JP2006201149A - Composition information acquisition method, composition information acquisition device, and sample stage for time-of-flight secondary ion mass spectrometry - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a protein detection method using a TOF-SIMS method in which non-specific adsorption inherent to a biological sample and protein denaturation are reduced.
In a method for analyzing an object using a TOF-SIMS method, an ionization promoting substance (metal such as silver or gold) is added to the object, and it corresponds to a parent molecule that can discriminate the type of the object. Next ions are generated. By using this to obtain a two-dimensional image image with high spatial resolution (˜1 μm), separation and purification techniques such as electrophoresis and thin layer chromatography can be used for mixed protein samples.
[Selection] Figure 1

Description

  The present invention relates to a method for acquiring information about a composition of a mixed organic substance, particularly a biological substance, using time-of-flight secondary ion mass spectrometry, a composition information acquisition device and a time-of-flight secondary used for the purpose. The present invention relates to a sample stage for ion mass spectrometry.

  With the recent progress of genome analysis, the importance of protein analysis, which is a gene product existing in the 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 basically (1) separation and purification by two-dimensional electrophoresis or high performance liquid chromatograph (HPLC),
(2) It has been carried out in combination with detection systems such as radiation analysis, optical analysis, and mass spectrometry.

  The development of protein analysis technology is broadly divided into database construction by proteome analysis (comprehensive analysis of intracellular proteins), which can be said to be the foundation, and diagnostic devices and drug discovery (drug candidate screening) devices based on the obtained database. The A device suitable for miniaturization, high speed, and automation different from the conventional methods having problems in analysis time, throughput, sensitivity, resolution and flexibility as described above has been demanded for any application form. . As a method for satisfying these requirements, development of a so-called protein chip in which proteins are densely integrated has attracted attention.

  A protein chip captures a specific protein expressed by utilizing an antigen-antibody reaction, etc., and uses it to detect fluorescence, surface plasmon resonance, radioisotope labeling, matrix-assisted laser desorption / ionization (MALDI) mass Detect by analysis method. In addition, there is a method using field emission as another mass spectrometry method for proteins (see Patent Document 1). In this method, the protein is covalently bonded or coordinated through an open group that can be split according to applied energy on a metal electrode, and a strong electric field is applied to guide the protein to a mass spectrometer. Is.

  One of the reasons why protein chips are used as a separation and purification technique for such detection methods is that the protein chips have just the right extent for the spatial resolution of these detection methods.

  For example, MALDI mass spectrometry and its improved SELDI mass spectrometry are the softest ionization methods currently known, and ionize proteins (also called proteins) that have a large molecular weight and are easily broken. It has an excellent feature that a parent ion or an ion equivalent thereto can be detected. It is now one of the standard ionization methods for analyzing protein mass. On the other hand, when these methods are applied to protein chip mass spectrometry, the spatial resolution of detection of two-dimensional protein distribution images (imaging using mass information) is limited due to the limitations of the presence of matrix substances. . That is, the laser beam itself as the excitation source can be easily condensed to a diameter of about 1 to 2 μm, but the matrix substance existing around the protein to be analyzed evaporates and ionizes, so that the two-dimensional distribution of the protein by the above method. The spatial resolution when measuring an image is generally about 100 μm. Further, in order to scan the condensed laser, it is necessary to operate the lens and the mirror in a complicated manner. That is, when a two-dimensional protein distribution image is measured by the above method, it is difficult to scan with a laser beam, and the method is limited to a method of moving a sample stage on which a sample to be analyzed is placed. When trying to obtain a two-dimensional distribution image of a protein with high spatial resolution, the method of moving the sample stage may generally not be preferable (the presence of a mechanical moving part is disadvantageous in terms of reliability. May be).

  In other conventional detection methods, it is difficult to obtain a two-dimensional distribution image of an object, and there is a limitation on the form of the object sample, such as the need to fix the object on a metal electrode.

  Thus, from the aspect of the spatial resolution of the detection method, a protein chip that can be separated and purified with sufficient spatial resolution has attracted attention.

On the other hand, in recent years, in protein mass spectrometry, time-of-flight secondary ion mass spectrometry (hereinafter, TOF-SIMS) is a highly sensitive mass analysis means or surface analysis means. ) Has come to be used. TOF-SIMS is an analysis method for examining what kind of atoms or molecules exist on the outermost surface of a solid sample, and has the following characteristics. In other words, it has the ability to detect trace components of 10 ⁹ atoms / cm ² (amount equivalent to 1/10 ^{5 of} one atomic layer on the outermost surface), can be applied to both organic and inorganic substances, and all that exists on the surface The ability to measure these elements and compounds, and the ability to image secondary ions from substances present on the sample surface.

  The principle of this method will be briefly described below.

  When a high-speed pulsed ion beam (primary ions) is irradiated on the surface of a solid sample in a high vacuum, surface components are released into the vacuum by a sputtering phenomenon. The positively or negatively charged ions (secondary ions) generated at this time are converged in one direction by an electric field and detected at a position separated by a certain distance. When the solid surface is irradiated with primary ions in a pulsed manner, secondary ions with various masses are generated depending on the composition of the sample surface, but lighter ions fly faster, and heavier ions fly faster. By measuring the time (time of flight) from when secondary ions are generated until they are detected, the mass of the generated secondary ions can be analyzed. When the primary ions are irradiated, only the secondary ions generated on the outermost surface of the solid sample surface are released into the vacuum, so that information on the outermost surface of the sample (depth of about several angstroms) can be obtained. In TOF-SIMS, since the amount of primary ion irradiation is extremely small, the organic compound is ionized while maintaining its chemical structure, and the structure of the organic compound can be known from the mass spectrum. However, when TOF-SIMS analysis of artificial polymers such as polyethylene and polyester, biopolymers such as protein under normal conditions, it becomes a small fragmented fragment ion, and it is generally known to know the original structure Is difficult. When the solid sample is an insulator, the insulator is analyzed because the positive charge accumulated on the solid surface can be neutralized by irradiating the gap between primary ions irradiated with the pulse with an electron beam. It is also possible. In addition, in TOF-SIMS, an ion image (mapping) on the sample surface can be measured by scanning a primary ion beam.

As an example of analyzing a protein by TOF-SIMS, a part of a specific protein is isotope-labeled with ¹⁵ N or the like, and the protein is imaged and detected using a secondary ion such as C ¹⁵ N- (non-patent document) 1), the type of fragment ion (secondary ion) corresponding to an amino acid residue, the type of protein estimated from its relative intensity (see Non-Patent Document 2), and the protein adsorbed on various substrates The thing which investigated the TOF-SIMS detection limit of (refer nonpatent literature 3) etc. is known.

  Since this TOF-SIMS method uses primary ions and can be easily focused and scanned, a secondary ion image (two-dimensional distribution image) with high spatial resolution can be obtained, and a spatial resolution of about 1 μm can be obtained. It is also possible to obtain. However, when TOF-SIMS measurement is performed on an object on a substrate under normal conditions, as described above, the secondary ions to be generated are mostly small fragmented fragment ions, and it is not possible to know the original structure. Generally difficult. Therefore, some ingenuity is required to obtain a high-resolution secondary ion image (two-dimensional distribution image) that can distinguish the type of protein from a sample such as a protein chip on which a plurality of proteins are arranged on a substrate. It becomes. The method of A. M. Belu et al. Is a method of isotopically labeling a part of a specific protein, and is a method that can fully utilize the high spatial resolution of TOF-SIMS. However, it is not common to label a specific protein every time. In addition, the method of estimating the type of protein from the fragment ion (secondary ion) type and the relative intensity corresponding to the amino acid residue shown by Mansat et al. (DS Mantus) contains proteins with similar amino acid composition. In this case, it becomes difficult to distinguish.

  Examples other than the above relating to protein separation and mass spectrometry include the following. In the separation of proteins using high performance liquid chromatography, the resolution is improved (see Patent Document 2). However, in this document, identification of separated proteins by mass spectrometry has not been achieved. Patent Document 3 discloses a specific protein detection method and detection by mass spectrometry as one of the detection methods, but is not intended for general proteins or mixtures thereof.

  In addition, as a method for diagnosing a health condition from analysis of a sample collected from a living body, for example, there is a method that discloses blood filtration, separation, and analysis (see Patent Document 4), but two-dimensionally developed protein There are few known methods for diagnosing health from mass information (two-dimensional distribution by mass).

  As described above, in the detection of proteins, protein chips are attracting attention for use as diagnostic devices or drug discovery (drug candidate screening) devices by separating and purifying proteins in a spatial region that can be identified by the detector.

However, the production cost of protein chips is high, and it is difficult to comprehensively investigate the diagnosis of unknown symptoms due to restrictions on the types of proteins placed on the chips. There may be a problem of denaturation.
JP-T-2001-521275 Japanese Patent No. 3376955 Japanese Patent No. 3035357 JP 2001-258868 A A. M.M. Belu et al. , Anal. Chem. 73, 143 (2001) D. S. Mantus et al. , Anal. Chem. , 65, 1431 (1993) M.M. S. Wagner el. Al. , J. et al. Biometer. Sci. Polymer Edn. , 13, 407 (2002)

  Therefore, an object of the present invention is to provide a protein detection method using the TOF-SIMS method in which nonspecific adsorption inherent to a biological sample and protein denaturation are reduced.

The composition information acquisition method of the present invention includes:
A method of acquiring a secondary ion mass spectrum of an object using time-of-flight secondary ion mass spectrometry,
A first step of separating the object;
A second step of applying an ionization promoting substance to the object;
A third step of obtaining a secondary ion mass spectrum of the object by the time-of-flight secondary ion mass spectrometry;
It is characterized by having.

  In the composition information acquisition method of the present invention, the method further comprises a step of decomposing the separated object by a chemical technique after the first step. In the present application, “decompose a separated object by a chemical technique” means DNA degradation by a restriction enzyme, protein degradation by a protease, or the like.

  Further, in the composition information acquisition method of the present invention, the first step of separating the target object is an electrophoresis method or thin layer chromatography using a separator that includes an ionization promoting substance and has a mechanism capable of separating the target object. It is the process of isolate | separating the said target object by.

  In the composition information acquisition method of the present invention, the method further comprises a step of decomposing the separated object by a chemical technique after the first step.

  The object is preferably a biological substance. Bio-related substances include nucleic acids and proteins such as DNA and RNA, but there are no restrictions on the molecular weight. In addition, the biological substance is preferably a substance selected from the group consisting of nucleic acids and proteins, and degradation products obtained by decomposing them by chemical techniques. Furthermore, the ionization promoting substance in the present invention is preferably a substance containing metal (may be a mixture), and in particular, the ionization promoting substance is preferably silver or gold or a mixture thereof.

  Furthermore, in the composition information acquisition method of the present invention, information on the two-dimensional distribution state of the object is acquired by scanning with a primary ion beam.

  On the other hand, the composition information acquisition apparatus of the present invention is a composition information acquisition apparatus including a time-of-flight secondary ion mass spectrometer, and the sample stage in the analyzer includes an ionization promoting substance and separates an object. It has the mechanism to do.

  The composition information acquisition apparatus of the present invention is a composition information acquisition apparatus including a time-of-flight secondary ion mass spectrometer, wherein the sample stage in the analyzer includes: an ionization promoting substance; and an object A mechanism for separating and a mechanism for decomposing the separated object by a chemical method.

  In addition, the sample stage for performing time-of-flight secondary ion mass spectrometry includes a mechanism that includes an ionization promoting substance and separates an object.

  Further, the sample stage is a thin layer chromatography plate containing an ionization promoting substance.

  Further, the sample stage includes an ionization promoting substance, and has a function of separating an object and a function of decomposing the separated object by a chemical method.

  Furthermore, the method for obtaining information on the health condition according to the present invention is the method for obtaining information on the health condition by the composition information acquisition method according to any one of claims 1 to 8, wherein the object is collected from a living body. It is a sample.

  In the method for obtaining information on the health condition of the present invention, the sample stage for fixing the sample is removable from the composition information acquisition device.

  Furthermore, the method for obtaining information on the health state of the present invention is characterized by comprising a step of analyzing the components of the sample based on the obtained secondary ion information and the separated pattern information. The step of analyzing the component of the sample is a step of comparing secondary ion information corresponding to various health conditions prepared in advance with library data of separated pattern information. .

  Using the method of the present invention, a sample composed of mixed organic substances is separated into positions characteristic of each organic substance, and imaging measurement is performed using the respective “mass information”, whereby the two-dimensional distribution state of the mixed organic substance can be obtained with high spatial resolution. It becomes possible to obtain “positional information” that is a specific clue of an organic substance in addition to “mass information”. From these two pieces of information, for example, for a specific biological sample such as blood, the detected organic matter and the health state are associated with each other to create a database, so that it is possible to obtain information on the health state.

  In addition, since the sample stage for fixing the sample can be detached from the composition information acquisition apparatus, the sample stage can be carried and the sample can be fixed under the subject.

  Hereinafter, the present invention will be described in more detail.

  In the TOF-SIMS method, which has not been a suitable analytical means for protein analysis while having high spatial resolution, a sample that promotes ionization, for example, a metal such as silver or gold, is firstly used in the present invention. Coexisting with the protein can promote the ionization of the protein, thereby making it possible to analyze the protein. Secondly, the spatially high-resolution measurement by TOF-SIMS is possible, so that separation is not possible with conventional protein chips, but is more cost effective and allows comprehensive protein separation. It is a feature of the present invention to use a technique such as electrophoresis or thin layer chromatography. In this method, separation is performed continuously with some protein components mixed in part. Therefore, conventional detection methods (for example, partial extraction, mass spectrometry after concentration, etc.) can sufficiently grasp the composition. There wasn't.

The composition information acquisition method of the present invention includes:
A method of acquiring a secondary ion mass spectrum of an object using time-of-flight secondary ion mass spectrometry,
A first step of separating the object;
A second step of applying an ionization promoting substance to the object;
A third step of obtaining a secondary ion mass spectrum of the object by the time-of-flight secondary ion mass spectrometry;
It is characterized by having.

  Here, the first step may be a step of separating an object by electrophoresis or thin layer chromatography. The second step may be a step of applying an ionization promoting substance by vapor deposition or chemical modification. Information relating to the object is obtained by performing the third step on the composition thus obtained.

  FIG. 2 illustrates the process of the present invention. In FIG. 2, 201 is a step of separating an object, 202 is a step of imparting an ionization accelerator to the object, and 203 is a secondary ion mass spectrum of the object by time-of-flight secondary ion mass spectrometry. The process of obtaining is shown. Reference numeral 204 denotes a process for decomposing an object. This process is performed by decomposing the object into a desired size as necessary before performing time-of-flight secondary ion mass spectrometry. Analysis accuracy can be increased by comprehensively judging the analysis results.

  The above steps will be described in detail below. First, the first step of separating the object will be described.

In order to take advantage of the feature of TOF-SIMS two-dimensional imaging, the method for separating an object in the present invention needs to be a method capable of separating components (spreading components in a two-dimensional direction) with the spread of position. It becomes. For example, examples of such a separation method include electrophoresis and thin layer chromatography. In addition, since it is necessary to put under high vacuum at the time of TOF-SIMS measurement, it is necessary to dry the separated sample and substrate sufficiently. A plurality of separation methods may be combined. For example, by separating a sample with a plate having a different separation mode such as C8 (a plate on which C ₈ H ₁₇ is fixed) and C18 (a plate on which C ₁₈ H ₃₇ is fixed), it becomes a specific clue of organic matter. "Location information" can be obtained more accurately.

Next, the second step of promoting ionization of the object by allowing the ionization promoting substance to coexist will be described.

  In the present invention, a substance that promotes ionization (ionization promoting substance) may be expressed as a sensitizer if necessary.

A method for applying a substance that promotes ionization of an object of the present invention includes:
(1) Apply after placing an object on the substrate,
(2) It gives beforehand with respect to the specific 1 type or multiple types of the target object arrange | positioned on a board | substrate.
(3) Before the object is placed on the substrate, it is applied to the substrate surface in advance.
One of them. Specific examples of the application method include vapor deposition and chemical modification.

  Among these, the method (1) can be applied to the analysis of objects of all forms. That is, this is a highly versatile system. On the other hand, when applying a substance that promotes ionization to an object distributed two-dimensionally on the substrate, care must be taken not to diffuse the object by this treatment. This is because the object of the present invention cannot be achieved if the two-dimensional distribution state of the object is changed by treatment such as chemical modification. Whether or not the two-dimensional distribution state of the object has changed can be determined from, for example, comparison with the result of TOF-SIMS analysis before and after chemical modification.

  Next, the method (2) is a method in which a substance (sensitizer) that promotes ionization of a target object and increases sensitivity in TOF-SIMS analysis is bound in advance to a specific part of the specific target object. However, this method has an advantage that a two-dimensional distribution state of a specific object can be selectively detected with high sensitivity. On the other hand, a chemical modification process or the like must be performed in advance for each object, and thus there is a disadvantage that the operation is somewhat complicated. In addition, as a coupling | bonding method of said sensitizer, a covalent bond, an ionic bond, the coordination bond in the case of using a metal complex as a sensitizer, etc. can be utilized, and there is no restriction | limiting in particular. However, since an object such as a chemically modified protein is placed on the substrate, the bond needs to be stable.

  Furthermore, in the method (3), a substance (sensitizing substance) that promotes ionization of an object and increases sensitivity in TOF-SIMS analysis is formed on the substrate surface in advance. In this method, it is important to thoroughly investigate whether or not a new nonspecific adsorption problem occurs due to the presence of the sensitizer. The sensitizer is not particularly limited as long as the sensitivity can be increased by TOF-SIMS analysis, and may be one that does not directly bind to an object (that is, a secondary ion is generated by TOF-SIMS analysis). In the process, it may be effective to increase the ionization efficiency of the object). Furthermore, it is preferable to form this sensitizer on the outermost surface of the substrate. However, in order to prevent non-specific adsorption, it is possible to dispose another substance such as a monomolecular film on the sensitizer. . For example, a gold vapor deposition film is formed as a sensitizer, and a self-assembled monomolecular film of 11-hydroxy-1-undecanethiol is formed thereon.

  The ionization promoting substance (sensitizing substance) of the present invention will be described.

  According to the knowledge of the present inventors, when the ionization promoting substance of the present invention is allowed to act on an object, the effect of improving secondary ionization production efficiency when the object is irradiated with primary ions is as follows. I think so.

  When the object is a sample collected from a living body, in biological tissue, peptide chains of protein molecules to be measured are entangled with each other, which causes a reduction in the generation efficiency of secondary ion species during measurement by the TOF-SIMS method. It has become. On the other hand, in the present invention, a solution containing an ionization promoting substance is allowed to act on the surface of a sample collected from a living body, and the generation efficiency of secondary ion species derived from protein molecules present on the surface can be improved. This ionization promoting substance is a substance that has a function of promoting / increasing the generation of secondary ion species derived from protein molecules present on the surface when irradiated with primary ions.

  As an example of a specific method of action, a solution containing a sensitizer is applied to the sample surface in order to cause the ionization promoting substance to directly act on protein molecules present on the sample surface collected from the living body, thereby covering the entire surface. Holding in a state to be performed.

  For example, when a dilute aqueous solution of silver nitrate is used as a solution containing an ionization promoting substance, the silver ions dissociated in the aqueous solution act on the peptide chain constituting the protein molecule and bind between the silver ion and the protein molecule. And the generation of secondary ion species is promoted. As described above, in the present invention, the ionization promoting substance itself or a component of the ionization promoting substance acts on the peptide chain constituting the protein molecule to generate a bond with the peptide chain. It is considered that the entanglement between the peptide chains of the molecule is released.

  Further, since silver ions themselves are substances that easily become ions, it is considered that from this point as well, they have an effect of promoting protein ionization by binding to protein molecules.

  Examples of the ionization-promoting substance that can be used in the present invention include the above-mentioned silver nitrate, salts such as sodium carbonate, substances containing metals such as gold and silver (metal complexes, etc.), metal colloids, and the like. The solution containing the ionization promoting substance is preferably in the form of an aqueous solution.

  As described above, the chemical modification of the present invention is a process in which the ionization efficiency of the protein is increased in the process of generating secondary ions by TOF-SIMS analysis, and the two-dimensional distribution state of the protein is not changed. If there is no particular limitation, it is preferable to use a substance containing a metal as a chemical modifier. As the kind of the metal, as long as the present inventors studied, it was particularly preferable to contain silver or gold, or both of them. May be.

  As one method of chemical modification, a method of adding silver or silver ions to a plurality of proteins arranged on a substrate using silver mirror reaction can be mentioned. The silver mirror reaction is a reaction in which a sample is added to an aqueous ammoniacal silver nitrate solution and then silver diamine (I) is reduced to precipitate silver. Since silver has a high affinity for sulfur, this reaction is particularly effective for proteins containing cysteine (Cysine). In addition, when this reaction is used for a protein distributed two-dimensionally on a substrate, care must be taken not to diffuse the protein by the same treatment. As a reagent used for this reaction, a commercially available product (for example, “Silver Staining II Kit Wako” manufactured by Wako Pure Chemical Industries, Ltd.) may be used. Moreover, the effect which accelerates | stimulates ionization of a target object can also be acquired by spraying silver nitrate aqueous solution directly.

  However, the method of chemical modification is not limited to the above, and any method can be used as long as it has an effect of increasing the secondary ionization efficiency of the object in TOF-SIMS analysis and does not change the two-dimensional distribution state of the object. May be.

  Finally, a third step of obtaining information (secondary ion mass spectrum) related to the mass of the object by time-of-flight secondary ion mass spectrometry will be described.

  The detection (imaging) of the two-dimensional distribution state of the object of the present invention is characterized by using a secondary ion that can identify the object, and the secondary ion is an ion having a mass / charge ratio of 500 or more. It is particularly preferable that the mass / charge ratio is 1000 or more. The reason is that the amino acid of a protein typically has a mass number of 100 to 200. Therefore, the ability to detect heavy secondary ions has a higher identification ability, and the mass / charge ratio is 1000 as useful data for protein identification. This is because 5 to 10 amino acid sequences can be known from the above, which is advantageous for identification.

As the primary ion species, gallium ions, cesium ions, and in some cases, gold (Au) ions are preferably used from the viewpoint of ionization efficiency, mass resolution, and the like. In addition, it is preferable to use Au ions in that extremely high sensitivity analysis is possible. At that time, not only Au ions but also Au ₂ ions and Au ₃ ions, which are gold polyatomic ions, can be used. In many cases, the sensitivity is increased in this order. Is a more preferred form.

  Furthermore, the primary ion beam pulse frequency is desirably in the range of 1 kHz to 50 kHz, the primary ion beam energy is in the range of 12 keV to 25 keV, and the primary ion beam pulse width is 0.5 ns. It is desirable to be in the range of 10 ns.

  In addition, since the present invention needs to complete measurement in a relatively short time while maintaining high mass resolution in order to improve quantitative accuracy (one measurement is on the order of several tens of seconds to several tens of minutes), The primary ion beam diameter is preferably measured at some sacrifice. Specifically, it is preferable to set the primary ion beam diameter in the range of 1 μm to 10 μm without narrowing to the submicron order. This is because the measurement can be completed in a short time due to the specifications of the apparatus.

  It is also possible to analyze the components of the sample based on the information regarding the mass number of the secondary ions obtained in this way and the pattern information obtained by imaging the secondary ions (the object is separated).

  That is, the component analysis in the present invention is characterized in that a substance for promoting ionization of an object (ionization promoting substance) is used to obtain information that can more accurately identify the sample by the second step of promoting ionization of the sample. And Furthermore, by using the separation position information peculiar to each separated component in the first step of separating the object as information for further identification, the mixed organic sample is identified with high accuracy. Yes. For example, in the case of thin layer chromatography, each organic sample is separated at a unique position with good reproducibility under the same separation conditions. For example, by performing TOF-SIMS measurement with silver atoms coexisting, secondary ions having a large mass / charge ratio advantageous for sample identification can be obtained. By combining these pieces of information, it is possible to accurately identify the mixed organic sample. In particular, if a database library of the mass number and separation position of these secondary ions is created for the protein composition in a specific part of a living body, compositional changes accompanying changes in health conditions can be quickly identified. Can be used for health condition diagnosis.

  Next, referring to FIG. 3, a sectional view of the sample stage of the present invention is shown. FIG. 3 shows a cross-sectional view of the sample stage of the present invention. In FIG. 3, 303 is a substrate, 302 is a separator, and 301 is an ionization promoting substance.

  The ionization promoting substance 301 may be provided only on the surface of the separator 302, but may be included in the separator. As a method for forming the ionization promoting substance 301 on the surface of the separator 302, it is preferable to use a method such as sputtering, vapor deposition, CVD, or electrodeposition.

When an ionization promoting substance is provided on the separator 302 as a function of separating the object, it is difficult to detect the object if the thickness is too thick. Therefore, the upper limit of the thickness is preferably It is 100 nm or less, more preferably 10 nm or less, and optimally 5 nm or less. On the other hand, the lower limit of the thickness of the ionization promoting substance is not particularly limited as long as the ionization promoting function is expressed and the target object can be analyzed. According to the knowledge of the present inventors, if the density of the ionization promoting substance on the separator is 10 ¹⁰ atoms / cm ² or more, the analysis of the present invention can be performed.

  In the case of performing separation by thin layer chromatography, as a separating body 302 as a function of separating an object, a glass sintered body having a desired void is fixed to a substrate 303. Similarly, cellulose is applied to a substrate 303. It is preferable to employ a fixed one.

  When separation is performed by electrophoresis, it is preferable to employ a gel-like substance having a desired void, such as a polyacrylamide gel or an agarose gel, as the separator 302 as a function of separating an object. When separating an object from a sample by electrophoresis, the object is applied by applying a predetermined electric field (for example, 100 V) after dropping the sample on the gel and maintaining the electric field applied for a predetermined time. Can be separated.

  Next, a method for separating an object using the sample stage of the present invention will be described with reference to FIG. In FIG. 4, 401 is a glass capillary, 402 is a sample solution, 403 is a sample stage having an ionization promoting substance, 404 is a reagent that decomposes an object (sprayed state), and 405 is a developed sample spot.

  In the present invention, the above sample stage refers to the whole plate itself such as a thin layer chromatography plate or the like fixed to a holder or the like for attaching the plate or the like to a time-of-flight secondary ion mass spectrometer.

  The sample stage of the present invention includes an ionization promoting substance and has a mechanism or function capable of separating an object. A specific example of the sample stage is not limited to the thin layer chromatography plate as long as it has such a function, and examples thereof include electrophoresis. Moreover, the process of decomposing | disassembling the isolate | separated target object as needed can be provided.

  For example, a sample stage (a thin layer chromatography plate on which silver is deposited) 403 is prepared by depositing silver, which is an ionization promoting substance, and a sample solution 402 is developed using a glass capillary 401 on the sample stage 402 so that an object is obtained from the sample solution 402. Things can be separated. The separated object can be decomposed by spraying the developed sample spot 405 with a reagent (spraying state) 404 that decomposes the object.

  The reagent (spraying state) 404 for decomposing the object can efficiently perform a series of operations by providing spraying means in the composition information acquisition apparatus of the present invention as necessary.

  Hereinafter, the present invention will be described more specifically with reference to examples. The specific example shown below is an example of the best embodiment according to the present invention, but the present invention is not limited to such specific form.

Preparation of sample plate for analysis from which biological samples are separated (1)
A plate for thin layer chromatography (Merck, RP-18, layer thickness 0.2 mm) is cut into 30 mm × 5 mm, and an Ag monoatomic layer is formed on the plate by sputter deposition.

Next, synthetic peptide I (peptide sequence: GGGGCGGGGG, C ₂₁ H ₃₄ N ₁₀ O ₁₁ S (average molecular weight: 634.61, mass of the molecule consisting of the element with the highest isotope abundance ratio: 634.21) sigmagenosis (Purchased from Japan) and synthetic peptide II (peptide sequence: GGGGCEGGGG, C ₂₄ H ₃₈ N ₁₀ O ₁₃ S (average molecular weight: 706.79, mass of the molecule consisting of the element with the highest isotope abundance ratio: 706.23) Each 10 μM mixed aqueous solution is prepared from Sigma Genosys Japan. 10 μl of this is dropped into the center of the plate, and this is developed by infiltrating a 1: 1 (volume ratio) solution of acetonitrile: distilled water containing 0.1 vol% trifluoroacetic acid through a glass capillary tube. A plurality of such samples are prepared.

TOF-SIMS analysis of the sample produced in Example 1 The sample produced in Example 1 is air-dried and analyzed using a TOF-SIMS type IV apparatus manufactured by ION TOF. The measurement conditions are summarized below.

Primary ion: 25 kV Ga +, 0.6 pA (pulse current value), random scan mode Primary ion pulse frequency: 2.5 kHz (400 μs / shot)
Primary ion pulse width: about 1 ns
Primary ion beam diameter: about 5 μm
Measurement area: Macro raster (30mm x 5mm)
Integration count: 256
When the positive and negative secondary ion mass spectra are measured under such conditions, Ag is added to the parent molecule of the synthetic peptide I in the positive secondary ion mass spectrum, and one carbon atom and one oxygen atom are added. Secondary ions corresponding to the added mass can be detected. Similarly, synthetic peptide II can detect a secondary ion corresponding to a mass in which Ag is added to its parent molecule in the positive secondary ion mass spectrum and carbon and oxygen atoms are added one by one. it can.

FIG. 1A shows an enlarged spectrum of this region in synthetic peptide I, and FIG. 1B shows a theoretical spectrum calculated based on the isotope abundance ratio. In FIG. 1, the peak with an arrow corresponds to the above-mentioned ion [(synthetic peptide I) + (Ag) + (CO)] ⁺ , and the two arrows indicate two Ag isotopes (mass number: 107, 109). The peak with the arrow on the right side contains ¹⁰⁹ Ag, and its m / z value (771.2) is almost identical to the theoretical value of [(synthetic peptide I) + ( ¹⁰⁹ Ag) + (CO)] ⁺ To do. A similar spectrum is obtained for synthetic peptide II. By using these secondary ions according to the parent ions of these synthetic peptides I and II, a two-dimensional image image reflecting the two-dimensional distribution state of the synthetic peptides I and II can be obtained.

  These synthetic peptides I and II are separated with good reproducibility on a thin layer chromatograph in which an Ag monoatomic layer is formed.

  In addition, in the plate for thin layer chromatography which does not perform Ag monoatomic layer formation, the secondary ion peak according to said parent ion is not observed. Also, no secondary ion peak is observed in the mass region corresponding to the parent molecule.

Preparation of sample plate for analysis from biological samples (2)
A plate for thin layer chromatography (Merck, RP-18, layer thickness 0.2 mm) is cut into 30 mm × 5 mm to obtain a sample separation plate.

Next, a 10 μM mixed aqueous solution of each of Synthetic Peptide I and Synthetic Peptide II similar to Example 1 is prepared. 10 μl of this is dropped into the center of the plate, and this is developed by infiltrating a 1: 1 (volume ratio) solution of acetonitrile: distilled water containing 0.1 vol% trifluoroacetic acid through a glass capillary tube. This is sprayed with a 10 μM silver nitrate aqueous solution to such an extent that the plate surface becomes slightly wet. For example, an amount of about 1 μL / cm ² is preferable. A plurality of such samples are prepared.

TOF-SIMS analysis of the sample produced in Example 3 The sample produced in Example 3 is air-dried and analyzed using a TOF-SIMS type IV apparatus manufactured by ION TOF. The measurement conditions are the same as in Example 2.

  As a result, the same peak as that shown in Example 2 is observed. Further, by using these secondary ions according to the parent ions of these synthetic peptides I and II, a two-dimensional image image reflecting the two-dimensional distribution state of the synthetic peptides I and II can be obtained.

  These synthetic peptides I and II are separated with good reproducibility on a thin layer chromatograph after spraying with an aqueous silver nitrate solution.

  In the thin layer chromatography plate that is not sprayed with an aqueous silver nitrate solution, no secondary ion peak according to the parent ion is observed. Also, no secondary ion peak is observed in the mass region corresponding to the parent molecule.

Preparation of sample plate for analysis from which biological samples are separated (3)
A plate for thin layer chromatography (Merck, RP-18, layer thickness 0.2 mm) is cut into 30 mm × 5 mm, and an Ag monoatomic layer is formed on the plate by sputter deposition.

  Next, 2 ml of healthy human blood is collected, and 2 ml of acetonitrile containing 0.2 vol% trifluoroacetic acid is added thereto. This is ground in a mortar and further subjected to ultrasonic treatment for 10 minutes. This is centrifuged (50,000 G × 60 minutes), and the resulting supernatant I is collected. Note that the above operation is performed with care so that all samples are held at around 4 ° C.

  1 ml of the collected supernatant I is lyophilized and dissolved in 20 μl of distilled water. This is centrifuged (50,000 G × 60 minutes), and finally 10 μl of supernatant is obtained. 10 μl of this is dropped into the center of the plate, and this is developed by infiltrating a 1: 1 (volume ratio) solution of acetonitrile: distilled water containing 0.1 vol% trifluoroacetic acid through a glass capillary tube. A plurality of such samples are prepared (Sample I).

Similarly, 1 ml of the supernatant I was freeze-dried as a sample for comparison, and cholesterol (C ₂₇ H ₄₆ O; average molecular weight: 386.73, mass of the molecule consisting of the element having the highest isotope abundance ratio: 386. A solution obtained by adding 200 μg of 35) is dissolved in 20 μl of distilled water. This is centrifuged (50,000 G × 60 minutes), and finally 10 μl of supernatant is obtained. 10 μl of this is dropped into the center of the plate, and this is developed by infiltrating a 1: 1 (volume ratio) solution of acetonitrile: distilled water containing 0.1 vol% trifluoroacetic acid through a glass capillary tube. A plurality of such samples are prepared (Sample II).

TOF-SIMS analysis of the sample produced in Example 5 The sample produced in Example 5 is air-dried and analyzed using a TOF-SIMS type IV apparatus manufactured by ION TOF. The measurement conditions are the same as in Example 2.

  As a result, for Sample I and Sample II, secondary ions corresponding to the mass of Ag added to the parent molecule of cholesterol molecules can be detected in the positive secondary ion mass spectrum. Further, by using this secondary ion according to the parent ion of cholesterol, a two-dimensional image image reflecting the two-dimensional distribution state of cholesterol in the samples I and II can be obtained. Both image images can be seen at the same position, but the intensity of sample II is about three times stronger than that of sample I.

Preparation of sample plate for analysis from which biological samples are separated (4)
A plate for thin layer chromatography (Merck, RP-18, layer thickness 0.2 mm) is cut into 30 mm × 5 mm, and an Ag monoatomic layer is formed on the plate by sputter deposition.

  Next, a 20 μM mixed aqueous solution of each of Synthetic Peptide I and Synthetic Peptide II similar to Example 1 is prepared. 100 μl of this was collected, and a 1.5 μg / μl solution (0.1 M sodium phosphate) of endoproteinase-Glu-C (serine protease, which specifically decomposes the C-terminal side of E and D of proteins and peptides) as a protease. 100 μl of buffer solution (pH: 8.0) is added and incubated at 37 ° C. for 18 hours. 10 μl of the treated solution is dropped on the center of the plate, and this is developed by infiltrating a 1: 1 (volume ratio) solution of acetonitrile: distilled water containing 0.1 vol% trifluoroacetic acid through a glass capillary tube. A plurality of such samples are prepared.

TOF-SIMS analysis of the sample produced in Example 7 The sample produced in Example 7 is air-dried and analyzed using a TOF-SIMS type IV apparatus manufactured by ION TOF. The measurement conditions are the same as in Example 2.

  As a result, the same peak as the synthetic peptide I shown in Example 2 is observed. Moreover, the peak corresponding to the synthetic peptide II is very weak. On the other hand, the peak according to the parent ion which added Ag to GGGGC and EGGGG is confirmed. By using these secondary ions, a two-dimensional image image reflecting the two-dimensional distribution state of the degradation products derived from the synthetic peptides I and II can be obtained.

  The degradation products derived from these synthetic peptides I and II are separated with good reproducibility on a thin-layer chromatograph in which an Ag monoatomic layer is formed.

  In addition, in the plate for thin layer chromatography which does not perform Ag monoatomic layer formation, the secondary ion peak according to said parent ion is not observed. Also, no secondary ion peak is observed in the mass region corresponding to the parent molecule.

Preparation of sample plate for analysis from which biological samples are separated (5)
A plate for thin layer chromatography (Merck, RP-18, layer thickness 0.2 mm) is cut into 30 mm × 5 mm, and an Ag monoatomic layer is formed on the plate by sputter deposition.

Next, a 10 μM mixed aqueous solution of each of Synthetic Peptide I and Synthetic Peptide II similar to Example 1 is prepared. 10 μl of this is dropped into the center of the plate, and this is developed by infiltrating a 1: 1 (volume ratio) solution of acetonitrile: distilled water containing 0.1 vol% trifluoroacetic acid through a glass capillary tube. To this, 1.5 μg / μl solution of endoproteinase-Glu-C (0.1 M sodium phosphate buffer, pH: 8.0) as a protease is slightly moistened, for example, about 1 μL / cm ² . Is preferred. And incubate at 100% humidity and 37 ° C. for 18 hours. A plurality of such samples are prepared.

TOF-SIMS analysis of the sample produced in Example 9 The sample produced in Example 9 is air-dried and analyzed using a TOF-SIMS type IV apparatus manufactured by ION TOF. The measurement conditions are the same as in Example 2.

  As a result, the same peak as the synthetic peptide I shown in Example 2 is observed. Moreover, the peak corresponding to the synthetic peptide II is very weak. On the other hand, a peak corresponding to the parent ion in which Ag is added to GGGGC and EGGGG is confirmed at the position corresponding to synthetic peptide II in Example 2. By using these secondary ions, a two-dimensional image image reflecting the two-dimensional distribution state of the degradation products derived from the synthetic peptides I and II can be obtained.

  The degradation products derived from these synthetic peptides I and II are separated with good reproducibility on a thin-layer chromatograph in which an Ag monoatomic layer is formed.

  In addition, in the plate for thin layer chromatography which does not perform Ag monoatomic layer formation, the secondary ion peak according to said parent ion is not observed. Also, no secondary ion peak is observed in the mass region corresponding to the parent molecule.

  The present invention is expected to be used as a method for obtaining information on health status, and its utility value is extremely high.

The partial enlarged view of the positive secondary ion mass spectrum in Example 2 is shown, (a) is an actually measured spectrum, and (b) shows a theoretical spectrum calculated based on the isotope abundance ratio. The process of this invention is shown. Sectional drawing of the sample stand of this invention is shown. The enlarged view of the sample stand of this invention is shown.

Explanation of symbols

201 Step of separating an object 202 Step of applying an ionization promoting substance to an object 203 Step of obtaining a secondary ion mass spectrum of the object by time-of-flight secondary ion mass spectrometry 204 Step of decomposing the object 301 Ionization Accelerating substance 302 Separator 303 Substrate 401 Glass capillary 402 Sample solution 403 Sample stage having ionization promoting substance 404 Reagent for decomposing the object (sprayed state)
405 Developed sample spot

Claims (17)

A method of acquiring a secondary ion mass spectrum of an object using time-of-flight secondary ion mass spectrometry,
A first step of separating the object;
A second step of applying an ionization promoting substance to the object;
A third step of obtaining a secondary ion mass spectrum of the object by the time-of-flight secondary ion mass spectrometry;
A method for obtaining composition information of the object.   The composition information acquisition method according to claim 1, further comprising a step of decomposing the separated object by a chemical technique after the first step.   The first step of separating the target object is a step of separating the target object by electrophoresis or thin layer chromatography using a separator that includes an ionization promoting substance and has a mechanism capable of separating the target object. The composition information acquisition method for the object according to claim 1.   The composition information acquisition method of the object according to claim 3, further comprising a step of decomposing the separated object by a chemical technique after the first step.   The composition information acquisition method for the object according to any one of claims 1 to 4, wherein the object is a biological material.   6. The method according to claim 5, wherein the biological substance is a substance selected from the group consisting of nucleic acids, proteins, and degradation products obtained by decomposing them by a chemical technique.   The composition information acquisition method for the object according to any one of claims 1 to 4, wherein the ionization promoting substance is silver, gold, or a mixture thereof.   The composition information acquisition method for the object according to any one of claims 1 to 7, wherein information on a two-dimensional distribution state of the object is acquired by scanning with a primary ion beam.   A composition information acquisition apparatus comprising a time-of-flight secondary ion mass spectrometer, wherein the sample stage in the analyzer includes an ionization promoting substance and has a mechanism for separating the object. Composition information acquisition device for a product. A composition information acquisition device comprising a time-of-flight secondary ion mass spectrometer,
The sample stage in the analyzer is:
An ionization-promoting substance is included; and a mechanism for separating an object and a mechanism for decomposing the separated object by a chemical method;
The composition information acquisition apparatus of the said target object characterized by the above-mentioned. In a sample stage for performing time-of-flight secondary ion mass spectrometry,
A sample stage for time-of-flight secondary ion mass spectrometry, comprising an ionization promoting substance and having a mechanism for separating an object.   The sample stage for time-of-flight secondary ion mass spectrometry according to claim 11, wherein the sample stage is a plate for thin layer chromatography containing an ionization promoting substance.   The time of flight type according to claim 11 or 12, wherein the sample stage includes an ionization promoting substance and has a function of separating an object and a function of decomposing the separated object by a chemical method. Sample stage for secondary ion mass spectrometry.   The method for obtaining information on a health condition by the composition information acquisition method according to any one of claims 1 to 8, wherein the object is a sample collected from a living body, and obtains information on a health condition. Method.   The method for obtaining information on health status according to claim 14, wherein the sample stage for fixing the sample is removable from the composition information acquisition device.   The health condition information according to claim 14 or 15, further comprising a step of analyzing a component of the sample based on secondary ion information and separated pattern information obtained from the result of the mass spectrometry. How to get.   The step of analyzing the component of the sample is a step of comparing secondary ion information corresponding to various health conditions prepared in advance with library data of separated pattern information. 16. A method for obtaining information on health status according to 16.

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