JP6156846B2 - Matrix for matrix-assisted laser desorption / ionization mass spectrometry - Google Patents

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, a sample solution is generally 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 widely used in the fields of life science and industrial materials (Non-Patent Document 1).

  Conventionally, compounds used as a matrix in the MALDI method include, for example, 2,5-dihydroxybenzoic acid (DHB), α-cyano-4-hydroxycinnamic acid (CHCA), disranol, 2- (4-hydroxylphenylazo So-called low molecular organic compounds such as benzoic acid (HABA) are common. 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 to 3 and 5).

  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, contaminant ion peaks derived from the matrix are remarkably observed in the low mass region (low 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 substance to be measured is a low molecular compound having a molecular weight of 500 or less, such as a bioactive amine, various molecular ion peaks derived from the target low molecular compound and the above interference peak are mixed on the mass spectrum. Or overlap in some cases, and 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 2 proposes a laser desorption / ionization method called DIOS (Desorption / Ionization on Silicon) using porous silicon (porous silicon) as a substrate. Further, Non-Patent Document 3 proposes a laser desorption ionization method using platinum nanoparticles named “nano flower” as an inorganic matrix. Patent Document 4 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 2010-204050 A JP 2004-347595 A JP 2008-261824 A JP 2006-201042 A JP 2013-234974 A

Shrivas K, and 4 others, “Ionic Matrix for Enhanced Mardi Imaging Mass Spectrometry for Identification of Phospholipids in Mouse River and Celebrum Tissue・ Sections (Ionic matrix for enhanced MALDI imaging mass spectrometry for identification of phospholipids in mouse liver and cerebellum tissue sections), Analytical Chemistry, 2010, Vol. 82 (21), pp.8800- 8806 J. Wei and two others, “Desorption-ionization mass spectrometry on porous silicon”, Nature, May 1999 20th, 339, pp.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, pp.16278-16283

  However, the above-described method using no matrix and the method using an inorganic matrix are considerably higher in analysis cost than the conventional MALDI method using a matrix of an organic compound. On the other hand, the method using a polymer organic matrix has a problem that it is difficult to handle due to the high viscosity of the matrix and it is difficult to prepare a sample. Furthermore, all of these conventional methods have a problem that it is difficult to ionize physiologically active amines.

  The present invention has been made in view of these points. The object of the present invention is to use a low molecular weight compound having a molecular weight of 500 or less as a measurement target substance for MALDI-TOFMS analysis so that the molecular ion peak of the measurement target substance is a matrix. The object is to provide a matrix for laser-assisted desorption / ionization mass spectrometry, which is a low molecular weight organic compound that is not disturbed by peaks derived from it.

  Compounds such as 2,5-dihydroxybenzoic acid (DHB) and α-cyano-4-hydroxycinnamic acid (CHCA) that are currently used as matrices contain hydroxyl groups and carboxylic acids based on the benzene ring. It is. Carboxylic acid in particular is considered to contribute to protonation to the molecule at the time of ionization of the molecule to be measured, and is considered to play an important role in ionization of the molecule. Therefore, the inventor 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, having a high boiling point so as to be suitable for MALDI-TOFMS analysis, and having a molecular weight as low as possible.

  The inventor of the present application pays attention to a 5-benzylidenethiazolidine-4-one ring having substantially the same pKa as a carboxylic acid as a compound that meets the above-mentioned conditions, and various low molecular weight 5-benzylidenethiazolidine-4-one derivatives are An analytical experiment was conducted. As a result, in a positive ion measurement mode of MALDI-TOFMS analysis, a low molecular weight compound such as a bioactive amine is measured with the same sensitivity as CHCA or higher sensitivity than CHCA without being disturbed by matrix-derived protonated ions. The present inventors have found a 5-benzylidenethiazolidine-4-one derivative that can be conveniently used as a matrix for matrix-assisted laser desorption / ionization mass spectrometry, and has led to the present invention.

で表される、５−ベンジリデンチアゾリジン−４−オン誘導体を有することを特徴とする。 That is, the matrix for laser-assisted laser desorption / ionization mass spectrometry according to the present invention, which has been made to solve the above problems, has the general formula (1)

In represented by, and having a 5-benzylidene thiazolidine-4-one derivatives.

Ｒ１が硫黄原子、Ｒ２がトリフルオロメチル基、Ｒ３〜Ｒ６が水素原子である、下記式（３）で表される５−（２−（トリフルオロメチル）ベンジリデン）−２−チオキソチアゾリジン−４−オン、

Ｒ１が酸素原子、Ｒ４がトリフルオロメチル基、Ｒ２、Ｒ３、Ｒ５、Ｒ６が水素原子である、下記式（４）で表される５−（４−（トリフルオロメチル）ベンジリデン）チアゾリジン−２，４−ジオン

は、ＭＡＬＤＩ−ＴＯＦＭＳ分析の正イオン測定モードにおいてマトリックス由来のプロトン付加イオンが殆ど検出されることはなく、ＭＡＬＤＩ質量分析用マトリックスとして有用である。 Here, R1 represents either an oxygen atom or a sulfur atom, one of R2 to R6 represents a trifluoromethyl group, and the rest represents a hydrogen atom. Particularly, 5- (3- (trifluoromethyl) benzylidene) thiazolidine-2 represented by the following formula (2), wherein R1 is an oxygen atom, R3 is a trifluoromethyl group, and R2 and R4 to R6 are hydrogen atoms. 4-dione,

5- (2- (trifluoromethyl) benzylidene) -2-thioxothiazolidine-4 represented by the following formula (3), wherein R1 is a sulfur atom, R2 is a trifluoromethyl group, and R3 to R6 are hydrogen atoms. -On,

5- (4- (trifluoromethyl) benzylidene) thiazolidine-2 represented by the following formula (4), wherein R1 is an oxygen atom, R4 is a trifluoromethyl group, and R2, R3, R5, and R6 are hydrogen atoms. 4-dione

Is useful as a matrix for MALDI mass spectrometry because almost no proton-added ions derived from the matrix are detected in the positive ion measurement mode of MALDI-TOFMS analysis.

  When the 5-benzylidenethiazolidine-4-one derivative according to the present invention is used as a matrix, in the positive ion measurement mode of MALDI-TOFMS analysis, ions derived from the matrix do not hinder the detection of molecular ions of the measurement target substance. Therefore, even when a low molecular compound having a molecular weight of 500 or less, such as a bioactive amine, is used as a measurement target sample, it is possible to accurately grasp the molecular ion peak derived from this sample. Therefore, MALDI-TOFMS analysis of a low molecular weight compound that has been difficult can be easily performed.

  Moreover, since the 5-benzylidenethiazolidine-4-one derivative according to the present invention can be synthesized in a relatively short step, it can be produced at low cost. Therefore, the analysis cost can be reduced as compared with the conventional method using an inorganic matrix or a polymer organic matrix. Furthermore, since the 5-benzylidenethiazolidine-4-one derivative according to the present invention has a molecular weight as low as 500 or less, the viscosity is lower than that of a conventional polymer organic matrix and the handling is easy.

Bioactive amines ((a) serotonin, (b) L-glutamic acid, (c) using 5- (3- (trifluoromethyl) benzylidene) thiazolidine-2,4-dione according to one embodiment of the present invention as a matrix Mass spectrum obtained by MALDI-TOFMS analysis in positive ion measurement mode using histamine (d) DL-adrenaline) as a measurement target substance. MALDI-TOFMS analysis was performed in the positive ion measurement mode using CHCA as a matrix and bioactive amines ((a) serotonin, (b) L-glutamic acid, (c) histamine, (d) DL-adrenaline) as a measurement target substance. Obtained mass spectrum.

一般式（１）において、Ｒ１は酸素原子又は硫黄原子のいずれかを表し、Ｒ２〜Ｒ６のうち一つはトリフルオロメチル基を、残りは水素原子を表す。
特に、Ｒ１が酸素原子、Ｒ３がトリフルオロメチル基、Ｒ２、Ｒ４〜Ｒ６が水素原子である５−（３−（トリフルオロメチル）ベンジリデン）チアゾリジン−２，４−ジオン、Ｒ１が硫黄原子、Ｒ２がトリフルオロメチル基、Ｒ３〜Ｒ６が水素原子である５−（２−（トリフルオロメチル)ベンジリデン）−２−チオキソチアゾリジン−４−オン、Ｒ１が酸素原子、Ｒ４がトリフルオロメチル基、Ｒ２、Ｒ３、Ｒ５、Ｒ６が水素原子である５−（４−（トリフルオロメチル）ベンジリデン）チアゾリジン−２，４−ジオンは、マトリックス支援レーザ脱離イオン化質量分析用マトリックスとして有用である。以下、これら５−ベンジリデンチアゾリジン−４−オン誘導体のうち５−（３−（トリフルオロメチル）ベンジリデン）チアゾリジン−２，４−ジオンの実施例について説明する。 The matrix-assisted laser desorption / ionization mass spectrometry matrix according to the present invention is a 5-benzylidenethiazolidine-4-one derivative represented by the following general formula (1).

In the general formula (1), R1 represents either an oxygen atom or a sulfur atom, one of R2 to R6 represents a trifluoromethyl group, and the rest represents a hydrogen atom.
In particular, 5- (3- (trifluoromethyl) benzylidene) thiazolidine-2,4-dione, wherein R1 is an oxygen atom, R3 is a trifluoromethyl group, R2, and R4 to R6 are hydrogen atoms, R1 is a sulfur atom, R2 Is a trifluoromethyl group, R3 to R6 are hydrogen atoms 5- (2- (trifluoromethyl) benzylidene) -2-thioxothiazolidine-4-one, R1 is an oxygen atom, R4 is a trifluoromethyl group, R2 , R3, R5, R6 are hydrogen atoms, 5- (4- (trifluoromethyl) benzylidene) thiazolidine-2,4-dione is useful as a matrix for matrix-assisted laser desorption / ionization mass spectrometry. Hereinafter, examples of 5- (3- (trifluoromethyl) benzylidene) thiazolidine-2,4-dione among these 5-benzylidenethiazolidine-4-one derivatives will be described.

(1) Method for producing 5- (3- (trifluoromethyl) benzylidene) thiazolidine-2,4-dione 1.0 g of thiazolidine-2,4-dione, 1.78 g of 3-trifluoromethylbenzaldehyde and 1 ml of piperidine in 20 ml of ethanol The mixture was heated to reflux for 18 hours, cooled to room temperature, and then 80 ml of water was added to make an aqueous solution. Concentrated hydrochloric acid was added to this aqueous solution to adjust to pH 2. The deposited precipitate was collected by filtration, dissolved in 4 ml of ethanol heated to 50 ° C., and left at room temperature for 4 hours. Thereafter, the precipitated crystals were collected by filtration and dried under reduced pressure for 12 hours to obtain 0.62 g of a product.
The melting point of this product was measured and found to be 183 ° C.
The analysis results of this product by ¹ H-NMR and matrix-assisted laser desorption / ionization (positive ion measurement mode) were as follows.
¹ H-NMR (solvent: deuterated chloroform, δ): 7.63 (1H), 7.67 (1H), 7.70 (1H), 7.88 (1H)
Mass spectrometry Matrix-assisted laser desorption / ionization (cation detection): m / z 274 [M + H] ⁺
From the above results, the obtained compound was 5- (3- (trifluoromethyl) benzylidene) thiazolidine-2,4-dione (molecular formula: C ₁₁ H ₆ F ₃ NO ₂ S, molecular weight: 273). Identified.

(2) MALDI-TOFMS analysis of bioactive amine A measurement sample prepared using 5- (3- (trifluoromethyl) benzylidene) thiazolidine-2,4-dione as a matrix and a bioactive amine as a measurement target sample was used as a MALDI- Measured with TOFMS.
Further, as a comparative example, a measurement sample prepared using a bioactive amine when CHCA was used as a matrix as a measurement target sample was measured by MALDI-TOFMS.
In addition, serotonin (molecular weight: 176.22), L-glutamic acid (molecular weight: 147.13), histamine (molecular weight: 111.15) and DL-adrenaline (molecular weight: 183.20) were used as physiologically active amines.

  Serotonin is a bioactive amine widely distributed in animals and plants, and is known to be mainly involved in biological rhythm, neuroendocrine, sleep, body temperature regulation and the like in humans. Glutamic acid is a protein-constituting amino acid and is known as an excitatory neurotransmitter in animals. Histamine is known to act as a mediator in the development of allergic reactions and inflammation when it is overexpressed in addition to neurotransmitters, and causes allergic diseases by binding to receptor proteins. Adrenaline is a hormone or neurotransmitter that plays a central role in stress responses and is known to increase heart rate and blood pressure when released into the blood. These physiologically active amines were all difficult to detect by analysis using a conventional matrix such as CHCA.

The measurement sample was prepared as follows.
After dropping 0.5 μl of a matrix (5- (3- (trifluoromethyl) benzylidene) thiazolidine-2,4-dione, CHCA) in a 20 μmol / ml acetonitrile: water 50:50 (v / v) solution onto each stainless steel sample plate Then, 0.5 μl of 2 μmol / ml measurement sample solution was added and dried. Among the samples to be measured, L-glutamic acid was dissolved in 0.01 mol hydrochloric acid aqueous solution, and the others were dissolved in acetonitrile: water 50:50 (v / v).

(2-1) Serotonin measurement results Obtained in positive ion mode when 5- (3- (trifluoromethyl) benzylidene) thiazolidine-2,4-dione is used as a matrix and when CHCA is used as a matrix. The mass spectra of serotonin are shown in FIG. 1 (a) and FIG. 2 (a), respectively.

As is clear from FIG. 1 (a), when 5- (3- (trifluoromethyl) benzylidene) thiazolidine-2,4-dione is used as a matrix, protonated molecular ion derived from serotonin [M + H] ⁺ Appears at the position of m / z 177. Several peaks of ions thought to be derived from the matrix are also observed, but these are all low in intensity and do not interfere with detection of serotonin molecular ions. Therefore, the molecular ion peak of serotonin can be clearly detected.
On the other hand, as is clear from FIG. 2 (a), when CHCA is used as a matrix, the peak of protonated molecular ion [M + H] ⁺ derived from serotonin appears at the position of m / z 177. However, many ion peaks that are thought to be derived from the matrix also appear. Moreover, since the ion peak derived from serotonin is comparatively low intensity | strength and it is hard to distinguish from the ion peak derived from a matrix, it turns out that identification is difficult.

(2-2) Detection result of L-glutamic acid Positive ion mode when 5- (3- (trifluoromethyl) benzylidene) thiazolidine-2,4-dione is used as a matrix and when CHCA is used as a matrix The mass spectrum of L-glutamic acid obtained in 1 is shown in FIG. 1 (b) and FIG. 2 (b).

As is apparent from FIG. 1B, when 5- (3- (trifluoromethyl) benzylidene) thiazolidine-2,4-dione is used as a matrix, a proton-added molecular ion derived from L-glutamic acid [M + H ] A ⁺ peak appears at m / z 148. In addition, some peaks of ions that are considered to be derived from the matrix are also observed, but these are either low in intensity or separated from the peak of the molecular ion of L-glutamic acid, so that the molecular ion peak of the L-glutamic acid Does not interfere with the detection. Therefore, the ion peak derived from L-glutamic acid can be clearly detected.

On the other hand, when CHCA is used as a matrix, as is clear from FIG. 2 (b), the peak of the protonated molecular ion [M + H] ⁺ of L-glutamic acid appears at the position of m / z 148. However, many ion peaks that are thought to be derived from the matrix also appear. Since the intensity of the molecular ion peak of L-glutamic acid is about the same or lower than the intensity of the ion peak derived from the matrix, it is difficult to distinguish from the ion peak derived from the matrix, and the identification is difficult.

(2-3) Histamine detection results Obtained in positive ion mode when 5- (3- (trifluoromethyl) benzylidene) thiazolidine-2,4-dione is used as a matrix and when CHCA is used as a matrix. The mass spectra of histamine are shown in FIG. 1 (c) and FIG. 2 (c), respectively.

As is clear from FIG. 1 (c), when 5- (3- (trifluoromethyl) benzylidene) thiazolidine-2,4-dione is used as a matrix, the protonated molecular ion [M + H] ⁺ of histamine A peak appears at m / z 112. On the other hand, the peak of ions considered to be derived from the matrix does not appear, or even if it appears, it has a very low intensity. Therefore, the molecular ion peak of histamine can be clearly detected.

On the other hand, as is clear from FIG. 2 (c), when CHCA is used as a matrix, the peak of proton-added molecular ion [M + H] ⁺ of histamine appears at the position of m / z 112. However, many ion peaks that are thought to be derived from the matrix appear. In addition, the molecular ion peak of histamine is relatively low in intensity, and it is difficult to distinguish from the ion peak derived from the matrix.

(2-4) Detection result of DL-adrenaline When 5- (3- (trifluoromethyl) benzylidene) thiazolidine-2,4-dione is used as a matrix and when CHCA is used as a matrix, in positive ion mode The mass spectra of the obtained DL-adrenaline are shown in FIG. 1 (d) and FIG. 2 (d), respectively.

As is apparent from FIG. 1 (d), when 5- (3- (trifluoromethyl) benzylidene) thiazolidine-2,4-dione is used as a matrix, proton-added molecular ion [M + H] of DL-adrenaline ^{A +} peak appears at m / z 184. Some peaks of ions thought to be derived from the matrix are also observed, but these are all low in intensity and do not interfere with detection of ions derived from DL-adrenaline. Therefore, the molecular ion peak of DL-adrenaline can be clearly detected.

On the other hand, as is clear from FIG. 2 (d), when CHCA is used as a matrix, a peak of proton-added molecular ion [M + H] ⁺ of DL-adrenaline appears at the position of m / z 184. However, many ion peaks that are thought to be derived from the matrix also appear. Moreover, since the molecular ion peak of DL-adrenaline is relatively low in intensity and is difficult to distinguish from the ion peak derived from the matrix, it can be seen that identification is difficult.

Thus, when 5- (3- (trifluoromethyl) benzylidene) thiazolidine-2,4-dione according to the present example was used as a matrix, MALDI-TOFMS analysis of a bioactive amine was performed. In the spectral region in which the peak of proton-added molecular ion [M + H] ⁺ appears, a peak that interferes with the observation of the molecular ion peak of the bioactive amine is hardly observed. For this reason, bioactive amine can be detected clearly and it turns out that 5- (3- (trifluoromethyl) benzylidene) thiazolidine-2,4-dione is useful as a matrix.

  Note that, here, 5- (3- (trifluoromethyl) benzylidene) thiazolidine-2,4-dione has been described as an example, but other 5-benzylidenethiazolidine-4-one derivatives are used as a matrix. The same action and effect can be obtained. Further, the above-described embodiments are merely some examples of the present invention, and any change, correction, or addition appropriately made within the spirit of the present invention is naturally included in the scope of the claims of the present application.

Claims (4)

A matrix for ionizing a sample to be subjected to matrix-assisted laser desorption / ionization mass spectrometry, having the general formula (1)

A matrix for laser-assisted laser desorption / ionization mass spectrometry having a 5-benzylidenethiazolidine-4-one derivative represented by:
Here, R1 represents either an oxygen atom or a sulfur atom, one of R2 to R6 represents a trifluoromethyl group, and the rest represents a hydrogen atom.   The matrix for laser-assisted desorption / ionization mass spectrometry according to claim 1, wherein R1 is an oxygen atom, R3 is a trifluoromethyl group, and R2 and R4 to R6 are hydrogen atoms.   The matrix for laser-assisted desorption / ionization mass spectrometry according to claim 1, wherein R1 is a sulfur atom, R2 is a trifluoromethyl group, and R3 to R6 are hydrogen atoms.   The matrix for laser-assisted desorption / ionization mass spectrometry according to claim 1, wherein R1 is an oxygen atom, R4 is a trifluoromethyl group, and R2, R3, R5, and R6 are hydrogen atoms.

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