JP2007309668A - Sample plate for laser desorption ionization mass spectrometry - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a sample plate for a laser desorption ionization mass spectrometry suitable for ionization in an LDI, a MALDI or the like, and usable repeatedly. <P>SOLUTION: This sample plate for the laser desorption ionization mass spectrometry includes a pyrolysis carbon layer having ≤2.0 μm of face roughness (Ra) as a holding face for an analytical object. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a sample plate for laser desorption / ionization (LDI) mass spectrometry that forms a fine and uniform crystal and can be used repeatedly.

  Laser desorption ionization (LDI), matrix-assisted laser desorption ionization (MALDI), and time-of-flight mass spectrometry (time-of-) using this for the analysis of macromolecular compounds such as proteins, peptides, polysaccharides, and organic compounds Mass spectrometry such as flight / Mass Spectrometry (TOF / MS) has become a useful tool.

  In these ionization techniques, for example, MALDI, a mixture of a matrix such as sinapinic acid and a protein is usually placed on a sample slide, crystallized, and then the protein is ionized by laser irradiation. As a sample plate used in such an ionization process, a metal plate such as stainless steel which is conductive for the subsequent mass analysis process is used.

  In addition, two-dimensionally arrayed molecules are plotted on a liquid-permeable non-conductive sample plate by electrophoresis or printing methods such as inkjet, and this sample plate is placed on a metal sample plate with conductive double-sided tape, etc. Ionization analysis is performed by pasting with (Patent Document 1).

Further, a highly accurate matrix-assisted laser desorption / ionization analysis has been performed using conductive glassy carbon as a sample plate to form fine and uniform crystals (Patent Document 2).
JP 2005-83784 A Japanese Patent Publication No. 3548769

  In mass spectrometry, chemical modification is often performed on an analysis target using Edman reaction or the like. However, a metal plate such as stainless steel has no resistance to such chemical modification, and it is necessary to transfer the modified product to the metal sample plate after performing the chemical modification separately, which makes the operation very complicated. Since the liquid-permeable sample plate disclosed in Patent Document 1 is not conductive, good ionization is difficult even if it is attached to a metal sample plate. Since it was necessary to affix on a metal sample plate, a thin film had to be used as a sample plate, and it was difficult to handle it. Furthermore, since functional groups and the like generated in the process of chemical modification in the sample plate corrode the metal plate, it has been difficult to repeatedly use the metal sample plate. In addition, in the case where a glass crystal carbon sample plate disclosed in Patent Document 2 that is strong against chemical modification is used and a matrix crystal including an object to be measured is deposited, the surface density at which the crystal is generated is reduced. There is a problem that unevenness is likely to occur and the peak intensity is not stable depending on the position of laser irradiation. For this reason, it is necessary to search for a region that gives a good signal, and there is a problem in that the reliability of the measured value is not stable every measurement.

  Therefore, an object of the present invention is to provide a sample plate for laser desorption ionization mass spectrometry that is suitable for ionization in LDI, MALDI, and the like and can be used repeatedly. Another object of the present invention is to provide a sample plate for laser desorption / ionization mass spectrometry capable of measuring with high accuracy.

  As a result of intensive studies to solve the above problems, it is possible to solve at least one of the above problems by providing a pyrolytic carbon layer having a surface roughness (Ra) of 2.0 μm or less as a holding surface of an analysis target. And the present invention was completed. That is, according to the present invention, the following means are provided.

According to the present invention, there is provided a sample plate for laser desorption ionization mass spectrometry, comprising a pyrolytic carbon layer having a surface roughness (Ra) of 2.0 μm or less as a holding surface for analysis. The The surface roughness (Ra) is preferably 1.4 μm or less.
The sample plate of the present invention is preferably used for MALDI mass spectrometry, and is preferably used for a time-of-flight mass spectrometer.

  In the sample plate of the present invention, the contact angle of the pyrolytic carbon layer with respect to a 20 (v / v)% acetonitrile aqueous solution is also preferably 45 ° or more and 70 ° or less. Furthermore, the thickness of the pyrolytic carbon layer is preferably 20 μm or more and 60 μm or less.

Moreover, the sample plate of the present invention can include the pyrolytic carbon layer on a ceramic substrate. The ceramic substrate is preferably isotropic graphite. The ceramic substrate preferably has a thermal expansion coefficient of 2.0 × 10 ⁻⁶ / ° C. or higher and 4.8 × 10 ⁻⁶ / ° C. or lower.

  According to the present invention, there is also provided a matrix-assisted laser desorption / ionization mass spectrometer comprising any one of the above sample plates. Furthermore, according to the present invention, there is also provided a matrix-assisted laser desorption / ionization mass spectrometry method using any one of the above sample plates.

  The sample plate for analysis of the present invention is characterized in that the holding surface to be analyzed is constituted by a pyrolytic carbon layer having a surface roughness (Ra) of 2.0 μm or less. According to the present invention, since the holding surface to be analyzed is composed of a pyrolytic carbon layer, it is useful as a sample plate when it is necessary to apply a voltage to the sample plate for sample supply, holding, or analysis. It is. For example, the sample plate for analysis of the present invention can be used in place of or as a part of a metal sample plate for mass spectrometry such as MALID-TOFMS.

  In addition, according to the present invention, the pyrolytic carbon layer has sufficient corrosion resistance, and can easily improve the corrosion resistance compared to a metal sample plate. For this reason, various chemical reactions for modifying the analysis target can be easily performed on the sample plate. As a result, conventionally, the product of the modification reaction that was separately performed prior to mass spectrometry was applied to the sample plate for mass spectrometry. Can be carried out on the same plate.

  Furthermore, according to the present invention, since the analysis target holding surface is composed of a pyrolytic carbon layer having a surface roughness of 2.0 μm or less, the sample solution containing the analysis target is dropped and held on the pyrolytic carbon layer. When this is done, a uniform surface density can be deposited on the analysis target. In particular, in MALDI, the surface density of crystals obtained by precipitating crystals from a sample solution containing a matrix and an analysis target on the pyrolytic carbon layer is unlikely to vary between the concave and convex portions of the pyrolytic carbon layer. Crystals can be generated with a more uniform surface density on the surface of the pyrolytic carbon layer. As a result, the problem that the peak intensity is not stable depending on the position where the laser is irradiated can be suppressed or avoided.

  The pyrolytic carbon in the present invention means a hydrocarbon gas such as methane or propane introduced at a high temperature (800 ° C. or higher and 2500 ° C. or lower) and deposited on a suitable substrate. Specifically, a carbon film formed by a thermal CVD method can be given. Therefore, DLC (diamond-like carbon) produced at a low substrate temperature (typically around room temperature) by the PVD method (low temperature plasma treatment, ion plating, ion sputtering, ECR plasma coating, etc.) is the heat of the present invention. Not included in cracked carbon. Hereinafter, embodiments of the present invention will be described in detail.

(Sample plate for laser desorption ionization mass spectrometry)
The sample plate of this invention can take the form according to the analysis objective etc. For example, it may be a flat plate such as a substrate, or a microtiter plate having a recess or a partition such as a well for forming one or a plurality of analysis target regions.

  The sample plate of the present invention includes a pyrolytic carbon layer as a holding surface for analysis. In the present invention, it is not excluded that the pyrolytic carbon layer constitutes the entire sample plate, but usually the pyrolytic carbon layer is formed on a suitable substrate surface.

(Base material)
The substrate for holding the pyrolytic carbon layer is not particularly limited as long as the rigidity as the sample plate is maintained. It may be porous or dense. The base material may be a metal material such as stainless steel or a conductive polymer material, but is preferably a substrate having a heat resistance of 800 ° C. or higher from the viewpoint of forming a pyrolytic carbon layer. As such a substrate material, a ceramic material is suitable. Examples of the ceramic material include carbon, silicon carbide, alumina, aluminum nitride, and boron nitride. Among these, graphite is preferable, and isotropic graphite is more preferable. Isotropic graphite has heat resistance against the temperature required for thermal CVD of pyrolytic carbon, and also has high conductivity itself. Furthermore, since the structure is dense, it is easy to produce a sample plate with a small surface roughness, so that stable measurement is possible. Further, since the difference in thermal expansion coefficient from the pyrolytic carbon film is small, there is an advantage that problems such as peeling of the coating film and cracks are unlikely to occur.

For example, when the substrate is used for TOFMS, a material having a volume resistance value (Ωcm) in the thickness direction of 0.2 to 1000 in the thickness direction can be used. Such a volume resistance value can be measured, for example, according to JIS R7222 for JISK7194 and particularly for a graphite material. Can be measured. In addition, a composite conductive ceramic material in which metal or conductive ceramic material particles are mixed in an insulating ceramic matrix may also be used. Further, the base material preferably has a thermal expansion coefficient of 2.0 × 10 ⁻⁶ / ° C. or more and 4.8 × 10 ⁻⁶ / ° C. or less. Since the thermal expansion coefficient in the direction perpendicular to the film-forming direction of pyrolytic carbon is about 2.2 × 10 ⁻⁶ / ° C., when the thermal expansion coefficient of the substrate exceeds 4.8 × 10 ⁻⁶ / ° C. There is a problem that the difference in thermal expansion coefficient from pyrolytic carbon becomes large, and it is easy to peel off. On the other hand, when the temperature is less than 2.0 × 10 ⁻⁶ / ° C., there is a possibility that cracks may occur in the pyrolytic carbon film. In addition, the measurement range of a thermal expansion coefficient is 50 degreeC-400 degreeC, and the calculation method shall calculate by dividing the elongation rate in 400 degreeC on the basis of the length in 50 degreeC by a temperature difference (350 degreeC). .

(Pyrolytic carbon layer)
As already described, examples of the pyrolytic carbon layer include a layer formed by decomposing and depositing hydrocarbons at least at 800 ° C. or higher, such as a layer formed by a thermal CVD method or the like. The pyrolytic carbon layer is preferably dense with suppressed liquid or gas permeability. In general, pyrolytic carbon film graphite (for example, Pyrocurve (manufactured by Ibiden Co., Ltd.), pyrograph (Toyo Tanso Co., Ltd.) is used to provide such a pyrolytic carbon layer on the surface of a substrate such as graphite (preferably isotropic graphite). Product) and Hitachi Chemical Co., Ltd. products).

  The pyrolytic carbon layer preferably has a surface roughness (Ra) of 2.0 μm or less. When the surface roughness exceeds 2.0 μm, the surface density of the crystals obtained when crystallized from the sample solution on the pyrolytic carbon film is not uniform due to the surface roughness (unevenness) of the surface. Cannot improve the accuracy. In addition, the unevenness causes a difference in flight time, and the spectral resolution decreases. On the other hand, if it exceeds 2.0 μm, the surface area becomes large and the laser irradiation energy is dispersed, so that the energy efficiency is lowered and the ion appearance irradiation intensity must be increased, and the object to be measured is easily damaged. Furthermore, from these viewpoints, the surface roughness is more preferably 1.4 μm or less. If it is 1.4 μm or less, the surface density of the crystal can be made more uniform, and the difference in flight time between the concave and convex portions can be suppressed, so that a decrease in spectral resolution can be suppressed or avoided. High measurement is possible. Furthermore, since the increase in the surface area is suppressed, it is possible to avoid the increase in the irradiation intensity of the laser that is irradiated while suppressing the dispersion of the laser irradiation energy. On the other hand, the surface roughness is preferably 0.05 μm or more. When it is 0.05 μm or more, when the solvent of the sample solution is volatilized and crystallized, it is easy to prevent the solution from moving to the center of the blot, and the crystals are unevenly distributed depending on the solvent volatilization condition at the time of crystallization. It is possible to suppress the surface density from becoming uneven. In addition, the surface roughness (Ra) was measured according to JISB0601 using Taylor Hobson's Form Talysurf S-5 type. The cut-off value is 0.25, the stylus radius is 2 μm, and the measurement length is 4 mm.

  The pyrolytic carbon layer preferably has a contact angle with respect to a 20 (v / v)% acetonitrile aqueous solution of 45 ° or more and 70 ° or less. When the contact angle is within this range, when a sample solution containing acetonitrile is dropped on the pyrolytic carbon layer, an appropriate spot diameter can be secured, the surface density can be maintained, and the SN ratio of measurement can be increased. On the other hand, when the contact angle is less than 45 °, the solution is diffused when the sample and the aqueous acetonitrile solution of the matrix are dropped on the sample plate, and the crystal formed after drying and air drying increases the spot diameter. Decreases, and the SN ratio of the measurement cannot be increased. In addition, when the contact angle exceeds 70 °, the wettability with respect to the sample plate is deteriorated, so that the sample solution measured by the pipette remains in the pipette and all the sample solutions are placed on the sample plate. Cannot be performed and the measurement accuracy is deteriorated. On the other hand, when the angle exceeds 70 °, the stability of the droplet on the sample plate is poor, the blot is enlarged by slight vibration and air flow, and the surface density of the crystal is not constant, so that the measurement accuracy is lowered. In addition, as for the component of acetonitrile aqueous solution, the remainder other than acetonitrile is water. The contact angle is measured by pipetting acetonitrile into the pyrolytic carbon layer with a pipette and taking a picture from the side within approximately 5 seconds. The tangent of the droplet and the sample are taken at the end where the sample plate and the droplet contact. It shall be done by measuring the angle of the plate.

  The pyrolytic carbon layer preferably has a thickness of 20 μm or more and 60 μm or less. Within this range, the pyrolytic carbon layer can be coated with a sufficient thickness corresponding to the unevenness of the substrate, and the pyrolytic carbon layer is suppressed in internal strain. On the other hand, if the thickness is less than 20 μm, the pyrolytic carbon layer may not be coated with a sufficient thickness on the bottom of the concave portion generated at the particle interface in a substrate such as ceramics. Accumulate distortion. For these reasons, the pyrolytic carbon layer is peeled off and cannot be used as a sample plate, or the sample plate is warped and high accuracy measurement cannot be performed.

(Sample plate manufacturing method)
Such a sample plate can be obtained by forming a pyrolytic carbon layer on a suitable substrate surface by a thermal CVD method or the like. The above-described surface roughness, thickness, and acetonitrile aqueous repellency of the pyrolytic carbon layer in the sample plate need only be provided as the characteristics of the pyrolytic carbon layer of the sample plate. That is, the pyrolytic carbon layer formed by the thermal CVD method or the like may have the same characteristics, or after the surface treatment such as shot blasting or polishing is performed on the pyrolytic carbon layer formed by the thermal CVD method or the like. It may be a characteristic.

(Analysis target)
The analysis target to be held in the sample plate of the present invention is not particularly limited, and is a polynucleotide and oligonucleotide in the form of DNA, RNA, DNA / RNA chimera, DNA / RNA hybrid, peptide, protein, polysaccharide, glycoprotein, Examples thereof include lipids and PNA (peptide nucleic acids). Such biopolymers may be derivatized by chemical modification for analysis. In addition to these biopolymers, natural or artificial organic compounds can be used as analysis targets. Such analysis objects are included in cell and fungal cell extracts, fermentation products, cell-free extracts, PCR products, artificially synthesized products, enzyme-treated products of proteins, and the like. In addition, the analysis target using the sample plate of the present invention is particularly preferably a compound that can be analyzed by MALDI or is suitable for analysis.

(Analysis method)
The mass spectrometric method using the sample plate of the present invention is characterized in that a sample plate holding an analysis target is used. That is, it is characterized in that a sample plate holding an analysis target is first prepared, and then various analysis techniques are performed on the sample plate to obtain analysis results. Hereinafter, first, a sample plate on which an analysis target is held and a manufacturing process thereof will be described.

(Sample plate holding the analysis target)
The holding of the analysis target on the sample plate means that the analysis target is held at a fixed position at least until the analysis target on the sample plate is analyzed. Therefore, even if the analysis target after analysis is in a fixed state that can be easily removed from the sample plate by washing or the like, it can be said that the analysis target is held on the sample plate. The holding form of the analysis target is not particularly limited. For example, it may be held by a non-covalent bond such as an ionic bond, a hydrogen bond, an electrostatic interaction, a hydrophobic interaction, or a chelate, or may be held by a covalent bond. In addition, other than such chemical bonds, even if the analyte is simply present in the recess on the surface of the sample plate, as long as it is retained in the recess until the time of analysis, this analyte Can be said to be retained. There are various fixed forms depending on the type of analysis target, the material of the sample plate, surface modification, and the like, but non-covalent binding is preferable in consideration of ionization in mass spectrometry.

  The analysis target may be held directly on the surface of the sample plate, or indirectly, for example, may be held on the sample plate via an inclusion held directly on the surface of the sample plate. For example, by holding an antibody or antigen on a sample plate and supplying the antigen or antibody to this sample plate and conducting an antigen-antibody reaction, the antibody or antigen to be analyzed can be converted into an antigen-antibody complex. In form, it may be held on the sample plate. Alternatively, a hybridizable compound such as an oligonucleotide or nucleic acid to be analyzed may be retained by complex formation (hybridization) by hydrogen bonding between the bases paired between the nucleotide chains. In this case, a nucleic acid or the like having a certain nucleotide sequence is held with respect to the sample plate. Such antigen-antibody specific interaction, receptor-ligand interaction, hybridization, protein-protein interaction, protein-nucleic acid interaction and other various interactions, inclusions and analysis previously held on the sample plate The analysis target may be held on the sample plate by utilizing a hydrogen bond, hydrophilic interaction, hydrophobic interaction, electrostatic interaction, chelation, or the like between the target.

  Further, the analysis target may be chemically modified as necessary. The type of chemical modification is not particularly limited. As will be described in detail later, for example, for proteins, reaction products obtained by the Edman method may be retained as analysis targets.

  Since the region (spot or the like) on the sample plate in which one or more types of analysis objects are held together includes the analysis object, a cell for analysis can be configured as it is. That is, the sample plate can have one or two or more analysis cells in which one or more analysis objects are held. Note that a cell is an area where one or more analysis objects are held, and means an area to be measured by an analysis method.

  In the case of having two or more types of analysis cells, these analysis cells are preferably arranged, and more preferably associated with unique position information. That is, it is preferable that the position of each analytical cell on each sample plate is specified. The analysis cell is associated with the specific position information and the position thereof is specified, so that the analysis process can be facilitated and the analysis after the analysis process can be facilitated. This makes it possible to speed up the analysis when a large number of analysis cells are held. For example, in the case of MALDI, the analysis cells arranged on the sample plate are sequentially irradiated with a laser to desorb and ionize the analysis target contained in the cell, thereby easily analyzing a large number of analysis cells. Can be done quickly.

(Preparation of sample plate holding analysis target)
In order to hold the analysis object directly on the sample plate, first, the analysis object is supplied onto the sample plate. The analysis target may be synthesized on the sample plate, or an analysis target prepared in advance may be supplied on the sample plate. Examples of the case where the analysis target is directly synthesized on the sample plate include the case where the protein is synthesized by a cell-free protein synthesis system and the case where the oligonucleotide is synthesized on the sample plate. Moreover, in order to supply an analysis object to a sample plate, the inkjet method, the pin method, etc. can be used, for example.

  Further, the mixture containing the analysis target may be supplied to the sample plate after being separated by a separation technique such as electrophoresis or chromatography. In particular, it is preferable to supply the components separated based on the patterns separated by electrophoresis or chromatography to the sample plate. By doing so, the analysis objects separated by the various separation means can be individually analyzed corresponding to the separation patterns. Specifically, the electrophoresis pattern can be directly transferred to the sample plate using blotting. In this case, the sample plate is preferably liquid permeable. In addition, various elution fractions can be held corresponding to the separation patterns by dropping the eluent from the liquid chromatography into dots or streams on the sample plate at regular intervals.

  In addition, when the analysis target is a polymer compound such as peptide or protein, the molecular weight can be analyzed by mass spectrometry and the information content obtained is taken into consideration on the sample plate after appropriately reducing the molecular weight with various degrading enzymes. You may supply. For example, the protein may be treated with a protease such as trypsin. In addition to analyzing fragmentation patterns, the accuracy of mass spectrometry is improved, and protein identification or protein sequencing is facilitated. Further, it is preferable that the fragmented protein is separated by electrophoresis or liquid chromatography and then supplied to the sample plate.

  Examples of electrophoresis include agarose gel electrophoresis, denaturing agarose gel electrophoresis, polyacrylamide electrophoresis, SDS polyacrylamide electrophoresis, isoelectric focusing, and two-dimensional electrophoresis. As liquid chromatography, liquid chromatography using a microcapillary or a nanocapillary is preferably used.

  After supplying the analysis target onto the sample plate in this way, the analysis target is held on the sample plate. Most simply, the analysis target can be held on the sample plate by evaporating and drying a medium such as water used for supplying the analysis target. In addition, when the functional group on the sample plate or the surface of the sample plate and the analysis target are held via a covalent bond, the necessary target reagent is supplied to the sample plate together with the analysis target or reacted separately to cause the analysis target to be the sample plate. Can be retained. In addition, necessary conditions are given according to the holding form.

  After the analyte is supplied or held on the sample plate, the analyte can be chemically modified on the sample plate. It can be said that the analysis target before chemical modification is also a precursor of the analysis target. The type and method of modification are not particularly limited. For example, when the analysis object is protein, peptide, etc., reaction with 1-fluoro-2,4-dinitrobenzene (FNDB) or 5-dimethylaminonaphthalene-1-sulfonyl chloride (dansyl chloride), Edman reagent ( Phenyl isothiocyanate) and the Edman method with anhydride.

  Examples of the analysis target in the present invention include reaction products and reaction intermediates of Edman reaction or similar reactions. For example, an N-terminal amino acid sequence of a protein or the like can be determined by a so-called ladder sequence method using a modified Edman method. That is, any one of a few percent of isocyanate (ITC), fluorescein isothiocyanate (FITC), methyl isothiocyanate (MITC), etc. is added to fanyl isothiocyanate (PITC) and coupled to form a modifying group for the N-terminal amino acid residue. Analyze the sample plate by changing it and then suppressing the cleavage reaction of the N-terminal amino acid residue by fluoroacetic acid or by adjusting the cleavage reaction time. On the region, (1) a derivative of a cleaved N-terminal amino acid residue that is one of these degradation reaction products, and (2) a fragment that is cleaved at the other N-terminal amino acid residue (Edman reaction) And (3) uncut protein or peptide that has been coupled but not cleaved. Kazaritai a (coupling mass) so as to predominantly remain.

  Here, the above (2) is a reaction product of the Edman reaction or a similar reaction, and the above (3) is a reaction intermediate of the Edman reaction or a similar reaction.

  Among these, (2) and (3) differ only in the N-terminal amino acid residue, and therefore, by measuring and comparing the mass difference of these fragments in the mass spectrometry step, the N-terminal amino acid residue is determined. The group can be determined. Further, the N-terminal amino acid sequence can be determined by repeating the reaction step and the mass spectrometry step as many times as necessary.

  From the above, the following examples can be given as operations for holding peptides and proteins on a sample plate and typical examples of such a sample plate. That is, after degrading the protein with a protease such as trypsin, the protein is separated by micro or nanocapillary liquid chromatography, and the eluent is arranged as a large number of spots on the sample plate, dried and dried. Then, the modified Edman method as described above is applied to react, and then washed and dried. In this way, one or two or more fragments obtained by degrading the protein with trypsin are separated into one or two or more eluent spots (for analysis) corresponding to the separation pattern by liquid chromatography. A sample plate provided as a cell) is obtained. At the same time, due to the application of the modified Edman method, each eluent spot was coupled with a fragment which is an Edman reaction product from which the N-terminal amino acid residue of the peptide or protein contained in the spot was cleaved. A sample plate is obtained in which an uncut coupling body in which the N-terminal is not cut is retained.

  Further, in order to indirectly hold the analysis target on the sample plate, first, a sample plate holding an inclusion for holding the analysis target indirectly is prepared. As described above, the inclusion is, for example, a protein or a nucleic acid. In order to supply and hold such inclusions on the sample plate, the above-described method can be appropriately used. In addition, in order to hold the analysis target, the sample containing the analysis target is supplied to the sample plate holding the inclusion in this way, and the intended interaction is generated to analyze the analysis target via the inclusion. Hold the subject on the sample plate.

(Analysis process)
An analysis process is performed on the sample plate holding the analysis target. In this analysis step, it is preferable to employ mass spectrometry. The ionization method in the mass spectrometric process is not particularly limited. From the viewpoint of using the sample plate for ionization as it is, laser desorption ionization (LDI) without using a matrix and matrix-assisted laser desorption ionization (MALDI) with a matrix are used. It is preferable to use it. Of these, MALDI is preferably used for peptides and proteins.

The laser used in _{MALDI, N 2, Nd-YAG} , CWCO, TEA-CO 2, argon and the like. The matrix used for MALDI is not particularly limited, but nicotinic acid, 2-pyrazinecarboxylic acid, sinapinic acid, 2,5-dihydroxybenzoic acid, 5-methoxysalicylic acid, α-cyano-4-hydroxycinnamic acid, 3-hydroxypicolinic acid, diaminonaphthalene, 2- (4-hydroxyphenylazo) benzoic acid, disranol, succinic acid, 5- (trifluoromethyl) uracil, glycerin, and the like can be used. The addition amount of the matrix with respect to the analysis target is not particularly limited and can be appropriately set within a conventionally known range.

  In addition, the mass spectrometric process in this mass spectrometric method can be analyzed by various mass spectrometers such as a magnetic polarization type, a quadrupole type, an ion trap type, a time-of-flight type, a Fourier transform ion cyclone type, and a tandem type. . In particular, in the case of LDI and MALDI, a mass analysis process using a time-of-flight analyzer is preferable.

  In the mass analysis step, the mass of one or more types of analysis objects existing in the analysis cell on the sample plate can be analyzed. For example, when an Edman method product is present in the analytical cell, that is, a derivative of a terminal amino acid residue (ATZ derivative or PTH derivative) that is an Edman reaction product, and a remaining fragment that is an Edman reaction product, When there is an uncut protein or peptide modification product in which an isocyanate coupling agent is coupled to the N-terminus but is not cleaved, according to the LDI method or MALDI method, The molecular weight of the uncleaved product can be easily measured, whereby the N-terminal amino acid can be identified along with the overall molecular weight.

  In addition, the analysis method implemented at an analysis process can implement various analysis methods besides mass spectrometry. In addition, when the analysis target is labeled with a fluorescent reagent, a coloring reagent, or the like, a method for detecting such fluorescence or coloring on the sample plate can be employed.

(Mass spectrometer)
The mass spectrometer of the present invention includes the sample plate of the present invention. The mass spectrometer of the present invention is LDI or MALDI, and is preferably a time-of-flight mass spectrometer.

EXAMPLES Hereinafter, although an Example is given and this invention is demonstrated concretely, this invention is not limited to these Examples.
(Sample plate making process)
Sample plate of Examples 1 and 2 by coating pyrolytic carbon (Pyrocurve manufactured by Ibiden Co., Ltd.) on a ceramic base material whose thickness is reduced by coating the pyrolytic carbon layer to be coated. Was made. When the appearance of the produced sample plate was checked, there was no problem such as peeling or cracking. Moreover, the material of the ceramic base material used at this time, the layer thickness of the pyrolytic carbon layer, the thermal expansion coefficient of the base material, and the surface roughness of the pyrolytic carbon layer were as shown in Table 1. In addition, these measurements were performed by the method as already demonstrated.

  In addition, sample plates of Comparative Examples 1 and 2 and Reference Examples 1 to 3 shown in Table 1 were prepared. The sample plate of Comparative Example 1 was produced in the same manner as in the example except that the surface roughness was 2.1 μm. In addition, the sample plate of Comparative Example 2 was prepared by impregnating isotropic graphite with a phenol resin, and processing a carbon plate that was vitrified carbonized through a curing and firing process into the shape of the sample plate. The sample plates of Comparative Examples 1 and 2 were free from peeling and cracks.

  The sample plates of Reference Examples 1 to 3 were produced in the same manner as in the Examples except that the layer thickness of the pyrolysis layer and the thermal expansion coefficient of the substrate were as shown in Table 1. In the sample plates of Reference Examples 1 to 3, problems such as peeling and cracking occurred, and it was difficult to use them as sample plates from this viewpoint.

  As for the sample plates of the comparative example and the reference example, similarly to the sample plate of the example, the material of the ceramic substrate, the layer thickness of the pyrolytic carbon layer, the thermal expansion coefficient of the substrate, and the surface roughness of the pyrolytic carbon layer are It was as Table 1.

(Sample preparation process)
The analysis target was held as follows for the sample plates of the examples and comparative examples. As an analysis object, angiotensin (MW1293) is diluted with 20 v / v% acetonitrile solution to 500, 250, 125, 62.5, 31.3, 15.6 fmol / μl, and 1 μl each is made of polypropylene. After placing the crystal using a pipette and depositing crystals, the surface of the pyrolytic carbon layer was observed. The matrix used was α-cyano-4-hydroxycinnamic acid, the concentration was 5 mg / ml, and 1 μm 1 was dropped. 1A, FIG. 1B, FIG. 1C, FIG. 1D, and Table 2 show the precipitation states of the matrix crystals containing angiotensin in Examples 1 and 2 and Comparative Examples 1 and 2, respectively. Moreover, the contact angle of 20 v / v% acetonitrile aqueous solution on these sample plates was measured. The contact angle with the acetonitrile aqueous solution is also shown in Table 1.

(Analysis process)
The sample plate holding the blot to be analyzed was analyzed using a MALDI-TOF mass spectrometer (ABIVoyager DE-STR MALDITF type mass spectrometer), a peak derived from angiotensin (angiotensin, MW1293), background noise, Comparison was carried out at each dilution rate to confirm the possibility of measurement. The measurement availability is shown in Table 2, and detailed measurement charts of Examples 1 and 2 and Comparative Examples 1 and 2 are shown in FIGS. 2A, 2B, 2C, and 2D, respectively. In addition, FIG. 3 shows the signal intensity distribution in arbitrary units on the sample plate blotted with 62.5 fmol / μl of 20 v / v% acetonitrile aqueous solution in Examples 1 and 2 and Comparative Examples 1 and 2.

  As shown in Table 1 and Table 2, in the sample plates shown in Examples 1 and 2 and Comparative Examples 1 and 2, it can be used as a sample plate of a MALDI mass spectrometer, and measurement by a MALDI mass spectrometer is performed. It was found to be possible in the same manner as conventional glassy carbon. As shown in FIGS. 1A and 1B and Table 2, the crystals on the pyrolytic carbon layer of the sample plates of Examples 1 and 2 are fine and the matrix crystals are uniformly dispersed at any position on the blot. It was confirmed that Furthermore, as shown in FIG. 3, according to the sample plates of Examples 1 and 2, it was found that the degree of dispersion of the signal intensity distribution is low, and measurement with excellent accuracy and reproducibility is possible. In contrast, as shown in FIGS. 1C and 1D and Table 2, in the sample plates of Comparative Examples 1 and 2, the dispersibility of the matrix crystals was poor, and crystal segregation was observed in the blot. Further, as shown in FIG. 3, in the sample plates of Comparative Examples 1 and 2, there was a tendency for the signal intensity to vary.

  In addition, since pyrolytic carbon is excellent in the corrosion resistance with respect to an acid and an alkali, and organic-solvent resistance, it can be chemically modified on a pyrolytic carbon layer, and has favorable repetitive usability. Therefore, it was found that the sample plate of this example is a sample plate for laser desorption ionization mass spectrometry that is suitable for ionization in LDI, MALDI, and the like, can be used repeatedly, and can measure with high accuracy.

FIG. 3 is a diagram showing a micrograph of a matrix crystal including an analysis target in Example 1. The figure which shows the microscope picture of the matrix crystal containing the analysis object in Example 2. FIG. The figure which shows the microscope picture of the matrix crystal containing the analysis object in the comparative example 1. FIG. The figure which shows the microscope picture of the matrix crystal containing the analysis object in the comparative example 2. FIG. FIG. 3 shows a measurement chart of Example 1. FIG. 6 shows a measurement chart of Example 2. The figure which shows the measurement chart of the comparative example 1. FIG. The figure which shows the measurement chart of the comparative example 2. FIG. The figure which shows the signal intensity distribution of arbitrary units in the sample plate which blotted the acetonitrile aqueous solution of 62.5 fmol / microliter of angiotensin in Example 1, 2 and Comparative Examples 1,2.

Claims (10)

A sample plate for laser desorption ionization mass spectrometry,
A sample plate comprising a pyrolytic carbon layer having a surface roughness (Ra) of 2.0 μm or less as a holding surface to be analyzed.   The sample plate according to claim 1, comprising a pyrolytic carbon layer having a surface roughness (Ra) of 1.4 μm or less.   The sample plate according to claim 1 or 2, wherein a contact angle with respect to a 20 (v / v)% acetonitrile aqueous solution is 45 ° or more and 70 ° or less.   The sample plate in any one of Claims 1-3 whose thickness of the said pyrolytic carbon layer is 20 micrometers or more and 60 micrometers or less.   The sample plate according to claim 1, wherein the pyrolytic carbon layer is provided on a ceramic substrate.   The sample plate according to claim 5, wherein the ceramic substrate is isotropic graphite. The sample plate according to claim 5 or 6, wherein the ceramic substrate has a coefficient of thermal expansion of 2.0 × 10 ^-6 / ° C or more and 4.8 × 10 ^-6 / ° C or less.   The sample plate according to any one of claims 1 to 7, which is used for a time-of-flight mass spectrometer.   A laser desorption / ionization mass spectrometer comprising the sample plate according to claim 1.   A laser desorption / ionization mass spectrometry method using the sample plate according to claim 1.