WO2005080960A1

WO2005080960A1 - Solid support and method of mass spectrometry through desorption/ionization of multiple substances or composites on solid support

Info

Publication number: WO2005080960A1
Application number: PCT/JP2005/002927
Authority: WO
Inventors: Mitsuyoshi Ohba; Hirofumi Yamano; Shuuichi Kamei; Hisashi Hirano
Original assignee: Toyo Kohan Co., Ltd.
Priority date: 2004-02-23
Filing date: 2005-02-23
Publication date: 2005-09-01
Also published as: JP2005233908A; JP4113139B2

Abstract

Means for speedy mass spectrometry of a multiplicity of samples; and a method of speedy analysis of biomolecules such as nucleic acids and proteins. There is provided a solid support having a carbon layer formed on its surface and further containing an aminated compound bonded to the carbon layer through covalent bond. There is further provided a method of mass spectrometry of multiple substances, comprising separating substances of a sample from each other by gel electrophoresis, transferring separated substances lying in a gel onto the solid support and conducting desorption/ionization of the substances on the solid support.

Description

Specification

Solid support and method for mass spectrometry by desorption of a plurality of substances or complexes on the solid support

Technical field

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

Background art

[0002] Many biomolecules such as peptides, proteins, nucleic acids, and sugar chains are formed by polymerizing a relatively small number of constituent units according to a certain rule. For example, peptides and proteins are molecules in which 20 L-a-amino acids are connected by peptide bonds. Most of the molecular structures of these structural units have already been elucidated, and of course, their precise molecular weights have also been elucidated. Therefore, if the molecular weight of a biomolecule or its fragment can be accurately measured, it can greatly contribute to the analysis of its structure (sequence, etc.) and various modification reactions received in the living body. It is positioned as an indispensable tool for structural analysis of biomolecules. Among mass spectrometers, laser desorption Z-ionization-time-of-flight mass spectrometers are attracting attention as useful analytical tools for biomolecules because they can ionize macromolecules such as DNA and proteins.

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

Then, ions with smaller m / z values, that is, lighter ions, fly faster and reach the detector. Laser desorption / ionization-time-of-flight mass spectrometry is a method of performing mass spectrometry by taking advantage of the fact that the flight time of ions differs due to such a difference in mass-to-charge ratio (m / z value).

On the other hand, for structural analysis / determination of biomolecules such as DNA and proteins by mass spectrometry, it is necessary to separate and purify the analysis target into a number of components, and to analyze individual components fragmented with restriction enzymes. , Have to analyze a very large number of samples. In addition, for DNA diagnosis, it is necessary to rapidly process samples obtained from a large number of humans. [0003] In contrast, a commercially available general laser desorption / ionization-one-time-of-flight mass spectrometer arranges each of the purified samples on a sample board and performs mass analysis on each of them. In other words, it was necessary to irradiate each point of the sampled sample with a laser and analyze it one by one. Therefore, when the unpurified sample was separated by electrophoresis, the gel after electrophoresis was cut out for each band, purified, and then separated one by one. Laser desorption Z ionization-time-of-flight mass spectrometry And it was very difficult to analyze many samples quickly.

[0004] In addition, a method of transferring an electrophoresed biomolecule from a gel onto a membrane such as nitrocellulose and analyzing the same is known. In the analysis on the membrane, mass spectrometry using a laser is performed. It cannot be performed, and is limited to antigen-antibody reaction and fluorescence detection using nucleic acid hybridization. This is because conventionally used membranes such as nitrocellulose and PVDF are likely to be decomposed by laser irradiation. That is, it was difficult to analyze the biomolecules transcribed on these membranes by the laser desorption / ionization-time-of-flight mass spectrometer as described above.

Disclosure of the invention

Problems to be solved by the invention

[0005] An object of the present invention is to provide a means for rapidly mass spectrometric analysis of a large number of samples and to provide a method for rapidly analyzing biomolecules such as nucleic acids and proteins. Means for solving the problem

[0006] The inventors of the present invention have conducted intensive studies to solve the above-described problems, and as a result, after separating a substance in a sample by gel electrophoresis, a carbon layer was formed on the surface of the substance separated and developed in the gel. It has been found that the above problem can be solved by a method of transferring onto a solid support, desorbing / ionizing this, and performing mass spectrometry, thereby completing the present invention.

That is, the present invention includes the following inventions.

(1) A solid support having a carbon layer on the surface, in which a substance in a sample is separated by gel electrophoresis, and the substance separated in the gel is transferred and held.

(2) After separating the substances in the sample by gel electrophoresis, separate the separated substances in the gel with a membrane. A solid support having a carbon layer on the surface, wherein the substance is immobilized by transferring and holding the substance transferred onto the membrane.

(3) A solid support obtained by adding another substance interacting 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 interacting substances is formed in a solution, and the complex is separated by gel electrophoresis.

(5) The solid support according to (1), 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 100 μm per monomolecular layer.

(7) The solid support according to any one of (1) to (6), wherein the solid support further includes 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. body.

(8) The solid support is present on the carbon layer on the surface of the substrate and further contains an amino group-containing compound which is covalently bonded to the carbon layer. Solid support.

(9) The solid support according to any one of (1) to (6), obtained by evaporating 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 is immersed in a solution containing a compound having an unsubstituted or monosubstituted amino group, and the solid support is obtained by (1) any one of (1) to (6). Solid support.

(11) The solid support according to any one of (1) to (10), wherein the substance held and transcribed is a nucleic acid, a peptide or a complex thereof.

(12) (1) A method for mass spectrometry by desorbing / ionizing a plurality of substances or complexes transferred and held on the solid support according to any one of (11) and (11).

(13) A solid support having a carbon layer on the surface for use in the method according to (12).

Brief Description of Drawings

[FIG. 1] FIG. 1 shows that the protein was transferred from the gel after electrophoresis to the solid support in Example 2. This shows the arrangement at the time of transfer.

[FIG. 2] FIG. 2 shows an arrangement in Example 4 when proteins are transferred from a gel after electrophoresis to a PVDF membrane.

[FIG. 3] FIG. 3 shows an arrangement when a protein is transferred from a PVDF membrane to a solid support 3 in Example 4.

BEST MODE FOR CARRYING OUT THE INVENTION

In the present invention, a sample separated by gel electrophoresis is brought into close contact with the gel after electrophoresis and a solid support having a carbon layer formed on the surface thereof, whereby an analyte to be separated and developed in the gel is separated. Transfer onto the solid support. Then, a plurality of substances are mass-analyzed by desorbing / ionizing the substances transferred and held on the solid support.

[0010] In the present invention, the substance that can be transcribed and held on a solid support and analyzed is not particularly limited, and examples thereof include nucleic acids such as DNA and RNA and biomolecules such as peptides. As used herein, the peptide includes oligopeptides, polypeptides, and proteins. It is particularly 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, Examples include synthetic DNA, synthetic RNA, and synthetic peptide.

The solid support for transferring and holding substances such as biomolecules separated in a gel by electrophoresis is particularly limited as long as it has a carbon layer on the surface of a substrate and can transfer and hold these biomolecules. What? Those with a specific chemical modification on the carbon layer are preferred. This is because the substance to be analyzed is easily retained by performing the specific chemical modification, and is stably transferred and retained.

In the present invention, the term “substrate” means a base material on which a carbon layer is formed, and such a base material is not particularly limited. For example, gold, silver, copper, aluminum, Metals such as tundast, molybdenum, chromium, platinum, titanium, and nickel; alloys such as stainless steel, hastelloy, inconel, monel, and duralumin; laminates of the above metals and ceramics; glass; silicon; fiber; wood; And mixtures of plastics and the above metals, ceramics, diamonds and the like. Gala It is also possible to use a material in which a metal layer made of platinum, titanium or the like is formed on the surface of metal or plastic. The metal layer can be formed by sputtering, vacuum deposition, ion beam deposition, electroplating, electroless plating, or the like.

[0012] When mass spectrometry is performed on an immobilized substance by laser desorption / ionization-time-of-flight mass spectrometry or the like, a high voltage is applied to the solid support, so that the substrate has conductivity. For example, stainless steel, ethanol, titanium and the like are preferable.

In the present invention, the carbon layer formed on the substrate is not particularly limited, but may be diamond, diamond-like carbon, amorphous carbon, graphite, hafnium carbide, niobium carbide, silicon carbide, tantalum carbide, thorium carbide, titanium carbide, Examples of the layer include uranium carbide, tungsten carbide, zirconium carbide, molybdenum carbide, chromium carbide, and vanadium carbide, and a diamond-like carbon (DLC) layer is preferable. The carbon layer has excellent chemical stability and can withstand subsequent reactions in chemical modification and binding with the analyte, and the bond is flexible due to the electrostatic binding with the analyte. This is advantageous in that it has the following properties, is transparent to the detection system UV due to the absence of UV absorption, and can be energized during electroblotting. It is also advantageous in that non-specific adsorption is small in the binding reaction with the analyte.

In the present invention, the formation of the carbon layer can be performed by a known method. Examples include microwave plasma CVD (Chemical vapor deposit), ECRCVD (Electric cyclotron on resonance chemical vapor deposit), ICP (Inductive coupled plasma), DC sputtering, ECR (Electric cyclotron resonance) sputtering, and ionization evaporation. , Arc deposition, laser deposition, EB (Electron beam) deposition, resistance heating deposition, and the like.

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

[0014] In the arc deposition method, a direct current voltage is applied between a solid graphite material (cathode evaporation source) and a vacuum vessel (anode) to cause an arc discharge in a vacuum to cause a plasma of carbon atoms from the cathode. Is generated, and by applying a more negative bias voltage to the substrate than the evaporation source, 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 target plate of graphite 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 about 100 μm per monolayer, and if it is too thin, the surface of the underlying solid support may be locally exposed. On the other hand, if the thickness is too large, the productivity becomes worse, so the thickness is preferably 2 nm lzm, more preferably 5 nm 500 nm. Note that all of the solid support may be made of a carbon material.

The solid support of the present invention preferably has a flat plate shape in order to directly perform laser desorption / ionization-time-of-flight mass analysis after transferring the substance from the gel after electrophoresis. Although the size is not particularly limited, it is usually about 10 to 200 mm in width × 10 to 200 mm in length × 0.1 to 20 mm.

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 chemically modifying the surface. Such surface electrification can be appropriately selected by those skilled in the art, and is not particularly limited. Examples thereof include introduction of an amino group, a carboxyl group, an epoxy group, a forminole group, and a hydroxyl group. Can be It is also effective to introduce metal chelates such as nickel chelates and cobalt chelates.

[0016] The introduction of an amino group can be carried out, for example, by irradiating the carbon layer with ultraviolet light in ammonia gas. Alternatively, the reaction can be carried out by reacting a polyvalent amine gas such as methylene diamine or ethylene diamine with a chlorinated carbon layer.

The carboxyl group can be introduced, for example, by reacting the aminated carbon layer with an appropriate polycarboxylic acid. The introduction of an epoxy group can be carried out, for example, by reacting an appropriate polyvalent epoxy conjugate with the carbon layer aminated as described above. Alternatively, it can be obtained by reacting an organic peracid with carbon = carbon double bond contained in the carbon layer. Examples of the organic peracid include peracetic acid, perbenzoic acid, diperoxyphthalic acid, formic acid, and trifluoroperacetic acid.

The introduction of a formyl group can be carried out, for example, by reacting the aluminized carbon layer with daltaraldehyde.

The introduction of the hydroxy group can be carried out, for example, by reacting the chlorinated carbon layer with water.

When nucleic acids such as DNA and RNA are retained, it is preferable to introduce an amino group, a carbodiimide group, an epoxy group, or a formyl group.

In the case of retaining a peptide, it is preferable to introduce an amino group, a nonolepositeimide 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, then aminating the substrate, and then adding a halocarbonic acid such as chloroacetic acid to introduce the chelate ligand. . Labels such as polyhistidine sequences can be introduced by methods known to those skilled in the art.

[0018] The surface electrostaticization may be performed by forming an electrostatic layer on the surface carbon layer. The electrostatic layer can be formed using a compound having a positive charge such as an amino group-containing compound.

The amino group-containing compound may be an unsubstituted amino group (1-NH 2) or a compound having 1 carbon atom.

Compounds having an amino group (one NHR; R is a substituent) monosubstituted with 26 alkyl groups, such as ethylenediamine, hexamethylenediamine, n-propylamine, monomethylamine, dimethinoleamine, Monoethylamine, getylamine, arinoleamine, aminoazobenzene, amino alcohol (eg, ethanolamine), atalinol, amino benzoic acid, amino anthraquinone, amino acids (glycine, alanine, valine, leucine, serine, threonine, cysteine, methionine, phenine) Nilalanine, tryptophan, tyrosine, proline, cystine, gnoretami Acid, aspartic acid, glutamine, asparagine, lysine, arginine, histidine), adiline, or a polymer (eg, polyallylamine, polylysine) or a copolymer thereof; 4,4 ', 4 "-triaminotriphenyl Examples include polyamines (polyamines) such as methane, triamterene, spermidine, spermine, and putrescine.

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.

When the electrostatic layer is formed without being covalently bonded to the substrate or the carbon layer, for example, by introducing the amino group-containing compound into a film forming apparatus when forming a carbon layer, A carbon-based film containing an amino group is formed. Ammonia gas may be used as the compound to be introduced into the film forming apparatus. Further, the carbon layer may be a multi-layer, such as when a film containing an amino group is formed after forming the adhesion layer, and 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.When the electrostatic layer is formed without being covalently bonded to the substrate or the carbon layer, the affinity between the electrostatic layer and the substrate or the carbon layer, that is, the adhesion is increased. From the viewpoint, it is preferable to deposit the above-mentioned compound having an unsubstituted or monosubstituted amino group and a carbon compound on a substrate. 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 ordinary temperature, are preferable. As for the method of vapor deposition, the conditions for ionization vapor deposition, which is preferred for ionization vapor deposition, are preferably an operating pressure of 0.1 to 50 Pa and an accelerating voltage of 200 to 1000 V. When the electrostatic layer is formed by covalent bonding to a substrate or a carbon layer, for example, the surface of the substrate or the substrate provided with the carbon layer is chlorinated by irradiating ultraviolet rays in a chlorine gas, and then the amino group-containing Among the compounds, for example, a polyamine such as polyallylamine, polylysine, 4,4 ', 4 "-triaminotriphenylmethane, or triamterene is reacted to bind to a substrate, and an amino group is added to the terminal on the other side. When forming an electrostatic layer by immersing a substrate in a solution containing a compound having an unsubstituted or monosubstituted amino group, In addition, when polyallylamine is used as the amino group-containing compound, the adhesion to the substrate is excellent, and the amount of immobilized biomolecules is further improved. An electrostatic layer can also be formed by immersing the substrate in a solution in which a silane coupling agent coexists with the group-containing compound.

The thickness of the electrostatic layer is preferably lnm-500 μm.

The electrophoresis method that can be used for sample separation in the present invention is not particularly limited. For example, agarose gel electrophoresis, sieving agarose gel electrophoresis, denaturing agarose gel electrophoresis, polyacrylamide gel electrophoresis, SDS polyacrylinoleamide gel Examples include electrophoresis, isoelectric gel electrophoresis, and two-dimensional electrophoresis. Those skilled in the art can appropriately select the type of electrophoresis to be used based on 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 agarose gel has a larger gel network structure than polyacrylamide gel, DNA fragments of several to several hundred Kbp can be separated due to differences in length and molecular structure. The mobility is proportional to the size of the DNA fragment, since the charge state of the entire DNA fragment mainly depends on the number of phosphate groups. When electrophoresis is performed with intermittent changes in the direction of the electric field, giant DNA such as yeast chromosomes can be separated (pulse field electrophoresis). Polyacrylamide gel electrophoresis of nucleic acids is mainly used for the analysis of DNA fragments. By utilizing the fine network structure of polyacrylamide gel, short-chain (1 Kbp) fragments are compared with agarose gel electrophoresis. This is a method of separating data based on length and structure. Due to the strong influence of DNA conformation, the estimation of DNA chain length is limited to double-stranded DNA migration. Since single-stranded DNA is expected to take various structures, there is no correlation between mobility and its DNA chain length, and it is often detected as multiple bands. Even slight differences in DNA bases cause structural changes, which are reflected in the migration pattern. A DNA fragment analysis method (SSCP: Single-Strand Conformation Polymorphism) using this method has also been developed and used for gene mutation analysis. Double-stranded DNA fragments containing special sequences (such as repetitive sequences and base bias) are known to distort the DNA structure, and polyacrylamide gel electrophoresis can be used to analyze the structure and function of DNA. In a denaturing gel containing urea or the like, single-stranded DNA can be separated according to the chain length without being affected by the structure. SDS (Sodium dodecyl sulfate) -polyacrylamide gel electrophoresis (SDS-PAGE) is a method of denaturing the higher-order structure of a target protein and separating it based on differences in molecular weight. Polyacrylamide gels are suitable for separating proteins and polypeptides of 100-200KDa or less due to the small pore size in the gel. It is the most commonly used method for protein electrophoresis because of its simple operation and high reproducibility. Normally, a reducing agent such as β-mercaptoethanol or DTT (Dithiothreitol) is added during the preparation of the electrophoresis sample to cut the S—S bond (disulphide bond) of the protein. Since the charge of the molecule is almost determined by the amount of SDS bound, the polypeptide molecules can be separated according to the molecular weight by electrophoresis. Since SDS is a strong anionic surfactant, it is also suitable for solubilizing insoluble proteins such as membrane proteins.

Isoelectric focusing is an electrophoresis method that separates proteins using the difference in isoelectric point (pi) and measures and analyzes the isoelectric point of the target protein. The charge of amino acid side chain amino terminal and carboxyl terminal constituting protein changes depending on pH conditions, and the value of pH at which the total charge becomes zero is the isoelectric point. To perform isoelectric focusing, it is necessary to create a pH gradient in the electrophoresis gel. When a sample is added to a running gel and an electric field is applied, each protein migrates through the gel, forming a pH gradient towards the same pH as the intrinsic pi. pH gradient gels can be prepared by adding an amphoteric carrier (carrier ampholite) to the gel and applying an electric field to form a pH gradient, or by preparing gels using acrylamide derivatives having various pi side chains. At the same time, there is a method for forming a pH gradient (IPG method: Immobilized pH gradient), and in proteomics research, the IPG method, which is excellent in all of the separation ability, reproducibility, and allowable amount of addition, is mainly used. A precast gel (Immobiline DryStrip Gel) dedicated to the IPG method is commercially available. The resolution of isoelectric focusing using carrier ampholite is 0.01-0.02 pH units, and the IPG method can separate even 0.01-pH units. Two-dimensional electrophoresis is a two-step electrophoresis. This is a method of separating proteins two-dimensionally by using. Generally, in the first dimension, proteins are separated by isoelectric focusing, and in the second dimension, molecular weight is separated by SDS-PAGE. Both methods have very high resolution and can separate whole cell proteins into thousands of spots. Again It is common to use the immobilized pH gradient method (IPG method), which has excellent realism and resolution, for the first dimension electrophoresis. In order to obtain more spots, only the target pH part can be separated using a narrow pH IPG gel based on the results of separation over a wide pH range, or second-dimensional electrophoresis can be performed using a large gel of 20 cm or more. You can do it too.

In the present invention, it is preferable to use agarose gel electrophoresis to separate DNA and RNA, and to use SDS polyacrylamide gel electrophoresis and two-dimensional It is preferred to use These electrophoresis methods can be performed by a method commonly used by those skilled in the art.

After the electrophoresis, the gel is cut into a size that can fit on the solid support to be used, and the gel and the solid support are brought into close contact with each other, and the analyte separated in the gel is transferred onto the solid support of the present invention. The method for transferring to a solid support is not particularly limited, and a method usually used in the art can be used. For example, a set of capillaries utilizing capillary action, vacuum-type blotting using a pump, and electro-blotting using an electric method can be used. When transcribe | transferring a nucleic acid, it is preferable to use the capillaries set | blotting. When transcribe | transferring a peptide, it is preferable to use elect opening blotting.

In electroblotting, any of tank type, semi-dry type and semi-wet type can be used.However, semi-dry type electroblotting is used from the viewpoint of the use of a small amount of knocker and short reaction time. It is preferred to use. As the blotting device, an electroblotting device commonly used in the art can be used. The energizing conditions in the electroblotting are a constant voltage, 200 V or less, preferably 0.1 to 10 V, preferably for 1 to 500 minutes, preferably 5 to 100 minutes. However, if the voltage is higher than the oxidation potential of the metal substrate, the metal is eluted. Therefore, it is preferable to perform the process at a voltage lower than the oxidation potential of the substrate metal.

Hereinafter, one embodiment of electrophoresis and transcription in the present invention when analyzing a protein 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 deactivate the proteolytic enzymes present in the sample and to effectively denature the protein with SDS and / 3-mercaptoethanol. Then SD Inject a fixed amount into each lane of the S-polyacrylamide gel, and run glycine-tris buffer containing SDS as a buffer for electrophoresis at a constant voltage for a fixed time. After the electrophoresis, the gel is immersed in a pre-cooled glycine-Tris buffer (transfer buffer 1) containing methanol for a certain period of time to equilibrate. Subsequently, the gel is attached to the electroblotting device with the cathode side and the transfer solid support 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 arrange a filter paper containing a buffer or ion-exchanged water between the cathode and the gel and between the anode and the solid support. Examples of the buffer to be 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 a buffer containing Tris and ε-aminocaproic acid is used, the concentration of ε-aminocaproic acid is usually 100 mM or less, preferably -1000 mM, more preferably 1-1300 mM. The filter paper on the anode side preferably contains ion-exchanged water.

[0024] In another embodiment of the present invention, the target substance is transferred from the gel after electrophoresis to a membrane as used in the related art, and further transferred from the membrane onto the solid support of the present invention. Thereby, the substance separated in the gel can be transferred and held on the solid support. Examples of the material of the membrane that can be used in this case include nitrocellulose, PVDF (polyvinylidene fluoride), nylon, and positively charged nylon. In the transcription of a protein, it is preferable to use PVDF having the highest binding ability of the protein. Even in the transcription of nucleic acid, it is preferable to use PVDF having less nonspecific adsorption of nucleic acid. Gel force of migrating substance Transfer to the membrane and transfer from the membrane to the solid support can be carried out by the same method as described above. Gel force When transferring to a membrane, it is preferable to use electroblotting. The energization conditions for electroblotting are 0.1 to 50 V, preferably for 5 to 120 minutes. In the transfer from the membrane to the solid support, it is preferable to use an electroblotting device.

[0025] In another embodiment of the present invention, a substance separated by electrophoresis is transferred and held on a solid support, and a substance that interacts with the substance is reacted to form a complex. Mass spectrometry can also be performed by ionizing the complex thus formed. As a mass spectrometry method, it is possible to use a method of analyzing atoms and molecules of ions based on the difference in mass using electric interaction. Such mass spectrometers have three different functions of ion production 'separation' detection. When a protein is transcribed and held on a solid support by the method described above, an antibody against the protein is reacted to form a complex, and the complex is irradiated with a laser or the like to be ionized. Enables mass spectrometry. When a nucleic acid such as DNA or RNA is transcribed 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. To perform mass spectrometry. As other interactions, for example, an enzymatic reaction, a biotin-streptavidin interaction, and 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 specifically interacting with the probe molecule can be analyzed.

In another embodiment of the present invention, for the purpose of analyzing interacting substances, after forming a complex of interacting substances in a solution, the complex is subjected to electrophoresis and separated by electrophoresis. The resulting composite can be transferred and held on a solid support, and mass spectrometry can be performed by ionizing the composite on the solid support. Since such an analysis method forms a complex in a solution, it is advantageous for an analysis that requires a high degree of retention of the three-dimensional structure of a substance to be analyzed, such as a 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. The types of ionization methods that can be used for mass spectrometry include matrix-assisted laser desorption (MALDI), ionization by electron impact (EI), photoionization, and radioisotope force. Ionization method, secondary ionization method, fast atom bombardment ionization method, field ionization ionization method, surface ionization ionization method, chemical ionization (CI) method, field ionization method (FI) method, ionization method using spark discharge And the like, and a matrix assisted laser desorption (MA LDI) method and an ion bombardment (EI) method by electron impact are preferable. Separation modes include linear or non-linear time-of-flight (TOF), single or multiple quadrupole, single or multiple magnetic sectors. First, Fourier Transform Ion Cyclotron Resonance (FTICR), ion capture, high frequency and ion capture / flight time, etc., which use linear or non-linear reflection time of flight (TOF), high frequency and ion capture / flight time. preferable. Mass spectrometry can be performed by combining the above-described ionization method with a separation mode including a separation mode or a combination thereof, and a detection mode such as an electrical record and a photographic record. Such mass spectrometers have three different functions of ion generation, separation and detection. From the viewpoint of ionizing a polymer 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 procedure of mass spectrometry using MALDI-TOF MS will be described.

A matrix such as para-cyanohydroxycinnamic acid or sinapinic acid is added to the solid support of the present invention on which the substance to be analyzed is immobilized, and dried. Next, the solid support is set on a flat target of MAL DI-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, create a parameter file for automatic measurement, a process file for data processing and database analysis performed after measurement, and a sample list. Data processing can be performed on MassLynx using ProteinLynx software. A mass spectrum is created from the acquired data, and the created statistic is converted to monoisotopic peak data after increasing the accuracy using MaxEnt 3 software (Micromass). Next, perform calibration to obtain final data with a mass error of about 50 ppm. From this data we can determine the exact mass of interacting proteins.

Following mass spectrometry, protein amino acid sequence analysis and identification can be performed. Set the analysis mode of MA LDI-TOF MS to a mode that can detect post-source decay (PSD) spectra, and analyze the amino acid sequences of interacting proteins. Then, search the SWISSPROT database based on the amino acid sequence data to identify the protein. Or MALDI—Analyte is fixed on TOF / TOF MS or MALDI Q-TOF MS By setting the solid support, the amino acid sequence IJ can be analyzed and the interacting proteins can be identified.

Hereinafter, the present invention will be described more specifically with reference to Examples, but the present invention is not limited to these Examples.

Example

(Example 1) Transfer of protein to Ti-Pt-DLC solid support

Creating a solid support

A Ti layer and a Pt layer were formed on a 76 mm X 26 mm X 1.1 mm slide glass by magnetron sputtering. The sputtering conditions are as follows. The thickness of the formed metal layer was 100 nm for each of the Ti layer and the Pt layer.

[0029] [Table 1]

[0030] Then, a diamond-like carbon layer was formed on the substrate on which the metal layer was formed. The formation of the diamond-like carbon layer was performed by ionization vapor deposition under the following conditions. The thickness of the generated diamond-like carbon layer was 20 nm.

[0031] [Table 2]

Vb: acceleration voltage, Va: anode voltage

After the diamond-like carbon layer is formed on the substrate as described above, the surface is chlorinated by irradiating ultraviolet rays for 1 minute in chlorine gas and irradiating ultraviolet rays for 10 minutes in ammonia gas. Amination was performed to produce a solid support.

[0033] SDS—electrophoresis by PAGE method Cy3_Protein A (1.5 g, SIGMA), E. coli protein (0.5 · ig / ig) and Michiruichi (Prestained Broad Range, 0.5 µl, BIO RAD) as samples Electrophoresis was performed using a device for SDS-PAGE (AE-7300 manufactured by ATTO). A 10% polyacrylamide gel was used as a swimming gel. Glycine tris buffer (pH 8.3) containing 0.1% SDS was used as the electrophoresis buffer, and electrophoresis was performed at 200 V for 35 minutes. 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.). Cy3_Protein A band was detected around 50 kDa.

[0034] Electroblotting

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

[Table 3]

[0036] Measurement of fluorescence intensity

The fluorescence intensity of the solid support after protein transfer was measured with FLA8000 (manufactured by Fuji Photo Film Co., Ltd.). The fluorescence intensity was measured as 28270, indicating that Cy3-protein A was immobilized on the surface of the solid support. confirmed. Subsequently, after the solid support was washed with PBS for 10 minutes, the fluorescence intensity was measured in the same manner. The fluorescence intensity was 9766, which was reduced to about 1/3. After blocking with a blocking reagent (Roche) for 1 hour and measuring the fluorescence intensity, there was no change in the fluorescence intensity. Next, 5001 of 0.05 / g // il Cy3-IgG was added and reacted at room temperature for 1 hour, washed with PBS at room temperature for 12 hours, and the fluorescence intensity was measured. Since the fluorescence intensity is 16448, which is increasing, the binding of protein A to IgG, that is, the binding ability of the transcribed protein is maintained. You can see that

(Example 2) Transfer of protein to stainless steel DLC solid support

Preparation of DLC solid support for stainless steel

A diamond-like carbon layer was formed on a stainless steel substrate. The stainless substrate was puff-polished in advance and further electropolished in order to reduce the smoothness and the fluorescent background. The formation of the diamond-like carbon layer was performed by ionization vapor deposition under the following conditions. The thickness of the generated diamond-like carbon layer was 20 nm. In addition, a solid support was prepared by introducing an amino group by ammonia plasma treatment.

[0038] [Table 4]

Vb: acceleration voltage, Va: anode voltage

[0039] Cy3_protein A (0.2 μg, SIGMA) was electrophoresed by SDS-PAGE (AT-6 AE-6530) in the same manner as in Example 1, and CBB staining was performed for 15 minutes after electrophoresis. After destaining, images were taken with LAS 1000 (manufactured by Fuji Photo Film Co., Ltd.).

[0040] The gel after electrophoresis was taken out, cut into a size to be placed on a solid support, and immersed in transfer buffer B (25 mM Tris, 5% methanol). The filter paper (manufactured by ATTO), which has been cut in advance to the size of the solid support, is transferred to a transfer buffer Α (0.3 M Tris, 5% methanol), C (25 mM Tris, 40 mM ε-aminocaproic acid, 5% ) Or ion-exchanged water. Next, filter paper, gel, and solid support were placed one on top of the other as shown in Fig. 1 in a semi-dried blotting device so that air bubbles did not enter, and electricity was applied at 8 V and 4 mA for 60 minutes to transfer proteins to the solid support. Transcribed. The solid support 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 a FLA8000 (manufactured by Fuji Photo Film Co., Ltd.).

[0041] (1) shows the result when the transfer was performed with both the cathode side and the anode side filter papers containing ion-exchanged water. Cy3_ protein A hardly escapes from the gel and No transfer was held on the carrier.

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

(3) shows the results when transfer was performed with filter paper I containing buffer C for transfer and filter papers II and III with ion-exchanged water. The gel was wiped of moisture before overlaying on a solid support. As a result, it was confirmed that Cy3_Protein A was not homogeneous but escaped from the gel. Further, transfer holding was observed on the solid support although the shape was not good.

(4) shows the results when the transfer buffer C was added to the filter paper I, the ion exchange water was added to the filter papers II and III, and the gel was transferred to the solid support without wiping the moisture. As a result, it was confirmed that Cy3_Protein A was not uniform but escaped from the gel. Cy3_protein A was well-transferred and retained on the solid support in good shape.

From the above, when transferring proteins in the gel to the solid support, the filter paper on the cathode side should contain a transfer buffer, and the filter paper on the anode side should contain ion-exchanged water, and the gel after the electrophoresis should be wiped off. It is believed that it is preferable to perform the transfer without the use.

(Example 3) Transfer of protein to stainless steel DLC solid support

Cy3-protein A (50 ng, SIGMA) and Cy3-IgA (100 ng, SIGMA) were electrophoresed by SDS-PAGE (ATTO AE-6530) in the same manner as in Example 1, and after completion of electrophoresis. After 15 minutes of CBB staining and destaining, images were taken with LAS1000 (Fuji Photo Film Co., Ltd.). For the migration of Cy3_IgA, a 12% polyacrylamide gel was used. In the same manner as in Example 2, a solid support was prepared.

The gel after electrophoresis was taken out, cut into a size on a solid support, and immersed in a transfer buffer (25 mM Tris, 5% methanol). Filter paper (manufactured by ATTO) previously cut to the size of the solid support was transferred to a transfer buffer CI (25 mM Tris, 40 mM ε-aminocaproic acid, 5% methanol), C2 (25 mM Tris, 400 mM ε-aminocapron). Acid, 5% methanol) or ion-exchanged water. Subsequently, filter paper, gel, and a solid support were stacked in the same manner as in FIG. 1 of Example 2 so as to prevent air bubbles from entering, and the filter and the solid support were placed in a semi-driving apparatus. At this time, use three filter papers on the cathode side that contain transfer buffer C1 or C2. For the three filter papers on the anode side, ion-exchanged water was used. Then, a current was applied at 2 V and 2 / i A for 60 minutes to transfer the protein to the solid support. The solid support after transfer was washed with ion-exchanged water at room temperature for 30 minutes and dried. The solid support and the gel after transfer were photographed with a FLA8000 (manufactured by Fuji Photo Film Co., Ltd.).

As a result, it was found that when the transfer buffer to be included in the filter paper on the cathode side during transfer to the solid support was Cl, that is, the concentration of ε-aminocaproic acid at 40 mM, the transfer retention efficiency was better. .

(Example 4) Transfer of protein from PVDF membrane to Ti-Pt_DLC solid support A diamond-like carbon layer was formed on the substrate in the same manner as in Example 1, and the surface was treated in an ammonia plasma using an ionization deposition apparatus. Was solidified to prepare a solid support 3.

Cy3_protein A was electrophoresed on a polyacrylamide gel in the same manner as in Example 1. After the electrophoresis, CBB staining was performed for 15 minutes, and after destaining, images were taken with LAS1000 (manufactured by Fuji Photo Film Co., Ltd.). Cy3_Protein A band was detected around 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 、, and gently shaken for 5 minutes. The PVDF membrane (manufactured by ATTO), which had been cut into gel size in advance, was immersed in a small amount of methanol for 5 seconds, immersed in about 100 ml of transfer buffer B, and shaken for 5 minutes or more. Two, one, and three filter papers, each of which was cut into gel size in advance, were immersed in 200 ml of each of transfer buffers A, B, and C. Subsequently, the filter paper, gel, and PVDF membrane were placed on a semi-driving device (Nihon Aid-Ichi) as shown in Fig. 2 so that air bubbles did not enter, and electricity was supplied at 15 V for 60 minutes. . 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, overlapped in the order shown in FIG. 3, and placed on a weight of 35 g / cm ² . After leaving at room temperature for 1 hour, the Cy3-Port Tin A on the membrane was transferred onto the solid support. Subsequently, the solid support was washed with PBS at room temperature for 20 minutes. After washing for minutes, it was dried. Then, the solid support was imaged with FLA8000 (manufactured by Fuji Photo Film Co., Ltd.). Fluorescence was detected at the corresponding position, indicating that Cy3_protein Tin A was transcribed and held on the solid support.

(Example 5) Transfer of protein to stainless steel DLC solid support

Cy3-labeled yeast protein (300 xg / lane) was electrophoresed by the SDS-PAGE method (ATTO AE-6530 type) in the same manner as in Example 1, and after the electrophoresis, FLA8000 (Fuji Photo Film Co., Ltd.) The image was taken with. In the same manner as in Example 2, a solid support was prepared.

The gel after electrophoresis was taken out, cut into a size to fit on the solid support, washed with 10% methanol, replaced with fresh 10% methanol, and further washed for 30 seconds. After washing, the gel is placed on a solid support, and a dialysis membrane and a filter containing 1 MHBO buffer (pH 8.0) are placed on the gel.

3 3

I put the paper. Then, current was applied at 2 V for 60 minutes to transfer the protein complex to the solid support. The solid support after transfer was washed with ultrapure water and dried. Then, an image of the solid support was taken with FL A8000 (manufactured by Fuji Photo Film Co., Ltd.).

As a result, it was found that the transfer efficiency was about 40%, and sufficient transfer was achieved.

(Example 6) Transfer of protein to stainless steel DLC solid support

Cy3_Protein A (50 ng, SIGMA) and Cy5_IgA (100 ng, SIGMA) were mixed in a solution, and electrophoresed by Native-PAGE method (ATTO AE-6530) as in Example 1. After the electrophoresis, images were taken with a FLA8000 (manufactured by Fuji Photo Film Co., Ltd.). In the same manner as in Example 2, a solid support was prepared.

The gel after electrophoresis was taken out, cut into a size on a solid support, and immersed in a transfer buffer (25 mM Tris, 5% methanol). The filter paper (manufactured by ATTO) cut in advance to the size of the solid support was immersed in a transfer buffer CI (25 mM Tris, 40 mM ε-aminocaproic acid, 5% methanol) or ion-exchanged water. Subsequently, filter paper, gel, and a solid support were stacked in the same manner as in FIG. 1 of Example 2 so as to prevent air bubbles from entering, and set in a semi-driving apparatus. At this time, the one containing the transfer buffer C1 was used for the three filter papers on the cathode side, and the one containing ion-exchanged water was used for the three filter papers on the anode side. Then, a current was applied at 2 V and 2 μA for 60 minutes to transfer the protein complex to the solid support. Turn The solid support after the transfer was washed with ion-exchanged water at room temperature for 30 minutes and dried. Then, the solid support and the gel after transfer were photographed with a FLA8000 (manufactured by Fuji Photo Film Co., Ltd.).

As a result, the fluorescence intensity of the image taken with the FLA8000 after transfer was about 35% compared to the fluorescence intensity of the image taken with the FLA8000 after the electrophoresis.

Industrial applicability

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

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

Claims

The scope of the claims

[I] A solid support having a carbon layer on the surface, on which a substance in a sample is separated by gel electrophoresis and then transferred and held in the gel.

[2] After separating the substance in the sample by gel electrophoresis, the substance separated in the gel is transferred to a membrane, and the substance transferred onto the membrane is further transferred and held, whereby the substance is immobilized. A solid support having a carbon layer on the surface.

[3] A solid support obtained by adding another substance interacting with the substance immobilized on the solid support according to claim 1 or 2 to form a complex.

[4] The solid support according to claim 1 or 2, wherein a complex of interacting substances is formed in a solution, and the complex is separated by gel electrophoresis.

[5] The solid support according to any one of claims 14 to 14, wherein the carbon layer is a diamond-like carbon layer.

[6] The solid support according to any one of [15] to [15], wherein the carbon layer has a thickness of 100 / im per monolayer.

7. The solid support according to claim 4, wherein the solid support further includes an amino group-containing compound that is present on the carbon layer on the surface of the substrate but is not covalently bonded to the carbon layer. .

[8] The solid support according to any one of [16] to [16], wherein the solid support further comprises an amino group-containing compound present on the carbon layer on the substrate surface and covalently bonded to the carbon layer.

[9] The solid support according to any one of [16] to [16], which is obtained by evaporating a compound having an unsubstituted or monosubstituted amino group and a carbon compound onto a substrate.

[10] The force described in claim 4, wherein the solid support having a carbon layer on the surface is immersed in a solution containing a compound having an unsubstituted or monosubstituted amino group. On the solid support.

[II] The solid support according to any one of claims 110, wherein the substance whose transcription is retained is a nucleic acid, a peptide or a complex thereof.

[12] A plurality of objects transferred and held on the solid support according to any one of claims 11 to 11 Mass spectrometry by desorbing / ionizing a substance or complex.

[13] A solid support having a carbon layer on the surface, for use in the method according to claim 12.