JP5837273B2 - Measurement sample for mass spectrometry and preparation method thereof - Google Patents

Description

  The present invention relates to a method for preparing a measurement sample for mass spectrometry, and more particularly to a method for preparing a measurement sample for mass spectrometry having a measurement sample surface having a specific structure.

  “Mass spectrometry (hereinafter sometimes abbreviated as“ MS ”)” means that a sample containing a molecule to be measured is ionized and ions derived from the molecule to be measured are mass-to-charge (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.

  Analysis of saccharides using MALDI-MS is considered to be difficult compared to peptides and proteins. One of the fundamental reasons is that the efficiency of ionization of saccharides is remarkably worse than that of peptides and the like, and high-sensitivity analysis is difficult. It has been reported that the ionization efficiency of a sample in MALDI-MS varies depending on various factors such as a matrix to be used, an additive to the matrix, a solvent, and the like. In addition, since it is affected by the properties of the sample itself, ionization efficiency can be improved by derivatizing the sample molecules.

  The ionization efficiency of saccharides and the like in MALDI-MS is significantly worse than that of peptides and the like. For example, when analyzing a small amount of sugar chain extracted from a living body, the hydroxyl group of the sugar chain is methylated to improve sensitivity. A technique and a technique of derivatizing the reducing end of the sugar chain are generally used. Examples include Non-Patent Documents 1, 2 and 3, and Patent Documents 1 and 2. Although the mechanism of sensitivity improvement by derivatization has not been completely elucidated, generally, a hydrophobic functional group such as an aromatic ring or an ionic functional group is introduced to improve sensitivity. Further, as an approach different from derivatization, special matrices have been developed to measure hydrophilic sugar chains with high sensitivity. Examples include Non-Patent Documents 4 and 5 and Patent Documents 3 and 4.

  Thus, since the ionization mechanism of MALDI has not been completely elucidated, selection of a matrix used for measurement, sample preparation method, and the like are often based on empirical knowledge. In general, it is considered that the matrix and the sample need to be mixed well and become a mixed crystal. However, it is non-uniform because it becomes a solid crystal, and ions derived from the measurement target molecule are not obtained from all the places where the crystal is generated, and only when a very small part of the generated crystal is irradiated with a laser Ions are obtained.

  This part is called “sweet spot”, and when measuring in MALDI, it is necessary to search for the sweet spot first to obtain a high-quality mass spectrum. Whether it is a part or not can only be searched by experience. Even if a sweet spot is identified and laser irradiation is performed on it, the sample prepared by the conventional sample preparation method has a low ion generation amount. The only measure was using a matrix.

  Such derivatization is performed prior to mass spectrometry, but the current situation is that the operation of the reaction and excessive reagent removal are included in multiple steps, thereby reducing the mass of the sample containing the molecule to be measured. .

On the other hand, in the case of a sugar chain or the like having a plurality of isomers having the same molecular weight and composition, the ion derived from the measurement target molecule is selected and fragmented as a precursor ion, and the generated ion is further selected as a precursor ion and fragmented. Repeated MS ⁿ (2 ≦ n) analysis is essential. In general, the signal intensity of ions obtained by MS / MS (MS ² ) measurement is reduced to about 1/10 of the previous stage ions. Therefore, by performing multi-stage MS (MS ⁿ (2 ≦ n)) measurement, the information on the chemical structure becomes extremely large, but the detection sensitivity decreases, so that the necessity for improving ionization efficiency increases more and more. It will be.

  As described above, a method for mass spectrometry of a measurement target molecule with high sensitivity and efficiency is strongly desired for a sample containing a very small amount of the measurement target molecule, but there is still no sufficient method. In addition, in order to obtain an excellent sweet spot, there was hardly anything that focused on the fine shape and surface structure of the measurement sample surface having a matrix.

JP 2008-051790 A WO 2006/109485 JP 2008-261824 A JP 2008-261825 A

Harvey, D.J., Mass Spectrom. Rev. 18 (1999) 349-451. Sugahara, D. et al., Anal. Sci. 19 (2003) 167-169. Kameyama, A. et al., Anal. Chem. 76 (2004) 4537-4542. Snovida, S.I. et al., Rapid Commun. Mass Spectrom. 21 (2007) 3711-3715. Snovida, S.I. et al., J. Am. Soc. Mass spectrom. 19 (2008) 1138-1146. Fukuyama, Y. et al., Anal. Chem. 80 (2008) 2172-2179.

  The present invention has been made in view of the above-mentioned background art, and its problem is applicable to commonly used matrices for MALDI methods, and includes molecules to be measured such as sugars and glycopeptides during laser irradiation. An object of the present invention is to provide a measurement sample for mass spectrometry that can dramatically increase the amount of ion generation and ionization efficiency of a sample as compared with conventional methods, and a method for preparing the same.

  Furthermore, specifically, it is to provide a measurement sample for mass spectrometry that makes a sweet spot easy to appear or a lot or a wide sweet spot appears, and a preparation method thereof, thereby including a molecule to be measured. An object of the present invention is to provide a mass spectrometry method capable of obtaining information on a highly reliable chemical structure by improving ionization efficiency and reproducibility of measurement even if the amount of the sample is extremely small.

  In particular, mass spectrometry that can be applied to trace amounts of "molecules derived from living organisms or molecules in biological samples" such as sugars, glycoproteins, glycopeptides, etc., to obtain useful information for elucidating their functions and pathological conditions An object of the present invention is to provide a method measurement sample, its preparation method and mass spectrometry.

  As a result of intensive studies to solve the above problems, the present inventor, when preparing a measurement sample on a measurement plate, at least the type of solvent in the solution in which the matrix is dissolved and the evaporation method (drying method) of the solvent By devising the method, etc., a specific structure can appear on the surface of the measurement sample, and the present invention has been completed by finding that the specific structure can be a sweet spot with excellent ionization efficiency. .

  In addition, when preparing a measurement sample on a measurement plate, by devising at least the type of solvent of the solution in which the matrix is dissolved, the evaporation method (drying method) of the solvent, etc. The present inventors have also found that a “specific structure that can be a sweet spot” can appear on the surface of a measurement sample in a large number and / or a wide range.

  That is, the present invention is a measurement sample for mass spectrometry prepared from a sample containing a molecule to be measured and a matrix, and has a wavy structure with an average period of 1 nm to 300 nm on at least a part of the surface. A measurement sample for an analytical method is provided.

The present invention also provides:
(A) a step of drying a solution of a sample containing a molecule to be measured on a plate, and thereafter
(B) Provided is a method for preparing a measurement sample for mass spectrometry, comprising a step of dropping a solution obtained by dissolving only a matrix in a solvent a onto the plate and drying the solution.

The present invention also provides:
(D) preparing a solution by mixing a sample containing a molecule to be measured and a matrix in a solvent a;
(E) A method of preparing a measurement sample for mass spectrometry including the step of dropping the solution prepared in (D) onto a plate and drying it.

  In the present invention, the solvent a is “an“ alcohol or phenol ”or an organic solvent that is compatible with water at 20 ° C. in an arbitrary ratio” and substantially does not contain water or water. The present invention provides a method for preparing the measurement sample for mass spectrometry, which is a solvent contained at 10% by mass or less.

  In the present invention, a solvent containing more than 10% by mass of water is used as the solvent a, and when the solution dissolved in the solvent a is dropped on the plate and dried, the drying is completed under heating and / or under reduced pressure. A method for preparing the measurement sample for mass spectrometry prepared as described above is provided.

  In addition, the present invention provides a measurement sample for mass spectrometry, which is prepared by the above-described method for preparing a measurement sample for mass spectrometry.

  Further, the present invention provides a measurement for mass spectrometry, characterized in that at least part of the surface of the measurement sample has a wave-like structure having an average period of 1 nm to 300 nm and twice the average amplitude of 1 nm to 40 nm. A sample is provided.

In addition, the present invention provides a mass spectrometry characterized by applying the MS ⁿ method (2 ≦ n) in which fragmentation is repeated n times to the measurement sample for mass spectrometry.

  According to the present invention, the above-mentioned problems can be solved and the above-mentioned problems can be solved, and it can be applied to a matrix generally used for the MALDI method, and the ion generation amount and ionization efficiency of a sample containing a molecule to be measured can be improved. A measurement sample for mass spectrometry and a preparation method thereof can be provided. That is, it is possible to provide a measurement sample for mass spectrometry having an excellent sweet spot and a preparation method thereof.

  Furthermore, specifically, since the present invention has revealed for the first time what kind of surface structure there is a lot of sweet spots with extremely good ionization efficiency on the measurement plate, it is easy to make such good sweet spots appear. Alternatively, it is possible to provide a method for preparing a measurement sample for mass spectrometry in which many good sweet spots appear or widely appear. As a result, mass spectrometry is possible even when there is only a small amount of sample that would be less than the amount that can be analyzed with the usual method for preparing a measurement sample, and even when there is a sufficient amount, it is more reliable. High chemical structure information can be obtained easily or reliably.

  In addition, since the present invention can be applied to a small amount of molecules derived from biological samples or molecules in biological samples, for example, molecules such as sugars, proteins (including peptides), glycoproteins (including glycopeptides), nucleic acids, glycolipids, etc. In addition to chemical structure analysis, it is possible to obtain useful information for elucidation of functions and disease states.

In Example 1, it is a positive ion mass spectrum when measuring neutral sugar chain NA2F of 100 fmol. In Example 1, (a) The confocal laser micrograph of a DHBA matrix, (b) The position where the sodium addition molecule | numerator of NA2F was detected, and the relative intensity | strength are shown. In Example 1, it is a positive ion mass spectrum when measuring neutral sugar chain NA2F of 5 fmol. In Example 1, it is a figure which shows the height in the cross section and the photograph (1000-times multiplication factor) of the DHBA matrix which was image | photographed with the scanning confocal laser microscope and was prepared with methanol using this method. In Example 1, it is the surface shape of the DHBA matrix which used methanol as the solvent a observed using the probe microscope (SPM) mode of nanosearch microscope SFT-3500 (made by OLYMPUS), and is a photograph which shows a wavy structure. is there. In Comparative Example 1, (a) a confocal laser micrograph of a DHBA matrix crystal prepared by a conventional method, and (b) a position where a sodium-added molecule of NA2F was detected and its relative intensity. In comparative example 1, it is a positive ion mass spectrum at the time of measuring neutral sugar chain NA2F of 100 fmol by the conventional method.

  Hereinafter, the present invention will be described, but the present invention is not limited to the following specific embodiments, and can be arbitrarily modified and implemented.

  The present invention relates to a mass spectrometry measurement sample prepared from a sample containing a molecule to be measured and a matrix, and having a wavy structure with an average period of 1 nm to 300 nm on at least a part of the surface. It is a measurement sample.

[Measurement sample for mass spectrometry]
<Wave structure and wave structure>
In the present invention, the “wave structure” present on at least a part of the surface of the measurement sample has an average period of 1 nm to 300 nm. Here, the “wave structure” refers to a structure in which linear uneven portions periodically exist as shown in FIG. 5, for example, and the “average period” refers to a nanosearch microscope (for example, SFT manufactured by OLYMPUS). -3500) probe microscope (SPM) mode, and the average value of the distances between the tops of adjacent waves, that is, between adjacent convex parts.

  The average period is essential to be in the range of 1 nm to 300 nm, preferably in the range of 5 nm to 200 nm, more preferably in the range of 20 nm to 150 nm, and in the range of 50 nm to 100 nm. Particularly preferred. A wavy structure with an average period shorter than this will not actually appear. On the other hand, if it is not a wavy structure or the average period is too long, the target molecule will be included even if it is irradiated with laser light. There may be a case where a sufficient amount of ion generation of the sample cannot be obtained. That is, it may not be a sweet spot with relatively good ionization efficiency from the surroundings, or even a sweet spot may not be an excellent sweet spot with extremely high ionization efficiency.

  In the case of a structure having no wavy structure (in the case of not having a wavy structure), for example, as shown in FIG. 6A, when the matrix molecule forms a large crystal, Moreover, even if it is a very small structure (for example, 1 μm or less), it has a crystal appearance peculiar to matrix molecules. When the sample surface does not have a wave-like structure, the crystal often has a stable crystal structure inherent to matrix molecules.

  The “wavy structure” in the present invention is not limited to the following, but is considered to be unique to the matrix molecule, that is, not having the most stable crystal structure in the matrix molecule. Also, it may be amorphous. Further, if the convex portions forming the wavy structure are linear or rod-shaped, the length in the longitudinal direction is not particularly limited, and for example, as shown in FIG. May be.

  The “wave structure” in the present invention refers to a structure in which uneven portions are periodically present as described above. Therefore, any structure in which irregularities are present periodically, such as a substantially sinusoidal structure, a substantially rectangular wave structure, or a structure in which bowl-shaped objects are arranged in parallel, is included. In the present invention, “twice the average amplitude” of the wavy structure is preferably 1 nm to 40 nm, more preferably 2 nm to 20 nm, and particularly preferably 5 nm to 10 nm. Outside this range, the ionization efficiency may not be sufficiently high when the point is irradiated with, for example, laser light. Here, “twice the average amplitude” means that “the structure in which irregularities are periodically present” is a sine wave-like structure, and when the structure can be approximated by a sine wave, the amplitude of the sine wave is When the average value is twice, but the structure cannot be approximated by a sine wave, “twice the average amplitude” means the average length between the uppermost point and the lowermost point of the uneven portion. . According to a specific method for preparing a measurement sample to be described later, such a wavy structure can appear on the surface of the measurement sample.

  The wavy structure may appear on the entire surface of the measurement sample surface placed on a plate for mass spectrometry or the like, or may appear on a part of the measurement sample surface. When the portion having the wavy structure is defined as a “wavy structure”, if the wavy structure exists only on a part of the surface of the measurement sample, it is irradiated with laser light (measurement spot). Mass spectrometry with high ionization efficiency of the molecule to be measured is possible.

It is essential that one or more wavy structures having the wavy structure exist on the surface of the measurement sample, but the larger the total area, the better. The sum of the areas is preferably 4 [mu] m ² to 3 mm ^2, particularly preferably ^{1 mm} 2 to 3 mm ^2. By preparing in this way, the existence probability of an excellent sweet spot with extremely high ionization efficiency is increased, and it becomes easier to find an excellent sweet spot, and the effect of the present invention is further obtained.

By preparing at least one of the wavy structures having the wavy structure so that the area when the measurement sample is viewed from above is 4 μm ² or more, an excellent sweet spot with extremely high ionization efficiency is included in the area. The probability of existence increases, and the effect of the present invention is further exhibited. In addition, by preparing as described above, an excellent sweet spot as a whole measurement sample can be easily present somewhere on the plate. The area is more preferably 10 μm ² or more, particularly preferably 100 μm ² or more, and still more preferably 1000 μm ² or more. Even if the area of the corrugated structure is smaller than the laser irradiation area described below, there may be a very excellent sweet spot there, in which case the above-described effects of the present invention can be achieved.

Usually, the beam diameter of the laser beam irradiated for ionization is about 200 μm, although it varies depending on the mass spectrometer to be used and its adjustment. However, not all of the irradiated area is desorbed. As far as we normally observe, the area desorbed by laser irradiation is about 50 μm in diameter, and the area is about 2000 μm ² . Therefore, from this point of view, the area of the wavy structure is preferably 2000 μm ² or more, and it is preferable to prepare the measurement sample so that at least one such portion exists. Furthermore, since it is more preferable that corrugated structure for greater than laser irradiation area is present, more preferably at least one of the corrugated structure is 10000 ² or more, particularly preferably at 30000Myuemu ² or more, therefore, It is preferable to prepare such a corrugated structure having such an area.

The larger the area of one wavy structure is, the easier it is to find an excellent sweet spot there. However, in consideration of the possibility (realism) of actual measurement sample preparation, more preferably 2000 μm ² to 3 mm ^2, particularly preferably ^{1 mm} 2 to 3 mm ^2. The ratio of the wave structure to the plate is not particularly limited, but is preferably 1/1000 to 1/1 (when the entire sample surface on the plate is a wave structure), more preferably 1/100 to 1, and 1 / 10 to 1 is particularly preferable.

  It is preferable to prepare the wavy structure on the surface of the measurement sample by preparing the measurement sample to have a thickness of 10 μm or less. The thickness of the measurement sample having a wave-like structure on the surface is more preferably 5 μm or less, and particularly preferably 3 μm or less. When the thickness is in the above range, not only is it easy to become a sweet spot with high ionization efficiency, but also when the sample is prepared so that the thickness is in the above range, the wavy structure is easily displayed on the sample surface, and the ionization efficiency is improved. A high sweet spot can be obtained. If the surface has a wave-like structure and is a wave-like structure, the inside (lower side) may be different. Said thickness shows the thickness of the whole measurement sample.

  In the present invention, the chemical structure of the matrix is not particularly limited as long as the above-described wavy structure appears, and any matrix used in mass spectrometry can be used in the present invention.

  There is no particular limitation on the plate to be used. Usually, any plate used in MALDI-MS can be used, but one having electrical conductivity is preferable. Specifically, a mirror plate, a stainless plate, a plate having a gold surface, or the like is preferable.

[Method of preparing measurement sample for mass spectrometry]
The preparation method of the measurement sample for mass spectrometry of the present invention is as follows:
(1) A step of drying a solution containing a molecule to be measured and a matrix dissolved in “solvent a” on a sample plate (hereinafter simply abbreviated as “plate”), or
(2) A step of drying a solution of a sample containing a molecule to be measured on a plate, and a step of drying a solution obtained by dissolving only the matrix in “solvent a” on the plate.
A method for preparing a measurement sample for mass spectrometry, comprising:
It is characterized in that it is prepared so that a wavy structure with an average period of 1 nm to 300 nm appears on at least a part of the surface of the measurement sample after the “step of drying the solution dissolved in the solvent a on the plate”.
Here, “solvent a” refers to a solvent that can dissolve both the molecule to be measured and the matrix, and the same applies in the present invention.

  In the MALDI method, the above (1) is generally called a “Dripple Droplet method”, and the above (2) is a method developed in the present invention. A sample containing a molecule to be measured is separately provided on a sample support. This is a method of preparing a measurement sample that is overlaid on a sample with a solution in which only the matrix is dissolved in the solvent a and is dried on the plate while dissolving the sample. The measurement sample preparation methods (1) and (2) above can cause a “wave structure”, which is a specific structure that can be a sweet spot with higher ionization efficiency, to appear on the surface of the measurement sample.

In the present invention, the wavy structure on the surface of the measurement sample was found as a portion where the ionization efficiency of the molecule to be measured by irradiating the laser beam is high, and further, a method for preparing the measurement sample that reveals the wavy structure was also found. It was. The method for preparing the measurement sample is the following (1) or (2).
(1) Method for preparing measurement sample for mass spectrometry including a step of drying a solution containing a sample containing a molecule to be measured and a matrix dissolved in solvent a on a plate (2) A solution of a sample containing the molecule to be measured A method for preparing a measurement sample for mass spectrometry, comprising a step of drying on a plate, and a step of subsequently drying on the plate a solution in which only the matrix is dissolved in solvent a

<"(1) Method" (Drived Droplet Method) to Appear Wave Structures>
The preparation method of the measurement sample of the above (1) is generally referred to as “Drived Droplet method” in the MALDI method, and more specifically, the following step (D) and the following step (E) Is a method for preparing a measurement sample including the essential step.
(D) A step of mixing a sample containing a molecule to be measured and a matrix in a solvent a to prepare a solution (E) A step of dropping the solution prepared in step (D) onto a plate and drying it

  The preparation method of the measurement sample of (1) above, that is, the “Driven Droplet method” including the steps (D) and (E) (hereinafter sometimes abbreviated as “(1) method”) is the wavy in the present invention. Used as one way to reveal the structure. However, the conventional solvent drying method (solvent evaporation method) using a conventional solvent cannot make the wave-like structure of the present invention appear on the surface of the measurement sample. The present inventor has found that a relatively large matrix crystal is generated under conditions where the matrix crystal grows gradually or at a normal rate as in the prior art, and the measurement target molecule is excluded from the matrix crystal in the process of crystal growth. Thought to be. In fact, even if the portion of the matrix having a crystal appearance is irradiated with laser light, the ionization efficiency is low and an excellent sweet spot is not obtained.

  Therefore, the present inventor considered that the conditions should be such that the matrix is rapidly precipitated. In order to achieve such conditions, the solvent is rapidly evaporated (dried), that is, a specific organic solvent is used as a solvent and / or a specific solvent drying method (solvent evaporation method) is used. I found. It was also found that it is more preferable to drop and dry a specific solution in a specific order (method (2) described later).

  Conventionally, for example, a 50 to 70% by mass aqueous solution of acetonitrile has been generally used as a solvent for the “Driven Droplet method”. However, in a solution in which “50 to 70 mass% aqueous solution of acetonitrile” is used as a solvent and the molecule to be measured and the matrix molecule are dissolved, a conventionally known ordinary solvent drying method (solvent evaporation method), that is, 15 to 30 is used. Under the conditions of 1 ° C., 1 atm and no wind, the solvent slowly evaporates (drys), so that the wavy structure in the present invention cannot appear on the surface of the measurement sample. Therefore, the wavy structure and the wavy structure having the wavy structure are novel as the structure of the surface of the measurement sample for mass spectrometry. Therefore, for the first time, the wavy structure having the wavy structure has been found to be a portion where the ionization efficiency of the molecule to be measured is high, and the method for preparing the measurement sample for mass spectrometry in order to reveal it is the wavy structure itself. Is a new sample preparation method.

  The concentration of the molecule to be measured and the concentration of the matrix in the solution in step (D) are not particularly limited, and the concentrations usually used in the Dred Droplet method are applicable. After the completion of the step (E) (after drying), the thickness of the layer containing the molecule to be measured and the matrix present on the plate is not particularly limited, but is preferably 10 μm or less, more preferably 5 μm or less, and particularly preferably 3 μm or less. preferable. If the thickness of such a layer is too thin, the matrix may be consumed by laser irradiation several times, and the amount of ions generated may be reduced. If the layer is too thick, needle-shaped crystals will easily grow and the amount of ions generated will be reduced. May decrease.

<<"Alcohols or phenols or an organic solvent that is compatible with water at 20 ° C in an arbitrary ratio" and substantially does not contain water or contains water at 10% by mass or less >>
The solvent a used in the step (D) is “alcohols or phenols” or “an organic solvent compatible with water at 20 ° C. in an arbitrary ratio” and substantially does not contain water or water. Is preferably 10% by mass or less because the solvent is rapidly evaporated (dried), and a wave-like structure with high ionization efficiency is easily displayed on the surface of the measurement sample. The “solvent a” is a solvent that can dissolve both the molecule to be measured and the matrix as described above.

  Alcohols or phenols that are not compatible with water at an arbitrary ratio at 20 ° C. are preferable because the above wavy structure is easy to appear. Here, “phenols” refers to compounds in which a hydrogen atom bonded to an aromatic ring is substituted with a hydroxyl group. The number of hydroxyl groups that the alcohols or phenols have is not particularly limited, but one is preferable from the viewpoint of the evaporation rate. The boiling point of alcohols or phenols at 1 atm is not particularly limited, but is preferably 100 ° C. or less, more preferably 90 ° C. or less, particularly preferably 80 ° C. or less, and further preferably 75 ° C. or less from the viewpoint of the evaporation rate. Further, the vapor pressure at 20 ° C. is preferably 4 mmHg or more, more preferably 10 mmHg or more, particularly preferably 20 mmHg or more, and further preferably 40 mmHg or more.

  Specifically, methanol, ethanol, propanol, butanol, pentanol, hexanol, cyclohexanol, heptanol, phenol, naphthol and the like are preferable. These are used alone or in combination. Particularly preferred is methanol or a mixed solvent containing methanol.

  The “organic solvent compatible with water at an arbitrary ratio at 20 ° C.” is not particularly limited as long as the conditions are satisfied. The boiling point of such an organic solvent at 1 atm is not particularly limited. However, the wavelike structure appears at a temperature of 100 ° C. or less from the point that the matrix is rapidly precipitated by rapidly evaporating (drying) the solvent. Preferably, 95 ° C. or lower is more preferable, 90 ° C. or lower is particularly preferable, and 85 ° C. or lower is further preferable. Further, the vapor pressure at 20 ° C. is preferably 20 mmHg or more, more preferably 40 mmHg or more, and particularly preferably 60 mmHg or more.

  Specific examples include acetonitrile and acetone. These are used alone or in combination. It can also be used by mixing with the alcohols or phenols described above.

  When the solvent a is alcohols or phenols or “an organic solvent compatible with water at an arbitrary ratio at 20 ° C.”, in particular, the sample containing the molecule to be measured is a mixture of a glycopeptide and a peptide. When the target molecule is a glycopeptide, the solvent a dissolves the glycopeptide and does not dissolve the peptide. Therefore, a solvent a solution of the glycopeptide and the matrix is formed, which is preferable in the wavy structure of the matrix (the ionization efficiency increases). In such a form / state, a measurement sample in which glycopeptide molecules exist in the matrix can be prepared. As a result, information on the chemical structure of the glycopeptide that is the molecule to be measured, particularly the chemical structure of the sugar chain contained therein, can be obtained. Easy to obtain by mass spectrometry.

  The above-mentioned solvent a is preferably a solvent that substantially does not contain water or contains 10% by mass or less of water from the viewpoint of rapidly evaporating the solvent. When water is contained in an amount of more than 10% by mass, a wavy structure may not appear on the surface of the measurement sample unless drying is completed under heating and / or reduced pressure (or natural drying described below). The solvent a described above preferably contains only 5% by mass or less of water, particularly preferably contains only 2% by mass or less of water, and more preferably contains substantially no water. Here, “substantially” means that the inclusion of a very small amount of water generally contained in a reagent is not excluded (allowed). However, it is most preferable that no water is contained. Under normal drying conditions, that is, 15 to 30 ° C., 1 atm and no wind, if there is too much water, a wavy structure may not appear. The reason is considered to be that the drying rate is slow and the matrix crystals gradually grow into needles.

  The solvent a is one type of solvent selected from the group consisting of methanol, ethanol, isopropanol, butanol and acetonitrile, or a mixed solvent of two or more types, and substantially does not contain water or contains 10% by mass or less of water. It is particularly preferable that the solvent is contained in the above because the wavy structure can be easily revealed in the above-described form and the effects of the present invention are further exhibited.

  The solvent a is ““ alcohols or phenols ”or“ an organic solvent compatible with water at an arbitrary ratio at 20 ° C. ”, and substantially does not contain water or contains 10% by mass or less of water. In the case of the solvent to be contained, evaporation (drying) of the solvent may be completed under normal conditions (for example, 15 to 30 ° C., 1 atm and no air). It is also particularly preferable to complete the drying under heating and / or reduced pressure when it is dropped and dried.

<< Solvent a containing more than 10% by mass of water >>
The solvent a may contain more than 10% by mass of water if it is rapidly evaporated (dried). That is, when a solvent containing more than 10% by mass of water is used as the solvent a, and the solution dissolved in the solvent a is dropped on the plate and dried, it is prepared to complete the drying under heating and / or reduced pressure. A sample preparation method is also preferred. Even if the solvent a contains more than 10% by weight of water, if the evaporation (drying) is completed (completed) faster under heating and / or under reduced pressure, the wavy structure appears in the above-described form. The effects of the present invention are exhibited.

  If the solvent a contains more than 10% by weight of water, when specifying the chemical structure of sugar chains such as sugars, glycopeptides, etc., they are easy to dissolve in water. There is a good point that the droplets are small and easily collected. From this point of view, when the solvent a contains more than 10% by mass of water, the content of water is determined based on the assumption that the drying is completed under heating and / or reduced pressure. On the other hand, 60 mass% or more is more preferable, and 90 mass% or more is especially preferable.

  Under normal drying conditions, i.e., 15 to 30 ° C., 1 atm and no wind, even if the solvent a is not capable of revealing a wavy structure, drying should be completed quickly under heating and / or under reduced pressure. Then, a wave-like structure appears. Samples prepared using a solvent containing more than 10% by weight of water as the solvent a and completing the drying in a shorter time than natural drying when the solution dissolved in the solvent a is dropped onto the plate and dried. A preparation method is preferred. Here, “natural drying” refers to drying performed at 20 ° C. under 1 atm and substantially no wind.

<"Method (2)" for revealing a wavy structure (method using a solution in which only the matrix is dissolved in the solvent a)>
As a method for preparing a measurement sample on a plate for mass spectrometry, in addition to the above-mentioned “Drived Droplet method”, “Crush Crystal method”, “Thin Layer method (thin film method)”, “Two Layer method ( Two-layer method). However, when the evaporation (drying) rate of the solvent is made normal, that is, when the solvent a containing more than 10% by mass of water is used and natural drying without heating or depressurization is performed, as described above, the “Dried Droplet” of the normal drying rate is used. In the same way that the “method” did not reveal a wavy structure, none of the above methods revealed a wavy structure at the normal solvent evaporation (drying) rate.

Therefore, the present inventor has found a novel sample preparation method that does not belong to any of the above-mentioned known methods, and can be explained as follows. That is, another one of the “measurement sample preparation method” for causing a wave-like structure to appear on the measurement sample surface is:
(2) A method for preparing a measurement sample for mass spectrometry, which includes a step of drying a solution of a sample containing a molecule to be measured on a plate, and a step of drying a solution obtained by dissolving only the matrix in solvent a on the plate. It is. Hereinafter, this method may be abbreviated as “(2) method”.

  (2) In the method, the step of drying the solution in which only the matrix is dissolved in the solvent a on the plate is the final step in the step of supplying the substance on the plate, so that the wavy structure is formed on the surface of the measurement sample. To appear. 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.

  If a step in which a solution in which only the matrix is dissolved in the solvent a is supplied onto the plate and dried is included as a substantial final step, the solution in which the molecule to be measured is dissolved in the final step may not be used. This is because the solvent a is a solvent that can dissolve both the molecule to be measured and the matrix, so that the molecule to be measured already in the lower layer can be dissolved and mixed with the matrix.

More specifically, the method (2) is a method for preparing a measurement sample including the following step (A) and step (B) after step (A) 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) A step of dropping a solution obtained by dissolving only the matrix in the solvent a onto the plate and drying the solution.

  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, when the good solvent of the sample containing the molecule to be measured is a poor solvent of the matrix and the matrix or solvent is to be used, the sample is in the solvent a in which the matrix is dissolved. 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 range of choice of solvent is widened, the sample is decomposed, and the sample is adsorbed on the container. It is preferable that the matrix is not dissolved in the solution at this time and only the sample containing the molecule to be measured is dissolved because of the point that it can be suppressed.

  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 signal may decrease.

  (2) In the method, since a solution of “sample containing a molecule to be measured” (preferably, a solution containing only “sample containing a molecule to be measured”) is first dropped onto a measurement plate, a very small amount of sample containing the molecule to be measured is present. In this case, it is particularly useful because almost the entire amount of a small amount of sample can be subjected to analysis without mixing with the matrix solution in a separate container and dropping a part of the solution onto 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.

  (2) In the method, after the step (A), (B) a step of dropping a solution obtained by dissolving only the matrix in the solvent a onto the plate and drying the solution. 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). In the step (B), a solution in which only the matrix is dissolved in the solvent a is dropped onto the plate. Here, the definition of the “solvent a” is the same as described above. Refers to a solvent that can also be dissolved. By using a solvent a that can dissolve both the molecule to be measured and the matrix, the lower molecule to be measured moves to the surface of the matrix, and if it has a wavy structure, it can be an excellent sweet spot. .

  (2) The type of solvent a in the method and the evaporation (drying) method of the solvent a are the same as those in (1) Method (Dried Droplet method) <<< alcohols or phenols or water at 20 ° C. at an arbitrary ratio "Compatible organic solvent", which is substantially free of water or contains a water a at a content of 10% by mass or less, and << a solvent a containing water at a content of more than 10% by mass It is the same as that described in the section >>, including the preferable range.

  Although the density | concentration of the matrix of the solution in a process (B) does not have limitation in particular, 0.1-1000 mg / mL is preferable and 1-100 mg / mL is especially preferable. After the completion of the step (B) (after drying), the thickness of the layer comprising the sample containing the molecule to be measured and the matrix present on the plate is not particularly limited, but is preferably 1 nm to 100 μm, particularly preferably 1 nm to 10 μm. If the thickness of such a layer is too thin, a wavy structure may not be formed. If it is too thick, a needle-like crystal that does not have a regular wavy structure may be formed.

<< When (2) Method has Step (C) >>
(2) In the method, between step (A) and step (B),
(C) In order to further increase the ionization efficiency, it is preferable to include a step of dropping a solution of a derivatizing agent that increases the ionization efficiency by reacting with the molecule to be measured and drying the solution on the plate.

  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

  In the case of having the step (C), after the completion of the step (B) (after drying), the thickness of the layer comprising the sample containing the molecule to be measured and the matrix present on the plate is not particularly limited, but 1 nm to 100 μm is preferable. 1 nm to 10 μm is particularly preferable. If the thickness of such a layer is too thin, a wavy structure may not be formed. If it is too thick, a needle-like crystal that does not have a regular wavy structure may be formed.

  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 ionization efficiency may be increased in the laser desorption ionization method, or the ionization efficiency may be increased in the electrospray ionization method. A compound having an effect as a matrix in mass spectrometry, or a compound further having a reactive functional group or a spacer portion described later is also preferable.

  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 more 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.

<Measurement molecule>
The “measuring molecule” 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, glycopeptide, nucleic acid, glycolipid, etc. It is preferable that the effect 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. 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, the mass spectrometric method of the present invention is preferable since the above-described effects are exerted particularly on the chemical structure analysis of these molecules.

<Measurement sample for mass spectrometry>
The measurement sample for mass spectrometry prepared by using the above-described method for preparing the measurement sample for mass spectrometry of the present invention exhibits the effects of the present invention described above. That is, if such a measurement sample is subjected to mass spectrometry, the amount of ion generation and ionization efficiency of the sample containing the molecule to be measured can be improved. That is, since an excellent sweet spot exists in such a measurement sample, sensitivity, accuracy, reproducibility and the like in mass spectrometry are improved. One embodiment of the present invention is characterized in that a wavy structure having an average period of 1 nm to 300 nm and twice an average amplitude of 1 nm to 40 nm appears on at least a part of a measurement sample surface. This is a measurement sample for mass spectrometry.

<Mass spectrometry>
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 (Hereinafter, sometimes abbreviated as “MS ⁿ method”) is preferable. In the present invention, it is preferable to apply the MS ⁿ method (2 ≦ n) in which fragmentation is repeated n times for a sample prepared using the method for preparing a measurement sample for mass spectrometry. By analyzing the ion containing the selected label by MS ⁿ method, the binding position in the molecule can be determined.

<Action and principle>
In the process where the sample containing the matrix and the molecule to be measured is deposited on the plate, a wave-like structure appears due to the rapid evaporation and drying of the solvent. As a result, the amount of ion production (ionization efficiency) is dramatically improved, and the wave-like structure is further improved. We found the surprising finding that almost every position in the structure is an excellent sweet spot, and especially when the molecule to be measured is sugar, a signal derived from sugar can be obtained regardless of where the laser is irradiated.

  This action / principle is not clearly clear, but it can be considered as follows. However, the present invention is not limited to the scope of the following actions and principles. It is considered that the amount of ion generation was improved by removing the solvent instantly to rapidly grow the matrix crystal and forcing the sample into the matrix crystal. The wavy structure is thought to be generated as a result of the rapid growth of the matrix crystal. And it may be advantageous for absorption of laser light or may be advantageous for desorption from the surface. Increasing the thickness of the sample to be measured inevitably increases the concentration of the matrix solution, but if this is done, acicular crystals grow, sweet spots cannot be formed, and the amount of ions generated decreases.

  If the surface of the matrix has a wavy structure, it is easy to absorb the laser, the matrix molecule does not form a stable crystal structure in the wavy structure, energy transfer easily occurs to the molecule to be measured, and By increasing the surface area ratio, efficient desorption of the measurement target molecule and accompanying ionization effectively occur, and as a result, the amount of ion generation of the measurement target molecule is considered to have increased dramatically.

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.
Further, among the results of using the neutral sugar chain NA2F and the DHBA matrix in the examples and comparative examples, the type of the solvent a, the temperature at which the solvent a was evaporated (experiment), the pressure, and the solvent a were dried. Table 1 summarizes the time required for the matrix to precipitate and the signal-to-noise ratio (S / N) of the signal derived from the molecule to be measured in the MALDI mass spectrum obtained from the sample.

Example 1
<(2) Process (A) in Method>
First, a neutral sugar chain (hereinafter abbreviated as “NA2F”) represented by the following formula (1) as a molecule to be measured in a spot having a diameter of about 2 mm on a mirror-finished MALDI sample plate (mirror plate). 1 μL of an aqueous solution in which 100 fmol / μL was dissolved in water was dropped, and the mixture was left to dry at room temperature (23 ° C.) under atmospheric pressure.
Galb1-4GlcNAcb1-2Mana1-6 (Galb1-4GlcNAcb1-2Mana1-3) Manb1-4GlcNAcb1-4GlcNAc) (1)

<(2) Process (B) in Method>
Next, 0.25 μL of a solution prepared by dissolving a commercially available 2,5-dihydroxybenzoic acid (hereinafter abbreviated as “DHBA”) in methanol to 40 mg / mL was added dropwise as a matrix. When allowed to stand at room temperature (23 ° C.) and atmospheric pressure and no wind, methanol as a solvent evaporates in about 8 to 10 seconds and uniformly spreads within the spot, and a measurement sample in which the matrix and the sample are deposited is obtained. .

  The crystal thickness of the obtained measurement sample was 1 μm to 10 μm, and most of the thickness was 1 μm to 3 μm.

  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 to the threshold at which the ion signal of the molecule to be measured begins, laser irradiation is performed at 100 μm intervals in order to obtain a mass spectrum from the entire area of the measurement sample deposit of one specimen in the 2 mm spot. Measured with

The obtained positive ion mass spectrum is shown in FIG. As a result, the neutral sugar chain NA2F was detected very strongly as a sodium adduct [M + Na] ⁺ . The signal-to-noise ratio (S / N) at this time was 220 (see No. (a) in Table 1), which was an improvement of about 50 times compared to the conventional method shown in Comparative Example 1. A large S / N for the entire measurement sample means that there are many excellent sweet spots. When the solvent evaporation method of the present invention is used, a wavy structure appears on the measurement sample surface. It shows that. The same applies to the following embodiments.

  FIG. 2 shows a confocal laser micrograph of the measurement sample deposit at this time (FIG. 2A) and a position where neutral sugar chain NA2F was detected in the measurement sample deposit (FIG. 2B), respectively. Show. It was confirmed that a high signal was obtained almost uniformly over the entire measurement sample precipitate, and the ionization efficiency was high throughout the measurement sample precipitate. In addition, the blackest part in FIG. 2B is an excellent sweet spot with extremely high ionization efficiency, and this part has the following wavy structure.

  From the above-mentioned aqueous solution of NA2F concentration of 100 fmol / μL, NA2F was serially diluted, and a measurement sample was prepared by the same procedure as described above. As a result of the measurement, the extremely dilute solution of 5 fmol / μL was obtained as shown in FIG. Even so, a signal could be obtained with 1 μL.

  Moreover, the measurement sample deposit prepared according to the procedure similar to the above was observed using a scanning confocal laser microscope OLS3100-LSAS (manufactured by OLYMPUS). Observation was performed at a total magnification of about 100 to 1000 times. In order to measure the height, the result of observing at 1000 times the part of the crystal that had been scraped to expose the plate surface is shown in FIG. The crystal spreads thinly, and it is within 1.6 μm when viewed only within the range of FIG. 4, but when looking at other places as a whole, most of the thickness is within 3 μm, and part of the crystal is thick However, it was confirmed to be 10 μm or less.

Furthermore, in order to observe a finer structure, it measured in the probe microscope (SPM) mode using nanosearch microscope SFT-3500 (made by OLYMPUS).
FIG. 5 shows the surface shape of a portion of the DHBA matrix sample prepared by the same procedure as described above, which had high ionization efficiency in the confocal laser micrograph. Regular irregularities, that is, wave structures having a height (twice the average amplitude of the wave structure) of 5 nm to 10 nm and an interval between adjacent protrusions (average period) of 50 nm to 100 nm were confirmed.

It was confirmed that at least one of the wavy structures having the wavy structure spread over about 64 μm ² as an area when the measurement sample was viewed from above. In addition, the corrugated structure having the corrugated structure was found in at least two places on the surface of the obtained measurement sample. From the observation result with a confocal laser microscope, such a corrugated structure is uniformly distributed. It was inferred that it spread throughout the measurement sample.

Instead of neutral sugar chain NA2F, 1 μL of an aqueous solution in which an acidic sugar chain represented by the following formula (2) (hereinafter abbreviated as “A1F”) as a measurement target molecule was dissolved in water to make 100 fmol / μL was dropped. And allowed to dry at room temperature (23 ° C.) under atmospheric pressure.
NeuNAca2-6Galb1-4GlcNAcb1-2Mana1-6 (Galb1-4GlcNAcb1-2Mana1-3) Manb1-4GlcNAcb1-4GlcNAc) (2)

When measured by the same method as described above, it was very strongly detected as a deprotonated product [M−H] ^{− in} the negative ion mode. The S / N was 500.

  In the measurement sample, many sweet spots with high ionization efficiency were observed, and a wavy structure was confirmed in that portion. In the wavy structure having the wavy structure, the area when the measurement sample was viewed from above and the number of wavy structures were almost the same as in the case of the neutral sugar chain NA2F.

<Effect of temperature during evaporation of solvent a>
Example 2
Next, in Example 1 using 100 fmol / μL of NA2F, instead of the solvent drying conditions “room temperature (23 ° C.), atmospheric pressure, no wind, no lid” in step (B), “4 ° C., Under atmospheric pressure, no wind, with lid ", the solvent methanol was evaporated.

  That is, the experiment was conducted in a cold room at 4 ° C. A mirror plate in which NA2F was dropped into the spot and dried in the same manner as in Example 1 and a matrix solution in which commercially purified DHBA was dissolved in methanol to 40 mg / mL were left in a cold room at 4 ° C. for 1 hour. did. In the cold room, 0.25 μL of the cooled matrix solution was dropped onto NA2F, and after dropping, the plate was covered with a plastic lid to delay the evaporation of the solvent.

  Methanol, which is a solvent, gradually evaporated and completely evaporated in about 2 to 4 minutes to obtain a precipitate of a measurement sample. The thickness of the portion having the corrugated structure on the surface of the obtained measurement sample was about 9 μm.

From this sample, the peak of S / N was 24 and the sodium adduct [M + Na] ⁺ of NA2F was detected (see No. (b) in Table 1). This result shows an improvement of about 5 times compared with the conventional method shown in Comparative Example 1. As described above, when methanol is used as the solvent a, an S / N superior to that of the conventional method can be obtained even when an operation for increasing the drying rate is not performed or even when an operation for decreasing the drying rate is performed. It turns out that it is obtained. In addition, since the normal drying example 1 obtained a larger S / N than the example 2 in which the drying rate was reduced, the higher the drying rate, the greater the S / N. It was found that the ionization efficiency of NA2F was large.

  In the measurement sample, many sweet spots with high ionization efficiency were observed, and a wavy structure was confirmed in that portion. The corrugated structure having the corrugated structure was slightly smaller or smaller in area when the measurement sample was viewed from above and the number of corrugated structures than when dried at 23 ° C. Obtained.

  In Example 1 using 100 fmol / μL of NA2F, instead of the solvent drying condition “room temperature (23 ° C.), atmospheric pressure, no wind” in step (B), “80 ° C., atmospheric pressure, In the absence of wind, the solvent methanol was evaporated.

  That is, the experiment was performed on a plate heated on a heat block at 80 ° C. A mirror plate in which NA2F was dropped into the spot and dried in the same manner as in Example 1 was left on a heat block heated to 80 ° C. for about 5 minutes. After about 5 minutes, 0.25 μL of a 40 mg / mL DHBA methanol solution was added dropwise to NA2F on the plate that had reached 80 ° C. Methanol as the solvent evaporated almost instantaneously (within 1 second) to obtain a measurement sample.

From this sample, the peak of S / N was 37 and the sodium adduct [M + Na] ⁺ of NA2F was detected (see No. (c) in Table 1). This result shows an improvement of about 7 times compared to the conventional method shown in Comparative Example 1.

  In the measurement sample, many sweet spots with high ionization efficiency were observed, and a wavy structure was confirmed in that portion. As for the corrugated structure having the corrugated structure, the area when the measurement sample was viewed from above and the number of corrugated structures were almost the same as those obtained by drying at 23 ° C.

  The experimental results shown in Example 1 and Example 2 could be reproduced in the same manner using a normal stainless steel MALDI plate other than the mirror plate, but the detection sensitivity was better for the mirror plate. Also, if the methanol did not spread to 2 mm or more by surface processing, good results were obtained even on the gold surface.

Comparative Example 1
Similarly to Example 1, 1 μL of 100 fmol / μL of NA2F was dropped on the mirror plate and dried. Next, 1 μL of a commercially available DHBA matrix dissolved in a 60% by mass acetonitrile aqueous solution and used at 10 mg / mL was added dropwise. When left in dry conditions (room temperature (23 ° C.), atmospheric pressure, no wind, no lid) in the same manner as in Example 1, the solvent gradually evaporates, taking 2 to 4 minutes, as shown in FIG. A measurement sample containing needle-like crystals of matrix DHBA was prepared.

A positive ion mass spectrum measured by the same method as in Example 1 is shown in FIG. Neutral sugar chain NA2F was detected as a sodium adduct [M + Na] ⁺ as in Example 1, but its peak intensity was low and S / N was about 5 (see No. (d) in Table 1). ), It was confirmed that ions of the neutral sugar chain NA2F were not sufficiently generated.

  FIG. 6 shows a scanning confocal laser micrograph (FIG. 6 (a)) of the measurement sample deposit observed before mass spectrum measurement, and the position where neutral sugar chain NA2F ions were detected (FIG. 6 (b)). ). It was confirmed that the deposited matrix was a relatively large needle-like crystal, and there was almost no sweet spot from which a sugar chain signal was obtained, and it was limited to a part. Moreover, there was no excellent sweet spot with high ion efficiency.

  The sweet spot portion was measured in the probe microscope (SPM) mode using a nanosearch microscope SFT-3500 (manufactured by OLYMPUS) in the same manner as in Example 1. No wavy structure was observed there. When the solvent a contains water, even if the measurement sample is prepared by the method (2), if the drying of the solvent a is not completed under heating and / or reduced pressure, the wave-like structure is not observed, and the ionization efficiency is excellent. It was found that a sweet spot could not be obtained.

Similarly, an experiment was performed in the same manner as described above using an aqueous solution of 100 fmol / μL of the acidic sugar chain A1F instead of the neutral sugar chain NA2F, and as a deprotonated product [M−H] ^{− in the} negative ion mode. Although detected, the intensity was much lower than that of Example 1.

  Furthermore, the measurement sample was prepared using the Dred Driplet method. That is, in a 60% by mass acetonitrile aqueous solution, NA2F was dissolved at 100 fmol / μL and DHBA as a matrix was 10 mg / mL, and 1 μL was dropped on a mirror plate, and the same drying conditions as in Example 1 ( When allowed to stand at room temperature (23 ° C.), atmospheric pressure, no wind, no lid, the solvent gradually evaporated and drying was completed over 2 to 4 minutes. Although not shown in the drawing, a measurement sample having many needle-like crystals of matrix DHBA as shown in FIG. 6A was prepared.

In the positive ion mass spectrum measured by the same method as in Example 1, the neutral sugar chain NA2F was detected as a sodium adduct [M + Na] ⁺ as in Example 1, but its peak intensity was low and neutral. It was confirmed that ions of sugar chain NA2F were not sufficiently generated.

  It was confirmed that there were almost no sweet spots from which sugar chain signals were obtained, and the sugar spots were limited to a part. Moreover, there was no excellent sweet spot with high ion efficiency. A portion of the sweet spot where a signal was detected was measured in the probe microscope (SPM) mode using a nanosearch microscope SFT-3500 (manufactured by OLYMPUS) in the same manner as in Example 1. No structure was observed.

<Influence of solvent type>
Example 3
In the same procedure as in Example 1, a measurement sample was prepared using ethanol, acetonitrile, isopropanol, and butanol as the solvent a instead of methanol, and measurement was performed in the same manner as in Example 1.

  Under the same drying conditions as in Example 1 (room temperature (23 ° C.), atmospheric pressure, no wind, no lid), the time required for each solvent to completely evaporate was about 30 seconds for ethanol and acetonitrile for It was about 8 to 10 seconds, isopropanol was about 60 seconds, and butanol was about 180 seconds. Even when these solvents were used, the same results as in Example 1 were obtained, and 100 fmol / μL of NA2F could be detected significantly. The strength was highest in ethanol, followed by isopropanol, acetonitrile, and butanol (see Nos. (D) to (h) in Table 1).

  The thickness of the portion having the corrugated structure on the surface of the obtained measurement sample was about 1 μm to 3 μm.

  Many sweet spots with high ionization efficiency were observed in the measurement samples using all the solvents described above, and a wavy structure was confirmed in that portion. The corrugated structure having the corrugated structure was slightly smaller or less in area when the measurement sample was viewed from above and the number of corrugated structures than in the case of methanol in Example 1, but almost the same results. was gotten.

  Similar results and effects were obtained even when a matrix solution was prepared by mixing these organic solvents. Similar results and effects were obtained even when 1 μL of an aqueous solution of 100 fmol / μL of acidic sugar chain A1F was used.

<Influence of water in solvent a>
Example 4
In the same manner as in Example 1, a measurement sample was prepared by adding water to a methanol solvent, and measurement was performed in the same manner as in Example 1. As the solvent a, a 98 mass% methanol aqueous solution, a 95 mass% methanol aqueous solution, and a 90 mass% methanol aqueous solution were used.

  In the case of a 98% by mass methanol aqueous solution and a 95% by mass methanol aqueous solution under the same dry conditions as in Example 1, the time required for the solvent to completely evaporate is the same as in the case of 100% by mass methanol in Example 1. The difference was about 10 seconds, but the time required for the 90% by mass aqueous methanol solution to completely evaporate was 15 to 20 seconds.

  When 98% methanol and 95% methanol were used, the measurement sample prepared with 100% methanol was not much different from the sample, but it was relatively uniform, but the matrix prepared with 90% methanol was somewhat close to needle crystals. became. When prepared with 98% methanol and 95% methanol to reflect the crystal shape, NA2F signal at 100 fmol / μL was detected strongly as in Example 1, but prepared with 90% methanol. In the matrix, the signal was lower than the result of Example 1, but the result was significantly better than that of Comparative Example 1 (see Nos. (I) to (k) in Table 1). As the solvent a, it may be confirmed that water may be contained in methanol, but it is preferably 10% by mass or less, more preferably 5% by mass or less.

  In all the measurement samples described above, many sweet spots with high ionization efficiency were observed, and a wavy structure was confirmed in that portion. The corrugated structure having the corrugated structure has extremely excellent results in which the area when the measurement sample is viewed from above and the number of corrugated structures are almost the same as those in the case of 100% by mass of methanol in Example 1. It was.

Comparative Example 2
In the same manner as in Example 3, 20% water was contained in a methanol solvent (with an 80% by mass aqueous methanol solution) to prepare a measurement sample, and measurement was performed in the same manner. Under the same drying conditions as in Example 1, the time required for the 80% by mass aqueous methanol solution to completely evaporate is 15 to 20 seconds, and the precipitate of the measurement sample becomes almost needle-like crystals, and the wavy structure is It was not confirmed.

The obtained mass spectrum was almost the same as that of Comparative Example 1. In the neutral sugar chain NA2F, the sodium adduct [M + Na] ⁺ ion had a low peak intensity, and the S / N was 7 (No. in Table 1). (See (l)).

<Influence of decompression>
Example 5
In the step (B), after dropping the matrix solution, an experiment was conducted in which the solvent a was evaporated and dried under reduced pressure conditions.

  Similarly to Example 1, 1 μL of commercially available NA2F, 100 fmol / μL was dropped on the mirror plate and dried. Next, 1.5 μL of a matrix prepared by dissolving DHBA in water to 6.7 mg / mL was dropped (the mass of the matrix is the same as in Example 1). The mirror plate was immediately placed in a desiccator having a diameter of about 25 cm, and the pressure was reduced using a vacuum pump with an exhaust capacity of 10 L / min. The measurement sample finished drying in 60 seconds.

From this sample, S / N was 13, and a sodium adduct [M + Na] ^{+ of} NA2F was detected (see No. (m) in Table 1). This indicates an improvement of about 5 times compared to the conventional method shown in Comparative Example 1.

  In the measurement sample described above, many sweet spots with high ionization efficiency were observed, and a wavy structure was confirmed in that portion. The corrugated structure having the corrugated structure was slightly smaller or less in area when the measurement sample was viewed from above, and the number of corrugated structures was 100% by mass of methanol in Example 1, normal drying. However, almost the same result was obtained.

  Separately, as in Example 1, NA2F was dropped on the spot on the plate, dried, the same amount of the same matrix solution as above was dropped, the plate was inserted into the mass spectrometer AXIMA-QIT-TOFMS, and the rotary pump The sample was rapidly depressurized using a turbo molecular pump.

From this sample, an S / N of 38 and a peak of a sodium adduct [M + Na] ⁺ of NA2F were detected (see No. (n) in Table 1). This shows an improvement of about 7 times compared with the conventional method shown in Comparative Example 1.

  In the measurement sample described above, many sweet spots with high ionization efficiency were observed, and a wavy structure was confirmed in that portion. The corrugated structure having the corrugated structure was slightly smaller or less in area when the measurement sample was viewed from above, and the number of corrugated structures was 100% by mass of methanol in Example 1, normal drying. However, almost the same result was obtained.

  Further, the same result as above was obtained even when “water” as the solvent a was replaced with “60 mass% acetonitrile aqueous solution”.

  From these facts, it became clear that even for a matrix solution containing water, it is preferable to prepare a measurement sample by evaporating the solvent a under reduced pressure.

<Influence of heating>
Example 6
In the same procedure as in Example 1, the mirror plate dried with NA2F was left on a heat block heated to 80 ° C. for 5 minutes. After 5 minutes, 1 μL of a matrix solution in which DHBA was dissolved in 60% by mass of acetonitrile and made 10 mg / mL was added dropwise to NA2F on the plate that had reached 80 ° C. The solvent was evaporated in about 8 to 10 seconds, and a measurement sample was obtained.

From this sample, a peak of the sodium adduct [M + Na] ⁺ with an S / N of 30 and NA2F was detected (see No. (o) in Table 1). This shows an improvement of about 6 times compared with the conventional method shown in Comparative Example 1.

  In the measurement sample described above, many sweet spots with high ionization efficiency were observed, and a wavy structure was confirmed in that portion. The corrugated structure having the corrugated structure was slightly smaller or less in area when the measurement sample was viewed from above, and the number of corrugated structures was 100% by mass of methanol in Example 1, normal drying. However, almost the same result was obtained.

  From the above, it became clear that even when the matrix solution contains water, a good measurement sample can be obtained by heating and quickly drying the solvent a.

<Influence of matrix type>
Example 7
In the same manner as in Example 1, measurement was performed using a matrix other than DHBA. As a molecule to be measured, 1 μL of 100 fmol / μL NA2F aqueous solution was used, and the matrix was ATT (6-az-2-thiothymine), THAP (2,4,6-trihydroxyacetophenone monohydrate), CHCA (a-cyano-hydrocinnamic acid). , Nor-harman (9H-pyrido [3,4-b] indole) was used. All matrices were dissolved in methanol at a concentration of 40 mg / mL. Only nor-harman was dissolved at a concentration of 4 mg / mL. As ATT and THAP, those dissolved in a saturated methanol solution of ACDB (ammonia citrate dibasic) at a concentration of 40 mg / mL were also used.

  The thickness of the measurement sample in the portion having the wave-like structure on the surface of the obtained measurement sample was 9 μm or less.

Using any matrix, the same mass spectrum as in Example 1 using DHBA was obtained, 100 fmol of NA2F was strongly observed as a sodium-added molecule [M + Na] ⁺ , and the sweet spot where the signal was detected was also the conventional method. Compared to

  Many sweet spots with high ionization efficiency were observed in the measurement samples using all the matrices described above, and a wavy structure was confirmed in that portion. With respect to the corrugated structure having the corrugated structure, the area when the measurement sample was viewed from above and the number of corrugated structures were the same as those obtained when DHBA of Example 1 was used.

  It was confirmed that the present invention is the same not only for DHBA but also for other matrices. In addition, it was confirmed that the method can be applied to CHCA, which is particularly unsuitable for the measurement of trace sugar chains in the conventional method.

<(1) Method (Drived Droplet Method)>
Example 8
First, the NA2F stock aqueous solution stored in the Eppendorf tube was dried to dryness with a centrifugal concentrator, and a 40 mg / mL DHBA methanol solution was directly added thereto to obtain a NA2F concentration of 400 fmol / μL (step (D )). Of that, 0.25 μL was dropped onto a MALDI plate (step (E)) to prepare a measurement sample. The evaporation and drying of the solvent a were all performed at room temperature (23 ° C.) and atmospheric pressure as in Example 1. The MALDI mass spectrum was measured using a MALDI-QIT-TOF type mass spectrometer in the same manner as in Example 1, and was performed in the positive ion mode.

  Also in the method (1) (Driven Droplet method), when the solvent a was methanol, the same strong signal and high S / N as those in “Method (2)” of Example 1 could be obtained.

  Many sweet spots with high ionization efficiency were observed in the measurement samples using all the matrices described above, and a wavy structure was confirmed in that portion. As for the corrugated structure having the corrugated structure, the area when the measurement sample was viewed from above and the number of corrugated structures were almost the same as those in Example 1.

  When the sample containing the molecule to be measured is only a trace amount, it is more useful to first dry the sample containing the molecule to be measured on the plate according to the method (2), but when methanol is used as the solvent a, It was confirmed that it was not necessary to dry the sample solution on the plate.

  The method for preparing a measurement sample for mass spectrometry according to the present invention provides a measurement sample containing a lot of sweet spots with good ionization efficiency, thereby providing a mass spectrometry method capable of obtaining information on a highly reliable chemical structure. Therefore, it can be widely used not only for the chemical structure analysis of a small amount of a biological sample-derived molecule or a molecule in a biological sample, but also for the field of function elucidation and pathophysiology.

Claims (11)

A method for preparing a measurement sample for mass spectrometry from a sample containing a molecule to be measured and a matrix,
(A) a step of drying a solution of a sample containing a molecule to be measured on a plate, and thereafter
(B) including a step of dropping a solution obtained by dissolving only the matrix in “the solvent a capable of dissolving both the molecule to be measured and the matrix” onto the plate and drying it,
The molecule to be measured is a sugar, glycopeptide or glycoprotein;
The “solvent a that can dissolve both the molecule to be measured and the matrix” is an alcohol or phenol, or an organic solvent that is compatible with water at 20 ° C. in an arbitrary ratio, and does not substantially contain water. A method for preparing a measurement sample for mass spectrometry, which is a solvent. Between step (A) and step (B),
(C) The method for preparing a measurement sample for mass spectrometry according to claim 1, comprising a step of dropping a solution of a derivatizing agent that increases ionization efficiency by reacting with a molecule to be measured, onto the plate and drying the solution.   The method for preparing a measurement sample for mass spectrometry according to claim 2, wherein the derivatizing agent is a condensed polycyclic compound having a condensed polycycle in the molecule.   The method for preparing a measurement sample for mass spectrometry according to claim 3, wherein the derivatizing agent is a pyrene ring compound. A method for preparing a measurement sample for mass spectrometry from a sample containing a molecule to be measured and a matrix,
(D) A step of preparing a solution by mixing a sample containing a molecule to be measured and a matrix in a “solvent a that can dissolve both the molecule to be measured and the matrix”;
(E) a step of dropping the solution prepared in (D) onto a plate and drying,
The molecule to be measured is a sugar, glycopeptide or glycoprotein;
The “solvent a that can dissolve both the molecule to be measured and the matrix” is an alcohol or phenol, or an organic solvent that is compatible with water at 20 ° C. in an arbitrary ratio, and does not substantially contain water. A method for preparing a measurement sample for mass spectrometry, which is a solvent.   The “solvent a that can dissolve both the molecule to be measured and the matrix” is one solvent selected from the group consisting of methanol, ethanol, isopropanol, butanol, and acetonitrile, or a mixed solvent of two or more, substantially The method for preparing a measurement sample for mass spectrometry according to any one of claims 1 to 5, which is a solvent that does not contain water.   7. The measurement sample for mass spectrometry prepared from a sample containing a molecule to be measured and a matrix has a wavy structure with an average period of 1 nm to 300 nm on at least a part of the surface. A method for preparing a measurement sample for mass spectrometry according to any one of the claims.   The method for preparing a measurement sample for mass spectrometry according to claim 7, wherein twice the average amplitude of the wavy structure is 1 nm to 40 nm. The method for preparing a measurement sample for mass spectrometry according to claim 7 or 8, wherein at least one of the wavy structures having the wavy structure has an area of 4 µm ² or more when the measurement sample is viewed from above.   The method for preparing a measurement sample for mass spectrometry according to any one of claims 7 to 9, wherein the thickness of at least a part is 10 µm or less. The MS ⁿ method in which fragmentation is repeated n times for a measurement sample for mass spectrometry prepared using the method for preparing a measurement sample for mass spectrometry according to any one of claims 1 to 10 ( 2. Mass spectrometry characterized by applying 2 ≦ n).