JP7382600B2 - Materials for preparing samples for mass spectrometry - Google Patents

Description

The present invention relates to materials for preparing samples for mass spectrometry.

Mass spectrometry is an important analytical technique for determining the molecular weight and structure of a target substance (analyte) contained in a sample. A wide range of target substances is possible, from synthetic substances to biological substances, and further improvements in the sensitivity of analysis techniques are being considered.

For example, matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) is a soft ionization method that causes less fragmentation of target substances, and the obtained molecular ions can be subjected to mass spectrometry. In the MALDI-MS method, conditions for realizing efficient ionization of a target substance are being studied (see, for example, Patent Document 1). However, it is known that hydrophobic substances such as hydrophobic peptides are difficult to ionize, and there is a problem in mass spectrometry that ionization efficiency is low and measurement sensitivity is low.

JP2013-68598A

An object of the present invention is to provide a material for a mass spectrometry sample that enables highly sensitive measurement of hydrophobic substances in mass spectrometry, particularly MALDI-MS.

The present inventors conducted extensive studies to solve the above problems. As a result, it was discovered that the above-mentioned problems could be solved by using the following materials, and the present invention was completed.
The present invention includes, for example, the following [1] to [14].

[1] A material containing a polymer for preparing a sample for mass spectrometry.
[2] The material according to [1] above, wherein the substance to be subjected to mass spectrometry is a hydrophobic substance.
[3] The material according to [2] above, wherein the hydrophobic substance is a hydrophobic peptide.
[4] The material according to any one of [1] to [3] above, wherein the polymer has a Mooney viscosity [ML ₁₊₄ (100° C.)] of 10 to 100.
[5] The material according to any one of [1] to [4] above, wherein the polymer is a crosslinked polymer.
[6] The material according to any one of [1] to [5], wherein the mass spectrometry sample is a matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) sample.
[7] A matrix composition for mass spectrometry containing the material according to any one of [1] to [6] above and a matrix for mass spectrometry.
[8] The matrix composition for mass spectrometry according to [7] above, wherein the matrix is a compound having at least one selected from a carboxy group, a hydroxy group, and an amino group.
[9] The matrix composition for mass spectrometry according to [8], wherein the compound having at least one selected from a carboxy group, a hydroxy group, and an amino group is a compound represented by formula (1).

［式（１）中、Ｒ¹は、炭素数１～２０の炭化水素基、前記炭化水素基中の少なくとも１つの炭素－炭素結合間に、－Ｏ－、－ＣＯＯ－、－ＯＣＯ－、－ＯＳＯ₂－、－ＳＯ₃－、－ＯＳＯ₂ＮＲ－、－ＮＲＳＯ₂－、－ＮＲＣＯ－、－ＮＲ－および－ＣＯＮＲ－から選ばれる少なくとも１種を有する基、またはこれらの基中の水素原子の一部が置換された基であり；Ｒ²は、直接結合、炭素数１～２０の２価の炭化水素基、前記２価の炭化水素基中の少なくとも１つの炭素－炭素結合間に、－Ｏ－、－ＣＯＯ－、－ＯＣＯ－、－ＯＳＯ₂－、－ＳＯ₃－、－ＯＳＯ₂ＮＲ－、－ＮＲＳＯ₂－、－ＮＲＣＯ－、－ＮＲ－および－ＣＯＮＲ－から選ばれる少なくとも１種を有する基、またはこれらの基中の水素原子の一部が置換された基であり；Ｒは、水素原子または炭素数１～２０の炭化水素基であり；Ｘは、カルボキシ基、ヒドロキシ基またはアミノ基であり；Ｒ³は、ニトロ基、ホルミル基、アシル基、アルコキシカルボニル基またはヒドロキシフェニルアゾ基であり；Ａｒは、（ｍ＋ｎ＋ｏ）価の芳香族基であり；ｍは０～５の整数であり、ｎは１～５の整数であり、ｏは０～５の整数であり、Ｒ¹が複数ある場合は、それぞれ同一でも異なっていてもよく、Ｒ²が複数ある場合は、それぞれ同一でも異なっていてもよく、Ｘが複数ある場合は、それぞれ同一でも異なっていてもよく、Ｒ³が複数ある場合は、それぞれ同一でも異なっていてもよい。］

[In formula (1), R ¹ is a hydrocarbon group having 1 to 20 carbon atoms, and between at least one carbon-carbon bond in the hydrocarbon group, -O-, -COO-, -OCO-, - A group having at least one selected from OSO ₂ -, -SO ₃ -, -OSO ₂ NR-, -NRSO ₂ -, -NRCO-, -NR- and -CONR-, or a hydrogen atom in these groups R 2 is a partially substituted group; R ² is a direct bond, a divalent hydrocarbon group having 1 to 20 carbon atoms, or a bond between at least one carbon-carbon bond in the divalent hydrocarbon group; At least one selected from O-, -COO-, -OCO-, -OSO ₂ -, -SO ₃ -, -OSO ₂ NR-, -NRSO ₂ -, -NRCO-, -NR- and -CONR- R is a hydrogen atom or a hydrocarbon group having 1 to 20 carbon atoms; X is a carboxy group, a hydroxy group, or a group in which some of the hydrogen atoms in these groups are substituted; group; R ³ is a nitro group, formyl group, acyl group, alkoxycarbonyl group, or hydroxyphenylazo group; Ar is an (m+n+o)-valent aromatic group; m is an integer of 0 to 5; , n is an integer from 1 to 5, and o is an integer from 0 to 5. If there are multiple R ^1s , they may be the same or different, and if there are multiple R ^2s , they may be the same or different. When there is a plurality of X's, they may be the same or different, and when there is a plurality of ^R3 's, they may be the same or different. ]

[10] The content of the polymer derived from the material is 0.1 to 10,000 parts by mass based on 100 parts by mass of the matrix, according to any one of [7] to [9] above. The mass spectrometry matrix composition described above.
[11] The method according to [7] to [10] above, wherein the content of the polymer derived from the material is 20 to 10,000 parts by mass per 100 parts by mass of the substance to be analyzed by mass spectrometry. The matrix composition for mass spectrometry according to any one of the items.
[12] A kit for preparing a sample for mass spectrometry, comprising the material according to any one of [1] to [6] above and a matrix for mass spectrometry.
[13] Forming a sample liquid containing a mass spectrometry target substance, a matrix, the material according to any one of [1] to [6] above, and a solvent on a mass spectrometry target plate; A method for preparing a sample for mass spectrometry, comprising the step of removing the solvent from the sample liquid.
[14] Forming a sample liquid containing a mass spectrometry target substance, a matrix, the material according to any one of [1] to [6] above, and a solvent on a mass spectrometry target plate; A mass spectrometry method comprising: removing the solvent from the sample liquid to obtain a sample for mass spectrometry; and irradiating the sample for mass spectrometry with a laser to detect the target substance.

According to the present invention, it is possible to provide a material for a sample for mass spectrometry that enables highly sensitive measurement of hydrophobic substances in mass spectrometry, particularly in MALDI-MS.

EMBODIMENT OF THE INVENTION Hereinafter, the form for implementing this invention is demonstrated.
〔material〕
The material of the present invention contains a polymer and is used to prepare a sample for mass spectrometry. That is, the prepared sample for mass spectrometry contains the polymer as a material. The material of the present invention is suitable for use in mass spectrometry samples containing a target substance for mass spectrometry and a mass spectrometry matrix (hereinafter simply referred to as "matrix"), particularly samples for matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). Preferably, it is used for the preparation.

The polymer can enhance the ionization of the target substance by the matrix and improve the detection limit of the target substance. As described above, in the present invention, target substances such as hydrophobic substances can be specifically detected and analyzed.

Examples of ionization for mass spectrometry include laser desorption ionization (LDI), electron ionization (EI), chemical ionization (CI), fast atom bombardment (FAB), and field desorption (FD). , electrospray ionization (ESI) method. Among these, the laser desorption ionization (LDI) method is preferred in the present invention, and the matrix-assisted laser desorption ionization (MALDI) method is particularly preferred.

[Polymer]
In the present invention, a polymer is used as a material for a mass spectrometry sample, particularly a MALDI-MS sample. It is presumed that the incorporation of a target substance such as a hydrophobic substance into the polymer indirectly promoted energy transfer from the matrix to the target substance for mass spectrometry.

Examples of the polymer include organic polymers such as conjugated diene polymers, (meth)acrylic polymers, and olefin polymers. The polymer may be a rubber or a resin.

Further, from the viewpoint of incorporating the target substance for mass spectrometry, the polymer is preferably a crosslinked polymer having a crosslinked structure in a part of the molecule, that is, a crosslinked rubber and/or a crosslinked resin. In order to obtain such a crosslinked polymer, it is preferable to copolymerize using a polyfunctional unsaturated compound (crosslinkable monomer) having two or more polymerizable unsaturated groups.

In one embodiment, the polymer is preferably not "fine particles in which a core made of a metal oxide is coated with a polymer."
A conjugated diene polymer is a polymer having a structural unit derived from a conjugated diene compound, and may be a single or copolymer of a conjugated diene compound, or a polymer composed of a conjugated diene compound and one or more other monomers. It may be a copolymer of The other monomer in the conjugated diene polymer is, for example, at least one compound selected from aromatic vinyl compounds, unsaturated carboxylic acids, unsaturated carboxylic acid esters, unsaturated nitrile compounds, and polyfunctional unsaturated compounds. .

The conjugated diene polymer is, for example, at least one compound selected from a conjugated diene compound, a polyfunctional unsaturated compound, an aromatic vinyl compound, an unsaturated carboxylic acid, an unsaturated carboxylic acid ester, and an unsaturated nitrile compound. A copolymer with is preferred.

The (meth)acrylic polymer is a polymer having a structural unit derived from (meth)acrylic ester, and may be a single or copolymer of (meth)acrylic ester, or may be a (meth)acrylic ester-based polymer. It may also be a copolymer of an acid ester and one or more other monomers. The other monomer in the (meth)acrylic polymer is, for example, at least one compound selected from aromatic vinyl compounds, unsaturated carboxylic acids, unsaturated nitrile compounds, conjugated diene compounds, and polyfunctional unsaturated compounds. .

The (meth)acrylic polymer is, for example, at least one selected from a (meth)acrylic ester, a polyfunctional unsaturated compound, an aromatic vinyl compound, an unsaturated carboxylic acid, an unsaturated nitrile compound, and a conjugated diene compound. Copolymers with species compounds are preferred.

Olefin polymers are polymers that have structural units derived from α-olefins, and may be monopolymers or copolymers of α-olefins, or polymers of α-olefins and one or more other monomers. It may also be a copolymer. The other monomer in the olefin polymer is, for example, at least one selected from non-conjugated diene compounds and (meth)acrylic acid. Specific examples of α-olefins are described in the <Other monomers> column.

Examples of olefin polymers include polyethylene, polypropylene, polybutylene, copolymers of ethylene and other α-olefins, copolymers of ethylene and other α-olefins, and non-conjugated diene compounds, and copolymers of ethylene and (meth) ) copolymers with acrylic acid.
Each of the above monomers will be explained below.

<Conjugated diene compound>
When the polymer has a structural unit derived from a conjugated diene compound, it can have better compatibility with other monomer components.

Examples of the conjugated diene compound include 1,3-butadiene, 2-methyl-1,3-butadiene, 2,3-dimethyl-1,3-butadiene, 2-chloro-1,3-butadiene, and 1,3-butadiene. Examples include pentadiene, 2-methyl-1,3-pentadiene, 1,3-hexadiene, and 4,5-diethyl-1,3-octadiene, with 1,3-butadiene being preferred. The conjugated diene compound can be used alone or in combination of two or more.

<Aromatic vinyl compound>
When the polymer has a structural unit derived from an aromatic vinyl compound, measurement sensitivity in mass spectrometry can be improved.
Examples of aromatic vinyl compounds include styrene, α-methylstyrene, 1,1-diphenylethylene; alkylated styrenes such as methylstyrene, t-butylstyrene, and vinylxylene; monofluorostyrene, difluorostyrene, monochlorostyrene, and dichlorostyrene; Examples include halogenated styrenes such as styrene, monobromostyrene and dibromostyrene; vinylnaphthalene; N,N-diethyl-p-aminomethylstyrene and N,N-diethyl-p-aminoethylstyrene, with styrene being preferred. Aromatic vinyl compounds can be used alone or in combination of two or more.

<Unsaturated carboxylic acid>
When the polymer has a structural unit derived from an unsaturated carboxylic acid, the dispersibility of the polymer in various solvents can be improved.
Examples of unsaturated carboxylic acids include unsaturated mono- or dicarboxylic acids such as (meth)acrylic acid, crotonic acid, cinnamic acid, maleic acid, fumaric acid, itaconic acid, citraconic acid, and mesaconic acid; It may also be an acid anhydride such as. Among these, unsaturated monocarboxylic acids are preferred. Unsaturated carboxylic acids can be used alone or in combination of two or more.

<Unsaturated carboxylic acid ester and (meth)acrylic acid ester>
When the polymer has a structural unit derived from an unsaturated carboxylic acid ester or a (meth)acrylic acid ester, the dispersibility of the polymer in various solvents can be improved.
Examples of unsaturated carboxylic esters include (meth)acrylic esters, specifically methyl (meth)acrylate, ethyl (meth)acrylate, n-propyl (meth)acrylate, and (meth)acrylate. ) Isopropyl acrylate, n-butyl (meth)acrylate, isobutyl (meth)acrylate, n-amyl (meth)acrylate, isoamyl (meth)acrylate, hexyl (meth)acrylate, (meth)acrylic acid 2 - (meth)acrylic acid alkyl esters such as ethylhexyl, octyl (meth)acrylate, nonyl (meth)acrylate, and decyl (meth)acrylate; (meth)acrylic acid cycloalkyl esters such as cyclohexyl (meth)acrylate; Hydroxyalkyl (meth)acrylates such as hydroxymethyl (meth)acrylate, hydroxyethyl (meth)acrylate, and hydroxybutyl (meth)acrylate; ethylene glycol mono(meth)acrylate, propylene glycol mono(meth)acrylate, etc. Alkylene glycol mono(meth)acrylate; Compounds in which one or more hydrogen atoms of these monomers are replaced with fluorine atoms are mentioned.

Among these, methyl (meth)acrylate, ethyl (meth)acrylate, n-butyl (meth)acrylate, 2-ethylhexyl (meth)acrylate, hydroxymethyl (meth)acrylate, hydroxyl (meth)acrylate At least one selected from ethyl and hydroxybutyl (meth)acrylate is preferred.
Unsaturated carboxylic acid esters can be used alone or in combination of two or more.

<Unsaturated nitrile compound>
When the polymer has a structural unit derived from an unsaturated nitrile compound, the dispersibility of the polymer in various solvents can be improved.
Examples of the unsaturated nitrile compound include α,β-unsaturated nitrile compounds such as (meth)acrylonitrile, α-chloroacrylonitrile, α-ethyl acrylonitrile, and vinylidene cyanide, with (meth)acrylonitrile being preferred. Unsaturated nitrile compounds can be used alone or in combination of two or more.

<Polyfunctional unsaturated compound>
When the polymer has a structural unit derived from a polyfunctional unsaturated compound, the measurement sensitivity in mass spectrometry can be improved.
Examples of polyfunctional unsaturated compounds include polyfunctional vinyl compounds such as divinylbenzene, divinyltoluene, diisopropenylbenzene, and trivinylbenzene; ethylene glycol di(meth)acrylate, propylene glycol di(meth)acrylate, and propanediol. Di(meth)acrylate, Butanediol di(meth)acrylate, Hexanediol di(meth)acrylate, Diethylene glycol di(meth)acrylate, Dipropylene glycol di(meth)acrylate, Polyethylene glycol di(meth)acrylate, Polypropylene glycol di(meth)acrylate Polyfunctional materials such as meth)acrylate, trimethylolpropane di(meth)acrylate, trimethylolpropane tri(meth)acrylate, pentaerythritol tri(meth)acrylate, pentaerythritol tetra(meth)acrylate, dipentaerythritol hexa(meth)acrylate, etc. (Meth) acrylate; polyfunctional allyl compounds such as diallyl phthalate can be mentioned. Among these, at least one selected from divinylbenzene and trimethylolpropane tri(meth)acrylate is preferred. The polyfunctional unsaturated compounds can be used alone or in combination of two or more.

<Other monomers>
The polymer may have other structural units derived from monomers other than those mentioned above.
Examples of monomers other than the above include ethylene, propylene, 1-butene, 1-pentene, 3-methyl-1-butene, 1-hexene, 3-methyl-1-pentene, 4-methyl-1-pentene, 3 -α-olefins such as ethyl-1-pentene, 1-octene, and 1-decene; fluorinated α-olefins such as vinylidene fluoride, ethylene tetrafluoride, and propylene hexafluoride; (meth)acrylamide, N-methylol ( (Meth)acrylamide compounds such as meth)acrylamide, aminoethyl (meth)acrylamide, dimethylaminomethyl (meth)acrylamide, methylaminopropyl (meth)acrylamide, dimethylaminopropyl (meth)acrylamide; vinyl acetate, vinyl propionate, etc. Examples include carboxylic acid vinyl esters.
Monomers other than those mentioned above can be used alone or in combination of two or more.

<Content ratio of structural units of conjugated diene polymer and (meth)acrylic polymer>
In 100% by mass of the conjugated diene polymer, the content of structural units derived from the conjugated diene compound is preferably 20 to 100% by mass, more preferably 30 to 90% by mass, and even more preferably 40 to 80% by mass. . Such an embodiment is preferable from the viewpoint of measurement sensitivity in mass spectrometry and dispersion stability of the resulting polymer in a solvent.

When a structural unit derived from an aromatic vinyl compound is contained in 100% by mass of the conjugated diene polymer, the content thereof is preferably 1 to 50% by mass, more preferably 5 to 30% by mass. Such an embodiment is preferable from the viewpoint of dispersion stability of the resulting copolymer in the solvent.

When a structural unit derived from an unsaturated carboxylic acid is contained in 100% by mass of the conjugated diene polymer, the content thereof is preferably 2 to 15% by mass, more preferably 3 to 14% by mass. Such an embodiment is preferable from the viewpoint of measurement sensitivity in mass spectrometry and dispersion stability of the resulting copolymer in a solvent.

When a structural unit derived from an unsaturated carboxylic acid ester is contained in 100% by mass of the conjugated diene polymer, the content thereof is preferably 1 to 30% by mass, more preferably 2 to 28% by mass, and even more preferably It is 3 to 25% by mass. Such an embodiment is preferable from the viewpoint of measurement sensitivity in mass spectrometry and dispersion stability of the resulting copolymer in a solvent.

When a structural unit derived from an unsaturated nitrile compound is contained in 100% by mass of the conjugated diene polymer, the content thereof is preferably 1 to 35% by mass, more preferably 10 to 30% by mass. Such an embodiment is preferable from the viewpoint of measurement sensitivity in mass spectrometry and dispersion stability of the resulting copolymer in a solvent.

When a structural unit derived from a polyfunctional unsaturated compound is contained in 100% by mass of the conjugated diene polymer, the content thereof is preferably 0.5 to 5% by mass, more preferably 0.5 to 3% by mass. %. Such an embodiment is preferable from the viewpoint of measurement sensitivity in mass spectrometry, dispersion stability in a solvent, and polymerization stability of the obtained copolymer.

In 100% by mass of the (meth)acrylic polymer, the content of structural units derived from (meth)acrylic acid ester is preferably 30 to 95% by mass, more preferably 40 to 90% by mass, and still more preferably 50 to 95% by mass. It is 90% by mass. Such an embodiment is preferable from the viewpoint of measurement sensitivity in mass spectrometry and dispersion stability of the resulting copolymer in a solvent.

In 100% by mass of the (meth)acrylic polymer, the total content of structural units derived from aromatic vinyl compounds, unsaturated carboxylic acids, unsaturated nitrile compounds, and conjugated diene compounds is preferably 1 to 65% by mass, and more It is preferably 5 to 58% by weight, more preferably 9 to 45% by weight. Such an embodiment is preferable from the viewpoint of measurement sensitivity in mass spectrometry and dispersion stability of the resulting copolymer in a solvent.

When a structural unit derived from a polyfunctional unsaturated compound is contained in 100% by mass of the (meth)acrylic polymer, the content thereof is preferably 0.5 to 5% by mass, more preferably 0.5 to 5% by mass. It is 3% by mass. Such an embodiment is preferable from the viewpoint of measurement sensitivity in mass spectrometry, dispersion stability in a solvent, and polymerization stability of the resulting copolymer.

The polymer may be composed of one type of polymer or two or more types of polymers, as long as the content ratio of each structural unit measured by NMR analysis satisfies the above requirements, for example. When the polymer is composed of two or more types of polymers (a blend), the content ratio (measured value) of each structural unit in the entire blend only needs to satisfy the above requirements.

In addition, during polymer synthesis, the proportion of each monomer used in 100% by mass of raw material monomers for the polymer is equivalent to the above numerical range of the content proportion of structural units derived from each monomer in 100% by mass of the polymer. can be in the range of

<Physical properties and content of polymer>
The Mooney viscosity [ML ₁₊₄ (100° C.)] of the polymer is preferably 10 to 100, more preferably 30 to 90, even more preferably 50 to 80. Mooney viscosity can be measured according to JIS K6300-1994. In this embodiment, the resulting polymer has excellent dispersion stability in the solvent.

The average particle diameter of the polymer is preferably 10 to 200 nm, more preferably 30 to 150 nm. Here, the average particle diameter is a volume average particle diameter determined by light scattering analysis, and can be measured using, for example, "Nanotrac Wave II (trade name)" manufactured by Microtrac Bell.

In the material of the present invention, the content of the polymer is usually 1% by mass or more, preferably 5 to 30% by mass, more preferably 5 to 20% by mass. Further, the content of the polymer in the solid content of the material of the present invention is usually 70 to 100% by mass, preferably 90 to 100% by mass, and more preferably 95 to 100% by mass. Here, in this specification, "solid content" means components other than the solvent described below.

<Production method of polymer>
Examples of methods for producing the polymer include radical polymerization and anionic polymerization. Examples of the radical polymerization method include suspension polymerization, emulsion polymerization, and bulk polymerization, and emulsion polymerization is particularly preferable because it provides a stable emulsion dispersion upon completion of polymerization.

Emulsion polymerization can be carried out by any conventional polymerization method. For example, monomers are emulsified in an aqueous medium in the presence of an emulsifier, polymerization is initiated with a radical polymerization initiator, and after a predetermined polymerization conversion rate is reached, the reaction is carried out. A method of terminating the polymerization using a terminator may be mentioned.
The aqueous medium usually includes water.

Examples of the emulsifier include anionic surfactants, nonionic surfactants, cationic surfactants, and amphoteric surfactants. Fluorine surfactants can also be used. Examples of anionic surfactants include sulfuric esters of higher alcohols, alkylbenzene sulfonates such as sodium dodecylbenzenesulfonate, aliphatic sulfonates such as sodium lauryl sulfate, capric acid, lauric acid, myristic acid, and palmitic acid. , higher aliphatic carboxylates (potassium salt, sodium salt, etc.) such as oleic acid and stearic acid, and rosinate salts. The emulsifier can be used alone or in combination of two or more. The amount of emulsifier is, for example, 1 to 10 parts by weight per 100 parts by weight of monomer.

Examples of the radical polymerization initiator include organic peroxides such as benzoyl peroxide, lauroyl peroxide, tert-butyl hydroperoxide, cumene hydroperoxide, paramenthane hydroperoxide, di-tert-butyl peroxide, and dicumyl peroxide; Diazo compounds such as butyronitrile; inorganic peroxides such as potassium persulfate; and redox catalysts made by combining these peroxides with ferrous sulfate. The radical polymerization initiator can be used alone or in combination of two or more. The amount of the radical polymerization initiator is, for example, 0.005 to 3 parts by weight per 100 parts by weight of the monomer.

Chain transfer agents can also be used to control the molecular weight of the polymer. Examples of chain transfer agents include alkyl mercaptans such as tert-dodecylmercaptan and n-dodecylmercaptan, carbon tetrachloride, thioglycols, diterpenes, terpinolenes, and γ-terpinenes.

In the production of polymers, each monomer, emulsifier, radical polymerization initiator, chain transfer agent, etc. may be added in their entirety to a reaction vessel at once to initiate polymerization, or may be added continuously or intermittently as the reaction continues. It may be added separately. This polymerization can be carried out at a temperature of 0 to 100°C, preferably at a polymerization temperature of 0 to 80°C. During the reaction, the temperature, manufacturing conditions such as stirring, etc. can be changed as appropriate. The polymerization method may be continuous or batchwise.

The polymerization time is usually about 0.01 to 30 hours, and can be terminated by adding a reaction terminator when a predetermined polymerization conversion rate is reached. Examples of the reaction terminator include amine compounds such as hydroxylamine and diethylhydroxylamine; and quinone compounds such as hydroquinone. After termination of the polymerization, unreacted monomers are removed from the reaction system by a method such as steam distillation, followed by coagulation, washing with water, drying, etc., to obtain a solid polymer.

[Components other than polymer]
The material of the present invention may contain, for example, an antioxidant and a surfactant in addition to the polymer.
Moreover, the material of the present invention may contain a solvent. In addition, in the material of the present invention, the solvent may function as a so-called dispersion medium for the polymer.

Examples of the solvent include alcohol solvents such as methanol, ethanol, isopropanol, and n-propanol; ether solvents such as diethyl ether, ethylpropyl ether, tetrahydrofuran, and 1,4-dioxane; methyl acetate, ethyl acetate, butyl acetate, and ethyl lactate. , butyl lactate, γ-butyrolactone and other ester solvents; acetone, methyl ethyl ketone, cyclohexanone and other ketone solvents; dimethyl sulfoxide and other sulfoxide solvents; acetonitrile, N,N-dimethylacetamide, N,N-dimethylformamide and other nitrogen-containing solvents; Examples include hydrocarbon solvents such as hexane, cyclohexane, benzene, toluene, and xylene; halogenated hydrocarbon solvents such as methylene chloride, chloroform, and carbon tetrachloride; and water.

Solvents can be used alone or in combination of two or more.
When the material of the present invention contains a solvent, its content is usually 10 to 99% by mass, preferably 30 to 95% by mass, and more preferably 50 to 95% by mass.

[Matrix composition for mass spectrometry, sample solution, sample]
The mass spectrometry matrix composition of the present invention contains the above-described material of the present invention and a mass spectrometry matrix. The mass spectrometry matrix composition of the present invention may contain a solvent. By blending a target substance for mass spectrometry into the matrix composition, if a solvent is included, it becomes a sample liquid for mass spectrometry, and if the solvent is removed, it becomes a sample for mass spectrometry.

[Matrix for mass spectrometry]
By irradiating a mass spectrometry sample with a matrix mixed with the target substance for mass spectrometry, the target substance is vaporized and ionized along with the matrix. For this reason, the matrix is usually a compound that has an absorption band in the wavelength range of the laser used. The matrix prevents direct laser irradiation of the target substance, which would lead to its decomposition, and promotes vaporization and ionization of the target substance by efficiently converting light energy into thermal energy.

The matrix is not particularly limited as long as it is a matrix used in mass spectrometry samples. Examples of the matrix include compounds having at least one selected from carboxy groups, hydroxy groups, and amino groups, and compounds represented by formula (1) are preferred.

式（１）中の各記号の意味は以下のとおりである。

The meaning of each symbol in formula (1) is as follows.

R ¹ is a hydrocarbon group having 1 to 20 carbon atoms, -O-, -COO-, -OCO-, -OSO ₂ -, -SO ₃ between at least one carbon-carbon bond in the hydrocarbon group; -, -OSO ₂ NR-, -NRSO ₂ -, -NRCO-, -NR- and -CONR- (hereinafter also referred to as "substituted hydrocarbon group 1"), or a group thereof This is a group in which some of the hydrogen atoms are substituted (hereinafter also referred to as "substituted hydrocarbon group 2").

R ² is a direct bond, a divalent hydrocarbon group having 1 to 20 carbon atoms, -O-, -COO-, -OCO- between at least one carbon-carbon bond in the divalent hydrocarbon group; , -OSO ₂ -, -SO ₃ -, -OSO ₂ NR-, -NRSO ₂ -, -NRCO-, -NR- and -CONR- (hereinafter referred to as "substituted hydrocarbon group 3") ), or a group in which some of the hydrogen atoms in these groups are substituted (hereinafter also referred to as "substituted hydrocarbon group 4").

The R is a hydrogen atom or a hydrocarbon group having 1 to 20 carbon atoms.
X is a carboxy group, a hydroxy group or an amino group.
R ³ is a nitro group, formyl group, acyl group, alkoxycarbonyl group or hydroxyphenylazo group.

Ar is an (m+n+o)-valent aromatic group.
m is an integer of 0 to 5, preferably an integer of 0 to 3, n is an integer of 1 to 5, preferably an integer of 1 to 3, and o is an integer of 0 to 5, preferably an integer of 0 to 3. is an integer. When there is a plurality of R ¹ s, they may be the same or different. When there is a plurality of R ² s, they may be the same or different. When there are multiple X's, they may be the same or different. When there is a plurality of R ³ s, they may be the same or different.

Examples of the hydrocarbon group having 1 to 20 carbon atoms in R ¹ and R include a chain hydrocarbon group having 1 to 20 carbon atoms and an alicyclic hydrocarbon group having 3 to 20 carbon atoms.
Examples of chain hydrocarbon groups include methyl group, ethyl group, isopropyl group, n-propyl group, isobutyl group, sec-butyl group, t-butyl group, n-butyl group, n-pentyl group, n-hexyl group. chain-like saturated hydrocarbon groups such as n-heptyl group, n-octyl group, n-decyl group; and chain-like unsaturated hydrocarbon groups such as ethenyl group, propynyl group, and ethynyl group. Among these, chain hydrocarbon groups having 1 to 6 carbon atoms are preferred.

Examples of the alicyclic hydrocarbon group include alicyclic saturated hydrocarbon groups such as a cyclobutyl group, cyclopentyl group, cyclohexyl group, cyclooctyl group, and adamantyl group; Examples include saturated hydrocarbon groups. Among these, alicyclic hydrocarbon groups having 4 to 8 carbon atoms are preferred.

The divalent hydrocarbon group having 1 to 20 carbon atoms in R ² is a group in which the hydrocarbon group having 1 to 20 carbon atoms in R ¹ and R is changed to a divalent group (for example, a methyl group is changed to a methanediyl group, A group in which an ethenyl group is changed to an ethendiyl group) can be mentioned.

Examples of the substituted hydrocarbon group 1 include alkyl ester alkyl groups such as a methyl ester methyl group, an ethyl ester methyl group, and a propyl ester methyl group, and alkyl ether alkyl groups such as a methyl ether methyl group, an ethyl ether methyl group, and a propyl ether methyl group. Examples include groups. Examples of the substituted hydrocarbon group 3 include a group obtained by changing the substituted hydrocarbon group 1 to a divalent group.

Examples of the substituent that replaces some of the hydrogen atoms in the substituted hydrocarbon groups 2 and 4 include a halogen atom (fluorine atom, chlorine atom, bromine atom, iodine atom), a halogenated alkyl group, and a cyano group.

Examples of the acyl group in R ³ include a group represented by -C(=O)R ³¹ , and examples of the alkoxycarbonyl group include a group represented by -C(=O)OR ³¹ In these formulas, R ³¹ is a hydrocarbon group having 1 to 20 carbon atoms, and specific examples thereof are the same as those for R ¹ and R.

Examples of the (m+n+o)-valent aromatic group in Ar include (m+n+o)-valent aromatic hydrocarbon groups having 6 to 20 carbon atoms, aromatic ketone groups, and heterocycles; Examples include a group obtained by removing (m+n+o) hydrogen atoms from a group hydrocarbon, an aromatic ketone, or a heterocyclic compound. The aromatic hydrocarbon preferably has 6 to 14 carbon atoms, and specifically includes benzene, naphthalene, and anthracene. Specifically, the aromatic ketone is anthrone. Examples of the above-mentioned heterocyclic compounds include nitrogen-containing heterocyclic aromatic compounds such as pyridine, pyridazine, pyrimidine, pyrazine, indole, quinoline, isoquinoline, quinoxaline, and acridine.
Specific examples of the matrix include compounds represented by the following formulas.

マトリックスは単独でまたは２種以上用いることができる。

The matrices can be used alone or in combination of two or more.

[Substances targeted for mass spectrometry]
Examples of substances to be analyzed by mass spectrometry include biological substances such as peptides, nucleic acids, carbohydrates, and lipids, and synthetic substances. The target substance does not necessarily have to be a purified substance. For example, biological materials such as cells, tissues, body fluids, extracts thereof, or cultured cells derived from animals, plants, or microorganisms; materials isolated from soil and wastewater may be used.

The target substance (analyte) for mass spectrometry is, for example, a molecule with a molecular weight of preferably 300 to 10,000, more preferably 500 to 5,000, and is preferably a polymer such as a peptide. When the molecular weight of the target substance has a distribution, it is preferable that the weight average molecular weight in terms of polystyrene measured by size exclusion chromatography is within the above range.

The target substance for mass spectrometry is preferably a hydrophobic substance, more preferably a hydrophobic peptide. In addition, the sample for mass spectrometry may contain, for example, a hydrophilic substance in addition to the hydrophobic substance, within a range that does not impair the effects of the present invention. In such cases, hydrophobic substances can be specifically detected and analyzed.

As an index of hydrophobicity, BB index, HPLC index, or SSRCalc Hydrophobicity can be used.
For example, the degree of hydrophobicity of the hydrophobic substance in the present invention may be any level that can be determined by a person skilled in the art to be hydrophobic based on an HPLC index. The HPLC index is described in Analytical Biochemistry, 124, 201-208, 1982 by CABrownw, HPJBennett, S.Solomon, using an acetonitrile aqueous solution containing 0.13% heptafluoro-n-butyric acid (HFBA) as the eluent. The hydrophobicity index is based on the reverse phase HPLC retention time used as The HPLC index of the hydrophobic substance is, for example, 30 or more, preferably 30 to 10,000, more preferably 30 to 1,000. In this case, the degree of hydrophilicity of the hydrophilic substance can be, for example, a value of less than 30.

Further, the degree of hydrophobicity of the hydrophobic substance in the present invention may be such that a person skilled in the art can judge that the substance is hydrophobic based on SSRCalcHydropobicity. SSRCalcHydropobicity is described in Oleg V. Krokhin, Analytical Biochemistry, 78, 7785-7795, 2006. In the present invention, when the target to be analyzed is a hydrophobic peptide, the degree of hydrophobicity is preferably determined using SSRCalcHydropobicity as an index. The SSRCalc Hydrophobicity (by the Manitoba Center for Proteomics and Systems Biology, available at http://hs2.proteome.ca/SSRCalc/SSRCalcX.html) of the hydrophobic substance can be, for example, 30-70. In this case, the degree of hydrophilicity of the hydrophilic substance can be, for example, a value of less than 30.

The target substance for mass spectrometry is preferably a hydrophobic peptide from the viewpoint of difficulty in detection using conventional techniques. "Peptide" is used in a broad sense including polypeptides and proteins. As the hydrophobic peptide, a peptide having a large number of highly hydrophobic amino acid residues is preferable. Examples of hydrophobic amino acid residues include glycine, alanine, valine, leucine, methionine, proline, phenylalanine, tryptophan, and isoleucine residues, and may also include cysteine and tyrosine residues.

[Cationation agent]
The mass spectrometry matrix composition of the present invention may contain a cationizing agent.
A cationizing agent can be used to more efficiently ionize a target substance. Examples of the cationizing agent include sodium chloride, potassium chloride, sodium iodide, potassium iodide, lithium iodide, ammonium acetate, trifluoroacetic acid, sodium trifluoroacetate, potassium trifluoroacetate, lithium trifluoroacetate, and trifluoroacetate. Silver acetate is mentioned.
The cationizing agent can be used alone or in combination of two or more.

[solvent]
The mass spectrometry matrix composition of the present invention may contain a solvent.
Examples of the solvent include alcohol solvents such as methanol, ethanol, isopropanol, and n-propanol; ether solvents such as diethyl ether, ethylpropyl ether, tetrahydrofuran, and 1,4-dioxane; methyl acetate, ethyl acetate, butyl acetate, and ethyl lactate. , butyl lactate, γ-butyrolactone and other ester solvents; acetone, methyl ethyl ketone, cyclohexanone and other ketone solvents; dimethyl sulfoxide and other sulfoxide solvents; acetonitrile, N,N-dimethylacetamide, N,N-dimethylformamide and other nitrogen-containing solvents; Examples include hydrocarbon solvents such as hexane, cyclohexane, benzene, toluene, and xylene; halogenated hydrocarbon solvents such as methylene chloride, chloroform, and carbon tetrachloride; and water.

Solvents can be used alone or in combination of two or more.
Specific examples of the solvent include, for example, methanol/water mixture, ethanol/water mixture, tetrahydrofuran/water mixture, dimethyl sulfoxide/water mixture, acetonitrile/water mixture, acetonitrile/ethanol/isopropanol/water mixture, Examples include organic solvent/water mixtures such as acetonitrile/ethanol/methyl ethyl ketone/water mixtures. The organic solvent concentration in the liquid mixture is, for example, 10 to 90% by volume, preferably 25 to 75% by volume.

[Content of each component]
In the matrix composition for mass spectrometry of the present invention, the content of the polymer derived from the material of the present invention is usually 0.1 to 10,000 parts by mass, preferably 5 parts by mass, based on 100 parts by mass of the matrix for mass spectrometry. ~8,000 parts by weight, more preferably 10~5,000 parts by weight.

When the matrix composition for mass spectrometry contains a cationizing agent, the content of the cationizing agent is preferably 1 to 100 parts by mass, more preferably 5 to 20 parts by mass, based on 100 parts by mass of the matrix for mass spectrometry. It is.

When the matrix composition for mass spectrometry contains a solvent, the total content of the polymer derived from the material of the present invention and the matrix for mass spectrometry is usually 0.1 to 1000 mg/mL, preferably 0.5 to 1000 mg/mL. 500 mg/mL, more preferably 1 to 100 mg/mL.

[Kit for preparing samples for mass spectrometry]
The kit for preparing a sample for mass spectrometry of the present invention is a stage before mixing each component of the matrix composition for mass spectrometry, and includes the material of the present invention and a matrix for mass spectrometry.

[Preparation method of sample for mass spectrometry]
For example, a sample for mass spectrometry is prepared by forming a sample liquid containing a target substance for mass spectrometry, a matrix, the material of the present invention, and a solvent on a target plate for mass spectrometry (sample table for measurement), and By removing the solvent from the sample liquid, the nonvolatile components in the sample liquid (that is, at least the target substance, the matrix, and the polymer derived from the material of the present invention) can be obtained as a residue. The residue is a sample for mass spectrometry, and is usually a crystal. In the crystal, for example, target substances (molecules) to be analyzed are uniformly dispersed in a matrix that efficiently absorbs laser energy.

As the mass spectrometry target, for example, a conductive metal plate used for MALDI-MS, specifically a stainless steel or gold plate, can be used.

The sample liquid may be prepared, for example, by separately preparing a target substance-containing liquid, a matrix-containing liquid, and the material of the present invention, and then mixing them. A matrix composition for mass spectrometry containing these materials may be prepared separately and then mixed together. Next, the obtained liquid mixture is dropped onto the target plate.

In addition, by dropping the target substance-containing liquid, the matrix-containing liquid, and the material of the present invention, or the target substance-containing liquid and the matrix composition for mass spectrometry at the same position on the target plate, You can also mix it with In this case, the order in which the liquids containing each component are dropped is not particularly limited.

In order to prepare a liquid containing each component, the above-mentioned solvents can be used.
In the sample solution and sample, the content of the polymer derived from the material of the present invention is usually 200 to 100,000 parts by mass, preferably 500 to 75,000 parts by mass, and more than 1 part by mass of the target substance. Preferably it is 500 to 50,000 parts by mass.

In the sample solution and sample, the content of the matrix for mass spectrometry is usually 50 to 5000 parts by mass, preferably 100 to 3000 parts by mass, and more preferably 200 to 2000 parts by mass, per 1 part by mass of the target substance. be.

In the sample liquid, the total content of the substance to be subjected to mass spectrometry, the matrix, and the polymer derived from the material of the present invention is usually 0.1 to 1000 mg/mL, preferably 0.5 to 500 mg/mL, More preferably 1 to 50 mg/mL.

The volume of the sample liquid droplet is not particularly limited. If wells are provided on the target plate, droplets of sample liquid can be formed within the wells. In this case, the droplet of the sample liquid has a volume that fits within the well, for example, a droplet of 0.1 to 2 μL.

Remove the solvent from the sample liquid droplet on the target plate. To remove the solvent, the solvent may be naturally evaporated, or the solvent may be evaporated by heating and/or reducing pressure.

[Mass spectrometry]
The mass spectrometry method of the present invention includes the step of obtaining the aforementioned sample for mass spectrometry, and the step of irradiating the sample for mass spectrometry with a laser to detect the target substance.

In the step of detecting the target substance by irradiating the sample for mass spectrometry with a laser, the target substance is usually ionized and the generated ions are separated by mass-to-charge ratio (m/z) using a magnetic field or an electric field. Separate and detect. This step preferably uses a mass spectrometer coupled with a matrix-assisted laser desorption ionization (MALDI) ion source. Examples of the devices include MALDI-time-of-flight (MALDI-TOF) type, MALDI-ion trap (MALDI-IT) type, MALDI-ion trap-time-of-flight (MALDI-IT-TOF) type, and MALDI-Fourier transform ion type. Examples include cyclotron resonance (MALDI-FTICR) type mass spectrometers. Among these, a MALDI-TOF mass spectrometer is preferred.

Hereinafter, the present invention will be explained in more detail based on Examples, but the present invention is not limited to these Examples. In the following description of Examples, etc., "parts" indicate "parts by mass" unless otherwise specified.

[Synthesis example 1]
An aqueous solution of 5 parts of sodium dodecylbenzenesulfonate dissolved in 200 parts of distilled water, 70 parts of butadiene as raw material monomers, 19 parts of acrylonitrile, 5 parts of 2-hydroxybutyl methacrylate, 5 parts of methacrylic acid, and 1 part of divinylbenzene were placed in an autoclave. 0.01 part of para-menthane hydroperoxide was added as a polymerization initiator, and emulsion polymerization was carried out. When the polymerization conversion rate reached 85%, diethylhydroxylamine was added as a reaction terminator to terminate the reaction and obtain a polymer product (polymer (A-1)). Then, after removing unreacted monomers by steam distillation, this solution was added to a 5% calcium chloride aqueous solution, the precipitated crumb-like polymer was washed with water, and then dried at 100°C using a hot air dryer. The polymer was isolated by. The yield of the obtained polymer was 82%, and the Mooney viscosity was measured according to JIS K6300-1994 and was 60 (ML ₁₊₄ , 100°C).

The obtained polymer (A-1) was poured into isopropanol as an organic solvent in a 1 L glass flask equipped with a stirrer so that the solid content concentration was 10% by mass, and the mixture was stirred at room temperature for 24 hours. A composition (J-1) was obtained. The average particle diameter of the polymer (A-1) was measured using "Nanotrac Wave II (trade name)" manufactured by Microtrac Bell Co., Ltd. using the obtained polymer composition. The measured volume average particle diameter results are also shown in Table 2.

[Synthesis Examples 2 to 5]
Polymer compositions (J-2) to (J-5) were obtained in the same manner as in Synthesis Example 1, except that each component listed in Table 1 was used as the raw material monomer and each component listed in Table 2 was used as the organic solvent. Ta. Tables 1 and 2 show the yield, Mooney viscosity, and average particle diameter of the obtained polymer.

[Examples 1 to 5]
The polymer compositions obtained in Synthesis Examples 1 to 5 were used as a material to be mixed into the matrix α-cyano-4-hydroxycinnamic acid (hereinafter referred to as "CHCA"), and the peptide components were measured.
(1) A matrix solution consisting of a 5 mg/mL (50 mass % acetonitrile/0.1 mass % trifluoroacetic acid (TFA) aqueous solution) solution of CHCA and 10 mass % of the polymer composition obtained in Synthesis Examples 1 to 5. The mixture was mixed with the dispersion liquid at the ratio (v/v) shown in Table 3, and stirred with a vortex for 1 minute to obtain a mixed liquid 1. Next, the obtained mixture 1 and a 20 μM ethanol solution of each peptide (P-1) were mixed at the mixing ratio (v/v) shown in Table 3, and the mixture was stirred for 1 minute by vortexing. 2 (sample solution) was obtained. In addition, equal amounts of two types of peptides, Angiotensin II and Amyloid β25-35 (SSRCalcHydrophobia are 27.79 and 38.34, respectively) were mixed, and a peptide solution (ethanol) was added so that the total peptide content was 20 μM. Solution (P-1)) was prepared.
(2) 1 μL of the prepared sample solution was applied onto a MALDI plate and dried.
(3) Measurement was performed using AXIMA Performance (Shimadzu Corporation) Linear TOF in positive mode. The results are shown in Table 3.

[Comparative example 1]
(1) A matrix solution consisting of a 5 mg/mL (50 mass% acetonitrile/0.1 mass% TFA aqueous solution) solution of CHCA and a 20 μM ethanol solution (P-1) of each peptide at the ratios shown in Table 3 (v/v ) and stirred with a vortex for 1 minute to obtain a sample solution. The subsequent operations (2) and (3) were performed in the same manner as in Examples 1 to 5. The results are shown in Table 3.
Table 3 shows that in Examples 1 to 5, Amyloid β25-35, which is a hydrophobic peptide, was specifically detected.

Claims (14)

A step of forming a sample liquid containing a material containing a conjugated diene polymer and a solvent for preparing a target substance for mass spectrometry, a matrix, and a sample for mass spectrometry on a target plate for mass spectrometry;
A method for preparing a sample for mass spectrometry, comprising the step of removing the solvent from the sample liquid. The conjugated diene polymer contains at least one compound selected from a conjugated diene compound, a polyfunctional unsaturated compound, an aromatic vinyl compound, an unsaturated carboxylic acid, an unsaturated carboxylic acid ester, and an unsaturated nitrile compound. The method for preparing a sample for mass spectrometry according to claim 1 , which is a copolymer of . The method for preparing a sample for mass spectrometry according to claim 1 or 2, wherein the conjugated diene polymer has a Mooney viscosity [ML ₁₊₄ (100° C.)] of 50 to 80. The method for preparing a sample for mass spectrometry according to any one of claims 1 to 3, wherein the sample for mass spectrometry is a sample for mass spectrometry of a hydrophobic substance. The method for preparing a sample for mass spectrometry according to any one of claims 1 to 3, wherein the sample for mass spectrometry is a sample for mass spectrometry of a hydrophobic peptide. The method for preparing a sample for mass spectrometry according to any one of claims 1 to 5, wherein the conjugated diene polymer is a crosslinked polymer. The method for preparing a sample for mass spectrometry according to any one of claims 1 to 6, wherein the sample for mass spectrometry is a sample for matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS). A step of forming a sample liquid containing a material containing a conjugated diene polymer and a solvent for preparing a target substance for mass spectrometry, a matrix, and a sample for mass spectrometry on a target plate for mass spectrometry;
removing the solvent from the sample liquid to obtain a sample for mass spectrometry;
A mass spectrometry method comprising the step of detecting the target substance by irradiating the sample for mass spectrometry with a laser. The conjugated diene polymer contains at least one compound selected from a conjugated diene compound, a polyfunctional unsaturated compound, an aromatic vinyl compound, an unsaturated carboxylic acid, an unsaturated carboxylic acid ester, and an unsaturated nitrile compound. The mass spectrometry method according to claim 8, which is a copolymer of . The mass spectrometry method according to claim 8 or 9, wherein the conjugated diene polymer has a Mooney viscosity [ML ₁₊₄ (100° C.)] of 50 to 80. The mass spectrometry method according to any one of claims 8 to 10, wherein the mass spectrometry sample is a mass spectrometry sample of a hydrophobic substance. The mass spectrometry method according to any one of claims 8 to 10, wherein the mass spectrometry sample is a hydrophobic peptide mass spectrometry sample. The mass spectrometry method according to any one of claims 8 to 12, wherein the conjugated diene polymer is a crosslinked polymer. The mass spectrometry method according to any one of claims 8 to 13, wherein the mass spectrometry sample is a matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) sample.