Patent Description:
Mass spectroscopy is an analytical method where a sample containing a target molecule is ionized to separate and detect ions derived from the target molecule with a mass-to-charge ratio (m/z), and information related to identification of a chemical structure of the target molecule is obtained.

Ionization of a sample of mass spectroscopy is a factor determining the quality of analysis, and numerous methods of ionization have been developed. Examples include matrix assisted laser desorption/ionization (MALDI) and electrospray ionization (ESI). Since ionization is easily performed in these methods even with a very small amount of a sample, these methods have been used in technical fields such as biotechnology and medicines.

In MALDI, a pulsed laser is applied to an area of a matrix including a sample, where the matrix is a material for assisting ionization of the sample, and the sample is ionized together with the matrix.

Light having wavelengths of the ultraviolet region is often used as such a pulsed laser, and light having wavelengths matched with light absorption properties of the matrix is preferable. Moreover, the matrix is a crystalline organic low-molecular-weight molecules, and needs to be co-crystalized with or formed into a mixture with the sample. It has been considered that homogeneousness or a degree of mixing of the co-crystal affects sensitivity or accuracy of analysis. Therefore, matrices have been developed correspondingly to samples.

Moreover, various methods for applying a matrix to a sample have been proposed. For example, proposed is a method for preparing a sample of mass spectroscopy where a matrix is vapor-deposited to form microcrystals, and a matrix solution is further sprayed to the microcrystals to make matrix crystals grow on the microcrystals (see, for example, <CIT>).

<NPL> discloses pre-coated MALDI targets having a coating of uniformly small crystals of matrix material. Reference may also be made to the disclosures of matrix deposition methods in <CIT> and <CIT>.

According to one aspect of the present disclosure, a method for preparing a measurement sample for MALDI mass spectrometry includes applying a laser beam to a base including a matrix used for preparing the measurement sample for MALDI mass spectrometry, the matrix being disposed on a surface of the base. The laser beam is applied to a surface of the base opposite to the surface including the matrix, to make the matrix fly from the base to be disposed at a predetermined position of an analyte of MALDI mass spectrometry. The base includes a laser energy absorbing material. Laser energy of the laser beam has a wavelength of <NUM> or longer.

A method of the present disclosure for preparing a measurement sample for MALDI mass spectrometry includes a step of applying a laser beam to a base including a matrix used for preparing the measurement sample for MALDI mass spectrometry, the matrix being disposed on a surface of the base, in a manner that the laser beam is applied to a surface of the base opposite to the surface including the matrix, to make the matrix fly from the base to be disposed at a predetermined position of an analyte of MALDI mass spectrometry, wherein the base includes a laser energy absorbing material and laser energy of the laser beam has a wavelength of <NUM> or longer. If necessary, the method of the present disclosure further includes other steps.

A device of the present disclosure for preparing a measurement sample for MALDI mass spectrometry is a device for preparing a measurement sample for MALDI mass spectrometry used in the method of the present disclosure for preparing a measurement sample for MALDI mass spectrometry. The device is configured to be used in the above mentioned method and comprises a base comprising a matrix and a laser energy absorbing material, the matrix being disposed on the surface of the base, and a laser beam irradiation unit configured to apply the laser beam to the base in a manner that the laser beam is applied to the surface of the base opposite to the surface comprising the matrix, the laser beam having a wavelength of <NUM> or longer.

The present disclosure has an object to provide a method for preparing a measurement sample for MALDI mass spectrometry, where the method is able to dispose two or more different kinds of matrix material on one sample in mass spectrometry using MALDI, and further perform MALDI mass spectrometry with small variation.

In the present disclosure, it is possible to provide a method for preparing a measurement sample for MALDI mass spectrometry, where the method is able to dispose two or more different kinds of matrix material on one sample in mass spectrometry using MALDI, and further perform MALDI mass spectrometry with small variation.

MALDI is an abbreviation of Matrix Assisted Laser Desorption/Ionization, which is one of methods of mass spectrometry.

In mass spectroscopy using the MALDI (which is referred to as "MALDI mass spectrometry" hereinafter), mass spectroscopy is performed by applying a pulsed laser to a position of a sample to which a matrix, which is a material for assisting ionization, is deposited to thereby ionize the sample together with the matrix.

The matrix for use is selected for components to be analyzed in the sample.

The method and device of the present disclosure for preparing a measurement sample for MALDI mass spectrometry have been accomplished based on the finding that only one kind of a matrix can be disposed according to methods known in the art, such as a method where a matrix is applied to a sample by a spray gun, and a method where a matrix is applied to a sample through gas-phase spray or vapor deposition. In other words, the method of the present disclosure has been accomplished based on the finding that although there are optimum matrices for components to be analyzed, the methods known in the art cannot separately apply such optimum matrices to a plurality of components to be analyzed in one sample.

Also, the methods known in the art for preparing a measurement sample for MALDI mass spectrometry is inefficient because they dispose only one kind of a matrix onto the entire sample, and they need the number of samples corresponding to matrices for a plurality of measurement targets in a sample.

The method of the present disclosure for preparing a measurement sample for MALDI mass spectrometry has been accomplished based on the finding that diameters of crystals of a matrix tend to be uneven and quantitativity is low, because mass spectroscopy of the method known in the art depends largely on skills of an operator, and therefore sensitivity or accuracy of analysis is largely affected.

The method of the present disclosure for preparing a measurement sample for MALDI mass spectrometry includes applying a laser beam having a wavelength of <NUM> or longer to a base including a laser energy absorbing material and a matrix used for preparing the measurement sample for MALDI mass spectrometry disposed on a surface of the base, in a manner that the laser beam is applied to a surface of the base opposite to the surface on which the matrix is disposed, to make the matrix fly from the base to be disposed at a predetermined position of an analyte of MALDI mass spectrometry.

According to the method of the present disclosure for preparing a measurement sample for MALDI mass spectrometry, even when there is only one sample, a plurality of kinds of matrices suitable for targets to be analyzed, such as a protein, a lipid, and a nucleotide, can be disposed at predetermined positions thereof. Therefore, even when there are a plurality of targets to be analyzed in one sample, the method of the present disclosure for preparing a measurement sample for MALDI mass spectrometry can perform imaging mass spectroscopy with high sensitivity for the targets to be analyzed.

Also, the method of the present disclosure for preparing a measurement sample for MALDI mass spectrometry can make the matrix fly from the base by applying the laser beam having a wavelength of <NUM> or longer (which is in the visible light wavelength region) to the base including the laser energy absorbing material and the matrix disposed on the surface of the base. This makes it possible to prevent change in properties (possible damage) due to application of a high-energy laser beam to a target to be analyzed, to thereby be able to achieve MALDI mass spectrometry with small variation. As used herein, "change in properties" means changes in the structure, molecular weight, etc. of the substance of interest.

One exemplary method for confirming the change in properties is confirming whether the proportion of low-molecular-weight components increases through mass spectrometry.

The base including a laser energy absorbing material and a matrix disposed on a surface of the base is not particularly limited and may be appropriately selected depending on the intended purpose. Hereinafter, the "base including a laser energy absorbing material and a matrix disposed on a surface of the base" is referred to as a "matrix plate.

The matrix plate includes a laser energy absorbing material, a matrix, and a base.

The laser energy absorbing material is not particularly limited and may be appropriately selected depending on the intended purpose as long as it can absorb energy of a laser beam having a wavelength of <NUM> or longer. It is, for example, a laser energy absorbing material having a transmittance of the laser beam having the wavelength of <NUM>% or less. The transmittance of the laser beam having the wavelength can be measured using a spectrophotometer such as an ultra violet-visible infrared spectrophotometer V-<NUM> (available from JASCO Corporation).

The shape of the laser energy absorbing material is not particularly limited and may be appropriately selected depending on the intended purpose. It is preferably a thin film having an average thickness of <NUM> or less.

A matter forming the laser energy absorbing material is preferably a matter that is inert to the matrix and that has a conductive surface when forming a matrix on the laser energy absorbing material. If it is inert to the matrix, there is not a risk that the laser energy absorbing material causes a chemical reaction with the matrix upon application of the laser beam. If it has a conductive surface when forming a matrix on the laser energy absorbing material, the matrix can be electrostatically coated on the laser energy absorbing material. This makes it possible to form a more uniform matrix and increase the yield of matrix coating.

The matter forming the laser energy absorbing material is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include metals.

Examples of the metals include gold, platinum palladium, and silver. Among them, gold is preferable because it is inert and absorbs a laser beam having a broad wavelength range.

When the laser energy absorbing material is a metal thin film, the average thickness thereof is preferably <NUM> or more but <NUM> or less. When the laser energy absorbing material is such a metal thin film having an average thickness of <NUM> or more but <NUM> or less, it is possible to suppress occurrence of variation in conductivity of the surface and suppress occurrence of variation in the thickness of the matrix when coating the matrix through electrostatic coating.

A method for forming the laser energy absorbing material on the base is, for example, vacuum vapor deposition or sputtering.

The matrix is not particularly limited and may be appropriately selected depending on the intended purpose, as long as the matrix is a material capable of suppressing photodecomposition and thermal decomposition of a sample and suppressing fragmentation (cleavage).

Examples of the matrix include matrices known in the art. Specific examples of the matrix include <NUM>,<NUM>-diaminonaphthalene (<NUM>,<NUM>-DAN), <NUM>,<NUM>-dihydroxybenzoic acid (which may be abbreviated as "DHBA" hereinafter), <NUM>,<NUM>-anthracenedicarboxylic acid dimethyl ester, leucoquinizarin, anthrarobin, <NUM>,<NUM>-diaminonaphthalene (<NUM>,<NUM>-DAN), <NUM>-aza-<NUM>-thiothymine, <NUM>,<NUM>-diaminoanthraquinone, <NUM>,<NUM>-diaminopyrene, <NUM>,<NUM>-diaminocarbazole, <NUM>,<NUM>-anthracenedicarboxylic acid, norharmane, <NUM>-pyrenepropylamine hydrochloride, <NUM>-aminofluorene hydrochloride, ferulic acid, dithranol, <NUM>-(<NUM>-hydroxyphenylazo)benzoic acid) (HABA), trans-<NUM>-[<NUM>-(<NUM>-tert-butylphenyl)-<NUM>-methyl-<NUM>-propenylidene]malononitrile) (DCTB), trans-<NUM>-phenyl-<NUM>-buten-<NUM>-one (TPBO), trans-<NUM>-indoleacrylic acid (IAA), <NUM>,<NUM>-phenanthroline, <NUM>-nitro-<NUM>,<NUM>-phenanthroline, α-cyano-<NUM>-hydroxycinnamic acid (CHCA), sinapic acid (SA), <NUM>,<NUM>,<NUM>-trihydroxyacetophenone (THAP), <NUM>-hydroxypicolinic acid (HPA), anthranilic acid, nicotinic acid, <NUM>-aminoquinoline, <NUM>-hydroxy-<NUM>-methoxybenzoic acid, <NUM>,<NUM>-dimethoxybenzoic acid, <NUM>,<NUM>-phenanthroline, p-coumaric acid, <NUM>-isoquinolinol, <NUM>-picolinic acid, <NUM>-pyrenebutanoic acid, hydrazide (PBH), <NUM>-pyrenebutyric acid (PBA), and <NUM>-pyrenemethylamine hydrochloride (PMA). Among the above-listed examples, matrix that is acicular-crystalized is preferable, and for example, <NUM>,<NUM>-dihydroxybenzoic acid (DHBA) is preferable.

As the matrix that is made fly from the matrix plate including the base in the method for preparing a measurement sample for MALDI mass spectrometry, one of the above-listed various kinds of the matrices can be selected, but the matrix is preferably two or more different kinds of the matrix material.

Moreover, the two or more different kinds of matrix material that are made fly from the matrix plate including the base are preferably disposed on mutually different predetermined positions of the sample for MALDI mass spectrometry. The above-mentioned configuration is advantageous because two or more different kinds of matrix material can be separately applied in one measurement sample, and two or more kinds of imaging mass spectrometry can be performed on one measurement sample.

A region in which the matrix is to be formed is not particularly limited as long as the region is present on the laser energy absorbing material. A shape, structure, and size thereof may be appropriately selected.

The matrix may coat the laser energy absorbing material entirely or partially.

When the matrix coats the laser energy absorbing material, a region in which the matrix is present may be referred to as a matrix layer.

The average thickness of the matrix is not particularly limited and may be appropriately selected depending on the intended purpose. For example, it is preferably <NUM> or more but <NUM> or less, more preferably <NUM> or more but <NUM> or less. When the average thickness of the matrix is <NUM> or more but <NUM> or less, it is possible to accurately deposit a sufficient amount of the matrix onto a measurement sample in one flying operation.

A shape, structure, size, material, and other features of the base are not particularly limited and may be appropriately selected depending on the intended purpose.

The shape of the base is not particularly limited and may be appropriately selected depending on the intended purpose, as long as the base includes a matrix on a surface thereof and a laser beam or an optical vortex laser beam can be applied onto a back surface of the base. Examples of a flat-plate base include a glass slide.

The material of the base is not particularly limited and may be appropriately selected depending on the intended purpose, as long as the material of the base transmits a laser beam or an optical vortex laser beam. Among the materials that transmit a laser beam or an optical vortex laser beam, inorganic materials, such as various glass including silicon oxide as a main component, and organic materials, such as transparent heat resistance plastics and elastomers, are preferable in view of transmittance and heat resistance.

A surface roughness Ra of the base is not particularly limited and may be appropriately selected depending on the intended purpose. The surface roughness Ra is preferably <NUM> or less both on a front surface and a back surface of the base in order to suppress refraction scattering of a laser beam or an optical vortex laser beam, and to prevent reduction in energy to be applied to the matrix. Moreover, the surface roughness Ra in the preferable range is advantageous because unevenness in an average thickness of the matrix deposited on the sample can be suppressed, and a desired amount of the matrix can be deposited.

The surface roughness Ra can be measured according to JIS B0601. For example, the surface roughness Ra can be measured by means of a confocal laser microscope (available from KEYENCE CORPORATION) or a stylus-type surface profiler (Dektak150, available from Bruker AXS).

A production method of the matrix plate is not particularly limited and may be appropriately selected depending on the intended purpose. Examples of the production method include a method where a matrix layer crystallized by a powder forming device as described below is placed on a glass slide to produce a matrix plate.

In an exemplified production method of the matrix layer, first, a matrix solution containing a matrix mixed in a solvent is prepared.

The solvent is not particularly limited and may be appropriately selected depending on the intended purpose. Examples of the solvent include TFA, TFA-acetonitrile, THF, and methanol.

Next, the prepared matrix solution is accommodated in a raw material container <NUM> of a powder forming device <NUM> illustrated in <FIG>.

<FIG> is a schematic view illustrating an example of a powder forming device as a whole. <FIG> is a schematic view illustrating a droplet forming head in a droplet forming unit in <FIG>. <FIG> is a cross-sectional view of a droplet forming unit in <FIG>, as taken along line A-A'.

The powder forming device <NUM> illustrated in <FIG> includes mainly a droplet forming unit <NUM> and a dry collection unit <NUM>. The droplet forming unit <NUM> is a liquid chamber having a liquid jetting region in communication with the outside through discharge holes, and has a plurality of droplet discharge heads <NUM> aligned, where the droplet discharge heads <NUM> are a droplet forming unit configured to jet from the discharge holes, droplets of the matrix solution inside a liquid column resonance liquid chamber in which liquid column resonance standing waves are generated under predetermined conditions. Both sides of each droplet discharge head <NUM> are provided with gas flow passages <NUM> through each of which a gas flow generated by a gas flow generating unit passes so that droplets of the matrix solution discharged from the droplet discharge head <NUM> are flown to the side of the dry collection unit <NUM>. Moreover, the droplet forming unit <NUM> includes a raw material container <NUM> storing therein a matrix solution <NUM> that is a matrix raw material, and a liquid circulation pump <NUM> configured to supply the matrix solution <NUM> accommodated in the raw material container <NUM> to the below-mentioned common liquid supply path <NUM> inside the droplet discharge head <NUM> through a liquid supply tube <NUM>, and to pump the matrix solution <NUM> inside the liquid supply tube <NUM> to return to the raw material container <NUM> through a liquid returning tube <NUM>. Moreover, the droplet discharge head <NUM> includes the common liquid supply path <NUM> and a liquid column resonance liquid chamber <NUM>, as illustrated in <FIG>. The liquid column resonance liquid chamber <NUM> is in communication with the common liquid supply path <NUM> provided at one of the wall surfaces at both edges in a longitudinal direction. The liquid column resonance liquid chamber <NUM> includes matrix discharge holes <NUM> disposed in one of the wall surfaces connected to the wall surfaces at both edges in the longitudinal direction and configured to discharge matrix droplets <NUM>. The liquid column resonance liquid chamber <NUM> further includes a vibration generating unit <NUM> disposed at the wall surface facing the matrix discharge holes <NUM> and configured to generate high frequency vibrations for forming liquid column resonance standing waves. Note that, a high frequency power source is connected to the vibration generating unit <NUM>.

The dry collection unit <NUM> illustrated in <FIG> includes a chamber <NUM> and a matrix collecting unit (not illustrated). Inside the chamber <NUM>, a gas flow generated by the gas flow generating unit and a downward gas flow <NUM> are merged to form a large downward gas flow. Matrix droplets <NUM> jetted from the droplet discharge head <NUM> of the droplet forming unit <NUM> are transported downwards by the downward gas flow <NUM> as well as by the action of gravity, and therefore the jetted matrix droplets <NUM> are prevented from slowing down due to air resistance. With this configuration, variation in crystal diameters of the matrix droplets <NUM> is prevented, where the variation would otherwise occur when the matrix droplets <NUM> are continuously jetted because the traveling speed of previously-jetted matrix droplets <NUM> is slowed down due to air resistance and subsequently-jetted matrix droplets <NUM> catch up with the previously-jetted matrix droplet <NUM> to cause cohesion between the matrix droplets <NUM>. The gas flow generating unit may employ a method where pressurization is performed by disposing a blower at an upstream section or a method where decompression is performed by vacuuming by the matrix collecting unit. The matrix collecting unit includes a rotary gas flow generating device configured to generate a rotary gas flow rotating around an axis parallel to the vertical direction. Powder of the dried and crystalized matrix is born on a base <NUM> disposed on the bottom of the chamber <NUM>.

The matrix powder obtained in the above-described manner has less variation in crystal diameters, and therefore analysis of high reproducibility becomes possible. Since the matrix powder includes almost no solvent as the solvent is evaporated by drying, biological tissue of the measurement sample is prevented from being destroyed by the solvent of the matrix solution applied to the sample, as seen in the methods known in the art by, for example, spraying. Since almost no solvent is evaporated in performing mass spectrometry, advantageously, the matrix powder can be used to perform mass spectrometry in medical fields or clinical trials, and analysis results can be obtained onsite.

Similarly, a method for forming the matrix layer is suitably a method using, for example, an electrostatic coating device illustrated in <FIG> (MICRO MIST COATER, available from Nagase Techno-Engineering Co.

The electrostatic coating device (MICRO MIST COATER) includes a power source <NUM>, a syringe <NUM>, and a coating stage <NUM>.

The electrostatic coating device (MICRO MIST COATER) utilizes a phenomenon of electrospray to form a liquid in the syringe <NUM> into a mist <NUM>-<NUM> (Rayleigh fission). More specifically, the electrospray is a phenomenon in which a voltage at several thousands of voltages is applied to a liquid in a nozzle <NUM>-<NUM> to form the liquid into a mist. The charged liquid <NUM> is finely formed into a mist through repulsion by an electrostatic force, and moves towards and adheres onto a conductive surface <NUM> (laser energy absorbing material) on a base <NUM> on the coating stage <NUM>.

By use of such electrostatic coating, the matrix solution formed into a fine mist or a powder thereof is attracted to the base by an electrostatic force. This makes it possible to minimize scattering the matrix solution or the powder, leading to a considerable increase in the rate of use of the matrix solution.

Also, the mist lands on the base softly and involves no rebounding after landing, which makes it possible to form a more uniform matrix layer.

Other methods usable for forming the matrix layer are vacuum vapor deposition and sputtering. These methods can form a uniform matrix layer.

A shape of the matrix disposed on the surface of the base is not particularly limited and may be appropriately selected depending on the intended purpose. Examples of the shape of the matrix include a single layer, a multiple layer, and dots.

Among the above-listed examples, when the matrix absorbs the laser having the wavelength, a single layer or dots, or both are preferable. The shape of the matrix being a single layer or dots, or both is advantageous because the matrix can be easily disposed on the surface of the sample.

In the present disclosure, in order to prevent damage to the sample caused by the laser to make the matrix fly, the matrix layer is formed on the laser energy absorbing layer that absorbs the laser having such a wavelength that is not absorbed in the matrix layer.

A method for applying a laser beam to the matrix plate (laser beam irradiation unit) is not particularly limited and may be appropriately selected depending on the intended purpose. For example, the method is preferably a method where a laser beam is applied to the matrix plate by the below-described laser beam irradiation unit.

Examples of the laser beam irradiation unit include a unit configured to apply an optical vortex laser beam, a unit configured to apply a uniformly heating irradiation laser beam, and a unit configured to apply a Gaussian laser beam.

<FIG> is a schematic view illustrating an example of a laser beam irradiation unit that can be used in the method of the present disclosure for preparing a measurement sample for MALDI mass spectrometry.

In <FIG>, the laser beam irradiation unit <NUM> is configured to apply a laser beam L to a matrix <NUM> and a laser energy absorbing material born on a base <NUM>, and the matrix <NUM> and the laser energy absorbing material are made fly by the energy of the laser beam L to deposit the matrix <NUM> and the laser energy absorbing material on a sample section <NUM> on a glass slide <NUM>.

The laser beam irradiation unit <NUM> includes, for example, a laser light source <NUM>, a beam diameter changing unit <NUM>, a beam wavelength changing unit <NUM>, an energy adjusting filter <NUM>, and a beam scanning unit <NUM>. The matrix plate <NUM> includes the base <NUM>, the matrix <NUM>, and the laser energy absorbing material, and the measurement sample <NUM> includes the sample section <NUM> and the glass slide <NUM>.

The laser light source <NUM> is configured to generate and apply a pulse-oscillated laser beam L to the beam diameter changing unit <NUM>.

Examples of the laser light source <NUM> include a solid laser, a gas laser, and a semiconductor laser.

The beam diameter changing unit <NUM> is disposed downstream of the laser light source <NUM> in an optical path of the laser beam L generated by the laser light source <NUM>, and is configured to change the diameter of the laser beam L.

Examples of the beam diameter changing unit <NUM> include a condenser lens.

The beam diameter of the laser beam L is not particularly limited and may be appropriately selected depending on the intended purpose. The beam diameter is preferably <NUM> or greater but <NUM> or less. The beam diameter of the laser beam L within the preferable range is advantageous because arrangement of a matrix corresponding to a beam diameter of the existing MALDI becomes possible.

The beam wavelength changing unit <NUM> is disposed downstream of the beam diameter changing unit <NUM> in an optical path of the laser beam L, and is configured to change the wavelength of the laser beam L to a wavelength that can be absorbed by the matrix <NUM> and the laser energy absorbing material.

In the present disclosure, the wavelength of the laser beam L becomes a wavelength that is not absorbed in the matrix <NUM> and is absorbed in the laser energy absorbing layer.

The beam wavelength changing unit is not particularly limited and may be appropriately selected depending on the intended purpose, as long as, for example, in the case of using the optical vortex laser beam described below, the total torque JL,S represented by the formula (<NUM>) below can satisfy the condition |JL,S| ≥ <NUM> when circular polarization is given to the laser beam. Examples of the beam wavelength changing unit include a quarter wave plate. In case of the quarter wave plate, oval circular polarization (elliptic polarization) may be given to the below-described optical vortex laser beam by setting an optical axis to an angle other than +<NUM>° or -<NUM>°, but preferably, circular polarization of a true circle is given to an optical vortex laser beam by setting the optical axis to +<NUM>° or -<NUM>° to satisfy the condition described above. As a result, the laser beam irradiation unit <NUM> can increase the effect of stably making the light-absorbing material fly to deposit the light-absorbing material on the deposition target with suppressed scattering.

In the formula (<NUM>), ε<NUM> is a dielectric constant in vacuum, ω is an angular frequency of light, L is a topological charge, I is an orbital angular momentum corresponding to the degree of vortex of a laser beam represented by the following mathematical formula (<NUM>), S is a spin angular momentum corresponding to circular polarization, and r is a radius vector of the cylindrical coordinates system.

In the formula (<NUM>), ω<NUM> is a beam waist size of light.

The topological charge is a quantum number appearing from the periodic boundary condition of the orientation direction in the cylindrical coordinates system of the laser beam. The beam waist size is the minimum value of the beam diameter of the laser beam.

L is a parameter determined by the number of turns of the spiral wavefront in the wave plate. S is a parameter determined by the direction of circular polarization in the wave plate. L and S are both integers. The symbols L and S represent directions of spiral; i.e., clockwise and anticlockwise, respectively.

When the total torque of the optical vortex laser beam is J, the relationship J = L + S is established.

Examples of the beam wavelength changing unit <NUM> include KTP crystals, BBO crystals, LBO crystals, and CLBO crystals.

The energy adjusting filter <NUM> is disposed downstream of the beam wavelength changing unit <NUM> in an optical path of the laser beam L, and is configured to transmit and convert the laser beam L to appropriate energy for making the matrix <NUM> fly. Examples of the energy adjusting filter <NUM> include an ND filter, and a glass plate.

The beam scanning unit <NUM> is disposed downstream of the energy adjusting filter <NUM> in an optical path of the laser beam L, and includes a reflector <NUM>.

The reflector <NUM> is movable in a scanning direction presented with an arrow S in <FIG> by a reflector driving unit, and is configured to reflect the laser beam L to an arbitrary position of the matrix <NUM> and the laser energy absorbing material born on the base <NUM>.

The matrix <NUM> and the laser energy absorbing material are irradiated with the laser beam L having passed through the energy adjusting filter <NUM>, and receives energy in the range of the diameter of the laser beam L to fly onto the sample section <NUM>.

The laser beam L is not particularly limited and may be appropriately selected depending on the intended purpose. Examples of the laser beam L include an optical vortex laser beam, a uniformly heating irradiation laser beam, and a Gaussian laser beam. Among the above-listed examples, an optical vortex laser beam is preferable because the optical vortex laser beam has such properties that can enhance robustness of conditions for transferring a sample without scattering a matrix. The laser beam L being an optical vortex laser beam is advantageous because the flying matrix <NUM> and laser energy absorbing layer are deposited on the sample section <NUM> while preventing the matrix <NUM> and laser energy absorbing layer from being scattered to the periphery by virtue of the Gyroscopic effect given by the optical vortex laser beam.

The optical vortex laser beam can be obtained by converting the Gaussian laser beam. The conversion to the optical vortex laser beam can be performed using, for example, a diffractive optical element, a multi-mode fiber, or a liquid crystal phase modulator.

The optical vortex laser beam will be described.

Since a typical laser beam has uniform phases, the laser beam has a planar equiphase surface (wavefront) as depicted in <FIG>. The direction of the pointing vector of the laser beam is the orthogonal direction of the planar equiphase surface. Accordingly, the direction of the pointing vector of the laser beam is identical to the irradiation direction of the laser beam. When the light-absorbing material is irradiated with the laser beam, therefore, a force acts on the light-absorbing material in the irradiation direction. However, the light intensity distribution in the cross-section of the layer beam is a normal distribution (Gaussian distribution) where light intensity is the maximum at the center of the beam as depicted in <FIG>. Therefore, the light-absorbing material tends to be scattered. Observation of the phase distribution confirms that there is no phase difference as depicted in <FIG>.

On the other hand, an optical vortex laser beam has a spiral equiphase surface as depicted in <FIG>. The direction of the pointing vector of the optical vortex laser beam is a direction orthogonal to the spiral equiphase surface. When the light-absorbing material is irradiated with the optical vortex laser beam, a force acts in the orthogonal direction. The light intensity distribution is a doughnut-shaped distribution where the center of the beam is <NUM> and recessed as depicted in <FIG>. The doughnut-shaped energy is applied as radiation pressure to the light-absorbing material irradiated with the optical vortex laser beam. As a result, the light-absorbing material irradiated with the optical vortex laser beam is made fly in the irradiation direction of the optical vortex laser beam and is then deposited on a deposition target with a less degree of scattering. Observation of the phase distribution confirms that a phase difference occurs as depicted in <FIG>.

A method for determining whether the laser beam is an optical vortex laser beam is not particularly limited and may be appropriately selected depending on the intended purpose. Examples of the method include observation of the above-described phase distribution, and measurement of interference. The measurement of interference is typically used.

The measurement of interference can be performed using a laser beam profiler (e.g., a laser beam profiler available from Ophir-Spiricon, Inc. , or a laser beam profiler available from Hamamatsu Photonics K. Examples of the results of the measurement of interference are depicted in <FIG> and <FIG>.

<FIG> is an explanatory view illustrating one example of a result of measurement of interference in an optical vortex laser beam. <FIG> is an explanatory view illustrating one example of a result of measurement of interference in a laser beam having a point of light intensity of <NUM> at the center thereof.

It can be confirmed from the measurement of interference in the optical vortex laser beam that the energy distribution is a doughnut shape as depicted in <FIG>, and the optical vortex laser beam is a laser beam having a point of light intensity of <NUM> at the center thereof, similar to <FIG>.

On the other hand, the measurement of interference in the typical laser beam having a point of light intensity of <NUM> at the center thereof gives a difference from the optical vortex laser beam. Specifically, the doughnut-shaped energy distribution of the typical laser beam is not uniform as depicted in <FIG> although it is similar to the energy distribution obtained by the measurement of interference in the optical vortex laser beam depicted in <FIG>.

The laser beam L being an optical vortex laser beam is advantageous because the flying matrix <NUM> is deposited on the sample section <NUM> while preventing the matrix <NUM> from being scattered to the periphery by virtue of the Gyroscopic effect given by the optical vortex laser beam.

The laser beam can be converted into the optical vortex laser beam using, for example, a diffractive optical element, a multi-mode fiber, or a liquid crystal phase modulator.

Next, the uniformly heating irradiation laser beam will be described below.

The uniformly heating irradiation laser beam is a laser beam that causes a uniformly heated region exhibiting an almost uniform temperature distribution equal to or higher than the melting point of a flying target material (the laser energy absorbing material and the matrix) at the interface between the base and the flying target material (the laser energy absorbing material and the matrix). By applying such a laser beam that causes a uniformly heated region exhibiting an almost uniform temperature distribution equal to or higher than the melting point of a flying target material (the laser energy absorbing material) at the interface between the base and the flying target material (in particular, the laser energy absorbing material), a binding force (intermolecular force) at the interface between the base and the flying target material decreases, so that the flying target material is made fly as powder or debris.

Here, the phenomenon that "causes a uniformly heated region exhibiting an almost uniform temperature distribution equal to or higher than the melting point of a flying target material at the interface between the base and the flying target material" in the uniformly heating irradiation laser beam will be described in detail with reference to the drawings.

The "uniformly heated region" means a region where the temperature distribution of the flying target material becomes almost uniform.

The "region where the temperature distribution of the flying target material becomes almost uniform" means a region of the flying target material disposed on the base where the temperature of the flying target material is not ununiform and becomes almost the same.

When the flying target material is a homogeneous material, it is important that the temperature (energy) distribution of the laser beam to be applied becomes almost uniform, in order to cause the "region where the temperature distribution of the flying target material becomes almost uniform". The temperature (energy) distribution of the laser beam to be applied being almost uniform will be described with reference to the drawings. In the following, a laser beam where the temperature (energy) distribution of the laser beam to be applied is almost uniform may be referred to as a "uniformly heating irradiation laser beam".

<FIG> is a view illustrating one example of a simulation image in which the temperature (energy) distribution of a commonly used Gaussian laser beam in the cross section perpendicular to the traveling direction of the laser beam is represented by contour lines. As illustrated in <FIG>, the Gaussian laser beam has a temperature (energy) distribution where the highest energy intensity is at the center (optical axis) of the laser beam and the energy intensity becomes lower towards the periphery in the cross section perpendicular to the traveling direction of the laser beam. <FIG> is a view illustrating one example of energy intensity distributions of the Gaussian laser beam (a dotted line) and the uniformly heating irradiation laser beam (a solid line) in the cross section perpendicular to the traveling direction of the laser beam. Like in <FIG>, it is found from <FIG> that for the Gaussian laser beam (a dotted line), the energy intensity is the maximum value at the center (optical axis) of the laser beam and the energy intensity becomes lower towards the periphery. Incidentally, the "energy intensity distribution of the laser beam in the cross section perpendicular to the traveling direction of the laser beam" may be referred to simply as a "cross-sectional intensity distribution of the laser beam".

<FIG> is a view illustrating one example of an image representing the temperature (energy) distribution of the uniformly heating irradiation laser beam. As illustrated in <FIG>, in the uniformly heating irradiation laser beam, a region with energy (a black region in the figure) and a region without energy (a gray region in the figure) are clearly divided. Also, as illustrated in <FIG>, it is found that the uniformly heating irradiation laser beam (a solid line) has an energy intensity distribution where the energy intensity of the laser beam is almost the same, differing from the Gaussian laser beam where the maximum value of energy is at the optical axis. Incidentally, a laser beam having a cross-sectional intensity distribution where the energy intensity of the laser beam is almost the same as described above may be referred to as a top-hat laser beam.

Hitherto, it is known that a top-hat laser beam is used for laser patterning of a thin film, but application of the top-hat laser beam in the LIFT method is not known (see, for example, <CIT>).

It is ideal that in the uniformly heating irradiation laser beam, the energy intensity of the laser beam is the same. Specifically, an ideal laser beam is a laser beam whose energy is almost uniform (almost constant) in the cross section perpendicular to the traveling direction of the laser beam.

Here, <FIG> is a view illustrating one example of the cross-sectional intensity distribution of the uniformly heating irradiation laser beam. <FIG> is a view illustrating another example of the cross-sectional intensity distribution of the uniformly heating irradiation laser beam. As illustrated in <FIG>, for example, an ideal uniformly heating irradiation laser beam appear to have the same energy intensity of the laser beam in the cross section perpendicular to the traveling direction of the laser beam. In reality, however, the energy intensity of the laser beam will not completely be constant as illustrated in <FIG>. Rather, as illustrated in <FIG>, the values of the energy intensity of the laser beam fluctuate, presenting an energy distribution that appears to be undulating. There are three or more points where the energy intensity of the uniformly heating irradiation laser beam is the same in the cross section perpendicular to the traveling direction of the laser beam. For example, in the cross-sectional intensity distribution of the uniformly heating irradiation laser beam illustrated in <FIG>, there are six points where the energy intensity of the laser beam is the same. Meanwhile, in the cross-sectional intensity distribution of an ideal Gaussian laser beam illustrated in <FIG>, the distribution of its energy intensity is a Gaussian distribution, and there are at most only two points where the energy intensity of the laser beam is the same.

Therefore, a laser beam having three or more points where the energy intensity of the laser beam in the cross-sectional intensity distribution of the laser beam is the same can be said otherwise as a laser beam whose energy distribution is almost uniform. In the present disclosure, the uniformly heating irradiation laser beam that forms the "uniformly heated region" mean a laser beam having three or more points where the energy intensity of the laser beam is the same in the cross-sectional intensity distribution of the laser beam.

Whether a laser beam is the uniformly heating irradiation laser beam can be judged by measuring the energy distribution of a laser beam to be applied using a beam profiler, and determining whether there are three points or more where the energy intensity of the laser beam is the same in the cross-sectional intensity distribution of the laser beam.

Next, advantages obtained by performing the LIFT method using the uniformly heating irradiation laser beam will be described with reference to the drawings.

<FIG> are schematic views each illustrating one example of the LIFT method using the existing Gaussian laser beam. <FIG> are schematic views each illustrating one example of the LIFT method using the uniformly heating irradiation laser beam in the present disclosure. In <FIG>, a transparent base <NUM> is used as the base and a solid film <NUM> is used as the flying target material.

<FIG> is a schematic view illustrating one example of the case of applying a Gaussian laser beam <NUM> to the base <NUM> from the side of a surface of the base opposite to the surface thereof provided with the flying target material <NUM>, which is provided on an at least part of the surface of the base <NUM>. As illustrated in <FIG>, when the Gaussian laser beam <NUM> is applied from the side of the surface of the base opposite to the surface thereof provided with the flying target material <NUM>, the Gaussian laser beam <NUM> is applied to the flying target material <NUM> through the base <NUM>. When the Gaussian laser beam <NUM> is applied to the flying target material <NUM>, the flying target material <NUM> is heated to a temperature equal to or higher than the melting point thereof by the energy of the laser beam to decrease the binding force (intermolecular force) at the interface between the base <NUM> and the flying target material <NUM>.

The cross-sectional intensity distribution <NUM> of the Gaussian laser beam <NUM> is that the maximum value is at the center of the Gaussian laser beam <NUM> and the intensity gradually becomes lower towards the periphery. As illustrated in <FIG>, therefore, a force tends to be easily generated in the flying target material <NUM> in a direction from the center of the Gaussian laser beam <NUM> towards the outside. As a result, as illustrated in <FIG>, the flying target material <NUM> is scattered during flying to attach onto a target <NUM> sparsely.

<FIG> is a schematic view illustrating one example of the case of applying a uniformly heating irradiation laser beam <NUM> to the base <NUM> from the side of a surface of the base opposite to the surface thereof provided with the flying target material <NUM>, which is provided on an at least part of the surface of the base <NUM>.

Also in the case of the uniformly heating irradiation laser beam like in the case of the Gaussian laser beam, the laser beam is applied to the flying target material <NUM> through the base <NUM>, the flying target material <NUM> is heated to a temperature equal to or higher than the melting point thereof by the energy of the laser beam to decrease the binding force at the interface between the base <NUM> and the flying target material <NUM>. In the present disclosure, however, the laser beam is applied so as to form a uniformly heated region in the flying target material <NUM>. Specifically, as described above, the uniformly heating irradiation laser beam <NUM> whose cross-sectional intensity distribution <NUM> is almost uniform is applied to the flying target material <NUM>. As illustrated in <FIG>, a force arises in the flying target material <NUM> in the same direction as the direction in which the uniformly heating irradiation laser beam <NUM> is applied. As a result, as illustrated in <FIG>, the flying target material <NUM> flies in the same direction as the direction in which the laser beam is applied, so that the flying target material <NUM> can attach onto the target <NUM> without scattering.

Some of the indicators representing the size (width) of a laser beam are "full width at half maximum (FWHM)" and "<NUM>/e<NUM> width".

The "full width at half maximum (FWHM)" means the width of a spectrum of a laser beam at half the maximum intensity of the laser beam (e.g., in <FIG>, the width of the spectrum at an intensity of A).

The "<NUM>/e<NUM> width" means an indicator of regarding as a laser beam size (diameter) the distance between two points of the intensity values corresponding to <NUM>% of the maximum intensity in the cross-sectional intensity distribution of the laser beam (e.g., in <FIG>, the width of the spectrum at an intensity of B).

When a ratio of the "full width at half maximum (FWHM)" to the "<NUM>/e<NUM> width" is ho (FWHM/(<NUM>/e<NUM> width)), the ho is "<NUM>" in an ideal Gaussian laser beam and the ho is "<NUM>" in an ideal top-hat beam.

In the case of the Gaussian laser beam, as the energy intensity of the laser beam is higher, an irradiated area at that intensity becomes smaller. Also, the intensity of the Gaussian laser beam becomes higher at a position closer to the center of the laser beam. In other words, the Gaussian laser beam is ununiform in energy intensity in an irradiated region.

Meanwhile, in the uniformly heating irradiation laser beam; i.e., the top-hat beam having the maximum intensity, the ho (FWHM/(<NUM>/e<NUM> width)) of the "full width at half maximum (FWHM)" to the "<NUM>/e<NUM> width" is theoretically "<NUM>". The energy intensity of the laser beam is uniform in an irradiated region ("<NUM>/e<NUM> width").

According to the present inventors, a laser beam is preferably applied to the flying target material so that the ratio ho (FWHM/(<NUM>/e<NUM> width)) of the full width at half maximum (FWHM) to the <NUM>/e<NUM> width in the energy intensity distribution of the laser beam in the cross section perpendicular to the traveling direction of the laser beam satisfies <NUM><ho<<NUM>, more preferably <NUM><ho<<NUM>. The ho (FWHM/(<NUM>/e<NUM> width) of the uniformly heating irradiation laser beam illustrated in <FIG> above was found to be <NUM>.

In the cross section perpendicular to the traveling direction of the laser beam, a shape of the energy intensity distribution of the uniformly heating irradiation laser beam when the <NUM>/e<NUM> width is assumed to be a bottom side is not particular limited and may be appropriately selected depending on the intended purpose. Examples thereof include a square, a rectangle, a parallelogram, a circle, and an oval.

A method for generating the uniformly heating irradiation laser beam is not particular limited and may be appropriately selected depending on the intended purpose. For example, the uniformly heating irradiation laser beam is generated by a uniformly heating irradiation laser beam converting unit.

The uniformly heating irradiation laser beam converting unit is not particularly limited as long as it can causes the above-described uniformly heated region. Examples thereof include an aspherical lens, a phase mask such as a diffractive optical element (DOE), and phase shifting units such as a spatial light modulator (SLM). These may be used alone or in combination.

A method using the aspherical lens is a method geometrically converting the Gaussian laser beam to the uniformly heating irradiation laser beam.

<FIG> is a schematic view illustrating one example of adjustment of the uniformly heating irradiation laser beam by a geometric method using an aspherical lens. As illustrated in <FIG>, a Gaussian laser beam is allowed to pass through an aspherical lens <NUM> to enlarge the central part <NUM> of a laser beam having a cross-sectional intensity distribution <NUM> of the Gaussian laser beam by the effect of a concave lens. A peripheral part <NUM> of the laser beam is condensed by the effect of a convex lens. On an irradiated surface (base) <NUM>, a laser beam having a cross-sectional intensity distribution <NUM> of the uniformly heating irradiation laser beam can be provided.

A method using the phase mask such as the diffractive optical element (DOE) is a method wave-optically converting the Gaussian beam to the uniformly heating irradiation laser beam.

<FIG> is a schematic view illustrating one example of adjustment of the uniformly heating irradiation laser beam by a wave optical method using the DOE. As illustrated in <FIG>, the Gaussian beam is allowed to pass through a DOE <NUM> to give a central portion of the laser beam a phase distribution by the effect of a concave lens and give a peripheral part of the laser beam a phase distribution by the effect of a convex lens, to be able to control the wavefront to generate the uniformly heating irradiation laser beam. In <FIG>, reference numeral <NUM> refers to a condenser lens and reference numeral <NUM> refers to the base.

A method using the phase shifting unit such as the spatial light modulator (SLM) can shift the phase distribution of the laser beam (temporal spatial light modulation). A wavefront of superimposed wavefronts may be changed temporally.

Another usable example than the above is a combination of a reflection-type liquid crystal phase shifting element and a prism.

<FIG> is a schematic view illustrating one example of adjustment of the uniformly heating irradiation laser beam by the combination of a reflection-type liquid crystal phase shifting element <NUM> and a prism <NUM>.

By the laser beam converting optical system and the fθ lens, conversion to the uniformly heating irradiation laser beam is performed and the uniformly heating irradiation laser beam is applied onto the flying target material. The size of the laser beam applied onto the base (diameter, <NUM>/e<NUM> width) is preferably <NUM> or more but <NUM> or less, more preferably <NUM> or more but <NUM> or less.

When the size of the laser beam is <NUM> or more but <NUM> or less, quality maintenance by laser scanning is made possible to enable high-resolution two- or three-dimensional printing.

Regarding the energy of the uniformly heating irradiation laser beam, the fluence FB (J/cm<NUM>) of the laser beam on the surface on which the flying targeting material is disposed is preferably <NUM>% or higher, more preferably <NUM>% or higher but <NUM>% or lower, of the fluence FF (J/cm<NUM>) of the laser beam on the surface of the base onto which the laser beam is applied.

The fluence (J/cm<NUM>) usually refers to a fluence on the incident side (front-side fluence, FF) and is often discussed with the absorption coefficient of a material. According to the studies by the present inventors, however, it is found to be important for flying quality to control the fluence on the film surface opposite to the light-absorbing film irradiated with light (back-side fluence, FB).

Next, an embodiment of the flying object generating device for performing the method of the present disclosure for preparing the measurement sample for MALDI mass spectrometry will be described with reference to the drawings.

<FIG> is a schematic view illustrating one example of the flying object generating device to perform the method of the present disclosure for preparing the measurement sample for MALDI mass spectrometry.

As illustrated in <FIG>, a flying object generating device <NUM> in the present disclosure includes a laser beam <NUM> emitted from an unillustrated light source, a beam converting optical system <NUM>, and a condensing optical system <NUM>. The flying object generating device <NUM> is used together with a base <NUM>, a flying target material <NUM>, and an adherent receiving medium <NUM>. The flying object generating device <NUM> is configured such that the laser beam <NUM> emitted from the unillustrated light source passes through the beam converting optical system <NUM> and the fθ as the condensing optical system <NUM> for conversion to a desired beam profile, and is applied to the flying target material <NUM> through the base <NUM>. The flying target material <NUM> after irradiation with the laser beam <NUM> flies towards the adherent receiving medium <NUM> provided to face, with an interval (a gap) <NUM>, the flying target material <NUM> disposed on the base <NUM>, and adhere to the adherent receiving medium <NUM> (adhered flying target material <NUM>). The interval (gap) <NUM> between the flying target material <NUM> and the adherent receiving medium <NUM> is adjusted with an unillustrated gap retaining unit. The position of the adherent receiving medium <NUM> in the plane direction can be adjusted by an unillustrated position adjusting unit.

<FIG> is a schematic view illustrating another example of a flying object generating device in the present disclosure.

As illustrated in <FIG>, the figure is drawn as an axially symmetrical model for the sake of convenience. As illustrated in <FIG>, the flying object generating device includes a light source <NUM>, a beam converting optical system <NUM>, an (X-Y) Galvano scanner <NUM> as a scanning optical system, and a condenser lens <NUM> as a condensing optical system. The flying object generating device is configured that a sample stage <NUM> can be provided thereon with a transparent object (base) <NUM> on at least part of a surface of which a flying object material <NUM> and a laser energy absorbing material (assist film) <NUM> are provided. The flying object generating device also includes a gap retaining member <NUM> configured to provide a gap between the transparent object (base) <NUM> and the adherent receiving medium (acceptor substrate) <NUM>. The flying object generating device converts a Gaussian beam <NUM>, which is emitted from a light source <NUM>, to a uniformly heating irradiation laser beam <NUM> in the beam converting optical system <NUM>.

In one example of the flying object generating device illustrated in <FIG>, a flying target material flying unit including the light source <NUM>, the beam converting optical system <NUM>, the Galvano scanner <NUM>, and the condenser lens <NUM> applies a laser beam <NUM> towards the transparent object (base) <NUM> from the side of the surface side opposite to the surface on which the a flying target material <NUM> is disposed, to make the flying target material <NUM> fly in the direction in which the laser beam <NUM> is applied. In one example of the flying object generating device illustrated in <FIG>, moreover, the flying target material <NUM> (flying object) made fly adheres to the adherent receiving medium (target) <NUM>.

A base of the present disclosure for preparing a measurement sample for MALDI mass spectrometry includes a substrate, a laser energy absorbing material that can absorb energy of a laser beam having a wavelength of <NUM> or longer, and a matrix, the laser energy absorbing material being on the substrate, the matrix being on the laser energy absorbing material; and if necessary, further includes other materials.

The base of the present disclosure for preparing a measurement sample for MALDI mass spectrometry is similar to the matrix plate in the method of the present disclosure for preparing a measurement sample for MALDI mass spectrometry.

The measurement sample for MALDI mass spectrometry is not particularly limited and may be appropriately selected depending on the intended purpose, as long as the measurement sample includes an analyte of MALDI mass spectrometry and one or more kinds of matrices disposed on the analyte at predetermined positions thereof. When MALDI mass spectrometry is performed, the measurement sample for MALDI mass spectrometry is desirably placed on a conductive substrate.

In the measurement sample for MALDI mass spectrometry, matrix material can be made fly twice or more from the base to the predetermined positions of the analyte of MALDI mass spectrometry. Making matrix material fly twice or more from the base is advantageous because the amount of the matrix can be adjusted.

The analyte of MALDI mass spectrometry is not particularly limited and may be appropriately selected depending on the intended purpose, as long as the analyte can be analyzed by MALDI mass spectrometry. Examples of the analyte include frozen brain tissue, whole animal sections, seeds, and printed images.

The MALDI mass spectrometry method of the present disclosure is not particularly limited and may be appropriately selected depending on the intended purpose, as long as the MALDI mass spectrometry method performs MALDI mass spectrometry using the measurement sample of the present disclosure for MALDI mass spectrometry.

The MALDI mass spectrometry method can be performed by, for example, MALDI-TOF-MS (available from Bruker Daltonics).

A program of the present disclosure for preparing a measurement sample for MALDI mass spectrometry causes a computer to execute a process including based on position information of an analyte of MALDI mass spectrometry, applying a laser beam to a base including a matrix used for preparing the measurement sample for MALDI mass spectrometry, the matrix being disposed on a surface of the base, in a manner that the laser beam is applied to a surface of the base opposite to the surface on which the matrix is disposed, to make the matrix fly from the base to be disposed at a predetermined position of the analyte of MALDI mass spectrometry.

The program of the present disclosure for preparing a measurement sample for MALDI mass spectrometry is preferably executed to perform the method of the present disclosure for preparing a measurement sample for MALDI mass spectrometry.

Specifically, the program of the present disclosure for preparing a measurement sample for MALDI mass spectrometry can execute the method of the present disclosure for preparing a measurement sample for MALDI mass spectrometry using, for example, a computer as a hardware resource. The program of the present disclosure for preparing a measurement sample for MALDI mass spectrometry may be executed by one or more computers or servers, or both thereof.

A process by the program of the present disclosure for preparing a measurement sample for MALDI mass spectrometry is performed in the state where a plurality of matrix plates on which mutually different kinds of matrices are disposed are set in predetermined positions in advance, and an ITO-coat glass slide on which a measurement sample is placed is fixed in a predetermined position.

The process by the program of the present disclosure for preparing a measurement sample for MALDI mass spectrometry can be executed, for example, by a device for preparing a measurement sample for MALDI mass spectrometry as illustrated in <FIG> and <FIG>.

<FIG> is a block diagram illustrating an example of hardware of a device for preparing a measurement sample for MALDI mass spectrometry.

As illustrated in <FIG>, a device <NUM> for preparing a measurement sample for MALDI mass spectrometry includes a mouse <NUM>, a CPU <NUM>, a display <NUM>, a laser beam irradiation unit <NUM>, a plate replacing mechanism <NUM>, and a memory unit <NUM>. The CPU <NUM> is coupled with each unit.

The mouse <NUM> is configured to receive irradiation data corresponding information of "a kind of a matrix" to information of "a position of a measurement sample irradiated with a laser beam" from a user with the below-described input unit 110a. The mouse <NUM> is configured to receive another input for the device <NUM> for preparing a measurement sample for MALDI mass spectrometry.

The CPU <NUM> is one kind of a processer and is a processing device configured to perform various controls and calculations. The CPU <NUM> realizes various functions, as the CPU <NUM> executes, for example, firmware stored in, for example, the memory unit <NUM>. The CPU <NUM> corresponds to the below-described control unit 120a.

The display <NUM> is configured to display a screen to receive various instructions with the below-described output unit 130a.

The laser beam irradiation unit <NUM> is similar to, for example, the laser beam irradiation unit illustrated in <FIG>, and can apply a laser beam to a predetermined position of the matrix plate with the below-described output unit 130a.

The plate replacing mechanism <NUM> is a mechanism configured to replace matrix plates, on which various matrices are disposed, stored in the device with the below-described plate replacing unit 150a.

The memory unit <NUM> stores various programs for operating the device <NUM> for preparing a measurement sample for MALDI mass spectrometry.

<FIG> is a block diagram illustrating an example of a function of the device for preparing a measurement sample for MALDI mass spectrometry.

As illustrated in <FIG>, the device for preparing a measurement sample for MALDI mass spectrometry <NUM> includes an input unit 110a, a control unit 120a, an output unit 130a, an irradiation unit 140a, a plate replacing unit 150a, and a memory unit 160a. The control unit 120a is coupled with each unit.

The input unit 110a is configured to, following the instructions of the control unit 120a, receive irradiation data corresponding information of "a kind of a matrix" to information of "a position of a measurement sample irradiated with a laser beam" from a user with the mouse <NUM>.

The receipt of the irradiation data may be performed by, for example, inputting a kind of a matrix and an irradiation position on an image that captures the measurement sample placed on the ITO-coat glass slide.

The input unit 110a is configured to receive another input from a user.

The control unit 120a is configured to store irradiation data received by the input unit 110a in a memory unit 160a. The control unit 120a is configured to control operations of the entire device <NUM> for preparing a measurement sample for MALDI mass spectrometry.

The output unit 130a is configured to display a screen to receive various instructions on the display <NUM>, following the instructions of the control unit 120a.

The irradiation unit 140a is configured to operate the laser beam irradiation unit <NUM>, following the instructions of the control unit 120a, and can apply a laser beam to a matrix plate disposed by the plate replacing unit 150a.

The plate replacing unit 150a is configured to replace a matrix plate, following the instructions of the control unit 120a based on the irradiation data. The device stores therein a plurality of matrix plates on which mutually different kinds of matrix powder are disposed. The matrix plates are replaced by the plate replacing mechanism <NUM>.

The memory unit 160a is configured to, following the instructions of the control unit 120a, store, for example, irradiation data received by the input unit 110a or various programs in the memory unit <NUM>.

<FIG> is a flowchart illustrating an example of a procedure of the program of the present disclosure for preparing a measurement sample for MALDI mass spectrometry.

In Step S101, the input unit 110a receives irradiation data corresponding information of "a kind of a matrix" to information of "a position of a measurement sample irradiated with a laser beam" from a user with the mouse <NUM>, and then moves the process to S102.

In Step S102, the control unit 120a moves an irradiation position of the laser beam irradiation unit <NUM> based on the irradiation data, and then moves the process to S103.

In Step S103, the irradiation unit 140a makes a matrix disposed on the matrix plate fly to dispose the matrix on a sample section with the laser beam irradiation unit <NUM>, and then moves the process to S104.

In Step S104, the control unit 120a determines whether all of the contents of the irradiation data have been completed. When determining that all of the contents of the irradiation data have been completed, the control unit 120a ends the process. When determining that not all of the contents of the irradiation data have been completed, the control unit 120a moves the process to S105.

In Step S105, the control unit 120a determines whether it is necessary to replace the matrix plate based on the irradiation data. When determining that replacement of the matrix plate is necessary, the control unit 120a moves the process to S106. When determining that replacement of the matrix plate is unnecessary, the control unit 120a returns the process to S102.

In Step S106, the replacing unit <NUM> replaces the matrix plate and then returns the process to S102.

As described above, the program of the present disclosure for preparing a measurement sample for MALDI mass spectrometry causes a computer to execute a process including based on position information of an analyte of MALDI mass spectrometry, applying a laser beam to a base including a matrix used for preparing the measurement sample for MALDI mass spectrometry, the matrix being disposed on a surface of the base, in a manner that the laser beam is applied to a surface of the base opposite to the surface on which the matrix is disposed, to make the matrix fly from the base to be disposed at a predetermined position of the analyte of MALDI mass spectrometry.

The present disclosure will be described below by way of Examples. The present disclosure should not be construed as being limited to these Examples.

The following presents Examples and Comparative Example, in which a pulse-oscillated laser beam is applied to Matrix A and Matrix B by the laser beam irradiation unit <NUM> illustrated in <FIG> to dispose dots of these two kinds of matrices on one sample section.

Sinapic acid (SA) as Matrix A was dissolved in THF (obtained from Tokyo Chemical Industry, Co. ) as a solvent to prepare a sinapic acid THF solution having a solid content of <NUM>% by mass as Matrix Solution A.

Next, α-cyano-<NUM>-hydroxycinnamic acid (HCCA) as Matrix B was dissolved in THF (obtained from Tokyo Chemical Industry, Co. ) as a solvent to prepare an α-cyano-<NUM>-hydroxycinnamic acid THF solution having a solid content of <NUM>% by mass as Matrix Solution B.

Next, <NUM>,<NUM>-dihydroxybenzonic acid (DHB) as Matrix C was dissolved in THF (obtained from Tokyo Chemical Industry, Co. ) as a solvent to prepare a <NUM>,<NUM>-dihydroxybenzonic acid THF solution having a solid content of <NUM>% by mass as Matrix Solution C.

Gold was vacuum vapor-deposited on one surface of a glass slide (S2441, Super frost white, obtained from Matsunami Glass Ind. ) as a base so as to give an average thickness of <NUM>. On the gold-deposited surface, the prepared Matrix Solution A was formed into powder of Matrix A having an average primary particle diameter of <NUM> in the longer sides of acicular crystals to form a powder layer of Matrix A so as to have an average thickness of <NUM>, using the powder forming technology illustrated in <FIG>. In this manner, Matrix Plate A having the structure depicted in <FIG> was prepared.

In the same manner as in the preparation of Matrix Plate A except that Matrix Solution A was changed to Matrix Solution B, powder of Matrix B having an average primary particle diameter of <NUM> was formed to form a powder layer of Matrix B so as to have an average thickness of <NUM>. In this manner, Matrix Plate B was prepared.

In the same manner as in the preparation of Matrix Plate A except that Matrix Solution A was changed to Matrix Solution C, powder of Matrix C having an average primary particle diameter of <NUM> was formed to form a powder layer of Matrix C so as to have an average thickness of <NUM>. In this manner, Matrix Plate C was prepared.

On a surface of a glass slide (S2441, Super frost white, obtained from Matsunami Glass Ind. ) as a base, the prepared Matrix Solution A was formed into powder of Matrix A having an average primary particle diameter of <NUM> to form a powder layer of Matrix A so as to have an average thickness of <NUM>, using the powder forming technology illustrated in <FIG>. In this manner, Matrix Plate D having the structure depicted in <FIG> was prepared.

In the same manner as in the preparation of Matrix Plate D except that Matrix Solution A was changed to Matrix Solution B, powder of Matrix B having an average primary particle diameter of <NUM> was formed to form a powder layer of Matrix B so as to have an average thickness of <NUM>. In this manner, Matrix Plate E was prepared.

In the same manner as in the preparation of Matrix Plate D except that Matrix Solution A was changed to Matrix Solution C, powder of Matrix C having an average primary particle diameter of <NUM> was formed to form a powder layer of Matrix C so as to have an average thickness of <NUM>. In this manner, Matrix Plate F was prepared.

Gold was vacuum vapor-deposited on one surface of a glass slide (S2441, Super frost white, obtained from Matsunami Glass Ind. ) as a base so as to give an average thickness of <NUM>. On the gold-deposited base, the prepared Matrix Solution A was formed into powder of Matrix A having an average primary particle diameter of <NUM> to form a powder layer of Matrix A so as to have an average thickness of <NUM>, using the electrostatic coating technology illustrated in <FIG>. In this manner, Matrix Plate G having the structure depicted in <FIG> was prepared.

In the same manner as in the preparation of Matrix Plate G except that Matrix Solution A was changed to Matrix Solution B, powder of Matrix B having an average primary particle diameter of <NUM> was formed to form a powder layer of Matrix B so as to have an average thickness of <NUM>. In this manner, Matrix Plate H having the structure depicted in <FIG> was prepared.

First, all of the contents of one vial of Peptide Calibration Standard II as a standard sample was dissolved in <NUM>µL of a <NUM>% THF solvent, followed by stirring with vibration for several seconds.

One microliter of the resultant solution <NUM> was dropped with a micro syringe (<FIG>) into each of the circles arranged vertically from A to P and horizontally from <NUM> to <NUM> of "MTP384 Target Ground Steel BC" <NUM> depicted in <FIG>, followed by drying, to prepare a standard sample group. The same standard sample group was prepared through the same procedure, to prepare two standard sample groups (standard sample group <NUM> and standard sample group <NUM>).

As a laser beam irradiation unit, the laser beam irradiation unit <NUM> illustrated in <FIG> was used.

Specifically, as a laser beam source, a YAG laser configured to excite YAG crystals to oscillate laser was used. The laser beam source was used to generate a one-pulse laser beam having a wavelength of <NUM>,<NUM>, a beam diameter of <NUM> × <NUM>, a pulse width of <NUM> nano seconds, and a pulse frequency of <NUM>. The generated one-pulse laser beam was applied to a condenser lens (YAG laser condenser lens, obtained from SIGMAKOKI CO. ) serving as a beam diameter changing member to adjust a beam diameter of the laser beam to be applied to a matrix to <NUM> × <NUM>. The laser beam having passed through the beam diameter changing member was applied to a LBO crystal (obtained from CESTEC) serving as the beam wavelength changing element to change the wavelength from <NUM>,<NUM> to <NUM>. The laser beam was passed through an energy adjusting filter (ND filter, obtained from SIGMAKOKI CO. ) so that the laser output upon application to a matrix was adjusted to <NUM> pJ/dot.

Laser beam irradiation unit B was the same as Laser beam irradiation unit A except for the following changes. Specifically, the laser beam converted by the wavelength changing unit was passed through a vortex phase plate (Vortex phase plate, obtained from Luminex Corporation) to convert into an optical vortex laser beam. Next, the optical vortex laser beam converted by the vortex phase plate was passed through a quarter wave plate (QWP, obtained from Kogakugiken Corp. ) disposed downstream of the vortex phase plate. The optical axis of the vortex phase plate and the optical axis of the quarter wave plate were set to +<NUM>° so that the total torque J represented by Formula (<NUM>) would be <NUM>. The converted optical vortex laser beam was passed through an energy adjusting filter (ND filter, obtained from SIGMAKOKI CO. ) so that the laser output upon application to a matrix was adjusted to <NUM> pJ/dot.

Laser beam irradiation unit C was the same as Laser beam irradiation unit B except for the following changes. Specifically, the laser beam of <NUM>,<NUM> and the laser beam of <NUM> were used in a LBO crystal to make a change to a laser beam of <NUM> using a wavelength changing unit configured to perform sum-frequency generation.

Laser beam irradiation unit D used was a laser irradiation unit exemplified in <FIG> or <FIG>. A laser having a wavelength of <NUM>,<NUM> emitted from a Nd:YAG laser light source was allowed to pass through a spatial isolator, a λ/<NUM> plate, and a collimator lens. An acousto-optic modulator (AOM) was used to control the frequency of the laser light source by temporally separating it into the zero-order light and the first-order light based on ON/OFF signals from a PC and a controller. The zero-order light was cut off when passing through the mirror and the lens. Only the first-order light passes through a nonlinear optical crystal (SHG element), and a second harmonic wave (SHG) was generated by the nonlinear optical effect, to generate green light having a wavelength of <NUM>.

Next, harmonic separator HS was used to separate it into a fundamental wave and a second harmonic wave, to obtain a laser beam of green as a single color (Green light).

The obtained Green light was corrected for phase distribution and intensity distribution by aberration correction and a vertical and horizontal scaling element, and was passed through a zoom lens so as to enter the laser beam converting unit illustrated in <FIG> configured for conversion to the uniformly heating irradiation laser beam.

After that, the light passed through the mirror, ND, and other optical elements, and was reflected by a light polarizer such as a Galvano mirror, to that the laser output was <NUM> pJ/dot upon application to the matrix via a condenser lens (focal length:<NUM>).

As illustrated in <FIG>, the powder layer (<NUM>) of Matrix A formed on the surface of Matrix Plate A (<NUM>) was allowed to face the sample surface of the standard sample group <NUM> so that a Gaussian laser beam could be vertically applied from Laser beam irradiation unit A to the back surface of Matrix Plate A (the surface including no matrix). In <FIG>, although unillustrated, the laser energy absorbing material was disposed between the powder layer (<NUM>) of Matrix A and the base <NUM> in Matrix Plate A (<NUM>). The gap between the standard sample section and the powder layer of Matrix G was set to <NUM>.

Next, as illustrated in <FIG> and <FIG>, the powder of Matrix A was allowed to fly from Matrix Plate A using Laser beam irradiation unit A for the back surface of Matrix Plate A, to thereby arrange it in sample position A-<NUM> in the form of a square of <NUM> dots × <NUM> dots with the matrix dot diameter being <NUM> and the dot-to-dot distance being <NUM>. In the same manner as described above, dots of Matrix A were formed in A-<NUM> to A-<NUM> to obtain measurement sample group A for MALDI mass spectrometry.

In the same manner as in the preparation of measurement sample group A for MALDI mass spectrometry except that Matrix Plate A was changed to Matrix Plate B to form matrix dots in B-<NUM> to B-<NUM>, measurement sample group B for MALDI mass spectrometry was obtained.

In the same manner as in the preparation of measurement sample group A for MALDI mass spectrometry except that Matrix Plate A was changed to Matrix Plate C to form matrix dots in C-<NUM> to C-<NUM>, measurement sample group C for MALDI mass spectrometry was obtained.

As illustrated in <FIG>, the powder layer of Matrix A formed on the surface of Matrix Plate A was allowed to face the sample surface of the standard sample group <NUM> so that an optical vortex laser beam could be vertically applied from Laser beam irradiation unit B to the back surface (the surface with no matrix) of Matrix Plate A. The gap between the standard sample section and the powder layer of Matrix A was set to <NUM>.

Next, as illustrated in <FIG> and <FIG>, the powder of Matrix A was allowed to fly from Matrix Plate A using Laser beam irradiation unit B for the back surface (the surface with no matrix) of Matrix Plate A, to thereby arrange it in sample position D-<NUM> in the form of a square of <NUM> dots × <NUM> dots with the matrix dot diameter being <NUM> and the dot-to-dot distance being <NUM>. In the same manner as described above, dots of Matrix A were formed in D-<NUM> to D-<NUM> to obtain measurement sample group D for MALDI mass spectrometry.

In the same manner as in the preparation of measurement sample group D for MALDI mass spectrometry except that Matrix Plate A was changed to Matrix Plate B to form matrix dots in E-<NUM> to E-<NUM>, measurement sample group E for MALDI mass spectrometry was obtained.

In the same manner as in the preparation of measurement sample group D for MALDI mass spectrometry except that Matrix Plate A was changed to Matrix Plate C to form matrix dots in F-<NUM> to F-<NUM>, measurement sample group F for MALDI mass spectrometry was obtained.

In the same manner as in the preparation of measurement sample group D for MALDI mass spectrometry except that Matrix Plate A was changed to Matrix Plate G to form matrix dots in G-<NUM> to G-<NUM>, measurement sample group G for MALDI mass spectrometry was obtained.

In the same manner as in the preparation of measurement sample group D for MALDI mass spectrometry except that Matrix Plate A was changed to Matrix Plate H to form matrix dots in H-<NUM> to H-<NUM>, measurement sample group H for MALDI mass spectrometry was obtained.

As illustrated in <FIG>, the powder layer of Matrix A formed on the surface of Matrix Plate A was allowed to face the standard sample on the standard sample group <NUM> so that a uniformly heating irradiation laser beam could be vertically applied from Laser beam irradiation unit D to the back surface (the surface with no matrix) of Matrix Plate A. The gap between the standard sample section and the powder layer of Matrix A was set to <NUM>.

Next, as illustrated in <FIG> and <FIG>, the powder of Matrix A was allowed to fly from Matrix Plate A using Laser beam irradiation unit D for the back surface (the surface with no matrix) of Matrix Plate A, to thereby arrange it in sample position A-<NUM> in the form of a square of <NUM> dots × <NUM> dots with the matrix dot diameter being <NUM> and the dot-to-dot distance being <NUM>. In the same manner as described above, dots of Matrix A were formed in A-<NUM> to A-<NUM> to obtain measurement sample group Q for MALDI mass spectrometry.

In the same manner as in the preparation of measurement sample group Q for MALDI mass spectrometry except that Matrix Plate A was changed to Matrix Plate B to form matrix dots in B-<NUM> to B-<NUM>, measurement sample group R for MALDI mass spectrometry was obtained.

As illustrated in <FIG>, the powder layer of Matrix A formed on the surface of Matrix Plate D was allowed to face the sample surface of the standard sample group <NUM> so that an optical vortex laser beam could be vertically applied from Laser beam irradiation unit B to the back surface (the surface with no matrix) of Matrix Plate D. The gap between the standard sample section and the powder layer of Matrix A was set to <NUM>.

Next, as illustrated in <FIG> and <FIG>, the powder of Matrix A was attempted to fly from Matrix Plate A using Laser beam irradiation unit A for the back surface (the surface with no matrix) of Matrix Plate D to form dots in sample position K-<NUM> each having a matrix dot diameter of <NUM>. As a result, Matrix A was not allowed to fly from Matrix Plate D, and matrix dots could not be formed in sample position K-<NUM>. However, this was used as measurement sample group K for MALDI mass spectrometry.

In the same manner as described above except that Matrix Plate D was changed to Matrix Plate E and dots each having a matrix dot diameter of <NUM> were attempted to be formed in sample position L-<NUM>. As a result, Matrix B was not allowed to fly from Matrix Plate E, and matrix dots could not be formed in sample position L-<NUM>. However, this was used as measurement sample group L for MALDI mass spectrometry.

In the same manner as described above except that Matrix Plate D was changed to Matrix Plate F and dots each having a matrix dot diameter of <NUM> were attempted to be formed in sample position M-<NUM>. As a result, Matrix C was not allowed to fly from Matrix Plate F, and matrix dots could not be formed in sample position M-<NUM>. However, this was used as measurement sample group M for MALDI mass spectrometry.

As illustrated in <FIG>, the powder layer of Matrix A formed on the surface of Matrix Plate D was allowed to face the sample surface of standard sample group <NUM> so that an optical vortex laser beam could be vertically applied by Laser beam irradiation unit C to the back surface (the surface with no matrix) of Matrix Plate D. The gap between the standard sample section and the powder layer of Matrix A was set to <NUM>.

Next, as illustrated in <FIG> and <FIG>, the powder of Matrix A was allowed to fly from Matrix Plate A using Laser beam irradiation unit A for the back surface (the surface with no matrix) of Matrix Plate A, to thereby arrange it in sample position N-<NUM> in the form of a square of <NUM> dots × <NUM> dots with the matrix dot diameter being <NUM> and the dot-to-dot distance being <NUM>. In the same manner as described above, dots of Matrix A were formed in N-<NUM> to N-<NUM> to obtain measurement sample group N for MALDI mass spectrometry.

In the same manner as in the preparation of measurement sample group N for MALDI mass spectrometry except that Matrix Plate D was changed to Matrix Plate E to form matrix dots in O-<NUM> to O-<NUM>, measurement sample group O for MALDI mass spectrometry was obtained.

In the same manner as in the preparation of measurement sample group N for MALDI mass spectrometry except that Matrix Plate D was changed to Matrix Plate F to form matrix dots in P-<NUM> to P-<NUM>, measurement sample group P for MALDI mass spectrometry was obtained.

Next, "presence or absence of flying of the matrix" and "detection sensitivity of MALDI mass spectrometry" were evaluated in the following manners.

Flying of a matrix was compared between use of Laser beam irradiation unit A and use of Laser beam irradiation unit B in the present disclosure.

In order to make a matrix fly, a dye plate was used which used red organic dye acid red <NUM> as a matrix layer instead of an invisible matrix because of its colorless transparency. The red organic dye acid red <NUM> is an organic low-molecular-weight substance that is visible and has crystallinity similar to the matrix of the present disclosure. Results of comparison of flying are presented in <FIG> and <FIG>.

When disposing the matrix to a predetermined position by the Gaussian laser <NUM> of Laser beam irradiation unit A, scattered dots are easily formed with the gap that is large between the standard sample section and the matrix layer (<FIG>), which needs to make the gap small. However, scattered dots are not easily formed by use of the optical vortex laser beam <NUM> of Laser beam irradiation unit B (<FIG>), which makes it possible to broaden the gap between the standard sample section and the matrix layer. As a result, it is possible for the sample section to receive a less extent of damage (e.g., a change in molecular weight) when making the matrix fly to the sample section.

In the experiments in the present disclosure, Laser beam irradiation unit B formed accurate dots without formation of scattered dots with the gap of <NUM> between the standard sample section and the matrix layer. Meanwhile, Laser beam irradiation unit B formed scattered dots without adjusting the gap to <NUM>, and could not form accurate dots equivalent to those formed by Laser beam irradiation unit B.

The measurement sample groups A to P for MALDI mass spectrometry, on which the powder or the matrix was disposed, were subjected to MALDI mass spectrometry using MALDI-TOF-MS (AUTOFLEX III LRF200-CID, obtained from Bruker Daltonics Co. Representative examples of the results are presented in <FIG> and <FIG>. In <FIG> and <FIG>, the horizontal axis denotes mass (m/z) and the vertical axis denotes detection intensity.

The measured data were evaluated as follows.

For detection data of <NUM> points of each of the measurement sample groups for MALDI mass spectrometry, scanning was performed per one point at a laser energy intensity of <NUM>% to measure a detection peak of the detection target at the time of <NUM> shots. A value of the detection peak is greatly different depending on the kind of a matrix. However, a component having a relatively high molecular weight is prone to the impact of the laser upon flying of the matrix. Larger damage cuts more molecular chains, and the detection peak ratio tends to decrease.

Damage of the standard sample was evaluated based on the ratio (Y/X), where X denotes the detection peak intensity of somatostatin <NUM> (the peak indicated by the arrow a1 in the figure) near a relatively small molecular weight of <NUM> and Y denotes the detection peak intensity of bradykinin (<NUM>-<NUM>) (the peak indicated by the arrow a2 in the figure) near a relatively large molecular weight of <NUM> among the representative peaks of the standard sample.

In the cases of using Matrices A and B, the lowest value of the above detection intensity ratio (Y/X) in the detection data of <NUM> points was evaluated based on the following evaluation criteria.

In the case of using Matrix C, the lowest value of the above detection intensity ratio (Y/X) in the detection data of <NUM> points was evaluated based on the following evaluation criteria. Matrix C is such a kind of matrix that does not easily give the detection intensity of a component having a relatively high molecular weight.

For detection data of <NUM> points of each of the measurement sample groups for MALDI mass spectrometry, the average value Ave, the maximum value Max, and the minimum value Min of the detection intensity ratio (Y/X) were evaluated based on the following evaluation criteria. Results are presented in Table <NUM>-<NUM> and Table <NUM>-<NUM>. The variation in the detection (fluctuation) was evaluated using the value calculated from the formula {(Max-Min)/(<NUM>×Ave)}×<NUM> described in the following evaluation criteria.

Claim 1:
A method for preparing a measurement sample for MALDI mass spectrometry, comprising
applying a laser beam to a base comprising a matrix used for preparing the measurement sample for MALDI mass spectrometry, the matrix being disposed on a surface of the base, in a manner that the laser beam is applied to a surface of the base opposite to the surface comprising the matrix, to make the matrix fly from the base to be disposed at a predetermined position of an analyte of MALDI mass spectrometry,
wherein the base comprises a laser energy absorbing material, and
wherein laser energy of the laser beam has a wavelength of <NUM> or longer.