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WO2005080960A1 - Support solide et procédé de spectrométrie de masse par désorption/ionisation de multiples substances ou composites sur le support solide - Google Patents

Support solide et procédé de spectrométrie de masse par désorption/ionisation de multiples substances ou composites sur le support solide Download PDF

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Publication number
WO2005080960A1
WO2005080960A1 PCT/JP2005/002927 JP2005002927W WO2005080960A1 WO 2005080960 A1 WO2005080960 A1 WO 2005080960A1 JP 2005002927 W JP2005002927 W JP 2005002927W WO 2005080960 A1 WO2005080960 A1 WO 2005080960A1
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WIPO (PCT)
Prior art keywords
solid support
carbon layer
gel
substance
electrophoresis
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PCT/JP2005/002927
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English (en)
Japanese (ja)
Inventor
Mitsuyoshi Ohba
Hirofumi Yamano
Shuuichi Kamei
Hisashi Hirano
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Toyo Kohan Co., Ltd.
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Application filed by Toyo Kohan Co., Ltd. filed Critical Toyo Kohan Co., Ltd.
Publication of WO2005080960A1 publication Critical patent/WO2005080960A1/fr

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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
    • 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 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.
  • 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.
  • 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.
  • 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.
  • a laser is irradiated to a sample portion to be analyzed, and ions desorbed therefrom are accelerated by an electric field.
  • 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).
  • 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
  • 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.
  • 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 the substance separated in the gel is transferred and held.
  • 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.
  • 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.
  • 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 is obtained by (1) any one of (1) to (6).
  • (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).
  • 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 shows an arrangement in Example 4 when proteins are transferred from a gel after electrophoresis to a PVDF membrane.
  • FIG. 3 shows an arrangement when a protein is transferred from a PVDF membrane to a solid support 3 in Example 4.
  • 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.
  • 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.
  • 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.
  • the term “substrate” means a base material on which a carbon layer is formed, and such a base material is not particularly limited.
  • a base material 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 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.
  • 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,
  • 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.
  • 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.
  • 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.
  • 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.
  • the DLC layer may be formed by an ionization vapor 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.
  • 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 ⁇ m 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.
  • 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.
  • the surface of the substrate on which the carbon layer is formed 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.
  • the introduction of an amino group can be carried out, for example, by irradiating the carbon layer with ultraviolet light in ammonia gas.
  • 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.
  • 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.
  • 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.
  • a metal chelate 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.
  • a solid support into which a metal chelate is introduced 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.
  • 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.
  • 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,
  • 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.
  • 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.
  • 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.
  • the affinity between the electrostatic layer and the substrate or the carbon layer, that is, the adhesion is increased.
  • 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.
  • the conditions for ionization vapor deposition are preferably an operating pressure of 0.1 to 50 Pa and an accelerating voltage of 200 to 1000 V.
  • 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.
  • an electrostatic layer 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 ⁇ m.
  • the electrophoresis method that can be used for sample separation in the present invention is not particularly limited.
  • agarose gel electrophoresis agarose gel electrophoresis, sieving agarose gel electrophoresis, denaturing agarose gel electrophoresis, polyacrylamide gel electrophoresis, SDS polyacrylinoleamide gel
  • 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.
  • 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 are known to distort the DNA structure, and polyacrylamide gel electrophoresis can be used to analyze the structure and function of DNA.
  • polyacrylamide gel electrophoresis can be used to analyze the structure and function of DNA.
  • SDS Sodium dodecyl sulfate
  • SDS-PAGE polyacrylamide gel electrophoresis
  • 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 ⁇ -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.
  • carrier ampholite carrier ampholite
  • IPG method Immobilized pH gradient
  • 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.
  • IPG method immobilized pH gradient method
  • agarose gel electrophoresis 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.
  • 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.
  • a set of capillaries utilizing capillary action, vacuum-type blotting using a pump, and electro-blotting using an electric method can be used.
  • transferring a nucleic acid it is preferable to use the capillaries set
  • transferring a peptide it is preferable to use elect opening blotting.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • transfer buffer 1 glycine-Tris buffer
  • Examples of the buffer to be contained 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 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.
  • 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.
  • the substance separated in the gel can be transferred and held on the solid support.
  • the material of the membrane that can be used in this case include nitrocellulose, PVDF (polyvinylidene fluoride), nylon, and positively charged nylon.
  • PVDF polyvinylidene fluoride
  • nylon nylon
  • 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.
  • an electroblotting device In the transfer from the membrane to the solid support, it is preferable to use an electroblotting device.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • a separation mode including a separation mode or a combination thereof
  • 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.
  • 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 set on a flat target of MAL DI-TOF MS.
  • mass spectrometry is started using MassLynx software or the like.
  • MassLynx can control all measurements and analyses.
  • create a parameter file for automatic measurement create 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.
  • protein amino acid sequence analysis and identification can be performed.
  • 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.
  • 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.
  • Vb acceleration voltage
  • Va anode voltage
  • 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.
  • 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.
  • 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.
  • a pre-cooled and cooled transfer buffer 25 mM Tris, 5% methanol
  • 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.
  • 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.
  • 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.
  • a solid support was prepared by introducing an amino group by ammonia plasma treatment.
  • Vb acceleration voltage
  • Va anode voltage
  • Cy3_protein A (0.2 ⁇ g, 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.).
  • 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.
  • Cy3-protein A 50 ng, SIGMA
  • Cy3-IgA 100 ng, SIGMA
  • SDS-PAGE SDS-PAGE
  • Example 2 so as to prevent air bubbles from entering, and the filter and the solid support were placed in a semi-driving apparatus.
  • ion-exchanged water was used for the three filter papers on the anode side.
  • 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.).
  • 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 ⁇ -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.
  • 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 .
  • the Cy3-Port Tin A on the membrane was transferred onto the solid support.
  • 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.
  • 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.
  • Cy3_Protein A 50 ng, SIGMA
  • Cy5_IgA 100 ng, SIGMA
  • 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 ⁇ -aminocaproic acid, 5% methanol) or ion-exchanged water.
  • CI 25 mM Tris, 40 mM ⁇ -aminocaproic acid, 5% methanol
  • 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.
  • 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 ⁇ A 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.).
  • 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.
  • 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.
  • the present invention is a very useful means in analyzing biomolecules such as nucleic acids and proteins.
  • biomolecules such as nucleic acids and proteins.
  • 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.

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Abstract

Moyen pour la spectrométrie de masse rapide d'une multitude d'échantillons ; et procédé d'analyse rapide de biomolécules telles que des acides nucléiques et des protéines. Il est fourni un support solide ayant une couche de carbone formée sur sa surface et contenant en plus un composé aminé lié à la couche de carbone par liaison covalente. Il est en plus fourni un procédé de spectrométrie de masse de multiples substances, comprenant de séparer les substances d'un échantillon les unes des autres par électrophorèse sur gel, de transférer les substances séparées présentes dans un portion de gel sur le support solide et d'effectuer la désorption/l'ionisation des substances sur le support solide.
PCT/JP2005/002927 2004-02-23 2005-02-23 Support solide et procédé de spectrométrie de masse par désorption/ionisation de multiples substances ou composites sur le support solide WO2005080960A1 (fr)

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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
JP4441653B2 (ja) 2005-08-31 2010-03-31 シャープ株式会社 自動化２次元電気泳動装置および装置構成器具
JP4846442B2 (ja) * 2006-05-16 2011-12-28 株式会社バイオロジカ レーザー脱離イオン化質量分析用サンプルプレート

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WO2010079919A3 (fr) * 2009-01-06 2010-10-21 한국생명공학연구원 Procédé de criblage de métalloprotéinases matricielles
KR101062828B1 (ko) 2009-01-06 2011-09-07 한국생명공학연구원 다층기질겔을 이용한 기질분해효소의 스크리닝 방법

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