JP4984900B2 - Mass spectrometry using polymer-coated microparticles - Google Patents

Description

  The present invention relates to a mass spectrometry method using polymer-coated fine particles that serve as an ion source when a sample is irradiated with laser light for ionization.

  In fields such as biochemistry, medicine, and genomic drug discovery, there is a high demand for structural analysis of proteins in living tissues and cells. Mass spectrometry can be cited as a response to such a demand. A mass spectrometer irradiates a sample with a laser having a small diameter, ionizes biomolecules distributed in the sample, and analyzes the mass of the generated ions (Non-Patent Document 1).

In such a mass spectrometer, it is necessary to ionize the sample by some method in order to perform analysis, but considering that the sample is a biological sample, it is desirable to minimize damage to the sample during ionization. Moreover, it is necessary to perform so-called soft ionization so that ions to be analyzed are not cleaved. From this point of view, the use of matrix-assisted laser desorption / ionization (MALDI) or secondary ion mass spectrometry (SIMS) can be considered as ionization methods, but in general, high S / N can be obtained with SIMS, but ionization is possible. The mass number is about 1000 at most. In addition, tandem mass spectrometry (MS ⁿ ) that determines the structure of biomolecules is also difficult. From the above, it is difficult to use for analysis of biological samples having a large molecular weight such as proteomics.

On the other hand, MALDI analyzes a sample that is difficult to absorb laser light or a sample that is easily damaged by laser light, such as protein, so that a substance that easily absorbs laser light and is easily ionized is used as a matrix (ionization aid) in advance. The sample is ionized by mixing and irradiating it with laser light. Examples of the matrix include a mixture of a metal oxide (cobalt) and glycerin, a chemically synthesized substance (hereinafter referred to as chemical) matrix, and the like. The chemical matrix includes 1,8-dihydroxy-9 (10H) -anthracenone (Dithranol), 2- (4-hydroxyphenylazo) benzoic acid (HABA), 2,5-dihydroxybenzoic acid (DHBA), α-cyano-4 -Hydroxycinnamic acid (CHCA) and sinapinic acid (SA) are listed, and they are selected according to the substance (protein, peptide, synthetic polymer) to be analyzed. MS ^{n is} also easy and suitable for structural analysis. However, when mass spectrometry is performed using MALDI, there are the following problems.

In general, the matrix is applied onto a sample by being dropped or sprayed in a liquid state, and takes up an analyte to be analyzed on the sample. When dried, crystal grains containing the substance to be analyzed are formed. At this time, the size of the crystal grains of the matrix is generally about 50 μm or more. Since the substance to be analyzed is dispersed between the crystal grains of this matrix, even if the irradiation diameter of the laser beam for ionization is reduced, a spatial resolution higher than the crystal grain diameter of this matrix cannot be obtained. For example, when it is desired to examine the distribution of proteins in human cells, if the cell size is small, it is about 10 to 30 μm, so sufficient results cannot be obtained unless a spatial resolution of about several μm is obtained. . On the other hand, it is technically possible to reduce the crystal grains of the matrix in MALDI, but if so, the ionization efficiency is extremely lowered and effective mass spectrometry cannot be performed.
Further, the matrix has a problem that ionization efficiency is lowered when salt components such as sodium and potassium are mixed.

If an in vivo substance such as a cell can be analyzed while being close to the in vivo state, a lot of information can be obtained, which leads to a simplified procedure. For this purpose, it is important that a matrix exists in the cell and that the cell is not toxic. However, such efforts and methods have not been established.
Further, MALDI has a problem that it is difficult to increase the S / N ratio of the detection signal because the matrix becomes background noise apart from the problem of the crystal grain size of the matrix. In order to solve this problem, a laser desorption ionization method that does not use a matrix has been proposed. For example, Patent Document 2 discloses an ionization method called DIOS (desorption / ionization on silicon) using porous silicon as a substrate. In this method, it is considered that a nano-scale fine uneven structure on the substrate surface contributes to ionization. Furthermore, as a laser desorption / ionization method using the same concavo-convex structure, a method using a carbon amorphous, Si / Ge dot plate or the like instead of porous silicon has been proposed (for example, Non-Patent Documents 2 to 4). reference). However, the laser desorption ionization method using the nano level uneven structure without using the matrix as described above was originally devised in order to avoid interference from matrix molecules appearing in the mass spectrum. In addition, even when cells are attached to such a substrate, it is difficult to detect even intracellular substances.

  The inventors already have technology for functionalizing ultra-nano-sized magnetic ultra-nanoparticles having a diameter of 1.3 to 3 nm and introducing them into living cells and tissues (Patent Document 1, Non-Patent Document 5). However, it has not been known to use these fine particles as an ionization aid for laser irradiation. In addition, it is considered that a metal oxide matrix can be introduced into cells by optimizing the particle size, but the matrix effect cannot be expected unless glycerin is present. It is difficult to introduce a liquid substance such as glycerin into a cell. Therefore, in order to identify intracellular substances, cells must be destroyed, and it is difficult to identify intracellular substances in a living state. For metal oxide matrices, a mixture of glycerin is applied to the cell surface. Only the analysis of cell surface membrane proteins can be expected. In addition, when an attempt is made to analyze by adding glycerin, intracellular substances are diluted, making it difficult to detect trace substances. Moreover, since two kinds of substances are used, the experimental operation becomes complicated. Next, since it is difficult to introduce a chemical matrix into cells, only the same effect as the metal oxide matrix can be expected. For these reasons, it was difficult to obtain detailed in vivo information.

Japanese Patent Application 2006-64689 US Pat. No. 6,288,390 Yasuhide Naito, “Mass Microscope for Biological Samples”, J. Mass Spectrom. Soc. Jpn., Vol. 53, No. 3, 2005, pp.125-132. Arakawa and two others, “Recent Advances in Soft Laser Desorption / Ionization Mass Spectrometry (LDI-MS)”, J. Mass. Spectrom. Soc. Jpn., Vol.52, No.1, 2004, pp.33 -38 Moriya and two others, “Usefulness of amorphous carbon substrates in laser desorption ionization”, 54th Annual Meeting of the Analytical Society of Japan (2005), Research Presentation Number: F2002 Kiyono and 4 others "Development of a new soft laser desorption ionization-mass spectrometry method using nanodot structures as an ionization substrate" 54th General Assembly of Mass Spectrometry (2006), poster number: 2P-P2-36 Moritake, Hira et al. “Functionalized Nano Magnetic Particles for an in vivo Delivery System” Journal of Nanoscience and Nanotechnology (2007) Vol.7

The object of the present invention is that the analyte can be ionized with high efficiency without using an existing matrix, and is included in a sample such as a living cell or tissue with a spatial resolution higher than the crystal grain size of the existing matrix. An object of the present invention is to provide a mass spectrometric method capable of analyzing an analysis target substance and a material that can be used therefor.

As a result of intensive studies to solve the above problems, the inventors of the present invention have a fine particle core coated with a polymer that absorbs laser energy because the fine particle core is a metal oxide. It has been found that it has matrix-like properties that support ionization of analysis target substances in the vicinity. In addition, when using fine particles with the metal oxide core covered with a silica (SiO ₂ ) network, affinity for in vivo substances (peptides, proteins, nucleic acids) using hydroxyl groups exposed on the surface Focusing on the improvement, it was found that when introduced into cells, glycerol is not required and the effect as a matrix can be obtained.

In addition, it was found that when extremely fine particles are used, the spatial resolution restriction is substantially eliminated as in the case of existing matrix crystal grains.
Furthermore, the present invention has found that the S / N ratio of the detection signal can be increased without increasing the background in order to assist in ionizing only the analysis target substance without ionizing the fine particle itself by laser irradiation. It came to complete.

That is, according to the present invention, the following inventions are provided.
(1) (a) a step in which a fine particle whose polymer is coated on a core made of a metal oxide and a substance to be analyzed are brought close to each other, and (b) ionization of the substance to be analyzed in the vicinity of the fine particle by performing laser irradiation. The process of supporting
Including mass spectrometry.
(2) (a) attaching a substance to be analyzed to a sample plate in which a fine particle in which a polymer is coated on a core made of a metal oxide is modified;
(B) a step of allowing the fine particles to assist ionization of the analyte in the vicinity of the fine particles by performing laser irradiation, and a mass spectrometry method comprising the steps of:
(3) The mass spectrometry method according to (1) or (2), wherein the step (a) is performed using a sample containing a substance to be analyzed.
(4) (a) a step of administering fine particles having a metal oxide core coated with a polymer to a non-human specimen, and (b) performing ionization of an analyte in the vicinity of the fine particles by performing laser irradiation. The process of supporting
Including mass spectrometry.
(5) (a) a step of administering fine particles having a metal oxide core coated with a polymer to a cell or tissue, and (b) performing ionization of an analyte in the vicinity of the fine particles by performing laser irradiation. The process of supporting
Including mass spectrometry.
(6) The mass spectrometric method according to any one of (1) to (5), wherein laser light having a diameter of 50 μm or less is condensed and laser irradiation is performed.
(7) An ionization assisting agent for laser irradiation for mass spectrometry, comprising, as an active ingredient, fine particles having a metal oxide core coated with a polymer.
(8) A sample plate for mass spectrometry in which fine particles in which a core made of a metal oxide is coated with a polymer are modified.
(9) A mass spectrometry kit for supporting ionization of an analysis target substance by laser irradiation, including the plate according to (8), and attaching a sample containing the analysis target substance on the upper surface of the plate by transfer, A kit for detecting analytes by irradiating a sample attached to the upper surface of a plate with a laser.

  As described above, the fine particles according to the present invention have a form in which a metal oxide is used for the fine particle core and the periphery thereof is coated with a polymer such as silica. The surface of the polymer made of silica is hydrophilic because it shows a hydroxyl group and has a good interaction with the substance to be analyzed. This makes it easier for the matrix and the analyte to come into contact, so that the energy required for ionization of the analyte can be efficiently transferred from the matrix to the analyte, and as a matrix that is an essential material in MALDI mass spectrometry. Very suitable for use. In addition, since the microparticles can be taken into living cells, the matrix is introduced into the living cells, and intracellular substances can be identified by analyzing the matrix-containing cells. Furthermore, by not using an existing chemical matrix, the S / N ratio and sensitivity of the detection signal can be improved, and the accuracy of detection of the target substance can be increased.

  The fine particle material according to the present invention functions as a matrix even if salt components such as sodium and potassium are mixed in the sample. Usually, the chemical matrix or the like does not function due to the mixing of salt or the like. This makes it possible to analyze specific substances in living cells, and can collect useful information particularly in the field of life science.

The present invention will be described in detail below, but these descriptions are examples (representative examples) of the embodiments of the present invention and do not limit the scope of the present invention.

(1) Polymer-coated fine particles First, the fine particles used in the mass spectrometry method of the present invention will be described.
In the mass spectrometry method of the present invention, fine particles in which a core made of a metal oxide is coated with a polymer are used.
Here, the metal oxide is preferably an oxide of a transition metal or a rare earth metal, more preferably an oxide of Ni, Fe, or Co, still more preferably an oxide of Fe or Co, and particularly preferably. Is an oxide of Fe.
Examples of the polymer include polyallylamine, polystyrene, polythiophene, polyacrylic acid, a copolymer of ethylene glycol glycidyl ester and lysine, and silica. Silica is particularly preferable.
The method of coating the metal oxide with the polymer may be appropriately selected according to the type of the metal oxide and the polymer, but a wet precipitation method (for example, JP-A-2003-252618) is preferable.
The size of the fine particles may be any size that can be used for mass spectrometry, but ultra-fine particles having a particle size of 3 nm or less, more preferably 1.3 to 3 nm are desirable.
The shape of the fine particles may be any shape such as a spherical shape, a needle shape, a disc shape, an elliptical sphere shape, or a rod shape, but a spherical shape is preferred.

上記式中、Mは遷移金属または稀土類金属を示し、Ni、FeまたはCoが使用され、好まし
くはFeまたはCoであり、より好ましくはFeである。XはF, Cl, Br, Iから選ばれるハロゲン元素を示す。pは２または３、nは０から９までの整数、mは９または０である。xおよびyはともに１未満の正数である。磁性を発揮するコアおよびその表面修飾層としてのシェルの役割分担の観点から、ｘ＞ｙ、かつ、ｘ／ｙとしては、通常１〜１００、好ましくは２〜２０程度の範囲から選定される。
より具体的には、超ナノ微粒子 [xM(OH)₂・ySiO₂]の構造は、図１のように模式的に表すことができる。 These ultra-nanofine particles are preferably prepared using a wet precipitation method (Japanese Patent Laid-Open No. 2003-252618). For example, ultranano fine particles [xM (OH) ₂ · ySiO ₂ ] given by the following formula (I) can be mentioned. Here, xM (OH) ₂ represents a core and ySiO ₂ represents a shell.

In the above formula, M represents a transition metal or a rare earth metal, and Ni, Fe, or Co is used, preferably Fe or Co, and more preferably Fe. X represents a halogen element selected from F, Cl, Br, and I. p is 2 or 3, n is an integer from 0 to 9, and m is 9 or 0. x and y are both positive numbers less than 1. From the viewpoint of role sharing of the core exhibiting magnetism and the shell as the surface modification layer, x> y and x / y are usually selected from the range of about 1 to 100, preferably about 2 to 20.
More specifically, the structure of ultranano fine particles [xM (OH) ₂ · ySiO ₂ ] can be schematically represented as shown in FIG.

The fine particles used in the mass spectrometry method of the present invention may be those obtained by surface-treating a polymer that coats a metal oxide. By applying the surface treatment, the affinity with the substance to be analyzed and the efficiency of introduction into cells can be improved.
The type of functional group introduced into the polymer surface is not particularly limited, and specific examples include a hydroxyl group, an amino group, an isocyanate group, or a mercapto group.
These functional groups can be introduced covalently through, for example, a silane coupling agent.
Examples of the treatment for introducing a hydroxyl group on the polymer surface include washing with an organic solvent such as ethanol, UV washing (Japanese Patent Laid-Open No. 1-146330), plasma treatment, autoclave treatment, and the like.

(2) Mass spectrometry using polymer-coated fine particles In the mass spectrometric method of the present invention, first, a step of bringing a substance to be analyzed close to the fine particles is performed.
Here, the microparticles and the analysis target substance may be brought close to each other so that the analysis target substance is ionized by laser irradiation. For example, the microparticle solution is mixed with the analysis target substance or a sample solution containing the same, or The fine particles can be immobilized on a carrier and brought close to each other by adding a substance to be analyzed or a sample containing the same. The ratio of the fine particles to the analysis target substance in the case of mass spectrometry is not particularly limited, but it is preferably adjusted so that the weight ratio is 1:10 to 10: 1, more preferably 1: 3 to 3: 1. desirable.

The type of the substance to be analyzed is not particularly limited, and examples thereof include biological substances such as proteins, peptides, nucleic acids, sugars, lipids, synthetic low molecular compounds, synthetic polymers, and the like.
Further, the analysis target substance is not necessarily a purified substance, and the analysis target substance and the fine particles may be brought close to each other by adding a sample containing the analysis target substance as it is to the fine particles. Examples of the sample containing the substance to be analyzed include biological samples such as cells, tissues and body fluids derived from animals, plants or microorganisms, or extracts from these, which contain the substance to be analyzed. Further, it may be a sample isolated from soil or drainage.
For example, a cell or tissue extract may be added to the fine particles, and mass analysis of the analysis target substance contained in the cells or tissue may be performed. Moreover, after adding microparticles | fine-particles to solutions, such as a culture medium containing a cultured cell or an isolated tissue, and incubating, the extract containing microparticles | fine-particles and a to-be-analyzed substance may be collect | recovered and mass spectrometry may be performed. Furthermore, after administering the microparticles to the non-human specimen, an extract containing the microparticles and the substance to be analyzed may be collected and subjected to mass spectrometry. Here, examples of the non-human specimen include laboratory animals such as mice and rats.

In the mass spectrometric method of the present invention, a step of using a sample plate with modified (immobilized) microparticles, and bringing the analyte to be close to the microparticles by attaching the analyte or a sample containing the same to the sample plate is performed. May be. For example, it is preferable that a sample prepared on a sheet such as a film is attached to the upper surface of the sample plate, and the sample is transferred from the sheet to the plate by transfer to be attached thinly on the plate. In this way, it is possible to comprehensively perform mass analysis on many analytes contained in a sample. The analysis target substance may be applied on a sample plate in which fine particles are modified.
The modification of the fine particles to the sample plate may be a chemical bond modification or a physical bond modification. For example, an ITO (indium synoxide) sheet or the like can be modified using a cross-linking agent such as Disuccinimidyl Glutarate.

In the mass spectrometric method of the present invention, next, a step of supporting the ionization of the analysis target substance on the fine particle by irradiating the analysis target substance close to the fine particle or a plate on which the fine particle and the analysis target substance are attached by laser irradiation is performed.
The laser light to be irradiated is preferably adjusted by a condensing optical system so that the irradiation diameter on the sample is 50 μm or less. Actually, when the sample to be analyzed is a cell, it is desirable to reduce the irradiation diameter of the laser light to 10 μm or less, for example, about several μm. As the laser irradiation apparatus, an apparatus used for ordinary MALDI (matrix-assisted laser desorption / ionization) mass spectrometry can be used. For example, TOF2 (Shimadzu Corporation), UltraFlex (Bruker Daltonics), ABI4800 (ABI) Etc. can be used.

In the case of a fine particle material in which a polymer is coated on a core made of a metal oxide, when a laser beam with a small diameter hits a sample in contact with the fine particle, the fine particle absorbs the laser light, and the fine particle composed of the metal oxide The sample molecules are ionized by the interaction between the core and the sample. Therefore, according to this fine particle material, it is possible to perform mass analysis by omitting selection and use from an existing matrix group, and it is possible to support ionization of only the analysis target substance.
For detection, a detection device used for ordinary mass spectrometry can be used, and for example, TOF2 (Shimadzu Corporation), UltraFlex (Bruker Daltonics), ABI4800 (ABI) and the like can be used.

  According to the mass spectrometric method of the present invention, an analyst can first grasp a substance to be analyzed from a living sample, and can perform mass spectrometry without using an existing matrix. As a result, the analyst can obtain the presence information of only the substance that he / she really wants to analyze, and it is not necessary to search for a substance suitable for the substance to be analyzed from various existing matrix groups, thus saving the time required for measurement. Can do.

The ionization assisting agent for laser irradiation according to the present invention comprises fine particles in which a polymer core is coated with a polymer, and is used for mass spectrometry.
The sample plate of the present invention is a sample plate for mass spectrometry in which fine particles obtained by coating a polymer on a core made of the above metal oxide are modified. The material of the sample plate is not particularly limited as long as it can withstand laser irradiation, and examples thereof include titanium, gold-coated steel, and ITO sheet.
A kit for supporting ionization of a substance to be analyzed by laser irradiation according to the present invention includes the above-described sample plate, a sample containing the substance to be analyzed is attached to the upper surface of the plate by transfer, and the sample is irradiated with a laser for analysis. A kit for detecting a target substance. Here, “attaching by transfer” includes, for example, a mode in which a sample is prepared on a sheet such as a film, and the sample is attached to the upper surface of the plate and moved from the sheet to the plate. The kit of the present invention is suitably used for mass spectrometry, and may contain other reagents for mass spectrometry.

  Next, specific examples of the present invention will be shown, but the present invention is not limited to these examples.

Example 1: Preparation of ultra-nano fine particles (γ-Fe system)
For the fine particles, a wet precipitation method in which fine particles were obtained in a monodisperse manner was used. During the preparation, FeCl ₂ .4H ₂ O and Na ₂ SiO ₃ .9H ₂ O were employed as materials to prepare γ-Fe ₂ O ₃ ultra-nano particles.
19.9 g of FeCl ₂ .4H ₂ O (10 mM) and 28.4 g of Na ₂ SiO ₃ .9H ₂ O (10 mM) were weighed and dissolved in 500 ml of distilled water to obtain two aqueous solutions. . After mixing the two with sufficient agitation at room temperature for about 5 hours to complete the reaction, throw the supernatant into a centrifuge for 20 minutes, discard the supernatant, re-disperse it with distilled water, and then apply to the centrifuge to remove the supernatant. The operation of discarding was repeated several times to wash the precipitate. Next, after drying in a thermostatic bath maintained at about 350K, pulverizing in a mortar, placing on an alumina boat, and firing in an electric furnace in an air atmosphere at a temperature range of 373 to 1273K for 4 to 10 hours Ultra-fine particles were obtained. The iron-based ultra-nanoparticles used in Example 3 and below are those annealed at 873K.

Example 2: Preparation of ultra-nano fine particles (Co system)
For the fine particles, a wet precipitation method in which fine particles were obtained in a monodisperse manner was used. In preparation, CoCl ₂ · 6H ₂ O and Na ₂ SiO ₃ · 9H ₂ O were used as materials to prepare ultra-nano particles.
1.19 g CoCl ₂ .6H ₂ O (10 mM) and 1.42 g Na ₂ SiO ₃ .9H ₂ O (10 mM) were weighed and dissolved in 500 ml distilled water to obtain two aqueous solutions. About 10 minutes at room temperature, the two are mixed with sufficient agitation to complete the reaction. Then, the supernatant is poured into a centrifuge for 20 minutes, and the supernatant is discarded. After re-dispersing with distilled water, the supernatant is put into a centrifuge. The operation of discarding was repeated several times to wash the precipitate. Next, after drying in a drying furnace maintained at about 350K, finely pulverizing in a mortar and placing on an alumina boat, firing in an electric furnace in an air atmosphere at a temperature range of 623 to 973K for 10 hours, Co ₃ O ₄ ultra-nanoparticles were obtained. The cobalt-based ultra-nanoparticles used in Example 3 and later are those annealed at 623K.

Example 3: Introduction of hydroxyl group onto fine particle surface As the fine particle surface cleaning method, since the fine particle surface is silica-coated, either ethanol cleaning, plasma treatment or autoclave treatment may be used. Washing, more preferably autoclaving is preferred.
Specifically, 500 μL of ethanol was used, gently stirred at room temperature for 1 minute, centrifuged at 5,000 rpm for 3 minutes, the supernatant was removed, and washed with 500 μL of ultrapure water in the same manner three times. Then, it was dried at 100 ° C. for 30 minutes to completely remove moisture.
Specifically, the fine particle powder is put in a heat-resistant bottle and processed by an autoclave apparatus for sterilization.

Example 4: Photographing of Fine Particle Image The obtained ultra-nanofine particle image was photographed with an electron microscope (JEM-1230: JEOL). The acceleration voltage was 100kV.
In the case of γ-Fe ₂ O ₃ ultra-nanoparticles (FIG. 2), the particle size was 3 nm (coefficient of variation (CV) 8%).
In the case of Co ₃ O ₄ (FIG. 3), the particle size was 3.6 nm (CV 21%).

Example 5: Matrix formation of ultra-fine nanoparticles (core composition: γ-Fe ₂ O ₃ )
γ-Fe ₂ O ₃ super-nanofine particle saturated solution, peptide sample (Substance P acetate salt hydrate; molecular weight 1346.7) mixed at a weight ratio of 2: 1 (1), water, peptide sample (Substance P A mixture of acetate salt hydrate (molecular weight 1346.7) at a weight ratio of 2: 1 was defined as (2) (control). 2 μL of each sample was dropped on a plate dedicated for mass spectrometry (MTP 384 target plate ground steel TF; Bruker Daltonics) and vacuum-dried. After drying, sample analysis was performed in a reflector positive mode using a mass spectrometer (Ultraflex; Bruker Daltonics). From the results, mass-to-charge ratios (m / z) of Substance P: 1347.7 (proton adduct [H ⁺ ]) and 1369.7 (sodium adduct [Na ⁺ ]) were obtained. In the absence of fine particles, the sample could not be ionized. S / N ratio, Substance P ⁺ [H +] at 1220: 1, Substance P + [ Na +] at 195: a: (particulate absence microparticles presence) 1. Since the peak of Na ⁺ adduct was also obtained at the same time, it was confirmed that only the sample supported ionization even in the presence of salt. (Fig. 4)

Example 6: Matrix formation of ultra-nano fine particles (core composition: γ-Fe ₂ O ₃ )
γ-Fe ₂ O ₃ super-nanofine particle saturated solution, peptide sample (Bradykinin [1-7]; molecular weight 756.4) mixed at a weight ratio of 2: 1 (1), water, peptide sample (Bradykinin [1-7]; A mixture of molecular weight 756.4) at a weight ratio of 2: 1 was designated as (2) (control). 2 μL of each sample was dropped on a plate dedicated for mass spectrometry (MTP 384 target plate ground steel TF; Bruker Daltonics) and vacuum-dried. After drying, sample analysis was performed in a reflector positive mode using a mass spectrometer (Ultraflex; Bruker Daltonics). From the results, a mass-to-charge ratio (m / z) 757.4 of Bradykinin [1-7] + [H ⁺ ] was obtained. In the absence of fine particles, the sample could not be ionized. The S / N ratio was 130: 1 in Bradykinin [1-7] (in the presence of microparticles: in the absence of microparticles), which was confirmed to support ionization of the sample. (Fig. 5)

Example 7: Matrix formation of ultra-nano fine particles (core composition: Co ₃ O ₄ )
Co ₃ O ₄ ultra-nano fine particle saturated solution, peptide sample (Substance P acetate salt hydrate; molecular weight 1346.7) mixed in a weight ratio of 2: 1 (1), water, peptide sample (Substance P acetate salt hydrate (molecular weight 1346.7) was mixed at a weight ratio of 2: 1 to make (2) (control). 2 μL of each sample was dropped on a plate dedicated for mass spectrometry (MTP 384 target plate ground steel TF; Bruker Daltonics) and vacuum-dried. After drying, sample analysis was performed in a reflector positive mode using a mass spectrometer (Ultraflex; Bruker Daltonics). From the results, mass-to-charge ratios (m / z) of Substance P: 1347.7 (proton adduct [H ⁺ ]) and 1369.7 (sodium adduct [Na ⁺ ]) were obtained. In the absence of fine particles, the sample could not be ionized. S / N ratio, Substance P ⁺ [H +] at 99: 1, Substance P + [ Na +] in 44: 1: (fine presence particulate absence). Since the peak of Na ⁺ adduct was also obtained at the same time, it was confirmed that only the sample supported ionization even in the presence of salt. (Fig. 6)

Example 8: Matrix formation of ultra-nano fine particles (core composition: Co ₃ O ₄ )
Co ₃ O ₄ ultra-nanofine particle saturated solution, peptide sample (Bradykinin [1-7]; molecular weight 756.4) in a weight ratio of 2: 1 (1), water, peptide sample (Bradykinin [1 -7]; a molecular weight of 756.4) mixed at a weight ratio of 2: 1 was defined as (2) (control). 2 μL of each sample was dropped on a plate dedicated for mass spectrometry (MTP 384 target plate ground steel TF; Bruker Daltonics) and vacuum-dried. After drying, sample analysis was performed in a reflector positive mode using a mass spectrometer (Ultraflex; Bruker Daltonics). From the results, a mass-to-charge ratio (m / z) of Bradykinin [1-7]: 757.4 was obtained. In the absence of fine particles, the sample could not be ionized. The S / N ratio was 376: 1 (in the presence of microparticles: in the absence of microparticles) as determined by Bradykinin [1-7], and thus was confirmed to support ionization of the sample. (Fig. 7)

The fine particles of the present invention have a function as an essential matrix in mass spectrometry, and support the ionization regardless of the type of analyte. Furthermore, since it can be easily taken into cells and tissues, it can be used for analysis of analysis target substances contained in samples such as living cells and tissues.

Predicted structure of ultra-nano particles Core composition: Electron micrograph of γ-Fe ₂ O ₃ ultra-fine particles. Core composition: Electron micrograph of ultra-fine particles of γ-Co ₃ O ₄ . Core composition: MS spectrum of γ-Fe ₂ O ₃ nanomatrix and peptide sample mixture. Core composition: MS spectrum of γ-Fe ₂ O ₃ nanomatrix and peptide sample mixture. Core composition: MS spectrum of a Co ₃ O ₄ nanomatrix and peptide sample mixture. Core composition: MS spectrum of a Co ₃ O ₄ nanomatrix and peptide sample mixture.

Claims (9)

(A) a step in which a core made of a metal oxide is coated with a polymer , and the surface-treated fine particles and the analyte are brought close to each other; and (b) ionization of the analyte in the vicinity of the fine particles by performing laser irradiation. step of support to the fine particles,
Including mass spectrometry. (A) a step of attaching an analyte to a sample plate in which a core made of a metal oxide is coated with a polymer and the surface-treated fine particles are modified; and (b) the vicinity of the fine particles by performing laser irradiation. step of the ionization of the analyte supported on the fine particles of,
Including mass spectrometry. The mass spectrometry method according to claim 1 or 2, wherein the metal oxide is an oxide of a transition metal or a rare earth metal. The mass spectrometry method as described in any one of Claims 1-3 which performs a process (a) using the sample containing a to-be-analyzed substance. (A) a step of administering to a non-human specimen fine particles having a metal oxide core coated with a polymer and surface-treated ;
(B) a step of supporting the ionization of analytes in the vicinity of the fine particles to the fine particles by a process for recovering an extract containing particulate and analytes from non-human subject, and (c) laser irradiation performed,
Including mass spectrometry. (A) a step of introducing into a cell or tissue microparticles coated with a polymer on a core made of a metal oxide and surface-treated ;
(B) supporting the ionization of analytes in the vicinity of the fine particles in the <br/> particles by performing step recovering the extract containing the microparticles and the analyte from the cells or tissue, and (c) laser irradiation The process of
Including mass spectrometry. The mass spectrometry method according to any one of claims 1 to 6 , wherein the laser beam is condensed so as to be a laser beam having a diameter of 50 µm or less. An ionization assisting agent for laser irradiation for mass spectrometry, comprising as an active ingredient fine particles having a metal oxide core coated with a polymer and surface-treated . A sample plate for mass spectrometry, in which a core made of a metal oxide is coated with a polymer and the surface-treated fine particles are modified.

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