はフッ化マグネシウム製である、請求項23又は24記載の方法。
- プレートに金、銀、プラチナ、パラジウム、アルミニウム、チタン及び銅からなる群より選択される金属ナノ粒子又は金属ナノ構造を用いる、請求項23~25のいずれか1項記載の方法。
- 分画された画分を、金属ナノ粒子又は金属ナノ構造を含む溶液と混合し、そのままラマン分光分析に供する請求項23に記載の方法。
- 分画された画分に、金属ナノ粒子又は金属ナノ構造と生体分子及び低分子化合物が結合した生体分子との均一な凝集体の形成を促進させる有機酸を添加する請求項26及び27の方法。
- 有機酸が、トリフルオロ酢酸、ジフルオロ酢酸、モノフルオロ酢酸、トリフルオロメタンスルホン酸、ジフルオロメタンスルホン酸、3,3,3-トリフルオロプロピオン酸、トリクロロ酢酸、ジクロロ酢酸、モノクロロ酢酸、トリクロロメタンスルホン酸、ジクロロメタンスルホン酸、3,3,3-トリクロロプロピオン酸、ギ酸、酢酸、プロピオン酸、メタンスルホン酸、及びこれらの組合せからなる群より選ばれる、請求項28の方法。
- 生体分子に結合する低分子化合物が、分子内にラマンスペクトルのサイレント領域に散乱スペクトルを有するアルキニル基、ニトリル基、ジアゾニオ基、イソシアン酸エステル基、イソニトリル基、ケテン基、カルボジイミド基、チオシアン酸エステル基、アジド基、ジアゾ基、アルキンジイル基及び重水素からなる群より選択される少なくとも1種の置換基を含む、請求項26~29のいずれか記載の方法。
- (1)目的分子と有機酸とを含む溶液に金属ナノ粒子又は金属ナノ構造を添加し、形成される目的分子と金属ナノ粒子又は金属ナノ構造との複合体を凝集させる工程、及び
(2)前記凝集体について表面増強ラマン分光(SERS)分析を行う工程、
を含む表面増強ラマン分光分析方法。 - (1)有機酸を含む溶液に金属ナノ粒子又は金属ナノ構造を添加して金属ナノ粒子又は金属ナノ構造を凝集させる工程、
(2)前記凝集体に、目的分子を含む溶液を添加する工程、
(3)工程(2)により得られた金属ナノ粒子又は金属ナノ構造と目的分子との複合体について表面増強ラマン分光(SERS)分析を行う工程、
を含む表面増強ラマン分光分析方法。 - 目的分子が、生体分子、生体分子の断片、サイレント領域にラマンピークを有する低分子化合物と結合した生体分子、又はサイレント領域にラマンピークを有する低分子化合物と結合した生体分子断片である、請求項31又は32に記載の方法。
- 該生体分子はタンパク質、ペプチド、核酸、糖及び脂質からなる群より選択される少なくとも1種の生体分子である、請求項33に記載の方法。
- 有機酸が、トリフルオロ酢酸、ジフルオロ酢酸、モノフルオロ酢酸、トリフルオロメタンスルホン酸、ジフルオロメタンスルホン酸、メタンスルホン酸、3,3,3-トリフルオロプロピオン酸、トリクロロ酢酸、ジクロロ酢酸、モノクロロ酢酸、トリクロロメタンスルホン酸、ジクロロメタンスルホン酸、3,3,3-トリクロロプロピオン酸、ギ酸、酢酸、プロピオン酸、メタンスルホン酸、及びこれらの組合せからなる群より選ばれる、請求項31~34のいずれか1項の方法。
- 生体分子に結合する低分子化合物が、分子内にラマンスペクトルのサイレント領域に散乱スペクトルを有するアルキニル基、ニトリル基、ジアゾニオ基、イソシアン酸エステル基、イソニトリル基、ケテン基、カルボジイミド基、チオシアン酸エステル基、アジド基、ジアゾ基、アルキンジイル基及び重水素からなる群より選択される少なくとも1種の置換基を含む、請求項33~35のいずれか1項に記載の方法。
- 目的分子を含む溶液が、液体クロマトグラフ又はキャピラリー電気泳動により分画された画分である、請求項31~36のいずれか1項に記載の方法。
- 表面増強ラマン分光(SERS)分析を行う前に、凝集体を含む溶液の液滴を、清浄化された面を有するプレート上に配列すること、及び前記液滴に含まれる溶媒を蒸発させることにより表面増強ラマン分光分析に供するスポットを調製することを含む、請求項31~37のいずれか1項に記載の方法。
- 請求項31~38のいずれか1項に記載の表面増強ラマン分光(SERS)分析法に供した溶液又は画分の全部又は一部を、さらに質量分析に供することを含む分析方法。
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CA2882003A1 (en) | 2014-02-20 |
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US10338078B2 (en) | 2019-07-02 |
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