JP2007078491A - Data acquisition method, data detecting method, pretreatment device and data acquisition device - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method for identifying the kind of protein contained in each of cells with a diameter size of 10 to several μm, a pretreatment device for detecting protein contained in a specific cell and a data acquisition device. <P>SOLUTION: A liquid droplet containing digestive enzyme is applied to the specific cell using an ink jet method and the mass of peptide subjected to limited decomposition is analyzed by a mass analyzing method such as a flight time type secondary ion mass analyzing method or the like. Various data bases are utilized to specify protein before limited decomposition from the peptide. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to an information acquisition method, a detection method, a preprocessing device, and an information acquisition device. In particular, the present invention relates to a method for detecting a protein at a specific site in a biological tissue, and a biological tissue pretreatment device and an information acquisition device therefor.

With the recent progress of genome analysis, the importance of protein analysis, which is a gene product existing in a living body, has been rapidly highlighted. Conventionally, the importance of protein expression and function analysis has been pointed out, and the development of analysis methods thereof has been promoted. These methods are
(1) Separation and purification by two-dimensional electrophoresis or high performance liquid chromatography (HPLC),
(2) Based on a combination of detection systems such as radiation analysis, optical analysis, and mass spectrometry.

The foundation of protein analysis technology is called proteome analysis, which analyzes proteins that are produced from genes and actually work in vivo, and investigates cell functions and causes of diseases. It is aimed. A typical analysis method includes a method including the following steps.
(1) Extraction of protein from a target biological tissue or cell.
(2) Protein separation by two-dimensional electrophoresis.
(3) Analysis of a protein or a fragment thereof by mass spectrometry such as MALDI method (matrix-assisted laser desorption-time-of-flight mass spectrometry: MALDI-TOFMS).
(4) Identification of proteins using databases such as genome projects.

In addition, a method including the following steps can be exemplified.
(1) Extraction of protein from a target biological tissue or cell.
(2) Digestion (or denaturation) of the extracted protein.
(3) Analysis of the digested (or denatured) protein using a system combining a liquid chromatograph (LC) and an ion trap mass spectrometer (Ion-trap MS).
(4) Database construction and protein identification.
(Toshiaki Isobe, Nobuhiro Takahashi, “Experimental Medicine, separate volume, Proteome Analysis”, Yodosha, 2000).

  These proteome analyzes have already begun to produce results, for example, cancers that are associated with recurrence and metastasis.

  On the other hand, the present inventors have developed an information acquisition method and apparatus based on the TOF-SIMS method (time-of-flight secondary ion mass spectrometry) for the purpose of visualizing a two-dimensional protein distribution in a protein chip or a biological tissue section. Proposed (International Publication No. 05/003715 pamphlet). This technique uses an inkjet method, etc., and applies an ionization promoting substance and / or digestive enzyme to the above protein chip or biological tissue slice, and information on the type of protein (including information on peptides that have been limitedly digested by the digestive enzyme) Is visualized by the TOF-SIMS method while maintaining the position information.

Further, as a protein chip repair method and apparatus, a method and apparatus for applying a proteolytic enzyme solution to a spot that requires repair under a microscope while using an inkjet method has been proposed (Japanese Patent Laid-Open No. 2002-365288). ).
Toshiaki Isobe, Nobuhiro Takahashi "Experimental Medicine Separate Volume Proteome Analysis", Yochisha, 2000 International Publication No. 05/003715 Pamphlet Japanese Patent Laid-Open No. 2002-365288

  However, the conventional proteome analysis method is intended for specific biological tissues, body fluids, blood, and the like, and is not directly directed to cells having a size of 10 to several tens of micrometers. If we can identify proteins contained in specific lesion cells such as cancer cells, proteins contained in cells adjacent to cancer cells, or both, we will contribute to the development of diagnostic devices and drug discovery (drug candidate screening) devices. It will be possible.

  In addition, the above information acquisition method proposed by the present inventors can acquire information on proteins in diseased tissues and normal tissues (including information on peptides that are limitedly digested by digestive enzymes), but is included in individual cells. In some cases, the protein was not sufficient to be obtained separately from that in adjacent cells.

  Furthermore, the protein chip repair method disclosed in Japanese Patent Application Laid-Open No. 2002-365288 is not intended for biological tissues that contain a large amount of water and are soft and easily denatured. Since it is not intended for mass spectrometry, it seems difficult to use the method as it is.

  An object of the present invention is to provide an information acquisition method for obtaining information that makes it possible to identify the type of protein contained in each cell having a size of 10 to several tens of micrometers. Another object of the present invention is to provide a method for detecting the presence or absence of a measurement object in a specimen using this information acquisition method. Another object of the present invention is to provide a preprocessing device and an information acquisition device for use in these methods.

The information acquisition method of the present invention includes:
An information acquisition method for measuring a mass of a constituent constituting an object in a specimen and acquiring information on the object from the mass,
Preparing a biological tissue section as a specimen,
Optically observing the specimen and determining a site for obtaining information about the object in the specimen;
A step of applying a droplet containing a digestive enzyme to the site using an inkjet method, and digesting the site with a digestive enzyme;
A step of measuring the mass of the constituent of the site obtained by digestive enzymatic degradation using a mass spectrometer, and a step of obtaining information on the object from the mass of the constituent;
It is the information acquisition method characterized by having.

  The method for detecting the presence or absence of an object in a sample according to the present invention detects the presence or absence of the object in the sample by acquiring information on the presence or absence of the object by the information acquisition method described above. Is a detection method characterized by

The pretreatment apparatus of the present invention is a pretreatment apparatus for measuring the mass of a constituent constituting an object in a specimen and acquiring information on the object from the mass,
A stage for fixing the substrate on which the specimen is placed,
Stage position control means for controlling the position of the stage;
Observation means for optically observing the specimen,
Means for applying a droplet containing a digestive enzyme to the site for obtaining information about the object determined in the optically observed specimen image, and the temperature and humidity of the region where the stage is arranged A pre-processing apparatus having temperature and humidity control means for controlling.

The information acquisition apparatus of the present invention is
An information acquisition device for measuring a mass of a constituent constituting an object in a specimen and acquiring information about the object from the mass,
A pre-processing apparatus having the above-described configuration for obtaining a constituent constituting an object in a specimen;
A mass spectrometer for analyzing the mass of the component;
It is an information acquisition apparatus characterized by having.

  According to the present invention, it is possible to provide a method for identifying the type of protein contained in each cell having a diameter of 10 to several tens of micrometers, and it is possible to provide a pretreatment device and an information acquisition device applicable to this method. More specifically, a method for identifying a protein contained in a specific lesion cell such as a cancer cell, a protein contained in a cell adjacent to a cancer cell, or both, can be provided. A processing device and an information acquisition device can be provided.

The information acquisition method of the present invention includes at least the following steps.
(1) A step of preparing a biological tissue section as a specimen.
(2) A step of optically observing the specimen and determining a site for obtaining information on the object in the specimen.
(3) A step of applying a droplet containing a digestive enzyme to the site using an inkjet method to decompose the site by digestive enzyme.
(4) The process of measuring the mass of the structure of this site | part obtained by the said digestive enzyme decomposition | disassembly using a mass spectrometer.
(5) A step of obtaining information on the object from the mass of the component.

  The preferable aspect of the information acquisition method of this invention which has said each process at least is shown in FIG.1 and FIG.2. The difference between the steps in FIG. 1 and FIG. 2 is that at least a part of the composition is measured by bringing a composition obtained by digestive enzymatic degradation of the target, specifically a protein degradation product, into contact with another substrate surface. Whether or not a process of moving to the base material side (also referred to as a transfer process) is included. In the process of FIG. 1, the enzyme degradation product on the specimen is directly analyzed, whereas in the process of FIG. 2, this is transferred to another substrate and analyzed.

  Hereinafter, “protein degradation product” is also referred to as “enzymatic degradation product” or “degradation peptide”.

  A feature of the information acquisition method of the present invention is that a slice of a diseased tissue can be used as a specimen, and in particular, a specific cell (for example, a diseased cell) in a lesion tissue slice can be selected as a site for obtaining information on an object. is there. Examples of specimens include a section of a diseased tissue collected during surgery, or a frozen portion of the diseased tissue, and a specific site sliced using a microtome or the like. The specimen is preferably prepared at a low temperature until it is decomposed by a digestive enzyme described later (including a cooling mechanism for a stage described later).

  The part for obtaining information on the object in the specimen under the optical observation of the specimen (hereinafter also referred to as the part for information acquisition) is determined by the observer visually in the specimen visual field image of the optical microscope. This can be done by selecting the part according to a preset criterion.

  Taking cancer cells as an example, the morphology of cancer cells differs depending on the site (esophageal cancer, breast cancer, bladder cancer, etc.), but cancer cells and normal cells generally differ greatly in morphology. This is thought to be because sugar chains on the cell surface change when normal cells become cancerous (many sugar chains are added to many proteins, and many post-translational modifications of proteins are caused by sugar chain modification reactions. is there). Therefore, it is relatively easy to distinguish cancer cells and normal cells from their morphology, and the area where cancer cells are present or the area where cancer cells and normal cells are mixed is set as the "site for obtaining information on the object" do it. Note that the presence of cancer cells can be automatically detected by preliminarily programming the characteristics of cancer cell morphology as image data.

  It is also possible to automate the determination of the part for acquiring information. For example, an image obtained by observing a specimen with an optical microscope is captured as a digital image, and the obtained image is processed according to a preset standard to automatically select a site for information acquisition.

The position of the information acquisition site determined in the above operation in the object is further converted into data, and the digestion enzyme application position is controlled based on this data to increase the accuracy of the digestion enzyme application position. Can do. Examples of the data conversion method include the following methods.
(1) Under observation with an optical microscope, an observer manually sets a measurement site in an object, and records this as a point (xy address) on an xy coordinate set in an observation field image.
(2) The part is automatically set using the contrast difference in the digital image obtained by the optical microscope observation, and this is recorded as a point (xy address) on the xy coordinate set in the observation image.

  The determination of the site for obtaining information and the data conversion thereof are common to the process diagrams of FIGS. 1 and 2 as the process of “determination of analysis target cell and xy addressing”.

  As the object in the lesion tissue section, a specific cell corresponding to the purpose of information acquisition is selected. And it is preferable that the site | part for information acquisition recorded as a point on xy coordinate mentioned above is the center point of this specific cell.

  On the other hand, it is preferable that a specimen to which a droplet containing a digestive enzyme is applied using an ink jet method is placed in a region where the temperature and humidity can be controlled by the temperature and humidity control means. Furthermore, it is preferable that the amount of droplets is in the range of 10al to 1pl. The humidity is preferably at least 80%.

  When applying the digestive enzyme to the specimen, it is preferable to control the position of applying the digestive enzyme by the ink jet method using the data related to the information acquisition site recorded as the points on the xy coordinates described above.

In the information acquisition method of the present invention, any of the following steps can be preferably used for the step of measuring the mass of the composition obtained by decomposing the target with digestive enzymes using a mass spectrometer. .
(1) The process which extracts the structure decomposed | disassembled by the digestive enzyme using a solvent, and measures the mass of the structure in this extract using various mass spectrometers.
(2) A step of directly measuring a component decomposed by a digestive enzyme on a digestion substrate as a two-dimensional distribution image for each mass / charge ratio by time-of-flight secondary ion mass spectrometry.
(3) The composition decomposed by the digestive enzyme on the substrate for digestion treatment is brought into contact with the substrate surface, and at least a part of the composition is moved to the substrate side, and then the substrate surface The process of measuring the structure of (2) using various mass spectrometers (including a secondary ion mass spectrometer).

  Above (1) and (2) follow the process diagram of FIG. 1, and (3) follows the process diagram of FIG.

  The information acquisition method of the present invention can be more suitably applied when a protein is an object. In the case where the protein is an object, the components obtained by digestive enzymatic degradation are mainly peptides. The mass number of this peptide is preferably in the range of about 500 to 20000. When secondary ion mass spectrometry is used for the above-described mass spectrometry, the mass number of the peptide is 500 to 5000, and may be about 500 to 2000. The upper limit of the mass number of the peptide to be measured may be based on a peptide group (a peptide having the largest mass number) that can identify the protein before decomposition.

  Process diagrams in the case of using secondary ion mass spectrometry for the above mass spectrometry are shown in FIGS. The difference between the steps of FIG. 3 and FIG. 4 is that a component decomposed by a digestive enzyme, specifically a protein degradation product, is directly analyzed or brought into contact with another substrate surface to Whether at least a part is moved to the substrate side and this transcript is to be analyzed. In addition, after decomposition | disassembly of the target protein by a digestive enzyme, you may provide an ionization promoting substance (it also expresses a sensitizer) using the inkjet method as needed.

  Further, as shown in FIGS. 3 and 4, in the time-of-flight secondary ion mass spectrometry, the measurement part and the measurement result are collated by using the point (xy address) in the xy coordinate of the information acquisition part. It is possible to carry out surely and easily.

  By using the information acquisition method of the present invention, the presence or absence of a specific target related to a disease in a sample can be detected. Specific examples include determining the presence or absence of a protein involved in metastasis or recurrence from the presence or absence of a specific peptide contained in a specific lesion cell such as a cancer cell.

An apparatus for performing pretreatment up to preparation of a sample for mass spectrometry in the information acquisition method of the present invention is configured to include at least the following elements.
(1) A stage for fixing a substrate on which a specimen is placed.
(2) Stage position control means for controlling the position of the stage.
(3) Observation means for optically observing a biological tissue section.
(4) Means for applying a droplet containing a digestive enzyme to a site for obtaining information on an object determined in the optically observed biological tissue slice image using an inkjet method.
(5) Temperature / humidity control means for controlling the temperature and humidity of the region where the stage is disposed.

  FIG. 5 shows a schematic diagram of a pretreatment apparatus according to the first aspect that can be applied when a biological tissue piece is used as a specimen. In FIG. 5, 1 is a container of the pretreatment apparatus, 2 is a temperature control mechanism, 3 is a humidity control mechanism, 4 is a sample, and 5 is a substrate for holding the sample. Reference numeral 6 denotes a stage for fixing the substrate 5 (including a movable and position-controllable mechanism, which may further include a cooling mechanism if necessary), 7 an optical observation means, and 8 a mechanism for moving the stage 6. It is. 9 is a mechanism for controlling the position of the stage 6, 10 is a mechanism for recording an optically observed image, 11 is an image processing of the optically observed image, and a droplet containing a digestive enzyme (and an ionization promoting substance) is applied. It is a mechanism (automatic or manual) that determines where to perform. 12 is an interface, 13 is an inkjet head, 14 is a mechanism for moving the inkjet head 13, and 15 is a control mechanism for ejecting droplets from the inkjet head 13 (in conjunction with the stage position control mechanism 9). is there. The determination of the location to which the droplet containing the digestive enzyme is automatically applied by the mechanism 10 can be performed in accordance with a preset program, and data relating to the determined location is input to the control mechanism 15 to obtain a predetermined value from the inkjet head. Applying droplets containing digestive enzymes to the location.

Furthermore, at least the following steps can be automatically performed according to a program incorporated in advance in the computer.
(1) A step of optically observing the specimen.
(2) A step of determining a site for obtaining information on the object based on the optically observed image.
(3) The process of providing the said site | part with the droplet containing a digestive enzyme using the inkjet method.
(4) The information acquisition apparatus according to claim 21, further comprising a control system for automatically performing the step of analyzing the component decomposed by the digestive enzyme by time-of-flight secondary ion mass spectrometry.

  Further, the temperature and / or humidity can be adjusted in the container 1 by a temperature control mechanism 2 and a humidity control mechanism 3 that constitute the temperature and humidity control means. Further, the mechanism 8 has a function as a position control unit for the stage 6, can control the position of the stage with respect to the optical observation unit 7, and further moves the stage 6 to the installation portion of the inkjet head 13. Further, the mechanism 8 may be provided with a function of adjusting the position of the stage 6 in the xy coordinate direction. In addition, the optical observation means 7 and the inkjet 13 can also be provided so that position control is possible in the xy coordinate direction and / or the z coordinate direction (vertical direction). By using both the position control on the stage side and the position control on the optical observation means and / or the ink jet head side, the position adjustment of the stage in each process can be performed accurately and efficiently.

  The pretreatment device shown in FIG. 5 is a device that measures the mass of a component that constitutes an object in a living tissue and performs preprocessing for acquiring information on the object from the measured mass.

  Moreover, the schematic diagram of the pre-processing apparatus concerning the 2nd aspect of this invention is shown in FIG. In FIG. 6, reference numerals 1 to 15 are basically the same as those in FIG. In FIG. 6, 17 is a transfer substrate, 16 is a mechanism for fixing and moving the substrate 17, 18 is a control mechanism for the substrate 17 (in conjunction with the stage position control mechanism 9 and the droplet discharge control mechanism 15). Is). The pretreatment device shown in FIG. 6 is another type of device that measures the mass of a component that constitutes an object in living tissue and performs preprocessing for acquiring information on the object from the mass. .

  In the pretreatment apparatus shown in FIGS. 5 and 6, the amount of the droplet containing the digestive enzyme is preferably in the range of 10al to 1pl, and the humidity is preferably maintained at 80% or more.

  The information processing apparatus of the present invention can be obtained by combining the preprocessing apparatus having the above configuration and the mass spectrometer. FIG. 5 shows a first example of the information acquisition apparatus of the present invention. This apparatus is characterized in that a mechanism capable of time-of-flight secondary ion mass spectrometry is connected to the pretreatment apparatus. The process of processing and analyzing the specimen using this information acquisition apparatus corresponds to the process shown in FIG.

  Moreover, the 2nd example of an information acquisition apparatus of this invention is shown in FIG. This apparatus is characterized in that a mechanism capable of time-of-flight secondary ion mass spectrometry is connected to the pretreatment apparatus. The process of processing and analyzing the specimen using the information acquisition apparatus corresponds to the process shown in FIG.

  Hereinafter, a specific procedure for processing and analyzing the specimen according to the steps shown in FIG. 3 using the information acquisition apparatus according to the first aspect described above will be described with reference to FIGS.

  FIG. 7A is a plan view and FIG. 7B is a perspective view schematically showing a specimen when a lesion tissue is used as the specimen. In FIG. 7, 31 indicates a lesion cell, and 32 indicates a normal cell. The size (average diameter) of these cells is about 10 to 20 μm. When a lesion cell is a cancer cell, the shape is different from that of a normal cell, and therefore, both can generally be easily distinguished by an optical microscope. It is also possible to automatically identify both by using an image processing technique.

  First, such a specimen (section or equivalent) is placed on a substrate. At that time, the substrate can be cooled as required. In the case where a specimen obtained by slicing a frozen lesion tissue with a microtome or the like is used as a specimen, it is generally preferable to handle the specimen under cooling in order to prevent the specimen from being altered before digestion with a digestive enzyme.

  Next, observation using an optical microscope is performed to obtain an image as shown in FIG. Subsequently, central coordinates for applying a droplet containing a digestive enzyme to a specific cell or all cells are determined by an inkjet method. FIG. 8 is a schematic diagram of the central coordinates when a droplet containing a digestive enzyme is applied to only the lesion cell 31. FIG. 8 shows the central coordinates when a droplet containing the digestive enzyme is applied to all cells. A schematic diagram is shown in FIG. 8 and 9, 33 indicates the center point of the lesion cell 31, and 34 indicates the center point of the normal cell 32. Here, the center point means a point substantially at the center of the cell. This center point can be set manually under the observation of an optical microscope, or can be set automatically by the above-described image processing. In an image observed with an optical microscope, lesion cells such as cancer cells and normal cells are observed as a two-dimensional image. Based on the information on the outline of the obtained cell image, the center of gravity is generally set as the “cell center point”.

  Further, as shown in FIG. 8, when a droplet containing a digestive enzyme is applied only to a lesion cell, only the protein in the lesion cell is limitedly decomposed. Therefore, methods other than time-of-flight secondary ion mass spectrometry can be used for mass analysis of degraded peptides. Specifically, as described above, extraction is performed using a solvent, and the mass of a constituent in the extract is measured using various mass spectrometers.

  Next, a droplet containing a digestive enzyme is applied to the center point determined above by an inkjet method. As the ink jet method, a bubble jet method, a piezo method, or the like can be used. In addition, a surfactant or the like can be used as necessary to stabilize the discharge of the droplets. When applying droplets, generally the movement of the sample stage or the movement of the optical microscope unit and the ink jet head is required (FIG. 5 shows a pretreatment apparatus when the stage is moved). Therefore, high positional accuracy is required for the moving mechanism. Specifically, the movement accuracy (reproducibility) of the stage is required to be several μm or less, preferably 1 μm.

  After applying the droplet containing the digestive enzyme, the specimen is preferably placed under high humidity for the purpose of advancing the digestion reaction and preventing drying. Therefore, it is preferable to keep the inside of the container shown in FIG. 5 at a high humidity, or to quickly move to a high humidity environment after applying droplets (FIG. 5 corresponds to the former). The latter has the disadvantage that the device becomes complicated, but has the advantage of reducing damage to the entire device (damage caused by high humidity). Here, if a preferable example of humidity is given, it will be about 95% when temperature fluctuation (especially when it falls) is considered. Condensation on the surface of the device (generation of water droplets) is not preferable.

  In addition, in order to obtain highly reproducible results by mass spectrometry to be described later, it is necessary to allow the digestion reaction to proceed quantitatively. For that purpose, it is necessary to apply droplets containing digestive enzymes and then place them at a constant temperature. . Therefore, it is preferable to provide a mechanism for controlling the temperature of the specimen.

  Subsequently, the ionization promoting substance (sensitizing substance) disclosed by the present inventors in the pamphlet of International Publication No. 05/003715 may be applied to the above cell center point as necessary. This is to improve the sensitivity of secondary ions of the degraded peptide by secondary ion mass spectrometry, which will be described later.

Next, the surface of the specimen is analyzed by time-of-flight secondary ion mass spectrometry, and a two-dimensional distribution image of the degraded peptide resulting from the enzymatically degraded protein is obtained. A general time-of-flight secondary ion mass spectrometer can be used for the time-of-flight secondary ion mass spectrometry. At this time, as a primary ion, in addition to general liquid metal ions such as Ga ⁺ , cluster ions such as Au ₃ ⁺ and Bi ₃ ⁺ can be used.

  The results when digestive enzymes are applied only to the lesion cells shown in FIG. 8 are shown in FIGS. Analysis FIG. 10 is a conceptual diagram of a two-dimensional distribution image of a degrading peptide and other secondary ions, and FIG. 11 is an overlay of the optical microscope image of the specimen and the two-dimensional distribution of secondary ions of FIG. Show. In FIGS. 10 and 11, (a) and (b) are peptides (secondary ions) in which a specific protein (for example, a malignant protein showing metastasis or recurrence in cancer cells) is limitedly digested by digestive enzymes. The two-dimensional distribution of is shown. (C) shows the two-dimensional distribution of secondary ions unrelated to the protein. From these results, it can be determined whether or not the specific protein is present in the lesion cell. In addition, various databases can be used for the peptide species generated upon limited decomposition with a specific protein.

  The results when digestive enzymes are added to all the cells shown in FIG. 9 are shown in FIGS. Analysis FIG. 12 is a conceptual diagram of a two-dimensional distribution image of a degrading peptide and other secondary ions, and FIG. 13 is an overlay of the optical microscope image of the specimen and the two-dimensional distribution of secondary ions of FIG. Show. 12 and 13, (a) shows a two-dimensional distribution of a peptide (secondary ion) in which a specific protein (for example, a malignant protein showing metastasis or recurrence in cancer cells) is limitedly digested by digestive enzymes. Show. (B) shows a two-dimensional distribution of peptides (secondary ions) that are not present in the diseased cells, and (c) shows a two-dimensional distribution of irrelevant secondary ions. From these results, it can be determined whether or not the specific protein is present in the lesion cell. In addition, about the peptide seed | species produced | generated when carrying out limited decomposition | disassembly with a specific protein, various databases can be utilized like the above.

  Next, a specific procedure for processing and analyzing a sample according to the steps shown in FIG. 4 using the information acquisition apparatus of the second aspect will be described with reference to FIGS.

  The steps from the placement of the specimen on the substrate to the application of the digestive enzyme by the inkjet method, the holding at a constant temperature and constant humidity, and the (application of the ionization promoting substance by the inkjet method) use the first information acquisition device described above. It is the same as the process to do.

  Thereafter, using the transcription mechanism shown in FIG. 6, the peptide decomposed by the digestive enzyme is transferred to the substrate for measurement. As a base material for measurement, a metal substrate such as gold or silver, a metal or glass substrate subjected to chemical treatment for improving the transfer efficiency of the peptide, or the like can be used.

  Subsequently, the peptide transferred onto the measurement substrate is analyzed by time-of-flight secondary ion mass spectrometry. The conceptual diagram of the obtained result is shown in FIGS. As described above, since the transfer to the substrate for measurement is performed, the images in FIGS. 14 to 17 are axisymmetric with respect to the original image shown in FIG. However, FIGS. 14 and 15 show images corresponding to FIGS. 10 and 11 obtained without transfer, and FIGS. 16 and 17 show images corresponding to FIGS. 12 and 13 obtained without transfer, respectively. It is done. From these results, it is possible to determine whether or not a specific protein is present in a diseased cell, as in the case of using the information acquisition device of the first aspect. When the peptide has a partial structure that is more easily transferred to a substrate than a protein or the like (for example, having many SH groups in a protein molecule), the transfer is more sensitive because the background is lowered. The analysis result can be expected. In addition, in time-of-flight secondary ion mass spectrometry, the substrate itself such as gold has an effect of increasing the secondary ionization efficiency, so when the transfer efficiency of a certain level or more can be expected, the information acquisition device of the second aspect is used. It is preferable to perform time-of-flight secondary ion mass spectrometry.

  The present invention has been described in two forms. The contents described in these forms are in a common sense range, and can be appropriately adopted in other forms, and are not limited to the respective forms described.

It is process drawing which shows an example of the pre-process of the sample for the information acquisition of the protein which is the target object in a lesion tissue piece. It is process drawing which shows an example of the pre-process of the sample for the information acquisition of the protein which is the target object in a lesion tissue piece. FIG. 6 is a process diagram showing an example of a procedure for pre-processing a specimen in order to detect a protein contained in a specific cell in a lesion tissue piece while retaining position information. FIG. 6 is a process diagram showing an example of a procedure for pre-processing a specimen in order to detect a protein contained in a specific cell in a lesion tissue piece while retaining position information. It is a mimetic diagram of the pretreatment device concerning the 1st mode which pretreats a sample in order to detect a subject in a sample. It is a schematic diagram of the pre-processing apparatus concerning the 2nd aspect which pre-processes a sample in order to detect the target object in a sample. It is a schematic diagram at the time of using a diseased tissue as a sample, (a) is a top view, (b) is a perspective view. It is a schematic diagram of central coordinates when a droplet containing a digestive enzyme is applied only to a lesion cell. It is a schematic diagram of the center coordinates when a droplet containing a digestive enzyme is applied to all cells. Shows two-dimensional distribution images of degraded peptides and other secondary ions when droplets containing digestive enzymes are applied only to lesion cells and then the surface is analyzed by time-of-flight secondary ion mass spectrometry It is a conceptual diagram. The optical microscope image of the specimen is superimposed on the two-dimensional distribution image of the secondary ions in FIG. Shows two-dimensional distribution images of degraded peptides and other secondary ions when all cells are subjected to droplets containing digestive enzymes and then the surface is analyzed by time-of-flight secondary ion mass spectrometry It is a conceptual diagram. FIG. 13 is an image obtained by superimposing an optical microscope image of a specimen and a two-dimensional distribution image of secondary ions in FIG. 12. A droplet containing a digestive enzyme was applied to only a lesion cell, and then the degradation peptide on the surface was transferred to another substrate surface, and then the substrate surface was analyzed by time-of-flight secondary ion mass spectrometry It is a conceptual diagram which shows the two-dimensional distribution image of the decomposition | disassembly peptide and other secondary ion at the time. FIG. 15 is an image obtained by superimposing an optical microscope image of a specimen and a two-dimensional distribution image of secondary ions in FIG. 14. All cells were subjected to droplets containing digestive enzymes, and then the surface degradation peptide was transferred to another substrate surface, which was then analyzed by time-of-flight secondary ion mass spectrometry. It is a conceptual diagram which shows the two-dimensional distribution image of the decomposition | disassembly peptide and other secondary ion at the time. FIG. 17 is an image obtained by superimposing an optical microscope image of a specimen and a two-dimensional distribution image of secondary ions in FIG. 16.

Explanation of symbols

DESCRIPTION OF SYMBOLS 1 Container of pre-processing apparatus 2 Temperature control mechanism 3 Humidity control mechanism 4 Specimen 5 Substrate holding the specimen 6 Fixed stage 7 Optical observation means 8 Mechanism for moving the stage 9 Mechanism 10 for controlling the position of the stage Optical The mechanism 11 for recording the observed image 11 The mechanism 12 for determining the location to which the droplet containing the digestive enzyme (and ionization promoting substance) is applied 12 The interface 13 The inkjet head 14 The mechanism 15 for moving the inkjet head The droplet from the inkjet head Control mechanism 16 for discharging mechanism 17 mechanism for fixing and moving base material 18 base material 18 base material control mechanism 31 lesion cell 32 normal cell 33 center point 34 of lesion cell normal cell 32

Claims (21)

An information acquisition method for measuring a mass of a constituent constituting an object in a specimen and acquiring information on the object from the mass,
Preparing a biological tissue section as a specimen,
Optically observing the specimen and determining a site for obtaining information about the object in the specimen;
A step of applying a droplet containing a digestive enzyme to the site using an inkjet method, and digesting the site with a digestive enzyme;
A step of measuring the mass of the constituent of the site obtained by digestive enzymatic degradation using a mass spectrometer, and a step of obtaining information on the object from the mass of the constituent;
An information acquisition method characterized by comprising:   2. The information acquisition method according to claim 1, wherein the specimen is a section of a diseased tissue.   2. The information acquisition method according to claim 1, wherein the site for obtaining information on the object is a specific cell in a diseased tissue section.   The information acquisition according to any one of claims 1 to 3, wherein the application of the digestive enzyme by the inkjet method is a step of applying a droplet containing the digestive enzyme to a specific cell in a lesion tissue section using the inkjet method. Method.   The information according to any one of claims 1 to 3, wherein the application of the digestive enzyme by the inkjet method is performed in a space in which temperature and humidity are controlled, and the amount of droplets is in the range of 10al to 1pl. Acquisition method.   The information acquisition method according to claim 7, wherein the humidity is at least 80%.   Determination and position specification of a part for obtaining information on the object is performed by determining the part in an observation field image obtained by optical microscope observation, and the observation field image is placed on xy coordinates, The information acquisition method according to claim 1, wherein the position is specified and recorded as a point on the xy coordinates.   The part for obtaining information on the object is determined by automatically determining the part based on a digital image obtained by optical microscope observation, and further, the part is positioned as a point on the xy coordinates. The information acquisition method according to claim 1, wherein the information acquisition method is specified and recorded.   The information acquisition method according to claim 7 or 8, wherein a part for obtaining information on the object is a specific cell in a lesion tissue section, and the point on the xy coordinates is a center point of the cell.   The information acquisition method according to any one of claims 7 to 9, wherein the application of the digestive enzyme by the inkjet method is performed based on the recorded points on the xy coordinates.   The measurement of the mass of the constituent by a mass spectrometer is performed by extracting the constituent obtained by the digestive enzyme decomposition treatment using a solvent, and the mass of the constituent in the extract using a mass spectrometer. The information acquisition method according to claim 1, wherein the information acquisition method is performed by measurement.   The step of measuring the mass of the composition using a mass spectrometer is the step of measuring the mass of the composition obtained by the digestive enzyme decomposition treatment using a mass spectrometer. The information acquisition method according to claim 1, wherein the information acquisition method is performed by measuring as a two-dimensional distribution image for each mass / charge ratio by time-of-flight secondary ion mass spectrometry.   Measurement of the mass of the constituent by a mass spectrometer moves the constituent obtained by the digestive enzyme decomposition treatment to the surface of the measuring substrate, and moves the constituent moved on the surface of the measuring base to the mass. The information acquisition method according to claim 1, wherein the information acquisition method is performed by measuring using an analyzer.   The information acquisition method according to claim 1, wherein the object is a protein.   The information acquisition method according to claim 14, wherein a mass number of a constituent obtained by digestive enzymatic degradation of the protein is in the range of 500 to 20,000.   A method for detecting the presence or absence of an object in a sample, wherein the sample is obtained by acquiring information on the presence or absence of the object by the information acquisition method according to any one of claims 1 to 3 and 11 to 13. A detection method characterized by detecting the presence or absence of the object inside. A pretreatment device for measuring a mass of a constituent constituting an object in a specimen and acquiring information on the object from the mass,
A stage for fixing the substrate on which the specimen is placed,
Stage position control means for controlling the position of the stage;
Observation means for optically observing the specimen,
Means for applying a droplet containing a digestive enzyme to the site for obtaining information about the object determined in the optically observed specimen image, and the temperature and humidity of the region where the stage is arranged A pretreatment apparatus having temperature and humidity control means for controlling.   18. The moving device according to claim 17, further comprising a moving unit configured to move a component obtained by decomposing a part of the target object on the substrate fixed to the stage with the digestive enzyme to a measurement substrate. Pre-processing device. An information acquisition device for measuring a mass of a constituent constituting an object of a specimen and acquiring information on the object from the mass,
The pretreatment device according to claim 17 or 18 for obtaining a constituent constituting an object in a specimen,
A mass spectrometer for analyzing the mass of the component;
An information acquisition apparatus comprising:   The information acquisition apparatus according to claim 19, wherein the mass spectrometer is a time-of-flight secondary ion mass spectrometer. Optically observing the specimen,
Determining a site for obtaining information on the object based on the optically observed image;
A step of applying a droplet containing a digestive enzyme to the site using an inkjet method;
21. The information acquisition apparatus according to claim 20, further comprising a control system for automatically performing the step of analyzing the constituent decomposed by the digestive enzyme by time-of-flight secondary ion mass spectrometry.

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