WO2004019025A1 - 固体支持体及び該固体支持体上に固定化された複数の物質又は複合体を脱離/イオン化することにより質量分析する方法 - Google Patents
固体支持体及び該固体支持体上に固定化された複数の物質又は複合体を脱離/イオン化することにより質量分析する方法 Download PDFInfo
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- WO2004019025A1 WO2004019025A1 PCT/JP2003/010406 JP0310406W WO2004019025A1 WO 2004019025 A1 WO2004019025 A1 WO 2004019025A1 JP 0310406 W JP0310406 W JP 0310406W WO 2004019025 A1 WO2004019025 A1 WO 2004019025A1
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/26—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating electrochemical variables; by using electrolysis or electrophoresis
- G01N27/416—Systems
- G01N27/447—Systems using electrophoresis
- G01N27/44704—Details; Accessories
- G01N27/44717—Arrangements for investigating the separated zones, e.g. localising zones
- G01N27/44739—Collecting the separated zones, e.g. blotting to a membrane or punching of gel spots
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N27/00—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means
- G01N27/62—Investigating or analysing materials by the use of electric, electrochemical, or magnetic means by investigating the ionisation of gases, e.g. aerosols; by investigating electric discharges, e.g. emission of cathode
- G01N27/622—Ion mobility spectrometry
- G01N27/623—Ion mobility spectrometry combined with mass spectrometry
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- H—ELECTRICITY
- H01—ELECTRIC ELEMENTS
- H01J—ELECTRIC DISCHARGE TUBES OR DISCHARGE LAMPS
- H01J49/00—Particle spectrometers or separator tubes
- H01J49/02—Details
- H01J49/04—Arrangements for introducing or extracting samples to be analysed, e.g. vacuum locks; Arrangements for external adjustment of electron- or ion-optical components
- H01J49/0409—Sample holders or containers
- H01J49/0418—Sample holders or containers for laser desorption, e.g. matrix-assisted laser desorption/ionisation [MALDI] plates or surface enhanced laser desorption/ionisation [SELDI] plates
Definitions
- the present invention relates to a solid support and a method for mass spectrometry by desorption of a plurality of substances or complexes immobilized on the solid support.
- the present invention provides a mass spectrometer for rapidly analyzing a solid support having a substance immobilized thereon by transferring the substance separated in a gel, and nucleic acids and proteins immobilized on the solid support. And to a method of analyzing. Background art
- 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.
- peptides and proteins are molecules in which 20 kinds of L-CK-amino acids are linked 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 in vivo. It is positioned as an indispensable means for structural analysis of biomolecules such as.
- mass spectrometers a laser desorption / ionization time-of-flight mass spectrometer is attracting attention as a useful means of analyzing biomolecules because it can ionize macromolecules such as DNA and proteins.
- Laser Desorption In a Z-ionization-time-of-flight mass spectrometer, a sample is irradiated with a laser and the ions desorbed therefrom are accelerated by an electric field. Then, ions with smaller mZ z values, that is, lighter ions, fly faster and reach the detector. Laser desorption Z ionization-time-of-flight mass spectrometry is based on the fact that ion flight time differs due to the difference in mass-to-charge ratio (mZ z value). It is a method of performing analysis.
- a general laser desorption / ionization-time-of-flight mass spectrometer on the market places each purified sample on a sample board and performs mass spectrometry one by one. In other words, it was necessary to irradiate each sampled sample with a laser and analyze it one by one. Therefore, if an unpurified sample is separated by electrophoresis, the gel after electrophoresis must be cut out for each band, purified, and then subjected to mass spectrometry using a laser desorption / ionization-time-of-flight mass spectrometer. Therefore, it was very difficult to analyze a large number of samples quickly.
- 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. Disclosure of the invention
- the present inventors have conducted intensive studies in order to solve the above-mentioned problems, and as a result, the substance in the sample was gelled. After separation by gel electrophoresis, the material separated and developed in the gel is immobilized on a solid support with a carbon layer formed on the surface, and this is desorbed.
- the present invention includes the following inventions.
- a solid support having a carbon layer on the surface in which a substance in a sample is separated by gel electrophoresis, and then the separated substance is immobilized by transferring the separated substance into the gel.
- the substance separated in the gel is transferred to a membrane, and the substance transferred on the membrane is further transferred to immobilize the substance.
- FIG. 9 A solid support having a carbon layer on the surface for use in the method according to (8).
- Figure 1 shows that in Example 1, Cy3-protein A and E. coli proteins were electrophoresed by SDS-PAGE, and the gel after electrophoresis was stained with CBB for 15 minutes and destained, followed by LAS 1000 ( The image was taken with Fuji Photo Film Co., Ltd.).
- FIG. 2 shows the gel after electrophoresis transferred to the solid support 1 in Example 1, and the fluorescence intensity of the transferred solid support was photographed with an FLA8000 (manufactured by Fuji Photo Film Co., Ltd.). .
- FIG. 1 shows that in Example 1, Cy3-protein A and E. coli proteins were electrophoresed by SDS-PAGE, and the gel after electrophoresis was stained with CBB for 15 minutes and destained, followed by LAS 1000 ( The image was taken with Fuji Photo Film Co., Ltd.).
- FIG. 2 shows the gel after electrophor
- Example 3 shows the solid support 1 to which the gel after electrophoresis was transferred in Example 1, washed with PBS for 10 minutes, dried, and then photographed.
- Figure 4 shows that in Example 1, the solid support 1 on which the gel after electrophoresis was transferred was washed with PBS for 10 minutes, blocked with a blocking reagent for 1 hour, and then added with Cy3-IgG. The reaction was carried out at room temperature for 1 hour, washed with PBS for 12 hours (room temperature) and photographed.
- Figure 5 shows that in Example 2, Cy3-protein A was electrophoresed by SDS-PAGE, and the gel after electrophoresis was subjected to CBB staining for 15 minutes and destained, followed by LAS 1000 (Fuji Photo Film Co., Ltd.). Image).
- FIG. 6 shows the arrangement in Example 2 when proteins are transferred from the gel after electrophoresis to the solid support 2.
- Figure 7 shows that in Example 2, the solid support 2 on which the protein was transferred from the gel after electrophoresis was washed with PBS for 30 minutes and dried, and the gel after transfer was labeled FLA8000 (Fuji Photo Film). (Manufactured by Co., Ltd.).
- Figure 8 shows that in Example 3, Cy3-protein A and Cy3-IgA were electrophoresed by the SDS-PAGE method, and the gel after electrophoresis was stained with CBB for 15 minutes and destained. After that, images were taken with LAS 1000 (manufactured by Fuji Photo Film Co., Ltd.).
- FIG. 1 shows that in Example 2, the solid support 2 on which the protein was transferred from the gel after electrophoresis was washed with PBS for 30 minutes and dried, and the gel after transfer was labeled FLA8000 (Fuji Photo Film). (Manufactured by Co.
- FIG. 9 shows that the solid support 2 on which the protein was transferred from the gel after electrophoresis was washed with PBS for 30 minutes and dried in Example 3, and the gel after transfer was FLA8000 (Fuji Photo File). (Ilm Co., Ltd.).
- Figure 10 shows that 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 LAS 1000 (Fuji Photo Film Co., Ltd.). It was taken.
- FIG. 11 shows the arrangement in Example 4 when proteins are transferred from a gel after electrophoresis to a PVDF membrane.
- FIG. 12 shows an arrangement when transferring a protein from the PVDF membrane to the solid support 3 in Example 4.
- FIG. 13 shows the solid support 3 on which the protein was transferred from the PV DF membrane in Example 4, washed with PBS for 20 minutes, dried, and imaged with a FLA8000 (Fuji Photo Film Co., Ltd.). It is.
- FIG. 14 is a diagram showing the results of a TOF-MS analysis of a stainless steel DLC solid support on which a leg insulin binding protein was immobilized in Example 5.
- FIG. 15 is a diagram showing the results of Example 5 in which leg insulin was allowed to interact with a stainless steel DLC solid support on which leg insulin binding protein was immobilized and analyzed by TOF-MS.
- the sample is separated by gel electrophoresis, and the gel after electrophoresis is brought into close contact with a solid support having a carbon layer formed on the surface thereof, whereby the substance to be analyzed separated and developed in the gel is converted to the solid.
- a solid support having a carbon layer formed on the surface thereof, whereby the substance to be analyzed separated and developed in the gel is converted to the solid.
- Transfer immobilization on a support Then, a plurality of substances are subjected to mass analysis by desorbing // ionizing the substance immobilized on the solid support.
- the substance that can be immobilized on a solid support and analyzed is not particularly limited, but PNA can be used for biomolecules such as nucleic acids such as DNA and RNA and peptides.
- peptide nucleic acid includes oligonucleotides, polypeptides, and proteins. It is particularly advantageous in that high molecular weight substances can be analyzed. Samples to be subjected to gel electrophoresis containing these substances are not particularly limited, and 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 is not particularly limited as long as it has a carbon layer on the surface of the substrate and can immobilize these biomolecules. It is preferable that the carbon layer is subjected to a specific chemical modification. This is because by applying a specific chemical modification, the substance to be analyzed can be easily bound and stably immobilized.
- the substrate means a base material on which a carbon layer is formed, and such a base material is not particularly limited.
- a base material is not particularly limited.
- gold, silver, copper, aluminum, tungsten, tungsten, Metals such as 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; paper; Plastics such as fluororesins; and mixtures of plastics with the above metals, ceramics, diamonds and the like.
- a material in which a metal layer made of platinum, titanium or the like is formed on the surface of glass or plastic can also be used.
- the metal layer can be formed by sputtering, vacuum evaporation, ion beam evaporation, electroplating, electroless plating, or the like.
- a high voltage is applied to the solid support, and the substrate has conductivity, for example, stainless steel or aluminum. And titanium are preferred.
- the carbon layer formed on the substrate is not particularly limited, but may be synthetic diamond, high-pressure synthetic diamond, natural diamond, soft diamond (for example, diamond-like carbon), amorphous carbon, carbon-based material (for example, Any of the followings: ait, fullerene, carbon nanotube), a mixture thereof, or a laminate thereof, hafnium carbide, niobium carbide, silicon carbide, tantalum carbide, thorium charcoal, titanium carbide, uranium carbide, tungsten carbide And a layer made of zirconium carbide, molybdenum carbide, chromium carbide or vanadium carbide, and a diamond-like carbon (DLC) layer is preferable.
- soft diamond means so-called diamond-like carbon (DLC: Diamond Li
- imperfect diamond structure which is a mixture of diamond and carbon, such as ke Carbon
- the carbon layer has excellent chemical stability and can withstand subsequent reactions in chemical modification and binding with the analyte, and the bond is stable due to the covalent bond with the analyte. It is advantageous in that it is transparent to the detection system UV because there is no UV absorption, and that it can be energized during electroblotting. It is also advantageous in that non-specific adsorption is small in the binding reaction with the analyte.
- the formation of the carbon layer can be performed by a known method.
- a known method microphone mouth-wave plasma C VD (Chemical vapordeposit) method, ECRCVD (Electriccyclotron resonance electromicavaordeposit) method, IPC (Inductively coupled plasma) method, DC sputtering method, ECR (E1 ectriecyclotronresonance) snorting method.
- a source gas (methane) is decomposed by a glow discharge generated between electrodes by high frequency, and a DLC (diamond-like carbon) layer is synthesized on a substrate.
- the source gas (benzene) is decomposed and ionized using thermionic electrons generated by the tungsten filament, and a carton layer is formed on the substrate by a bias voltage.
- the DLC layer may be formed by ionization vapor deposition in a mixed gas consisting of 1 to 99% by volume of hydrogen gas and 99 to 1% by volume of methane gas remaining.
- a DC 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 generate a plasma of carbon atoms from the cathode.
- a solid graphite material cathode evaporation source
- anode a vacuum vessel
- carbon ions in the plasma are accelerated toward the substrate to form a carbon layer.
- 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.
- 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 a monolayer to about 100 ⁇ . If the thickness is too small, the surface of the underlying solid support may be locally exposed, On the other hand, when the thickness is increased, the productivity is deteriorated. Therefore, the thickness is preferably 2 ⁇ to 1 ⁇ , more preferably 5 nm to 500 nm. Note that all of the solid support may be made of a carbon material.
- the solid support of the present invention is preferably in the form of a flat plate for direct laser desorption / ionization / time-of-flight mass spectrometry after transferring the substance from the gel after electrophoresis.
- the size is not particularly limited, it is usually about 10 to 20 mm in width ⁇ 10 to 200 111111 in thickness and about 0.1 to 20 mm in thickness.
- the surface of the substrate on which the carbon layer is formed is chemically modified to be activated 1 ".
- a person skilled in the art can appropriately select the substance that promotes the immobilization of the substance to be formed, and is not particularly limited. Examples thereof include an amino group, a carboxyl group, an epoxy group, a formyl group, a hydroxyl group, and a carpoimide group. It is also effective to introduce a metal chelate such as nickel chelate and coparto chelate.
- an amino group can be carried out, for example, by irradiating the carbon layer with ultraviolet light in chlorine gas to chlorinate and then irradiating the carbon layer with ultraviolet light in ammonia gas.
- it can be carried out by reacting a polyvalent amine such as methylenediamine and ethylenediamine with the chlorinated carbon layer.
- it is also performed by treating the surface of the carbon layer with ammonia plasma or ethylenediamine plasma.
- the carboxyl group can be introduced, for example, by reacting a suitable polyvalent rubonic acid with the carbon layer aminated as described above.
- the organic peracid include peracetic acid, perbenzoic acid, diperoxyphthalic acid, formic acid, and trifluoroperacetic acid.
- the formyl group can be introduced, for example, by reacting glutaraldehyde with the carbon layer aminated as described above.
- the introduction of the hydroxyl group can be carried out, for example, by reacting the chlorinated carbon layer with water.
- the introduction of the carpoimide group can be carried out, for example, by reacting the carbon layer aminated as described above with a carposimid.
- the active ester group can be introduced, for example, by irradiating the carbon layer with ultraviolet light in chlorine gas to chlorinate the surface, then irradiating the carbon layer with ultraviolet light in ammonia gas, aminating the surface, and then applying an appropriate acid chloride or dicarboxylic acid. It can be carried out by decarboxylation using an anhydride, and dehydration-condensing the terminal carboxyl group with carposimide or hexylcarbodiimide and N-hydroxysuccinimide. By this treatment, a group in which an active ester group such as an N-hydroxysuccinimide ester group is bonded to the terminal of the hydrocarbon group via an amide bond can be formed.
- nucleic acids such as DNA and RNA are immobilized
- immobilizing a peptide it is preferable to introduce an N-hydroxysuccinimide group, a carbodiimide group, an epoxy group, a formyl group, or a metal chelate.
- metal chelates such as polyhistidine sequences can be used.
- a peptide having a label having affinity with the protein can be immobilized effectively and stably.
- the metal chelate is introduced by, for example, chlorinating the substrate on which the carbon layer is formed, then aminating the substrate, and then adding a halocarboxylic acid such as chloroacetic acid to introduce a chelating ligand. It can.
- a label such as a polyhistidine sequence can be introduced by a method known to those skilled in the art.
- the electrophoresis method that can be used for separating a sample in the present invention is not particularly limited. Examples include SDS polyacrylamide gel electrophoresis, isoelectric focusing gel electrophoresis, and two-dimensional electrophoresis. Those skilled in the art can appropriately select the type of the electrophoresis method 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 H ⁇ to several hundred Kbp can be separated based on 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.
- electrophoresis is performed with intermittent changes in the electric field direction, large DNAs 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.
- short chains ⁇ lKbp
- agarose gel electrophoresis This is a method to separate fragments based on length and structure. Due to the strong influence of the three-dimensional structure (conformation) of the DNA, the estimation of the DNA chain length is limited to the case of electrophoresis of double-stranded DNA. Since single-stranded DNAs are expected to take various structures, there is no correlation between mobility and the length of the DNA chain, and it is often detected as multiple bands. Even slight differences in DNA bases cause structural changes, which are reflected in migration patterns .
- S SCP Single-Strand Configuration Polymorphism 3 ⁇ 4 Developed; ⁇ Used for gene mutation analysis.
- Double-stranded DNA fragments containing special sequences are known to distort the DNA structure, and polyacrylamide gel electrophoresis is also used for structural and functional analysis of DNA. it can.
- polyacrylamide gel electrophoresis is also used for structural and functional analysis of DNA. it can.
- single-stranded DNA can be separated according to the chain length without being affected by the structure.
- SDS-PAGE Single polyacrylamide gel electrophoresis
- SDS-PAGE Single polyacrylamide gel electrophoresis
- Polyacrylamide gels are suitable for separating proteins and polypeptides of 100 to 200 KDa or less due to the small pore size in the gel. It is the most commonly used technique for protein electroswimming because of its simple operation and high reproducibility.
- a reducing agent such as 3-mercaptoethanol or DTT (Dithiothrelite) is added to cleave the S—S bond (disulphide bond) of the protein.
- 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 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 terminus and lipoxyl terminus constituting protein changes depending on the pH condition, and the pH value at which the sum of the charges 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 the running gel and an electric field is applied, each protein migrates through the gel, forming a pH gradient towards the same pH as the unique pI.
- an amphoteric carrier carrier
- IPG method Imm obilized pH gradient
- IPG method Imm obilized pH gradient
- proteomics research resolution and reproducibility
- a precast gel (Immobiline Dry Strip Gel) dedicated to the IPG method is commercially available.
- the resolution of isoelectric focusing using carrier ampholyte is from 0.01 to 0.02 pH units, and the IPG method can separate even 0.001 pH units.
- Two-dimensional electrophoresis is a method of separating proteins two-dimensionally by two-step electrophoresis.
- proteins are separated by isoelectric focusing
- molecular weight is separated by SDS-PAGE.
- Both methods have very high resolution, and can separate total cellular proteins into thousands of spots.
- IPG method immobilized pH gradient method
- the target pH part can be separated using an N arrow pH I PG gel based on the results of separation over a wide pH range, or the second dimension can be obtained using a large gel of 20 cm or more.
- Electrophoresis can also be performed.
- agarose gel electrophoresis when separating DNA and RNA, it is preferable to use agarose gel electrophoresis, and when separating peptides, use SDS polyacrylamide gel electrophoresis and two-dimensional electrophoresis. Is preferred. These electrophoresis methods can be performed by a method commonly used by those skilled in the art.
- the gel is cut into a size that fits 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.
- a set of capillaries utilizing the capillary phenomenon, suction by a pump Vacuum-type blotting and electroblotting using an electric technique are included.
- transcribing a nucleic acid it is preferable to use a set of capillaries, and when transcribing a peptide, it is preferable to use electroblotting.
- any of tank type, semi-dry type and semi-jet type can be used.However, semi-dry type electro-plotting is used from the viewpoints of small buffer usage and short reaction time. It is preferred that As the blotting device, an electroblotting device commonly used in the art can be used.
- the energization conditions in electroblotting are constant voltage, 200 V or less, preferably 0.1 to 10 V, for 1 to 500 minutes, and 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.
- the substance to be analyzed may be immobilized by direct spotting on the solid support of the present invention without electrophoresis, and this may be analyzed by TOF-MS or the like.
- the substance on the solid support may be immobilized, and the substance interacting with the substance may be further immobilized by spotting, and the interacting substance may be analyzed by TOF-MS or the like.
- the following describes one embodiment of electrophoresis and transcription in the present invention when analyzing a protein in a sample.
- 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.
- a fixed amount is injected into each lane of the SDS-polyacrylamide gel, and glycine-tris buffer containing SDS is used as a buffer for electrophoresis and electrophoresed at a constant voltage for a fixed time.
- the gel After the electrophoresis, the gel is immersed in a pre-cooled glycine tris buffer (transfer buffer) containing methanol for a certain period of time to equilibrate. I do. Subsequently, the gel is mounted on an electro-cutting apparatus 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 and a constant 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.
- transfer buffer glycine tris buffer
- Examples of the buffer to be included in the filter paper on the cathode side include those containing Tris, ⁇ -aminocaproic acid, acetic acid, EDTA, phosphoric acid, boric acid, tartaric acid, SDS, and the like.
- the concentration of aminocaproic acid is preferably about 100 OmM or less.
- the filter paper on the positive electrode side preferably contains ion-exchanged water.
- 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.
- the material separated in the gel can be immobilized on a solid support.
- examples of the material of the membrane that can be used include nitrocellulose, PVDF (polyvinylidene fluoride), nylon, and positive-chargeable resin.
- PVDF polyvinylidene fluoride
- nylon nylon
- positive-chargeable resin In the transcription of a protein, it is preferable to use PVDF, which has the highest binding ability of the protein, and in the transcription of nucleic acid, it is preferable to use PVDF, which causes less nonspecific adsorption of nucleic acid.
- the transfer of the migrating substance from the gel to the membrane and the transfer from the membrane to the solid support can be carried out by the same method as described above.
- the transfer from the gel to the membrane it is preferable to use electroblotting, and the energizing condition in electroblotting is 0.1 to 50 V, preferably about 5 to 120 minutes.
- the energizing condition in electroblotting is 0.1 to 50 V, preferably about 5 to 120 minutes.
- an electroblotting device For transfer from the membrane to the solid support, it is preferable to use an electroblotting device.
- the method comprises immobilizing a substance separated by electrophoresis on a solid support, reacting a substance interacting with the substance to form a complex, and ionizing the formed complex.
- mass spectrometry is a technique for analyzing atomic and molecular ions based on their differences in mass using electrical interaction. Mass spectrometers have three different functions: ion generation, separation, and detection. When a protein is immobilized on a solid support by the method described above, an antibody against the protein is reacted to form a complex, and the complex is ionized by irradiation with a laser or the like. Enables mass spectrometry.
- a nucleic acid such as DNA or RNA
- a nucleic acid complementary to the nucleic acid is hybridized to the nucleic acid on the solid support, and the formed double strand is formed.
- Mass spectrometry can be performed by ionization. As other interactions, for example, an enzyme 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 that specifically interacts with the probe molecule can be analyzed. In addition, mass spectrometry can be performed by ionizing only molecules that interact with the molecules immobilized on the solid support.
- 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.
- mass spectrometry by means such as laser desorption / ionization-time-of-flight mass spectrometry.
- the types of ionization methods that can be used in mass spectrometry include matrix-assisted laser desorption (MAL DI), ionization by electron impact (EI), photoionization, and large ⁇ or LET emitted from radioisotopes.
- ionization method using j3 line secondary ionization method, fast atom impact ionization method, field ionization method, surface ionization method, chemical ionization (CI) method, field ionization (FI) method, spark discharge Matrix-assisted laser desorption (MALD I) and ionization by electron impact (EI) are preferred.
- Separation modes include linear or nonlinear time-of-flight (TOF), single or multiple quadrupole, single or multiple magnetic sectors, Fourier transform ion cyclotron resonance (FT ICR), ion capture, high frequency and Ion capture Z flight time, etc., and those using linear or non-linear reflection time of flight (TOF), high frequency and ion capture Z flight time are preferred.
- Ions as above Mass spectrometry can be performed by a combination of the separation method and a separation mode including the separation mode or a combination thereof, an electrical record, and a detection mode such as a photo record. From the viewpoint of ionizing a high molecular substance such as a biomolecule and analyzing a plurality of substances on a solid support, it is preferable to use laser desorption Z-ionization-time-of-flight mass spectrometry.
- 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.
- the solid support is placed on a flat target of MALD I-TOF MS.
- mass spectrometry is started using Mass Lynx software or the like.
- MassLynx controls all measurements and analysis. During 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 the PlotteLynx software.
- a mass spectrum is created from the acquired data, and the created spectrum is converted to monoisotopic / peak data after its accuracy is improved by MaXEnt3 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.
- protein amino acid sequence analysis and identification can be performed.
- a solid support on which the analyte is immobilized can be placed in MALD I-TOF / TOF MS or M ALD IQ-TOF MS.
- the amino acid sequence can be analyzed to identify interacting proteins.
- a Ti layer and a Pt layer were formed on a 76 mmX 26 mmX 1.1 mm slide glass by magnetron sputtering.
- the sputtering conditions are as follows.
- the thickness of the generated metal layer is 100 nm for each of the Ti layer and the Pt layer.
- 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 the ionization evaporation method under the following conditions.
- the thickness of the generated diamond-like carbon layer was 20 nm. Table 2 Conditions for forming one layer
- Vb Accelerating voltage
- Va Anode voltage
- Cy3-protein A (1.5 ⁇ SI GMA), E. coli protein (0.5 g) and Prestained B road Range (0.5 ⁇ BIO RAD) And electrophoresis was performed using an SDS-PAGE device (AE-7300, manufactured by ATTO).
- SDS-PAGE device AE-7300, manufactured by ATTO.
- 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 the electrophoresis was completed, the cells were stained with CBB for 15 minutes, destained, and photographed with RAS 1000.0 (manufactured by Fuji Photo Film Co., Ltd.) (FIG. 1).
- a band of Cy3-protein A was detected around 50 kDa.
- the polyacrylamide gel after the electrophoresis was immersed in a pre-cooled transfer buffer (25 mM Tris, 5% methanol) for 30 minutes to equilibrate. Next, the polyacrylamide gel is cut into a size corresponding to the solid support 1, and the polyacrylamide gel is cut out.
- the polyacrylamide gel was placed on the cathode, the solid support was placed on the anode, and electricity was supplied under the following conditions. Table 3 Energizing conditions
- the fluorescence intensity was 16448, indicating an increase, indicating that protein A and IgG bound, that is, the binding ability of the immobilized protein was maintained.
- a diamond-like carbon layer was formed on a stainless steel substrate.
- Stainless steel substrate In order to lower the smoothness and the fluorescent packing ground, puff polishing was performed in advance, followed by electrolytic polishing.
- the formation of the diamond-like carbon layer was performed by ionization evaporation under the following conditions.
- the thickness of the generated diamond-like carbon layer was 20 nm. Table 4 'Conditions for forming one-bon layer
- Vb acceleration voltage
- Va anode voltage
- Cy3_protein A (0.2 ⁇ g, SI GMA) was electrophoresed by the SDS-PAGE method (ATTO AE-653) in the same manner as in Example 1, and after the electrophoresis, 1 After staining with CBB for 5 minutes and destaining, images were taken with LAS1000 (manufactured by Fuji Photo Film Co., Ltd.) (FIG. 5).
- the filter paper on the cathode side contains a transfer buffer and the filter paper on the anode side contains ion-exchanged water, and the gel after the electrophoresis is wiped off. It is believed that it is preferable to perform the transfer without performing the transfer.
- Example 3 Transfer of protein to stainless steel DLC solid support Cy3—Protein A (50 ng, manufactured by SI GMA) and Cy3—IgA (100 ng, manufactured by SI GMA) were subjected to the SDS-PAGE method (ATTO After electrophoresis, the cells were stained with CBB for 15 minutes, destained, and photographed with LAS1000 (manufactured by Fuji Photo Film Co., Ltd.) (Fig. 8). For the migration of Cy 3-1 g A, a 12% polyacrylamide gel was used. In the same manner as in Example 2, a solid support 2 was prepared.
- Example 2 so as to prevent air bubbles from entering, and the filter was set in a semi-driving apparatus.
- the one containing the transfer buffer C1 or C2 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.
- a current was applied at 2 V and 2 ⁇ 60 for 60 minutes to transfer the protein to the solid support 2.
- the solid support 2 after the 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 (Fuji Photo Film Co., Ltd.) (FIG. 9).
- the transfer buffer to be included in the filter paper on the cathode side during transfer to the solid support is C1, that is, the one with a concentration of ⁇ -aminocaproic acid of 4 OmM has better immobilization efficiency. I was separated.
- a diamond-like carbon layer was formed on the substrate 1 in the same manner as in Example 1, and the substrate surface was chlorinated by irradiating the substrate surface with ultraviolet light for 1 minute in a chlorine gas, followed by ionization.
- the surface was aminated by treatment in an ammonia plasma using a vapor deposition device. Thereafter, the resultant was treated with succinic anhydride and further treated with polyacrylic acid. Then, it was activated with N-hydroxysuccinimide 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 LAS 1000 (manufactured by Fuji Photo Film Co., Ltd.) (FIG. 10). A Cy3-protein A band was detected at 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 ⁇ -aminocaproic acid, 5% methanol) were prepared. .
- the gel after electrophoresis was taken out, immersed in about 20 Om1 of transfer buffer ⁇ ⁇ ⁇ ⁇ ⁇ , and gently shaken for 5 minutes.
- 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 a transfer buffer B of about 100 ml, and shaken for 5 minutes or more.
- the PVDF membrane after the transfer was cut into a size to be placed on the solid support 3, and superimposed in the order shown in FIG. 12 to place a weight of 35 g / cm 2 .
- Cy3-protein A on the membrane was transferred onto solid support 3.
- the solid support 3 was washed with PBS at room temperature for 20 minutes, and then dried.
- the solid support was imaged with FLA8000 (manufactured by Fuji Photo Film Co., Ltd.).
- Fig. 13 shows the captured images. The part enclosed in the image is the part where the membrane is adhered. Cy3-Protein A is supported on solid because fluorescence was detected at the corresponding position It can be seen that it is immobilized on the body.
- leginsulin-binding protein (12.5 ⁇ g / ⁇ 1) was spotted and left for 10 minutes. Subsequently, the plate was washed by shaking with ultrapure water for 10 minutes, and then dried to prepare a solid support on which leginsulin-binding protein was immobilized.
- leg insulin-binding protein-immobilized solid support 0.5 ⁇ l of the matrix solution ( ⁇ -cyanohydroxycinnamic acid solution) was added to the leg insulin-binding protein-immobilized solid support and the leg insulin-binding protein-reg insulin-immobilized solid support obtained as described above. Added and dried.
- Fig. 14 shows the results of TOF-MS analysis of the solid support on which legulin-binding protein was immobilized.
- Figure 15 shows the results of TOF-MS analysis of the leginsulin-binding protein-leginsulin-immobilized solid support.
- the substances contained in individual bands can be immobilized on a solid support, and a plurality of substances can be purified. Since mass spectrometry can be performed simultaneously and directly without using any other method, a large number of samples can be analyzed quickly.
- the present invention is a very useful means in analyzing biomolecules such as nucleic acids and proteins.
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Description
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Priority Applications (3)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP03792698A EP1542001A1 (en) | 2002-08-21 | 2003-08-18 | Solid support and method of mass spectrometry of multiple substances or composites immobilized on the solid support through desorption/ionization |
US10/525,394 US20060121473A1 (en) | 2002-08-21 | 2003-08-18 | Solid support and method of mass spectrometry of multiple substances or composites immobilized on the solid support through desorption/ionization |
AU2003257859A AU2003257859A1 (en) | 2002-08-21 | 2003-08-18 | Solid support and method of mass spectrometry of multiple substances or composites immobilized on the solid support through desorption/ionization |
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JP2002240905 | 2002-08-21 | ||
JP2002-240905 | 2002-08-21 | ||
JP2003-042491 | 2003-02-20 | ||
JP2003042491A JP3860130B2 (ja) | 2002-08-21 | 2003-02-20 | 固体支持体及び該固体支持体上に固定化された複数の物質又は複合体を脱離/イオン化することにより質量分析する方法 |
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PCT/JP2003/010406 WO2004019025A1 (ja) | 2002-08-21 | 2003-08-18 | 固体支持体及び該固体支持体上に固定化された複数の物質又は複合体を脱離/イオン化することにより質量分析する方法 |
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US (1) | US20060121473A1 (ja) |
EP (1) | EP1542001A1 (ja) |
JP (1) | JP3860130B2 (ja) |
KR (1) | KR20050058398A (ja) |
CN (1) | CN1682110A (ja) |
AU (1) | AU2003257859A1 (ja) |
WO (1) | WO2004019025A1 (ja) |
Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO2005080960A1 (ja) * | 2004-02-23 | 2005-09-01 | Toyo Kohan Co., Ltd. | 固体支持体及び該固体支持体上に複数の物質又は複合体を脱離/イオン化することにより質量分析する方法 |
WO2005096346A2 (de) * | 2004-04-02 | 2005-10-13 | Physikalisches Büro Steinmüller Gmbh | Target für laser desorptions/ionisations-massenspektrometrie |
JP2008180728A (ja) * | 2008-03-07 | 2008-08-07 | Toyo Kohan Co Ltd | 固体支持体及び該固体支持体上に複数の物質又は複合体を脱離/イオン化することにより質量分析する方法 |
US20100130380A1 (en) * | 2005-06-17 | 2010-05-27 | Kiyoshi Nokihara | Biochip substrate and biochip |
WO2010079919A2 (ko) * | 2009-01-06 | 2010-07-15 | 한국생명공학연구원 | 다층기질겔을 이용한 기질분해효소의 스크리닝 방법 |
Families Citing this family (8)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
JP2006170857A (ja) * | 2004-12-16 | 2006-06-29 | Toyo Kohan Co Ltd | 固体支持体上の生体分子を質量分析する方法およびそのための固体支持体 |
JP4576606B2 (ja) * | 2005-01-21 | 2010-11-10 | 独立行政法人産業技術総合研究所 | 質量分析用イオン化基板及び質量分析装置 |
JP2006292680A (ja) * | 2005-04-14 | 2006-10-26 | Toyo Kohan Co Ltd | 固体支持体上において相互作用した生体分子を分析する方法およびそのための固体支持体 |
KR20090115930A (ko) * | 2006-12-26 | 2009-11-10 | 브라이엄 영 유니버시티 | 혈청 단백질체학 시스템 및 관련 방법 |
JP4802156B2 (ja) * | 2007-08-15 | 2011-10-26 | ハイモ株式会社 | 粘着性ピッチの分析方法 |
KR101156795B1 (ko) * | 2010-03-10 | 2012-06-18 | 삼성전기주식회사 | 그래핀 코팅된 말디-토프 질량분석용 샘플분석 타겟 및 이를 포함하는 말디-토프 질량분석장치 |
JP2014232055A (ja) * | 2013-05-29 | 2014-12-11 | 株式会社島津製作所 | Maldi質量分析用測定基板 |
FR3024465B1 (fr) * | 2014-07-30 | 2018-03-23 | Biomerieux | Caracterisation de micro-organismes par maldi-tof |
Citations (1)
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WO2000045168A1 (en) * | 1999-01-28 | 2000-08-03 | Universite De Geneve | Method and kit for identifying or characterising polypeptides |
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US6051380A (en) * | 1993-11-01 | 2000-04-18 | Nanogen, Inc. | Methods and procedures for molecular biological analysis and diagnostics |
US6221626B1 (en) * | 1998-06-30 | 2001-04-24 | University Of Geneva | Kit for electroblotting polypeptides separated on an electrophoresis gel |
US6287783B1 (en) * | 1999-03-18 | 2001-09-11 | Biostar, Inc. | Optical assay device and method |
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2003
- 2003-02-20 JP JP2003042491A patent/JP3860130B2/ja not_active Expired - Fee Related
- 2003-08-18 EP EP03792698A patent/EP1542001A1/en not_active Withdrawn
- 2003-08-18 CN CNA038223791A patent/CN1682110A/zh active Pending
- 2003-08-18 WO PCT/JP2003/010406 patent/WO2004019025A1/ja not_active Application Discontinuation
- 2003-08-18 KR KR1020057002897A patent/KR20050058398A/ko not_active Application Discontinuation
- 2003-08-18 US US10/525,394 patent/US20060121473A1/en not_active Abandoned
- 2003-08-18 AU AU2003257859A patent/AU2003257859A1/en not_active Abandoned
Patent Citations (1)
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WO2000045168A1 (en) * | 1999-01-28 | 2000-08-03 | Universite De Geneve | Method and kit for identifying or characterising polypeptides |
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BIENVENUT W.V. ET AL.: "Toward a clinical molecular scanner for proteome research: parallel protein chemical processing before and during western blot", ANALYTICAL CHEMISTRY, vol. 71, no. 21, 1 November 1999 (1999-11-01), pages 4800 - 4807, XP002973873 * |
MITSUYOSHI OBA ET AL.: "Diamond like corbon (DAC) no tokucho to kanosei", DAI 6 KAI KAGAKU TO MICRO.NANO SYSTEM KENKYUKAI KOEN YOKUSHU, 6 August 2002 (2002-08-06), pages 26, XP002973874 * |
Cited By (9)
Publication number | Priority date | Publication date | Assignee | Title |
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WO2005080960A1 (ja) * | 2004-02-23 | 2005-09-01 | Toyo Kohan Co., Ltd. | 固体支持体及び該固体支持体上に複数の物質又は複合体を脱離/イオン化することにより質量分析する方法 |
WO2005096346A2 (de) * | 2004-04-02 | 2005-10-13 | Physikalisches Büro Steinmüller Gmbh | Target für laser desorptions/ionisations-massenspektrometrie |
WO2005096346A3 (de) * | 2004-04-02 | 2006-10-26 | Physikalisches Buero Steinmuel | Target für laser desorptions/ionisations-massenspektrometrie |
US20100130380A1 (en) * | 2005-06-17 | 2010-05-27 | Kiyoshi Nokihara | Biochip substrate and biochip |
US9778256B2 (en) * | 2005-06-17 | 2017-10-03 | Hipep Laboratories | Biochip substrate and biochip |
JP2008180728A (ja) * | 2008-03-07 | 2008-08-07 | Toyo Kohan Co Ltd | 固体支持体及び該固体支持体上に複数の物質又は複合体を脱離/イオン化することにより質量分析する方法 |
JP4644263B2 (ja) * | 2008-03-07 | 2011-03-02 | 東洋鋼鈑株式会社 | 固体支持体及び該固体支持体上に複数の物質又は複合体を脱離/イオン化することにより質量分析する方法 |
WO2010079919A2 (ko) * | 2009-01-06 | 2010-07-15 | 한국생명공학연구원 | 다층기질겔을 이용한 기질분해효소의 스크리닝 방법 |
WO2010079919A3 (ko) * | 2009-01-06 | 2010-10-21 | 한국생명공학연구원 | 다층기질겔을 이용한 기질분해효소의 스크리닝 방법 |
Also Published As
Publication number | Publication date |
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EP1542001A1 (en) | 2005-06-15 |
CN1682110A (zh) | 2005-10-12 |
JP3860130B2 (ja) | 2006-12-20 |
KR20050058398A (ko) | 2005-06-16 |
JP2004138596A (ja) | 2004-05-13 |
US20060121473A1 (en) | 2006-06-08 |
AU2003257859A1 (en) | 2004-03-11 |
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