JP2007051957A - Direct mass spectrometry of living tissue - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a pretreatment method of a sample for mass spectrometry capable of performing mass spectrometry of a protein constituting a tissue with high analysis sensitivity and high analysis efficiency, while keeping an anatomical structure of the living tissue, and capable of performing multi-stage mass spectrometry, while keeping the anatomical structure in the living tissue. <P>SOLUTION: This direct mass spectrometry method of the living tissue includes a process for denaturing a protein in the living tissue by supplying a a denaturation agent to a living tissue specimen, a process for staining the protein by supplying a stain, a process for digesting the protein by dispensing a digestive enzyme, and a process for performing mass spectrometry by dispensing a matrix, and by using a MALDI mass spectrometer. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to the life science field, specifically to the medical / biological field such as cell biology, pathology, biochemistry and the like. In particular, the present invention relates to a method for mass spectrometry of biological tissue directly on a biological tissue specimen.

  Examples of direct mass spectrometry of biological tissues include, for example, Stoeckli, M .; Chaurand, P .; Hallahan, ED; Caprioli, MR Nature Med. 7, 493-496 (2001), Chaurand, P .; Schwartz, AS; Caprioli, MR Anal. Chem., 76, 86A-93A (2004), etc., frozen biological tissue sections were thawed directly on a mass spectrometry target plate and washed with a 70 (v / v)% aqueous ethanol solution, A technique for acquiring an MS spectrum without performing enzyme digestion has been reported.

  Further, for example, in Chaurand, P .; Schwartz, AS; Billheimer, D .; Xu, J. B; Crecelius, A .; Caprioli, MR Anal. Chem., 76, 1145-1155 (2004) There has been a report of obtaining an MS spectrum similar to that in the above technique using a slide glass.

  Furthermore, for example, Japanese Patent Application Laid-Open No. 2004-347594 reports that an MS spectrum was obtained by subjecting a biological tissue specimen to protein staining, trypsin digestion, and MALDI mass spectrometry.

  On the other hand, in two-dimensional electrophoresis, after isoelectric focusing (IEF) is performed, separation is performed based on molecular weight by SDS (sodium dodecyl sulfate) polyacrylamide gel electrophoresis (SDS-PAGE). When performing SDS-PAGE, the protein in the gel is converted to SDS by equilibrating the first-dimensional gel with a solution containing SDS.

Stoeckli, M., Chaurand, P., Hallahan, ED, and Caprioli, MR, “Nature Medicine ) "Volume 7, p. 493-496, 2001 Chaurand, P., Schwartz, A. S., and Caprioli, M. R., “Analytical Chemistry”, Vol. 76, p. 86A-93A, 2004 Chaurand, P., Schwartz, AS, Billheimer, D., Xu, JB, Crecelius, A ), And Caprioli, MR, “Analytical Chemistry”, Vol. 76, p. 1145-1155, 2004 JP 2004-347594 A

  In the above conventional method, an MS spectrum can be acquired, but a meaningful MS / MS spectrum cannot be acquired because enzyme digestion is not performed. In addition, since digestion cannot be performed even by simply applying a digestive enzyme, a meaningful MS / MS spectrum cannot be obtained.

  Therefore, in order to identify a protein in a living tissue using mass spectrometry, the tissue must be separated and purified by a conventional biochemical technique. However, when a very small amount of material is used as a sample to be analyzed, the sample may be lost during the operation. Moreover, since the addition of the matrix is not controlled, there is a problem in resolution and reproducibility in mass spectrometry.

  In general, in order to identify a protein by mass spectrometry, spectral information obtained by multistage mass spectrometry is required. For this purpose, the sample protein is purified by a technique such as electrophoresis, then denatured and then peptideized with a digestive enzyme. Therefore, in order to identify a protein in a living tissue by mass spectrometry, it is necessary to perform multi-stage mass spectrometry by a method according to the above. However, since the protein maintains a three-dimensional structure in the tissue, digestion efficiency by digestive enzymes is poor.

  Accordingly, an object of the present invention is to provide a pretreatment method for a sample for mass spectrometry, which can perform mass spectrometry of proteins constituting a tissue with high analysis sensitivity and high analysis efficiency while maintaining the anatomical structure of a living tissue. It is to provide. Another object of the present invention is to provide a pretreatment method for a sample for mass spectrometry, which can perform multi-stage mass spectrometry while maintaining an anatomical structure in a living tissue.

  The present inventor has found that the object of the present invention can be achieved by applying a technique used for equilibration in two-dimensional electrophoresis, and has completed the present invention.

The present invention includes the following inventions.
(1) For biological tissue specimens
Denaturing the protein in the biological tissue by supplying a denaturant;
Staining the protein by supplying a staining agent;
Digesting the protein by dispensing a digestive enzyme;
A method of directly mass-analyzing a biological tissue, comprising dispensing a matrix and performing mass spectrometry by using a MALDI mass spectrometer.

The following (2) and (3) describe the biological tissue specimen.
(2) The mass spectrometry method according to (1), wherein the biological tissue specimen is a frozen section.
(3) The mass spectrometric method according to (1) or (2), wherein the biological tissue specimen is fixed to the surface of a film selected from a polyvinylidene difluoride film, a nitrocellulose film, and a nylon film.

The following (4) and (5) describe the modifier.
(4) The mass spectrometric method according to any one of (1) to (3), wherein the denaturing agent is selected from a surfactant, urea, and guanidine hydrochloride.
(5) The surfactant is sodium dodecyl sulfate, 3-[(3-cholamidopropyl) dimethylammonio] -1-propanesulfonic acid, polyoxyethylene-pt-octylphenyl ether, sorbitan monolaurate, And the mass spectrometric method according to (4), selected from 1-O-n-octyl-β-D-glucopyranoside.

The following (6) and (7) describe a reducing agent that can be used with the above modifier.
(6) The mass spectrometry method according to any one of (1) to (5), wherein the modifier is used together with a reducing agent.
(7) The mass spectrometric method according to (6), wherein the reducing agent is selected from 2-mercaptoethanol, dithiothreitol, tris [2-carboxyethyl] phosphine, and 2-mercaptoethanolamine.

The following (8) describes the above dispensing operation.
(8) The mass spectrometry method according to any one of (1) to (7), wherein a dispensing operation is performed using an inkjet technique in the step of digesting the protein and / or the step of performing the mass spectrometry.

The following (9) describes the mass spectrometry.
(9) The mass spectrometry method according to any one of (1) to (8), wherein multistage mass spectrometry is performed in the step of performing mass spectrometry.

  According to the present invention, there is provided a pretreatment method for a sample for mass spectrometry, capable of performing mass spectrometry of proteins constituting the tissue with high analysis sensitivity and high analysis efficiency while maintaining the anatomical structure of a biological tissue. be able to. Moreover, according to the present invention, it is possible to provide a pretreatment method for a sample for mass spectrometry, which can perform multi-stage mass spectrometry while maintaining an anatomical structure in a living tissue.

  In the present invention, “directly” means that the anatomical structure of the biological tissue specimen is maintained. In the present invention, each treatment solution is directly acted on a biological tissue specimen, whereby the protein in the tissue is processed in the tissue, and the obtained processed biological tissue is directly subjected to mass spectrometry as a sample for mass spectrometry. . Specifically, in the present invention, each treatment of tissue protein denaturation, protein staining, and protein digestion is performed, and MALDI mass spectrometry is performed.

  The biological tissue specimen in the present invention is not particularly limited as long as it has a state of maintaining an anatomical structure in the living body. In order to obtain such a state, a living tissue embedded with an appropriate embedding agent can be used. The embedding agent can be used without particular limitation depending on the suitability of the biological sample. For example, water, paraffin, celloidin, carbowax, gelatin, albumin, agarose, epoxy resin, polyester resin, water-soluble resin such as glycol methacrylate, etc. can be used, and those embedding agents are appropriately selected by those skilled in the art. can do. As such a biological specimen, for example, an unfixed sample such as a frozen section, an immobilized sample such as a paraffin-embedded section, or the like can be used.

  The biological specimen can be used by being fixed to the surface of the membrane and / or the support. Examples of the film include films made of organic synthetic polymers such as polyvinylidene difluoride (PVDF), nitrocellulose, polyamide, polyethylene, and derivatives thereof. Nylon etc. are mentioned as polyamide. Examples of the support include a glass support, a resin support, and a metal support. In the present invention, since analysis is performed using mass spectrometry, a metal support, for example, a sample plate for mass analysis, is preferably used as the support, and a biological specimen fixed to a membrane is attached to the sample plate. Alternatively, a biological specimen can be fixedly used on the sample plate. When a frozen section is used as a biological tissue specimen, fixation can be performed by placing the frozen section on a membrane and melting it. In addition, a conductive double-sided adhesive tape or the like may be used for fixing to the sample plate.

  Said biological specimen can be produced using a well-known method. After the preparation, the biological specimen can be appropriately washed and dried.

<Modification process>
By supplying an appropriate denaturing agent to such a biological tissue specimen, the protein in the biological tissue is denatured. The denaturing agent is not particularly limited as long as it destroys only the higher order structure while maintaining the primary structure of the protein. For example, the denaturing agent is selected from various surfactants, urea, guanidine hydrochloride and the like. As the surfactant, any of an anionic surfactant, a cationic surfactant, an amphoteric surfactant, and a nonionic surfactant can be used. For example, examples of the anionic surfactant include sodium dodecyl sulfate (SDS). Examples of amphoteric surfactants include 3-[(3-cholamidopropyl) dimethylammonio] -1-propanesulfonic acid (CHAPS). Nonionic surfactants include Triton X-100 (polyoxyethylene-pt-octylphenyl ether), Tween 20 (sorbitan monolaurate), 1-O-n-octyl-β-D-glucopyranoside, etc. Can be mentioned.

  In addition, these modifiers can select 1 type or multiple types. When multiple types are selected, the combination is not particularly limited as long as the selected modifiers do not interact with each other. Moreover, a modifier | denaturant can be normally supplied with the form of aqueous solution.

  The denaturant concentration may be 1 to 10M, preferably 4 to 8M. When the concentration is lower than the above concentration, protein denaturation tends to be insufficient. When the concentration is higher than the above concentration, the tissue section tends to peel off. The surfactant concentration can be 0.05% to 2%, preferably 0.1 to 1% (unit% can be w / v% or v / v% depending on the form of the surfactant). When the concentration is lower than the above concentration, protein denaturation tends to be insufficient. Further, when the concentration is higher than the above concentration, the surfactant remains on the tissue section, and the polymer-derived peak tends to be detected in the mass spectrum.

  The aqueous denaturant solution usually contains a buffer selected from Tris-HCl buffer, phosphate buffer, phosphate buffered saline, acetate buffer and the like. Tris-HCl can be used at a concentration of 10 mM to 100 mM, a phosphate buffer of 0.1 M to 0.3 M, a phosphate buffered saline of 0.05 M to 0.2 M, and an acetate buffer of 0.1 M to 0.2 M. When the concentration is lower than the above concentration, the tissue section tends to peel off. When the concentration is higher than the above concentration, the background tends to increase due to the influence of salt in mass spectrometry. In addition, 1 type or multiple types can be selected for these buffers. The pH of the buffer can be adjusted to 6 to 9, preferably 6.5 to 8.0 in Tris-HCl, phosphate buffer, and phosphate buffered saline, and pH 3 to 4 in acetate buffer.

  The denaturant aqueous solution may further contain a reducing agent for the purpose of cleaving disulfide bonds or protecting the SH group. Examples of the reducing agent include 2-mercaptoethanol, DTT (dithiothreitol), TCEP (Tris [2-carboxyethyl] phosphine), 2-MEA (2-mercapto). And ethanolamine (2-Mercaptoethanolamine). One or a plurality of these reducing agents can be selected.

  The concentration of the reducing agent in the aqueous denaturant solution can be 10 to 70 mM, preferably 40 mM to 50 mM. In the case of a concentration lower than the above concentration, the reaction efficiency tends to decrease in the enzyme digestion. When the concentration is higher than the above concentration, the background tends to increase due to the influence of salt in mass spectrometry.

  The modifier aqueous solution may further contain a stabilizer. Examples of the stabilizer include glycerol (glycerin).

  The stabilizer concentration in the aqueous denaturant solution can be 0.1 to 50 (v / v)%, preferably 20 to 50 (v / v)%. In the case of a concentration higher than the above concentration, in the case of a concentration higher than the above concentration, handling tends to be difficult due to the viscosity of the modifier.

  The protein in the biological specimen is denatured by supplying the aqueous denaturant solution to the biological tissue specimen. Specifically, the biological tissue specimen may be immersed in the above-described aqueous denaturant solution and shaken, or an aqueous denaturant aqueous solution sprayed with an airbrush or the like may be sprayed onto the biological tissue specimen and left under wet conditions. The shaking time and the standing time under wet conditions can be 3 to 24 hours. Denatured biological specimens can be appropriately washed.

<Dyeing process>
By supplying an appropriate staining agent to the biological tissue specimen in which the protein has been denatured by the above steps, the protein in the biological tissue is stained. When a hydrophobic embedding agent such as paraffin is used in the preparation of a biological tissue specimen, the embedding agent is removed prior to the staining step.

As the staining agent, those usually used for protein staining can be used without any particular limitation. For example, monoazo dyes such as methyl red; diazo dyes such as Ponceau S; triazo dyes such as Direct Blue 71; azoic dyes such as fast red; diphenylmethane dyes such as auramine; diaminotriphenylmethane dyes such as brilliant green; Triaminotriphenylmethane dye; xanthene dye such as rhodamine B; fluorone dye such as rhodamine 3G; acridine dye such as acridine orange; oxazine dye such as cresyl fast violet; thiazine dye such as toluidine blue; anthraquinone such as alizarin red S Dye: Natural dyes such as hematoxylin can be used. Dyeing can be performed by using these dyeing agents and performing operations according to ordinary methods. For example, the operation of immersing and shaking the biological tissue specimen in an aqueous stain solution dissolved in an ethanol-acetic acid aqueous solution at an appropriate concentration, and then washing may be repeated. When Direct Blue 71 is used, the ethanol-acetic acid aqueous solution is preferably an aqueous solution containing 10 to 70 (v / v)% ethanol-1 to 20 (v / v)% acetic acid. -It is preferable to set it as 0.005-0.01 (w / v)% in acetic acid aqueous solution, It is preferable to perform the said shaking for 2.5 to 7 minutes, and it is preferable to perform the operation of shaking and washing | cleaning 3-6 times repeatedly.
The stained biological specimen should be sufficiently dried.

<Digestion process>
The protein in the living tissue is digested by dispensing an appropriate protein digestion enzyme to the living tissue specimen in which the protein is stained by the above process. In the present invention, dispensing refers to supplying a reagent solution to a specific region on a biological tissue specimen (the same applies to a mass spectrometry step described later). That is, in the digestion step, an appropriate protein digestion enzyme is supplied to a specific region of the biological tissue specimen in which the protein is stained in the above step, thereby digesting the protein in the biological tissue in the specific region. From the viewpoint of effective use of biological tissue specimens and reduction of reagent consumption, it is preferable that the area and dispensing amount of a specific region be suppressed as much as possible and be sufficient to enable mass spectrometry described later. . In the present invention, a known apparatus capable of supplying a small amount of reagent solution can be used without any particular limitation in order to perform dispensing. In particular, it is preferable to use a dispensing device having a mechanism based on an ink jet technique. Examples of such a dispensing apparatus include a chemical printer CHIP-1000 (manufactured by Shimadzu Corporation).

  In order to prevent the enzyme from adsorbing to the membrane, it is preferable to perform a treatment for dispensing polyvinylpyrrolidone or the like prior to the dispensing of the enzyme. In this case, an aqueous polyvinyl pyrrolidone solution dissolved at an appropriate concentration in an aqueous methanol solution may be used. As polyvinyl pyrrolidone, it is preferable to use PVP-40 or the like, the methanol aqueous solution is preferably 10 to 80 (v / v)%, and polyvinyl pyrrolidone is 0.1 to 0.5 (w / v)% in the methanol aqueous solution. It is preferable that The dispensing amount of the polyvinylpyrrolidone aqueous solution can be 0.1 to 10 nl.

  Although it does not specifically limit as a protein digestive enzyme, Usually, trypsin is used. A person skilled in the art can appropriately determine the digestion conditions. The amount of trypsin solution dispensed can be 10 to 50 nl.

  In order to perform digestion in the tissue efficiently, the sample supplied with the digestive enzyme is preferably stored in a thermostat set at 20 to 40 ° C. for 1 to 24 hours. For example, the storage temperature and time are preferably 37 ° C. and 12 hours, respectively. When the temperature is lower than the above temperature, the enzyme activity tends to be low, and when the temperature is higher than the above temperature, the enzyme activity tends to be lost. In addition, when the storage time is shorter than the above storage time, digestion is not sufficiently performed and ionization efficiency tends to be low. When the storage time is longer than the above storage time, the enzyme self-digestion product increases and the detection intensity of the target substance is low. Tend to be.

  In the present invention, since the protein in the tissue is denatured, the digestion efficiency by the digestive enzyme is superior to the conventional one.

<Mass spectrometry process>
First, the matrix is overlaid and dispensed on the digested region on the biological tissue specimen. As the matrix, those used for MALDI mass spectrometry of proteins can be used without any particular limitation. For example, 2,5-dihydroxybenzoic acid (DHBA), DHBA (sDHB) mixed with 5-methoxysalicylic acid, a-cyano-4-hydroxycinnamic acid (a-CHCA), 3,5-dimethoxy-4-hydroxy Cinnamic acid (sinapic acid) or the like can be used. These matrices are dissolved in a suitable solvent such as acetonitrile-trifluoroacetic acid (TFA) aqueous solution and used as a matrix solution. For example, when DHBA is used, the acetonitrile-TFA aqueous solution is preferably an aqueous solution containing 25-50 (v / v)% acetonitrile-0.05-1 (v / v)% TFA, and the matrix concentration is acetonitrile- It is preferably 5 to 30 mg / ml in an aqueous TFA solution. The dispensing amount of the matrix solution can be 50 to 200 nl.

  Next, the portion where the matrix solution is dispensed is irradiated with laser to perform MALDI mass spectrometry. As the MALDI mass spectrometer, for example, AXIMA-QIT (manufactured by Shimadzu Corporation) can be used. As described above, in the present invention, since the protein in the tissue is denatured, the digestion efficiency by the digestive enzyme is superior to the conventional one. For this reason, it is possible to detect a protein peak with relatively strong intensity in MS analysis. This also allows MS / MS and higher multi-stage mass spectrometry. In the present invention, since multistage mass spectrometry can be performed, spectral information necessary for determination of amino acid sequence of protein, analysis of post-translational modification, and the like can be obtained.

<Example 1>
A frozen section of mouse brain tissue was thawed on a PVDF membrane and fixed. The membrane to which the frozen section was fixed was shaken in a 70 (v / v)% ethanol aqueous solution for 30 seconds. After washing twice, it was dried. After drying, the mixture was shaken in the denaturant solution for 12 hours. Here, the composition of the denaturant solution was 0.5 M Tris / HCl (pH 6.8) 2 ml, 10 (v / v)% SDS aqueous solution 4 ml, 50 (v / v)% glycerol aqueous solution 7 ml, urea 8.4 g. , And DTT 0.1 g. In this denaturant solution, a PVDF membrane having a frozen section melted and bonded was immersed for 12 hours. After the tissue section became transparent, it was washed with a washing solution (a solution containing 40 (v / v)% ethanol and 10 (v / v)% acetic acid in water) for 30 seconds. Thereafter, the tissue sections were stained for 7 minutes in a staining solution. Here, the staining solution is a solution obtained by dissolving Direct blue (Aldrich, 12, 240-7) in the washing solution so that the final concentration is 0.008 (v / v)%. Stained tissue sections were shaken in washing solution for 5 minutes. This series of dyeing and washing operations was performed three times in total. Thereafter, the tissue section was shaken in ultrapure water for 5 minutes and sufficiently dried.

The following treatment was performed on the tissue sections subjected to the above treatment using a chemical printer (CHIP-1000, Shimadzu Corporation).
A polyvinylpyrrolidone solution (a solution containing polyvinylpyrrolidone in a 60% (v / v) aqueous methanol solution at a concentration of 0.25% (v / v)) was printed. The total print volume was 7 nl.
A digestive enzyme solution (a solution containing trypsin in an aqueous solution containing 25 mM sodium bicarbonate and 10 (v / v)% 2-propanol at a concentration of 200 μg / ml) was printed. The total print volume was 50 nl. Thereafter, the tissue section was stored under humid conditions for 12 hours in a thermostatic chamber set at a temperature of 37 ° C.
A matrix solution (a solution containing 2,5-dihydroxybenzoic acid at a concentration of 8 mg / ml in an aqueous solution containing 25% (v / v) acetonitrile and 0.1% (v / v) TFA) was printed. The total print volume was 100 nl.
The place where the reagent was printed was measured with a mass spectrometer (AXIMA-QIT, Shimadzu Corporation).

<Comparative Example 1>
The same operation as in Example 1 was performed except that a 70 (v / v)% aqueous ethanol solution was used instead of the denaturant solution.

<Result>
FIG. 1A shows the MS spectrum obtained in Example 1, and FIG. 1B shows the MS spectrum obtained in Comparative Example 1. In FIG. 1, the horizontal axis represents mass / charge (Mass / Charge), the vertical axis represents relative intensity, and the asterisk indicates that the peak to which it is attached is a peak derived from a protein in the tissue. . As shown in FIG. 1, it can be seen that in (a) where the denaturation treatment was performed, many peaks with relatively strong intensity were detected.

  Since many peaks were detected in the MS spectrum as described above by the method of the present invention, MS / MS measurement was performed on these peaks. FIG. 2 shows the result of MS / MS measurement using m / z 1602 as a precursor ion in FIG. 1A, and FIG. 3 shows the result of MS / MS measurement using m / z 1902 as a precursor ion. 2 and 3, the horizontal axis represents mass / charge (Mass / Charge), and the vertical axis represents relative intensity. As these spectra show, it can be seen that the present invention has made it possible to obtain very good MS / MS spectra.

MS spectrum (a) obtained in Example 1 which was pretreated by immersing in a denaturant solution for 12 hours, and MS spectrum obtained in Comparative Example 1 which was pretreated by immersing in an ethanol solution for 12 hours ( b). It is a MS / MS spectrum which used m / z 1602 in Fig.1 (a) as a precursor ion. It is a MS / MS spectrum which used m / z 1902 in Fig.1 (a) as a precursor ion.

Claims (9)

For biological tissue specimens,
Denatures the protein in the living tissue by supplying a denaturant;
Staining the protein by supplying a staining agent;
Digesting the protein by dispensing a digestive enzyme;
A method of directly mass-analyzing a living tissue, comprising dispensing a matrix and performing mass spectrometry by using a MALDI mass spectrometer. The mass spectrometry method according to claim 1, wherein the biological tissue specimen is a frozen section. The mass spectrometry method according to claim 1 or 2, wherein the biological tissue specimen is fixed to the surface of a film selected from a polyvinylidene difluoride film, a nitrocellulose film, and a nylon film. The mass spectrometric method according to claim 1, wherein the denaturing agent is selected from a surfactant, urea, and guanidine hydrochloride. The surfactant is sodium dodecyl sulfate, 3-[(3-cholamidopropyl) dimethylammonio] -1-propanesulfonic acid, polyoxyethylene-pt-octylphenyl ether, sorbitan monolaurate, and 1- The mass spectrometric method according to claim 4, which is selected from On-octyl-β-D-glucopyranoside. The mass spectrometric method according to any one of claims 1 to 5, wherein the modifier is used together with a reducing agent. The mass spectrometry method according to claim 6, wherein the reducing agent is selected from 2-mercaptoethanol, dithiothreitol, tris [2-carboxyethyl] phosphine, and 2-mercaptoethanolamine. The mass spectrometry method according to any one of claims 1 to 7, wherein a dispensing operation is performed using an inkjet technique in the step of digesting the protein and / or the step of performing mass spectrometry. The mass spectrometry method according to claim 1, wherein multistage mass spectrometry is performed in the step of performing mass spectrometry.

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