JP2005140755A - Probe for mass spectrometry and mass spectrometry method using the same - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a new probe for mass spectrometry capable of increasing measurement precision as compared with a known mass spectrometry probe, when applying to the determination of an objective material, especially a biological sample, such as protein and DNAs. <P>SOLUTION: The probe for mass spectrometry has a structure expressed by a general expression [I] R<SP>1</SP>-X-A-R<SP>2</SP>. In this case, R<SP>1</SP>indicates a mass probe section that becomes an ion in a solvent; X indicates a photocleavage section made of structure cleaved by light irradiation; A indicates a spacer section that may exist or not and comprises an arbitrary structure when the spacer section exists; and R<SP>2</SP>indicates a connection section that can be connected to other substances. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

  The present invention relates to a novel mass spectrometry probe capable of mass spectrometry of various substances with ease and high sensitivity, and a mass spectrometry method using the probe. The present invention is useful for analysis of proteins, DNA, etc., and particularly for comprehensive analysis of multiple types of proteins, DNA, etc.

  Currently, mass spectrometry is a method that can quantify a sample in a specimen with the highest sensitivity and accuracy. When measuring a liquid sample using mass spectrometry, it is important to select an ionization method in accordance with the properties of the compound. For example, when using properly depending on the instability factor of the compound, it can be roughly divided into two types, hard ionization and soft ionization. Examples of the former method include electron ionization, chemical ionization, and atmospheric pressure ionization, and examples of the latter include electrospray ionization, matrix-assisted laser desorption ionization, and fast atom bombardment ionization.

  On the other hand, recently, a method (LC / MS) for identifying a sample separated by high performance liquid chromatography using mass spectrometry online is frequently used in the fields of chemistry and biology. In this case, since the ionization method having good compatibility with liquid chromatography is a method of directly ionizing a solution sample flowing out from the capillary tip, the latter method corresponds to the method described above. Among these, electrospray ionization mass spectrometers and high-performance liquid chromatography devices connected on-line are currently the most widely used, and are essential hormones such as proteins and carbohydrates. The power is fully demonstrated to identify. In the case of electrospray ionization, the molecular ion peak of the sample is very rarely detected and is usually observed as a pseudo-molecular ion peak added with sodium or the like, or in LC / MS, ammonium acetate, formic acid, A method of assisting ionization of a sample by mixing a protic solvent such as acetic acid is employed.

  As a means for assisting ionization in mass spectrometry of a liquid sample, a technique of adding a protic solvent to a mobile phase of liquid chromatography is generally used in LC / MS. However, i) When ammonium acetate is in negative ion mode, the sample ion and ammonium ion are paired and the sensitivity decreases. Ii) When trifluoroacetic acid is in positive ion mode, sample ion and trifluoroacetate ion are paired. In the negative ion mode, ionization is obstructed with some exceptions. Iii) In acetonitrile, acid must be added, and in this case, ammonium acetate does not dissolve, so it should be used. Some disadvantages such as inability to do so have been pointed out.

  As a means for efficiently ionizing a sample without adding a protic solvent to the mobile phase in the ionization method for solving the above problems and mass spectrometry of a liquid sample, International Publication No. WO 02/04936 A1 (Patent Document 1) discloses a probe for mass spectrometry in which an ionic functional group that becomes an ion in a solvent and a structure capable of binding to another substance are connected via a spacer portion. By using this probe, it is possible to efficiently ionize the target substance (sample compound) and greatly improve the sensitivity and accuracy of mass spectrometry.

  Although the probe of Patent Document 1 is excellent, it goes without saying that it is advantageous to provide a probe that can further improve the measurement accuracy than this probe when applied to the determination of a target substance. In addition, with the mass spectrometry probes including the mass spectrometry probe described in Patent Document 1, the substance bound to the solid phase could not be quantified. If a mass spectrometric probe capable of quantifying the target substance bound to the solid phase is obtained, the label measurement in the conventional immunoassay can be performed by mass spectrometry, and the measurement accuracy Is thought to improve.

International publication number WO 02/04936 A1

  Accordingly, the object of the present invention is to provide a novel probe for mass spectrometry that can provide higher measurement accuracy than known mass spectrometry probes when applied to the quantification of target substances, particularly biological samples such as proteins and DNA. It is to be. Another object of the present invention is to provide a novel probe for mass spectrometry that can be used for quantification of a substance bound to a solid phase.

  As a result of diligent research, the inventors of the present application provided a structure in the probe that cleaves when irradiated with light between a structure that can bind to another substance and a structure that becomes an ion in a solvent. Are directly or indirectly bonded, and then a part of the probe molecule is liberated by irradiating light, and only a part of the probe molecule is subjected to mass spectrometry, the molecule subjected to mass spectrometry has a structure and ionization efficiency. In addition, since the molecules are liberated, almost the entire amount can be recovered, so that it is thought that high-accuracy measurement is possible. It was conceived that the substance bound to can be quantified, and the present invention was completed.

That is, the present invention provides the following general formula [I]
R ¹ -XAR ²
(However, R ¹ is a mass probe part consisting of a structure that becomes an ion in a solvent, X is a photocleavage part consisting of a structure that is cleaved by light irradiation, and A may or may not exist. Spacer part composed of any structure, R ² represents a joint part composed of a structure capable of binding to other substances)
The probe for mass spectrometry which has a structure represented by these is provided. In addition, the present invention provides the probe of the present invention directly or indirectly bound to the target substance via the binding portion of the probe, and separates the bound substance between the probe and the target substance, and light is emitted to the bound substance. Is provided to cleave the photocleavable part, and to measure a molecule containing the mass probe part liberated by cleavage by mass spectrometry. Furthermore, the present invention provides the probe according to the present invention, wherein at least two types of probes having different masses of the mass probe portion of each probe are directly or indirectly connected to each object via the coupling portion of each probe. Including the mass probe part of each probe released by cleavage, by binding to a substance, separating a bond between each probe and each target substance, irradiating the bond with light to cleave the photocleavage part Provided is a method for measuring a plurality of target substances, including measuring molecules by mass spectrometry.

  According to the present invention, there has been provided a novel probe for mass spectrometry that can provide higher measurement accuracy than known mass spectrometry probes when applied to quantification of a target substance, particularly a biological sample such as protein or DNA. In the probe of the present invention, after the probe and the target substance are directly or indirectly bound, the conjugate is separated, and the photocleavable part is cleaved by irradiating with light, and the mass probe part released by the cleavage is separated. Since the contained molecules (hereinafter referred to as “mass probe part-containing molecules”) can be subjected to mass spectrometry, the molecules subjected to mass spectrometry have the same structure and ionization efficiency, and the molecules are released. Since almost the entire amount can be collected, high-precision measurement is possible. Performing mass analysis of a biopolymer itself such as a protein using a known probe has a large molecular weight, so many peaks derived from one protein appear, and quantification is difficult because ionization efficiency differs depending on the protein. Moreover, it is not easy to separate a substance that interacts with a protein, and in order to perform a separation reaction, the protein itself is decomposed, and a plurality of peptide chains are generated from one protein, and finally identified. There are many cases where this is not possible. Therefore, it is advantageous if there is a site where the protein can be solid-phased and easily separated (by light irradiation only), and that identification and quantification can be obtained only by that (as will be understood from the following examples) The photocleavage has a very high efficiency of almost 100%). Further, in the probe of the present invention, at least two types of probes having different masses of the mass probe part are directly or indirectly coupled to each target substance via the coupling part of each probe, and each probe and each target are coupled. Separating a bound substance with a substance, irradiating the bound substance with light to cleave the photocleavage part, and measuring the molecule including the mass probe part of each probe released by the cleavage by mass spectrometry, Multiple substances can be quantified. As described above, by using the probe of the present invention, comprehensive quantitative analysis in which various proteins and DNAs are simultaneously quantified becomes possible. Furthermore, when the probe of the present invention is used, the mass probe portion-containing molecule that is released by light irradiation is measured. Therefore, even when the target substance is immobilized on a solid phase, the mass probe is directly or indirectly connected to the target substance. Quantitative analysis of the target substance bound to the solid phase is also possible by mass-analyzing the mass probe-containing molecules released by irradiation with light after washing and removing unbound molecules and the like. As described above, by using the probe of the present invention, it is possible to quantify solid-phase-bound substances that could not be obtained with known probes at all, such as peptide chips and DNA chips in which various proteins and DNAs are immobilized on the chip. Even in the case of using, it is possible to perform quantification by mass spectrometry, and it is considered that quantification can be performed with higher accuracy than conventional label measurement.

As described above, the mass spectrometric probe of the present invention is represented by the above general formula [I]. In the general formula [I], R ¹ is a mass probe portion having a structure that becomes an ion in a solvent. Here, “mass” refers to mass, and as will be described in detail later, in the present invention, since the target substance is quantified using the mass of this portion as a mark, the mass of this portion (“mass”) Is called "mass probe part" in the sense that works as a probe. In order to increase the measurement sensitivity of mass spectrometry, the mass probe part has a structure that becomes ions in a solvent, preferably in water. Such a structure may be any structure as long as it is ionized in the solvent to be used, and may be positively charged or negatively charged. Examples of R ¹ include amines, carboxylic acids or salts thereof, sulfonic acids or salts thereof, and preferred examples include structures represented by the following general formula [V], [VI] or [VII]. At the end opposite to the side bonded to the photocleavable part.

（ただし、一般式[V]ないし[VII]において、R⁵、R⁶及びR⁷は、それぞれ独立に、水素又は任意の基を示す）

(However, in the general formulas [V] to [VII], R ⁵ , R ⁶ and R ⁷ each independently represent hydrogen or an arbitrary group)

R ⁵ , R ⁶ and R ⁷ are preferably hydrogen, halogen, or a linear or branched alkyl group having 1 to 20 carbon atoms, more preferably hydrogen or a lower alkyl group having 1 to 6 carbon atoms. (In this specification, unless otherwise specified, the “lower alkyl group” means a linear or branched alkyl group having 1 to 6 carbon atoms). Of these, quaternary amines represented by the above general formula [V] are particularly preferred, and those in which R ⁵ , R ⁶ and R ⁷ are hydrogen or a lower alkyl group are particularly preferred. Since the chain lengths of the alkyl chains of R ⁵ , R ⁶ and R ⁷ can be easily extended, various alkyl chain lengths, and thus mass probes having various masses can be easily prepared. Thus, it is convenient when the probe of the present invention is used for simultaneous quantitative analysis of a plurality of types of target substances.

  Groups represented by the above general formulas [V] to [VII] that become ions in a solvent may be directly bonded to a photocleavage part described later, or a spacer region such as an alkylene group. It may couple | bond with the photocleavage part. The structure of such a spacer region is not particularly limited, and examples thereof include an alkylene group having 1 to 6 carbon atoms. Such a spacer region is also included in the free molecule after photocleavage, and thus is a part of the mass probe portion.

In the general formula [I], R ² represents a structure capable of binding to another substance. As a preferred group of examples of R ² , there can be mentioned a functional group capable of reacting with a functional group in another substance and covalently bonding. In this case, R ² may be any functional group as long as it can react with a functional group in another substance to form a covalent bond. Samples that are analyzed in liquid by mass spectrometry are often biological substances such as proteins and sugars, and therefore functional groups often included in these, that is, —NH ₂ groups, —SH groups, —COOH groups, A functional group that reacts with an —OH group, —CHO group or the like to form a covalent bond is preferred. As an example of such a functional group,

SCN-, ClO ₂ S-, BrH ₂ C, ClOC-, CH ₃ CH (NH ₂ ) = CH-, -CH ₂ ONH ₂ -HCl, -NH ₂ , -NHNH ₂ and -CH ₂ I Can do.

The functional groups (R ′) in other substances that react with these functional groups and the bonds formed as a result of the reactions are shown in the following Table 1 (in the case where R ² is —NH ₂ , they are shown in Table 1). R ² when the functional group (R ′) in the other substance is —NH ₂ can be bonded as described in Table 1).

Preferable R ² is further represented by the general formula [IX]

（ただし、Halはハロゲンを示す）、

(However, Hal indicates halogen),

（ただし、ｎは１〜５の整数を示す）
で表される基を挙げることができる。一般式[IX]中、ハロゲンとしては、好ましくは臭素である。 And general formula [X]

(However, n represents an integer of 1 to 5)
The group represented by these can be mentioned. In general formula [IX], the halogen is preferably bromine.

R ² represented by the above general formula [X] reacts with cytosine in other substances and binds as follows.

（ただし、式中、Ｒは、例えばヌクレオチドや核酸の糖部分のような任意の基を示す）

(However, in the formula, R represents an arbitrary group such as a nucleotide or a sugar moiety of a nucleic acid)

Since this R ² can bind to the cytosine moiety in other substances, cytidine monophosphate, cytidine diphosphate, cytidine triphosphate, deoxycytidine monophosphate, deoxycytidine diphosphate and deoxycytidine tri It can bind to nucleic acids such as DNA and RNA, including phosphate and cytosine.

R ² is the above

の場合は、他の物質中のグアニンと反応して次のように結合する。

In the case of, it reacts with guanine in other substances and binds as follows.

（ただし、式中、Ｒは、例えばヌクレオチドや核酸の糖部分のような任意の基を示す）

(However, in the formula, R represents an arbitrary group such as a nucleotide or a sugar moiety of a nucleic acid)

Since this R ² can bind to the guanine moiety in other substances, guanosine monophosphate, guanosine diphosphate, guanosine triphosphate, deoxyguanosine monophosphate, deoxyguanosine diphosphate and deoxyguanosine tri It can bind to nucleic acids such as DNA and RNA containing phosphate and guanine.

When R ² is a group represented by the above general formula [X], it reacts with a phosphate ester moiety in another substance and bonds as follows (however, in the following example, n is 2) Show).

In this reaction formula, the other substance is deoxyadenosine monophosphate (that is, the base in the nucleotide is adenine and the sugar is deoxyribose), but R ² is bonded to the phosphate ester moiety. Thus, the base may be a base other than adenine such as cytosine, guanine, thymine or uracil, and the sugar may be deoxyribose or ribose. In addition, the number of phosphate esters is one in the above reaction formula, but R ² is bonded to the terminal phosphate ester, so the number of condensed phosphate esters is any one of 1 to 3. It may be. Accordingly, R ² represented by the general formula [X] can bind to all kinds of nucleoside monophosphates, nucleoside diphosphates and nucleoside triphosphates. Furthermore, since a free phosphate ester exists at the end of the nucleic acid, R ² represented by the general formula [X] can also bind to a nucleic acid such as DNA or RNA.

Note that R ² is not limited to a functional group that can be covalently bonded by reacting with a functional group of another substance, and a group that binds to another substance by intercalation or coordination bond is also used as R ^2. be able to.

That is, examples of R ² include those having a structure that intercalates into a double-stranded nucleic acid. Specific examples of such R ² include a group represented by the following general formula [XI].

（ただし、Ｒ^１１及びＲ^１２は、それぞれ独立に、水素、ハロゲン、炭素数１〜５のアルキル基又は炭素数１〜５のN,N-ジアルキルアミノ基を示す）

(However, R ¹¹ and R ¹² each independently represent hydrogen, halogen, an alkyl group having 1 to 5 carbon atoms, or an N, N-dialkylamino group having 1 to 5 carbon atoms.)

Such R ² intercalates and binds to a double-stranded nucleic acid, and thus a probe having such R ² can be preferably used for measurement of a double-stranded nucleic acid.

Furthermore, as R ² , a group in which one hydrogen atom is removed from a complex-forming compound such as EDTA, which is coordinated with another substance, can be used.

In the general formula [I], A is an optional spacer portion. A is not essential, and R ² may be directly bonded to the photocleavage part described later. The probes of the invention, the R ² is bound with other substances, after cleavage of the light cleavage X by light irradiation, because the mass probe unit containing molecules is to mass spectrometry, between R ¹ and X The located A can take any structure. However, when it is assumed to be used in an aqueous medium, it is preferable to include a hydrophilic group such as amide, ether, amine, or ketone. Therefore, when assuming use in an aqueous medium, a structure in which an amide, ether, amine, ketone or the like is inserted or added to an alkylene group having about 1 to 6 carbon atoms (hereinafter referred to as a lower alkylene group) is preferable. For example, in the examples described later, the spacer portion is —C ₂ H ₄ —CO—NH—CH ₂ —, in which an amide group —CO—NH— is inserted into a propylene group. When it is desired to impart hydrophobicity, an aryl group (such as phenyl) can be introduced into the lower alkylene group. It is also possible to make both the hydrophobic part and the hydrophilic part amphiphilic.

  In the general formula [I], X is a photocleavage part composed of a structure that is cleaved by light irradiation. The photocleavage part is not particularly limited as long as it is a structure that is cleaved by light irradiation. Preferred examples of the photocleavable part include divalent structures represented by the following general formula [II], [III] or [IV].

(However, in the general formulas [II] to [IV], R ³ and R ⁴ each independently represent hydrogen, a lower alkoxyl group or a carboxy lower alkyl group).

Among these, in the general formula [II], R ³ and R ⁴ are hydrogen, and in the general formula [III], R ³ and R ⁴ are both (1) hydrogen or (2) a nitro group. 4,5-dimethoxy as the position, and in the above general formula [IV], R ³ and R ⁴ are either (1) 4,5-dimethoxy with the nitro group as the 2-position or (2) carboxy with the hydrogen and nitro group as the 2-position A methyl group is particularly preferable, and a structure represented by the general formula [II] in which R ³ and R ⁴ are both hydrogen is particularly preferable.

For example, when light is irradiated on a structure represented by the general formula [II] in which R ³ and R ⁴ are both hydrogen, the carbon atom to which the nitrophenyl group is bonded and the oxygen atom adjacent thereto are cut. Is done.

  Among the probes of the present invention described above, particularly preferred are those represented by the following general formula [VIII].

（ただし、ｎ及びｍは、互いに１〜６の整数、R⁵、R⁶及びR⁷は、それぞれ独立に、水素又は任意の基を示し、好ましくは水素又は炭素数１〜６のアルキル基を示す）

(However, n and m are each an integer of 1 to 6, R ⁵ , R ⁶ and R ⁷ each independently represent hydrogen or an arbitrary group, preferably hydrogen or an alkyl group having 1 to 6 carbon atoms. Show)

  The probe of the present invention can be easily synthesized by those skilled in the art based on a known method. The following examples also specifically describe a plurality of preferred probe manufacturing methods.

The binding reaction between the probe and another substance to be bound to the probe is appropriately performed based on a known technique according to the R ² of the probe and the type of the functional group to be bound to R ² in the other substance. Can be performed under the following conditions. Also in the examples below, conditions for the binding reaction between a preferred probe and another substance are specifically described.

For each specific combination described above of R ² and functional groups in the other substances that bind to the R ² (R '), describes an example of a specific binding reaction conditions below. However, these are merely examples, and these functional groups can be bonded to each other under other reaction conditions, and it will be apparent to those skilled in the art that the bonding reaction conditions are not limited to the following. is there. Moreover, those skilled in the art can easily carry out the binding reaction for R ² not described here according to common chemical knowledge.

(1) When R ² is SCN- and R 'is -NH ₂
Add 0.2 ml (ethanol: water: triethylamine = 2: 2: 1 v / v) to a sample of 2-3 mg and dry under reduced pressure, then 0.5 ml (ethanol: water: triethylamine: probe = 7: 7: 1: Add 1 v / v) and react at room temperature for 20 minutes. After evaporating the solvent under reduced pressure, the solvent is dissolved in the eluent to give a sample solution (see BA Bidlingmeyer, et al, J. Chromatogr., 336, 93 (1984)).

(2) When R ² is ClO ₂ S— and R ′ is —NH ₂
Add 500 μL of 1 M NaHCO ₃ aqueous solution and 200 μL of 1 mg / mL probe / acetone solution to 2-3 mg sample and heat at 60 ° C. for 30 minutes. Acetone is distilled off, and then dissolved in an eluent to obtain a sample solution (see Meffin, PJ, et al, J. Pharm. Sci., 66, 583 (1977)).

(3) R ² is

で、Ｒ’が−ＮＨ_２の場合

And R ′ is —NH ₂

  Dissolve 1.5 mg of sample in 5.0 mL of THF, add 50 mg of probe, and heat at 60 ° C. for 30 minutes. After THF is distilled off, it is dissolved in the eluent to obtain a sample solution (see Jupill, T.H., Am. Lab., 8 (5), 85-92 (1976)).

(4) R ² is

で、Ｒ’が−ＳＨの場合

And R 'is -SH

  Dissolve 2-3 mg of sample in 500 μL of water, add 200 μL of 1 mg / mL probe / acetonitrile solution, and heat at 60 ° C. for 30 minutes. After distilling off the solvent, dissolve in the eluent to obtain a sample solution (see Nakashima K., et al, Talanta., 32, 167 (1985)).

(5) When R ² is BrH ₂ C— or IH ₂ C— and R ′ is —COOH
Dissolve 2-3 mg of sample in 500 μL of acetone, add 200 μL of 1 mg / mL probe / acetone solution and 1 mg of K ₂ CO ₃ and heat at 60 ° C. for 30 minutes. After the solvent is distilled off, the sample is dissolved in the eluent to obtain a sample solution (see Dunges, W., Anal. Chem., 49, 442 (1977)).

(6) When R ² is ClOC- and R 'is -OH
Dissolve 2-3 mg of sample in 0.5 ml of pyridine, add 0.2 mL of 1 mg / mL probe / pyridine solution, and heat at 40 ° C for 1 hour. After distilling off the solvent, it is dissolved in an eluent to obtain a sample solution (see Suzuki, A., et al., J. Biochem., 82, 1185 (1977)).

(7) R ² is

で、Ｒ’が−ＣＨＯの場合

And R ′ is —CHO

  Add 2 to 3 mg of sample with 1.0 ml of methanol, 0.1 ml of acetic acid and 0.2 mL of 1 mg / mL probe / methanol solution and heat at 40 ° C. for 30 minutes. After distilling off the solvent, the sample solution is dissolved in the eluent.

(8) R ² is

で、Ｒ’が−ＮＨ_２の場合

And R ′ is —NH ₂

  Add 1.5 mL of 10 mg / mL probe / ethanol solution to 0.1 mL of the sample ethanol solution (10-4 to 10-3 M), and stir at room temperature for 5 to 10 minutes. After evaporating the solvent under reduced pressure, the solvent is dissolved in the eluent to prepare a sample solution (see Roth, M., Anal. Chem., 43, 880 (1971)).

(9) R ² is —CH ₂ ONH ₂ .HCl and R ′ is

の場合

in the case of

  Heat 1-5 mg sample, 2 drops of triethylamine, 50 mg of probe at 50 ° C. for 30 minutes. After distilling off the solvent under reduced pressure, the sample is dissolved in the eluent to prepare a sample solution (see Jupille, T.H., Am. Lab., 8 (5), 85-92 (1976)).

(10) When R ² is —NHNH ₂ and R ′ is —CHO
After dissolving 1 to 5 mg of sample in 0.5 ml of water, a solution in which the probe was dissolved in 30% HClO ₄ aqueous solution at a rate of 20 mg / mL was added and stirred at room temperature for 10 minutes. After evaporating the solvent under reduced pressure, the sample is dissolved in the eluent to prepare a sample solution (see Newberg, C., et al., Anal. Chim. Acta, 7, 238 (1952)).

(11) R ² is

で、Ｒ’が

And R 'is

の場合

in the case of

  Dissolve 0.1-5.0 μg of sample in 50 μL of phosphate buffer solution (pH 7.0), add 200 μL of 1 mg / mL probe / phosphate buffer solution (pH 7.0), and stir at room temperature for 5 minutes. After evaporating the solvent under reduced pressure, the solvent is dissolved in the eluent to obtain a sample solution.

(12) R ² is

で、Ｒ’が

And R 'is

の場合

in the case of

  Dissolve 0.1-5.0 μg of sample in 50 μL of phosphate buffer solution (pH 7.0), add 200 μL of 1 mg / mL probe / phosphate buffer solution (pH 7.0), and stir at room temperature for 5 minutes. After evaporating the solvent under reduced pressure, the solvent is dissolved in the eluent to obtain a sample solution.

(13) R ² is —CH ₂ CH ₂ NH ₂ and R ′ is

の場合

in the case of

  After dissolving 0.1-5.0 μg of sample in 50 μL of phosphate buffer solution (pH 7.0), 200 μL of 1 mg / mL probe / phosphate buffer solution (pH 7.0) and 1-ethyl-3- (3- Add (dimethylaminopropyl) carbodiimide and stir at room temperature for 30 minutes. After evaporating the solvent under reduced pressure, the solvent is dissolved in the eluent to obtain a sample solution.

When the target substance is quantified using the probe of the present invention, R ² of the above general formula [I] may be directly bonded to the target substance, but as will be described in detail later, the probe is cleaved at the photocleavage site. In order to make the most of the characteristics of the probe of the present invention, in which only the released mass probe-containing molecule is subjected to mass spectrometry, a substance that specifically binds R ² to the target substance (hereinafter, for convenience) It is preferable to bind the produced probe-specific corresponding substance conjugate specifically to the target substance. Here, “specifically binds” means antigen-antibody reaction, hybridization between single-stranded nucleic acids having complementary base sequences, biotin-streptavidin binding reaction, enzyme-substrate binding reaction, etc. However, this does not necessarily mean that it does not react with anything other than the target substance. For example, even an antibody that cross-reacts with other antigens is interpreted as a “specific binding reaction”.

When such a specific counterpart is used, the probe is indirectly bound to the target substance via the specific counterpart. In this case, R ² of the general formula [I] is bound to a specific corresponding substance by the above method. Examples of specific corresponding substances include, but are not limited to, antigens, haptens, antibodies, nucleic acids, streptavidin, enzymes, and the like.

  Next, the probe-specific counterpart substance and the target substance are reacted to bind the specific counterpart substance and the target substance. This reaction can be easily carried out by employing known reaction conditions suitable for the binding reaction between each specific corresponding substance and the target substance. By this binding reaction, the probe is bound to the target substance via the specific corresponding substance.

  Next, the probes indirectly bound to the target substance are separated from other unrelated substances including unbound probes. This can be easily performed by washing the solid phase when the target substance is immobilized on the solid phase. In addition, when the target substance is liberated, the target substance is indirectly bound to the probe, and then reacted with a solid phase that can capture the target substance to capture the target substance on the solid phase. Can be achieved by washing. The fact that the target substance bound to the solid phase can be quantified as described above is a remarkable effect obtained by using the probe of the present invention having a photocleavage. An advantageous method for measuring the target substance using the probe of the present invention will be described later.

  After separating the probe bonded to the target substance, light is irradiated to cleave the photocleavage in the probe. The light irradiation conditions can be appropriately selected according to the type of the photocleavage used. Usually, ultraviolet rays are used, and the amount of irradiation can be set as appropriate. For example, when performing on a solution in a well of a microplate, a 100 W commercial high-pressure mercury lamp is usually irradiated for about 1 to 10 minutes. That's fine.

  By photocleavage, the probe is cleaved at the photocleavage part, and the mass probe part-containing molecule is released. This free mass probe-containing molecule is measured by mass spectrometry. The molecular weight of the mass probe-containing molecule is clearly known in advance, and since the mass probe is ionized as described above, mass analysis can be performed without requiring preliminary purification.

  Mass analysis of the mass probe-containing molecule can be performed in exactly the same manner as mass analysis of a normal liquid sample. The probe of the present invention includes electrospray ionization mass spectrometry, atmospheric pressure chemical ionization mass spectrometry, thermospray ionization mass spectrometry, particle beam mass spectrometry, frit fast atom bombardment mass spectrometry, and the like as examples of preferable mass spectrometry methods for liquid samples. However, it is not limited to these. Further, it can be applied not only to normal mass spectrometry but also to mass spectrometry performed in an analyzer integrated with a reaction vessel. For example, in an analytical apparatus in which a reaction tank or reaction coil is incorporated in an analytical apparatus, such as a post-column reaction type high-performance liquid chromatography apparatus or a flow injection analytical apparatus, for mass spectrometry performed downstream of the reaction tank or reaction coil. The probe of the present invention can be applied. In this case, the probe can be mixed with the sample upstream of the reaction vessel or reaction coil and the binding reaction can be performed in the reaction vessel or reaction coil. In addition, mass spectrometry itself can be easily performed using a commercially available apparatus according to the instructions attached to the apparatus.

  Since the probe of the present invention has a photocleavage, it is possible to cleave the photocleavage by irradiating light after the predetermined reaction as described above, thereby releasing the mass probe part-containing molecule. In addition, the molecular weight of the mass probe part is changed, for example, by changing the chain length of the alkyl group in the ionic group as represented by the general formulas [V] to [VII]. The method is characterized in that it can be easily changed. By making the best use of these distinctive features, it becomes possible to simultaneously quantitatively analyze a plurality of target substances in a sample. This is because at least two types of probes having different masses of the mass probe parts of the probes are directly or indirectly coupled to the target substances via the coupling parts of the probes, Is performed by irradiating the conjugate with light to cleave the photocleavage portion, and measuring the molecule including the mass probe portion of each probe released by cleavage by mass spectrometry. it can. Hereinafter, this method will be described more specifically with reference to an example.

  Of the various anti-allergen IgE antibodies in human serum, the case of quantifying anti-mite antibody, anti-pollen antibody and anti-ovalbumin antibody will be described as an example. First, three types of probes of the present invention with different molecular weights in the mass probe part are prepared. For example, it is assumed that three types of probes having molecular weights of 170, 180, and 190 having mass probe portion-containing molecules after photocleavage were produced (each probe is referred to as “probe-170”, “probe-180”, “probe” Called Probe-190). Next, each probe is separately bound to each corresponding antigen (ie, mite antigen, pollen antigen, ovalbumin) that specifically reacts with each target substance (ie, anti-tick antibody, anti-pollen antibody and anti-ovalbumin antibody). Let That is, for example, probe-170 is bound to mite antigen, probe-180 is pollen antigen, and probe-190 is bound to ovalbumin. On the other hand, beads in which an anti-human IgE antibody is immobilized on a solid phase such as beads are prepared, and this is reacted with human serum as a specimen and washed. Then, the human IgE antibody in serum is immobilized on the beads by the antigen-antibody reaction. Next, the obtained beads are reacted with probe-170-mite antigen conjugate, probe-180-pollen antigen conjugate and probe-190-ovalbumin conjugate prepared separately. The probe-170-mite antigen conjugate then binds to the anti-tick antibody on the bead, the probe-180-pollen antigen conjugate binds to the anti-pollen antibody on the bead, and the probe-190-ovalbumin conjugate is Bind to the anti-ovalbumin antibody on the beads. The beads are then washed to remove material that is not bound to the beads. Thereafter, light is irradiated to cleave the photocleavage part of the probe, and the released mass probe part-containing molecule is subjected to mass spectrometry. If it does so, the peak of the height according to the quantity of the mass probe part containing molecule which exists will be obtained. Since the mass of each mass probe portion-containing molecule is known in advance, it can be accurately measured by mass spectrometry how much mass probe portion-containing molecule is present. Since it is also known what antibody each probe is indirectly bound to, three types of anti-allergen antibodies in human serum can be quantified with high accuracy by the mass spectrometry.

  In the above example, each antigen is changed to a nucleic acid having a unique base sequence, and each target substance is a nucleic acid having a complementary base sequence (immobilized on a solid phase such as a chip by physical adsorption or covalent bond). In the same manner, three types of target nucleic acids having different base sequences can be simultaneously quantified.

  As described above, when the probe of the present invention is used, quantitative analysis of a plurality of target substances can be performed simultaneously and comprehensively, which is impossible at all using a conventional probe.

  Further, when mass analysis of a protein itself, for example, using a conventional probe is performed, the molecular weight of the protein is too large, and many peaks derived from one protein appear. Moreover, since ionization efficiency changes with things, fixed_quantity | quantitative_assay is difficult. Moreover, it is not easy to separate a substance that interacts with a protein, and in order to perform a separation reaction, the protein itself is decomposed, and a plurality of peptide chains are generated from one protein, and finally identified. There are many cases where this is not possible. On the other hand, according to the present invention, if the probe of the present invention is used and the protein is solid-phased, the mass probe-containing molecule can be easily separated (by light irradiation alone). Thus, identification and quantification can be obtained only by measuring the above, and the use of the probe of the present invention enables the quantification of protein and DNA biopolymers, which was difficult with conventional probes.

  Hereinafter, the present invention will be described more specifically based on examples. However, the present invention is not limited to the following examples.

Reagents and solvents used were Wako Pure Chemicals, Tokyo Kasei, Kanto Chemical, Aldrich and Merck's commercially available special grades. In addition, dry dichloromethane was distilled for industrial use, and dried molecular sieves were added to remove water. The abbreviations are as follows.
BO: (Benzotriazol-1-yloxy) tris (dimethylamino) phosphonium hexafluorophosphate
4-DMAP: (4-Dimethylaminopyridine)
DCC: (N, N'-dicyclohexylcarbodiimide)

Separation and purification column chromatography Merck silica gel 60 was used as a stationary phase. As the mobile phase, primary grade chloroform and methanol were used.

Compound identification method
(1) Thin layer chromatography TLC plates silica gel 60 F ₂₄₅ manufactured by Merck was used for the stationary phase. As the mobile phase, primary chloroform corresponding to chloroform, methanol and ethyl acetate were used. The compound was detected using iodine and UV absorption (254 nm).

(2) Nuclear magnetic resonance spectrometer (NMR)
As the NMR apparatus, LNM-LA 300 FT-NMR and JNM-GSX 270 FT-NMR manufactured by JEOL Ltd. were used. Deuterium chloroform (chloroform-d ₁ ), heavy methanol (methanol-d ₄ ) and heavy water (Deuterium oxide-d ₂ ) manufactured by Merck were used as the heavy solvent, and tetramethylsilane (TMS) was used as an internal standard. The signal abbreviations are as follows.
s: singlet, d: doublet, t: triplet, q: quartet, m: multiplet

Measurement conditions in the mass spectrometer The ionization method of the mass spectrometer is the ESI method, and the mass spectrometer is TOF-MS. A Mariner Biospectrometry Workstation from Applied Biosystems was used. TOF is a reflector type.
The main equipment requirements for ESI-TOF MS are as follows.
Spray tip potential: 3451.76 [V]
Nozzle potential: 184.81 [V]
Quad RF voltage: 999.76 [V]
Gas pressure: 0.5 to 0.6 [MPa]
Solvent: Methanol Flow rate: 11.5 μL / min

  The spray tip potential, nozzle potential and quad RF voltage are the potentials applied to the spray tip, nozzle and quadrupole, respectively. The spray tip potential represents a potential that directly affects the ionization of the sample solution on the tip of the capillary. The nozzle potential affects the size of fragments, peak stability, and sensitivity. Quadrupole is a quadrupole, and the range of detectable molecular weight can be changed by changing the Quad RF voltage.

UV source for photocleavage
The UV source used was a 100 W UVL-100 HA high-pressure mercury lamp from Riko Kagaku Sangyo Co., Ltd. The lamp was cooled by running tap water.

Synthesis method 1. 1- [2- (2-Nitro-phenyl) -2-oxo-ethyl] -3,5,7-triaza-1-azonia-tricyclo [3.3.1.13,7] decane; synthesis of bromide

<Experiment method>
Under a stream of Ar, hexamethylenetetraamine (6.31 g, 45 mmol, 1.1 eq.) Was placed in a 200 ml three-necked flask as a suspension of chlorobenzene (40 ml). To the system was added dropwise a solution of 2-bromo-2'-nitroacetophenone (1; 10.0 g, 41.0 mmol, 1.0 eq.) In chlorobenzene (35 ml), stirred at room temperature for 60 minutes, and then stirred at 50 ° C for 3 hours . The reaction system is returned to room temperature, suction filtered, and the crystals are washed three times with cold ethanol to give 1- [2- (2-nitro-phenyl) -2-oxo-ethyl] -3,5,7 -Triaza-1-azonia-tricyclo [3.3.1.13,7] decane; bromide (2; crude yield 16.5 g) was obtained as a white solid.

¹ H-NMR (300 MHz, D ₂ O, TMS, rt) δ: 4.52 (6 H, s, -NCH ₂ N-), 4.60 (2 H, s, ArCOCH _2- ), 5.38 (6 H, s , -NCH ₂ N ⁺ -), 7.47 (1 H, J = 6.6 Hz, d, ArH), 7.67 (1 H, J = 8.0 Hz t, ArH), 7.78 (1 H, J = 7.7 Hz, t, ArH), 8.17 (1H, J = 8.4 Hz, d, ArH)

TLC; Rf = 0.2 (chloroform / methanol = 5/1, v / v)

MS (ESI-TOF)
[M] ⁺ = 304.3

2. Synthesis of 2-amino-1- (2-nitro-phenyl) -ethanone

<Experiment method>
In a 500 ml eggplant flask, put 95% ethanol / concentrated HCl = 5/1 (v / v, 120 ml), 1- [2- (2-nitro-phenyl) -2-oxo-ethyl] -3,5, 7-Triaza-1-azania-tricyclo [3.3.1.13,7] decane; bromide (2, 15.8 g, 41.0 mmol, 1.0 eq) was dissolved and stirred at room temperature for 16 hours. After cooling the reaction system in an ice bath, a white solid was obtained by suction filtration. This white solid was added to ethanol, stirred at room temperature for 5 minutes, cooled in an ice bath, and then suction filtered to give 2-amino-1- (2-nitro-phenyl) -ethanone (3; 7.1 g, yield) 95.7%) was obtained as a white solid.

¹ H-NMR (300 MHz, CD ₃ OD, TMS, rt) δ: 4.46 (2 H, s, ArCOCH ₂ NH ₂ ), 7.75 (1 H, J = 7.5 Hz, d, ArH), 7.83 (1 H , J = 7.8 Hz, t, ArH), 7.91 (1 H, J = 7.5 Hz, t, ArH), 8.23 (1 H, J = 7.8 Hz, d, ArH)

TLC; Rf = 0.4 (chloroform / methanol = 5/1, v / v)

MS (ESI-TOF)
[M + H] ⁺ = 181.2

3. Synthesis of 2-amino-1- (2-nitro-phenyl) -ethanol

<Experiment method>
2-Amino-1- (2-nitro-phenyl) -ethanone (3; 5.0 g, 28 mmol, 1.0 eq.) In ethanol / H ₂ O = 7/3 (v / v, 70 NaBH ₄ (4.2 g, 111 mmol, 4.0 eq.) was added while stirring in an ice bath and stirred for 60 minutes. The reaction system was returned to room temperature and stirred for 5 hours.

  A large amount of ethanol was added to the suspension, suction filtration was performed, and the filtrate was concentrated. Suction was filtered again, and the filtrate was washed three times with cold ethanol, and 2-amino-1- (2-nitro-phenyl) -ethanol (4; 3.28 g, 64.8% yield) was obtained as a yellow solid. Got as.

¹ H-NMR (300 MHz, CD ₃ OD, TMS, rt) δ: 4.87 (2 H, J = 6.6 Hz, d, ArCH (OH) CH ₂ NH ₂ ), 5.42 (1 H, J = 6.6 Hz, dd, ArCH (OH) CH ₂ NH ₂ ), 7.58 (1 H, J = 7.7 Hz, t, ArH), 7.80 (1 H, J = 7.6 Hz, t, ArH), 8.00 (1 H, J = 7.8 Hz, d, ArH), 8.04 (1 H, J = 7.3 Hz, d, ArH)

TLC; R _f = 0.1 (chloroform / methanol = 10/1, v / v)

MS (ESI-TOF)
[M + H] ⁺ = 183.2

4). Synthesis of 3-maleimidopropionic acid

<Experiment method>
In a 1 L eggplant flask, maleic anhydride (5; 50 g, 0.5 mol, 1.0 eq.) Is added as a suspension of acetic acid (300 ml) and stirred with 3-aminopropionic acid (55 g, 0.6 mol, 1.2 eq. .) Was added dropwise and stirred at 140 ° C. for 4 hours.

  By distilling under reduced pressure, the obtained crystal was washed with cold ethanol to obtain 3-maleimidopropionic acid (6; 18 g, yield 21.0%) as a white solid.

¹ H-NMR (300 MHz, CDCl ₃ , TMS, rt) δ: 2.71 (2 H, J = 7.2 Hz, t, -COCH ₂ CH ₂ N-), 3.84 (2 H, J = 7.2 Hz, t, -COCH ₂ CH ₂ N-), 6.73 (2 H, s, NCOCH = CHCO-)

MS (ESI-TOF)
[M + H] ⁺ = 170.4

5). Synthesis of 3- (dioxo-2,5-dihydro-pyrrol-1-yl) -N- [2-hydroxy-2- (2-nitro-phenyl) -ethyl] -propionamide

<Experiment method>
Under a stream of Ar, put a solution of 2-amino-1- (2-nitro-phenyl) -ethanol (4; 2.1 g, 11.3 mmol, 1.0 eq.) In dichloromethane (120 ml) in a 500 ml three-necked flask and stir. While adding triethylamine (2.0 ml, 14.1 mmol, 1.3 eq.), BOP (6.3 g, 14.1 mmol, 1.3 eq.) And 3-maleimidopropionic acid (3.1 g, 17.0 mmol, 1.5 eq.). After 24 hours, molecular sieve 4A dried under reduced pressure was added, and the mixture was further stirred for 24 hours.

  The obtained crude product was separated and purified by silica gel chromatography (eluent: chloroform / methanol = 40/1, v / v), and 3- (dioxo-2,5-dihydro-pyrrol-1-yl) -N -[2-Hydroxy-2- (2-nitro-phenyl) -ethyl] -propionamide (7; 1.7 g, 45.2% yield) was obtained as a yellow syrup.

¹ H-NMR (300 MHz, CDCl ₃ , TMS, rt) δ: 2.60 (2 H, J = 6.9 Hz, t, -NHCOCH ₂ CH _2- ), 3.63 (2 H, J = 4.4 Hz, d,- CH (OH) CH ₂ NHCO), 3.86 (2 H, J = 6.9Hz, t, -NHCOCH ₂ CH _2- ), 4.72 (1 H, m, -CH (OH) CH ₂ NHCO-), 6.73 (2 H, s, NCOCH = CHCO-), 7.45 (1 H, J = 7.8 Hz, t, ArH), 7.68 (1 H, J = 7.4 Hz, t, ArH), 7.91 (1 H, J = 7.8 Hz, d, ArH), 7.97 (1 H, J = 8.3 Hz, d, ArH)

TLC; R _f = 0.3 (Chloroform / Ethyl acetate = 1/1, v / v)

MS (ESI-TOF)
[M + Na] ⁺ = 356.1

6). Synthesis of 4-bromo-butyric acid 2- [3- (2,5-dioxo-2,5-dihydro-pyrrol-1-yl) -propionylamino] -1- (2-nitro-phenyl) -ethyl ester

<Experiment method>
3- (Dioxo-2,5-dihydro-pyrrol-1-yl) -N- [2-hydroxy-2- (2-nitro-phenyl) -ethyl] -propione in a 50 ml two-necked flask under Ar flow A solution of amide (7; 238 mg, 713 μmol, 1.0 eq.) In dichloromethane (10 ml) was added and stirred in an ice bath, triethylamine (200 μL, 1.4 mmol, 2.0 eq.), 4-dimethylaminopyridine (28.0 mg). , 214 μmol, 0.3 eq.), 4-bromobutyryl chloride (85 μL, 713 μmol, 1.0 eq.) Was added dropwise, and the mixture was stirred for 3 hours.

  The obtained crude product was separated and purified by silica gel chromatography (eluent; chloroform / methanol = 40/1, v / v), and 4-bromo-butyric acid 2- [3- (2,5-dioxo- 2,5-Dihydro-pyrrol-1-yl) -propionylamino] -1- (2-nitro-phenyl) -ethyl ester (12; 40 mg, 11.6% yield) was obtained as a yellow syrup.

¹ H-NMR (300 MHz, CDCl ₃ , TMS, rt) δ: 2.16 (2 H, J = 6.2 Hz, t, -COCH ₂ CH ₂ CH _2- ), 2.48-2.63 (2 H, m, -NHCOCH _2- ), 2.48-2.63 (2 H, m, -OCOCH _2- ), 3.41-3.48 (2 H, m, -OCOCH ₂ CH _2- ), 3.70-3.90 (2 H, m, -NHCOCH ₂ CH ₂ -), 3.70-3.90 (2 H, m, -CONHCH _2- ), 6.33-6.37 (1 H, m, -CH (OH) CH ₂ NHCO-), 6.71 (2 H, s, NCOCH = CHCO-) , 7.48-7.52 (1 H, m, ArH), 7.65-7.67 (2 H, m, ArH), 8.01 (1 H, J = 8.1 Hz, d, ArH)

TLC; R _f = 0.3 (chloroform / methanol = 30/1, v / v)

MS (ESI-TOF)
[M + Na] ⁺ = 504.1, 506.1

7). Synthesis of 4-bromo-butyric acid 2- [3- (2,5-dioxo-2,5-dihydro-pyrrol-1-yl) -propionylamino] -1- (2-nitro-phenyl) -ethyl ester

<Experiment method>
3- (Dioxo-2,5-dihydro-pyrrol-1-yl) -N- [2-hydroxy-2- (2-nitro-phenyl) -ethyl] -propione in a 50 ml two-necked flask under Ar flow Add a solution of amide (7; 1.0 g, 3.0 mmol, 1.0 eq.) In dichloromethane (15 ml) and stir in an ice bath while adding DCC (930 mg, 4.5 mmol, 1.5 eq), 4-bromobutyryl acid (0.75 g, 4.5 mmol, 1.5 eq.) was added. After stirring for 24 hours, 4-dimethylaminopyridine (73 mg, 0.6 mmol, 0.2 eq.) Was added, and the mixture was further stirred for 24 hours.

  The obtained crude product was separated and purified by silica gel chromatography (eluent; chloroform / methanol = 40/1, v / v), and 4-bromo-butyric acid 2- [3- (2,5-dioxo- 2,5-Dihydro-pyrrol-1-yl) -propylamino] -1- (2-nitro-phenyl) -ethyl ester (12; 683 mg, 47.1% yield) was obtained as a yellow syrup.

¹ H-NMR (300 MHz, CDCl ₃ , TMS, rt) δ: 2.16 (2 H, J = 6.2 Hz, t, -COCH ₂ CH ₂ CH _2- ), 2.48-2.63 (2 H, m, -NHCOCH _2- ), 2.48-2.63 (2 H, m, -OCOCH _2- ), 3.41-3.48 (2 H, m, -OCOCH ₂ CH _2- ), 3.70-3.90 (2 H, m, -NHCOCH ₂ CH ₂ -), 3.70-3.90 (2 H, m, -CONHCH _2- ), 6.33-6.37 (1 H, m, -CH (OH) CH ₂ NHCO-), 6.71 (2 H, s, NCOCH = CHCO-) , 7.48-7.52 (1 H, m, ArH), 7.65-7.67 (2 H, m, ArH), 8.01 (1 H, J = 8.1 Hz, d, ArH)

TLC; R _f = 0.3 (chloroform / methanol = 30/1, v / v)

MS (ESI-TOF)
[M + Na] ⁺ = 504.1, 506.1

8). {3- [2- [3- (2,5-Dioxo-2,5-dihydro-pyrrol-1-yl) -propionylamino] -1- (2-nitro-phenyl) -ethoxycarbonyl] -propyl}- Diethyl-methyl-ammonium; synthesis of bromide

<Experiment method>
Under a stream of Ar, 4-bromo-butyric acid 2- [3- (2,5-dioxo-2,5-dihydro-pyrrol-1-yl) -propionylamino] -1- (2 A toluene solution of -nitro-phenyl) -ethyl ester was added, diethylmethylamine was added in large excess, and the mixture was stirred at 50 ° C for 12 hours. The crude product {3- [2- [3- (2,5-dioxo-2,5-dihydro-pyrrol-1-yl) -propionyl-amino] -1- (2-nitro- Phenyl) -ethoxycarbonyl] -propyl} -diethyl-methyl-ammonium; bromide was obtained as a yellow solid.

MS (ESI-TOF)
[M] ⁺ = 489.3

9. {3- [2- [3- (2,5-Dioxo-2,5-dihydro-pyrrol-1-yl) -propionylamino] -1- (2-nitro-phenyl) -ethoxycarbonyl] -propyl}- Triethyl-ammonium; synthesis of bromide

<Experiment method>
4-Bromo-butyric acid 2- [3- (2,5-dioxo-2,5-dihydro-pyrrol-1-yl) -propionylamino] -1- (2-nitro-phenyl) in a 30 ml eggplant type flask ) -Ethyl ester (8; 150 mg, 0.31 mmol, 1.0 eq.) In toluene (2.0 ml) was added, triethylamine (430 μL, 3.1 mmol, 10 eq.) Was added dropwise, and oil bath at 50 ° C. for 12 hours. Stir.

  Toluene and triethylamine were distilled off under reduced pressure and purified by reverse-phase liquid chromatography (developing solvent; methanol), and {3- [2- [3- (2,5-dioxo-2,5-dihydro-pyrrole-1- Yl) -propionyl-amino] -1- (2-nitro-phenyl) -ethoxycarbonyl] -propyl} -triethyl-ammonium; bromide (10; 60.0 mg, 38.2% yield) was obtained as a white solid.

MS (ESI-TOF)
[M] ⁺ = 503.3

  The 9 compounds and 10 compounds shown in the above reaction formulas are hereinafter referred to as KMP-174 and KMP-188. KMP-174 and KMP-188 were subjected to photocleavage experiments. Irradiate UV light from a mercury xenon lamp (L8868 manufactured by Hamamatsu Photonics Co., Ltd., wavelength 365 nm, intensity set to 50%) to 150 μL of an aqueous solution with a concentration of 5, 15 or 25 μM for each compound, and perform mass spectrometry using the above method. It was. The results are shown in FIGS. A peak corresponding to the predicted molecular weight was obtained, confirming that photocleavage occurred.

Reference example 1
Confirmation of binding property to target substance KMP series, which is a probe of the present invention, has a maleimide site that specifically binds to cysteine contained in a molecule constituting a protein. Therefore, we observed the actual binding ability using D-cysteine. Since D-cysteine was in the form of hydrochloride, the pH was adjusted with aqueous NaHCO ₃ solution to make pH 5-8 (male pH 7 in the experiment) where maleimide can specifically react with thiol.

  The experiments were performed at 5, 10, 15, 30 when the concentrations of KMP-174 '(without the terminal diethylmethylaminomethyl group of KMP-174) and D-cysteine were 500, 50, 5, and 0.5 μM, respectively. In 60 minutes, the progress of the reaction was confirmed by mass spectrometry.

<Experimental conditions>
Solvent: water
pH: 6-7
Reaction temperature: room temperature

  At concentrations of 500 and 50 μM, stirring for 5 minutes at room temperature eliminates the peak of raw material KMP-174 'in MS and the reaction product KMP 174'-cysteine peak is detected, confirming that the reaction proceeds sufficiently (FIG. 3). Even at a concentration of 5 μM, it was confirmed that the reaction proceeded sufficiently when stirred for 15 minutes. On the other hand, at 0.5 μM, the reaction product KMP 174'-cysteine peak (molecular weight 531.48) was detected in MS even after 60 minutes had passed, but the peak of the raw material KMP-174 'was also detected, and at room temperature. It was confirmed that the progress of the reaction was insufficient.

From the above, it was found that the reaction between maleimide and thiol proceeds sufficiently by stirring for about 15 minutes at a concentration of several tens of μM or more.

Concentration measurement 10 mM KMP-188 was diluted 3 times from 10 mM in 9 stages, and benzyltriethylammonium bromide was added 10 μM each as an internal standard substance. After photocleavage, analysis was performed with a mass spectrometer as described above. A standard curve was created based on the ratio of the peak area derived from -188 and the peak area of the internal standard. When the vertical axis shows the peak area ratio with the internal standard and the horizontal axis shows the concentration of KMP-188, it was shown that the correlation coefficient exceeded 0.99 and was used quantitatively for concentration measurement (FIG. 4). .

Sample measurement using a mixture
In order to confirm the quantitativeness when KMP-174 and KMP-188 were mixed, they were mixed in a certain amount, and 10 μM of benzyltriethylammonium bromide was added as an internal standard substance, and the mass spectrometer was subjected to photocleavage in the same manner as above. The analysis was performed. A standard curve was prepared based on the ratio between the peak area of each molecule and the peak area of the internal standard. FIGS. 5 and 6 below show the case where the concentration of KMP-174 is changed and the case where the concentration of KMP-188 is changed. In any case, even if it is a mixture, the quantitative property is not lost.

It is a figure which shows the result of the mass spectrometry performed after carrying out photocleavage of KMP-174 which is the probe of this invention produced in the Example of this invention. It is a figure which shows the result of the mass spectrometry performed after carrying out photocleavage of KMP-188 which is the probe of this invention produced in the Example of this invention. It is a figure which shows the result of the mass spectrometry of the reaction product of the thing which does not have the diethylmethylaminomethyl group of the terminal of KMP-174 which is the probe of this invention produced in the Example of this invention, and cysteine. It is a figure which shows the standard curve created by carrying out serial dilution of KMP-188 which is the probe of this invention produced in the Example of this invention, and carrying out mass spectrometry after photocleavage. It is a figure which shows the standard curve of KMP-174 produced by carrying out serial dilution of the mixture of KMP-174 and KMP-188 which is the probe of this invention produced in the Example of this invention, and carrying out mass spectrometry after photocleavage. It is a figure which shows the standard curve of KMP-188 prepared by serially diluting a mixture of KMP-174 and KMP-188, which are probes of the present invention prepared in the examples of the present invention, and performing mass spectrometry after photocleavage.

Claims (13)

The following general formula [I]
R ¹ -XAR ²
(However, R ¹ is a mass probe part consisting of a structure that becomes an ion in a solvent, X is a photocleavage part consisting of a structure that is cleaved by light irradiation, and A may or may not exist. Spacer part composed of any structure, R ² represents a joint part composed of a structure capable of binding to other substances)
A probe for mass spectrometry having a structure represented by: The probe according to claim 1, wherein the photocleavable part has a divalent structure represented by the following general formula [II], [III] or [IV].

(However, in the general formulas [II] to [IV], R ³ and R ⁴ each independently represent hydrogen, a lower alkoxyl group or a carboxy lower alkyl group). In the general formula [II], R ³ and R ⁴ are hydrogen, and in the general formula [III], R ³ and R ⁴ are both (1) hydrogen or (2) a nitro group in the 2-position, 5-dimethoxy, in the above general formula [IV], R ³ and R ⁴ are (1) 4,5-dimethoxy with the nitro group in the 2-position or (2) a carboxymethyl group with the hydrogen and nitro groups in the 2-position The probe according to claim 2. The probe according to claim 3, wherein the photocleavable part is represented by the general formula [II] (wherein R ³ and R ⁴ are hydrogen). The mass probe section according to any one of claims 1 to 4, wherein the mass probe section has a structure represented by the following general formula [V], [VI] or [VII] at an end opposite to a side coupled to the photocleavage section. The probe according to item 1.

(However, in the general formulas [V] to [VII], R ⁵ , R ⁶ and R ⁷ each independently represents hydrogen or an arbitrary group).   The probe according to claim 5, wherein the mass probe portion has a structure represented by the general formula [V] at an end portion on a side opposite to a side coupled to the photocleavage portion. In the general formulas [V] to [VII], R ⁵ , R ⁶ and R ⁷ are each independently hydrogen or an alkyl group having 1 to 6 carbon atoms. R ² in the general formula [I] is

SCN-, ClO ₂ S-, BrH ₂ C-, ClOC-, CH ₃ CH (NH ₂ ) = CH-, -CH ₂ ONH ₂ -HCl, -NH ₂ , -NHNH ₂ or -CH ₂ I Item 8. The probe according to any one of Items 1 to 7. R ² in the general formula [I] is

The probe according to claim 8. The following general formula [VIII]

(Where n and m are each an integer of 1 to 6, R ⁵ , R ⁶ and R ⁷ are each independently hydrogen or an arbitrary group)
The probe of Claim 1 which has a structure represented by these. The probe according to claim 10, wherein R ⁵ , R ⁶ and R ^{7 in the} general formula [VIII] are hydrogen or an alkyl group having 1 to 6 carbon atoms.   The probe according to any one of claims 1 to 11 is directly or indirectly bound to a target substance via the binding portion of the probe, and a binding substance between the probe and the target substance is separated, and the binding is performed. A mass spectrometric method comprising irradiating an object with light to cleave the photocleavage part and measuring a molecule containing the mass probe part liberated by cleaving by mass spectrometry. The probe according to any one of claims 1 to 12, wherein at least two types of probes having different masses of the mass probe portion of each probe are directly or indirectly connected via the coupling portion of each probe. The mass probe of each of the probes released by cleavage is bonded to each target substance, the bonded product of each probe and each target substance is separated, and the combined product is irradiated with light to cleave the photocleavage part. A method for measuring a plurality of target substances, comprising measuring a molecule containing a moiety by mass spectrometry.

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