JP2006133098A - Method for detecting protein or dna and detection chip for same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method of detecting a protein or a nucleic acid not using a stain, a decolorizer and a fluorescence-labeling reagent, having a process simplified thereby, dispensing with waste liquid treatment, and easily shifted to analysis by a mass spectrometer in a subsequent process. <P>SOLUTION: In the method of detecting a protein or a nucleic acid, a working electrode is inserted into a separated medium acquired by electrophoresing a protein sample or a nucleic acid sample, and a functional group in the protein or the nucleic acid is oxidized, and a generated current is measured, to thereby detect existence of the protein or the nucleic acid. The method of detecting the protein or DNA and this detection chip of the protein or DNA, not using the stain toxic to an objective human body and polluting environment, the decolorizer and the expensive fluorescence-labeling reagent, having the process simplified thereby, dispensing with the waste liquid treatment, and transferable easily to analysis by the mass spectrometer in the subsequent process, are provided by using the method. The method and the chip are used properly for analysis of proteome or genome. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to a protein or DNA detection method and a detection chip. More specifically, the present invention relates to a protein or DNA detection method and a detection chip that are suitably used for proteome or genome analysis.

  With the progress of analysis of large-scale genetic information such as the genome project, a vast amount of genetic information is being revealed. The current major challenge is to proceed with the functional analysis of genes by clarifying the relationship between genetic information clarified in this way and proteins that interact in a complex manner in cells. . This is called the proteome, but the proteome is a new concept expressed by combining PROTEin and genOME. Proteome analysis can be said to be an attempt to comprehensively understand the relationship between various proteins.

  In addition, protein chips were developed for the purpose of efficiently purifying or identifying the target protein (protein), and performing functional analysis such as protein expression, interaction, and post-translational modification. .

  By the way, it is said that there are about 5000 to 7000 types of proteins constituting cells. Thus, it is required to quickly grasp changes in the proteome, which is a collection of diverse proteins, and various protein chips have been proposed for this purpose.

  As a conventionally used protein chip, for example, there is a method in which an antibody specific to a specific protein is immobilized on a substrate and the binding between the protein and the antibody is identified by fluorescence or the like. In this case, there is a limit that only known proteins that already exist can be identified. The same applies to the DNA chip, and a substrate on which complementary DNA is immobilized is used to identify the bound DNA by fluorescence or the like. In this case as well, only DNA having complementary DNA can be identified.

  In addition, chemical chips with various chemical properties suitable for use in protein purification or identification, expression analysis, etc., such as hydrophobicity, cation exchange, anion exchange, etc. Biological chip to see the action), specifically, antibodies, receptors, DNA immobilized on the chip and time-of-flight mass spectrometer using peptide mass fingerprint method used for measurement, There is also a commercially available protein chip consisting of a computer installed with software used for measurement and analysis. In this case, so-called peptide mapping is performed in which the purified protein is digested with a proteolytic enzyme such as trypsin, the digested fragment is captured, and the molecular weight is measured with a time-of-flight mass spectrometer. Furthermore, the protein is identified by inputting the molecular weight of the fragment measured in the protein identification software and performing database analysis. In this method, identification is impossible with a chip alone, and a proteolytic enzyme and a time-of-flight mass spectrometer are always required, so that the operation becomes complicated.

  In addition, two-dimensional electrophoresis of proteins, a type of other protein chip, is separated based on the isoelectric point (pI) of the protein in the one-dimensional direction and developed based on the molecular weight of the protein in the two-dimensional direction. It is to be done. Specifically, the procedure is as follows. First, isoelectric focusing (first-dimensional separation) is performed using a capillary gel or a commercially available strip gel as a separation medium. The gel that has finished the first-dimensional migration is placed on the second gel for the second-dimensional migration. Since the second gel must migrate in a direction perpendicular to the direction of development in the first dimension, a planar slab gel is used. When isoelectric focusing is performed in the first dimension, SDS-polyacrylamide electrophoresis (SDS-PAGE) is generally selected as the second dimension electrophoresis. Based on the above method, two-dimensional electrophoresis has already been performed for many proteomes. Analysis results of amino acid sequence determination and peptide mass fingerprinting are recorded along with coordinate information for past protein gel spots, and the information is stored in databases such as SWISS-2DPAGE. Yes.

  Coordinate information is obtained by labeling the protein with staining or fluorescence. That is, a protein is identified by analyzing the position of a spot obtained by staining a gel on which a protein has been run or a spot labeled with fluorescence using a database. For unknown proteins that are not in the database, cut out unknown protein spots from the gel, digest the proteins with proteolytic enzymes in the gel (In-gel digestion), extract peptide fragments from them, and time-of-flight mass spectrometry By peptide mass fingerprinting using a meter or the like, peptide mapping is performed for identification. In the case of this method, at the time of protein identification, at least a staining step or a fluorescence labeling step is required even if the protein is a known protein that can process the position (coordinates) of the gel spot by database analysis. Furthermore, when identifying by fluorescence, in addition to these, a fluorescence image analyzer is required. In the case of an unknown protein that cannot be processed by database analysis, a spot of the unknown protein is cut out from the gel, and in the case of a stained band, a decolorization process is required. Will be identified. Further, the spot subjected to the fluorescence labeling cannot be analyzed as it is with a time-of-flight mass spectrometer.

  On the other hand, SDS-PAGE is common for the second dimension of electrophoresis. However, when preparing polyacrylamide gels, it is difficult to maintain uniform quality over a large area. The non-uniformity of the second-dimensional gel appears as a difference in mobility, and even for the same protein, for example, it is often the case that the mobility near the center of the gel is different from that at the periphery. Such a phenomenon is called “smiling” and causes a distortion of the electrophoretic image, and easily causes an error in coordinate information. Thus, current technology requires time and effort to analyze proteins.

On the other hand, a protein identification method that does not use the above-described affinity effect or molecular sieving effect, that is, a technique for measuring a protein only with an electrode has already been proposed. Specifically, an electrode in which a nickel thin film is formed on a ceramic substrate by sputtering is manufactured and protein is measured, but there is a problem that there is no protein selectivity.
Japanese Patent No. 3,500,772

  As described above, in the conventional protein chip, for example, in the case of two-dimensional electrophoresis, first, a staining agent, a decoloring agent, an expensive fluorescent labeling reagent, and the like are required. The dyeing process and the decoloring process are necessary and the operation is complicated, and many chemicals used for them are harmful to the human body, and handling is required, and there is also a problem of environmental pollution due to disposal. On the other hand, when a fluorescent labeling reagent is used, an expensive fluorescence image analyzer is required, and if the fluorescent labeling reagent is covalently bonded to a protein, there is a problem that it cannot be applied to a mass spectrometer as it is. . Furthermore, when preparing a gel, it is difficult to maintain a uniform quality over a large area, the reproducibility of mobility is poor, and it tends to lead to errors in coordinate information. The DNA chip has almost the same problem.

  In this way, both protein chips and DNA chips require expensive reagents and analyzers for identification, some reagents are harmful to the human body, require waste liquid treatment to prevent environmental pollution, and are complicated to operate. There is also a problem in the reproducibility of the data obtained. Further, in the subsequent process used for identification of the unknown sample, there may be a problem in the analysis by the mass spectrometer.

  The object of the present invention is not to use a staining agent, a decoloring agent, a fluorescent labeling reagent, etc., so that the process is simplified, no waste liquid treatment is required, and it can be easily transferred to analysis by a mass spectrometer in the subsequent process. It is to provide a method for detecting a protein or nucleic acid.

  The object of the present invention is to measure the current generated by oxidizing a functional group of a protein or nucleic acid by inserting a working electrode into a separation medium obtained by electrophoresis of a protein sample or a nucleic acid sample. This is achieved by a protein or nucleic acid detection method for detecting the presence or absence. More preferably, the protein or nucleic acid is detected by immersing the gel after two-dimensional electrophoresis in an alkaline aqueous solution, and immersing the working electrode in an alkaline aqueous solution portion on the gel so as not to contact the gel, and applying a constant potential. Thereafter, the working electrode is inserted into the gel and the current generated by oxidizing the functional group of the protein or nucleic acid is measured.

  By using the method of the present invention, it is not necessary to use stains, decoloring agents and expensive fluorescent labeling reagents that are harmful to the target human body and pollute the environment, so that the process is simplified and no waste liquid treatment is required, Furthermore, a protein or DNA detection method and a detection chip that can be easily transferred to subsequent analysis by a mass spectrometer are provided. These are suitably used for proteome or genome analysis.

  The method of the present invention can be applied to either a proteome or a genome, but will be described in detail below with a focus on proteome analysis.

  The proteome sample to be analyzed is first subjected to the first dimension. Two-dimensional electrophoresis analysis of proteins is generally performed by isoelectric focusing in the first dimension and SDS-PAGE in the second dimension. Therefore, isoelectric focusing is performed using strip gel or capillary gel as a medium. Specifically, a proteome sample is placed at the center of a medium having a pH gradient, both ends are immersed in an anode buffer and a cathode buffer, and electrophoresed by energization. At this time, the isoelectric point marker is also migrated under the same conditions.

  The electrophoresis medium obtained as a result of the first dimension electrophoresis is brought into contact with the medium for the second dimension SDS-PAGE. For example, the gels are placed in the well so that the gels are in contact with each other side by side, or are placed so as to overlap each other. First-dimensional electrophoresis medium is brought into close contact with one opening of the gel medium, and SDS-PAGE is performed in this state. Individual proteins constituting the proteome developed in the first-dimensional gel move to the second-dimensional gel and develop according to the molecular weight. In the second dimensional electrophoresis, the molecular weight marker is electrophoresed under the same conditions.

  As the separation medium, gels such as polyacrylamide and agarose, and media usually used for separating proteins or nucleic acids such as filter paper, nitrocellulose, and cellulose acetate are used. Although the shape is not particularly limited, it is generally a planar rectangle, and a rod-like shape or a circular shape is also used.

  In the gel after the two-dimensional electrophoresis, at least a working electrode sensitive to protein or an electrode sensitive to nucleic acid and a counter electrode are inserted when analyzing a genomic sample.

  As a working electrode, when analyzing a functional group such as an amino group, a thiol group, or a hydroxyl group of a protein or a genomic sample, an electrode that is sensitive to a functional group such as an amino group or a hydroxyl group of a nucleic acid is used. Specifically, nickel, cobalt, silver, and copper are preferable, and these may be used by themselves, or a thin film of these metals is formed on the surface of other metals or conductive plastics by sputtering or vapor deposition. In general, a film formed with a film thickness of about 1000 to 10000 mm, preferably about 1000 to 6000 mm by a method, a screen printing method, a plating method, metal foil bonding, or the like may be used. The number of working electrodes may be any number and can be changed according to the shape and size of the gel, the desired resolution of the electrophoresis field, and the like.

  For example, if a gel having a width of 50 mm, a depth of 50 mm, and a thickness of 3 mm is to be detected with a resolution of several mm, the presence or absence of protein or nucleic acid may be detected by inserting several hundred working electrodes into the gel. By installing a large number of electrodes in a small area, the presence or absence of protein or nucleic acid can be detected more accurately. Therefore, the number of electrodes is determined by the desired resolution.

  The arrangement, number, size, and shape of each electrode are arbitrary. The required number of working electrodes and one counter electrode may be provided, or a reference electrode may be provided in addition to the working electrode and the counter electrode in order to ensure the accuracy of the potential. 1 and 2 show an example of the electrode-equipped substrate 1 in which a large number of electrodes are provided on the substrate. Although only the working electrode 2 is shown in the figure, the counter electrode or the reference electrode and the reference electrode are formed at arbitrary positions. Further, the shape of the electrode is not basically limited, but preferably the hollow shape 3 is used as illustrated in FIG. By adopting a hollow shape, the contact area with the sample is widened, an improvement in sensitivity can be expected, and the effect of being easy to insert into a gel or the like as a separation medium is exhibited. Furthermore, the sample in the gel can be collected by utilizing the hollow portion, which is useful for later analysis. In addition, as a substrate on which a large number of electrodes are provided, plastic such as polyethylene terephthalate, glass, ceramics, etc. are used, and the formation of electrodes on these substrates is performed by a mold forming method, vapor deposition, sputtering, etching, plating, etc. It is performed by the method of forming by.

  The counter electrode material is arbitrary, but platinum is preferred. In addition, the counter electrode may be a metal itself, but as in the case of the working electrode, a thin film of the metal is deposited on the surface of other metals or conductive plastics by sputtering, vapor deposition, screen printing, plating, etc. In general, a film having a film thickness of about 1000 to 10000 mm, preferably about 1000 to 6000 mm is used by a method, a metal foil bonding method, or the like. As the reference electrode, a silver / silver chloride electrode is preferable.

  To detect a protein or nucleic acid, a predetermined voltage is sequentially applied to the working electrode, and a functional group such as an amino group, thiol group, hydroxyl group or nucleic acid amino group or hydroxyl group of the protein trapped in the gel is brought into contact therewith. The presence or absence of a protein or nucleic acid at a specific coordinate of the gel is detected by the current generated at that time. Therefore, no current resulting from these substances is generated at the working electrode that is not in contact with the protein or nucleic acid.

  As a measuring method, differential measurement is preferable. This is a method of taking the difference between the working electrode generation current that is in contact with protein or nucleic acid and the working electrode generation current that is not in contact with protein or nucleic acid, and there are functional groups such as amino groups and hydroxyl groups in the gel. Therefore, in order to accurately grasp the current caused by the protein or nucleic acid, it is possible to measure accurately rather than simply judging based on the magnitude of the current. In this case, a differential working electrode is installed at a position where it does not come into contact with protein or nucleic acid.

The reaction of an organic substrate such as an amino group, thiol group or hydroxyl group with a metal M such as nickel, cobalt, silver or copper is generally as follows.
Under alkaline atmosphere

^{M + mOH - → M (OH} ) m + me -

When a predetermined voltage is applied here,

^{OH - + lower oxide → higher oxide} + H 2 O + e -

It becomes. Then when in contact with an organic substrate,
kc
higher oxide + organic substrate → lower oxide + organic substrate intermediate (radical)

And this reaction is rate limiting. Then

Organic substrate intermediate (radical) → product + (n−1) e ⁻

This reaction is generally fast.

For example, when the working electrode sensitive part is nickel, when this nickel comes into contact with a strong alkaline solution, nickel hydroxide Ni (OH) ₂ is generated there.

Ni + 2OH ^- _{→ Ni (OH) 2 + 2e} -

Here, when a predetermined voltage is applied,

^{_{Ni (OH) 2 + OH -}} → NiO (OH) + H 2 O + e -

When the generated NiO (OH) comes into contact with a functional group such as an amino group, a thiol group, or a hydroxyl group that is a reducing residue of a protein, or a functional group such as an amino group or a hydroxyl group that is a reducing residue of a nucleic acid. Oxidize this residue. At that time, since a current is generated, the presence of protein or nucleic acid can be detected by measuring the output of the generated current.

Protein reducing residues + NiO (OH) → Ni ( OH) 2 + protein oxidation residues + (n-1) e ^-

Nucleic reducing residues + NiO (OH) → Ni ( OH) 2 + nucleic oxide residues + (n-1) e ^-

Here, the formula of the limit current i based on these oxidation reactions is shown by the following formula.

i = n × F × A × β × kc × Corg

Where n is the number of electrons involved in the reaction, F is the Faraday constant, A is the electrode area, β is the roughness factor, kc is the rate constant of the rate-limiting step, and Corg is the concentration of the reactant.

Specifically, the oxidation of amino groups and hydroxyl groups is as shown in the following example, for example.

_{RCH 2 NH 2 → RCN + 4} e -

_{RCH 2 OH → RCO 2 H +} 4 e -

  The electrode and the second dimensional separation medium usually exist separately, and the operation of inserting the electrode into the second dimensional separation medium is performed as described above. However, the electrode is formed on the same substrate together with the second dimensional separation medium. Can also be used. Producing a detection chip in one chip in this way is convenient because the sample can be bent and used after electrophoresis.

When measuring the gel after two-dimensional electrophoresis of proteins or nucleic acids by the above-described method, for example, with a nickel working electrode, the gel after two-dimensional electrophoresis is about 0.1-10 mM NiSO ₄ and pH 7-13. It is preferable to immerse in an aqueous solution in which an alkaline amount of NaOH or KOH is dissolved at room temperature to 60 ° C. At this time, when a silver / silver chloride electrode is used as a reference electrode by adding about 10 to 500 mM inorganic chloride such as potassium chloride or sodium chloride, the dissociation equilibrium of AgCl used is maintained. It acts to stabilize the potential of the reference electrode and increase its durability. The potential applied at this time is about 0.01 to 1.0 V, preferably about 0.1 to 0.5 V.

^{AgCl + e - ← → Ag +} Cl -

  After the gel after two-dimensional electrophoresis is immersed in an alkaline aqueous solution, the electrode is immersed in an alkaline aqueous solution portion on the gel for a certain time, for example, about 10 seconds to 30 minutes so as not to contact the gel. Under this condition, the electrode metal is in a lower oxide state. Next, a constant potential, for example, a potential of 0.01 to 1.0 V is applied to the working electrode. In this state, the electrode surface becomes a higher oxide state. Thereafter, the working electrode is inserted into the gel, and at the same time, the counter electrode and the reference electrode are inserted into the gel. In this state, the working electrode comes into contact with the organic substrate, and the organic substrate becomes an organic substrate intermediate (radical) and immediately becomes a product. At this time, the current generated by the oxidation of the organic substrate is measured.

  The protein or nucleic acid separated by confirming the protein or nucleic acid spot on the gel after the second-dimension migration is compared with the coordinate information by comparing it with the result of two-dimensional electrophoresis performed under the same conditions. Based on the identification or unknown proteome, the identification of proteins or nucleic acids in each spot is further advanced and analyzed.

  The analysis of the measured value is performed by specifying the migration position of protein or nucleic acid based on the position of the working electrode that has generated a significant current, and expressing the presence / absence position in two-dimensional coordinates of XY.

  Further, the present invention can detect the spread of the sample from the measured values of a large number of working electrodes, and it is considered that a correction method for improving reproducibility in terms of software can be easily applied.

  On the other hand, protein identification is performed based on amino acid sequence analysis or peptide mass fingerprinting. The amino acid sequence can be determined from the terminal by a technique such as Edman degradation. The entire amino acid sequence of the protein is not necessarily determined at this stage. Even obtaining a partial sequence is effective for protein identification. Many proteins are blocked at the N-terminus, which may be inconvenient for sequencing, but one of the methods to ensure the amino acid sequence of this type of protein is proteolytic enzyme ( There are methods for sequencing digested fragments by proteases. Digestion with a protease occurs in various places depending on the protein, and the protein can be identified by comparing the partial amino acid sequence obtained by sequencing with a known amino acid sequence database. In addition, the peptide mass fingerprint method can obtain much information for protein identification more quickly than amino acid sequencing which requires complicated work. This is because the protein contained in the spot separated as a result of two-dimensional electrophoresis is digested with a certain protease, and the fragment is analyzed with a mass spectrometer to obtain the mass pattern (fingerprint) of the protease digested fragment. How to get. The peptide mass finger print is unique information about each protein, and the database Protein Prospector and the like can be used on the Internet, so that the protein can be identified based on the peptide mass finger print.

  By using the method of the present invention, in proteome analysis, it is possible to compare the two-dimensional electrophoresis patterns of proteomes in various states, and confirm the fluctuation of the spot related to a specific function. A protein that constitutes a spot that disappears in the proteome of a cell in a specific state or appears specifically on the contrary, is likely to be related to the function of interest. If identification is possible based on the partial amino acid sequence of the protein and the peptide mass fingerprint, the relevance to the function of interest can be estimated. If it is an unknown protein, the isolation of the protein, the gene encoding the protein, and the cloning of the factor that regulates the expression are promoted, so that the gene or protein that supports the function of interest Can elucidate how it works.

It is an example of a substrate with electrodes in which a large number of electrodes are provided in a substrate shape It is an example of a substrate with electrodes in which a large number of hollow electrodes are provided in a substrate shape

Explanation of symbols

1 Substrate with electrodes 2 Working electrode 3 Hollow working electrode

Claims (14)

  Detecting the presence or absence of protein or nucleic acid by measuring the current generated by oxidizing the functional group of protein or nucleic acid by inserting a working electrode into a separation medium obtained by electrophoresis of protein or nucleic acid sample A method for detecting a protein or nucleic acid.   2. The protein or nucleic acid detection method according to claim 1, wherein the electrophoresis is two-dimensional electrophoresis, and the separation medium into which the working electrode is inserted is a second-dimensional separation medium.   2. The method for detecting a protein or nucleic acid according to claim 1, wherein the working electrode is an electrode sensitive to an amino group, a thiol group or a hydroxyl group.   2. The protein or nucleic acid detection method according to claim 1, wherein the working electrode structure is hollow.   The protein or nucleic acid extraction method according to claim 4, wherein the protein sample or nucleic acid sample in the separation medium is collected in the hollow portion of the working electrode.   2. The method for detecting a protein or nucleic acid according to claim 1, wherein the working electrode material is a nickel, copper, silver or cobalt thin film formed on the surface of nickel, copper, silver or cobalt, or other metal or conductive plastic. .   The protein or nucleic acid detection according to claim 1, wherein the separation medium is immersed in an aqueous solution having a pH of 7 to 13 containing a metal salt used as a working electrode material of 0.1 to 10 mM and an inorganic chloride of 10 to 500 mM at room temperature to 60 ° C. Method.   The gel after two-dimensional electrophoresis is immersed in an alkaline aqueous solution, and the working electrode is immersed in the alkaline aqueous solution portion on the gel so that it does not come into contact with the gel. After applying a constant potential, the working electrode is inserted into the gel. The method for detecting a protein or nucleic acid according to claim 1, wherein an electric current generated by oxidizing a functional group of the protein or nucleic acid is measured.   The method for detecting a protein or nucleic acid according to claim 8, wherein the counter electrode is also inserted into the gel when the working electrode is inserted into the gel.   The method for detecting a protein or nucleic acid according to claim 8, wherein the counter electrode and the reference electrode are also inserted into the gel when the working electrode is inserted into the gel.   The protein according to claim 8, 9 or 10, wherein the current value is measured as a differential measurement that takes a difference between a working electrode generation current in contact with a protein or nucleic acid and a working electrode generation current not in contact with the protein or nucleic acid. Alternatively, a method for detecting a nucleic acid.   2. The protein or nucleic acid according to claim 1, wherein the presence or absence of a protein or nucleic acid is measured by inserting a working electrode sensitive to a functional group into an electrophoretic separation medium, and the position of the presence or absence is expressed by two-dimensional coordinates of XY. Detection method.   Insert a working electrode that is sensitive to functional groups into the electrophoresis separation medium, measure the presence or absence of protein or nucleic acid, take out the protein or nucleic acid from a site, measure its molecular weight with a mass spectrometer, The method for detecting a protein or nucleic acid according to claim 1, wherein a substance is specified from the distribution pattern. A protein or nucleic acid detection chip in which a working electrode and a separation medium for electrophoresis of a protein sample or a nucleic acid sample are arranged on the same substrate.

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