JP2007170870A - Insitu detection method using mass analysis - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a high sensitivity insitu detection method of a target molecule using mass analysis. <P>SOLUTION: The insitu detection method of the target molecule present in tissue or a cell includes a process (1) for bringing the tissue or the cell into contact with the surface of a support to transfer the molecule contained in the tissue or the cell to the surface of the support, a process (2) for subjecting the support to mass analysis and a process (3) for detecting the target molecule on the basis of the result of mass analysis. In the process (3), the target molecule is dyed by image processing. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

The present invention relates to a novel method for detecting a target molecule present in a tissue or cell in situ using mass spectrometry.

  As a method for identifying the position of a specific molecule contained in a tissue, for example, a histochemical staining method or an immunostaining method is known. However, in the histochemical staining method, only the difference in the degree of staining and the difference in color distribution between the tissues to be compared were observed, and confirmation of the exact distribution of a single molecule remained unclear. On the other hand, the immunostaining method is applied only when the protein to be stained (ie, the target protein) is known and the corresponding antibody is present in advance, and the target protein is a variant that has undergone alternative splicing or post-translational modification. That identification was difficult. In addition, the ligand staining method in the case where the target protein is a receptor has been pointed out the same problem as the immunostaining method.

  As one of means for solving the above problems, tissue imaging using mass spectrometry (hereinafter also referred to as “MS imaging”) has been proposed (see Non-Patent Documents 1 to 4). However, in conventional MS imaging, a biological tissue piece is placed on a plate for mass spectrometry, and then the tissue is dried, a matrix is applied thereon, and MALDI-type mass spectrometry is performed. There are various difficulties in differential analysis. For example, (1) protein ionization during laser irradiation becomes non-uniform due to the unevenness of the dry tissue surface, and (2) cells, proteins, lipids, sugars, and low molecular compounds contained in different tissues. Since all other components are different, the difference in their component ratios has a serious effect on the crystallization of the matrix. As a result, many proteins cannot be detected. I couldn't consider it.

  Furthermore, in the conventional method, when a tissue piece is placed on a plate after tissue staining, dye molecules are mixed with protein, so that the peak intensity of the protein is reduced or noise due to a large amount and many types of stained molecules is generated. There were also drawbacks such as making the analysis extremely difficult.

The present inventors have so far grouped membrane proteins (eg, receptors) and compounds capable of interacting with them (eg, soluble ligands), and collectively analyzed them using mass spectrometry. A proteome analysis method that can be used is proposed (Patent Document 1), and further, a plate for mass spectrometry suitable for its implementation is described (Patent Document 2). In the method, preferably, molecules contained in the soluble fraction of the biological sample are separated by gel electrophoresis, and transferred onto the plate by directly contacting the gel with a plate for mass spectrometry.
However, the application of the method to MS imaging is not suggested.
International Application Publication No. 02/56026 Pamphlet International Application Publication No. 2004/031759 Pamphlet Shimma, S., & Setoh, M., J. Mass Spectrom. Soc. Jpn., 53: 230 (2005) Karasawa, K., Komatsu, M., & Yamazaki, T., J. Mass Spectrom. Soc. Jpn., 53: 284 (2005) Stoeckli M., Chaurand P., Hallahan DE & Caprioli RM, Nat. Med., 7: 493-6 (2001) Chaurand P, Schwartz SA, Billheimer D, Xu BJ, Crecelius A, Caprioli RM, Anal. Chem., 76 (4): 1145-55 (2004)

  An object of the present invention is to provide a novel MS imaging technique that overcomes the disadvantages of the conventional methods, and thus provides a means for clarifying the location and amount of a specific molecule contained in a tissue. It is.

As a result of intensive research to achieve the above-mentioned object, the present inventors have directly disrupted tissues and cells and extracted and separated fractions containing the target protein without directly extracting the tissue or cells. The molecule can be transferred to the plate unexpectedly and efficiently, so that mass spectrometry can be performed without holding the tissue / cell on the plate. Found that can be done. In addition, when proteins are transferred to a plate by actually contacting tissue pieces from various organisms, quantitative analysis of the target protein between different tissues can be performed without affecting the identity of the tissues being compared (differential analysis). ) Can be implemented with high accuracy.
As a result of further studies based on these findings, the present inventors have completed the present invention.

That is, the present invention
A method for detecting a target molecule present in a tissue or cell in situ ,
(1) A step of bringing a tissue or cell into contact with the support surface and transferring a molecule contained in the tissue or cell onto the support surface;
(2) A method including the step of subjecting the support to mass spectrometry; and (3) a method of detecting a target molecule based on the result of mass spectrometry is provided. The method may also include staining the target molecule by image processing in the step (3). Alternatively, the step (3) may include profiling a tissue or cell based on the result of mass spectrometry, and further differentially analyzing profiles in two or more different tissues or cells. .

  In the method of the present invention, the matrix is applied after the protein distributed in the tissue is once transferred to the plate for mass spectrometry, instead of directly applying the matrix to the tissue piece. Problems such as non-uniform ionization and the influence on the crystallization of the matrix due to the difference in the composition ratio of the tissue are solved, and more proteins can be detected and quantitative examination can be performed. Furthermore, after transferring the test tissue to the plate for mass spectrometry, the tissue can be peeled off from the plate to stain the tissue, so that trouble caused by mixing of dye molecules does not occur.

  Any tissue or cell may be used as a sample in the method of the present invention, and any animal or plant cell [for example, hepatocyte, spleen cell, nerve cell, glial cell, pancreatic β cell, bone marrow cell, Mesangial cells, Langerhans cells, epidermal cells, epithelial cells, goblet cells, endothelial cells, smooth muscle cells, fibroblasts, fiber cells, muscle cells, adipocytes, immune cells (eg macrophages, T cells, B cells, natural killer) Cells, mast cells, neutrophils, basophils, eosinophils, monocytes), megakaryocytes, synoviocytes, chondrocytes, bone cells, osteoblasts, osteoclasts, mammary cells or stromal cells, or Progenitor cells of these cells, stem cells, cancer cells, etc.] or tissues [eg, brain, brain parts (eg, olfactory bulb, amygdala, basal sphere, hippocampus, hypothalamus, cerebral cortex, medulla, cerebellum) Spinal cord, pituitary, stomach, pancreas, kidney, liver, gonad, thyroid, gallbladder, bone marrow, adrenal gland, skin, lung, gastrointestinal tract (eg, large intestine, small intestine), blood vessel, heart, thymus, spleen, submandibular gland, peripheral Blood, prostate, testis, ovary, placenta, uterus, bone, joint, adipose tissue (eg, brown adipose tissue, white adipose tissue), muscle, etc.] or sections thereof. Preferably, it includes, but is not limited to, a sliced tissue sample such as a cross section of tissue. Examples of the transverse section include those having a width of about 0.1 μm to about 10 mm. As a method for preparing the cross section, the methods described in Non-Patent Documents 1 to 4 and the like can be appropriately modified as necessary.

  The target molecule to be detected in the method of the present invention may be any molecule as long as it exists in a tissue or cell used as a sample. For example, a protein (eg, enzyme, transcription factor (glycoprotein, lipoprotein) Complex proteins such as proteins)), peptides, nucleic acids (eg, polynucleotides, oligonucleotides), sugars (eg, polysaccharides, oligosaccharides), lipids (eg, glycolipids, phospholipids, cholesterol, neutral fats), Examples include, but are not limited to, free fatty acids, toxins, viral epitopes, hormones, enzyme substrates or inhibitors, cofactors, drugs, lectins and antibodies. Preferably, the target molecule is a protein.

There is no particular limitation on the number of target molecules in the method of the present invention. That is, since the method of the present invention uses mass spectrometry for detection of a target molecule, it goes without saying that it can be used to detect one or several types of target molecules, but more various types (for example, 10 types). As described above, it can also be used for comprehensive detection of 100 or more types of molecules.
Therefore, the present invention also provides a method for comprehensively detecting molecules contained in a tissue or cell by mass spectrometry and profiling the tissue or cell. By applying the method to two or more different tissues or cells (eg, diseased and normal tissues such as cancer, cells collected from drug-treated and non-treated patients, etc.) and comparing their profiles to each other It is possible to discover and identify a molecule whose expression is remarkably varied in a specific test tissue or cell, and is useful for, for example, searching for a disease marker such as a tumor marker or a toxicity marker such as phospholipidosis. Such comparative analysis can be performed by visually comparing two or more profiles, but differential expression is obtained by subtracting the same mass number peak in the control tissue or cell from the mass analysis data of the target tissue or cell. This profile is preferably output by outputting data by computer processing.

  Unlike the conventional MS imaging method, the method of the present invention is not applied to mass spectrometry by applying a matrix on a plate for mass spectrometry (support) and applying mass analysis to the tissue or tissue piece. Alternatively, after the cells are brought into contact with the support surface and the molecules contained in the tissue or cells are transferred onto the support surface, the matrix is applied to the support with the tissue or cells removed, and the mass It is used for analysis.

The support used as the plate for mass spectrometry may be any material as long as it has a surface structure that can efficiently adsorb molecules contained in tissues or cells. Such surface structures include, for example, functionalized glass, Si, Ge, GaAs, GaP, SiO ₂ , SiN ₄ , modified silicon, a wide range of gels or polymers (eg, (poly) tetrafluoroethylene, (poly And vinylidene difluoride, polystyrene, polycarbonate, or combinations thereof). Examples of surface structures having a plurality of monomer or polymer sequences include linear and cyclic polymers of nucleic acids, polysaccharides, lipids, peptides having α-, β-, or ω-amino acids, gel surfaces used in chromatography Carriers (anionic / cationic compounds, hydrophobic compounds composed of carbon chains 1-18, hydrophilic compounds (eg, carriers cross-linked with silica, nitrocellulose, cellulose acetate, agarose, etc.), artificial homopolymers) (For example, polyurethane, polyester, polycarbonate, polyurea, polyamide, polyethyleneimine, polyarylene sulfide, polysiloxane, polyimide, polyacetate, etc.) Any known drug or natural compound is bound to any of the above compounds (covalent and non-covalent bonds). ) By heteropolymer etc. Coating, and the like.

  In a preferred embodiment, the support used as the plate for mass spectrometry is a substrate coated with a thin layer of polyvinylidene difluoride (PVDF), nitrocellulose or silica gel, particularly preferably PVDF [usually a plate for mass spectrometry For example, insulators (glass, ceramics, plastics, resins, etc.), metals (aluminum, stainless steel, etc.), conductive polymers, composites thereof, etc. An aluminum plate is preferably used. The shape of the support can be appropriately devised, for example, in the shape of a multi-titer plate, or in a shape suitable for the sample analyzer, particularly the sample inlet.

Preferably, the coating refers to a thin layer formed by depositing on a support in a state where coating molecules are dispersed, instead of overlaying a pre-formed structure like a membrane on the support. The manner in which the coating molecules are deposited is not particularly limited, but means exemplified in a method for preparing a plate for mass spectrometry described later is preferably used.
The thickness of the thin layer can be appropriately selected within a range that does not adversely affect the transfer efficiency of the molecules contained in the tissue or cells, the measurement sensitivity of mass spectrometry, etc., for example, about 0.001 to about 100 μm , Preferably about 0.01 to about 30 μm.

Although the plate for mass spectrometry (support) can be prepared by a method known per se, for example, the above preferred mass spectrometry plate is prepared by thinly coating the surface of the support with a coating molecule such as PVDF. The Preferred examples of the coating means include application, spraying, vapor deposition, dipping, printing (printing), and sputtering.
In the case of “application”, the coating molecule is dissolved in an appropriate solvent, for example, an organic solvent such as dimethyl formamide (DMF) at an appropriate concentration (eg, about 1 to about 100 mg / mL) (coating) The molecule-containing solution) can be applied to the substrate using a suitable tool such as a brush.
In the case of “spraying”, the coating molecule-containing solution prepared in the same manner as described above may be put in a sprayer and sprayed so that PVDF is uniformly deposited on the substrate.
In the case of “evaporation”, the coating molecule (which may be solid or solution) is heated and vaporized in a vacuum tank containing the base material using a normal vacuum deposition apparatus for producing an organic thin film. A thin layer of molecules can be formed.
In the case of “immersing”, the substrate may be immersed in a coating molecule-containing solution prepared in the same manner as described above.
In the case of “printing”, various printing techniques that can be normally used depending on the material of the substrate can be appropriately selected and used. For example, screen printing or the like is preferably used.
In the case of “sputtering”, for example, a DC high voltage is applied between the substrate and the coating molecule while introducing an inert gas (eg, Ar gas) in a vacuum, and the ionized gas is caused to collide with the molecule. The repelled coating molecules can be deposited on the substrate to form a thin layer.
The coating may be applied to the entire surface of the substrate, or may be applied only to one surface subjected to mass spectrometry.

The coating molecule can be used in a suitable form depending on the coating means. For example, the coating molecule can be applied to the substrate in the form of a coating molecule-containing solution, a coating molecule-containing vapor, a solid coating molecule, etc. It is preferable to apply in the form. “Apply” refers to bringing a coating molecule into contact with the support so that the coating molecules remain and deposit on the support after contact. The amount of application is not particularly limited, and examples of the coating molecular weight include about 10 to about 100,000 μg / cm ² , preferably about 50 to about 5,000 μg / cm ² . After the application, the solvent is removed by natural drying, vacuum drying or the like.

  The substrate of the mass spectrometric plate may be modified (processed) in advance by an appropriate physical or chemical technique before coating with the coating molecule. Specifically, techniques such as polishing the plate surface, scratching, acid treatment, alkali treatment, glass treatment (tetramethoxysilane, etc.) are exemplified.

  Transfer of the molecules contained in the tissue or cells to the mass spectrometry plate (support) can be achieved by bringing the tissue or cells into contact with the surface of the support. Molecules present in the tissue or cells including the target molecule migrate to the support surface by diffusion. The time for which the tissue or cell is kept in contact with the support surface is not particularly limited as long as it is at least sufficient for the target molecule to migrate to the support surface, and is, for example, about 1 second to about 180 minutes. Also, the transfer temperature is not particularly limited, but for example, it can be carried out at about -80 to about 60 ° C, preferably about 4 to about 37 ° C.

  In addition, after the transfer, a reagent called a matrix may be added so as to absorb the laser beam and promote ionization of the analyte molecule through energy transfer, so as to be advantageous for later mass spectrometry (by MALDI method). it can. As the matrix, those known in mass spectrometry can be used. For example, sinapinic acid (SPA (= 3,5-dimethoxy-4-hydoroxycinammic acid)), indoleacrylic acid (IAA), 2,5-dihydroxybenzoic acid (DHB) ), Α-cyano-4-hydroxycinammic acid (CHCA), and the like, but is not limited thereto. Preferably, it is DHB or CHCA.

By analyzing the molecule derived from the test tissue or cell transferred onto the support surface by the above method, the location and amount of the target molecule can be identified from the information on the molecular weight.
A mass spectrometer ionizes a gaseous sample, then puts the molecule or molecular fragment into an electromagnetic field, separates it by mass number / charge number from its movement state, and determines the molecular weight of the substance by obtaining the spectrum of the substance. It is a device that measures and detects. Matrix-assisted laser deionization (MALDI), in which a sample and a matrix that absorbs laser light are mixed, dried and crystallized, and ionized analytes are brought into vacuum by ionization by energy transfer from the matrix and instantaneous heating by laser irradiation. And the MALDI-TOFMS method using time-of-flight mass spectrometry (TOFMS), which analyzes the mass number based on the time-of-flight difference of sample molecular ions by initial acceleration. Principles of ionization, nanoelectrospray mass spectrometry (nano-ESMS), etc., in which sample solutions are electrically sprayed into the atmosphere and individual analyte multivalent ions are unfolded into the gas phase A mass spectrometer based on can be used.
A method for mass spectrometry of molecules on a plate for mass spectrometry is known per se. For example, the methods described in any of Patent Documents 1 and 2 and Non-Patent Documents 1 to 4 can be used with appropriate modifications as necessary.

  From the result of mass spectrometry, based on the molecular weight information of the target molecule, the presence / absence, location and amount of the target molecule in the test tissue or cell can be identified. In this step, the information from the mass spectrometer is subjected to image processing using an arbitrary program, whereby the peak information of a specific target molecule is stained, as if observing a histochemical staining image, It is also possible to obtain a stained image of the target molecule. Such image processing programs are known, and examples thereof include, but are not limited to, those described in Caprioli et al. (Non-Patent Documents 3 and 4 above). It will be understood that those skilled in the art can easily construct or modify a program to suit a specific tissue or cell or a specific target molecule using known information processing techniques.

  The present invention can also be used for the detection and identification of molecules that can interact with the molecules present in the tissue or cells transferred onto the plate for mass spectrometry as described above. That is, in the collective identification method of molecules that can interact as described in Patent Document 1, contact is made with the above tissue or cell instead of the plate for mass spectrometry transferred from the gel after electrophoresis of the sample. Thus, by using a mass spectrometry plate to which molecules contained in the tissue or cells are transferred, the molecules and molecules that can interact with the molecules can be collectively detected and collectively identified by mass spectrometry. The sample containing molecules that can interact with molecules contained in tissues or cells may be any sample that contains or is expected to contain such molecules, for example, the same In the same or different species, derived from the same tissue or cell as the tissue or cell (eg, crude extract, soluble fraction, membrane fraction, fraction of subcellular organelle, etc.) Examples include, but are not limited to, those derived from tissues or cells different from the tissues or cells, artificial compound libraries, random peptide libraries, and the like in different individuals.

  The present invention will be described more specifically with reference to the following examples. However, these are merely examples and do not limit the present invention.

Example 1 Mass Analysis of Tissue Section Transcript of Mouse Liver and Brain After slicing mouse liver at 1 mm width and slicing mouse brain at 2 mm width, PVDF-coated mass spectrometry plate In accordance with the method described in Example 1, the tissue piece was laminated at room temperature for 5 minutes, the tissue piece was removed with tweezers, and the plate surface was further washed with PBS to completely remove the tissue piece. Thereafter, two types of matrices (CHCA, SPA) were applied to the plate surface, and mass spectrometry was performed with MALDI-TOF (Ultraflex II manufactured by Bruker).
The results are shown in FIGS. Mouse brain and liver data by Richard M. Caprioli et al. [See Non-Patent Documents 3 and 4 above. They first stack a frozen section on a mass spectrometric carrier (magnetic glass, gold coated plate), apply a matrix on it, and perform direct laser irradiation on the section] It was found that both the number of detected peaks and peak intensity were superior to those of the conventional method [see FIGS. 1 and 2; all peaks (indicated by arrows) obtained by the method of Caprioli et al. Were also detected by the method of the present invention. The peak intensity was also high. In addition, a large number of peaks (denoting mass numbers) were detected only in the method of the present invention].

  According to the method of the present invention, the disadvantages of low sensitivity and non-quantitative in conventional MS imaging are eliminated, so that the molecular weight (M / z) of the molecule can be accurately determined regardless of whether the target molecule is known or unknown. ) Can be distinguished and stained, and the advantages inherent in mass spectrometry, such as selective splicing and post-translational modification can be detected accurately. Moreover, since a target protein can be quantitatively compared between different tissues, differential analysis can be performed with high accuracy. Therefore, it is extremely useful as a more accurate and sensitive analysis method to replace the histochemical staining method and immunostaining method.

It is a figure which shows the mass spectrometry result (matrix: CHCA) of the molecule | numerator group transcribe | transferred from the mouse liver section to the plate for mass spectrometry coated with PVDF. The vertical axis represents relative intensity, and the horizontal axis represents mass number (M / z value). Arrows indicate peaks detected by the method of Caprioli et al. It is a figure which shows the mass spectrometry result (matrix: CHCA) of the molecule | numerator group transcribe | transferred from the mouse brain section to the PVDF-coated mass spectrometry plate. The vertical axis represents relative intensity, and the horizontal axis represents mass number (M / z value). Arrows indicate peaks detected by the method of Caprioli et al. It is a figure which shows the mass spectrometry result (matrix: SPA) of the molecule | numerator group transcribe | transferred from the mouse liver section to the plate for mass spectrometry coated with PVDF. The vertical axis represents relative intensity, and the horizontal axis represents mass number (M / z value). It is a figure which shows the mass spectrometry result (matrix: SPA) of the molecule | numerator group transcribe | transferred from the mouse brain section to the PVDF-coated mass spectrometry plate. The vertical axis represents relative intensity, and the horizontal axis represents mass number (M / z value).

Claims (10)

A method for detecting a target molecule present in a tissue or cell in situ ,
(1) A step of bringing a tissue or cell into contact with the support surface and transferring a molecule contained in the tissue or cell onto the support surface;
(2) subjecting the support to mass spectrometry; and (3) detecting the target molecule based on the results of mass spectrometry.   Between the steps (1) and (2), the method includes a step of bringing a sample containing a molecule capable of interacting with a target molecule into contact with the support surface, and in step (3), the target molecule and a molecule capable of interacting with the target molecule. The method according to claim 1, comprising: collectively detecting.   The method according to claim 1 or 2, comprising staining the target molecule by image processing in step (3).   The method according to claim 1, wherein the target molecule is a protein.   The method according to claim 1, comprising profiling a tissue or a cell in step (3) based on the result of mass spectrometry.   6. The method of claim 5, comprising differentially analyzing profiles in two or more different tissues or cells.   The method according to claim 5 or 6, comprising displaying the profiling result after image processing.   The method according to claim 1, wherein the support surface is coated with polyvinylidene difluoride, nitrocellulose or silica gel.   9. The method of claim 8, wherein the support surface is coated with polyvinylidene difluoride.   10. A method according to claim 8 or 9, wherein the coating means is application, spraying, vapor deposition, dipping, printing or sputtering.

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