JP2021153044A - Measurement sample preparation method for maldi mass spectroscopy and measurement sample preparation device for maldi mass spectroscopy, and substrate for measurement sample preparation for maldi mass spectroscopy - Google Patents

Abstract

To provide a measurement sample preparation method for MALDI mass spectroscopy that, in performing mass spectroscopy with MALDI, can arrange two or more types of matrices in one sample and further can perform MALDI mass spectroscopy with a small variation.SOLUTION: A measurement sample preparation method for MALDI mass spectroscopy includes: irradiating, with a laser beam, a surface of a substrate having matrices arranged on its surface used for preparation of a measurement sample for MALDI mass spectroscopy, the surface of the substrate on the opposite side of the surface having the matrices, to cause the matrices to fly from the substrate to be arranged at a predetermined position of a sample to be subjected to the MALDI mass spectroscopy. The substrate has a laser energy absorbing material, and the wavelength of laser energy for the laser beam is 400 nm or more.SELECTED DRAWING: Figure 4A

Description

The present invention relates to a method for preparing a measurement sample for MALDI mass spectrometry, a device for preparing a measurement sample for MALDI mass spectrometry, and a substrate for preparing a measurement sample for MALDI mass spectrometry.

Mass analysis is an analytical method capable of ionizing a sample containing a target molecule, separating and detecting ions derived from the target molecule by mass-to-charge ratio (m / z), and obtaining information on identification of a chemical structure in the target molecule.

In mass spectrometry, sample ionization is a factor that affects the quality of analysis, and many methods have been developed in the past. For example, MALDI (Matrix Assisted Laser Deposition / Ionization), ESI (Electrospray ionization), and the like can be mentioned. These methods are used in technical fields such as biotechnology and medical treatment because they can be easily ionized even if the amount of sample is small.

In MALDI, a sample is ionized together with the matrix by irradiating a portion where a matrix, which is a substance for assisting ionization of the sample, is applied to the sample with a pulsed laser beam.
As the pulsed laser beam, a wavelength in the ultraviolet region is often used, and it is preferable to use a wavelength that matches the light absorption characteristics of the matrix. Further, it is said that the matrix is a crystalline organic small molecule and needs to be co-crystal or a mixture with a sample. Since it is considered that the uniformity of the co-crystals and the degree of mixing affect the sensitivity and accuracy of the analysis, those according to the sample have been developed.

In addition, various proposals have been made for a method of applying these matrices to a sample. For example, a sample preparation method for mass spectrometry has been proposed in which a matrix is vapor-deposited to form microcrystals, and then a matrix solution is spray-sprayed to grow matrix crystals on the microcrystals (see, for example, Patent Document 1).

The present invention provides a method for preparing a measurement sample for MALDI mass spectrometry, which can arrange two or more types of matrices on one sample when performing mass spectrometry by MALDI, and can further perform MALDI mass spectrometry with small variation. The purpose is to provide.

The method for preparing a measurement sample for MALDI mass spectrometry of the present invention as a means for solving the above-mentioned problems is that the side having the matrix in the substrate on which the matrix used for preparing the measurement sample for MALDI mass spectrometry is arranged on the surface is By irradiating the surface of the base material on the opposite side with a laser beam, the matrix is caused to fly from the base material and placed at a predetermined position of a sample to be subjected to MALDI mass spectrometry, and the base material absorbs laser energy. It has a material, and the wavelength of the laser energy of the laser beam is 400 nm or more.

According to the present invention, when performing mass spectrometry by MALDI, two or more types of matrices can be arranged on one sample, and further, a measurement sample preparation method for MALDI mass spectrometry that can perform MALDI mass spectrometry with small variation. Can be provided.

FIG. 1A is a schematic view showing an overall example of the powder forming apparatus. FIG. 1B is a schematic view showing a droplet forming head in the droplet forming unit of FIG. 1A. FIG. 1C is a cross-sectional view taken along the line AA'of the droplet forming unit of FIG. 1A. FIG. 1D is a schematic view showing an example in which a matrix is applied onto a substrate by an electrostatic coating method. FIG. 2 is a schematic view showing an example of a laser beam irradiation means that can be used in the measurement sample preparation method for MALDI mass spectrometry of the present invention. FIG. 3A is a block diagram showing an example of the hardware of the measurement sample preparation device for MALDI mass spectrometry. FIG. 3B is a block diagram showing an example of the function of the measurement sample preparation device for MALDI mass spectrometry. FIG. 3C is a flowchart showing an example of the processing procedure of the measurement sample preparation program for MALDI mass spectrometry of the present invention. FIG. 4A is a schematic view showing the layer structure of the matrix plate in the examples. FIG. 4B is a schematic view showing the layer structure of the matrix plate in the comparative example. FIG. 5A is a diagram showing an example of a base material used in Examples and Comparative Examples. FIG. 5B is a diagram showing an example of a base material on which a sample used in Examples and Comparative Examples is placed. FIG. 6A is a schematic view showing the preparation of a measurement sample for MALDI mass spectrometry in an example. FIG. 6B is a schematic view showing the preparation of a measurement sample for MALDI mass spectrometry in an example. FIG. 6C is a schematic view showing the preparation of a measurement sample for MALDI mass spectrometry in an example. FIG. 7A is a graph showing an example of the result of MALDI mass spectrometry in the example. FIG. 7B is a graph showing an example of the result of MALDI mass spectrometry in the example. FIG. 7C is a graph showing an example of the result of MALDI mass spectrometry in the example. FIG. 8A is a graph showing an example of the result of MALDI mass spectrometry in the comparative example. FIG. 8B is a graph showing an example of the result of MALDI mass spectrometry in the comparative example. FIG. 8C is a graph showing an example of the result of MALDI mass spectrometry in the comparative example. FIG. 9A is a conceptual diagram showing a method of preparing a measurement sample for MALDI mass spectrometry when a general laser beam is used. FIG. 9B is a photograph showing an example of a method for preparing a measurement sample for MALDI mass spectrometry when a general laser beam is used. FIG. 9C is a conceptual diagram showing a method of preparing a measurement sample for MALDI mass spectrometry when an optical vortex laser beam is used. FIG. 9D is a photograph showing an example of a method for preparing a measurement sample for MALDI mass spectrometry when an optical vortex laser beam is used. FIG. 10A is a schematic view showing an example of a wavefront (equal phase plane) in a general laser beam. FIG. 10B is a diagram showing an example of a light intensity distribution in a general laser beam. FIG. 10C is a diagram showing an example of the phase distribution in a general laser beam. FIG. 11A is a schematic view showing an example of a wavefront (equal phase plane) in the optical vortex laser beam. FIG. 11B is a diagram showing an example of the light intensity distribution in the optical vortex laser beam. FIG. 11C is a diagram showing an example of the phase distribution in the optical vortex laser beam. FIG. 11D is a diagram showing an example of the result of interference measurement in the optical vortex laser beam. FIG. 11E is a diagram showing an example of the result of interference measurement in a laser beam having a point having a light intensity of 0 in the center. FIG. 12A is a diagram showing an example of a simulation image in which the temperature (energy) distribution of the Gaussian laser beam is represented by contour lines. FIG. 12B is a diagram showing an example of an image showing the temperature (energy) distribution of the soaking heat irradiation laser beam. FIG. 13 is a diagram showing an example of the cross-sectional intensity distribution of the laser beam in the Gaussian laser beam (dotted line) and the soaking heat irradiation laser beam (solid line). FIG. 14A is a schematic view showing an example of the cross-sectional intensity distribution of the soaking heat irradiation laser beam. FIG. 14B is a schematic view showing another example of the cross-sectional intensity distribution of the soaking heat irradiation laser beam. FIG. 15A is a schematic view showing an example of the LIFT method using a conventional Gaussian laser beam. FIG. 15B is a schematic view showing another example of the LIFT method using a conventional Gaussian laser beam. FIG. 15C is a schematic diagram showing another example of the LIFT method using a conventional Gaussian laser beam. FIG. 15D is a schematic view showing an example of the LIFT method using the soaking heat irradiation laser beam in the present invention. FIG. 15E is a schematic view showing another example of the LIFT method using the soaking heat irradiation laser beam in the present invention. FIG. 15F is a schematic view showing another example of the LIFT method using the soaking heat irradiation laser beam in the present invention. FIG. 16A is a schematic view showing an example of adjusting a soaking laser beam by a geometric method using an aspherical lens. FIG. 16B is a schematic view showing an example of adjusting a soaking laser beam by a wave optical technique using DOE. FIG. 16C is a schematic view showing an example of adjusting a soaking laser beam by combining a reflective liquid crystal phase conversion element and a prism. FIG. 17A is a schematic view showing an example of a flying object generator that implements the measurement sample preparation method for MALDI mass spectrometry of the present invention. FIG. 17B is a schematic view showing another example of a projectile generator that implements the measurement sample preparation method for MALDI mass spectrometry of the present invention.

(Measurement sample preparation method for MALDI mass spectrometry and measurement sample preparation device for MALDI mass spectrometry)
In the method for preparing a measurement sample for MALDI mass spectrometry of the present invention, a laser is applied to the surface of the base material on the side opposite to the side having the matrix in the base material on which the matrix used for preparing the measurement sample for MALDI mass spectrometry is arranged on the surface. By irradiating the beam, the matrix is caused to fly from the base material and arranged at a predetermined position of a sample to be subjected to MALDI mass spectrometry, the base material having a laser energy absorbing material, and a laser of the laser beam. The wavelength of the energy is 400 nm or more, and if necessary, other steps are included.
The measurement sample preparation device for MALDI mass spectrometry of the present invention is a measurement sample preparation device for MALDI mass spectrometry used in the measurement sample preparation method for MALDI mass spectrometry of the present invention, and is a laser that irradiates the surface of the base material with a laser beam. It has a beam irradiating means and, if necessary, other means.

MALDI is an abbreviation for Matrix Assisted Laser Desorption / Ionization, and is a method of mass spectrometry called a matrix-assisted laser desorption / ionization method.
In mass spectrometry using this MALDI (hereinafter referred to as "MALDI mass spectrometry"), a sample is ionized together with the matrix by irradiating a portion where a matrix, which is a material for assisting ionization, is applied to the sample with a pulse laser. Let it perform mass spectrometry.
This matrix is used properly according to the component to be analyzed in the sample.

The method for preparing a measurement sample for MALDI mass spectrometry and the method for preparing a measurement sample for MALDI mass spectrometry of the present invention are described in the conventional methods such as a method of applying a matrix to a sample with a spray gun and a method of applying by vapor phase spraying or vapor deposition. It is based on the finding that only one matrix can be placed in one sample. In other words, there is an optimum matrix depending on the component to be analyzed, but the conventional method is based on the finding that even if there are a plurality of components to be analyzed, the optimum matrix cannot be applied separately in one sample.
Further, in the method of preparing a measurement sample for MALDI mass spectrometry in such a conventional technique, only one type of matrix is arranged in the entire sample, and for a sample having a plurality of measurement targets, the corresponding matrix is divided. There was a problem that it was inefficient because it required only a sample.
The method for preparing a measurement sample for MALDI mass spectrometry of the present invention often depends on the skill of the operator in the conventional method, so that the crystal diameter of the matrix tends to be non-uniform and the quantification is low. It is based on the finding that it may affect sensitivity and accuracy.

The method for preparing a measurement sample for MALDI mass analysis of the present invention is a substrate on which a laser energy absorbing material and a matrix are arranged on the surface, which is used for preparing a measurement sample for MALDI mass analysis, on the side opposite to the side on which the matrix is arranged. By irradiating the surface of the base material with a laser beam having a wavelength of 400 nm or more, the matrix is made to fly from the base material and arranged at a predetermined position of the sample to be MALDI mass-analyzed.
Thereby, in the measurement sample preparation method for MALDI mass spectrometry of the present invention, even if there is only one sample, for example, a matrix suitable for the target to be analyzed such as protein, lipid, and nucleotide is arranged at each predetermined position. Can be done. Therefore, the method for preparing a measurement sample for MALDI mass spectrometry of the present invention can perform highly sensitive imaging mass spectrometry for each target to be analyzed even if there are a plurality of targets to be analyzed for one sample.
Further, the method for preparing a measurement sample for MALDI mass spectrometry of the present invention has a laser energy absorbing material and can fly a matrix from a substrate by irradiating a laser beam having a wavelength of 400 nm or more (visible light wavelength region). Therefore, it is possible to prevent the target to be analyzed from being altered (damaged) by irradiating it with a high-energy laser beam. As a result, MALDI mass spectrometry with small variation can be performed. Here, "alteration" means that the structure, molecular weight, etc. of the substance change.
Examples of the method for confirming the deterioration include a confirmation method depending on whether or not the proportion of the low molecular weight component is increased by mass spectrometry.

<Base material with laser energy absorbing material and matrix on the surface>
The base material on which the laser energy absorbing material and the matrix are arranged on the surface is not particularly limited and may be appropriately selected depending on the intended purpose. In the following, the "base material on which the laser energy absorbing material and the matrix are arranged on the surface" will be referred to as a "matrix plate".
The matrix plate has a laser energy absorbing material, a matrix, and a base material.

<< Laser Energy Absorption Material >>
The laser energy absorbing material is not particularly limited as long as it can absorb the energy of a laser beam having a wavelength of 400 nm or more, and can be appropriately selected depending on the intended purpose. For example, the transmittance of the wavelength of the laser beam is 60%. The following are examples. The transmittance of the wavelength of the laser beam can be measured using a spectrophotometer such as an ultraviolet visible infrared spectrophotometer V-660 (manufactured by JASCO Corporation).
The shape of the laser energy absorbing material is not particularly limited and may be appropriately selected depending on the intended purpose, and a thin film having an average thickness of 3 μm or less is preferable.

As the material of the laser energy absorbing material, a material that is inert to the matrix and a material whose surface is conductive when the matrix is formed on the laser energy absorbing material is preferable. If the material is inert to the matrix, there is no risk of chemical reaction with the matrix when irradiated with a laser beam. Further, if the surface is a conductive material when forming the matrix on the laser energy absorbing material, the matrix can be electrostatically applied, so that a more uniform matrix can be formed and the matrix can be formed. The matrix application yield can be improved.

The material of the laser energy absorbing material is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include metal.
Examples of the metal include gold, platinum-palladium, and silver. Among these, gold, which absorbs a wide range of laser beam wavelengths and is inert, is preferable.

When the laser energy absorbing material is a thin metal film, the average thickness thereof is preferably 20 nm or more and 200 nm or less. When the average thickness of the metal thin film of the laser energy absorbing material is 20 nm or more and 200 nm or less, the variation in the conductivity of the surface is suppressed, and the matrix thickness varies when the matrix is applied by electrostatic coating. Can be suppressed.
Examples of the method for forming the laser energy absorbing material on the substrate include a vacuum vapor deposition method and a sputtering method.

<< Matrix >>
The matrix is not particularly limited as long as it is a material capable of suppressing photodecomposition and thermal decomposition of the sample and suppressing fragmentation (cleavage) of the sample, and can be appropriately selected depending on the intended purpose. ..
Examples of the matrix include known matrices such as 1,8-diaminonaphthalene (1,8-DAN) and 2,5-dihydroxybenzoic acid (2,5-Dihydroxybenzoic acid) ( (Hereinafter, it may be abbreviated as "DHBA"), 1,8-anthracene dicarboxylic acid dimethyl ester (1,8-Anthracenedicarcoxyacid Dimethylester), leucoquinizarin, anthralobin, 1,5- (1,5-Diaminonaphthracene) (1,5-DAN), 6-aza-2-thiothymine, 1,5-diaminoanthraquinone, 1,6- Diaminopyrene, 3,6-diaminocarbazole, 1,8-anthracene dicarboxylic acid (1,8-Anthracenedicarboxylacid), Norharmane, 1-pyrenepropylamine Hydrochloride (1-Pyrenepropylamine hydrochloride), 9-aminofluorene hydrochloride (9-Aminofluorene Hydrochloride), ferulic acid, dithranol, 2- (4-hydroxyphenylazo) benzoic acid -Hydroxyphenylazo) bentoic acid) (HABA), trans-2- [3- (4-tert-butylphenyl) -2-methyl-2-propenilidene] malonnitrile (trans-2- [3- (4-tert-Butylphenyl) ) -2-methyl-2-propenylidene] malononirile) (DCTB), trans-4-phenyl-3-buten-2-one (trans-4-Phenyl-3-buten-2-one) (TPBO), trans- 3-Indole acrylic acid (IAA), 1,10-phenanthroline (1,10-phenylanthr) olive), 5-nitro-1,10-phenanthroline (5-Nitro-1,10-phenanthroline), α-cyano-4-hydroxycinnamic acid (CHCA), synapic acid (CHCA) Sinapic acid (SA), 2,4,6-trihydroxyacetophenone (2,4,6-Trihydroxyacetophenone) (THAP), 3-Hydroxypicolic acid (HPA), Anthranic acid. , Nicoticicid, 3-aminoquinoline, 2-hydroxy-5-methoxybenzoic acid (2-Hydroxy-5-methoxybenzoic acid), 2,5-dimethoxybenzoic acid (2,5-Dimethoxybenzoic) acid), 4,7-phenanthroline (4,7-Phenanthrline), p-coumaric acid, 1-isoquinolinol, 2-picolinic acid, 1- Pyrenebutanoic acid (PBH), 1-Pyrenebutyric acid (PBA), 1-Pyrenemethylamine hydrochloride (1-Pyrenemethylamine hydrochlide, etc.) (PM). Among these, a matrix having a property of crystallizing in a needle shape is preferable, and for example, 2,5-dihydroxybenzoic acid (DHBA) is preferable.

In this method for preparing a measurement sample for MALDI mass spectrometry, one of the various matrices described above can be selected as the matrix to be flown from the matrix plate containing the base material, but two or more of them may be selected. preferable.
Further, as the two or more kinds of matrices to be flown from the matrix plate containing the base material, it is preferable to arrange them at predetermined positions different from each other in the sample to be MALDI mass spectrometry. This is advantageous in that two or more types of matrices can be applied separately to one measurement sample, and two or more types of imaging mass spectrometry can be performed on one measurement sample.

The region forming the matrix is not particularly limited as long as it exists on the laser energy absorbing material, and its shape and structural size can be appropriately selected.
The matrix may be fully coated or partially coated on the laser energy absorbing material.
When the matrix covers the laser energy absorbing material, the region where the matrix exists may be referred to as the 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 3 μm or more and 50 μm or less, and more preferably 5 μm or more and 30 μm or less. When the average thickness of the matrix is 5 μm or more and 50 μm or less, a sufficient amount of matrix can be accurately attached to the measurement sample in one flight.

<< Base material >>
The base material is not particularly limited in shape, structure, size, material, etc., and can be appropriately selected depending on the intended purpose.

The shape of the base material is not particularly limited as long as the matrix is supported on the front surface and the laser beam or the optical vortex laser beam can be irradiated from the back surface, and can be appropriately selected depending on the intended purpose. Further, as the shape of the flat plate-shaped base material, for example, slide glass and the like can be mentioned.

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

The surface roughness Ra of the base material is not particularly limited and may be appropriately selected depending on the intended purpose. However, it suppresses the refraction scattering of the laser beam and the optical vortex laser beam and does not reduce the energy applied to the matrix. Therefore, it is preferable that both the front surface and the back surface are 1 μm or less. Further, when the surface roughness Ra is within a preferable range, it is possible to suppress the variation in the average thickness of the matrix attached to the sample, and it is advantageous in that a desired amount of matrix can be attached.
The surface roughness Ra can be measured according to JIS B0601, for example, using a confocal laser scanning microscope (manufactured by KEYENCE Co., Ltd.) or a stylus type surface shape measuring device (Dektake 150, manufactured by Bruker AXS Co., Ltd.). Can be measured.

[Method of making matrix plate]
The method for producing the matrix plate is not particularly limited and may be appropriately selected depending on the intended purpose. For example, a matrix layer crystallized by the following powder forming apparatus is arranged on a slide glass to form a matrix plate. There is a method of producing.

As a method for producing the illustrated matrix layer, first, a matrix solution in which a matrix is mixed with a solvent is prepared.
The solvent is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include TFA, TFA-acetonitrile, THF and methanol.
Next, the prepared matrix solution is stored in the raw material container 13 of the powder forming apparatus 1 shown in FIGS. 1A to 1C.

FIG. 1A is a schematic view showing an overall example of the powder forming apparatus. FIG. 1B is a schematic view showing a droplet forming head in the droplet forming unit of FIG. 1A. FIG. 1C is a cross-sectional view taken along the line AA'of the droplet forming unit of FIG. 1A.
The powder forming apparatus 1 shown in FIG. 1A mainly includes a droplet forming unit 10 and a dry collecting unit 30. The droplet forming unit 10 is a liquid chamber having a liquid injection region communicating with the outside by a discharge hole, and a matrix solution in the liquid column resonance liquid chamber in which a liquid column resonance standing wave is generated under a predetermined condition. A plurality of droplet ejection heads 11 which are means for forming droplets to be ejected from the ejection holes as droplets are arranged. Airflow passages 12 through which the airflow generated by the airflow generating means passes so that the droplets of the matrix solution discharged from the droplet ejection head 11 flow out to the dry collection unit 30 side are provided on both sides of each droplet ejection head 11. Has been done. Further, the droplet forming unit 10 passes the raw material container 13 containing the matrix solution 14 which is the matrix raw material and the matrix solution 14 contained in the raw material container 13 into the droplet ejection head 11 through the liquid supply pipe 16. It includes a liquid circulation pump 15 that supplies the liquid to the liquid common supply path 17 described later and further pumps the matrix solution 14 in the liquid supply pipe 16 to return to the raw material container 13 through the liquid return pipe 22. Further, as shown in FIG. 1B, the droplet ejection head 11 includes a liquid common supply path 17 and a liquid column resonance liquid chamber 18. The liquid column resonance liquid chamber 18 communicates with a liquid common supply path 17 provided on one of the wall surfaces at both ends in the longitudinal direction. Further, the liquid column resonance liquid chamber 18 is provided in the matrix discharge hole 19 for discharging the matrix droplet 21 to one of the wall surfaces connected to the wall surfaces at both ends and the wall surface facing the matrix discharge hole 19 and liquid. It has a vibration generating means 20 that generates high-frequency vibration in order to form a column resonance standing wave. A high frequency power supply is connected to the vibration generating means 20.

Further, the dry collection unit 30 shown in FIG. 1A includes a chamber 31 and a matrix collection unit 32. In the chamber 31, a large downdraft is formed in which the airflow generated by the airflow generating means and the downdraft 33 merge. Since the matrix droplet 21 ejected from the droplet ejection head 11 of the droplet forming unit 10 is conveyed downward not only by gravity but also by the downdraft 33, the ejected matrix droplet 21 is air. It is possible to suppress deceleration due to resistance. As a result, when the matrix droplets 21 are continuously ejected, the previously ejected matrix droplets 21 are decelerated by air resistance, and the later ejected matrix droplets 21 are the previously ejected matrix droplets 21. By catching up with, it is possible to prevent the matrix droplets 21 from coalescing with each other and causing the crystal diameter of the matrix droplets 21 to vary. As the airflow generating means, either a method of providing a blower in the upstream portion to pressurize the airflow or a method of sucking the airflow from the matrix collecting unit 32 to reduce the pressure can be adopted. Further, the matrix collecting unit 32 is provided with a rotary airflow generator that generates a rotary airflow that rotates around an axis parallel to the vertical direction. Then, the dried and crystallized matrix powder is supported on the base material 201 arranged below the chamber 31.

Since the matrix powder thus obtained has little variation in crystal diameter, highly reproducible analysis is possible. In addition, since the solvent of this matrix powder is volatilized by drying, it contains almost no solvent. Therefore, the matrix solution is applied to the sample and the solvent is applied as in the conventional method such as spray spraying. Is unlikely to destroy the biological tissue of the measurement sample. Furthermore, since the solvent is hardly volatilized by mass spectrometry, it is possible to perform it in medical practice or clinical trials by using the powder of this matrix, which is advantageous in that the analysis result can be obtained on the spot. be.

Similarly, as a method for forming the matrix layer, an electrostatic coating device (micro mist coater, manufactured by Nagase Techno Engineering) shown in FIG. 1D or the like can be preferably used.
The electrostatic coating device (micro mist coater) has a power supply 41, a syringe 42, and a coating stage 44.
The electrostatic coating device (micro mist coater) uses a phenomenon called electrospray to mist the liquid in the syringe 42 (Rayleigh split). More specifically, electrospray is a phenomenon in which a voltage of several thousand volts is applied to the liquid in the nozzle 42-1 to form a mist. The charged liquid 43 is finely misted by the repulsion of the electrostatic force, and advances toward the conductive surface 46 (laser energy absorbing material) on the base material 45 on the coating stage 44 and adheres.
By using such electrostatic coating, the matrix solution or powder finely misted by electrostatic force is attracted to the substrate, so that the scattering of the matrix solution or powder can be minimized, and the matrix can be minimized. The solution usage rate can be greatly improved.
Further, since the mist lands softly on the base material, there is no rebound after landing, and a more uniform matrix layer can be formed.
Further, as a method for forming the matrix layer, a vacuum vapor deposition method or a sputtering method can also be used. By using these methods, a uniform matrix layer can be formed.

The shape of the matrix arranged on the surface of the base material is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a single layer, a plurality of layers, and a dot shape.
Among these, when the matrix absorbs the laser wavelength, it is preferably at least one of a single layer and a dot shape. It is advantageous that the shape of the matrix is at least one of a single layer and a dot shape, in that the matrix can be easily arranged on the surface of the sample.
In the present invention, in order to prevent the laser that flies the matrix from damaging the sample, the matrix layer is formed on the laser energy absorbing layer that absorbs the laser having a wavelength that is not absorbed by the matrix layer.

[Method of irradiating the matrix plate with a laser beam (laser beam irradiation means)]
The method of irradiating the matrix plate with a laser beam (laser beam irradiating means) is not particularly limited and may be appropriately selected depending on the intended purpose. For example, a method performed by the following laser beam irradiating means is preferable. ..

-Laser beam irradiation means-
Examples of the laser beam irradiating means include a means for irradiating an optical vortex laser beam, a soaking laser beam, and a Gauss laser beam.

Here, FIG. 2 is a schematic view showing an example of a laser beam irradiation means that can be used in the measurement sample preparation method for MALDI mass spectrometry of the present invention.
In FIG. 2, the laser beam irradiating means 140 irradiates the matrix 202 and the laser energy absorbing material supported on the base material 201 with the laser beam L, and the matrix 202 and the laser energy absorbing material fly by the energy of the laser beam L. And adhere to the sample section 301 on the slide glass 302.

The laser beam irradiating means 140 includes, for example, a laser light source 141, a beam diameter changing means 142, a beam wavelength changing means 143, an energy adjusting filter 144, and a beam scanning means 145. The matrix plate 200 is composed of a base material 201, a matrix 202, and a laser energy absorbing material, and the measurement sample 300 is composed of a sample section 301 and a slide glass 302.

The laser light source 141 generates a pulse-oscillated laser beam L and irradiates the beam diameter changing means 142.
Examples of the laser light source 141 include a solid-state laser, a gas laser, and a semiconductor laser.

The beam diameter changing means 142 is arranged downstream of the laser light source 141 in the optical path of the laser beam L generated by the laser light source 141, and changes the diameter of the laser beam L.
The beam diameter changing means 142 is, for example, a condenser lens or the like.
The beam diameter of the laser beam L is not particularly limited and may be appropriately selected depending on the intended purpose, but is preferably 20 μm or more and 300 μm or less. When the beam diameter of the laser beam L is within a preferable range, it is advantageous in that a matrix corresponding to the existing MALDI beam diameter can be arranged.

The beam wavelength changing means 143 is arranged downstream of the beam diameter changing means 142 in the optical path of the laser beam L, and changes the wavelength of the laser beam L to a wavelength that can be absorbed by the matrix 202 and the laser energy absorbing material.
In the present invention, the wavelength of the laser beam L is not absorbed by the matrix 202 but is absorbed by the laser energy absorption layer.

As the beam wavelength changing means, for example, when the optical vortex laser beam described below is used, the total rotational moment J _{L represented by the following equation (1) is applied by applying circularly polarized light to the laser beam. , S} is not particularly limited as long as it can satisfy the condition that | J _{L, S} | ≧ 0, and can be appropriately selected according to the purpose. Examples of the beam wavelength changing means include a 1/4 wave plate and the like. In the case of a 1/4 wave plate, the optical axis may be installed at a position other than + 45 ° or −45 ° to impart elliptical circularly polarized light (elliptical polarized light) to the optical vortex laser beam described below. It is preferable to set the axis at + 45 ° or −45 ° to impart circularly polarized light to the laser beam and satisfy the above conditions. As a result, the image forming apparatus can increase the effect of stably flying the light absorber and adhering it to the object to be adhered in a shape in which scattering is suppressed.

ただし、式（１）において、ε_０は真空中の誘電率であり、ωは光の角周波数であり、Ｌはトポロジカルチャージであり、Ｉは下記数式（２）で表されるレーザビームの渦次数に対応する軌道角運動量であり、Ｓは円偏光に対するスピン角運動量であり、ｒは円筒座標系の動径である。

ただし、式（２）において、ω_０は光のビームウエストサイズである。
なお、トポロジカルチャージとは、レーザビームの円筒座標系における方位方向の周期的境界条件から現れる量子数を意味する。また、ビームウエストサイズとは、レーザビームにおけるビーム径の最小値を意味する。

However, in equation (1), ε ₀ is the permittivity in vacuum, ω is the angular frequency of light, L is the topology culture, and I is the vortex of the laser beam represented by the following equation (2). It is the orbital angular momentum corresponding to the order, S is the spin angular momentum with respect to circularly polarized light, and r is the radius of the cylindrical coordinate system.

However, in equation (2), ω ₀ is the beam waist size of light.
The topological culture means the quantum number that appears from the periodic boundary condition in the directional direction in the cylindrical coordinate system of the laser beam. The beam waist size means the minimum value of the beam diameter in the laser beam.

L is a parameter determined by the number of turns of the spiral wave front in the wave plate. S is a parameter determined by the direction of circularly polarized light on the wave plate. Both L and S are integers. The symbols L and S represent the directions of the spiral (clockwise and counterclockwise), respectively.
If the total rotational moment in the laser beam is J, it can be expressed as J = L + S.
Examples of the beam wavelength changing means 143 include a KTP crystal, a BBO crystal, an LBO crystal, and a CLBO crystal.

The energy adjustment filter 144 is arranged downstream of the beam wavelength changing means 143 in the optical path of the laser beam L, and when the laser beam L is transmitted, the energy adjustment filter 144 is changed to an appropriate energy for flying the matrix 202. Examples of the energy adjusting filter 144 include an ND filter and a glass plate.

The beam scanning means 145 is arranged downstream of the energy adjusting filter 144 in the optical path of the laser beam L and includes a reflecting mirror 146.
The reflecting mirror 146 is moved by the reflecting mirror driving means in the scanning direction indicated by the arrow S in FIG. 2, and reflects the laser beam L at an arbitrary position of the matrix 202 supported by the base material 201 and the laser energy absorbing material.

The matrix 202 and the laser energy absorbing material are irradiated with the laser beam L that has passed through the energy adjusting filter 144, receive energy within the diameter range of the laser beam L, fly, and adhere to the sample section 301.

The laser beam L is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include an optical vortex laser beam, a soaking laser beam, and a Gauss laser beam. Among these, the optical vortex laser beam is preferable because it can improve the robustness of the condition of transferring to the sample without scattering the matrix due to the characteristics of the laser. When the laser beam L is an optical vortex laser beam, the flying matrix 202 and the laser energy absorption layer adhere to the sample section 301 while being suppressed from scattering to the periphery by the gyro effect imparted by the optical vortex laser beam. It is advantageous.
The optical vortex laser beam can be obtained by converting a Gaussian laser beam. The conversion to an optical vortex laser beam can be performed by using, for example, a diffractive optical element, a multimode fiber, a liquid crystal phase modulator, or the like.

--Optical vortex laser beam ---
Here, the optical vortex laser beam will be described.
Since a general laser beam has the same phase, it has a planar equiphase plane (wavefront) as shown in FIG. 10A. Since the direction of the pointing vector of the laser beam is orthogonal to the planar equiphase plane, the direction is the same as the irradiation direction of the laser beam. Therefore, when the laser beam is irradiated to the light absorber, the light is absorbed. A force acts on the material in the irradiation direction. However, since the light intensity distribution in the cross section of the laser beam has the strongest normal distribution (Gaussian distribution) at the center of the beam as shown in FIG. 10B, the light absorber tends to scatter. Further, when the phase distribution is observed, it is confirmed that there is no phase difference as shown in FIG. 10C.
On the other hand, the optical vortex laser beam has a spiral equiphase plane as shown in FIG. 11A. Since the direction of the pointing vector of the optical vortex laser beam is orthogonal to the spiral equiphase plane, when the optical vortex laser beam is applied to the light absorber, a force acts in the orthogonal direction. Therefore, as shown in FIG. 11B, the light intensity distribution becomes a concave donut-shaped distribution in which the center of the beam becomes zero, and the light absorber irradiated with the optical vortex laser beam applies donut-shaped energy as radiation pressure. Will be done. Then, the light absorber irradiated with the optical vortex laser beam flies along the irradiation direction of the optical vortex laser beam and adheres to the adhered object in a state of being difficult to scatter. Further, when the phase distribution is observed, it is confirmed that the phase difference is generated as shown in FIG. 11C.

The method for determining whether or not the beam is an optical vortex laser beam is not particularly limited and can be appropriately selected according to the purpose. Examples thereof include the above-mentioned observation of the phase distribution and interference measurement, and interference measurement is common. Is the target.
The interference measurement can be observed using a laser beam profiler (a laser beam profiler manufactured by Spiricon, a laser beam profiler manufactured by Hamamatsu Photonics Co., Ltd., etc.), and examples of the results of the interference measurement are shown in FIGS. 11D and 11E.
FIG. 11D is an explanatory diagram showing an example of the result of interference measurement in the optical vortex laser beam, and FIG. 11E is an explanatory diagram showing an example of the result of interference measurement in the laser beam having a point of light intensity 0 in the center. ..
When the optical vortex laser beam is subjected to interference measurement, it can be confirmed that the laser beam has a donut-shaped energy distribution and has a point with a light intensity of 0 in the center as in FIG. 9C, as shown in FIG. 11D.
On the other hand, when a general laser beam having a point of light intensity 0 in the center is subjected to interference measurement, as shown in FIG. 11E, it is similar to the interference measurement of the optical vortex laser beam shown in FIG. Since the energy distribution of is not uniform, the difference from the optical vortex laser beam can be confirmed.

When the laser beam L is an optical vortex laser beam, the flying matrix 202 is advantageous in that it adheres to the sample section 301 while being suppressed from scattering to the periphery by the gyro effect imparted by the optical vortex laser beam.
The conversion to an optical vortex laser beam can be performed by using, for example, a diffractive optical element, a multimode fiber, a liquid crystal phase modulator, or the like.

--Same heat irradiation laser beam ---
Next, the soaking laser beam will be described below.
The soaking heat irradiation laser beam shows a substantially uniform temperature distribution above the melting point of the flying target material (laser energy absorbing material and matrix) at the interface between the base material and the flying target material (laser energy absorbing material and matrix). A laser beam that creates a region.
By irradiating the base material with a laser beam so as to generate a region above the melting point of the flight target material (laser energy absorbing material) at the interface between the base material and the flight target material (particularly, the laser energy absorbing material), the base material and the flying target material (particularly, the laser energy absorbing material) are formed. The bonding force (intermolecular force) at the interface with the flight target material decreases, and the flight target material flies as powder or small debris.

Here, with reference to the drawing, regarding "a soaking region showing a substantially uniform temperature distribution above the melting point of the flying target material is generated at the interface between the base material and the flying target material" in the soaking heat irradiation laser beam. This will be described in detail.
The “heat equalizing region” means a region in which the temperature distribution of the material to be flown becomes substantially uniform.
The "region where the temperature distribution of the flight target material becomes substantially uniform" means that the temperature of the flight target material is not uneven and becomes substantially the same within the region where the flight target material is arranged on the base material. do.
When the material to be flown is a homogeneous material, it is important that the temperature (energy) distribution of the irradiated laser beam is substantially uniform in order to create a "region where the temperature distribution of the material to be flown is substantially uniform". be. The fact that the temperature (energy) distribution of the laser beam to be irradiated is substantially uniform will be described with reference to the drawings. In the following, a laser beam having a substantially uniform temperature (energy) distribution of the irradiated laser beam may be referred to as a "uniform heat irradiation laser beam".

FIG. 12A is a diagram showing an example of a simulation image in which the temperature (energy) distribution in a cross section orthogonal to the traveling direction of the laser beam of a Gaussian laser beam, which is a commonly used laser beam, is represented by contour lines. .. As shown in FIG. 12A, in the Gaussian laser beam, the temperature at which the energy intensity at the center (optical axis) of the laser beam is maximum and the energy intensity decreases toward the edge in a cross section orthogonal to the traveling direction of the laser beam. It has an (energy) distribution. FIG. 13 is a diagram showing an example of energy intensity distribution of a Gaussian laser beam (dotted line) and a soaking laser beam (solid line) in a cross section orthogonal to the traveling direction of the laser beam. As shown in FIG. 13, similarly to FIG. 12A, in the Gaussian laser beam (dotted line), the energy intensity shows the maximum value at the center (optical axis) of the laser beam, and the energy intensity decreases toward the edge. I understand. The "energy intensity distribution of the laser beam in the cross section orthogonal to the traveling direction of the laser beam" may be simply referred to as the "cross-section intensity distribution of the laser beam".
FIG. 12B is a diagram showing an example of an image showing the temperature (energy) distribution of the soaking heat irradiation laser beam. As shown in FIG. 12B, in the soaking laser beam, the region having energy (black region in the figure) and the region having no energy (gray region in the figure) are clearly separated. ing. Further, as shown in FIG. 13, the soaking laser beam (solid line) does not take the maximum energy value on the optical axis unlike the Gaussian laser beam, but the energy intensity of the laser beam is substantially the same. It can be seen that it has an energy intensity distribution. A laser beam having a cross-sectional intensity distribution such that the energy intensity of the laser beam is substantially the same is sometimes referred to as a top hat laser beam.
Conventionally, it is known that a top hat laser beam is used for thin film laser patterning, but it is not known to be applied in the LIFT method (see, for example, Japanese Patent Application Laid-Open No. 2012-143787).

In the soaking heat irradiation laser beam, it is ideal that the energy intensity of the laser beam is the same. That is, a laser beam in which the energy of the laser beam is substantially uniform (substantially constant) in a cross section orthogonal to the traveling direction of the laser beam is ideal.
Here, FIG. 14A is a schematic view showing an example of the cross-sectional intensity distribution of the soaking heat irradiation laser beam, and FIG. 14B is a schematic view showing another example of the cross-sectional intensity distribution of the soaking heat irradiation laser beam. For example, as shown in FIG. 14A, in a cross section orthogonal to the traveling direction of the laser beam, the ideal soaking irradiation laser beam appears to have the same energy intensity of the laser beam. However, in reality, the energy intensity of the laser beam is not completely constant as shown in FIG. 14A, and the value of the energy intensity of the laser beam oscillates and appears to undulate as shown in FIG. 14B. Is shown. Therefore, there are three or more points where the energy intensity of the soaking heat irradiation laser beam is the same in the cross section orthogonal to the traveling direction of the laser beam. For example, in the cross-sectional intensity distribution of the soaking heat irradiation laser beam shown in FIG. 14B, there are six points where the energy intensity of the laser beam is the same. On the other hand, in the ideal cross-sectional intensity distribution of the Gaussian laser beam shown in FIG. 13, the energy intensity distribution is a Gaussian distribution, so that the energy intensity of the laser beam is the same at only two points at the maximum. not exist.
Therefore, in the cross-sectional intensity distribution of the laser beam, those having three or more points where the energy intensity of the laser beam is the same can be rephrased as having a substantially uniform energy distribution of the laser beam. In the present invention, the soaking irradiation laser beam that produces the "smoothing region" means that the laser beam has three or more points having the same energy intensity in the cross-sectional intensity distribution of the laser beam.

The fact that the laser beam is a soaking laser beam means that the energy distribution of the irradiated laser beam is measured by a beam profiler, and whether or not the laser beam has three or more energy intensities of the same laser beam in the cross-sectional intensity distribution of the laser beam. You can judge.

Next, the advantages of carrying out the LIFT method using a soaking laser beam will be described with reference to the drawings.
15A to 15C are schematic views showing an example of a LIFT method using a conventional Gaussian laser beam, and FIGS. 15D to 15F show an example of a LIFT method using a soaking heat irradiation laser beam in the present invention. It is a schematic diagram. In FIGS. 15A to 15F, a case where a transparent base material 411 is used as a base material and a solid film 421 is used as a flight target material will be described.
FIG. 15A shows a case where the Gaussian laser beam 431 is applied to the base material 411 from the surface side facing the surface on which the flight target material 421 is arranged in the base material 411 in which the flight target material 421 is arranged on at least a part of the surface. It is a schematic diagram which shows an example. As shown in FIG. 15A, when the Gaussian laser beam 431 is irradiated from the surface side facing the surface on which the flight target material 421 is arranged, the Gaussian laser beam 431 is irradiated to the flight target material 421 via the base material 411. When the Gaussian laser beam 431 irradiates the flight target material 421, the flight target material 421 is heated to a melting point or higher by the energy of the laser beam, and the bonding force (intermolecular force) at the interface between the base material 411 and the flight target material 421. To reduce.
The cross-sectional intensity distribution 432 of the Gaussian laser beam 431 reaches a maximum value at the center of the Gaussian laser beam 431, and its intensity gradually decreases toward the edge. Therefore, as shown in FIG. 15B, a force is likely to be generated in the flight target material 421 in the direction from the center of the Gaussian laser beam 431 to the outside. As a result, as shown in FIG. 15C, the flying target material 421 is scattered and adheres to the target 441 in a scattered manner.
FIG. 15D shows that in the base material 411 in which the flight target material 421 is arranged on at least a part of the surface, the base material 411 is irradiated with the soaking laser beam 433 from the surface side facing the surface on which the flight target material 421 is arranged. It is a schematic diagram which shows an example of the case.
Even in the case of the soaking heat irradiation laser beam, the flight target material 421 is irradiated with the laser beam through the base material 411, the flight target material 421 is heated to the melting point or higher by the energy of the laser beam, and the base material 411 and the flight target material are heated. The point of reducing the coupling force at the interface with 421 is the same as in the case of the Gaussian laser beam. However, in the present invention, the flight target material 421 is irradiated with a laser beam so as to generate a soaking region. That is, as described above, since the soaking target material 421 is irradiated with the soaking irradiation laser beam 433 having a substantially uniform cross-sectional intensity distribution 434, as shown in FIG. 15E, the flying target material 421 is subjected to the soaking irradiation laser beam. A force is generated in the same direction as the irradiation direction of 433. As a result, as shown in FIG. 15F, the flight target material 421 flies in the same direction as the irradiation direction of the laser beam, and the flight target material 421 can be suppressed from scattering and adhered to the target 441.

Further, as one of the indexes indicating the size (width) of the laser beam, there are "full width at half maximum (FWHM)" and "1 / e ² width".
The "full width at half maximum (FWHM)" means the spectral width of the laser beam at half the maximum intensity of the laser beam (for example, the spectral width at the intensity of A in FIG. 13).
The “1 / e ² width” means an index in which the distance between two points having an intensity value corresponding to 13.5% of the maximum intensity in the cross-sectional intensity distribution of the laser beam is regarded as the laser beam diameter (diameter) ( For example, in FIG. 13, the spectral width at the intensity of B).
When the ratio of this "full width at half maximum (FWHM)" and "1 / e ² width" is ho (FWHM / (1 / e ² width)), ho is "" in the case of an ideal Gaussian laser beam. It becomes "0.6", and in the case of an ideal tophat beam, ho becomes "1".
In the case of a Gaussian laser beam, as the energy intensity of the laser beam increases, the irradiation area at that intensity decreases. Further, the intensity of the Gaussian laser beam increases as it approaches the center. That is, the Gaussian laser beam has a non-uniform energy intensity in the irradiation region.
On the other hand, in the soaking laser beam, the tophat beam having the maximum intensity is the ratio ho (FWHM / (1 / e ² width)) of ^{"full width at half maximum (FWHM)" and "1 / e 2 width".} There is theoretically is intended to be a "1", the energy intensity of the laser beam in the irradiation region ( "1 / e ² width") is uniform.
The present inventors, the full width at half maximum of the energy intensity distribution of the laser beam in a cross section perpendicular to the traveling direction of the laser beam (FWHM), the ratio of the 1 / ^{e 2} width ho (FWHM / (1 / ^{e 2} width)) However, it is preferable to irradiate the flight target material with a laser beam so as to satisfy 0.6 <ho <1, and more preferably 0.7 ≦ ho ≦ 0.9. Incidentally, ho of soaking the irradiated laser beam shown in FIG. ^{12B (FWHM / (1 / e} 2 width) was 0.85.

^{The shape of the energy intensity distribution of the soaking irradiation laser beam when the 1 / e 2} width is the base in the cross section orthogonal to the traveling direction of the laser beam is not particularly limited and may be appropriately selected according to the purpose. It can be made, for example, square, rectangular, parallelogram, circular, elliptical and the like.

The method for generating the soaking heat irradiation laser beam is not particularly limited and may be appropriately selected depending on the intended purpose. Examples thereof include a soaking heat irradiation laser beam conversion means.
The heat equalizing irradiation laser beam conversion means is not particularly limited as long as the above-mentioned heat equalizing region can be generated, and for example, an aspherical lens, a phase mask such as a diffractive optical element (DOE), or a liquid crystal phase conversion. Examples include a phase conversion means such as an element (SLM). These may be used alone or in combination of two or more.

The method using an aspherical lens is a method of geometrically converting a Gaussian laser beam into a soaking laser beam.
FIG. 16A is a schematic view showing an example of adjusting a soaking laser beam by a geometric method using an aspherical lens. As shown in FIG. 16A, by passing the Gaussian laser beam through the aspherical lens 511, the laser beam having the cross-sectional intensity distribution 432 of the Gaussian laser beam is magnified by the concave lens effect at the central portion 521 of the laser beam, and the laser beam. The peripheral portion 522 is converged by the convex lens effect, and the laser beam having the cross-sectional intensity distribution 434 of the soaking heat irradiation laser beam can be adjusted on the irradiation surface (base material) 512.

A method using a phase mask such as a diffractive optical element (DOE) is a method of wave-optically converting a Gaussian laser beam into a soaking laser beam.
FIG. 16B is a schematic view showing an example of adjusting a soaking laser beam by a wave optical technique using DOE. As shown in FIG. 16B, by passing the Gaussian laser beam through DOE531, the central part of the laser beam is given a phase distribution having a concave lens effect, and the peripheral part of the laser beam is given a phase distribution having a convex lens effect to control the wavefront. This makes it possible to obtain a soaking laser beam. In FIG. 16B, 541 represents a condenser lens and 551 represents a base material.

Since the method using a phase conversion means such as a liquid crystal phase conversion element (SLM) can convert the phase distribution of the laser beam (spatial light modulation in time), the wave surface on which the wave surface is superimposed is changed in time. May be good.

Further, as an example other than the above, a combination of a reflective liquid crystal phase conversion element and a prism can also be used.
FIG. 16C is a schematic view showing an example of adjusting a soaking laser beam by combining a reflective liquid crystal phase conversion element 561 and a prism 562.

The laser beam conversion optical system and the fθ lens convert the laser beam into a soaking irradiation laser beam, and irradiate the flight target material with the soaking irradiation laser beam. The size of the laser beam irradiated on the substrate (diameter, 1 / ^{e 2} width) is preferably 20μm or more 200μm or less, more preferably 30μm or more 150μm or less.
By setting the size of the laser beam to 20 μm or more and 200 μm or less, it is possible to maintain the quality by laser scanning, and it is possible to perform high-resolution two-dimensional drawing or three-dimensional printing.

The energy of soaking the irradiated laser beam, the fluence of the laser beam, the fluence F _B of the laser beam in a flying target material disposed surface _(J / cm ²⁾ is, on the surface of the substrate to a laser beam is irradiated preferably the laser beam is the fluence _{F F} (J / cm ²⁾ of 20% or more, more preferably 20% to 80%.
The fluence (J / cm ² ) usually refers to the incident side fluence (front side fluence, _FF ). Also, we often discuss the absorption coefficient of materials. However, the fluence of the opposite side of the membrane surface and the light irradiation of the light absorbing material layer (backside fluence, F _B) to control the important in flying quality has been found by the study of the present inventors.

Next, an embodiment of a projectile generator that implements the measurement sample preparation method for MALDI mass spectrometry of the present invention will be described with reference to the drawings.
FIG. 17A is a schematic view showing an example of a flying object generator that implements the measurement sample preparation method for MALDI mass spectrometry of the present invention.
As shown in FIG. 17A, the projectile generator 700 of the present invention has a laser beam 711, a beam conversion optical system 721, and a condensing optical system 731 emitted from a light source (not shown), and has a base material 741 and a flying object. Used with material 751 and adherent medium 761. In the projectile generator 700, the laser beam 711 irradiated from a light source (not shown) passes through a beam conversion optical system 721, an fθ lens as a condensing optical system 731, and the like in order to convert to a desired beam profile, and is a flight target. The material 751 is irradiated via the base material 741. The flight target material 751 irradiated with the laser beam 711 flies toward the adhered medium 761 provided so as to face the flight target material 751 arranged on the base material 741 through the gap 771. Adheres (material to be flown after attachment 752). The gap 771 between the flight target material 751 and the adhered medium 761 is adjusted by a gap holding means (not shown), and the position of the adhered medium 761 in the plane direction can be adjusted by a position adjusting means (not shown).
FIG. 17B is a schematic view showing another example of the flying object generator of the present invention.
As shown in FIG. 17B, the figure is an axisymmetric model for convenience. As shown in FIG. 17B, the projectile generator includes a light source 811, a beam conversion optical system 821, a scanning optical system (XY) galvano scanner 831 and a condensing lens 841 which is a condensing optical system. A transparent body (base material) 851 in which the flight target material 853 and the laser energy absorbing material (assist film) 852 are arranged on at least a part of the surface can be installed on the sample table 881. There is. Further, it has a Gap (gap) holding member 871 for providing a gap between the transparent body (base material) 851 and the object to be adhered (acceptor substrate) 861. The flying object generator converts the Gaussian laser beam 812 emitted from the light source 811 into a soaking laser beam 813 in the beam conversion optical system 821.
Further, in an example of the flying object generator shown in FIG. 17B, a flying target material having a light source 811, a beam conversion optical system 821, a galvano scanner 831 as a scanning optical system, and a condenser lens 841 is used to fly a target material. By irradiating the transparent body (base material) 851 with the laser beam 813 from the surface side facing the surface on which the material 853 is arranged, the material 853 to be flown is made to fly in the irradiation direction of the laser beam 813. Then, in the example of the flying object generator shown in FIG. 17B, the flight target material 853 (flying object) that has been flown adheres to the adhered object (object) 861.

(Base material for preparing measurement sample for MALDI mass spectrometry)
The base material for preparing a measurement sample for MALDI mass spectrometry of the present invention has a laser energy absorbing material capable of absorbing the energy of a laser beam having a wavelength of 400 nm or more and a matrix in this order on the substrate, and further is required. Has other materials accordingly.
The base material for preparing a measurement sample for MALDI mass spectrometry of the present invention is the same as the matrix plate in the method for preparing a measurement sample for MALDI mass spectrometry of the present invention.

(Measurement sample for MALDI mass spectrometry)
The measurement sample for MALDI mass spectrometry is not particularly limited as long as it has a sample to be subjected to MALDI mass spectrometry and one or more types of matrices arranged at predetermined positions on the sample, and is appropriately selected according to the purpose. can do. When performing MALDI mass spectrometry, the measurement sample for MALDI mass spectrometry needs to be placed on a conductive substrate.

As the measurement sample for MALDI mass spectrometry, the matrix can be arranged by flying the matrix from the base material a plurality of times with respect to a predetermined position of the sample to be subjected to MALDI mass spectrometry. It is advantageous that the amount of the matrix can be adjusted if the matrix can be flown and arranged a plurality of times from the base material.

<Sample>
The sample to be analyzed by MALDI mass spectrometry is not particularly limited as long as it can be analyzed by MALDI mass spectrometry, and can be appropriately selected depending on the intended purpose. Examples thereof include frozen brain tissue, whole body sections of animals, seeds, and printed images. ..

(MALDI mass spectrometry method)
The MALDI mass spectrometry method of the present invention is not particularly limited as long as MALDI mass spectrometry is performed using the measurement sample for MALDI mass spectrometry of the present invention, and can be appropriately selected depending on the intended purpose.
As the MALDI mass spectrometry method, for example, MALDI-TOF-MS (manufactured by Bruker Daltonics Co., Ltd.) can be used.

(Measurement sample preparation program for MALDI mass spectrometry)
In the measurement sample preparation program for MALDI mass spectrometry of the present invention, the matrix is arranged on the substrate on which the matrix used for preparing the measurement sample for MALDI mass spectrometry is arranged on the surface based on the position information of the sample to be MALDI mass spectrometry. By irradiating the surface of the base material on the side opposite to the side with a laser beam, the matrix is caused to fly from the base material and a computer is made to perform a process of arranging the sample to be subjected to MALDI mass spectrometry at a predetermined position.

The measurement sample preparation program for MALDI mass spectrometry of the present invention is suitably executed for carrying out the measurement sample preparation method for MALDI mass spectrometry of the present invention.
That is, the measurement sample preparation program for MALDI mass spectrometry of the present invention can execute the measurement sample preparation method for MALDI mass spectrometry of the present invention by using a computer or the like as a hardware resource. In addition, the measurement sample preparation program for MALDI mass spectrometry of the present invention may be executed by at least one of one or more computers or servers.

In the process according to the measurement sample preparation program for MALDI mass spectrometry of the present invention, a plurality of matrix plates on which different types of matrices are arranged are pre-installed at predetermined positions, and an ITO-coated slide glass on which the measurement sample is placed is placed in a predetermined position. It is done in a fixed position.

The processing by the measurement sample preparation program for MALDI mass spectrometry of the present invention can be carried out by, for example, the measurement sample preparation apparatus for MALDI mass spectrometry as shown in FIGS. 3A and 3B.

FIG. 3A is a block diagram showing an example of the hardware of the measurement sample preparation device for MALDI mass spectrometry.
As shown in FIG. 3A, the measurement sample preparation device 100 for MALDI mass spectrometry includes a mouse 110, a CPU 120, a display 130, a laser beam irradiation means 140, a plate exchange mechanism 150, and a storage means 160. The CPU 120 is connected to each part.

The mouse 110 receives from the user irradiation data in which the information of the "matrix type" and the "position of the measurement sample to be irradiated with the laser beam" are associated with each other by the input unit 110a described later. In addition, the mouse 110 accepts other inputs to the measurement sample preparation device 100 for MALDI mass spectrometry.

The CPU 120 is a kind of processor, and is a processing device that performs various controls and calculations. The CPU 120 realizes various functions by executing firmware or the like stored by the storage means 160 or the like. The CPU 120 corresponds to the control unit 120a described later.

The display 130 displays a screen for receiving various instructions by the output unit 130a described later.

The laser beam irradiating means 140 is, for example, the same as the laser beam irradiating means shown in FIG. 2, and the laser beam can be irradiated to a predetermined position on the matrix plate by the output unit 130a described later.

The plate exchange mechanism 150 is a mechanism for exchanging a matrix plate in which various matrices stored in the apparatus are arranged by a plate exchange unit 150a described later.

The storage means 160 stores various programs for operating the measurement sample preparation device 100 for MALDI mass spectrometry.

FIG. 3B is a block diagram showing an example of the function of the measurement sample preparation device for MALDI mass spectrometry.
As shown in FIG. 3B, the measurement sample preparation device 100 for MALDI mass spectrometry includes an input unit 110a, a control unit 120a, an output unit 130a, an irradiation unit 140a, a plate exchange unit 150a, and a storage unit 160a. Have. The control unit 120a is connected to each unit.

The input unit 110a receives irradiation data corresponding to the information of "matrix type" and "position of the measurement sample to be irradiated with the laser beam" from the user by the mouse 110 according to the instruction of the control unit 120a.
For receiving the irradiation data, for example, the type of the matrix and the irradiation position may be input on the image obtained by capturing the measurement sample placed on the ITO-coated slide glass.
The input unit 110a accepts other inputs from the user.

The control unit 120a stores the irradiation data received by the input unit 110a in the storage unit 160a. Further, the control unit 120a controls the operation of the entire measurement sample preparation device 100 for MALDI mass spectrometry.

The output unit 130a displays a screen for receiving various instructions on the display 130 in accordance with the instructions of the control unit 120a.

The irradiation unit 140a can operate the laser beam irradiation means 140 according to the instruction of the control unit 120a to irradiate the matrix plate arranged by the plate replacement unit 150a with the laser beam.

The plate exchange unit 150a exchanges the matrix plate according to the instruction of the control unit 120a based on the irradiation data. A plurality of matrix plates are stored in the apparatus, and powders of different types of matrices are arranged on the plates and exchanged by the plate exchange mechanism 150.

The storage unit 160a stores the irradiation data and various programs received by the input unit 110a in the storage means 160 in accordance with the instruction of the control unit 120a.

FIG. 3C is a flowchart showing an example of the processing procedure of the measurement sample preparation program for MALDI mass spectrometry of the present invention.

In step S101, when the input unit 110a receives the irradiation data corresponding to the information of the "matrix type" and the "position of the measurement sample to be irradiated with the laser beam" from the user by the mouse 110, the process shifts to S102. ..

In step S102, when the control unit 120a moves the irradiation position of the laser beam irradiation means 140 based on the irradiation data, the process shifts to S103.

In step S103, the irradiation unit 140a flies the matrix arranged on the matrix plate by the laser beam irradiation means 140, and when the matrix is arranged on the sample section, the process shifts to S104.

In step S104, the control unit 120a determines whether or not all the contents of the irradiation data have been completed. When the control unit 120a determines that all the contents of the irradiation data are completed, the control unit 120a ends this process, and when it determines that all the contents of the irradiation data are not completed, the control unit 120a shifts the process to S105.

In step S105, the control unit 120a determines whether or not the matrix plate needs to be replaced based on the irradiation data. When the control unit 120a determines that the matrix plate needs to be replaced, the process shifts to S106, and when it determines that the matrix plate does not need to be replaced, the control unit 120a returns the process to S102.

In step S106, when the exchange unit 120 exchanges the matrix plate, the process returns to S102.

As described above, the measurement sample preparation program for MALDI mass spectrometry used in the present invention is based on the position information of the sample to be MALDI mass spectrometry, and is a substrate on which the matrix used for preparing the measurement sample for MALDI mass spectrometry is arranged on the surface. By irradiating the surface of the base material on the side opposite to the side on which the matrix is arranged with a laser beam, the matrix is made to fly from the base material and a computer is made to perform a process of arranging the sample at a predetermined position for MALDI mass spectrometry. ..

Hereinafter, examples of the present invention will be described, but the present invention is not limited to these examples.
In the following, the laser beam irradiating means 140 shown in FIG. 2 irradiates the two types of matrices A and B with the pulse-oscillated laser beam, respectively, and arranges the dots of the two types of matrices on one sample section. The examples and comparative examples described in the above will be described.

(Example 1)
[Preparation of matrix solution]
<Preparation of matrix solution A>
Sinapinic acid (SA) as matrix A was dissolved in THF (manufactured by Tokyo Kasei Kogyo Co., Ltd.) as a solvent, and a solution of 1% by mass of solid content in THF was prepared as matrix solution A.

<Preparation of matrix solution B>
Next, α-cyano-4-hydroxycinnamic acid (HCCA) as matrix B was dissolved in THF (manufactured by Tokyo Kasei Kogyo Co., Ltd.) as a solvent, and the solid content was 1 mass. % Α-Cyano-4-hydroxycinnamic acid THF solution was prepared as matrix solution B.

<Preparation of matrix solution C>
Next, 2,5-dihydroxybenzoic acid (DHB) as matrix C was dissolved in THF (manufactured by Tokyo Kasei Kogyo Co., Ltd.) as a solvent, and 2,5-dihydroxy with a solid content of 1% by mass was dissolved. A THF solution of benzoate was prepared as a matrix solution C.

[Preparation of matrix plate]
<Creation of matrix plate A>
Gold was vacuum-deposited on one side of a slide glass (S2441, Super Frost White, manufactured by Matsunami Glass Ind.) As a base material so that the average thickness was 50 nm. The prepared matrix solution A is used to form a matrix A powder having a primary average particle diameter of 20 μm on the long side of the acicular crystal using the powder forming techniques shown in FIGS. 1A to 1C on a gold-deposited paper surface. Then, a powder layer of Matrix A was formed so that the average thickness was 5 μm, and Matrix Plate A having the configuration shown in FIG. 4A was prepared.

<Creation of matrix plate B>
Similar to the matrix plate A, the matrix B powder having a primary average particle diameter of 10 μm was formed except that the matrix solution A was changed to the matrix solution B, and the powder layer of the matrix B was formed so that the average thickness was 5 μm. It was formed to prepare a matrix plate B.

<Creation of matrix plate C>
Similar to the matrix plate A, the matrix C powder having a primary average particle diameter of 15 μm is formed except that the matrix solution A is replaced with the matrix solution C, and the powder layer of the matrix C is formed so that the average thickness is 5 μm. It was formed to prepare a matrix plate C.

<Creation of matrix plate D>
On the surface of a slide glass (S2441, Super Frost White, manufactured by Matsunami Glass Ind.) As a base material, the prepared matrix solution A is subjected to primary average particles using the powder forming techniques shown in FIGS. 1A to 1C. A matrix A powder having a diameter of 20 μm was formed, and a matrix A powder layer was formed so that the average thickness was 5 μm, to prepare a matrix plate D having the configuration shown in FIG. 4B.

<Creation of matrix plate E>
Similar to the matrix plate D, the powder of the matrix B having a primary average particle diameter of 10 μm is formed except that the matrix solution A is changed to the matrix solution B, and the powder layer of the matrix B is formed so that the average thickness is 5 μm. It was formed and a matrix plate E having the configuration shown in FIG. 4B was prepared.

<Creation of matrix plate F>
Similar to the matrix plate D, the matrix C powder having a primary average particle diameter of 15 μm was formed except that the matrix solution A was changed to the matrix solution C, and the powder layer of the matrix C was formed so that the average thickness was 5 μm. It was formed and a matrix plate F having the configuration shown in FIG. 4B was prepared.

<Creation of matrix plate G>
The matrix solution A prepared on a base material vacuum-deposited with gold so that the average thickness is 50 nm on one side surface of a slide glass (S2441, Super Frost White, manufactured by Matsunami Glass Ind. Co., Ltd.) as a base material is shown in FIG. Using the electrostatic coating technique shown in 1D, a matrix A powder having a primary average particle diameter of 10 μm was formed, and a matrix A powder layer was formed so that the average thickness was 5 μm. Matrix plate G was prepared.

<Creation of matrix plate H>
Similar to the matrix plate G, the matrix B powder having a primary average particle diameter of 10 μm was formed except that the matrix solution A was changed to the matrix solution B, and the powder layer of the matrix B was formed so that the average thickness was 5 μm. It was formed and a matrix plate H having the configuration shown in FIG. 4A was prepared.

[Preparation of standard sample group]
First, the content of one peptide calibration standard II as a standard sample is dissolved in 125 μL of 0.1% THF solvent, and the mixture is vibrated and stirred for several seconds.
This solution 53 is dropped every 1 μL with a microsyringe 52 into a circle arranged vertically A to P and horizontally 1 to 24 of the “MTP384 Group Group Steel BC” 51 shown in FIG. 5A, and dried. A standard sample group was prepared. The same standard sample group was prepared by the same operation, and two standard sample groups (standard sample group 1 and standard sample group 2) were prepared.

[Preparation of laser beam irradiation means]
<Preparation of laser beam irradiation means A>
As the laser beam irradiating means, the laser beam irradiating means 140 shown in FIG. 2 was used.
Specifically, as the laser beam source (YAG), a YAG laser that excites a YAG crystal to oscillate the laser was used. Using this laser beam source, a 1-pulse laser beam having a wavelength of 1,064 nm, a beam diameter of 1.25 mm × 1.23 mm, a pulse width of 2 nanoseconds, and a pulse frequency of 20 Hz in the generated laser beam is generated. Generated. The generated 1-pulse laser beam is applied to a condenser lens (YAG laser condenser lens manufactured by Sigma Kogyo Co., Ltd.) as a beam diameter changing member, and the beam diameter when irradiated to the matrix is 100 μm × 100 μm. I tried to be. The laser beam passed through the beam diameter changing member was irradiated to an LBO crystal (manufactured by CESTEC) used as the beam wavelength changing element to change the wavelength from 1,064 nm to 532 nm. By passing the laser beam through an energy adjustment filter (ND filter manufactured by SIGMA KOKI Co., Ltd.), the laser output when the matrix was irradiated was adjusted to 50 μJ / dot.

<Preparation of laser beam irradiation means B>
In the laser beam irradiating means A, the laser beam irradiating means B passes the laser beam changed by the wavelength changing means through a spiral phase plate (Vortex phase plate manufactured by Luminex) to convert it into an optical vortex laser beam. Next, the optical vortex laser beam converted by the spiral phase plate was passed through a 1/4 wave plate (QWP; manufactured by Kogaku Giken Co., Ltd.) arranged downstream of the spiral phase plate. At this time, the optical axes of the spiral phase plate and the 1/4 wave plate were set to + 45 ° so that the total rotational moment J represented by the equation (1) was 2. By passing the converted optical vortex laser beam through an energy adjustment filter (ND filter manufactured by Sigma Kouki Co., Ltd.), the laser output when the matrix was irradiated was adjusted to 50 μJ / dot. Other than this change, it is the same as the laser beam irradiation means A.

<Preparation of laser beam irradiation means C>
The laser beam irradiating means C is changed to a 355 nm laser beam in the laser beam irradiating means B by using a wavelength changing means for generating a sum frequency by using a laser beam of 1,064 nm and a laser beam of 532 nm in the LBO crystal. It is the same as the laser beam irradiating means B except that the laser beam irradiating means B is used.

<Preparation of laser beam irradiation means D>
As the laser beam irradiating means D, the laser irradiating means using FIGS. 17A and 17B as examples was used.
A laser emitted from an Nd: YAG laser light source unit having a wavelength of 1064 nm was passed through a spatial isolator, a λ / 4 plate, and a collimating lens. The acoustic-optical deflection element (AOM) controls the frequency of the laser light source by temporally separating the 0th-order light and the 1st-order light based on the ON / OFF signals from the PC and the controller. The 0th order light is cut when passing through the mirror and the lens, only the 1st order light passes through the nonlinear optical crystal (SHG element), and the second harmonic (SHG) is generated by the nonlinear optical effect, and the wavelength is 532 nm. Green light was generated.
Next, the fundamental wave and the second harmonic were separated by the harmonic separator HS to obtain a Green monochromatic laser beam (Green light).
The obtained Green light is incident on the laser beam conversion means shown in FIG. 16A, in which the phase distribution and the intensity distribution are corrected by an aberration correction or a longitudinal / horizontal magnification element, the light passes through a zoom lens, and is converted into a soaking laser beam. I tried to be done.
After that, it passes through a mirror, ND, and other optical elements, is reflected by a light deflector such as a galvano mirror, and irradiates the matrix through a condenser lens (focal length: 100 mm) with a laser output of 50 μJ / It was set as dot.

[Preparation of measurement sample group for MALDI mass spectrometry]
(Example 1)
As shown in FIG. 6A, the powder layer (202) of the matrix A formed on the surface of the matrix plate A (200) is opposed to the sample surface of the standard sample group 1, and the back surface (having a matrix) of the matrix plate A is provided. It was installed so that the Gaussian laser beam could be vertically irradiated by the laser beam irradiating means A from the surface). Although not shown in FIG. 6A, in the matrix plate A (200), a laser energy absorbing material is arranged between the powder layer (202) of the matrix A and the base material 201. The gap G between the standard sample section and the powder layer of matrix A was set to 100 μm.
Next, as shown in FIGS. 6B and 6C, the powder of the matrix A is flown from the matrix plate A by the laser beam irradiating means A from the back surface of the matrix plate A, and the matrix dot diameter is 200 μm at the sample position A-1. , The dots were arranged in a square shape with 10 dots in each of the vertical and horizontal directions at a distance of 10 μm. Further, dots of matrix A were formed in the same manner from A-2 to A-12 to obtain a measurement sample group A for MALDI mass spectrometry.

(Example 2)
Similar to the measurement sample group A for MALDI mass spectrometry, the matrix plate A was changed to the matrix plate B and matrix dots were formed from B-1 to B-12 to obtain the measurement sample group B for MALDI mass spectrometry.

(Example 3)
Similar to the measurement sample group A for MALDI mass spectrometry, the matrix plate A was changed to the matrix plate C and matrix dots were formed from C-1 to C-12 to obtain the measurement sample group C for MALDI mass spectrometry.

(Example 4)
As shown in FIG. 6A, the powder layer of the matrix A formed on the surface of the matrix plate A is opposed to the sample surface of the standard sample group 1, and the laser beam is emitted from the back surface of the matrix plate A (the surface having no matrix). It was installed so that the optical vortex laser beam could be vertically irradiated by the irradiation means B. The gap between the standard sample section and the powder layer of Matrix A was set to 100 μm.
Next, as shown in FIGS. 6B and 6C, the powder of the matrix A is flown from the matrix plate A by the laser beam irradiation means B from the back surface (the surface having no matrix) of the matrix plate A to the sample position. D-1 was arranged in a square shape with a matrix dot diameter of 200 μm, a dot-to-dot distance of 10 μm, and 10 dots in each of the vertical and horizontal directions. Further, dots of matrix A were formed in the same manner from D-2 to D-12 to obtain a measurement sample group D for MALDI mass spectrometry.

(Example 5)
Similar to the measurement sample group D for MALDI mass spectrometry, the matrix plate A was changed to the matrix plate B and matrix dots were formed from E-1 to E-12 to obtain the measurement sample group E for MALDI mass spectrometry.

(Example 6)
Similar to the measurement sample group D for MALDI mass spectrometry, the matrix plate A was changed to the matrix plate C and matrix dots were formed from F-1 to F-12 to obtain the measurement sample group F for MALDI mass spectrometry.

(Example 7)
Similar to the measurement sample group D for MALDI mass spectrometry, the matrix plate A was changed to the matrix plate G and matrix dots were formed from G-1 to G-12 to obtain the measurement sample group G for MALDI mass spectrometry.

(Example 8)
Similar to the measurement sample group D for MALDI mass spectrometry, the matrix plate A was changed to the matrix plate H and matrix dots were formed from H-1 to H-12 to obtain the measurement sample group H for MALDI mass spectrometry.

(Example 9)
As shown in FIG. 6A, the powder layer of the matrix A formed on the surface of the matrix plate A is opposed to the standard sample on the standard sample group 2, and the laser is viewed from the back surface of the matrix plate A (the surface having no matrix). The beam irradiating means D was installed so that the soaking laser beam could be vertically irradiated. The gap between the standard sample section and the powder layer of Matrix A was set to 100 μm.
Next, as shown in FIGS. 6B and 6C, the powder of the matrix A is flown from the matrix plate A by the laser beam irradiation means D from the back surface (the surface having no matrix) of the matrix plate A to the sample position. A-1 was arranged in a square shape with a matrix dot diameter of 200 μm, a dot-to-dot distance of 10 μm, and 10 dots in each of the vertical and horizontal directions. Further, dots of matrix A were formed in the same manner from A-2 to A-12 to obtain a measurement sample group Q for MALDI mass spectrometry.

(Example 10)
Similar to the measurement sample group Q for MALDI mass spectrometry, the matrix plate A was changed to the matrix plate B and matrix dots were formed from B-1 to B-12 to obtain the measurement sample group R for MALDI mass spectrometry.

(Comparative Example 1)
As shown in FIG. 6A, the powder layer of the matrix A formed on the surface of the matrix plate D is opposed to the sample surface of the standard sample group 1, and the laser beam is emitted from the back surface of the matrix plate D (the surface having no matrix). It was installed so that the optical vortex laser beam could be vertically irradiated by the irradiation means B. The gap between the standard sample section and the powder layer of Matrix A was set to 100 μm.
Next, as shown in FIGS. 6B and 6C, the powder of the matrix A is flown from the matrix plate A by the laser beam irradiating means A from the back surface (the surface having no matrix) of the matrix plate D to the sample position. An attempt was made to form a dot having a matrix dot diameter of 200 μm on K-1, but the matrix A did not fly from the matrix plate D, and the matrix dot at the sample position K-1 could not be formed. It was designated as the measurement sample group K for MALDI mass analysis.

(Comparative Example 2)
The matrix plate D was changed to the matrix plate E, and an attempt was made to form a dot having a matrix dot diameter of 200 μm at the sample position L-1, but the matrix B did not fly from the matrix plate E and the sample position L-1. Although it was not possible to form the matrix dots of MALDI, this was designated as the measurement sample group L for MALDI mass analysis.

(Comparative Example 3)
The matrix plate D was changed to the matrix plate F, and an attempt was made to form a dot having a matrix dot diameter of 200 μm at the sample position M-1, but the matrix C did not fly from the matrix plate F and the sample position M-1. Although it was not possible to form the matrix dots of MALDI, this was designated as the measurement sample group M for MALDI mass analysis.

(Comparative Example 4)
As shown in FIG. 6A, the powder layer of the matrix A formed on the surface of the matrix plate D is opposed to the sample surface of the standard sample group 1, and the laser beam is emitted from the back surface of the matrix plate D (the surface having no matrix). It was installed so that the optical vortex laser beam could be vertically irradiated by the irradiation means C. The gap between the standard sample section and the powder layer of Matrix A was set to 100 μm.
Next, as shown in FIGS. 6B and 6C, the powder of the matrix A is flown from the matrix plate A by the laser beam irradiation means A from the back surface (the surface having no matrix) of the matrix plate A, and the sample position N -1 was arranged in a square shape with a matrix dot diameter of 200 μm, a dot-to-dot distance of 10 μm, and 10 dots in each of the vertical and horizontal directions. Further, dots of matrix A were formed in the same manner from N-2 to N-12 to obtain a measurement sample group N for MALDI mass spectrometry.

(Comparative Example 5)
Similar to the measurement sample group N for MALDI mass spectrometry, the matrix plate D was changed to the matrix plate E and matrix dots were formed from O-1 to O-12 to obtain the measurement sample group O for MALDI mass spectrometry.

(Comparative Example 6)
Similar to the measurement sample group N for MALDI mass spectrometry, the matrix plate D was changed to the matrix plate F and matrix dots were formed from P-1 to P-12 to obtain the measurement sample group P for MALDI mass spectrometry.

Next, "presence or absence of matrix flight" and "detection sensitivity of MALDI mass spectrometry" were evaluated as follows.

[Comparison of matrix flight by laser]
The matrix flight by the laser beam irradiating means A and the laser beam irradiating means B of the present invention was compared.
In order to fly the matrix, instead of the matrix that cannot be visualized because it is colorless and transparent, the red organic dye Acid Red 151, which is an organic low molecular weight substance that can be visualized and has the same crystallinity as the matrix of the present invention, is used as a substitute for the matrix layer. The results of flight comparison using the above dye plate are shown in FIGS. 9B and 9D.
In the Gaussian laser 91 of the laser beam irradiation means A, when the matrix is arranged at a predetermined position, dust is likely to occur if the gap between the standard sample section and the matrix layer is wide (FIG. 9B), so that the interval needs to be narrowed. However, by using the light vortex laser beam 92 of the laser beam irradiating means B, dust is less likely to be generated (FIG. 9D), so that the gap between the standard sample section and the matrix layer can be widened, and the sample section can be used. Damage caused by flying the matrix (for example, change in molecular weight) can be further reduced.
In the experiment of the present invention, the laser beam irradiating means B was able to form accurate dots without generating dust when the distance between the matrix layer and the standard sample was 200 μm, but in the laser beam irradiating means A, dust was generated unless the distance was set to 100 μm. It was generated, and dots as accurate as the laser beam irradiating means B could not be formed.

[MALDI mass spectrometry]
MALDI mass spectrometry was performed using MALDI-TOF-MS (autoflex III LRF200-CID manufactured by Bruker Daltonix Co., Ltd.) from the measurement sample group A to P for MALDI mass spectrometry in which the matrix powder was arranged. Representative examples of the results are shown in FIGS. 7A-7C and 8A-8C. In FIGS. 7A to 7C and 8A to 8C, the horizontal axis represents mass (m / z) and the vertical axis represents detection intensity.
[Evaluation method]
The following evaluations were performed on the data obtained by the measurement.
-Impact on the sample-
The detection data of 12 points of each MALDI mass spectrometric measurement sample group was scanned with a laser energy intensity of 50% per point, and the detection peak of the detection target when 1500 shots were taken was measured. Although the detection peak value differs depending on the type of matrix, the influence of the laser during the flight of the matrix is likely to be affected by a component having a relatively large molecular weight, and the larger the damage, the more the molecular chain is broken and the ratio of the detection peaks tends to decrease.
Among the typical peaks of the standard sample, the detection peak intensity X of somatostatin 28 (the peak indicated by the arrow a1 in the figure) having a molecular weight of about 759, which has a relatively small molecular weight, and bradykinin (1-) having a molecular weight of about 3149 having a relatively large molecular weight. 7) The damage of the standard sample was evaluated by the ratio (Y / X) of the detected peak intensity Y (the peak indicated by the arrow a2 in the figure).
[Evaluation criteria]
-When matrices A and B were used, evaluation was made based on the following evaluation criteria based on the lowest value of the detection intensity ratio (Y / X) in the detection data of 12 points.
Good: Minimum detection intensity ratio (Y / X) is 3 or more Detectable: Minimum detection intensity ratio (Y / X) is 1 or more and less than 3 Undetectable: Minimum detection intensity ratio (Y / X) is In the case of less than 1 and matrix C, evaluation was performed based on the following evaluation criteria based on the lowest value of the detection intensity ratio Y / X in the detection data of 12 points. Matrix C is a matrix species in which the detection intensity of relatively high molecular weight components is difficult to obtain.
Good: Minimum value of detection intensity ratio (Y / X) is 0.1 or more Detectable: Minimum value of detection intensity ratio (Y / X) is 0.05 or more and less than 0.1 Detectable: Detection intensity ratio (Y / X) The minimum value of X) is less than 0.05-Detection variation (fluctuation width)-
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 with respect to the detection data of 12 points of each measurement sample group for MALDI mass spectrometry. The results are shown in Table 1. The detection variation (fluctuation width) was evaluated using a numerical value obtained from the following equation, {(Max-Min) / (2 × Ave)} × 100, described in the following evaluation criteria.
[Evaluation criteria]
-When Matrix A and Matrix B are used Excellent: {(Max-Min) / (2 x Ave)} x 100 is less than 10% Good: {(Max-Min) / (2 x Ave)} x 100 is 10 % Or more and less than 15% Possible: {(Max-Min) / (2 × Ave)} × 100 is 15% or more and less than 20% Impossible: {(Max-Min) / (2 × Ave)} × 100 is 20% or more -When matrix C is used Excellent: {(Max-Min) / (2 x Ave)} x 100 is less than 10% Good: {(Max-Min) / (2 x Ave)} x 100 is 10% or more 20 % Less than Possible: {(Max-Min) / (2 × Ave)} × 100 is 20% or more and less than 40% Impossible: {(Max-Min) / (2 × Ave)} × 100 is 40% or more

Examples of aspects of the present invention are as follows.
<1> In a base material on which a matrix used for preparing a measurement sample for MALDI mass spectrometry is arranged on the surface, the matrix is provided by irradiating the surface of the base material on the side opposite to the side having the matrix with a laser beam. Including flying from the substrate and arranging it in a predetermined position of the sample to be MALDI mass spectrometry.
The substrate has a laser energy absorbing material and
This is a method for preparing a measurement sample for MALDI mass spectrometry, characterized in that the wavelength of the laser energy of the laser beam is 400 nm or more.
<2> The method for preparing a measurement sample for MALDI mass spectrometry according to <1>, wherein the laser energy absorbing material and the matrix are provided on the substrate in this order.
<3> The method for preparing a measurement sample for MALDI mass spectrometry according to any one of <1> to <2>, wherein the laser beam transmittance of the laser energy absorbing material is 60% or less.
<4> The method for preparing a measurement sample for MALDI mass spectrometry according to any one of <1> to <3>, wherein the laser energy absorbing material is a thin metal film.
<5> The method for preparing a measurement sample for MALDI mass spectrometry according to <4>, wherein the metal is gold.
<6> The method for preparing a measurement sample for MALDI mass spectrometry according to any one of <1> to <5>, wherein the matrix to be flown from the base material is two or more.
<7> The MALDI mass according to any one of <1> to <6>, wherein two or more kinds of the matrix to be flown from the base material are arranged at different predetermined positions in the sample to be subjected to the MALDI mass spectrometry. This is a method for preparing a measurement sample for analysis.
<8> The measurement for MALDI mass spectrometry according to any one of <1> to <7>, wherein the matrix is dispersed from the base material a plurality of times with respect to a predetermined position of the sample to be analyzed for MALDI mass spectrometry. This is a sample preparation method.
<9> The method for preparing a measurement sample for MALDI mass spectrometry according to any one of <1> to <8>, wherein the laser beam is an optical vortex laser beam.
<10> The method for preparing a measurement sample for MALDI mass spectrometry according to any one of <1> to <8>, wherein the laser beam is a soaking laser beam.
<11> The MALDI mass spectrometry measurement sample preparation device used in the method for preparing a measurement sample for MALDI mass spectrometry according to any one of <1> to <10>.
A measurement sample preparation device for MALDI mass spectrometry, which comprises a laser beam irradiating means for irradiating the surface of the base material with a laser beam.
<12> A base material for preparing a measurement sample for MALDI mass spectrometry, which comprises a laser energy absorbing material capable of absorbing the energy of a laser beam having a wavelength of 400 nm or more and a matrix in this order on a substrate. be.
<13> The base material for preparing a measurement sample for MALDI mass spectrometry according to <12>, wherein the laser energy absorbing material is a thin film of metal.
<14> The base material for preparing a measurement sample for MALDI mass spectrometry according to <13>, wherein the metal is gold.

The method for preparing a measurement sample for MALDI mass spectrometry according to any one of <1> to <10>, the device for preparing a measurement sample for MALDI mass spectrometry according to <11>, and any of the above <12> to <14>. According to the base material for preparing a measurement sample for MALDI mass spectrometry described in 1), the above-mentioned problems in the prior art can be solved and the object of the present invention can be achieved.

100 Measurement sample preparation device for MALDI mass spectrometry 140 Laser beam irradiation means 200 Matrix plate 201 Base material 202 Matrix 203 Laser energy absorbing material 301 Sample section (sample)
L laser beam

Japanese Unexamined Patent Publication No. 2014-206389

Claims (14)

In a substrate on which a matrix used for preparing a measurement sample for MALDI mass spectrometry is arranged on the surface, the matrix is subjected to the substrate by irradiating the surface of the substrate on the side opposite to the side having the matrix with a laser beam. Including flying from and placing in a predetermined position on the sample to be MALDI mass spectrometry,
The substrate has a laser energy absorbing material and
A method for preparing a measurement sample for MALDI mass spectrometry, wherein the wavelength of the laser energy of the laser beam is 400 nm or more. The method for preparing a measurement sample for MALDI mass spectrometry according to claim 1, wherein the laser energy absorbing material and the matrix are provided on the substrate in this order. The method for preparing a measurement sample for MALDI mass spectrometry according to any one of claims 1 to 2, wherein the laser beam transmittance of the laser energy absorbing material is 60% or less. The method for preparing a measurement sample for MALDI mass spectrometry according to any one of claims 1 to 3, wherein the laser energy absorbing material is a thin film of metal. The method for preparing a measurement sample for MALDI mass spectrometry according to claim 4, wherein the metal is gold. The method for preparing a measurement sample for MALDI mass spectrometry according to any one of claims 1 to 5, wherein the matrix to be flown from the substrate is two or more. The method for preparing a measurement sample for MALDI mass spectrometry according to any one of claims 1 to 6, wherein two or more kinds of the matrix to be flown from the base material are arranged at different predetermined positions in the sample to be MALDI mass spectrometry. .. The method for preparing a measurement sample for MALDI mass spectrometry according to any one of claims 1 to 7, wherein the matrix is dispersed from the base material a plurality of times with respect to a predetermined position of the sample to be subjected to MALDI mass spectrometry. The method for preparing a measurement sample for MALDI mass spectrometry according to any one of claims 1 to 8, wherein the laser beam is an optical vortex laser beam. The method for preparing a measurement sample for MALDI mass spectrometry according to any one of claims 1 to 8, wherein the laser beam is a soaking laser beam. A MALDI mass spectrometry measurement sample preparation apparatus used in the method for preparing a measurement sample for MALDI mass spectrometry according to any one of claims 1 to 10.
A measurement sample preparation device for MALDI mass spectrometry, which comprises a laser beam irradiating means for irradiating the surface of the base material with a laser beam. A base material for preparing a measurement sample for MALDI mass spectrometry, which comprises a laser energy absorbing material capable of absorbing the energy of a laser beam having a wavelength of 400 nm or more and a matrix in this order on a substrate. The base material for preparing a measurement sample for MALDI mass spectrometry according to claim 12, wherein the laser energy absorbing material is a thin film of metal. The base material for preparing a measurement sample for MALDI mass spectrometry according to claim 13, wherein the metal is gold.

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