JP5283178B2 - Matrix for matrix-assisted laser desorption / ionization mass spectrometry - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To properly implement a MALDI mass spectrometry of a compound having the lower molecular weight (especially the molecular weight of 500 or less) which is hard to be properly detected if a conventional matrix is used. <P>SOLUTION: The matrix uses a 1H-tetrazole derivative having a structure formula shown in the figure. R is (a) an alkyl group, or (b) a benzene system aromatic series which may have a hydroxyl group, the alkyl group or a halogen element. Since a molecular ion peak derived from the matrix does not appear in a low mass region, the molecular ion peak derived from a to-be measured object is easily identified. <P>COPYRIGHT: (C)2010,JPO&amp;INPIT

Description

  The present invention relates to a matrix used for ionization of a sample in matrix-assisted laser desorption ionization mass spectrometry.

  As one of ionization methods in mass spectrometry, a matrix-assisted laser desorption / ionization (MALDI) method is known. In the MALDI method, a sample is irradiated with laser light for a short time to vaporize the sample instantaneously, thereby ionizing the molecules of the measurement target substance in the sample without decomposing.

  In the MALDI method, in general, a sample solution is prepared by mixing a solution of a substance to be measured with a matrix solution and, if necessary, mixing with another ionization aid, applying the solution on a sample plate, and removing the solvent. . The sample thus prepared is in a state in which the substance to be measured is mixed almost uniformly with a large amount of matrix. When this sample is irradiated with laser light, the matrix absorbs the energy of the laser light and converts it into thermal energy. At this time, a part of the matrix is rapidly heated and vaporizes together with the substance to be measured. In the process, the substance to be measured is ionized.

  Mass spectrometers utilizing such MALDI methods for ionization, particularly matrix-assisted laser desorption / ionization time-of-flight mass spectrometers (MALDI-TOFMS), can cleave high molecular compounds such as proteins. In recent years, it has been used extensively in the fields of life science and industrial materials.

  Conventionally, compounds generally used as a matrix in the MALDI method are, for example, 2,5-dihydroxybenzoic acid (DHB), α-cyano-4-hydroxycinnamic acid (CHCA), disranol, 2- (4- So-called low molecular organic compounds such as hydroxylphenylazo) benzoic acid (HABA). In addition, in order to improve ionization efficiency and ionization stability in the MALDI method, attempts have been made to improve compounds used as matrices (see, for example, Patent Documents 1 and 2).

  Until now, the MALDI method has been used particularly for ionization of polymer compounds. However, since the MALDI method is a very simple and highly sensitive ionization method, there has been a great demand in recent years for application to low-molecular compounds. It is growing. When MALDI-TOFMS analysis is performed using the conventional matrix as described above, the impurity ion peak derived from the matrix is remarkably observed in the low mass (m / z) region in the mass spectrum. When the substance to be measured is a polymer compound, the presence of such an interference peak in the low mass region does not matter. However, when the measurement target substance is a low-molecular compound, on the mass spectrum, various molecular ion peaks derived from the target low-molecular compound and the interference peak may coexist or overlap in some cases. The target peak cannot be accurately grasped. For these reasons, it has been difficult to appropriately analyze low molecular weight compounds by MALDI-TOFMS using a conventional matrix.

  On the other hand, some proposals have conventionally been made as techniques for analyzing low molecular weight compounds by MALDI-TOFMS without using a matrix which is an organic compound. For example, Non-Patent Document 1 proposes a laser desorption ionization method called DIOS (Desorption / ionization on silicon) using porous silicon as a substrate. Non-Patent Document 2 proposes a laser desorption ionization method using platinum nanoparticles named “nano-flower” as an inorganic matrix. Patent Document 3 proposes a laser desorption ionization method using a plate in which Ge nanodots are formed on a silicon single crystal using a molecular beam epitaxy method. Furthermore, an example of a laser desorption ionization method using a so-called polymer matrix, which has a considerably higher molecular weight than conventional ones, has been reported so as not to cause a matrix-derived peak in a low mass region on a mass spectrum.

JP 2004-347595 A JP 2008-261824 A JP 2006-201042 A

J. Wei and two others, “Desorption-ionization mass spectrometry on porous silicon”, Nature, May 1999 20th, 339, p. 243-246 H. Kawasaki and three others, "Platinum Nanoflowers for Surface-Assisted Laser Desorption" / Ionization Mass Spectrometry of Biomolecules), The Journal of physical chemistry C, 2007, Vol. 11, p. 16278-16283

  However, the method using no matrix or the method using an inorganic matrix described above has a considerably higher analysis cost than the MALDI method using a conventional matrix. In addition, there is a problem that it is difficult to use because methods have not been established for various types of substances to be measured. On the other hand, the method using a polymer organic matrix has a problem that it is difficult to handle because the viscosity of the matrix is high, and the practicality is poor because sample preparation is difficult.

  The present invention has been made in view of these points, and the object of the present invention is to provide a low molecular organic compound capable of performing MALDI mass spectrometry practically sufficient particularly for a low molecular compound having a molecular weight of 500 or less. Is to provide a matrix that is

  Currently, compounds such as DHB and CHCA that are used as a matrix are based on a benzene ring and contain hydroxyl groups, carboxylic acids, and the like. In particular, hydroxyl groups and carboxylic acids are thought to contribute to protonation of the substance to be measured during ionization, and are considered to play an important role in ionization. Therefore, the inventors of the present application searched for a low molecular organic compound having a functional group having the same properties as carboxylic acid. Specifically, since the carboxylic acid is an acidic group, the search conditions for the functional group were determined to be acidic and have a high boiling point and a low molecular weight so as to be suitable for MALDI-TOFMS analysis.

  The inventor of the present application has shown the same degree of acidity as a carboxylic acid as a compound that satisfies the above conditions, paying attention to a tetrazole ring regarded as an equivalent of carboxylic acid, and repeated experiments based on actual analysis on various tetrazole derivatives. . As a result, the following 1H-tetrazole derivatives have been found as low molecular organic compounds that are not themselves detected as ions in the positive ion measurement mode.

ここで、Ｒは、（ａ）アルキル基、（ｂ）水酸基、アルキル基、若しくはハロゲン元素を有するベンゼン系芳香族、（ｃ）フェニル基、（ｄ）ビフェニル基、（ｅ）ナフチル基、（ｆ）アントラセン基、（ｇ）テトラセン基、又は、（ｈ）ピレン基、のいずれかである。
That is, the present invention made to solve the above problems is a matrix for ionizing a sample to be subjected to MALDI mass spectrometry, and is characterized by being a 1H-tetrazole derivative having the following structural formula.

Wherein, R is, (a) alkyl groups, (b) a hydroxyl group, an alkyl group, or a benzene-based aromatic which have a halogen element, (c) phenyl, (d) biphenyl group, (e) naphthyl, ( f) any one of anthracene group, (g) tetracene group, or (h) pyrene group .

  When the 1H-tetrazole derivative having the above structure is used as a matrix, in the positive ion measurement mode of the MALDI-TOFMS analysis, the proton-added ions derived from the matrix are substantially not detected (not detected at all or negligible even if detected). Is). On the other hand, when MALDI-TOFMS analysis in the negative ion measurement mode is performed using the 1H-tetrazole derivative having the above structure as a matrix, proton-desorbed ions derived from the matrix are detected very clearly. This indicates that the proton contained in the 1H-tetrazole derivative is easily separated from the tetrazole ring and moves to the measurement target substance in the positive ion measurement mode and contributes to the generation of proton-added ions of the substance. Yes. Therefore, it can be said that the matrix according to the present invention is a very useful matrix for positive ionization of low molecular compounds.

  In particular, when R is any of methyl, phenyl, phenol, or methylbenzene, matrix-derived protonated ions are hardly detected in the positive ion measurement mode of MALDI-TOFMS analysis. Therefore, 1H-tetrazole derivatives having R as these are particularly preferable matrices in MALDI mass spectrometry of low molecular weight compounds.

  In MALDI mass spectrometry using a conventional matrix, a molecular ion peak derived from the matrix appears remarkably in a low mass region of m / z 100 to 400 on the mass spectrum. Therefore, especially when a low molecular compound having a molecular weight of 500 or less is used as a measurement target sample, the molecular ion peak derived from this sample and the molecular ion peak derived from the matrix are mixed or overlapped, and the intended former peak is accurately obtained. It is difficult to grasp. On the other hand, according to the matrix for MALDI mass spectrometry according to the present invention, molecular ion peaks derived from the matrix are hardly observed on the mass spectrum in the positive ionization measurement mode. Therefore, even when a low molecular compound is used as a measurement target sample, it is easy to accurately grasp the molecular ion peak derived from this sample. This makes it possible to easily and accurately perform MALDI-TOFMS analysis of low molecular weight compounds, which has been difficult until now.

  The matrix for MALDI mass spectrometry according to the present invention is a compound that is generally distributed as a reagent or the like, and is easily available and inexpensive. In addition, handling is easy, and the sample preparation procedure is the same as that of a conventional matrix. Therefore, the analysis cost can be suppressed.

Examples of mass spectrum (a) in positive ion measurement mode and mass spectrum (b) in negative ion measurement mode obtained when 5-phenyl-1H-tetrazole as an embodiment of the present invention is used as a matrix FIG. Examples of mass spectrum (a) in positive ion measurement mode and mass spectrum (b) in negative ion measurement mode, obtained when 5-methyl-1H-tetrazole as an embodiment of the present invention is used as a matrix FIG. A mass spectrum in the positive ion measurement mode (a) and a mass spectrum in the negative ion measurement mode obtained when 5- (1-naphthyl) -1H-tetrazole, which is an embodiment of the present invention, is used as a matrix ( The figure which shows an example of b). Mass spectrum in positive ion measurement mode (a) and mass spectrum in negative ion measurement mode obtained when 5- (2-bromophenyl) -1H-tetrazole, which is an embodiment of the present invention, is used as a matrix. The figure which shows an example of (b). Examples of mass spectrum (a) in positive ion measurement mode and mass spectrum (b) in negative ion measurement mode obtained when 5-biphenyl-1H-tetrazole as an embodiment of the present invention is used as a matrix FIG. Mass spectrum in positive ion measurement mode (a) and mass spectrum in negative ion measurement mode obtained when 5- (4-hydroxyphenyl) -1H-tetrazole, which is an embodiment of the present invention, is used as a matrix. The figure which shows an example of (b). The figure which shows an example of the mass spectrum (a) in the positive ion measurement mode, and the mass spectrum (b) in the negative ion measurement mode obtained when CHCA used conventionally is used as a matrix. The figure which shows an example of the mass spectrum (a) in the positive ion measurement mode and the mass spectrum (b) in the negative ion measurement mode obtained when DHB used conventionally is used as a matrix.

[Example 1]
As a matrix, 5-phenyl-1H-tetrazole (molecular weight: 146.15): 68 mM / methanol solution was used, and propranolol hydrochloride: 0.1 mM / 50% acetonitrile aqueous solution was used as a measurement target sample. A sample was prepared by mixing an equal amount of both and placing 1 μL on a stainless steel target plate (manufactured by Shimadzu Corporation) and air drying. This sample was measured by MALDI-TOFMS (AXIMA-CFR manufactured by Shimadzu Corporation). An example of the mass spectrum obtained in the positive ion measurement mode and the mass spectrum obtained in the negative ion measurement mode is shown in FIGS.

As is clear from FIG. 1 (a), the peaks of the proton-added molecular ion [M + H] ⁺ , sodium-added molecular ion [M + Na] ⁺ , and potassium-added molecular ion [M + K] ⁺ derived from propranolol, which is the measurement target sample, Appears at positions m / z 260, 282, and 298. On the other hand, except for ions that cannot be identified such as m / z 72, no peaks are clearly observed in the low mass region other than the peaks of sodium ions and potassium ions observed at m / z 23 and 39. This means that a proton-added ion [m + H] ⁺ or the like derived from 5-phenyl-1H-tetrazole as a matrix is not detected, and thus the matrix does not prevent detection of ions derived from the sample to be measured. I understand. In addition, ions that cannot be identified such as m / z 72 are considered to be inevitable ions caused by the target plate or the like. The same applies to the following embodiments.

On the other hand, in the mass spectrum shown in FIG. 1 (b), conspicuously m / z1 4 5, 5- phenyl -1H- tetrazole proton desorption ionization [m-H] ^- peak of is observed. This means that one proton is easily desorbed from 5-phenyl-1H-tetrazole by laser desorption ionization. In other words, it is shown that protons derived from this matrix are added to the sample to be measured during positive ionization, and proton-added ions are easily generated. Thus, a high assurance of propranolol from the protonated molecular ions observed in positive ion measurement mode [M + H] ^+, protons H ⁺ is derived from a matrix is obtained. Therefore, it can be said that 5-phenyl-1H-tetrazole is a useful matrix for measuring positive ions of low molecular weight compounds in MALDI mass spectrometry.

[Example 2]
As a matrix, 5-methyl-1H-tetrazole (molecular weight: 84.08)): 200 mM / methanol solution was used, and propranolol hydrochloride: 10 mM / 50% acetonitrile aqueous solution was used as a measurement target sample. Propranol hydrochloride has a molecular weight of 295.80. Equal amounts of both were mixed, and in the same manner as in Example 1, 1 μL was placed on a stainless steel target plate and air-dried, and this sample was measured by MALDI-TOFMS. An example of the mass spectrum obtained in the positive ion measurement mode and the mass spectrum obtained in the negative ion measurement mode is shown in FIGS.

As is clear from FIG. 2 (a), the proton-added molecular ion [M + H] ⁺ , sodium-added molecular ion [M + Na] ⁺ , and potassium-added molecular ion [M + K] ⁺ peaks derived from the sample to be measured, propranolol. Observed. On the other hand, proton-added ion [m + H] ⁺ derived from 5-methyl-1H-tetrazole as a matrix has not been detected. Thereby, like Example 1, it turns out that a matrix does not prevent the detection of the ion derived from a measuring object sample. On the other hand, in the mass spectrum shown in FIG. 2B, proton desorption ions [mH] ^{− in} the matrix are remarkably observed. Therefore, it can be confirmed that this matrix is useful in measuring positive ions of low molecular weight compounds.

  In addition, it is conceivable that substantially the same result is obtained even with a 1H-tetrazole derivative using another alkyl group such as an ethyl group in place of the methyl group.

[Example 3]
As a matrix, 5- (1-naphthyl) -1H-tetrazole (molecular weight: 196.21): 10 mM / methanol solution was used, and propranolol hydrochloride: 10 mM / 50% acetonitrile aqueous solution was used as a measurement target sample. Equal amounts of both were mixed, and in the same manner as in Example 1, 1 μL was placed on a stainless steel target plate and air-dried, and this sample was measured by MALDI-TOFMS. An example of the mass spectrum obtained in the positive ion measurement mode and the mass spectrum obtained in the negative ion measurement mode is shown in FIGS.

As is clear from FIG. 3 (a), the proton-added molecular ion [M + H] ⁺ , sodium-added molecular ion [M + Na] ⁺ , and potassium-added molecular ion [M + K] ⁺ peaks derived from the sample to be measured, propranolol. Observed. On the other hand, the protonated ion [m + H] ⁺ derived from 5- (1-naphthyl) -1H-tetrazole as a matrix has not been detected. Thereby, like Example 1, it turns out that a matrix does not prevent the detection of the ion derived from a measuring object sample. On the other hand, in the mass spectrum shown in FIG. 3B, proton desorption ions [m—H] ⁻ of the matrix are remarkably observed. Therefore, it can be confirmed that this matrix is useful in measuring positive ions of low molecular weight compounds.

  The naphthyl group has a structure in which two benzene rings share one side, but anthracene, tetracene, or pyrene in which three or four benzene rings are bonded in a linear or rhombus shape can be used instead of naphthyl. Even with the 1H-tetrazole derivative, almost the same result can be easily obtained.

[Example 4]
As a matrix, 5- (2-bromophenyl) -1H-tetrazole (molecular weight: 222.05): 10 mM / methanol solution was used, and propranolol hydrochloride: 10 mM / 50% acetonitrile aqueous solution was used as a measurement target sample. Equal amounts of both were mixed, and in the same manner as in Example 1, 1 μL was placed on a stainless steel target plate and air-dried, and this sample was measured by MALDI-TOFMS. An example of the mass spectrum obtained in the positive ion measurement mode and the mass spectrum obtained in the negative ion measurement mode is shown in FIGS.

As is clear from FIG. 4A, the peak of the proton-added molecular ion [M + H] ⁺ derived from propranolol as the measurement target sample is clearly observed. On the other hand, the proton-added ion [m + H] ⁺ derived from the matrix 5- (2-bromophenyl) -1H-tetrazole is not detected. Thereby, like Example 1, it turns out that a matrix does not prevent the detection of the ion derived from a measuring object sample. On the other hand, in the mass spectrum shown in FIG. 4B, proton desorption ions [mH] ^{− in} the matrix are remarkably observed. Therefore, it can be confirmed that this matrix is useful in measuring positive ions of low molecular weight compounds.

  Bromophenyl is a halogenated phenyl whose halogen element is bromine. However, it is easily considered that a 1H-tetrazole derivative using a halogenated phenyl whose halogen element is chlorine or fluorine will give almost the same result. .

[Example 5]
As a matrix, 5-biphenyl-1H-tetrazole (molecular weight: 222.25): 10 μM / methanol solution was used, and propranolol hydrochloride: 10 μM / 50% acetonitrile aqueous solution was used as a measurement target sample. Equal amounts of both were mixed, and in the same manner as in Example 1, 1 μL was placed on a stainless steel target plate and air-dried, and this sample was measured by MALDI-TOFMS. An example of the mass spectrum obtained in the positive ion measurement mode and the mass spectrum obtained in the negative ion measurement mode is shown in FIGS.

As is clear from FIG. 5A, the peak of proton-added molecular ion [M + H] ⁺ derived from propranolol, which is the measurement target sample, is clearly observed. On the other hand, proton-added ion [m + H] ⁺ derived from 5-biphenyl-1H-tetrazole as a matrix has not been detected. Thereby, it turns out that a matrix does not prevent the detection of the ion derived from a measuring object sample. On the other hand, in the mass spectrum shown in FIG. 5B, proton desorption ions [mH] ^{− in} the matrix are remarkably observed. Therefore, it can be confirmed that this matrix is useful in measuring positive ions of low molecular weight compounds.

[Example 6]
As a matrix, 5- (4-hydroxyphenyl) -1H-tetrazole (molecular weight: 162.15): 10 μM / methanol solution was used, and propranolol hydrochloride: 10 μM / 50% acetonitrile aqueous solution was used as a measurement target sample. Equal amounts of both were mixed, and in the same manner as in Example 1, 1 μL was placed on a stainless steel target plate and air-dried, and this sample was measured by MALDI-TOFMS. An example of the mass spectrum obtained in the positive ion measurement mode and the mass spectrum obtained in the negative ion measurement mode is shown in FIGS.

As is clear from FIG. 6 (a), the peaks of the proton-added molecular ion [M + H] ⁺ , the sodium-added molecular ion [M + Na] ⁺ , and the potassium-added molecular ion [M + K] ⁺ derived from propranolol, which is the measurement target sample. Observed. On the other hand, proton-added ion [m + H] ⁺ derived from 5- (4-hydroxyphenyl) -1H-tetrazole as a matrix has not been detected. Thereby, it turns out that a matrix does not prevent the detection of the ion derived from a measuring object sample. On the other hand, in the mass spectrum shown in FIG. 6B, proton desorption ions [mH] ^{− in} the matrix are remarkably observed. Therefore, it can be confirmed that this matrix is useful in measuring positive ions of low molecular weight compounds.

[Comparative Example 1]
As a comparison object with each of the above examples, the same measurement was performed on CHCA (molecular weight: 188.16) that has been conventionally used as a matrix. That is, CHCA 10 mM as a matrix and propranolol 100 μM as a sample to be measured are mixed in an equal amount, and in the same manner as in Example 1, 1 μL is placed on a stainless steel target plate and air-dried to prepare a sample. Measured with TOFMS. An example of the mass spectrum obtained in the positive ion measurement mode and the mass spectrum obtained in the negative ion measurement mode is shown in FIGS.

As is clear from FIG. 7 (a), the proton-added molecular ion [M + H] ⁺ derived from propranolol as the measurement target sample is detected, and the proton-added ions [m + H] ⁺ and [2m + H derived from CHCA as the matrix are detected. ] ⁺ , Sodium addition ion [m + Na] ⁺ , potassium addition ion [m + K] ⁺ , and H ₂ O elimination ion [m + H−H ₂ O] ⁺ . Since these peaks appear in a low mass region, it is difficult to identify the molecular ion peak derived from the target sample to be measured.

[Comparative Example 2]
As another comparison target, DHB 10 mM as a matrix and propranolol 100 μM as a measurement target sample were mixed in equal amounts, and in the same manner as in Example 1, 1 μL was placed on a stainless steel target plate and air-dried to prepare a sample. The sample was measured by MALDI-TOFMS. An example of the mass spectrum obtained in the positive ion measurement mode and the mass spectrum obtained in the negative ion measurement mode is shown in FIGS.

As is clear from FIG. 8 (a), proton-added molecular ion [M + H] ⁺ , sodium-added ion [M + Na] ⁺ , potassium-added ion [M + K] ⁺ , and H ₂ O derived from propranolol as the measurement target sample. The desorbed ion [m + H-H ₂ O] ⁺ is detected, and the proton-added ion [m + H] ⁺ , sodium-added ion [m + Na] ⁺ , and potassium-added ion [m + K] ⁺ derived from the matrix DHB , Has also been detected. Since these peaks appear in a low mass region, it is difficult to identify the molecular ion peak derived from the target sample to be measured.

  From the above results, it can be confirmed that by using the matrix for MALDI mass spectrometry according to the present invention, molecular ions derived from a low molecular weight compound sample, which was difficult to detect with a conventional matrix, can be detected well. .

  The above-described embodiment is an example of the present invention, and it is a matter of course that modifications, corrections, and additions may be appropriately made within the scope of the present invention, and included in the scope of the claims of the present application.

Claims (2)

A matrix for laser-assisted desorption / ionization mass spectrometry, which is a matrix for ionizing a sample to be subjected to matrix-assisted laser desorption / ionization mass spectrometry, which is a 1H-tetrazole derivative having the following structural formula.

Wherein, R is, (a) alkyl groups, (b) a hydroxyl group, an alkyl group, or a benzene-based aromatic which have a halogen element, (c) phenyl, (d) biphenyl group, (e) naphthyl, ( f) any one of anthracene group, (g) tetracene group, or (h) pyrene group .
  The matrix-assisted laser desorption / ionization mass spectrometry matrix according to claim 1, wherein R is methyl, phenyl, phenol, or methylbenzene. .

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