JP2006098266A - Sample dyeing method and sample analyzing method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method which enables the judgment and mass analysis of the intermolecular interaction of a substance set to a solid phase on this support. <P>SOLUTION: This sample dyeing method includes a process for judging the interaction of a target substance A, which is set to the solid phase on the support, with a substance B having a specific bonding capacity with respect to the target substance A using the substances A and B. The blocking reagent shows no mass spectrum peak imparting trouble to the analytic result due to the mass analysis of the substance A and/or the substance B. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to all fields related to clinical, diagnosis, biochemistry, and molecular biology. In particular, the present invention relates to analysis of biomolecules using immunochemical reactions.

  The following methods are known as analysis methods for biomolecules separated by chromatography such as two-dimensional electrophoresis.

  (1) Usefulness of matrix-assisted laser desorption / ionization mass spectrometry (MALDI-MS) has been recognized (J. Mass Spectrom. Soc. Jpn (2003), Vol. 51, pp. 530-537). In addition, the target substance is immobilized on a membrane (blotted) directly from the advantage that it can be stored in a target sample or applied to a microdispensing / analysis system using inkjet technology such as a piezo element. A system for analysis on a membrane has been proposed (Japanese Patent Publication No. 2001-521623, Molecular & Cellular Proteomics (2002) vol. 1, pp. 490-499).

  (2) A method is widely used in which a target substance is developed by electrophoresis or the like, solidified on a solid support, reacted with a molecule that interacts with the target substance, and its presence is detected. (Mainly immunoblotting).

JP-T-2001-521623 Journal of the Mass Spectrometry Society of Japan, Volume 51, 2003, p. 530-537 Molecular & Cellular Proteomics Volume 1, 2002, p. 490-499

  Reagents and the like used to determine the intermolecular interaction of the substance immobilized on the support interferes with the analysis when the substance is analyzed by mass spectrometry. For this reason, in the conventional analysis methods for biomolecules and the like, a process for determining an intermolecular interaction of a solid phased substance and a step for performing an identification analysis of the solid phased substance are performed. In some cases, each step is performed on a separate support. That is, it is necessary to prepare a plurality of supports in which the same sample is solid-phased under the same conditions, or to separate one support solidified in such a manner. Thus, in the conventional method, the number of processes that can be performed on the same sample on the support is limited, and the analysis efficiency is low.

  Therefore, an object of the present invention is to provide a method capable of performing determination of intermolecular interaction and mass spectrometry of a solid-phased substance on the same support. Moreover, the objective of this invention is providing the system which aims at the labor saving of a series of work by applying a micro amount dispensing technique and mass spectrometry technique.

The present invention includes the following inventions.
The following <1> to <16> relate to a sample staining method using a blocking reagent that does not interfere with mass spectrometry. Among these, the following <1> to <3> relate to a form in which the substance A and the specific binding substance B immobilized on a support are related. Further, the following <5> to <10> relate to a form in which the substance A immobilized on the support, the substance A ′ prepared therefrom, and the specific binding substance B are related.

<1> For a sample containing the target substance A, which is solid-phased on a support,
Determining the interaction between the substance A and the substance B by using a blocking reagent and the substance B having a specific binding ability to the target substance A,
The method for staining a sample, wherein the blocking reagent does not exhibit a mass spectrum peak that interferes with an analysis result of the substance A and / or the substance B by mass spectrometry.

<2> The blocking reagent is
Any fragment of the fragment generated from the substance A and the fragment generated from the substance B, which has a molecular weight greater than that of the substance A and / or the substance B and depending on the conditions for fragmenting the substance A and / or the substance B <1> The method for staining a sample according to <1>, wherein the sample has a structure that does not produce a fragment having a molecular weight equal to or lower than the molecular weight.

<3> The method for staining a sample according to <1> or <2>, wherein the substance A is selected from proteins, nucleic acids, lipids, sugar chains, and complexes thereof.
<4> The method for staining a sample according to any one of <1> to <3>, wherein the substance B is selected from proteins, nucleic acids, lipids, sugar chains, and complexes thereof.

<5> For a sample containing the target substance A, which is solid-phased on a support,
Preparing a substance A ′ from the target substance A on the support;
A step of determining an interaction between the substance A ′ and the substance B by using a blocking reagent and a substance B having specific binding ability to the substance A ′ prepared on the support from the target substance A Including
The sample staining method, wherein the blocking reagent does not exhibit a mass spectrum peak that interferes with an analysis result of the substance A, the substance A ′, and / or the substance B by mass spectrometry.

<6> The blocking reagent is
The substance has a molecular weight greater than that of the substance A, the substance A ′, and / or the substance B, and the substance A, the substance A ′, and / or the substance B can be fragmented according to the conditions. <5> The method for staining a sample according to <5>, wherein the sample has a structure that does not produce a fragment having a molecular weight smaller than that of the fragment produced from A, the substance A ′, and / or the substance B.

<7> The sample staining method according to <5> or <6>, wherein the substance A is selected from proteins, nucleic acids, lipids, sugar chains, and complexes thereof.
<8> The method for staining a sample according to any one of <5> to <7>, wherein the substance B is selected from proteins, nucleic acids, lipids, sugar chains, and complexes thereof.

<9> The method for staining a sample according to any one of <5> to <8>, wherein the substance A ′ is a protein.
<10> For a sample containing nucleic acid immobilized on a support,
By simultaneously using a transcription reagent and / or a translation reagent and a labeling substance having a labeling part and an acceptor part capable of binding to the N-terminal or C-terminal of the protein,
<9> The method for staining a sample according to <9>, wherein a protein is prepared from the nucleic acid by transcription and / or translation, and the simultaneously prepared protein is used as a labeled protein by binding of the labeling substance.

<11> The method for staining a sample according to any one of <1> to <10>, wherein the blocking reagent is polyvinylpyrrolidone or n-octyl-β-D-glucopyranoside.

<12> The sample staining method according to any one of <1> to <11>, wherein the substance B is a labeled molecule labeled with a labeling substance.
<13> The sample staining method according to <12>, wherein the labeling substance is a fluorescent labeling substance.

<14> The method for staining a sample according to any one of <1> to <13>, wherein the interaction is determined by immunoblotting.

<15> The method for staining a sample according to any one of <1> to <14>, wherein at least one selected from a membrane, a plate, nonmagnetic particles, and magnetic particles is used for the support.
<16> The support is a plate for mass spectrometry,
The sample according to <15>, wherein the substance A solid-phased on the mass spectrometry plate is obtained by contacting a gel obtained by electrophoresis of the sample containing the substance A and a mass spectrometry plate. Dyeing method.

  The following <17> to <20> relate to a sample analysis method using the staining method of <1> to <16> above.

<17> (1) determining the interaction between the target substance A and the specific binding substance B by performing the method for staining a sample according to any one of <1> to <16>;
(2) fragmenting the substance A, the substance A ′, and / or the substance B determined to interact on the support;
(3) A method for analyzing a sample, comprising identifying the fragmented substance A, substance A ′, and / or substance B by mass spectrometry on the support.

<18> The sample analysis method according to <17>, wherein in the step (2), a reagent for fragmentation is added using a microdispensing device.

<19> In the step (3), MS analysis is performed on at least one fragment obtained in the step (2), and further MS / MS analysis is performed on at least one fragment in the data obtained by the MS analysis. The sample analysis method according to <17> or <18>.

<20> The sample analysis method according to any one of <17> to <19>, wherein in the step (3), mass spectrometry is performed by a MALDI method.
<21> The sample analysis method according to <20>, wherein the matrix solution is added using a microdispensing device.

  ADVANTAGE OF THE INVENTION According to this invention, the method which can perform the determination of the molecular interaction of the solid-phased substance and mass spectrometry on the same support body can be provided. Another object of the present invention is to provide a system that can save labor in a series of operations by applying a micro-dispensing technique and a mass spectrometry technique.

  The present invention is a method for staining a sample using a blocking reagent that does not interfere with mass spectrometry, mainly in immunoblotting. The staining method of the present invention is a particularly useful method when mass spectrometry is performed on the same support as the support on which the sample for immunostaining is immobilized. In the present invention, the term “dyeing” is not limited to the form of coloring, but includes a form that can be recognized by visualization under specific conditions. Further, in the present invention, solidifying on a support means to include both a state immobilized on a support by a covalent bond or the like and a state resting on the support regardless of a covalent bond or the like. Used in.

In the present invention, the blocking reagent is a substance that prevents nonspecific binding of a specific binding substance to a substance on a support on which the substance to be analyzed is solid-phased. A blocking reagent that does not interfere with mass spectrometry is a blocking reagent that does not exhibit a mass spectrum peak that hinders mass spectrometry. That is, the blocking reagent used in the present invention is not detected within the peak range derived from the substance to be identified and analyzed. Specifically, the blocking reagent of the present invention may be generated from a substance having a molecular weight larger than that of the substance to be identified and analyzed, under the condition that the substance to be identified and analyzed can be fragmented. A substance having a structure that does not produce a fragment having a molecular weight equal to or lower than the molecular weight of the fragment.
Examples of such a blocking reagent include polyvinylpyrrolidone (PVP) and n-octyl-β-D-glucopyranoside.

The blocking reagent is used after being dissolved in an appropriate solvent. Usually, it is used by dissolving together with the substance B (antibody etc.) described later. As the solvent, a TBS buffer or the like may be used. The concentration of the blocking reagent can be 0.1 to 5% (w / v) depending on the amount of the blocking reagent used. For example, in the case of polyvinyl pyrrolidone (PVP), about 0.25% (w / v) may be used. Further, the amount of the blocking reagent solution to be used is not particularly limited because it depends on the kind of the sample, the amount of the dissolved blocking reagent or substance B, etc., but usually the amount does not exceed 10 ml per 1 cm ² of membrane. Use. Usually, since a small amount of the blocking reagent solution is preferably used, the lower limit of the amount is not particularly limited, but is, for example, about 0.2 ml, more preferably about 0.09 ml.

  On the other hand, blocking reagents conventionally used in Western blots and the like are BSA, skim milk, casein, serum, surfactants and the like. These are low molecular weights, or are decomposed by enzymatic digestion to produce low molecular weight fragments. For this reason, when a substance to be identified is subjected to mass spectrometry on the same support after performing these conventional Western blotting methods, it is detected as a contaminant within the peak range of the substance to be identified and analyzed.

  As other conditions of the staining method of the sample in the present invention, the conditions in a commonly performed staining method may be used. Specifically, a support on which a sample containing the target substance A is solid-phased is prepared; a blocking reagent and a substance B having a specific binding ability to the target substance A are prepared for the sample on the support. Adding; by determining the interaction between substance A and substance B. Therefore, the blocking reagent used in the present invention can block a site on the support where the substance B can bind nonspecifically, and the analysis result of the substance A and / or the substance B by mass spectrometry It does not exhibit a mass spectrum peak that impedes the process. Further, the blocking reagent in the present invention has a molecular weight larger than that of the substance A and / or the substance B, and the substance A and / or the substance B under the condition that the substance A and / or the substance B can be fragmented. It has a structure that does not produce a fragment having a molecular weight equal to or lower than that of a fragment that can be produced from

Specific examples of the target substance A are selected from proteins, nucleic acids, lipids, sugar chains, and complexes thereof.
The substance B is a substance that can specifically bind to the target substance A, and is usually a substance that can form a substance A-substance B complex by an immunochemical reaction with the target substance A. Specific examples are selected from proteins, nucleic acids, lipids, sugar chains, and complexes thereof. Furthermore, the substance B may be a labeled molecule that is labeled with a labeling substance. Examples of the labeling substance include fluorescent labeling substances. Such a substance B is used by being appropriately contained in a solution or the like.

  As the support, at least one selected from a membrane, a plate, nonmagnetic particles, magnetic particles and the like is used. When using a membrane, the sample containing the substance A can be used by being electrically transferred to the membrane. Examples of the membrane include organic synthetic polymers such as polyvinylidene difluoride (PVDF), nitrocellulose, polyamide, polyethylene, and derivatives thereof. Nylon etc. are mentioned as polyamide.

  Examples of the plate include a glass plate, a resin plate, a metal plate, and the like. When such a plate is used, a gel after electrophoresis of a sample containing the substance A or a sample containing the substance A is transferred. The membrane can be used in contact with the surface of the plate.

  When the present invention is used for mass spectrometry, a metal plate such as a sample plate for mass spectrometry is preferably used as the support. When a sample plate for mass spectrometry is used as a support, it is preferable in that mass analysis can be performed on the same support after determining the interaction. At this time, the substance A solid-phased on the sample plate for mass spectrometry can be obtained by bringing a gel obtained by electrophoresis of a sample containing the substance A into contact with the sample plate for mass spectrometry. Further, it may be obtained by adhering a sample containing the substance A to a sample plate for mass spectrometry (from a gel or the like after electrophoresis) by attaching it to a membrane. In addition, fixing using a conductive double-sided adhesive tape or the like is preferable.

  Furthermore, as a support using nonmagnetic particles, a support using a polysaccharide gel or a synthetic polymer is used. As a support using magnetic particles, a support using a conductive metal as the basic structure of a support as a transfer support after electrophoresis is used. Furthermore, the thing which used the support | carrier for the magnetic particle which couple | bonded the spacer for the purpose of improving avidity etc. is mentioned.

  The present invention can be particularly useful for immunoblotting. Therefore, in this case, specific examples of the substance A immobilized on the support include those obtained by electrophoresis of proteins and then transferred to a membrane, and specific examples of the substance B include antibodies. Then, the interaction between the two substances is determined by confirming the antigen-antibody reaction between the substance A and the substance B.

  In the present invention, the determination of the interaction between substances means that the substance A in the sample solid-phased on the support is assumed in advance, and a specific binding substance for the assumed substance A A form in which the position of the substance A on the support is confirmed by acting B; and the substance A is an unknown substance, and various possible substances B are allowed to act on the substance A. It is used in the meaning including both of determining the specific binding of the substance B and confirming the position of the substance A.

  In addition, examples of the method for staining a sample in the present invention include the following methods. In this embodiment, a support on which a sample containing the target substance A is solid-phased is prepared; a substance A ′ synthesized from the target substance A on the support is prepared; prepared on the support. A blocking reagent and a specific binding substance B for substance A ′ are added to substance A ′; the interaction between substance A ′ and substance B is determined.

  The blocking reagent used at this time does not exhibit a mass spectrum peak that hinders the analysis result of the substance A, the substance A ′, and / or the substance B by mass spectrometry. Further, the blocking reagent in this case has a higher molecular weight than substance A, substance A ′, and / or substance B, and can fragment substance A, substance A ′, and / or substance B. It has a structure that does not generate a fragment having a molecular weight equal to or lower than that of a fragment that can be generated from substance A, substance A ′, and / or substance B under conditions.

  The substance A ′ is a protein or the like. Such a protein can be prepared from a nucleic acid immobilized as a substance A on a support by cell-free protein synthesis on the support. That is, it can be prepared by expressing in a transcription system and / or translation system constructed by adding a transcription reagent and / or translation reagent to a sample containing nucleic acid immobilized on a support. .

  Such proteins can also be obtained as labeled proteins. Such a labeled protein can be prepared by using a transcription reagent and / or a translation reagent and a labeling reagent. The labeling reagent used at this time has a label part and an acceptor part capable of binding to the N-terminal or C-terminal of the protein. And this labeled reagent couple | bonds with the protein expressed with the transcription | transfer system and / or the translation system, and labeled protein is prepared.

  In the present invention, the solution may be added onto the support using a microdispensing device, or the support may be immersed in the solution. As a microdispensing device, a device equipped with a mechanism of an ink jet method can be used. An example of such an apparatus is CHIP-1000 (manufactured by Shimadzu Corporation).

  In the present invention, the amount of the solution added can be greatly controlled by using the microdispensing device. For example, in the case of an apparatus equipped with a mechanism of an ink jet method, the amount of reagent added per dispensing operation from one ink jet discharge unit can be controlled to, for example, about 100 pl. Depending on the ink jet mechanism, the amount can be controlled to be even smaller. Further, since the discharge can be repeated, the upper limit of the amount for dispensing the reagent amount is not particularly limited. For a specific region, for example, a reagent amount of the nanoliter level, usually about 100 nl, can be dispensed. Therefore, even a rare and expensive reagent such as an antibody solution consumes the minimum necessary amount, so that a significant cost reduction can be achieved.

By using a micro-dispensing device, for example, an extremely small dispensing range of about 7800 μm ² is generated by ejection of about 100 pl. This dispensing range can be limited to a smaller range by an inkjet mechanism. Furthermore, since the dispensing position can be shifted by changing the relative position of the inkjet discharge unit on the support, the solution can be dispensed in an arbitrary region. For this reason, the upper limit of the dispensing range is not particularly limited. When dispensing as an array to a specific area, for example, ^{4 to} 100 spots can be dispensed per 1 mm ² .

  It is also possible to set two or more regions where the solution should be added on the same support. Therefore, it is possible to add different types of solutions in different regions even on the same support, and the interaction can be determined efficiently.

  As described above, since the microdispensing apparatus can add a trace amount of solution, it is possible to process only a desired minimal region on the support. For this reason, it becomes easy to process a large amount of specimens, and consequently, high-throughput (high efficiency) interaction screening for reliably identifying interacting molecules becomes possible.

  The staining method of the present invention described so far can be used to obtain physical information and chemical information of the substance A and / or the substance B. In order to obtain such information, mass spectrometry, fluorescence, RI, surface plasmon resonance, and the like are used. In particular, since the staining method of the present invention uses a blocking reagent that does not interfere with mass spectrometry, mass spectrometry can be performed on the same support as the support on which the sample is immobilized. This is useful when performing the analysis.

  Furthermore, the present invention is a method for analyzing a sample by performing the above-described staining on a sample immobilized on a support. The analysis method of the present invention is as follows. (1) Substance A in a sample (or substance A ′ prepared on a support from substance A by the above-described staining method of the sample; hereinafter, these are collectively referred to simply as substance A. And (2) fragmenting the substance A in the sample determined to interact on the same support; (3) fragment Identifying the converted substance A by mass spectrometry on the same support.

  In the analysis method of the present invention, in step (1), a blocking reagent that does not exhibit a mass spectrum peak that hinders mass spectrometry of a substance to be identified and analyzed is used. As described above, the blocking reagent used in the present invention is not detected within the peak range derived from the substance to be identified and analyzed. Specifically, it may be generated from a substance having a molecular weight larger than that of the substance to be analyzed for identification and a condition capable of fragmenting the substance to be identified and analyzed, that is, the condition in the step (2). It has a structure that does not produce a fragment having a molecular weight equal to or lower than the molecular weight of the fragment.

  In the fragmentation in step (2), fragmentation based on a known method can be performed. For example, trypsin digestion may be performed. In the present invention, a blocking reagent having a structure that does not produce a low molecular weight fragment depending on the conditions of this step is used, so that the substance that has interacted in step (1) is fragmented at the same position on the same support. Even if the treatment is performed, the substance to be analyzed can be appropriately selected from the blocking reagent that may be present at that position without generating a fragment exhibiting a mass spectrum peak that hinders mass spectrometry in the subsequent step (3). It can be fragmented. Thus, since the present invention can perform a plurality of different processes on the same sample, analysis efficiency is high.

  Further, the fragmentation reagent used in this step, such as a trypsin solution, can be added using a microdispensing device. The microdispensing device is as described in the above-described staining method. Therefore, the fragmentation process can be performed on a desired minute region where the target substance that has interacted in step (1) is present.

  In the mass analysis of the step (3), the measurement can be performed at the same position on the same support as the steps (1) and (2). Therefore, the blocking reagent that may be present at that position by this step may be measured at the same time. However, in this step, the peak of the blocking reagent is not detected within the peak range of the mass spectrum necessary for the analysis of the substance to be identified. This is because the blocking reagent used in the present invention has a higher molecular weight than the substance to be identified and analyzed, and the molecular weight of the fragment generated from the substance to be identified and analyzed by the fragmentation conditions in step (2) This is because it has been devised to have a structure that does not produce the following molecular weight fragments. Therefore, in this step, the blocking reagent does not interfere with the analysis of the substance to be identified. As described above, the analysis method of the present invention can perform steps (1) to (3) at the same position on the same support, and therefore the analysis efficiency is very high.

  Thus, by devising the blocking reagent in step (1), analysis can be performed with high sensitivity in this step. Therefore, not only MS analysis but also MS / MS analysis is possible. That is, it is possible to perform MS analysis on at least one fragment obtained in the step (2), and further perform MS / MS analysis on at least one fragment in the data obtained by the MS analysis. By enabling MS / MS analysis in this way, it becomes possible to identify substances for which sufficient data cannot be obtained by MS analysis alone.

  Substances for which sufficient data cannot be obtained by MS analysis alone are related to the molecular weight and properties of the substance, and particularly those with low molecular weight. For example, a substance having a molecular weight of about 10 kDa to 60 kDa, or a molecular weight lower than that. Specific examples include GroES (10 kDa), which is a low molecular weight protein. For example, when MS measurement is performed after performing the operations of steps (1) and (2) for GroES, there are few peaks of peptide fragments derived from GroES in the obtained mass spectrum, and sufficient identification cannot be achieved. However, if MS / MS measurement is performed on one of these peptide fragments, further information can be obtained, so that sufficient identification analysis can be performed. That is, even if the molecular weight of the substance A is a low molecular weight of about 10 kDa to 60 kDa or less, sufficient identification analysis can be performed by MS / MS measurement. Needless to say, even if the molecular weight of the substance A is larger than that shown here, the analysis is possible.

  In this step, it is preferable to use laser desorption ionization mass spectrometry. In this case, the position where the fragmented substance exists is irradiated with a laser to obtain a mass spectrum. Furthermore, it is preferable to use matrix-assisted laser desorption / ionization mass spectrometry (MALDI method) in this step. In this case, the treated substance is preferably dispensed by superimposing the matrix solution using a micro-dispensing device, and the dispensing position is irradiated with a laser to obtain a mass spectrum. When using a micro-dispensing device, the target of mass spectrometry is limited to a very small range. Therefore, in the mass spectrum obtained at this time, unnecessary signals from areas other than the desired region, that is, the background must be reduced. Can do. In addition, in the process (1) and / or (2), when a plurality of treatments are performed on the same support, dispensing of the matrix solution and laser irradiation are performed on each of the plurality of positions where the treatment is performed. By doing so, it is possible to save labor for a series of operations for analysis.

  Thus, the staining method and the analysis method of the present invention are very efficient in analysis. A series of work steps in the method of the present invention can be automatically controlled. For example, (a) an apparatus that solidifies a sample containing the target substance A on a support, and (b) an apparatus that captures the target substance A by contact with a specific binding substance B for the target substance A. (C) The target substance A and / or the specific binding substance B can be automatically controlled as an analysis apparatus including at least a device for obtaining physical and / or chemical information. In this case, the apparatus (c) can use a method selected from mass spectrometry, fluorescence, RI, and surface plasmon resonance. Such automatic control makes it possible to further improve throughput and save labor.

<Example 1>
Bovine serum albumin (BSA) was transferred to a hydrophobic membrane after SDS-PAGE, and the transferred membrane was fixed to a sample plate. FIG. 1 schematically shows the process of transferring the gel after electrophoresis onto the membrane. Western blotting was performed on the transferred membrane according to the protocol of Rapid Immunodetection Method (Millipore Application Note RP562). However, in this example, polyvinyl pyrrolidone (PVP) was used without using the surfactant actually used in the Rapid Immunodetection Method. That is, the solid support was blocked using PVP, and a positive band was detected by immunoblotting using a fluorescently labeled BSA antibody. The results obtained at this time are shown in FIG. The positive band was subjected to enzyme digestion using a microdispensing device (CHIP-1000 manufactured by Shimadzu Corporation). Thereafter, PMF analysis was directly performed on the membrane using a mass spectrometer (AXIMA manufactured by Shimadzu Corporation). The mass spectrum obtained at this time is shown in FIG. In FIG. 3, the horizontal axis represents mass / charge (Mass / Charge), and the vertical axis represents the relative intensity of ions (% Int.). All ion peaks detected in FIG. 3 were searched using the mascot database (http://www.matrixscience.com/). As a result of the search, BSA was identified with a high score (score 72). Hereinafter, the protocol of Western blot performed in this example will be specifically described.

[Drying the membrane]
1. The membrane was immersed in 100% methanol for 10 seconds.
2. The blot was placed on filter paper for 15 minutes.
3. The blot was left in the vacuum chamber for 30 minutes.
4). The blot was incubated for 1 hour at 37 ° C.
5. The blot was placed on a lab bench and allowed to dry at room temperature for 2 hours.

[Immunodetection]
1. The blot was immersed in a TBS buffer containing 0.25% (w / v) polyvinylpyrrolidone (PVP-40; Sigma) and a primary antibody for 1 hour. At this time, 0.09 ml of TBS buffer was used for 1 cm ^{2 of} blot.
2. The blot was washed with TBS for 10 seconds. At this time, 0.9 ml of TBS buffer was used for 1 cm ^{2 of} blot. This washing operation was repeated twice.
3. The blot was immersed in a TBS buffer containing 0.25% (w / v) polyvinylpyrrolidone (PVP-40; Sigma) and a secondary antibody for 1 hour. At this time, 0.09 ml of TBS buffer was used for 1 cm ^{2 of} blot.
4). The blot was washed with TBS for 10 seconds. At this time, 0.9 ml of TBS buffer was used for 1 cm ^{2 of} blot. This washing operation was repeated twice.
5. The blot was rinsed with Milli-Q water (Millipore).
6). The blot was placed on a lab bench and allowed to dry at room temperature.

<Example 2>
Isoelectric focusing (pH 4.0-7.0) was performed using a soluble fraction (100 mg) of E. coli. SDS-PAGE (12.5%) was performed in the second dimension. Then, it transferred to the membrane. The transferred membrane was dried by performing the same operation as the staining protocol in Example 1 above.

1. The dried membrane was shaken in a TBS buffer containing 0.25% (w / v) PVP and a primary antibody (Molecular Probe, anti-GroEL) for 1 hour. At this time, 0.09 ml of TBS buffer was used for 1 cm ^{2 of} blot.
2. The blot was washed with TBS for 10 seconds. At this time, 0.9 ml of TBS buffer was used for 1 cm ^{2 of} blot. This washing operation was repeated twice.
3. Blots were shaken for 30 minutes in TBS buffer containing 0.25% (w / v) PVP and secondary antibody (using fluorescent label). At this time, 0.09 ml of TBS buffer was used for 1 cm ^{2 of} blot.
4). The blot was washed with TBS for 10 seconds. At this time, 0.9 ml of TBS buffer was used for 1 cm ^{2 of} blot. This washing operation was repeated twice.
5. GroEL protein spots were detected using a fluorescence image analyzer (Fuji Film). The results obtained at this time are shown in FIG.
6). To confirm by visualization, the protein was stained using Direct Blue 71 (manufactured by Sigma). The results obtained at this time are shown in FIG.
7). The spot detected with the fluorescence image analyzer and the spot stained with Direct Blue 71 were overlapped, and the overlapped spot was added in the order of enzyme digestion and matrix using CHIP-1000, and measured with a mass spectrometer . The results obtained at this time are shown in FIG. In FIG. 6, the horizontal axis represents mass / charge (Mass / Charge), and the vertical axis represents the relative intensity of ions (% Int.). A mascot search (http://www.matrixscience.com/) was performed on the ion peaks detected in FIG. (Search conditions are as follows: Taxonomy: All entries, Enzyme: Trypsin, Allow up to 1 missed cleavage, Peptide tol .: ± 0.4 Da, Variable modification: Oxidation (M)) And sequence coverage (score 97, 12% sequence coverage).

In Example 1, it is the figure which showed typically the process of transcribe | transferring the gel after electrophoresis on a membrane. In this Example 1, it is the result of having detected BSA by the Western blot. It is a mass spectrometry result obtained in the present Example 1. In this Example 2, it is the result of having detected the protein spot of E. coli soluble fraction with the fluorescence image analyzer. In this Example 2, it is the result of having dye | stained the protein spot of an E. coli soluble fraction. It is a mass spectrometry result obtained in the present Example 2.

Claims (21)

For a sample containing the target substance A, which is immobilized on a support,
Determining the interaction between the substance A and the substance B by using a blocking reagent and the substance B having a specific binding ability to the target substance A,
The method for staining a sample, wherein the blocking reagent does not exhibit a mass spectrum peak that interferes with an analysis result of the substance A and / or the substance B by mass spectrometry. The blocking reagent is
Any fragment of the fragment generated from the substance A and the fragment generated from the substance B, which has a molecular weight greater than that of the substance A and / or the substance B and depending on the conditions for fragmenting the substance A and / or the substance B The sample staining method according to claim 1, wherein the sample has a structure that does not produce a fragment having a molecular weight equal to or lower than that of the sample. The method for staining a sample according to claim 1 or 2, wherein the substance A is selected from proteins, nucleic acids, lipids, sugar chains, and complexes thereof. The method for staining a sample according to any one of claims 1 to 3, wherein the substance B is selected from proteins, nucleic acids, lipids, sugar chains, and complexes thereof. For a sample containing the target substance A, which is immobilized on a support,
Preparing a substance A ′ from the target substance A on the support;
A step of determining an interaction between the substance A ′ and the substance B by using a blocking reagent and a substance B having specific binding ability to the substance A ′ prepared on the support from the target substance A Including
The method for staining a sample, wherein the blocking reagent does not exhibit a mass spectrum peak that interferes with an analysis result of the substance A, the substance A ′, and / or the substance B by mass spectrometry. The blocking reagent is
The substance has a molecular weight greater than that of the substance A, the substance A ′, and / or the substance B, and the substance A, the substance A ′, and / or the substance B can be fragmented according to the conditions. The sample staining method according to claim 5, wherein the sample has a structure that does not produce a fragment having a molecular weight smaller than that of a fragment produced from A, the substance A ′, and / or the substance B. The sample staining method according to claim 5 or 6, wherein the substance A is selected from proteins, nucleic acids, lipids, sugar chains, and complexes thereof. The method for staining a sample according to any one of claims 5 to 7, wherein the substance B is selected from proteins, nucleic acids, lipids, sugar chains, and complexes thereof. The method for staining a sample according to any one of claims 5 to 8, wherein the substance A 'is a protein. For a sample containing nucleic acid immobilized on a support,
By simultaneously using a transcription reagent and / or a translation reagent and a labeling substance having a labeling part and an acceptor part capable of binding to the N-terminal or C-terminal of the protein,
The method for staining a sample according to claim 9, wherein a protein is prepared from the nucleic acid by transcription and / or translation, and the simultaneously prepared protein is used as a labeled protein by binding of the labeling substance. The method for staining a sample according to any one of claims 1 to 10, wherein the blocking reagent is polyvinylpyrrolidone or n-octyl-β-D-glucopyranoside. The sample staining method according to claim 1, wherein the substance B is a labeled molecule labeled with a labeling substance. The sample staining method according to claim 12, wherein the labeling substance is a fluorescent labeling substance. The method for staining a sample according to any one of claims 1 to 13, wherein the interaction is determined by immunoblotting. The method for staining a sample according to claim 1, wherein at least one selected from a membrane, a plate, nonmagnetic particles, and magnetic particles is used for the support. The support is a plate for mass spectrometry,
The sample according to claim 15, wherein the substance A solid-phased on the mass spectrometry plate is obtained by bringing a gel obtained by electrophoresis of the sample containing the substance A into contact with a mass spectrometry plate. Dyeing method. (1) A step of determining an interaction between the target substance A and the specific binding substance B by performing the sample staining method according to any one of claims 1 to 16;
(2) fragmenting the substance A, the substance A ′, and / or the substance B determined to interact on the support;
(3) A method for analyzing a sample, comprising identifying the fragmented substance A, substance A ′, and / or substance B by mass spectrometry on the support. The sample analysis method according to claim 17, wherein in the step (2), a reagent for fragmentation is added using a microdispensing device. In the step (3), MS analysis is performed on at least one fragment obtained in the step (2), and further MS / MS analysis is performed on at least one fragment in the data obtained by the MS analysis. Item 19. The sample analysis method according to Item 17 or 18. The sample analysis method according to any one of claims 17 to 19, wherein in the step (3), mass spectrometry is performed by a MALDI method. The sample analysis method according to claim 20, wherein the matrix solution is added using a microdispensing device.