JP4644263B2 - Solid support and method for mass spectrometry by desorption / ionization of a plurality of substances or complexes on the solid support - Google Patents

Description

  The present invention relates to a method for analysis and analysis by transferring biomolecules such as nucleic acids and proteins separated in a gel to a solid support and performing rapid mass spectrometry.

  Many biomolecules such as peptides, proteins, nucleic acids and sugar chains are formed by polymerizing a relatively small number of structural units according to a certain rule. For example, peptides and proteins are molecules in which 20 types of L-α-amino acids are linked by peptide bonds. Most of the molecular structures of these structural units have already been clarified, and naturally their exact molecular weights have also been clarified. Therefore, if the molecular weight of biomolecules or fragments thereof can be accurately measured, mass spectrometry can greatly contribute to the analysis of the structure (sequence, etc.) and various modification reactions received in vivo. It is positioned as an indispensable means for molecular structure analysis. Among mass spectrometry, a laser desorption / ionization-time-of-flight mass spectrometer has attracted attention as a useful means for analyzing biomolecules because it can ionize macromolecules such as DNA and proteins.

  In a laser desorption / ionization-time-of-flight mass spectrometer, a sample site to be analyzed is irradiated with a laser, and ions desorbed therefrom are accelerated by an electric field.

  Then, ions having a smaller m / z value, that is, lighter ions fly at a higher speed and arrive at the detector. Laser desorption / ionization-time-of-flight mass spectrometry is a method of performing mass analysis by utilizing the fact that the time of flight of ions varies depending on such a difference in mass-to-charge ratio (m / z value).

  On the other hand, in the structural analysis / determination of biomolecules such as DNA and protein by mass spectrometry, it is necessary to separate and purify the analysis target into a number of components, and further analyze the individual components fragmented with restriction enzymes A very large number of samples must be analyzed. In DNA diagnosis, it is necessary to rapidly process samples obtained from a large number of people.

  On the other hand, a general laser desorption / ionization-time-of-flight mass spectrometer commercially available places each purified sample on a sample board and performs mass analysis one by one. In other words, it was necessary to irradiate each sampled sample with laser and analyze it one by one. Therefore, when an unpurified sample is separated by electrophoresis, it is necessary to cut out the gel after electrophoresis for each band and purify each, and then perform mass analysis using a laser desorption / ionization-time-of-flight mass spectrometer. It was very difficult to analyze a large number of samples quickly.

  In addition, a method is also known in which electrophoretic biomolecules are transferred from a gel onto a membrane such as nitrocellulose and analyzed. However, mass spectrometry using a laser cannot be performed for analysis on the membrane. And fluorescence detection using antigen-antibody reaction or nucleic acid hybridization. This is because conventionally used membranes such as nitrocellulose and PVDF are highly likely to decompose when irradiated with a laser. That is, it is difficult to analyze the biomolecules transferred on these membranes as they are with the above-described laser desorption / ionization-time-of-flight mass spectrometer.

  An object of the present invention is to provide a means for rapidly mass-analyzing a large number of samples, and to provide a method for rapidly analyzing biomolecules such as nucleic acids and proteins.

  As a result of intensive studies to solve the above problems, the present inventors have separated a substance in a sample by gel electrophoresis, and then separated and developed the substance in the gel, and a solid support having a carbon layer formed on the surface. It has been found that the above problem can be solved by a method of mass spectrometry by transferring it onto and desorbing / ionizing it, and the present invention has been completed.

That is, the present invention includes the following inventions.
(1) A solid support having a carbon layer on the surface, wherein a substance in a sample is separated by gel electrophoresis, and then the substance separated in the gel is transferred and held.
(2) After the substance in the sample is separated by gel electrophoresis, the substance separated in the gel is transferred to the membrane, and the substance transferred on the membrane is further transferred and held to immobilize the substance. A solid support having a carbon layer on the surface.
(3) A solid support obtained by adding another substance that interacts with the substance immobilized on the solid support according to (1) or (2) to form a complex.
(4) The solid support according to (1) or (2), wherein a complex of substances interacting in a solution is formed, and the complex is separated by gel electrophoresis.
(5) The solid support according to any one of (1) to (4), wherein the carbon layer is a diamond-like carbon layer.
(6) The solid support according to any one of (1) to (5), wherein the thickness of the carbon layer is a monomolecular layer to 100 μm.
(7) The solid support according to any one of (1) to (6), further comprising an amino group-containing compound that is present on the carbon layer on the substrate surface but is not covalently bonded to the carbon layer. .
(8) The solid support according to any one of (1) to (6), wherein the solid support further comprises an amino group-containing compound that exists on the carbon layer on the substrate surface and is covalently bonded to the carbon layer.
(9) The solid support according to any one of (1) to (6), obtained by vapor-depositing a compound having an unsubstituted or monosubstituted amino group and a carbon compound on a substrate.
(10) The solid support having a carbon layer on the surface thereof is obtained by immersing in a solution containing a compound having an unsubstituted or monosubstituted amino group, according to any one of (1) to (6) Solid support.
(11) The solid support according to any one of (1) to (10), wherein the transferred and retained substance is a nucleic acid, a peptide, or a complex thereof.
(12) A method of mass spectrometry by desorption / ionization of a plurality of substances or complexes transferred and held on the solid support according to any one of (1) to (11).
(13) A solid support having a carbon layer on its surface for use in the method according to (12).

  According to the method of the present invention, after a large number of substances contained in a sample are separated by electrophoresis, substances contained in individual bands can be transferred and held on a solid support without purifying a plurality of substances. Since mass spectrometry can be performed simultaneously and directly, a large number of samples can be analyzed quickly.

  Therefore, the present invention is a very useful means in the analysis of biomolecules such as nucleic acids and proteins.

  In the present invention, a sample is separated by gel electrophoresis, and the analyte after separation and development in the gel is supported by bringing the gel after electrophoresis into close contact with a solid support having a carbon layer formed on the surface. Transfer on the body. A plurality of substances are subjected to mass spectrometry by desorption / ionization of the substances transferred and held on the solid support.

  In the present invention, substances that can be transferred and retained on a solid support and analyzed are not particularly limited, and examples thereof include nucleic acids such as DNA and RNA, and biomolecules such as peptides. In the present specification, the peptide includes oligopeptides, polypeptides, and proteins. In particular, it is advantageous in that a high molecular weight substance can be analyzed. Samples to be subjected to gel electrophoresis containing these substances are not particularly limited, but include cell extracts, bacterial cell extracts, cell-free synthetic products, PCR (Polymerase chain reaction) products, enzyme-treated products, synthetic DNAs , Synthetic RNA, synthetic peptide and the like.

  The solid support for transferring and holding substances such as biomolecules separated in gel by electrophoresis is not particularly limited as long as it has a carbon layer on the surface of the substrate and can transfer and hold these biomolecules. . What gave the carbon layer a specific chemical modification is preferable. This is because by applying a specific chemical modification, the substance to be analyzed can be easily retained, and can be stably transferred and retained.

  In the present invention, the substrate means a base material from which the carbon layer is formed, and such a base material is not particularly limited. For example, gold, silver, copper, aluminum, tungsten, molybdenum, chromium, Metals such as platinum, titanium and nickel; alloys such as stainless steel, hastelloy, inconel, monel and duralumin; laminates of the above metals and ceramics; glass; silicon; fibers; wood; paper; plastics such as polycarbonate and fluororesin; Mention may be made of a mixture of plastic and the above metals, ceramics, diamond and the like. What formed the metal layer which consists of platinum, titanium, etc. on the surface of glass or plastic etc. can also be used. The metal layer can be formed by sputtering, vacuum deposition, ion beam deposition, electroplating, electroless plating, or the like.

  When mass analysis is performed on the immobilized substance by laser desorption / ionization-time-of-flight mass spectrometry or the like, since a high voltage is applied to the solid support, the substrate has conductivity, such as stainless steel, aluminum, Titanium or the like is preferable.

  The carbon layer formed on the substrate in the present invention is not particularly limited, but diamond, diamond-like carbon, amorphous carbon, graphite, hafnium carbide, niobium carbide, silicon carbide, tantalum carbide, thorium carbide, titanium carbide, uranium carbide. , Tungsten carbide, zirconium carbide, molybdenum carbide, chromium carbide, vanadium carbide and the like, and a diamond-like carbon (DLC) layer is preferable. The carbon layer has excellent chemical stability and can withstand subsequent chemical modification and reaction in the binding to the analyte, and the binding is flexible because it binds to the analyte by electrostatic binding. This is advantageous in that it has no UV absorption, is transparent to the detection system UV, and can be energized during electroblotting. Further, it is advantageous in that nonspecific adsorption is small in the binding reaction with the analyte.

  In the present invention, the carbon layer can be formed by a known method. For example, a microwave plasma CVD (Chemical vapor deposition) method, an ECRCVD (Electrical cyclotron resonance) method, an ICP (Inductive coupled plasma) method, a direct current sputtering method, an ICP (Inductive coupled plasma method) Examples thereof include a vapor deposition method, a laser vapor deposition method, an EB (Electron beam) vapor deposition method, and a resistance heating vapor deposition method.

  In the high-frequency plasma CVD method, a source gas (methane) is decomposed by glow discharge generated between electrodes by a high frequency to synthesize a DLC (diamond-like carbon) layer on a substrate. In the ionization vapor deposition method, the source gas (benzene) is decomposed and ionized using thermoelectrons generated by a tungsten filament, and a carbon layer is formed on the substrate by a bias voltage. The DLC layer may be formed by ionized vapor deposition in a mixed gas composed of 1 to 99% by volume of hydrogen gas and 99 to 1% by volume of remaining methane gas.

  In the arc evaporation method, an arc discharge is generated in a vacuum by applying a DC voltage between a solid graphite material (cathode evaporation source) and a vacuum vessel (anode), and a plasma of carbon atoms is generated from the cathode to generate an evaporation source. Further, by applying a negative bias voltage to the substrate, carbon ions in the plasma can be accelerated toward the substrate to form a carbon layer.

  In the laser vapor deposition method, for example, a carbon layer can be formed by irradiating a graphite target plate with Nd: YAG laser (pulse oscillation) light and melting it, and depositing carbon atoms on a glass substrate.

  The thickness of the carbon layer on the surface of the solid support of the present invention is usually a monomolecular layer to about 100 μm, and if it is too thin, the surface of the underlying solid support may be locally exposed. Since productivity deteriorates, it is preferably 2 nm to 1 μm, more preferably 5 nm to 500 nm. Note that all of the solid support may be made of a carbon material.

  In order to perform direct laser desorption / ionization-time-of-flight mass spectrometry after transferring the substance from the gel after electrophoresis, the solid support of the present invention preferably has a flat plate shape. The size is not particularly limited, but is usually about 10 to 200 mm wide × 10 to 200 mm long × 0.1 to 20 mm thick.

  In order to immobilize biomolecules such as nucleic acids and peptides, it is preferable to electrostatically charge the surface of the substrate on which the carbon layer is formed by chemical modification. Such surface electrostaticization can be appropriately selected by those skilled in the art and is not particularly limited, and examples thereof include introduction of an amino group, a carboxyl group, an epoxy group, a formyl group, and a hydroxyl group. It is also effective to introduce metal chelates such as nickel chelates and cobalt chelates.

  Introduction of amino groups can be carried out, for example, by irradiating the carbon layer with ultraviolet rays in ammonia gas. Alternatively, the reaction can be carried out by reacting a polyvalent amine gas such as methylenediamine or ethylenediamine with a chlorinated carbon layer.

  The introduction of the carboxyl group can be carried out, for example, by reacting a suitable polycarboxylic acid with the carbon layer aminated as described above.

  Introduction of an epoxy group can be carried out, for example, by reacting a suitable polyvalent epoxy compound with the carbon layer aminated as described above. Or it can obtain by making an organic peracid react with the carbon = carbon double bond which a carbon layer contains. Examples of the organic peracid include peracetic acid, perbenzoic acid, diperoxyphthalic acid, performic acid, and trifluoroperacetic acid.

  Introduction of the formyl group can be carried out, for example, by reacting glutaraldehyde with the carbon layer aminated as described above.

  Introduction of hydroxyl groups can be carried out, for example, by reacting water with the carbon layer chlorinated as described above.

  When nucleic acids such as DNA and RNA are retained, amino groups, carbodiimide groups, epoxy groups, and formyl groups are preferably introduced.

  When holding a peptide, it is preferable to introduce an amino group, a carbodiimide group, an epoxy group, a formyl group, or a metal chelate. When a solid support into which a metal chelate is introduced is used, a peptide having a label having an affinity for a metal ion such as a polyhistidine sequence can be immobilized effectively and stably. The metal chelate can be introduced, for example, by chlorinating the substrate on which the carbon layer is formed and then aminating it, and then adding a halocarboxylic acid such as chloroacetic acid to introduce a chelate ligand. Labels such as polyhistidine sequences can be introduced by methods known to those skilled in the art.

  Further, the surface electrostaticization may be performed by forming an electrostatic layer on the surface carbon layer. The electrostatic layer can be formed using a positively charged compound such as an amino group-containing compound.

Examples of the amino group-containing compound include an unsubstituted amino group (—NH ₂ ), or an amino group monosubstituted by an alkyl group having 1 to 6 carbon atoms (—NHR; R is a substituent), for example, Ethylenediamine, hexamethylenediamine, n-propylamine, monomethylamine, dimethylamine, monoethylamine, diethylamine, allylamine, aminoazobenzene, aminoalcohol (eg, ethanolamine), acrinol, aminobenzoic acid, aminoanthraquinone, amino acid (glycine, alanine) , Valine, leucine, serine, threonine, cysteine, methionine, phenylalanine, tryptophan, tyrosine, proline, cystine, glutamic acid, aspartic acid, glutamine, asparagine, lysine, arginine, histidine , Aniline, or a polymer thereof (for example, polyallylamine, polylysine) or a copolymer; a polyamine (polyvalent amine) such as 4,4 ′, 4 ″ -triaminotriphenylmethane, triamterene, spermidine, spermine, or putrescine Is mentioned.

  The electrostatic layer may be formed without being covalently bonded to the substrate or the carbon layer, or may be formed to be covalently bonded to the substrate or the carbon layer.

  In the case where the electrostatic layer is formed without being covalently bonded to the substrate or the carbon layer, for example, when the carbon layer is formed, the amino group-containing compound is introduced into the film forming apparatus, thereby containing an amino group. A carbon-based film is formed. Ammonia gas may be used as the compound introduced into the film forming apparatus. In addition, the carbon layer may be a multi-layer structure in which a film containing an amino group is formed after the adhesion layer is formed. In this case, the carbon layer may be formed in an atmosphere containing ammonia gas. Film formation can be performed by, for example, a plasma method.

  Further, when the electrostatic layer is formed without being covalently bonded to the substrate or the carbon layer, the above-mentioned non-existence is formed on the substrate in order to increase the affinity, that is, the adhesion between the electrostatic layer and the substrate or the carbon layer. It is preferable to deposit a compound having a substituted or monosubstituted amino group and a carbon compound. The carbon compound used here is not particularly limited as long as it can be supplied as a gas. For example, methane, ethane, and propane which are gases at normal temperature are preferable. As a method of vapor deposition, ionized vapor deposition is preferable, and as conditions for ionized vapor deposition, it is preferable that an operating pressure is 0.1 to 50 Pa and an acceleration voltage is in a range of 200 to 1000 V.

  When the electrostatic layer is formed by covalently bonding to the substrate or the carbon layer, for example, the surface of the substrate or the substrate provided with the carbon layer is chlorinated by irradiating ultraviolet rays in chlorine gas, and then the amino group-containing compound. Among them, for example, polyallylamine, polylysine, 4,4 ′, 4 ″ -triaminotriphenylmethane, triamterene and other polyvalent amines are reacted to introduce an amino group at the terminal not bonded to the substrate. Thereby, an electrostatic layer can be formed.

  When forming an electrostatic layer by immersing the substrate in a solution containing a compound having an unsubstituted or monosubstituted amino group, when polyallylamine is used as the amino group-containing compound, the substrate is in close contact with the substrate. This improves the amount of immobilized biomolecules. The electrostatic layer can also be formed by immersing the substrate in a solution in which the silane coupling agent coexists with the amino group-containing compound.

  The thickness of the electrostatic layer is preferably 1 nm to 500 μm.

  The electrophoresis method that can be used for separation of the sample in the present invention is not particularly limited. For example, agarose gel electrophoresis, sieving agarose gel electrophoresis, denatured agarose gel electrophoresis, polyacrylamide gel electrophoresis, SDS Examples thereof include polyacrylamide gel electrophoresis, isoelectric point gel electrophoresis, and two-dimensional electrophoresis. A person skilled in the art can appropriately select the type of electrophoresis method to be used from the type and molecular weight of the substance to be separated.

  Agarose gel electrophoresis is the most commonly used technique for separating nucleic acids. Since an agarose gel has a larger network structure than a polyacrylamide gel, DNA fragments of several tens to several hundreds Kbp can be separated by differences in length and molecular structure. Since the charge state of the entire DNA fragment mainly depends on the number of phosphate groups, the mobility is proportional to the size of the DNA fragment. When electrophoresis is carried out by changing the electric field direction intermittently, it is possible to separate large DNA such as yeast chromosomes (pulse field electrophoresis).

  Polyacrylamide gel electrophoresis of nucleic acids is mainly used for the analysis of DNA fragments. By using the fine network structure of polyacrylamide gel, fragments of short chains (~ 1 Kbp) compared to agarose gel electrophoresis are used. Is a method of separating the images based on length and structure. Since it is strongly influenced by the three-dimensional structure (conformation) of DNA, the estimation of the DNA chain length is limited to the case of migrating double-stranded DNA. Since single-stranded DNA is expected to have various structures, there is no correlation between mobility and the length of the DNA strand, and it is often detected as a plurality of bands. Even slight differences in DNA bases cause structural changes that are reflected in the electrophoretic pattern. A DNA fragment analysis method (SSCP: Single-Strand Conformation Polymerism) using this has been developed and used for gene mutation analysis. Double-stranded DNA fragments containing special sequences (repetitive sequences, base biases, etc.) are known to distort DNA structures, and polyacrylamide gel electrophoresis can be used for DNA structure / function analysis. In a denaturing gel containing urea or the like, single-stranded DNA can also be separated according to the chain length without being affected by the structure.

  SDS (Sodium dodecyl sulfate) -polyacrylamide gel electrophoresis (SDS-PAGE method) is a technique in which a higher-order structure of a target protein is denatured and separated by a difference in molecular weight. Polyacrylamide gel is suitable for separating proteins and polypeptides of 100 to 200 KDa or less because the pore diameter in the gel is dense. It is the most commonly used technique for protein electrophoresis because of its simple operation and high reproducibility. Usually, when preparing an electrophoresis sample, a reducing agent such as β-mercaptoethanol or DTT (Dithiothreitol) is added to cleave the S—S bond (disulfide bond) of the protein. Since the molecular charge is substantially determined by the amount of SDS bound, the polypeptide molecules can be separated by electrophoresis according to the molecular weight. Since SDS is a strong anionic surfactant, it is also suitable for solubilizing insoluble proteins such as membrane proteins.

  Isoelectric focusing is an electrophoretic technique in which the isoelectric point (pI) of proteins is separated to measure and analyze the isoelectric point of the target protein. The charge at the amino acid side chain, amino terminal, and carboxyl terminal constituting the protein varies depending on the pH condition, and the pH value at which the total charge becomes zero is the isoelectric point. In order to perform isoelectric focusing, it is necessary to create a pH gradient in the electrophoresis gel. When a sample is added to an electrophoresis gel and an electric field is applied, each protein moves through the gel that forms a pH gradient toward the same pH as the unique pI. For the preparation of a pH gradient gel, an amphoteric carrier (carrier ampholite) is added to the gel and an electric field is applied to form a pH gradient, and acrylamide derivatives having various pI side chains are used simultaneously with gel preparation. There is a method of forming a pH gradient (IPG method: Immobilized pH gradient), and in the proteomics research, the IPG method which is excellent in resolution, reproducibility, and addition tolerance is mainly used. A precast gel (Immobiline Dry Strip Gel) dedicated to the IPG method is commercially available. The resolution of isoelectric focusing using carrier ampholite is 0.01 to 0.02 pH unit, and the IPG method can be separated even by a difference of 0.001 pH unit.

  Two-dimensional electrophoresis is a method of separating proteins in two dimensions by two-stage electrophoresis. In general, the first dimension separates proteins by isoelectric focusing, and the second dimension separates by molecular weight by SDS-PAGE. Since both methods have very high resolution, it is possible to separate the whole cell protein into thousands of spots. In general, an immobilized pH gradient method (IPG method) excellent in reproducibility and resolution is used for the first-dimensional electrophoresis. In addition, in order to obtain more spots, only the target pH portion is separated with a narrow pH IPG gel based on the separation results in a wide pH range, or the second dimension electrophoresis is performed using a large gel of 20 cm or more. You can also.

  In the present invention, agarose gel electrophoresis is preferably used for separating DNA and RNA, and SDS polyacrylamide gel electrophoresis and two-dimensional electrophoresis are used for separating peptides. Is preferred. These electrophoresis methods can be carried out by methods commonly used by those skilled in the art.

  After electrophoresis, the gel is cut into a size on the solid support to be used, the gel and the solid support are brought into close contact with each other, and the analyte to be analyzed separated in the gel is transferred onto the solid support of the present invention. The transfer method to the solid support is not particularly limited, and a method usually used in this technical field can be used. For example, capillary blotting using capillary action, vacuum blotting by suction with a pump, and electroblotting using an electrical technique may be mentioned. When transferring nucleic acids, it is preferable to use capillary blotting. When transferring peptides, it is preferable to use electroblotting.

  In electroblotting, any of tank type, semi-dry type and semi-wet type can be used, but it is preferable to use semi-dry type electro-blotting from the viewpoint of a small amount of buffer used and a short reaction time. . As a blotting apparatus, an electroblotting apparatus usually used in this technical field can be used. The energization conditions in electroblotting are a constant voltage, 200 V or less, preferably 0.1 to 10 V, and 1 to 500 minutes, preferably 5 to 100 minutes. However, if the voltage is higher than the oxidation potential of the metal substrate, metal elution occurs. Therefore, it is preferable that the voltage be lower than the oxidation potential of the substrate metal.

  Hereinafter, one embodiment of electrophoresis and transcription in the present invention when analyzing proteins in a sample will be described. First, the protein in the sample is solubilized. That is, heat treatment is performed for a certain period of time in boiling water in order to inactivate the proteolytic enzyme present in the sample and effectively denature the protein with SDS and β-mercaptoethanol. Next, a certain amount is injected into each lane of the SDS-polyacrylamide gel, and electrophoresis is performed at a constant voltage for a certain time using a glycine-Tris buffer containing SDS as a migration buffer. After electrophoresis, the gel is immersed in glycine-Tris buffer (transfer buffer) containing methanol, which has been cooled in advance, for equilibration. Subsequently, the gel is attached to the electroblotting apparatus with the cathode side and the solid support for transfer as the anode side. A transfer buffer is added to the transfer tank, and transfer is performed at a constant voltage for a certain time under ice cooling. At this time, from the viewpoint of increasing the transfer efficiency, it is preferable to dispose a filter paper containing a buffer or ion exchange water between the cathode and the gel and between the anode and the solid support. Examples of the buffer contained in the filter paper on the cathode side include those containing Tris, ε-aminocaproic acid, acetic acid, EDTA, phosphoric acid, boric acid, tartaric acid, SDS and the like. When using a buffer containing Tris and ε-aminocaproic acid, the concentration of ε-aminocaproic acid is usually 1000 mM or less, preferably 1 μM to 1000 mM, more preferably 1 to 300 mM. The filter paper on the anode side preferably contains ion exchange water.

  In another aspect of the present invention, the target substance is transferred from the gel after electrophoresis to a membrane as used in the prior art, and further transferred from the membrane onto the solid support of the present invention. It is also possible to transfer and hold the separated material on the solid support. Examples of membrane materials that can be used in this case include nitrocellulose, PVDF (polyvinylidene fluoride), nylon, and positively charged nylon. In protein transcription, PVDF having the highest protein binding ability is preferably used, and in nucleic acid transcription, PVDF with less non-specific adsorption of nucleic acid is preferably used. Transfer of the electrophoretic substance from the gel to the membrane and transfer from the membrane to the solid support can be carried out by the same method as described above. In the transfer from the gel to the membrane, electroblotting is preferably used, and the energization conditions in electroblotting are 0.1 to 50 V, preferably about 5 to 120 minutes. In the transfer from the membrane to the solid support, an electroblotting device is preferably used.

  In another aspect of the present invention, a substance separated by electrophoresis is transferred and held on a solid support, a substance that interacts with the substance is reacted to form a complex, and the formed complex is ionized. Therefore, mass spectrometry can be performed. As a mass spectrometric method, a technique of analyzing atomic / molecular ions based on a difference in mass using electrical interaction can be used. Such a mass spectrometer has three different functions of ion generation / separation / detection. When a protein is transferred and held on a solid support by the method described above, a complex is formed by reacting an antibody against the protein, and the complex is ionized by irradiating a laser or the like. Thus, mass spectrometry can be performed. When a nucleic acid such as DNA or RNA is transferred and held on a solid support, a nucleic acid complementary to the nucleic acid is hybridized to the nucleic acid on the solid support, and the formed double strand is ionized. Mass spectrometry can be performed. As other interactions, for example, an enzyme reaction, biotin-streptavidin interaction, or the like can be used. By performing mass spectrometry of the complex formed by the interaction, the base sequence or amino acid sequence of the target molecule that specifically interacts with the probe molecule can be analyzed.

  In another embodiment of the present invention, for the purpose of analyzing interacting substances, a complex of interacting substances in solution is formed and then subjected to electrophoresis and separated by electrophoresis. Mass spectrometry can also be performed by transferring and holding the complex on a solid support and ionizing the complex on the solid support. Since such an analysis method performs complex formation in a solution, it is advantageous for an analysis that needs to maintain a high degree of three-dimensional structure of a substance to be analyzed, such as protein analysis.

  The substance immobilized on the solid support can be directly subjected to mass spectrometry by means such as laser desorption / ionization-time-of-flight mass spectrometry. Examples of ionization methods that can be used for mass spectrometry include matrix-assisted laser desorption (MALDI), electron impact ionization (EI), photoionization, and large LET α or β rays emitted from radioisotopes. Ionization method using a secondary ionization method, fast atom collision ionization method, field ionization ionization method, surface ionization ionization method, chemical ionization (CI) method, field ionization (FI) method, ionization method by spark discharge, etc. The matrix-assisted laser desorption (MALDI) method and the ionization (EI) method by electron impact are preferable. In addition, separation modes include linear or nonlinear reflection time-of-flight (TOF), single or multiple quadrupole, single or multiple magnetic sector, Fourier transform ion cyclotron resonance (FTICR), ion capture, radio frequency and ion capture / flight. Examples include time or the like, and those using linear or non-linear reflection time of flight (TOF), high frequency and ion capture / time of flight are preferred. Mass spectrometry can be performed by combining ionization methods such as those described above with separation modes including separation modes or combinations thereof, electrical recording and detection modes such as photographic recording. Such a mass spectrometer has three different functions of ion generation / separation / detection. From the viewpoint of ionizing a macromolecular substance such as a biomolecule and analyzing a plurality of substances on a solid support, it is preferable to use laser desorption / ionization-time-of-flight mass spectrometry.

  Hereinafter, as one embodiment of the present invention, a mass spectrometric procedure using MALDI-TOF MS will be described.

  A matrix such as α-cyanohydroxycinnamic acid and sinapinic acid is added to the solid support of the present invention on which the substance to be analyzed is immobilized, and dried. Next, this solid support body is installed in the flat target of MALDI-TOF MS. Then, mass spectrometry is started using MassLynx software or the like. MassLynx can control all measurements and analyses. At the time of measurement, a parameter file for automatic measurement, a process file for data processing and database analysis performed after measurement, a sample list, and the like are created. Data processing can be performed on MassLynx using ProteinLynx software. A mass spectrum is created from the acquired data, and the created spectrum is converted to monoisotopic peak data after the accuracy is improved by MaxEnt 3 software (Micromass). Subsequently, calibration is performed to obtain final data with a mass error of about 50 ppm. From this data, the exact mass of the interacting protein can be determined.

  Mass spectrometry can be followed by amino acid sequence analysis and identification of the protein. The analysis mode of MALDI-TOF MS is changed to a mode capable of detecting a post-source decay (PSD) spectrum, and the amino acid sequence of the interacted protein is analyzed. Subsequently, the SWISSPROT database is searched based on the amino acid sequence data to identify the protein. Alternatively, a solid support on which a substance to be analyzed is immobilized is placed on MALDI-TOF / TOF MS or MALDI Q-TOF MS, and the amino acid sequence is analyzed to identify the interacted protein.

  EXAMPLES Hereinafter, although an Example demonstrates this invention more concretely, this invention is not limited to these Examples.

Example 1 Protein Transfer to Ti-Pt-DLC Solid Support
Preparation of solid support A Ti layer and a Pt layer thereon were formed by magnetron sputtering on a 76 mm × 26 mm × 1.1 mm glass slide. The sputtering conditions are as follows. The thickness of the generated metal layer was 100 nm for each of the Ti layer and the Pt layer.

  Then, a diamond-like carbon layer was formed on the substrate on which the metal layer was formed. The diamond-like carbon layer was formed by the ionization vapor deposition method under the following conditions. The thickness of the produced diamond-like carbon layer was 20 nm.

  After the diamond-like carbon layer is formed on the substrate 1 as described above, the surface is chlorinated by irradiating ultraviolet rays in chlorine gas for 1 minute, and aminated and solidified by irradiating ultraviolet rays in ammonia gas for 10 minutes. A support 1 was prepared.

SDS-PAGE electrophoresis Cy3-protein A (1.5 μg, manufactured by SIGMA), E. coli protein (0.5 μg) and marker (Prestained Broad Range, 0.5 μl, manufactured by BIO RAD) were used as samples. -Electrophoresis was performed using a device for PAGE (AE-7300, manufactured by ATTO). A 10% polyacrylamide gel was used as the gel for electrophoresis. The electrophoresis buffer was glycine-Tris buffer (pH 8.3) containing 0.1% SDS, and electrophoresis was performed at 200 V for 35 minutes. After the completion of electrophoresis, the sample was stained with CBB for 15 minutes, destained, and then imaged with LAS1000 (manufactured by Fuji Photo Film Co., Ltd.) (FIG. 1). A Cy3-Protein A band was detected in the vicinity of about 50 kDa.

The polyacrylamide gel after the electroblotting electrophoresis was immersed in a transfer buffer (25 mM Tris, 5% methanol) cooled in advance for 30 minutes to equilibrate. Next, the polyacrylamide gel was cut to a size on the solid support 1 and brought into close contact with the solid support, and the polyacrylamide gel was placed on the cathode and the solid support was placed on the anode, and energized under the following conditions.

Measurement of fluorescence intensity When the fluorescence intensity of the solid support 1 after protein transfer was measured with FLA8000 (manufactured by Fuji Photo Film Co., Ltd.), the fluorescence intensity was measured as 28270, and Cy3-Protein A was immobilized on the surface of the solid support. Subsequently, the solid support was washed with PBS for 10 minutes, and the fluorescence intensity was measured in the same manner. The fluorescence intensity was 9766, which was reduced to about 1/3. (FIG. 3). When blocking with a blocking reagent (Roche) for 1 hour and measuring the fluorescence intensity, there was no change in the fluorescence intensity. Next, 500 μl of 0.05 μg / μl Cy3-IgG was added and reacted at room temperature for 1 hour, then washed with PBS for 12 hours at room temperature, and the fluorescence intensity was measured (FIG. 4). Since the fluorescence intensity is 16448 and increases, it can be seen that protein A and IgG were bound, that is, the binding ability of the transcriptionally retained protein was maintained.

(Example 2) Transfer of protein to stainless steel-DLC solid support
Preparation of stainless steel-DLC solid support A diamond-like carbon layer was formed on a stainless steel substrate. The stainless steel substrate was subjected to electrolytic polishing after buffing in advance in order to lower the smoothness and the fluorescent background. The diamond-like carbon layer was formed by the ionization vapor deposition method under the following conditions. The thickness of the produced diamond-like carbon layer was 20 nm. Moreover, the solid support body 2 was created by introduce | transducing an amino group by carrying out ammonia plasma processing.

  Cy3-Protein A (0.2 μg, manufactured by SIGMA) was electrophoresed by SDS-PAGE (AE-6530, manufactured by ATTO) in the same manner as in Example 1, and after completion of the electrophoresis, CBB staining was performed for 15 minutes, followed by destaining. Then, an image was taken with LAS1000 (manufactured by Fuji Photo Film Co., Ltd.) (FIG. 5).

  After the electrophoresis, the gel was taken out, cut into a size to be placed on the solid support 2, and then immersed in transfer buffer B (25 mM Tris, 5% methanol). Filter papers (manufactured by ATTO) previously cut to the size of the solid support 2 were transferred to transfer buffer A (0.3 M Tris, 5% methanol) and C (25 mM Tris, 40 mM ε-aminocaproic acid, 5%, respectively. Methanol) or ion-exchanged water. Subsequently, the filter paper, the gel, and the solid support were overlapped as shown in FIG. 6 and placed in a semi-driving device so that air bubbles would not enter, and the protein was transferred to the solid support 2 by applying current at 8 V and 4 mA for 60 minutes. . The solid support 2 after the transfer was washed with ion-exchanged water at room temperature for 30 minutes and dried, and then the solid support and the gel after the transfer were photographed with FLA8000 (manufactured by Fuji Photo Film Co., Ltd.) (FIG. 7). ).

  (1) is the result when transfer is performed with ion-exchanged water included in both the cathode-side and anode-side filter papers. Cy3-Protein A hardly escaped from the gel and was not transferred and retained on the solid support.

  (2) shows the results when transfer is performed with the filter paper I containing the transfer buffer C, the filter paper II containing the transfer buffer B, and the filter paper III containing the transfer buffer A. Cy3-Protein A was somewhat reduced from the gel, but was not transferred and retained on the solid support.

  (3) shows the results when transfer was performed with filter paper I containing transfer buffer C and filter papers II and III containing ion-exchanged water. The gel was wiped off moisture before being overlaid on the solid support. As a result, it was confirmed that Cy3-Protein A was not uniform but escaped from the gel. Further, although the shape was not good on the solid support, transfer holding was observed.

  (4) shows the results when the transfer paper C contains transfer buffer C, and the filter papers II and III contain ion-exchanged water, and the transfer is performed on the solid support without wiping off the moisture of the gel. As a result, it was confirmed that Cy3-Protein A was not uniform but escaped from the gel. Cy3-protein A was transferred and held in a good shape on the solid support.

  From the above, when transferring the protein in the gel to the solid support, the filter paper on the cathode side should contain the transfer buffer, and the filter paper on the anode side should contain ion-exchanged water, and wipe off the moisture of the gel after electrophoresis. It is considered preferable to perform the transfer without any problems.

(Example 3) Transfer of protein to stainless steel-DLC solid support Cy3-Protein A (50 ng, manufactured by SIGMA) and Cy3-IgA (100 ng, manufactured by SIGMA) were subjected to SDS-PAGE method in the same manner as in Example 1 ( AE-6530 (manufactured by ATTO), and after completion of the electrophoresis, CBB staining was performed for 15 minutes, and after destaining, an image was taken with LAS1000 (manufactured by Fuji Photo Film Co., Ltd.) (FIG. 8). For the migration of Cy3-IgA, a 12% polyacrylamide gel was used. Further, a solid support 2 was prepared in the same manner as in Example 2.

  The gel after the electrophoresis was taken out, cut into a size to be placed on the solid support 2, and then immersed in a transfer buffer (25 mM Tris, 5% methanol). Filter papers (manufactured by ATTO) cut in advance to the size of a solid support were transferred to transfer buffers C1 (25 mM Tris, 40 mM ε-aminocaproic acid, 5% methanol) and C2 (25 mM Tris, 400 mM ε-aminocaproic acid), respectively. 5% methanol) or ion-exchanged water. Subsequently, the filter paper, the gel, and the solid support were stacked in the same manner as in FIG. At this time, the three filter papers on the cathode side contained transfer buffer C1 or C2, and the three filter papers on the anode side contained ion exchange water. Then, electricity was applied at 2 V and 2 μA for 60 minutes to transfer the protein to the solid support 2. The solid support 2 after the transfer was washed with ion exchange water at room temperature for 30 minutes and dried. The solid support and the gel after transfer were photographed with FLA8000 (manufactured by Fuji Photo Film Co., Ltd.) (FIG. 9).

  As a result, it was found that the transfer buffer contained in the cathode-side filter paper at the time of transfer to the solid support had better transfer retention efficiency when C1, that is, the concentration of ε-aminocaproic acid was 40 mM.

(Example 4) Protein transfer from PVDF membrane to Ti-Pt-DLC solid support A diamond-like carbon layer is formed on the substrate 1 in the same manner as in Example 1, and is processed in ammonia plasma using an ionization deposition apparatus. Thus, the solid support 3 was prepared by amination of the surface.

  Further, Cy3-protein A was electrophoresed on a polyacrylamide gel in the same manner as in Example 1. After completion of the electrophoresis, CBB staining was performed for 15 minutes, and after destaining, images were taken with LAS1000 (manufactured by Fuji Photo Film Co., Ltd.) (FIG. 10). A Cy3-Protein A band was detected in the vicinity of about 50 kDa.

  Transfer buffers A (0.3 M Tris, 5% methanol), B (25 mM Tris, 5% methanol) and C (25 mM Tris, 40 mM ε-aminocaproic acid, 5% methanol) were prepared. The gel after electrophoresis was taken out, immersed in about 200 ml of transfer buffer B, and gently shaken for 5 minutes. A PVDF membrane (manufactured by ATTO) that had been cut into a gel size in advance was immersed in a small amount of methanol for 5 seconds, then immersed in about 100 ml of transfer buffer B, and shaken for 5 minutes or more. Two, one, and three filter papers that had been cut to the size of the gel in advance were immersed in 200 ml each of the transfer buffer A, B, or C. Subsequently, the filter paper, the gel and the PVDF membrane were placed on a semi-driving device (Nippon Aido) so as not to enter bubbles as shown in FIG. 11, and energized for 60 minutes at a voltage of 15V. After the transfer, the PVDF membrane was immersed in 200 ml of PBS and permeated for 5 minutes.

  The PVDF membrane after the transfer was cut into a size to be placed on the solid support 3 and overlapped in the order as shown in FIG. 12, and a weight of 35 g / cm 2 was placed. After standing at room temperature for 1 hour, Cy3-protein A on the membrane was transferred onto the solid support 3. Subsequently, the solid support 3 was washed with PBS at room temperature for 20 minutes and then dried. The solid support was imaged with FLA8000 (Fuji Photo Film Co., Ltd.). A captured image is shown in FIG. The part enclosed in the image is the part where the membrane is in close contact. Since fluorescence was detected at the corresponding position, it can be seen that Cy3-protein A was transferred and held on the solid support.

(Example 5) Transfer of protein to stainless steel-DLC solid support Cy3-labeled yeast protein (300 µg / lane) was electrophoresed in the same manner as in Example 1 using the SDS-PAGE method (AE-6530, manufactured by ATTO). After completion of the electrophoresis, images were taken with FLA8000 (Fuji Photo Film Co., Ltd.) (FIG. 14 (1)). Further, a solid support 2 was prepared in the same manner as in Example 2.

After the electrophoresis, the gel was taken out, cut into a size to be placed on the solid support 2, and the gel was washed with 10% methanol, then replaced with fresh 10% methanol, and further washed for 30 seconds. After washing, the gel was placed on the solid support 2, and a dialysis membrane and a filter paper containing 1M H ₃ BO ₃ buffer (pH 8.0) were placed thereon. Then, electricity was applied at 2 V for 60 minutes to transfer the protein complex to the solid support 2. The solid support 2 after the transfer was washed with ultrapure water and dried. The solid support was imaged with FLA8000 (Fuji Photo Film Co., Ltd.) (FIG. 14 (2)).
As a result, the transfer efficiency was about 40%, and it was found that the transfer was sufficient.

(Example 6) Transfer of protein to stainless steel-DLC solid support Cy3-Protein A (50 ng, manufactured by SIGMA) and Cy5-IgA (100 ng, manufactured by SIGMA) were mixed in a solution, and the same as in Example 1 The sample was electrophoresed using a Native-PAGE method (AE-6530, manufactured by ATTO). After the electrophoresis was completed, an image was taken using FLA8000 (manufactured by Fuji Photo Film Co., Ltd.). Further, a solid support 2 was prepared in the same manner as in Example 2.

The gel after the electrophoresis was taken out, cut into a size to be placed on the solid support 2, and then immersed in a transfer buffer (25 mM Tris, 5% methanol). Filter paper (manufactured by ATTO) that had been cut in advance to the size of a solid support was immersed in transfer buffer C1 (25 mM Tris, 40 mM ε-aminocaproic acid, 5% methanol) or ion-exchanged water, respectively. Subsequently, the filter paper, the gel, and the solid support were stacked in the same manner as in FIG. At this time, the three filter papers on the cathode side contained transfer buffer C1, and the three filter papers on the anode side contained ion-exchanged water. Then, the protein complex was transferred to the solid support 2 by applying electricity at 2 V and 2 μA for 60 minutes. The solid support 2 after the transfer was washed with ion exchange water at room temperature for 30 minutes and dried. The solid support and the gel after transfer were photographed with FLA8000 (Fuji Photo Film Co., Ltd.).
As a result, the fluorescence intensity of the image taken with the FLA8000 after the transfer was about 35% compared to the fluorescence intensity of the image taken with the FLA8000 after completion of the electrophoresis.

  According to the method of the present invention, after a large number of substances contained in a sample are separated by electrophoresis, substances contained in individual bands can be transferred and held on a solid support without purifying a plurality of substances. Since mass spectrometry can be performed simultaneously and directly, a large number of samples can be analyzed quickly.

  Therefore, the present invention is a very useful means in the analysis of biomolecules such as nucleic acids and proteins. In addition, since a complex of substances that interact in a solution can be formed, it is advantageous for analysis that requires a high degree of retention of the three-dimensional structure of the substance to be analyzed, such as protein analysis.

In Example 1, Cy3-protein A and E. coli protein were electrophoresed by SDS-PAGE, the gel after electrophoresis was stained with CBB for 15 minutes, destained, and then imaged with LAS1000 (Fuji Photo Film Co., Ltd.). It is a thing. In Example 1, the gel after electrophoresis was transferred to the solid support 1, and the fluorescence intensity of the solid support after transfer was imaged with FLA8000 (Fuji Photo Film Co., Ltd.). In Example 1, the solid support 1 onto which the gel after electrophoresis was transferred was washed with PBS for 10 minutes, dried, and then imaged. In Example 1, the solid support 1 onto which the gel after electrophoresis was transferred was washed with PBS for 10 minutes, further blocked with a blocking reagent for 1 hour, and then Cy3-IgG was added and reacted at room temperature for 1 hour. Images were taken after washing with PBS for 12 hours (room temperature). In Example 2, Cy3-protein A was electrophoresed by SDS-PAGE, the gel after electrophoresis was stained with CBB for 15 minutes, destained, and then imaged with LAS1000 (manufactured by Fuji Photo Film Co., Ltd.). is there. In Example 2, arrangement | positioning when protein is transcribe | transferred from the gel after electrophoresis to the solid support body 2 is represented. In Example 2, the solid support 2 onto which the protein was transferred from the gel after electrophoresis was washed with PBS for 30 minutes and dried, and the transferred gel was imaged with FLA8000 (manufactured by Fuji Photo Film Co., Ltd.). Is. In Example 3, Cy3-Protein A and Cy3-IgA were electrophoresed by SDS-PAGE, the gel after electrophoresis was stained with CBB for 15 minutes, destained, and then imaged with LAS1000 (Fuji Photo Film Co., Ltd.). It was taken. In Example 3, the solid support 2 onto which the protein was transferred from the gel after electrophoresis was washed with PBS for 30 minutes and dried, and the transferred gel was imaged with FLA8000 (manufactured by Fuji Photo Film Co., Ltd.). Is. In Example 4, Cy3-protein A was electrophoresed by SDS-PAGE, the gel was stained with CBB for 15 minutes, destained, and then imaged with LAS1000 (Fuji Photo Film Co., Ltd.). In Example 4, arrangement | positioning when protein is transferred to a PVDF membrane from the gel after electrophoresis is represented. In Example 4, arrangement | positioning when protein is transcribe | transferred from the PVDF membrane to the solid support body 3 is represented. In Example 4, the solid support 3 onto which the protein was transferred from the PVDF membrane was washed with PBS for 20 minutes, dried, and then imaged with FLA8000 (manufactured by Fuji Photo Film Co., Ltd.). FIG. 14 (1) shows the image taken in FLA8000 (manufactured by Fuji Photo Film Co., Ltd.) after electrophoresis by SDS-PAGE in Example 5, and FIG. 14 (2) shows the solid to which the protein was transferred. The support was washed with ion-exchanged water for 30 minutes, dried, and then imaged with FLA8000 (Fuji Photo Film Co., Ltd.).

Claims (7)

A method for mass spectrometry of a substance in a sample,
Separating the substances in the sample by gel electrophoresis,
A step of transferring the separated material in the gel to a membrane, and the transferred material on the membrane, seen including the step of transferring the holding to a solid support with a carbon layer on the surface, the solid having a carbon layer on the surface support is present on the carbon layer on the substrate surface is further including an amino group-containing compound which is not covalently bonded to the carbon layer, said method.   The method according to claim 1, wherein the solid support is obtained by vapor-depositing a compound having an unsubstituted or monosubstituted amino group and a carbon compound on a substrate. The method according to claim 1 or 2 , further comprising the step of adding another substance that interacts with the substance immobilized on the solid support to form a complex. The method according to any one of claims 1 to 3 , wherein a complex of substances interacting in a solution is formed, and the complex is separated by gel electrophoresis. The method according to any one of claims 1 to 4 , wherein the carbon layer is a diamond-like carbon layer. The method according to any one of claims 1 to 5 , wherein the thickness of the carbon layer is a monomolecular layer to 100 µm. The method according to any one of claims 1 to 6 , wherein the substance transferred and held on the solid support is a nucleic acid, a peptide, or a complex thereof.