JP2012177689A - Measurement sample preparation method for maldi mass analysis method - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a measurement sample preparation method for a MALDI mass analysis method for preparing a measurement sample for the MALDI mass analysis method having a high ion generation amount to efficiently obtain a high quality MS spectrum in short time, and the MALDI mass analysis method with high throughput is attained, for allowing high sensitivity MS(2≤n) analysis required for structure identification and allowing application to automatic analysis.SOLUTION: In a measurement sample preparation method for a MALDI mass analysis method, mixed crystal of a sample containing a measurement object molecule and a matrix is deposited, and ionization efficiency is raised by utilizing specific polymorphism structure of the mixed crystal in the MALDI mass analysis method.

Description

  The present invention relates to a measurement sample preparation method for MALDI mass spectrometry that can increase ionization efficiency and detect signals with high sensitivity in MALDI mass spectrometry, and MALDI mass spectrometry prepared using the measurement sample preparation method The present invention relates to MALDI mass spectrometry using a measurement sample.

  The “mass spectrometry (hereinafter sometimes abbreviated as“ MS ”) method” refers to ionizing a sample containing a molecule to be measured and converting ions derived from the molecule to be measured into a mass-to-charge ratio (mass / charge ( This is a method for obtaining information on the chemical structure of the molecule to be measured by separating and detecting by m / z)).

  In MS, sample ionization is an important process that determines whether analysis is possible and the quality of the spectrum obtained, and many ionization methods have been developed so far in order to efficiently ionize the sample. At present, for the ionization of biopolymers, matrix-assisted laser desorption / ionization (hereinafter sometimes abbreviated as “MALDI”) or electrospray ionization (soft ionization). The electrospray ionization (hereinafter sometimes abbreviated as “ESI”) method is mainly used. Mass spectrometers using these ionization methods are widely used in the bio field because they can be measured with a smaller amount of sample than NMR.

  In MALDI, molecules to be measured (for example, proteins, peptides, saccharides, etc.) are dispersed in a substance that absorbs light called a matrix, and the target to be measured together with the matrix is irradiated with a pulse laser. It is a technology to ionize the molecule.

  In many cases, the wavelength of the laser to be used has a wavelength in the ultraviolet region, and a wavelength in the infrared region may be used. However, it is general to use a laser that matches the light absorption characteristics of the matrix. At present, the most frequently used laser is a nitrogen laser, which has a wavelength of 337 nm.

  On the other hand, since the selection of the matrix to be used determines the success or failure of the analysis, many matrices have been developed and used in MALDI. In general, the matrix is a crystalline organic molecule, and a co-crystal with an analysis sample is generated, and then ionized by irradiation with the pulse laser. In recent years, various liquid matrices have been developed, and there are various options depending on the sample to be measured.

  Among the matrices that are actually used, there is versatility, and standard matrices are CHCA (α-cyano-hydroxycinnamic acid), SA (sinamic acid), DHBA (2,5-dihydroxybenzoic acid), etc. DHBA is an excellent matrix suitable for ionization of low molecular weight organic compounds, peptides, saccharides, proteins and the like.

  In general, it is considered that the matrix and the sample are well mixed and need to be a mixed crystal or mixture. The quality of the mixed crystal or mixture of sample and matrix affects sensitivity and spectral quality. Furthermore, even though it appears to be a homogeneous mixture, it is actually inhomogeneous. In particular, in the case of a solid crystal, ions derived from the molecule to be measured cannot be obtained from all the places where the crystal is generated. Only when a part is irradiated with a laser, ions derived from the molecule to be measured are obtained.

  This part is called “sweet spot”, and in order to obtain a high-quality mass spectrum by MALDI mass spectrometry, especially in automatic analysis, a lot of sweet spots must be created in the target measurement sample. Is effective.

  In particular, DHBA produces relatively large needle-like crystals of about 0.1 to 1.5 mm, but the difference in the amount of ions generated between the sweet spot and the place where it is not so different. Nonuniformity in crystal size is considered to be a major cause of this phenomenon. Examples of this are Non-Patent Document 1 and Non-Patent Document 2.

  For example, DHBA is known to have two types of crystal polymorphs, and each crystal has been reported to have a different melting point. Examples of this are Non-Patent Document 3 and Non-Patent Document 4. According to Non-Patent Document 3, two types of polymorphs are prepared using different solvents. When water is used as the solvent, stable ordered is produced, and the volume ratio of acetone to chloroform is 1: It is reported that a metastable type Disordered is formed from the mixed solvent of No. 3.

  In addition, the spectrum database of organic compounds of the National Institute of Advanced Industrial Science and Technology (AIST) does not specify crystal polymorphism, but includes the Raman spectrum and IR spectrum of DHBA. However, not only has not been studied in detail about the crystalline polymorphs of the matrix produced under the conditions of standard sample preparation methods in MALDI, but also the relationship between the crystalline polymorphs and the signal intensity in MALDI, the crystal structure and The relationship between sweet spots was not known at all.

  Furthermore, DHBA is widely used for the measurement of sugar chains and glycopeptides. However, since sugar chains and relatively hydrophilic glycopeptides have poor ionization efficiency, a sample is consumed to obtain a high-quality mass spectrum. This requires time-consuming operations such as finding a sweet spot manually by laser pre-scanning and experience, and performing laser irradiation, and is currently not suitable for automatic analysis of the entire measurement sample.

  In order to solve this problem, for example, a technique for forcibly generating microcrystals by rapidly drying a matrix solution and limiting a region that can be a sweet spot (Non-Patent Document 5), or a liquid matrix that does not crystallize is used. Although there is a technique to be used, it may lead to a decrease in the amount of ion generation or promote the generation of additional ions such as contaminated salts, and it is not a technique that can directly use the high ion generation amount of the sweet spot of the measurement sample. It was. Even in a liquid matrix, there is no variation as much as a solid matrix, but the spectral reproducibility may be inferior due to the influence of a contaminated salt or the like, and it has not been a fundamental solution.

  Further, Non-Patent Document 6 describes a method of reducing physical irregularities by breaking the surface after crystallization of DHBA. Non-Patent Document 6 also describes that a high signal can be obtained if the generated crystal is broken after crystallization is performed in the refrigerator, but the details of the effect of crystallizing the matrix in the refrigerator are clear. is not. Further, there are problems that the polymorphic structure is changed by breaking the crystal, and the sweet spot in the produced measurement sample is unclear. Therefore, the gist of the present invention is different from the present invention, which aims to localize sweet spots with good reproducibility at the time of crystal generation and to efficiently acquire a high-quality mass spectrum.

  In addition, several techniques have been developed for the purpose of controlling the formation of sweet spots, and there are Patent Document 1 and Patent Document 2 as examples. However, in order to execute these techniques, a dedicated device or sample support member is used. is necessary. In addition, these technologies are based on the idea of concentrating a sample in as narrow a region as possible. By devising the crystallization conditions of the matrix, a sweet instrument can be used without using a special instrument, device, sample support member, etc. This was fundamentally different from the present invention in which a larger number of crystals serving as spots were formed over a wide range, or formed with good reproducibility.

JP 2003-098115 A JP 2003-098149 A

Revecca W. et al., Anal. Chem. 72 (18), 30-36 (2000) Luxembourg S.L. et al., Anal. Chem. 75 (10), 2333-2341 (2003) Haisa M. et al., Acta. Cryst. B38, 1480-1485 (1982) Cohen D.E. et al., Crystal Growth & Design 7 (3), 492-495 (2007) Williams T.I. et al., Rapid Commun. Mass Spectrom. 21, 807-811 (2007) Allwood D.A. et al., Appl. Surf. Sci. 103 (3), 231-244 (1996)

  The present invention has been made in view of the above-mentioned background art, and its problem is to prepare a measurement sample having a high ion production amount in MALDI mass spectrometry, and to efficiently and high-quality MS spectrum in a short time. The object of the present invention is to provide a method for preparing a measurement sample for MALDI mass spectrometry.

Also, it is possible to provide a method that enables high-sensitivity MS ⁿ (2 ≦ n) analysis necessary for structure identification, enables application to automatic analysis, and realizes high-throughput MALDI mass spectrometry. It is in.

  The present inventor has unexpectedly analyzed the crystal structure of the matrix alone using a spectroscopic method and a thermal analysis method and, as a result of performing a local analysis on the crystal on the sample support member, unexpectedly, the sample support member The DHBA matrix crystal produced above has a different physicochemical property from the standard recrystallized matrix of the matrix (in the spectrum database of organic compounds of the National Institute of Advanced Industrial Science and Technology). I found that the structure was mixed.

  Furthermore, in the mixed crystal of the sample to be measured and the matrix, the difference between the portion of the mixed crystal that creates a sweet spot having a high ion generation amount and the portion of the mixed crystal that does not become a sweet spot depends on the difference in the crystal structure of the matrix. The portion of the mixed crystal that creates a sweet spot has a crystal structure that exhibits physicochemical properties different from those of the matrix standard product (in the spectral database of organic compounds of the National Institute of Advanced Industrial Science and Technology). Identified.

  From this, by controlling the crystallization conditions of the matrix in order to control the formation of the mixed crystals that create sweet spots, that is, become sweet spots, and the mixed crystals that do not become sweet spots, As a result of intensive studies with the idea that it may be possible to produce a measurement sample that gives a high MS spectrum, it was found that the above problems can be solved.

  That is, the polymorphic structure of the matrix crystal can be easily formed by setting the temperature conditions when removing the solvent during the preparation of the measurement sample or when the crystals are precipitated. For the first time, the present inventors have found that the position of the mixed crystal that can be a sweet spot can be clearly controlled by only setting, and completed the present invention. Then, when acetonitrile and / or water commonly used for the preparation of a matrix solution is used as a solvent, it is found that a specific polymorphic structure is formed differentially, particularly by setting temperature conditions, and the present invention is completed. It came to. Note that acetone and chloroform used in Non-Patent Document 3 are not suitable as solvents for matrix solutions in MALDI mass spectrometry because molecules to be measured often do not dissolve.

Usually, the solubility of molecules to be measured, such as glycopeptides, decreases as the temperature of the solution decreases, so cooling the matrix solution or sample support member decreases the miscibility of the matrix crystals and the molecules to be measured, resulting in mixed crystals. It seems to be a disadvantage against the formation of sweet spots.
However, surprisingly, the cooling of the matrix solution does not increase the non-uniformity of the mixed crystal, and it is possible to control the formation of the sweet spot by setting only the temperature condition. By controlling the formation of the sweet spot, The inventors have found that it is possible to obtain a high-quality mass spectrum without performing pre-scanning or manual measurement, and have reached the present invention.

  In particular, by further combining the derivatizing agent for increasing the ionization efficiency of the measurement target molecule and the washing process thereof, it is possible to control the position of the sweet spot etc. by setting only the temperature condition, By controlling the position of the sweet spot, etc., it was found that it was possible to easily obtain a high-quality mass spectrum without performing a pre-scan or manual measurement, and the present invention was achieved. .

That is, the present invention provides the following measurement sample preparation method for MALDI mass spectrometry.
[1] In MALDI mass spectrometry, a mixed crystal of a sample containing a molecule to be measured and a matrix is precipitated, and a specific polymorphic structure of the mixed crystal is used to increase ionization efficiency. Method for preparing measurement sample for analytical method.
[2] In MALDI mass spectrometry, the crystallization conditions for precipitating the mixed crystal of the sample containing the molecule to be measured and the matrix are controlled to form a specific polymorphic structure that can be a sweet spot in the mixed crystal, The measurement sample preparation method for MALDI mass spectrometry according to [1], wherein ionization efficiency is increased by utilizing a specific polymorphic structure that can be a sweet spot.
[3] The matrix according to [1] or [2], wherein the matrix is 2,5-dihydroxybenzoic acid (DHBA), and the polymorphic structure that can be a sweet spot is a metastable crystal β crystal. Measurement sample preparation method for MALDI mass spectrometry.
[4] In MALDI mass spectrometry, a specific polymorphic structure that can be a sweet spot is formed in the mixed crystal by controlling the temperature condition when the mixed crystal of the sample containing the molecule to be measured and the matrix is precipitated. The method for preparing a measurement sample for MALDI mass spectrometry according to any one of [1] to [3].
[5] In MALDI mass spectrometry, a matrix solution is placed after placing a sample containing a molecule to be measured on a sample support member, or a sample containing the molecule to be measured and a matrix solution are placed simultaneously on the sample support member. And controlling the temperature condition when the mixed crystal of the sample and the matrix is precipitated by drying the solution to form a specific polymorphic structure that can be a sweet spot in the mixed crystal [1. ] To MALDI mass spectrometry measurement sample preparation method according to any one of [4] to [4].
[6] The matrix is 2,5-dihydroxybenzoic acid (DHBA), and the temperature at which the mixed crystal of the sample containing the molecule to be measured and the matrix is precipitated is set in the range from the freezing point of the matrix solution to 15 ° C. The measurement sample preparation method for MALDI mass spectrometry according to any one of [1] to [5], wherein the measurement sample preparation method is set.
[7] In MALDI mass spectrometry, the temperature of the sample support member is set in the range from the freezing point of the matrix solution to 15 ° C. or less when the mixed crystal of the sample containing the molecule to be measured and the matrix is precipitated. The measurement sample preparation method for MALDI mass spectrometry according to any one of [1] to [6].
[8] MALDI mass spectrometry as described in any one of [1] to [7], wherein the solvent used for precipitating the mixed crystal of the sample containing the molecule to be measured and the matrix is water and / or acetonitrile. Preparation method for legal measurement sample.
[9] After placing the sample containing the molecule to be measured on the sample support member and before placing the matrix solution, a solution of the derivatizing agent that increases the ionization efficiency by reacting with the molecule to be measured is added to the solution. The method for preparing a measurement sample for MALDI mass spectrometry according to any one of [1] to [8], wherein a step of dropping and drying on a sample support member is inserted.

The present invention also provides the following MALDI mass spectrometry.
[10] The ionization efficiency is increased using the MALDI mass spectrometry measurement sample prepared by using the MALDI mass spectrometry measurement sample preparation method according to any one of [1] to [9] MALDI mass spectrometry.

  According to the present invention, work for solving the above-mentioned problems and problems, judging whether the measurement sample is good or bad, and finding out where the sweet spot is on the sample support member for MALDI mass spectrometry by experience Therefore, anyone can efficiently obtain a high-quality MS spectrum in a short time. Thereby, application to automatic analysis is also possible, and a high-throughput MS analysis technique can be provided.

In particular, high-sensitivity MS ⁿ (2 ≦ n) analysis necessary for structure identification is enabled, and the conventional measurement could not be performed due to a small amount of sample containing the molecule to be measured and low ionization efficiency of the molecule to be measured. Even if it is a sample, anyone can determine a chemical structure efficiently in a short time.

It is a figure which shows the chemical structure of the glycopeptide which is a measuring object molecule | numerator used in the Example. It is a figure which shows the confocal laser micrograph of the deposit on the sample support member in the reference example 1. FIG. It is a figure which shows two types of Raman spectroscopy spectra. (A) Raman spectrum measured by a stable crystal α crystal (standard DHBA powder) on a plate (sample support member). (B) Raman spectrum measured by metastable crystal β crystal on a plate (sample support member). On plate (specimen support member), represents white stable crystalline α crystals with a strong signal in the vicinity of 1199cm ^-1, metastable crystalline β crystal of the black represents Raman imaging image having no strong signal in the vicinity of 1199cm ^-1 is there. It is a figure which shows the mass spectrum of the part (metastable crystal (beta) crystal) shown black in FIG. It is the image which scanned the sample support member with MS signal, and is a figure which shows the part with high ionization efficiency. 2 is a diagram showing a microscopic Raman spectrum measured in Example 1. FIG. It is a figure which shows the micro Raman spectrum measured in Reference Example 2. 4 is a diagram showing powder X-ray diffraction measured in Reference Example 2. FIG. 6 is a diagram showing a microscopic Raman spectrum measured in Comparative Example 1. FIG. 4 is a diagram showing powder X-ray diffraction measured in Comparative Example 1. FIG. It is a figure which shows two types of MS spectra. (A) It is the MS spectrum which measured on the sample support member of Example 2. FIG. (B) MS spectrum measured on the sample support member of Comparative Example 2. It is the image scanned with the confocal laser micrograph of Example 2 and the comparative example 2, and MS signal. (A) It is the image scanned with the confocal laser micrograph on the sample support member of Example 2, and MS signal, and is a figure which shows the part with high ionization efficiency from dark gray to black. (B) It is the image scanned with the confocal laser scanning micrograph and MS signal on the sample support member of the comparative example 2, and is a figure which shows the part with high ionization efficiency from dark gray to black. It is a confocal laser scanning micrograph on the sample support member of comparative example 3, and an image scanned with MS signal, and is a figure showing a portion with high ionization efficiency from dark gray to black. It is a figure which shows two types of MS spectra. (A) It is the MS spectrum which measured on the sample support member of Example 3. FIG. (B) MS spectrum measured on the sample support member of Comparative Example 4. It is a figure which shows two types of MS spectra. (C) MS spectrum measured on the sample support member of Example 4. (D) It is the MS spectrum which measured on the sample support member of Example 5. FIG. It is the image scanned with the confocal laser micrograph and the MS signal of Example 3 and Comparative Example 4. (A) It is the image scanned with the confocal laser micrograph on the sample support member of Example 3, and MS signal, and is a figure which shows the part with high ionization efficiency in black. (B) It is the image scanned with the confocal laser microscope picture and MS signal on the sample support member of the comparative example 4, and is a figure which shows the part with high ionization efficiency in black. It is the image scanned with the confocal laser micrograph of Example 4 and Example 5, and MS signal. (C) It is the image scanned with the confocal laser micrograph on the sample support member of Example 4, and MS signal, and is a figure which shows the part with high ionization efficiency in black. (D) It is the image scanned with the confocal laser micrograph and MS signal on the sample support member of Example 5, and is a figure which shows the part with high ionization efficiency in black. It is a figure which shows two types of MS spectra. (A) It is the MS spectrum which measured on the sample support member of Example 6. FIG. (B) MS spectrum measured on the sample support member of Comparative Example 5. It is the image scanned with the confocal laser micrograph and the MS signal of Example 6 and Comparative Example 5. (A) It is the image scanned with the confocal laser micrograph on the sample support member of Example 6, and MS signal, and is a figure which shows the part with high ionization efficiency in black. (B) It is the image scanned with the confocal laser scanning micrograph and MS signal on the sample support member of the comparative example 5, and is a figure which shows the part with high ionization efficiency in black. It is a figure which shows two types of MS spectra. (A) It is the MS spectrum which measured on the sample support member of Example 7. FIG. (B) MS spectrum measured on the sample support member of Comparative Example 6. It is the image scanned with the confocal laser micrograph of Example 7 and the comparative example 6, and MS signal. (A) It is the image scanned with the confocal laser micrograph on the sample support member of Example 7, and MS signal, and is a figure which shows the part with high ionization efficiency in black. (B) It is the image scanned with the confocal laser micrograph on the sample support member of the comparative example 6, and MS signal, and is a figure which shows the part with high ionization efficiency in black. It is the confocal laser micrograph of the measurement sample of Example 8 and Comparative Example 7, and the MS signal scan image of four types of peptides. (A) It is the image scanned with the confocal laser micrograph on the sample support member of Example 8, and MS signal, and is a figure which shows the part with high ionization efficiency in black. (B) It is the image scanned with the confocal laser micrograph on the sample support member of the comparative example 7, and MS signal, and is a figure which shows the part with high ionization efficiency in black.

  Hereinafter, although this invention is demonstrated, this invention is not limited to the specific aspect of the following implementation, It can implement arbitrarily deform | transforming.

<Preparation method of measurement sample for MALDI mass spectrometry>
The matrix is not limited as long as it absorbs the light energy of the laser and achieves desorption and ionization of the coexisting molecules to be measured, and examples thereof include the following.

1,8-diaminonaphthalene (1,8-DAN), 2,5-dihydroxybenzoic acid (hereinafter sometimes abbreviated as “DHBA”), 1,8-Anthracenedicarboxylic Acid Dimethyl ester, Leucoquinizarin, Anthrarobin, 1,5-Diaminonaphthalene (1,5-DAN) ), 6-Aza-2-thiothymine, 1,5-diaminoanthraquinone, 1,6-diaminopyrene, 3,6 -Diaminocarbazole (3,6-Diaminocarbazole), 1,8-Anthracenedicarboxylic Acid, Norharmane, 1-pyrenepropylamine hydrochloride (1-Pyrenepropylamine hydrochloride), 9-Aminofluorene Hydrochloride, Ferrulic acid, Dithranol, 2- (4-Hydroxyphenylazo) benzoic acid (2- (4-Hydroxyphenylazo) ) benzoic acid) (HABA), trans-2- [3- (4-tert-butylphenyl) -2-methyl-2-propenylidene] malononitrile (trans-2- [3- (4-tert-Butylphenyl)- 2-methyl-2-propenylidene] malononitrile) (DCTB), trans-4-phenyl-3-buten-2-one (TPBO), trans-3- Trans-3-Indoleacrylic acid (IAA), 1,10-phenanthroline, 5-nitro-1,10-phenanthroline, α- Cyano-4-hydroxycinnamic acid CHCA), Sinapic acid (SA), 2,4,6-trihydroxyacetophenone (THAP), 3-hydroxypicolinic acid (HPA), Anthranilic acid, Nicotinic acid
acid), 3-Aminoquinoline, 2-Hydroxy-5-methoxybenzoic acid, 2,5-Dimethoxybenzoic acid, 4 , 7-phenanthroline, p-coumaric acid, 1-isoquinolinol, 2-picolinic acid, 1-pyrenebutanoic hydrazide ( 1-Pyrenebutanoic acid, hydrazide (PBH), 1-Pyrenebutyric acid (PBA), 1-Pyrenemethylamine hydrochloride (PMA).

  Among the above-mentioned matrices, a matrix having a property of crystallizing in a needle shape is preferable. Furthermore, 2,5-dihydroxybenzoic acid (DHBA) is most preferred.

<Precipitation of mixed crystal of sample and matrix or generation of mixture>
In the method for preparing a measurement sample for MALDI mass spectrometry of the present invention, a method for producing a mixed crystal of a sample containing the molecule to be measured and a matrix is not particularly limited. For example, as a preferable method, the following (1 ) Method to (3) method.
(1) A method of depositing a mixed crystal by placing a matrix solution on a sample support member, placing a matrix solution after drying or before drying, and then drying the solution.
(2) A method in which a sample containing a molecule to be measured and a matrix solution are simultaneously placed on a sample support member, and then the mixed crystal is precipitated by drying the solution.
(3) The matrix solution is placed on a sample support member, and then the solution is dried to precipitate crystals of the matrix, and then a solution containing a matrix having the same or different chemical structure and a molecule to be measured is overlaid. And then drying the solution to form a mixture.

The method (1) or (2) is particularly preferable as a method for producing a mixed crystal of a sample and a matrix. That is,
(1) Place the matrix solution after placing the sample containing the molecule to be measured on the sample support member, or
(2) A method in which a sample containing a molecule to be measured and a matrix solution are simultaneously placed on a sample support member, and then the mixed crystal is precipitated by drying the solution.

  In the present invention, the “mixed crystal of a sample containing a molecule to be measured and a matrix” means a crystal containing both the molecule of the sample containing the molecule to be measured and the molecule of the matrix as a substance. By replacing it with a molecule, it does not mean a crystal having a different crystal structure from that of the matrix molecule alone (which is unique to the matrix molecule). Usually, on the sample support member, the number of molecules to be measured is very small compared to the number of molecules in the matrix.

<< (1) Method >>
The above method (1) is a particularly preferable method in the present invention, in which a sample containing a molecule to be measured is first present on the sample support member, and then a matrix solution (preferably a solution in which only the matrix is substantially dissolved). ) Is dried on a sample support member (hereinafter sometimes abbreviated as “plate”) while the sample is dissolved, and a crystal is precipitated.

  In the method (1), the step of drying the matrix solution on the plate is the final step in the step of supplying the substance onto the plate. In general, in order to obtain a high ion production amount, the sample and the matrix must be well mixed, but the molecule to be measured is not dissolved (contained) in the solution supplied to the plate in the final step. The present inventor has found that this is acceptable.

  In the method (1), in order to form a large number of crystals serving as sweet spots, the temperature of the matrix solution (preferably a solution in which only the matrix is substantially dissolved) is preferably lower than the temperature of the sample support member. Better.

  Specifically, the temperature of the matrix solution (preferably a solution in which only the matrix is dissolved) is specifically preferably 20 ° C. or lower, more preferably 15 ° C. or lower when being placed on the sample support member. A temperature of not higher than ° C is particularly preferred. Further, the temperature of the matrix solution maintained during crystal precipitation is preferably 20 ° C. or less, more preferably 15 ° C. or less, and particularly preferably 4 ° C. or less. In addition, the temperature of the matrix solution needs to be equal to or higher than the freezing point of the matrix solution. The matrix solution is particularly preferably used after sufficiently cooled in a refrigerator or the like.

  Furthermore, it is preferable that the sample support member is also cooled in advance. Specifically, when the matrix solution is placed on the sample support member, the temperature of the sample support member is preferably 20 ° C. or less, and particularly preferably 15 ° C. or less. Moreover, the temperature of the sample support member maintained during crystal precipitation is preferably 20 ° C. or less, and particularly preferably 15 ° C. or less. If it is the conditions which can prevent dew condensation, 4 degrees C or less is especially preferable. Therefore, when the sample support member is cooled, condensation may occur on the sample support member depending on the humidity at the place where the measurement sample is prepared. Therefore, it is preferable to work in a dry environment. Moreover, it is preferable that the temperature of a sample support member is more than the freezing point of a matrix solution.

  In the present invention, “crystallization conditions for precipitating a mixed crystal of a sample containing a molecule to be measured and a matrix” refers to temperature, degree of supersaturation (concentration), solvent, pH, additive, interfacial tension, and the like. Temperature and supersaturation (concentration) are the main controlling factors affecting the formation of polymorphic structures of crystals, and solvent, pH, additives and interfacial tension influence as cofactors. Accordingly, the “method for forming a crystalline polymorph that becomes a sweet spot” in the present invention is not particularly limited, but the temperature, supersaturation (concentration), solvent, pH, additive, interfacial tension, etc. are optimized and controlled. A method is mentioned.

  When the matrix is 2,5-dihydroxybenzoic acid (hereinafter sometimes abbreviated as “DHBA”) as the above-mentioned “method for forming a polymorph as a sweet spot”, the temperature of the plate and the matrix solution However, the method of cooling to a low temperature as described above is the most simple and effective, and is particularly preferable because the measurement sample can be prepared with good reproducibility. It is preferable to lower the temperature when the solvent is distilled off (when crystals are precipitated), and in order to achieve this reliably, the matrix solution and / or the sample support member should be kept at a low temperature when placed. Is preferred.

  It is particularly preferable to lower the temperature of the sample support member when the solvent is distilled off (when the mixed crystal is precipitated) in order to exert the effect of the present invention. The “sample support member” or “both the sample support member and the matrix solution” is preferably kept at a low temperature.

  When a solution in which only a matrix is dissolved is used in a substantial final step for placing a substance on a plate, the solvent of the solution must be a solvent that can dissolve both the molecule to be measured and the matrix. preferable. This is because the molecule to be measured already existing in the lower layer can be dissolved and mixed with the matrix.

The method (1) preferably includes the following steps (A) and (B) as essential steps.
(A) a step of drying a solution of a sample containing a molecule to be measured on a plate, and thereafter
(B) The process of dripping the solution which melt | dissolved only the matrix in the solvent to the said plate

  In step (A), a sample solution containing the molecule to be measured is dried on a plate. The solution at this time only needs to contain the molecule to be measured, and may or may not contain the matrix. However, when the sample containing the molecule to be measured is extremely small, the good solvent of the sample containing the molecule to be measured is a poor solvent for the matrix. If the sample is unstable and cannot be stored or used as a stock solution or is difficult to handle, the preparation of the measurement sample is easy, the selection range of the solvent is widened, the decomposition of the sample and the adsorption to the container are suppressed Because of the possible points, it is preferable to dissolve only the sample containing the molecule to be measured without dissolving the matrix in the solution at this time.

  The solvent at that time is not particularly limited as long as the sample containing the molecule to be measured is dissolved. The solvent may not have a property of dissolving the matrix, and has a high boiling point at normal pressure and is evaporated (dried). The speed may be slow. If the measurement target molecule or the sample containing the measurement target molecule is water-soluble, the solvent used in the step (A) is particularly preferably water alone. If the sample containing the molecule to be measured is sugar, the solvent used in step (A) is most preferably water alone. Since the matrix is not dissolved, the solubility in the matrix may be low. Therefore, the most suitable solvent can be selected for the measurement target molecule or the sample containing the measurement target molecule.

  The concentration of the molecule to be measured in the solution in the step (A) is not particularly limited, but is preferably 1 amol / μL to 1 μmol / μL, particularly preferably 100 amol / μL to 1 nmol / μL. After completion of the step (A) (after drying), the thickness of the layer containing the molecule to be measured present on the plate is not particularly limited, but is preferably 5 μm or less, particularly preferably 1 μm or less. If the thickness of such a layer is too thick, when the matrix solution is dropped, it may not mix well with the matrix molecules, and the MS signal intensity may decrease.

  (1) In the method, since the solution of “sample containing the molecule to be measured” is first dropped on the plate, when the sample containing the molecule to be measured is very small, it is mixed with the matrix solution in a separate container and part of it. This is particularly useful because almost the entire amount of a small amount of sample can be subjected to analysis without being dropped on the plate. More useful when the sample containing the molecule to be measured is 1000 fmol or less, particularly useful when the sample is 300 fmol or less, and less than 100 fmol (for example, placing 100 fmol on a plate by dropping 1 μL of an aqueous solution dissolved in 100 fmol / μL) or less In this case, it is further useful.

  (1) In the method, after step (A), (B) a step of dropping a solution obtained by dissolving only the matrix in a solvent onto the plate is included. The meaning of “after step (A)” is not limited to “immediately after step (A)”, and another step may be inserted between step (A) and step (B). . Examples of the “other process” include a process (C) described later.

  In the step (B), a solution in which only the matrix is dissolved in the solvent is dropped onto the plate. By using a solvent that can dissolve both the measurement target molecule and the matrix, the lower measurement target molecule is transformed into the matrix. When mixed with molecules, the mixed crystals appear as the solvent evaporates.

<<< derivatization >>>
(1) In the method, between the step (A) and the step (B),
(C) It is preferable to perform a step of dropping a solution of a derivatizing agent that enhances ionization efficiency by reacting with a molecule to be measured onto the plate and drying the solution to further increase ionization efficiency.

  That is, (A) After the sample containing the molecule to be measured is placed on the sample support member and before (B) the matrix solution is placed, (C) the ionization efficiency is increased by reacting with the molecule to be measured. MALDI mass spectrometry, in which a step of dropping a solution of the derivatizing agent to be enhanced onto the sample support member and drying it, is preferable because the ionization efficiency is further increased.

  If a derivatizing agent that increases ionization efficiency by reacting with the molecule to be measured (hereinafter simply abbreviated as “derivatizing agent”) is reacted with the molecule to be measured in advance and then supplied to the plate, the loss of the sample to be measured May be connected. Accordingly, when mass spectrometry is applied to a small amount of “molecules derived from a living body or molecules in a biological sample” such as sugars, glycoproteins, glycopeptides, the process (C) between the process (A) and the process (B). It is particularly preferred to insert

  The derivatizing agent is not particularly limited as long as it enhances the ionization efficiency of the derivatized molecule to be measured, that is, the molecule to be subjected to mass spectrometry. The derivatizing agent is also preferably a compound having an effect as a matrix in MALDI mass spectrometry, or a compound further having a reactive functional group or a spacer portion described later.

  The chemical structure of such a derivatizing agent is not particularly limited as long as it exhibits the above effects, but a condensed polycyclic compound having a condensed polycycle such as naphthalene, anthracene, and pyrene in the molecule preferably exhibits the above effects. Therefore, it is particularly preferable. Here, the “condensed polycyclic compound” means a reactivity capable of binding a molecule to be measured with a condensed polycyclic moiety which may partially contain a heterocyclic ring containing nitrogen, sulfur or oxygen molecules. The compound which has a functional group and the spacer part which connects this condensed polycyclic part and this reactive functional group as needed. In particular, a compound having an aromatic ring is preferable.

  It is particularly preferable that the derivatizing agent is a substance that makes it possible to control the ionization cleavage position of the derivatized molecule, that is, the molecule subjected to mass spectrometry, by reacting with the molecule to be measured.

  The derivatizing agent preferably has a reactive functional group such as an amino group, a hydrazide group, a diazomethyl group, a succinimidyl ester group, a sulfonyl chloride group, or an iodo group (-I). As a particularly preferred derivatizing agent, specifically, a condensed polycyclic derivative compound in which the above group is bonded to a condensed polycycle such as a naphthalene ring, an anthracene ring, or a pyrene ring directly or through another group (spacer portion) Methyl iodide; diazomethane; trimethylsilyldiazomethane and the like.

  Of these, a pyrene ring compound is particularly preferable in terms of increasing ionization efficiency of a derivatized molecule, that is, a molecule subjected to mass spectrometry, and controlling the ionization cleavage position. Here, the “pyrene ring compound” means a pyrene ring, a reactive functional group that can be bonded to the “molecule to be measured”, and, if necessary, a spacer that connects the pyrene ring and the reactive functional group. And a compound having a moiety.

  Specifically, 1-pyrenebutanoic acid, hydrazide (hereinafter abbreviated as “PBH”), 1-pyreneacetic acid, hydrazide, 1-pyrenepropionic acid hydrazide ( 1-pyrenepropionic acid, hydrazide), 1-pyreneacetic acid, succinimidyl ester, 1-pyrenepropionic acid, succinimidyl ester, 1-pyrenepropionic acid, succinimidyl ester Sinimidyl ester (1-pyrenebutanoic acid, succinimidyl ester), N- (1-pyrenebutanoyl) cysteic acid succinimidyl ester (N- (1-pyrenebutanoyl) cysteic acid, succinimidyl ester), N- (1-pyrene) iodoacetamide (N- (1-pyrene) iodoacetamide), N- (1-pyrene) iodomaleimide (N- (1-pyrene) maleimide), N- (1-pyrenemethyl) Iodoacetamide (N- (1-pyrenemethyl) iodoacetamide), 1-pyrenemethyl iodoacetate, aminopyrene, 1-pyrenemethylamine, 1-pyrenepropylamine (3 -(1-pyrenyl) propylamine), 1-pyrenebutylamine (4- (1-pyrenyl) butylamine), 1-pyrenesµLfonyl chloride, 1-pyrenyldiazomethane (1-pyrenyldiazomethane) Preferred examples include 1-pyrenecarbaldehyde hydrazone, 1-pyrenylthiocyanate, 1-pyrenylisothiocyanate and the like. . Of these, PBH or PDAM is most preferred.

  Preferred examples of the derivatizing agent include those obtained by replacing the pyrene ring with a naphthalene ring or an anthracene ring in the above specific compound. Also preferred are methyl iodide, diazomethane or trimethylsilyldiazomethane.

  As a preferable “combination of a molecule to be measured and a derivatizing agent”, the molecule to be measured is a molecule having a sugar chain containing an aldehyde group, and the derivatizing agent has an amino group or a hydrazide group. Can be mentioned. In addition, as a preferable combination, there is a case where the molecule to be measured is a protein or glycoprotein having a carboxyl group, an amino group or a mercapto group, and the derivatizing agent has an amino group, a hydrazide group or a diazomethyl group. In addition, the measurement target molecule may be a protein or glycoprotein having a carboxyl group, and the derivatizing agent may be methyl iodide or trimethylsilyldiazomethane. In these combinations, the molecule to be measured and the derivatizing agent can be easily reacted on the plate, the ionization is not inhibited, the reaction is selective, and generally there is a high necessity for analysis in a small amount. Therefore, it is preferable from the viewpoint of easily achieving the effect.

<< (2) Method >>
(2) A method in which a sample containing a molecule to be measured and a matrix solution are simultaneously placed on a sample support member, the solution is dried, and the mixed crystal is precipitated on the sample support member is also preferable. The preparation method of the measurement sample of the method (2) is known as “Drived Droplet method”. In the preparation method (1), the method of placing the matrix solution on the sample support member and placing the sample on the sample support member before drying is also the same.

  Also in the method (2), by preliminarily cooling the matrix solution, and preferably by preliminarily cooling the matrix solution and the sample support member, formation of a crystal structure that can be a sweet spot can be promoted. it can. The preferable temperature of the matrix solution, the preferable temperature of the sample support member, the relationship between them and the “atmospheric temperature for preparing the measurement sample”, the temperature setting means, and the like are the same as in the case of the method (1) described above.

  The portion of the mixed crystal that can become a sweet spot shows an analysis result different from the standard product of the matrix when the mixed crystal is locally analyzed using an analysis method other than the MS analysis method. It can be detected as a mixed crystal of a sample containing molecules and a matrix. When the matrix is DHBA, the above “matrix standard product” means a state consisting mostly of stable crystals α crystals of DHBA. “A mixed crystal of a sample containing a matrix and a matrix” ”refers to a metastable crystal β crystal of DHBA.

  In the present invention, “crystallization conditions for precipitating a mixed crystal of a sample containing a molecule to be measured and a matrix” refers to temperature, supersaturation (concentration), solvent, pH, additive, interfacial tension, and the like. The influence, the control method, the preferable range, the reason, and the like are the same as in the above method (1).

  In the method (2), the molecule to be measured may be derivatized. The derivatization is the same as the method (1) described above.

<< General method for precipitation of mixed crystals of sample and matrix >>
In the method for preparing a measurement sample for MALDI mass spectrometry of the present invention, there is no particular limitation on the method for preparing a mixed crystal of a “sample containing the molecule to be measured” and a matrix. It is preferable to form a specific polymorphic structure that can be a sweet spot by controlling the crystallization conditions when the mixed crystal of the sample to be included and the matrix is precipitated. The formation of a specific polymorphic structure that can be a sweet spot in the mixed crystal is promoted.

  Regardless of the method of preparing the mixed crystal of the “sample containing the molecule to be measured” and the matrix, the “crystallization conditions” can be used in any of the methods (1), (2), and (3). The “control” is preferably performed by “control of temperature conditions during deposition”.

  In the present invention, the specific polymorphic structure that can be a sweet spot is formed by controlling the temperature condition of the sample support member when the solution is dried to precipitate the mixed crystal of the sample and the matrix at a temperature lower than room temperature. To promote.

  In the present invention, “room temperature” refers to the temperature (atmosphere temperature) of the place where the sample is prepared. The effect of the present invention is suitably achieved by keeping the temperature of the sample support member at a low temperature, and does not utilize the temperature difference from the place where the sample is prepared.

When the matrix is 2,5-dihydroxybenzoic acid (DHBA) and water is contained in the solvent, the temperature at which the mixed crystal of the sample containing the molecule to be measured and the matrix is precipitated is specifically, It is preferably 20 ° C. or lower, more preferably 15 ° C. or lower, particularly preferably 10 ° C. or lower, and further preferably 4 ° C. or lower. Moreover, it is necessary to be above the freezing point of the matrix solution.
“Temperature when precipitating mixed crystals” refers to the temperature of a solution immediately before and / or during precipitation on a plate in any “method for preparing a measurement sample”.

The principle of operation in which the effect of the present invention is achieved is considered as follows. However, the present invention is not limited only to a method for preparing a measurement sample in which the phenomenon described below occurs reliably.
That is, in the above temperature range, the saturation solubility of DHBA decreases depending on the temperature, the concentration gradient that becomes the driving force for matrix crystallization increases, and the DHBA solution becomes an unstable high energy state. Since DHBA crystallization proceeds kinetically, a large number of metastable crystal nuclei having a metastable type that can become sweet spots are formed.
When a large number of metastable crystal nuclei are formed, the DHBA crystal nuclei generated and the molecules to be measured interact to increase the number of target molecules incorporated into the matrix crystal. In particular, in the case of a target molecule having a low ionization efficiency, it is necessary that the target molecule is not ionized only by being adsorbed on the surface of the matrix crystal and is taken into the crystal. The target molecule is difficult to be taken into the crystal at the stage of crystal growth.

  Also, by keeping the temperature low, the viscosity of the matrix aqueous solution becomes high, the fluidity of the solution on the sample support member is suppressed, and the initial wetted area of the matrix aqueous solution on the sample support member is finally maintained. As a result, the sweet spot is formed at a fixed position of the support member.

  When precipitating the mixed crystal of the sample containing the molecule to be measured and the matrix, the temperature of the sample support member is preferably 20 ° C. or less, more preferably 15 ° C. or less, particularly preferably 10 ° C. or less, and further preferably 4 ° C. or less. is there. Moreover, it is preferable that a minimum is more than the freezing point of a matrix solution.

  The fact that the solvent for precipitating the mixed crystal of the sample containing the molecule to be measured and the matrix is water and / or acetonitrile can be widely used for sugars, proteins, peptides, glycoproteins, glycopeptides, nucleic acids, glycolipids, etc. Is preferable. In other words, the measurement target molecules such as sugars, proteins, peptides, glycoproteins, glycopeptides, nucleic acids, glycolipids, etc., which are extremely small molecules compared to the number of molecules in the matrix, are sufficiently dissolved, and at the same time It is particularly preferable in that a specific polymorphic structure that can be a spot can be rapidly produced and a molecule to be measured can be efficiently incorporated into a crystal. The effect described above is particularly exerted when water or acetonitrile, or a mixed solvent of water and acetonitrile. These are solvents that can easily form a specific polymorphic structure that can be a sweet spot, particularly when the matrix is DHBA.

The present invention is characterized in that a portion of a mixed crystal that can be a sweet spot is formed and laser desorption ionization is performed on a sample on which a large number of the sweet spots are formed.
There are no particular limitations on the type of matrix used in the present invention, and any matrix can be used as long as it is used in MALDI mass spectrometry. In particular, a substance that crystallizes in a needle shape keeps the temperature during crystallization low. This facilitates the incorporation of the measurement sample into a specific crystal structure that can be a sweet spot, and thus can be suitably used in the present invention.
The present invention is also the above-described measurement sample preparation method for MALDI mass spectrometry, wherein the matrix is a substance that crystallizes in a needle shape.
Among them, 2,5-dihydroxybenzoic acid (DHBA) is most preferable as the matrix.

  When the matrix is 2,5-dihydroxybenzoic acid (DHBA), the polymorphic structure that can be a sweet spot is a metastable crystalline β crystal. The “metastable crystal β crystal” is indicated as “Disordered” in Non-Patent Document 3, and is registered in the Cambridge database (CCDC No. BESKAL01, lattice constant a = 111.299 (2) Å, b = 11.830 (3) Å, c = 4.966 (4) Å, β = 90.50 (3) °) crystal structure.

The Raman spectrum of the “metastable crystal β crystal” of DHBA is the Raman spectrum of the stable crystal α crystal (observed in the “reference Raman spectrum” registered in the spectrum database of the National Institute of Advanced Industrial Science and Technology, 1199 cm ⁻ There is no strong signal such as around ¹ , 1307 cm ⁻¹ , or around 376 cm ⁻¹ , and there is a broad signal around 3265 cm ⁻¹ .

  According to the powder X-ray diffraction by CuKα ray, “metastable crystal β crystal” is 2θ = 11.0 degrees, 16.0 degrees, 17.5 degrees, 24.8 degrees, 28.3 degrees and 31.2. It has a diffraction peak characteristic to the degree, and gives the powder X-ray diffraction pattern shown in FIG.

  When the matrix is 2,5-dihydroxybenzoic acid (DHBA), the polymorphic structure that does not become a sweet spot is a stable crystalline α crystal. The “stable crystal α crystal” is a crystal structure that gives a Raman spectrum of DHBA registered in the spectrum database of the National Institute of Advanced Industrial Science and Technology, and is shown as “Ordered” in Non-Patent Document 3. Registered in the Cambridge database (CCDC No. BESKAL, lattice constant a = 23.945 (2) Å, b = 4.908 (1) Å, c = 5.621 (1) Å, β = 100.98 (0) °) Crystal structure.

  According to powder X-ray diffraction by CuKα ray, “stable crystal α crystal” is 2θ = 16.3 degrees, 19.8 degrees, 23.8 degrees, 27.3 degrees, 31.3 degrees and 37.0 degrees. Has a characteristic diffraction peak, and gives the powder X-ray diffraction pattern shown in FIG.

<Measurement molecule>
The molecule to be measured to which the MALDI mass spectrometry method of the present invention is applied is not particularly limited, but is preferably a molecule derived from a living body or a molecule in a biological sample, specifically, sugar, protein, peptide, glycoprotein Glycopeptides, nucleic acids, glycolipids, and the like are preferable because the effects of the present invention can be further exhibited. As a “molecule to be measured”, those prepared from natural products, those prepared by partially modifying natural products chemically or enzymatically, and those prepared chemically or enzymatically are also preferred. . Moreover, what has the partial structure of the molecule | numerator contained in the biological body, and the thing produced by imitating the molecule | numerator contained in the biological body are also preferable. Moreover, derivatization may already be performed before mounting on a plate.

  Further, the sample placed on the plate used for mass spectrometry, that is, the sample containing the molecule to be measured may be only the “measuring molecule” itself, or one containing the “measuring molecule”, for example, a tissue of a living body, It may be a cell, body fluid or secretion (eg, blood, serum, urine, semen, saliva, tears, sweat, stool, etc.). That is, a biological sample may be used directly. Alternatively, the molecule to be measured may be prepared by placing a sample on a plate and performing enzyme treatment or the like.

  The above-mentioned molecules are often obtained in a small amount of sample to be analyzed, and in particular, obtained by chemically or enzymatically liberating complex carbohydrates such as sugars, glycoproteins, glycolipids, etc. Since there are a plurality of isomers having the same molecular weight and composition, there are many cases where the sweet spot was not easily found and the measurement was abandoned, and the sweet spot prediction method of the present invention and MALDI mass spectrometry were Since the said effect is especially show | played with respect to the chemical structure analysis of those molecules, it is preferable.

<Mass spectrometer>
Examples of the laser used for ionization include a nitrogen laser (337 nm), a YAG laser triple wave (355 nm), an NdYAG laser (256 nm), a carbon dioxide gas laser (2940 nm), and a nitrogen laser is preferable. The ion separation and detection method is not particularly limited. Double focusing method, quadrupole focusing method (quadrupole (Q) filter method), tandem quadrupole (QQ) method, ion trap method, time of flight (TOF) ) Method or the like to separate and detect ionized molecules according to the mass / charge ratio (m / z). QIT-TOF is preferable.

Since molecules such as sugars, proteins, peptides, glycoproteins, glycopeptides, nucleic acids, and glycolipids contain many isomers having the same molecular weight and composition, this method improves ion generation efficiency and repeats molecular fragmentation n times (MS ⁿ method) is preferred. In the present invention, the MS ⁿ method (2 ≦ n) in which fragmentation is repeated n times is preferably applied to the detected “mixed crystal of sample and matrix containing the molecule to be measured”. For example, the binding position in the molecule can be determined by the MS ⁿ method.

<Action and principle>
In the present invention, the action / principle that an excellent sweet spot exists in the metastable crystal β crystal part is not clear, but it is also considered as follows. However, the present invention is not limited to the scope of the following actions and principles.
That is, in the “mixed crystal of the sample containing the molecule to be measured and the matrix” serving as the sweet spot, it is considered that the molecule to be measured is incorporated in a preferable state in order to efficiently desorb and ionize. In particular, when crystal nuclei are formed at the initial stage of matrix crystallization, if the temperature of the matrix solution is set lower, the formation of matrix crystal nuclei is promoted, and metastable crystal β crystals are crystallized in a wide range at the same time. This is considered to have a favorable influence on the formation.

  EXAMPLES Hereinafter, the present invention will be described more specifically with reference to examples and comparative examples. However, the present invention is not limited to these examples unless it exceeds the gist.

Reference example 1
<Drying at 23 ° C. on plate, detecting stable α crystal and metastable β crystal>
First, an aqueous solution in which a glycopeptide 1 (chemical structure is shown in FIG. 1), which is a molecule to be measured, is dissolved in water on a gold substrate plate (sample support member) obtained by depositing gold on a silicon substrate to 500 fmol / μL. 1 μL was added dropwise and allowed to dry at room temperature (23 ° C.) and atmospheric pressure.

  Next, a labeling reagent PDAM (1-pyryldiazomethan; manufactured by Molecular Probes), which is a derivatizing agent, is dissolved in DMSO (dimethylsulfoxide; manufactured by SIGMA), and 0.25 μL of a solution having a concentration of 2 nmol / μL is added dropwise to the heat block. Above, it was allowed to dry at 80 ° C. under atmospheric pressure. In order to remove the excess derivatizing agent on the plate, it was immersed in toluene (manufactured by SIGMA) to remove excess PDAM and dried sufficiently.

  Next, 0.75 μL of an aqueous solution in which high-purity DHBA (manufactured by Shimadzu Biotech) was dissolved in water to make 10 mg / mL was dropped, and the mixture was left to dry at room temperature (23 ° C.) at atmospheric pressure. A confocal laser scanning micrograph of this measurement sample deposit is shown in FIG.

  In order to obtain a Raman imaging image of the DHBA crystal on this plate (sample support member), Raman measurement was performed using an inVia Raman microscope (manufactured by Renishaw Co., Ltd.) under excitation laser conditions of a wavelength of 532 nm.

  Regarding the Raman spectrum of the “measurement sample deposit”, first, the spectrum patterns were compared by multivariate analysis. As a result, a unique Raman spectrum was obtained for each crystal of DHBA deposited on the plate (sample support member). And in the “measurement sample deposit”, the same Raman spectrum as the Raman spectrum of DHBA registered in the National Institute of Advanced Industrial Science and Technology (hereinafter referred to as “reference Raman spectrum”). Can be divided into at least two types of α crystal (also referred to as “stable crystal α crystal”) and β crystal (also referred to as “metastable crystal β crystal”) from which a Raman spectrum different from the reference Raman spectrum can be obtained. found.

  Note that the same Raman spectrum as the reference Raman spectrum is obtained from the recrystallized and purified powder (matrix standard product) that does not contain the molecule to be measured. FIG. 3 shows the Raman spectra obtained from the stable crystal α crystal and metastable crystal β crystal of the measurement sample precipitate.

Raman spectra of metastable crystalline β crystal is around reference 1199Cm ^-1 which has been observed in the Raman spectrum, 1307Cm around ^-1, a strong signal of like around 376Cm ^-1 had disappeared. Conversely, a broad signal was observed around 3265 cm ⁻¹ .

Of DHBA crystals of the "measurement sample precipitates" represents white stable crystalline α crystals with a strong signal in the vicinity of 1199cm ^-1, expressed in black metastable crystalline β crystal having no strong signal in the vicinity of 1199cm ^-1 A Raman imaging image is shown in FIG. That is, in FIG. 4, the crystal expressed in white is a stable crystal α crystal from which the same Raman spectrum as the reference Raman spectrum was obtained, and the crystal expressed in black is a Raman spectrum different from the reference Raman spectrum (FIG. 3 ( b)) is the obtained metastable crystalline β crystal.

  About the said "measurement sample deposit", the mass spectrum was acquired by the MALDI mass spectrometry. The measurement was performed using a MALDI-QIT-TOF type mass spectrometer (AXIMA-QIT, manufactured by Shimadzu Biotech) as a mass spectrometer. Measurement is performed in the positive ion mode, and the laser power is optimized over the entire area of the above “measurement sample precipitate” to the threshold value at which the ion signal of the molecule to be measured begins. It was measured.

As a result, it was not possible to detect the signal from “all the places where there are crystals” shown in FIG. 2, but from the metastable crystal β crystal which was not expressed in white but expressed in black (having no strong signal in the vicinity of 1199 cm ⁻¹ ). Only a strong signal could be detected. In addition, a particularly strong signal could be detected from the root on the circumference of the metastable crystal β crystal.

On the other hand, a strong signal could not be detected from anywhere in the stable crystal α crystal expressed in white (having a strong signal near 1199 cm ⁻¹ ). That is, there was no sweet spot in the portion of the stable crystal α crystal showing the same Raman spectrum as the reference Raman spectrum.

  FIG. 5 shows a mass spectrum obtained from the root on the circumference of the metastable crystal β crystal. As shown in FIG. 6, it was found that a measurement point (sweet spot) where a good MS signal was obtained was present in the β crystal, and a very good mass spectrum could be obtained from this crystal.

  Further, comparing FIG. 4 and FIG. 6, the correlation between the Raman imaging image and the position of the sweet spot became clearer. That is, the stable crystal α crystal that generates the same Raman spectrum as the reference Raman spectrum at the root of the metastable crystal β crystal that generates a Raman spectrum that is different from the reference Raman spectrum, which is displayed in black in FIG. Compared to (displayed in white), the amount of ions generated was significantly higher, indicating that it was an excellent sweet spot.

Example 1
<Deposit at 4 ° C. in a sealed container, confirming that it is metastable crystalline β crystal>
3 mL of acetonitrile was added to 200 mg of high-purity matrix DHBA (manufactured by LaserBio Labs) and dissolved by heating to 50 ° C. The insoluble material was filtered off while hot, then the solution was put in a sealed container, cooled to 4 ° C. and allowed to stand for 30 minutes, and then the precipitated needle-like white crystals were collected by filtration and washed with acetonitrile. Drying at room temperature gave a crystalline material.

In order to evaluate the crystal structure of the obtained sample, microscopic Raman spectroscopic analysis was performed on a plurality of points of the needle crystal. For the measurement, the measurement was performed at a laser wavelength of 532 nm and a laser intensity of 50 mW using a Holab SERIES 5000 type microscopic Raman spectroscopic analyzer manufactured by KAISER SYSTEM. A microscopic Raman spectrum of this crystalline sample is shown in FIG. As shown in FIG. 7, the acicular crystal obtained by the above method agrees very well with the Raman spectrum of the metastable crystal β crystal obtained in Reference Example 1 (FIG. 3B), and C = It was found that a signal around 1325 cm ⁻¹ derived from O and C═C stretching vibrations was observed very strongly, and a broad signal was shown around 3261 cm ⁻¹ . Unlike the reference Raman spectrum, this spectrum did not have a strong signal around 1199 cm ⁻¹ .

The Raman spectrum of this crystal shown in FIG. 7 was found to agree very well with the crystal of Reference Example 2 described below. Therefore, the polymorph of the needle crystal obtained in Example 1 is a metastable crystal β crystal having the same crystal structure as the needle crystal obtained in Reference Example 2.
Note that it is considered that the same crystal structure as that in the sealed container can be easily obtained on the sample support member if the temperature conditions are the same.

Reference example 2
<Deposit at 4 ° C. in a sealed container, confirming that it is metastable crystalline β crystal>
To 200 mg of high-purity matrix DHBA (manufactured by LaserBio Labs), 6 mL of a solvent in which chloroform and acetone were mixed at a volume ratio of 3: 1 was added and heated to 50 ° C. to dissolve. The insoluble material is filtered off while hot, then the solution is put into a sealed container, cooled to 4 ° C. and allowed to stand for 30 minutes, and then the precipitated white crystals are collected by filtration and washed with a solvent having the same composition as the mother liquor. did. It dried under room temperature and obtained the acicular crystalline substance.

In order to evaluate the Raman spectrum exhibited by this crystalline sample, microscopic Raman spectroscopic analysis was performed by the same method as in Example 1. The measurement result of the obtained Raman shift is shown in FIG. As shown in FIG. 8, the needle-like crystal obtained by the above method has a feature that a signal around 1325 cm ⁻¹ is observed very strongly like the crystal obtained in Example 1, and 3261 cm. ^A broad signal was found around ^-1 .

In order to evaluate the crystal structure of the obtained crystalline sample, powder X-ray diffraction using CuKα rays was measured.
For the powder X-ray diffraction measurement apparatus, XRD-6100 manufactured by Shimadzu Corporation was used, and CuKα ray was used as the X-ray source under the following measurement conditions.
Step width: 0.02 deg
Scan speed: 2.0 deg / min
Acceleration voltage: 40 kV
Acceleration current: 40 mA
Scanning range: 5-50deg

  The powder X-ray diffraction pattern of this crystalline sample is shown in FIG. As shown in FIG. 9, the diffraction angles (2θ) of this crystal are characteristically about 11.0 degrees, 16.0 degrees, 17.5 degrees, 24.8 degrees, 28.3 degrees, and 31. It had a peak twice. In the Cambridge database, a polymorph of a DHBA crystal indicated as “Disordered” in Non-Patent Document 3 is registered (CCDC No. BESKAL01). A good agreement was found.

Therefore, the needle-like crystal obtained in Reference Example 2 has a crystal structure indicated as “Disordered” in Non-Patent Document 3, and this needle-like crystal does not show a strong signal near 1900 cm ⁻¹ in the reference spectrum. It has been found.
Therefore, the “metastable crystal β crystal” has a crystal structure indicated as “Disordered” in Non-Patent Document 3.

  Furthermore, since the Raman spectrum obtained in Example 1 coincided with the Raman spectrum obtained in Reference Example 2, acicular crystals obtained by the method of Example 1 are described as “Disordered” in Non-Patent Document 3. Was found to be a metastable crystalline β crystal.

Comparative Example 1
<Confirmation that it is a stable crystal α crystal, precipitated at 25 ° C. in a sealed container>
3 mL of acetonitrile was added to 200 mg of high-purity matrix DHBA (manufactured by LaserBio Labs) and dissolved by heating to 50 ° C. The insoluble material was removed by filtration while hot, and the solution was placed in a sealed container and allowed to stand at 25 ° C. overnight at room temperature. The precipitated plate-like white crystals were collected by filtration and washed with acetonitrile. Drying at room temperature gave a crystalline material.

Next, micro Raman spectroscopic analysis was performed by the same method as in Example 1. The measurement result of the Raman shift is shown in FIG. As shown in FIG. 10, the plate-like crystal obtained by the above method showed a strong signal in the vicinity of 1199 cm ⁻¹ , showing good agreement with the reference Raman spectrum, and the quasi obtained in Example 1 and Reference Example 2 A Raman spectrum different from the measurement result of the stable crystal β crystal was shown.

  In order to evaluate the crystal structure of the crystalline sample obtained in Comparative Example 1, powder X-ray diffraction was measured by the same method as in Reference Example 2. The powder X-ray diffraction pattern of this crystal is shown in FIG.

  As shown in FIG. 11, the diffraction angles (2θ) of this crystal are characteristically about 16.3 degrees, 19.8 degrees, 23.8 degrees, 27.3 degrees, 31.3 degrees, and 37.0. It had a peak at a degree, and showed a different characteristic from the powder X-ray diffraction pattern (FIG. 9) of the crystal obtained in Reference Example 2.

In the Cambridge database, the polymorph of DHBA crystal shown as Ordered in Non-Patent Document 1 is registered (CCDC No. BESKAL), and the result is a very good agreement with the result of simulating powder X-rays from its theoretical structure. Was recognized. Therefore, it was confirmed that the crystal obtained in Comparative Example 1 had a crystal structure indicated as Ordered in Non-Patent Document 1.
Therefore, the “stable crystal α crystal” has a crystal structure indicated as “Ordered” in Non-Patent Document 1.

From the results of Example 1 and Comparative Example 1, regarding crystallization of DHBA using acetonitrile as a solvent, when it was cooled and precipitated in a short time, it became a crystallized form of Disordered (metastable crystal β crystal) (implementation) Example 1) When it was crystallized over a relatively long time at a temperature of room temperature or higher, it was found that an ordered (stable crystal α crystal) crystal form was obtained (Comparative Example 1).
Even on the sample support member, it is considered that the same crystal structure as that in the sealed container can be easily obtained if the temperature conditions are the same.

Example 2
<Drying at 15 ° C. on a plate, preferentially preparing metastable crystalline β crystals>
First, 1 μL of an aqueous solution in which a glycopeptide (chemical structure is shown in FIG. 1), which is a molecule to be measured, is dissolved in water and made 100 fmol / μL is dropped on a plate for mass spectrometry (sample support member), and room temperature (23 C.) and left to dry under atmospheric pressure.

  Next, a labeling reagent PDAM (1-pyrenyldiazomethan; manufactured by Molecular Probes), which is a derivatizing agent, is dissolved in DMSO (dimethylsulfoxide; manufactured by SIGMA), and 0.25 μL of a solution made to 10 nmol / μL is added dropwise to the heat block. Above, it was allowed to dry at 80 ° C. under atmospheric pressure. In order to remove excess derivatizing agent, it was immersed in xylene (manufactured by SIGMA) to remove excess PDAM and dried sufficiently.

  Next, an aqueous solution prepared by dissolving high-purity DHBA (manufactured by Shimadzu Biotech) in 60% acetonitrile aqueous solution to 10 mg / mL is cooled to 4 ° C., and 1.00 μL is dropped on a plate set at 15 ° C. in advance as a matrix. did. Next, the solvent was dried under atmospheric pressure while maintaining the plate temperature at 15 ° C., and the matrix was crystallized. That is, this condition corresponds to a condition for advantageously producing the metastable crystal β crystal by lowering the temperature of the plate and the matrix solution. In a dry environment, it is better to cool to a lower temperature, but it is preferable not to cool below the temperature at which the plate causes condensation.

  About this measurement sample, the mass spectrum was acquired by the MALDI mass spectrometry. The measurement was performed in positive ion mode using a MALDI-QIT-TOF type mass spectrometer (AXIMA-QIT, manufactured by Shimadzu Biotech) as a mass spectrometer. Further, the laser power was optimized over the entire area of the measurement sample to a threshold value at which the signal of the ion of the molecule to be measured begins, and then laser irradiation was performed at 50 μm intervals for automatic measurement.

  The obtained mass spectrum is shown in FIG. 12 (a), and FIG. 13 (a) shows a position where a glycopeptide ion signal is detected in the measurement sample and a confocal laser micrograph of the measurement sample. Further, the signal intensity shown in FIG. 13 (a) was evaluated in 5 stages, and the number of detection points of signals corresponding to the top 4 stages was tabulated and shown in Table 1.

Comparative Example 2
<Drying at 23 ° C. on plate, detecting stable α crystal and metastable β crystal>
In the same manner as in Example 2, a glycopeptide (chemical structure is shown in FIG. 1), which is a molecule to be measured, is dissolved in water, 1 μL of an aqueous solution made to 100 fmol / μL is dropped, dried, and derivatized with PDAM. After that, it was immersed in xylene (manufactured by SIGMA) to remove excess PDAM and sufficiently dried. The same operation as in Example 2 was performed until the DHBA solution was dropped.

  Next, 1.00 μL of an aqueous solution prepared by dissolving high-purity DHBA (manufactured by Shimadzu Biotech) in 60% acetonitrile aqueous solution to 10 mg / mL is dropped, and left at room temperature (23 ° C.) under atmospheric pressure. Dried.

  About this measurement sample, the mass spectrum was acquired by the MALDI mass spectrometry similar to Example 2. FIG. The measurement was performed using a MALDI-QIT-TOF type mass spectrometer (AXIMA-QIT, manufactured by Shimadzu Biotech) as a mass spectrometer. The measurement was performed in positive ion mode. After optimizing the laser power over the entire area of the measurement sample to the threshold value at which the ion signal of the molecule to be measured begins to appear, laser irradiation was performed at 50 μm intervals and automatic measurement was performed.

  The obtained mass spectrum is shown in FIG. 12 (b), and FIG. 13 (b) shows the position where the glycopeptide ion signal was detected in the measurement sample and the confocal laser micrograph of the measurement sample. Further, the signal intensity shown in FIG. 13 (b) was evaluated in 5 stages, and the number of detection points of signals corresponding to the top 4 stages was tabulated and shown in Table 2.

<Results of Example 2 and Comparative Example 2>
Comparing the mass spectra of FIGS. 12 (a) and 12 (b), the signal intensity of the molecule to be measured is clearly about twice as good as that of FIG. 12 (a). Further, comparing FIGS. 13 (a) and (b), in any case, there is a tendency that a signal is obtained from the root on the circumference, but the point in FIG. 13 (a) is clearly detected. There were many. That is, the measurement sample prepared in Example 2 can obtain a good signal from most crystals on the circumference, but the measurement sample prepared in Comparative Example 2 has a signal generated from only a part of the crystal. There wasn't.

  Furthermore, when comparing Table 1 and Table 2, the number of points detected in the whole measurement sample is about 3 times more in the case of Example 2 (Table 1) than in the case of Comparative Example 2 (Table 2), It is also clear that high intensity signals can be obtained at many measurement points.

Comparative Example 3
<Drying at 25 ° C. on a plate and preferentially preparing stable crystalline α crystals>
In the same manner as in Example 2, the glycopeptide (chemical structure is shown in FIG. 1), which is a molecule to be measured, was dissolved in water, 1 μL of an aqueous solution adjusted to 100 fmol / μL was dropped, dried, and derivatized with PDAM. Then, it was immersed in xylene (manufactured by SIGMA) to remove excess PDAM and sufficiently dried. The same operation as in Example 2 was performed until the DHBA solution was dropped.

  Next, 1.00 μL of an aqueous solution (40 ° C.) prepared by dissolving high-purity DHBA (manufactured by Shimadzu Biotech) in 60% acetonitrile aqueous solution to 10 mg / mL was dropped onto the plate as a matrix. Next, the solvent was dried at room temperature (25 ° C.) under atmospheric pressure to crystallize the matrix. That is, this condition corresponds to the condition for advantageously forming the stable crystal α-crystal of Comparative Example 1.

  With respect to this measurement sample, a mass spectrum was obtained by MALDI mass spectrometry similar to that of Example 2, and the result was similar to that of Comparative Example 2. The measurement was performed using a MALDI-QIT-TOF type mass spectrometer (AXIMA-QIT, manufactured by Shimadzu Biotech) as a mass spectrometer. The measurement was performed in positive ion mode. After optimizing the laser power over the entire area of the measurement sample to the threshold value at which the ion signal of the molecule to be measured begins to appear, laser irradiation was performed at 50 μm intervals and automatic measurement was performed.

  FIG. 14 shows a position where a glycopeptide ion signal was detected in the measurement sample and a confocal laser micrograph of the measurement sample. As shown in FIG. 14, when a matrix solution heated to about 40 ° C. was dropped on the plate, the uniform evaporation rate of the solvent was not maintained due to the high evaporation rate of the solvent, and the evaporation of the solvent was biased. . For this reason, locations other than the circumference became the starting point for crystallization of DHBA, and the shape of the matrix crystal was not reproducible.

  Further, the signal intensity of the obtained mass spectrum was the same as in Comparative Example 2. The signal intensity shown in FIG. 14 was evaluated in five stages by the same method as in Example 2, and the result of totaling the number of signal points corresponding to the top four stages was similar to that in Comparative Example 2.

Example 3
<With PDAM label / plate cooling / matrix solution cooling>
First, 1 μL of an aqueous solution in which a glycopeptide (chemical structure is shown in FIG. 1), which is a molecule to be measured, is dissolved in water and made 100 fmol / μL is dropped on a plate for mass spectrometry (sample support member), and room temperature (23 C.) and left to dry under atmospheric pressure.

  Next, a labeling reagent PDAM (1-pyryl diazomethan; manufactured by Molecular Probes), which is a derivatizing agent, is dissolved in DMSO (dimethyl sulfoxide; manufactured by SIGMA), and 0.25 μL of a solution made to 10 nmol / μL is dropped to the heat block. Above, it was left to dry at 70 ° C. under atmospheric pressure. In order to remove excess derivatizing agent, it was immersed in xylene (manufactured by SIGMA) to remove excess PDAM and dried sufficiently.

  Next, an aqueous solution prepared by dissolving high-purity DHBA (manufactured by Shimadzu Biotech) in 60% acetonitrile aqueous solution to 10 mg / mL was cooled to 4 ° C., and 1 μL thereof was dropped onto a plate set at 15 ° C. in advance as a matrix. Next, the solvent was dried under atmospheric pressure while keeping the temperature of the plate at 15 ° C. or lower, and the matrix was crystallized. That is, this condition corresponds to a condition for advantageously producing the metastable crystal β crystal by lowering the temperature of the plate and the matrix solution. In a dry environment, it is better to cool to a lower temperature, but it is preferable not to cool below the temperature at which the plate causes condensation.

  About this measurement sample, the mass spectrum was acquired by the MALDI mass spectrometry similar to Example 1. FIG.

  The obtained mass spectrum is shown in FIG. 15 (a), and FIG. 17 (a) shows a position where a glycopeptide ion signal was detected in the measurement sample and a confocal laser micrograph of the measurement sample.

Comparative Example 4
<With PDAM label / Plate room temperature / Matrix solution room temperature>
In the same manner as in Example 2, a glycopeptide (chemical structure is shown in FIG. 1), which is a molecule to be measured, is dissolved in water, 1 μL of an aqueous solution made to 100 fmol / μL is dropped, dried, and labeled reagent PDAM is used. Reaction was performed and washing was performed. The same operation as in Example 3 was performed until the DHBA solution was dropped.

  Next, 1 μL of an aqueous solution prepared by dissolving high-purity DHBA (manufactured by Shimadzu Biotech) in 60% acetonitrile aqueous solution to 10 mg / mL is dropped, and left at room temperature (23 ° C.) under atmospheric pressure to dry the solvent. It was.

  About this measurement sample, the mass spectrum was acquired by the MALDI mass spectrometry similar to Example 3. FIG.

  The obtained mass spectrum is shown in FIG. 15 (b), and FIG. 17 (b) shows the position where the glycopeptide ion signal was detected in the measurement sample and the confocal laser micrograph of the measurement sample.

<Results of Example 3 and Comparative Example 4>
Comparing the mass spectra of FIG. 15 (a) and FIG. 15 (b), the signal intensity of the molecule to be measured is clearly better by about 3 times in FIG. 15 (a). Further, when FIG. 17A is compared with FIG. 17B, a signal tends to be obtained from the root portion of the crystal along the circumference of the sample support member in any case, but FIG. ) Clearly had a large number of detection points. That is, in the measurement sample prepared in Example 3, the base portion of the majority of crystals from which a good signal is obtained is formed along the circumference of the sample support member, and the crystal portion can be shortened by laser irradiation. Although a good signal was obtained with time, in the case of the measurement sample prepared in Comparative Example 4, it was difficult to obtain a good signal even when the edge of the crystal was irradiated with laser.

Example 4
<With PDAM label / Plate cooling / Matrix solution at room temperature>
As in Example 3, 1 μL of an aqueous solution in which a glycopeptide (chemical structure is shown in FIG. 1), which is a molecule to be measured, is dissolved in water and made 100 fmol / μL is dropped, dried, and labeled reagent PDAM is used. Reaction was performed and washing was performed. The same operation as in Example 3 was performed until the DHBA solution was dropped.

  Next, 1 μL of a room temperature aqueous solution in which high purity DHBA (manufactured by Shimadzu Biotech) was dissolved in 60% acetonitrile aqueous solution to 10 mg / mL was dropped onto a plate set at 15 ° C. in advance. Next, the solvent was dried under atmospheric pressure while keeping the temperature of the plate at 15 ° C. or lower, and the matrix was crystallized.

  About this measurement sample, the mass spectrum was acquired by the MALDI mass spectrometry similar to Example 3. FIG.

  The obtained mass spectrum is shown in FIG. 16 (c), and FIG. 18 (c) shows the position where the glycopeptide ion signal was detected in the measurement sample and the confocal laser micrograph of the measurement sample.

Example 5
<With PDAM label / Plate room temperature / Matrix solution cooling>
As in Example 3, 1 μL of an aqueous solution in which a glycopeptide (chemical structure is shown in FIG. 1), which is a molecule to be measured, is dissolved in water and made 100 fmol / μL is dropped, dried, and labeled reagent PDAM is used. Reaction was performed and washing was performed. The same operation as in Example 3 was performed until the DHBA solution was dropped.

  Next, an aqueous solution prepared by dissolving high-purity DHBA (manufactured by Shimadzu Biotech) in 60% acetonitrile aqueous solution to 10 mg / mL was cooled to about 20 ° C. under zero, and then 1 μL was dropped on a plate kept at room temperature. 23 ° C.) and left under atmospheric pressure to dry the solvent.

  About this measurement sample, the mass spectrum was acquired by the MALDI mass spectrometry similar to Example 3. FIG.

  The obtained mass spectrum is shown in FIG. 16 (d), and FIG. 18 (d) shows the position where the glycopeptide ion signal was detected in the measurement sample and the confocal laser micrograph of the measurement sample.

<Results of Example 4 and Example 5>
When the mass spectra of FIG. 16 (c) and FIG. 16 (d) are compared with FIG. 15 (a) and FIG. 15 (b), the signal intensity of the measurement target molecule is as shown in FIG. 15 (a)> FIG. 16 (c)>. High and favorable in the order of FIG. 16 (d)> FIG. 15 (b). Further, comparing FIGS. 17 and 18 (a) to (d), the number of sweet spots is in the order of FIG. 17 (a)> FIG. 18 (c)> FIG. 18 (d)> FIG. 17 (b). There were many. That is, the measurement samples prepared in Examples 4 and 5 are not as effective as cooling both of the plates or the matrix solution, but they are not as effective as when the samples are prepared at normal room temperature. Gives a good signal.

  In both Example 3 and Comparative Example 4, measurement was performed after labeling the glycopeptide, which is the molecule to be measured, with PDAM, which is the derivatizing agent. Differences were observed with or without PDAM labeling. That is, even when a derivatizing agent (labeling reagent) is not used, when DHBA is crystallized by setting to 15 ° C., compared to the case where DHBA is crystallized by setting to 23 ° C., The MS signal intensity improved more than twice.

Example 6
<Plate cooling / Matrix solution cooling>
First, in a plate for mass spectrometry (sample support member) (manufactured by Shimadzu Corp., 384 well sample plate), a glycopeptide (chemical structure is shown in FIG. 1) as a molecule to be measured is dissolved in water to 100 fmol / μL. 1 μL of the prepared aqueous solution was dropped, and the mixture was left to dry at room temperature (23 ° C.) and atmospheric pressure.

  Next, an aqueous solution prepared by dissolving high-purity DHBA (manufactured by Shimadzu Biotech) in 60% acetonitrile aqueous solution to 10 mg / mL was kept in a refrigerator at 4 ° C. It was dripped. Next, the solvent was dried under atmospheric pressure while keeping the temperature of the plate at 15 ° C. or lower, and the matrix was crystallized. That is, this condition corresponds to a condition for advantageously producing the metastable crystal β crystal by lowering the temperature of the plate and the matrix solution. In a dry environment, it is better to cool to a lower temperature, but it is preferable not to cool below the temperature at which the plate causes condensation.

  About this measurement sample, the mass spectrum was acquired by the MALDI mass spectrometry. The measurement was performed in positive ion mode using a MALDI-QIT-TOF type mass spectrometer (AXIMA-QIT, manufactured by Shimadzu Biotech) as a mass spectrometer. Moreover, after optimizing the laser power with respect to the whole area of the measurement sample to the threshold value at which the ion signal of the molecule to be measured begins, laser irradiation was performed at 50 μm intervals, and the entire measurement sample was automatically measured.

  The obtained mass spectrum is shown in FIG. 19 (a), and FIG. 20 (a) shows the position where the signal of glycopeptide ions was remarkably detected in the measurement sample and the confocal laser micrograph of the measurement sample. Furthermore, the signal intensity shown in FIG. 20 (a) was evaluated in 5 stages, and the number of detection points of signals corresponding to the top 4 stages was tabulated and shown in Table 3.

Comparative Example 5
<Plate room temperature / Matrix solution room temperature>
In the same manner as in Example 6, 1 μL of an aqueous solution in which a glycopeptide (chemical structure is shown in FIG. 1) as a measurement target molecule was dissolved in water to make 100 fmol / μL was dropped and dried. The same operation as in Example 6 was performed until the DHBA solution was dropped.

  Next, 1 μL of an aqueous solution prepared by dissolving high-purity DHBA (manufactured by Shimadzu Biotech) in 60% acetonitrile aqueous solution to 10 mg / mL is dropped, and left at room temperature (23 ° C.) under atmospheric pressure to dry the solvent. It was.

  About this measurement sample, the mass spectrum was acquired by the MALDI mass spectrometry similar to Example 6. FIG.

  The obtained mass spectrum is shown in FIG. 19 (b), and FIG. 20 (b) shows the position where the glycopeptide ion signal was detected in the measurement sample and the confocal laser micrograph of the measurement sample. Furthermore, the signal intensity shown in FIG. 20 (b) was evaluated in five stages, and the number of detection points of signals corresponding to the top four stages was tabulated and shown in Table 4.

<Results of Example 6 and Comparative Example 5>
Comparing the mass spectra of FIG. 19 (a) and FIG. 19 (b), the signal intensity of the molecule to be measured is clearly about twice as good as that of FIG. 19 (a). Furthermore, comparing FIG. 20 (a) and FIG. 20 (b), in any case, there is a tendency that a good signal is obtained from the base portion of the crystal, but clearly FIG. 20 (a) is detected. There were a lot of points. That is, the measurement sample prepared in Example 6 can obtain a good signal from the base portion of most of the crystals, but in the case of the measurement sample manufactured in Comparative Example 5, the signal is generated only from the base portion of some crystals. Did not occur. In addition, when the detection limit of the mass spectrum obtained by the automatic analysis is S / N> 3, in FIG. 20A of Example 6, it was possible to detect up to 10 fmol of the measurement sample. In 20 (b), 50 fmol could not be detected.

  Furthermore, when comparing Table 3 and Table 4, the number of points detected in the whole measurement sample was about twice as large in the case of Example 6 (Table 3) than in the case of Comparative Example 5 (Table 4), It is also clear that a strong signal can be obtained at about twice as many measurement points.

Example 7
<Plate without well boundary / With PDAM label / Plate cooling / Matrix solution cooling>
First, an aqueous solution in which a glycopeptide (chemical structure shown in FIG. 1), which is a molecule to be measured, is dissolved in water on a flat surface without a well boundary of a plate for mass spectrometry (sample support member) to 100 fmol / μL. 1 μL was dropped, dried, reacted with a labeling reagent PDAM, and washed. The same operation as in Example 3 was performed until the DHBA solution was dropped.

  Next, an aqueous solution prepared by dissolving high-purity DHBA (manufactured by Shimadzu Biotech) in 60% acetonitrile aqueous solution to 10 mg / mL was cooled to 4 ° C., and 1 μL thereof was dropped onto a plate set at 15 ° C. in advance as a matrix. Next, the solvent was dried under atmospheric pressure while keeping the temperature of the plate at 15 ° C. or lower, and the matrix was crystallized. That is, this condition corresponds to a condition for advantageously producing the metastable crystal β crystal by lowering the temperature of the plate and the matrix solution. In a dry environment, it is better to cool to a lower temperature, but it is preferable not to cool below the temperature at which the plate causes condensation.

  About this measurement sample, the mass spectrum was acquired by the MALDI mass spectrometry similar to Example 3. FIG.

  The obtained mass spectrum is shown in FIG. 21 (a), and FIG. 22 (a) shows the signal position of the glycopeptide ion detected in the automatic analysis and the confocal laser micrograph of the measurement sample.

Comparative Example 6
<Unbounded plate / PDAM label / Plate room temperature / Matrix solution room temperature>
Similarly to Example 7, a glycopeptide (chemical structure is shown in FIG. 1), which is a molecule to be measured, is dissolved in water, and an aqueous solution having a concentration of 100 fmol / μL is free from the boundary of the mass spectrometry plate (sample support member) 1 μL was dropped on a flat surface, dried, reacted with a labeling reagent PDAM, and washed. The same operation as in Example 7 was performed until the DHBA solution was dropped.

  Next, 1 μL of an aqueous solution prepared by dissolving high-purity DHBA (manufactured by Shimadzu Biotech) in 60% acetonitrile aqueous solution to 10 mg / mL is dropped, and left at room temperature (23 ° C.) under atmospheric pressure to dry the solvent. It was.

  About this measurement sample, the mass spectrum was acquired by the MALDI mass spectrometry similar to Example 1. FIG.

  The obtained mass spectrum is shown in FIG. 21 (b), and FIG. 22 (b) shows the position where the glycopeptide ion signal was detected in the measurement sample and the confocal laser micrograph of the measurement sample.

<Results of Example 7 and Comparative Example 6>
When comparing the mass spectra of FIG. 21 (a) and FIG. 21 (b), the signal intensity of the molecule to be measured is clearly about twice as good as that of FIG. 21 (a). Furthermore, comparing FIG. 22 (a) and FIG. 22 (b), in any case, a signal tends to be obtained from the base portion of the crystal, but in the case of FIG. 22 (a), it is clearly detected. There were many sweet spots. That is, in the measurement sample produced in Example 7, the number of sweet spots at the edge of the crystal was controlled as a result of controlling the formation position of the mixed crystal by setting the plate temperature during crystallization of the matrix to 15 ° C. Increased. Comparing Example 3 and Example 7, the effect of this matrix crystal on sweet spot formation is due to the temperature of the support member and the matrix solution being controlled to a temperature lower than room temperature. It is clear that it is unrelated to general boundaries.

Example 8
<Peptide mixture / plate cooling / matrix solution cooling>
First, 20 μg of bovine serum albumin (manufactured by Sigma-Aldrich) was added to sodium 3-{(2-methyl-2-undecyl-1,3-dioxol-4-yl) methyl} -1-propansulfonate as a surfactant. RapiGest SF (manufactured by Waters)) was added to a final concentration of 0.1% and heated at 100 ° C. for 5 minutes. After cooling, it was dissolved in an aqueous solution containing 10 mmol / L ammonium bicarbonate and 10 mmol / L dithiothreitol and incubated at 55 ° C. for 45 minutes.
After the reaction, 5 μL of 135 mmol / L iodoacetamide was added and allowed to stand in the dark for 45 minutes. Next, 1 μg of trypsin was added and reacted at 37 ° C. overnight.

  Cellulose powder is added to the solution after the reaction, followed by adsorption and elution, followed by concentration and purification. Then, 1 μL of the resulting solution is dropped on a plate for mass spectrometry (sample support member), at room temperature (23 ° C.) and under atmospheric pressure. Left to dry.

  The measurement sample was crystallized by the same operation as in Example 1, and a mass spectrum in the positive ion mode was obtained by MALDI mass spectrometry.

  FIG. 23A shows the positions of about 25 points showing high values among the peptide ion signals detected in the automatic analysis, and confocal laser scanning micrographs of the measurement samples.

Comparative Example 7
<Peptide mixture / plate room temperature / matrix solution room temperature>
In the same manner as in Example 8, the peptide mixture, which is the molecule to be measured, was dissolved in water, and 1 μL was dropped onto the plate for mass spectrometry and dried. The same operation as in Example 8 was performed until the DHBA solution was dropped.

  Next, 1 μL of an aqueous solution prepared by dissolving high-purity DHBA (manufactured by Shimadzu Biotech) in 60% acetonitrile aqueous solution to 10 mg / mL is dropped, and left at room temperature (23 ° C.) under atmospheric pressure to dry the solvent. It was.

  About this measurement sample, the mass spectrum was acquired by the MALDI mass spectrometry method similar to Example 8. FIG.

  FIG. 23B shows the positions of about 25 points showing high values among the peptide ion signals detected in the automatic analysis, and confocal laser micrographs of the measurement samples.

<Results of Example 8 and Comparative Example 7>
Comparing FIG. 23 (a) and FIG. 23 (b), in the case of FIG. 23 (a), the detected sweet spots are clearly distributed along the edge of the matrix crystal. That is, in the measurement sample prepared in Example 8, the sweet spot of all peptides was uniformly positioned by setting the plate temperature during crystallization of the matrix to 15 ° C. Therefore, if a laser is irradiated along the edge of the matrix crystal, the sweet spots of all peptides can be easily detected simultaneously, and an MS spectrum can be acquired efficiently.
On the other hand, in Comparative Example 7, the sweet spot of each peptide is not necessarily present at the edge of the matrix crystal, and the distribution differs depending on the type of peptide, so it is difficult to determine the sweet spot in advance, In order to acquire a high MS spectrum, it is necessary to measure the entire measurement sample.

<Summary of Examples, Comparative Examples, and Reference Examples>
Therefore, in the present invention, in order to obtain a good MS spectrum in sample preparation for MALDI mass spectrometry, it is effective to form a crystal polymorph of a matrix that becomes a large number of sweet spots in the measurement sample. The difference in whether a crystal becomes a sweet spot depends on the difference in the crystal polymorphism of the matrix.In addition, rather than rapidly drying the solvent of the matrix solution, the method of lowering the temperature of the plate and the solution is the sweet spot. It proved to be most effective with respect to spot formation.

The method for preparing a measurement sample for MALDI mass spectrometry, in which a mixed crystal of a sample containing a molecule to be measured and a matrix according to the present invention is precipitated and ionization efficiency is increased by utilizing a specific polymorphic structure of the mixed crystal is short. It is possible to obtain high-quality MS spectra efficiently in time, enabling high-sensitivity MS ⁿ (2 ≦ n) analysis necessary for structure identification, and further enabling application to automatic analysis. The present invention is widely used in all analysis fields using spectra, particularly in the field of biological analysis where only a small amount of sample may be available.

Claims (10)

  In MALDI mass spectrometry, a mixed crystal of a sample containing a molecule to be measured and a matrix is precipitated, and a specific polymorphic structure of the mixed crystal is used to increase ionization efficiency. Measurement sample preparation method.   In MALDI mass spectrometry, the crystallization conditions for precipitating the mixed crystal of the sample containing the molecule to be measured and the matrix are controlled, and a specific polymorphic structure that can be a sweet spot is formed in the mixed crystal. The method for preparing a measurement sample for MALDI mass spectrometry according to claim 1, wherein the ionization efficiency is increased by utilizing a specific polymorphic structure to be obtained.   The MALDI mass spectrometry according to claim 1 or 2, wherein the matrix is 2,5-dihydroxybenzoic acid (DHBA), and the polymorphic structure that can be a sweet spot is a metastable crystalline β crystal. Preparation method for legal measurement sample.   In MALDI mass spectrometry, a specific polymorphic structure that can be a sweet spot is formed in the mixed crystal by controlling the temperature conditions when the mixed crystal of the sample containing the molecule to be measured and the matrix is precipitated. The measurement sample preparation method for MALDI mass spectrometry according to any one of claims 1 to 3.   In MALDI mass spectrometry, a sample containing a molecule to be measured is placed on a sample support member and then a matrix solution is placed, or a sample containing a molecule to be measured and a matrix solution are placed on a sample support member at the same time, 2. A specific polymorphic structure capable of forming a sweet spot in the mixed crystal is formed by controlling a temperature condition when the mixed crystal of the sample and the matrix is precipitated by drying the solution. The measurement sample preparation method for MALDI mass spectrometry according to any one of claims 4 to 5.   The matrix is 2,5-dihydroxybenzoic acid (DHBA), and the temperature at which the mixed crystal of the sample containing the molecule to be measured and the matrix is precipitated is set in the range of the freezing point of the matrix solution to 15 ° C. or less. The method for preparing a measurement sample for MALDI mass spectrometry according to any one of claims 1 to 5, wherein:   2. In MALDI mass spectrometry, the temperature of the sample support member is set in a range from the freezing point of the matrix solution to 15 ° C. or less when the mixed crystal of the sample containing the molecule to be measured and the matrix is precipitated. The measurement sample preparation method for MALDI mass spectrometry according to any one of claims 6 to 6.   The MALDI mass spectrometry according to any one of claims 1 to 7, wherein the solvent for precipitating the mixed crystal of the sample containing the molecule to be measured and the matrix is water and / or acetonitrile. Preparation method for legal measurement sample.   After the sample containing the molecule to be measured is placed on the sample support member and before placing the matrix solution, a solution of the derivatizing agent that increases the ionization efficiency by reacting with the molecule to be measured is placed on the sample support member. The method for preparing a measurement sample for MALDI mass spectrometry according to any one of claims 1 to 8, wherein a step of dropping and drying is inserted.   A method for increasing ionization efficiency using a measurement sample for MALDI mass spectrometry prepared by using the measurement sample preparation method for MALDI mass spectrometry according to any one of claims 1 to 9. MALDI mass spectrometry.