RNA analysis method

Abstract

To enable detection of molecular weight-related ions with higher sensitivity and uniformity when analyzing RNA contained in a sample using MALDI mass spectrometry.SOLUTION: An RNA analysis method disclosed herein comprises using a mixed matrix containing 2,4-dihydroxyacetophenone (DHAP) and 2,4,6-trihydroxyacetophenone monohydrate (THAP) as a matrix to analyze RNA contained in a sample using a matrix-assisted laser desorption ionization mass spectrometer.SELECTED DRAWING: None

Description

[Technical field]

[0001] The present invention relates to a method for analyzing RNA. [Background technology]

[0002] One of the mass spectrometry methods for nucleic acids (DNA, RNA) is known to use matrix-assisted laser desorption / ionization (MALDI) (hereafter referred to as MALDI mass spectrometry). In MALDI mass spectrometry, an ionization assistant called a matrix is ​​used to ionize materials that do not easily absorb laser light or are easily damaged by laser light.

[0003] For example, in the MALDI mass spectrometry of nucleic acids, Non-Patent Document 1 describes the use of 3-hydroxypicolinic acid (3-HPA) and 1,5-diaminonaphthalene (1,5-DAN) as matrices. Non-Patent Document 2 describes the use of 2,4-dihydroxyacetophenone (2,4-DHAP) as a matrix. Non-Patent Document 3 describes the use of a mixed matrix containing 2,4,6-trihydroxyacetophenone (2,4,6-trihydroxyacetophenone: 2,4,6-THAP) and 2,3,4-trihydroxyacetophenone (2,3,4-THAP), and a mixed matrix containing anthranilic acid (AA) and nicotinic acid (NA) as matrices.

[0004] A matrix is ​​usually prepared as a matrix solution dissolved in a specific solvent, and mixed with a sample containing an analyte by various methods. For example, a matrix solution and a sample solution are mixed in advance to prepare a sample / matrix mixed solution, and the sample / matrix mixed solution is dropped into wells (sample dropping sites. For example, there are a circle with a diameter of about 2 mm with the outer edge scraped off, and a circle with a diameter of about 2 mm with the entire inside surface processed) formed in a metal plate called a sample plate, and dried to form a sample / matrix mixed crystal in which the analyte and the matrix are mixed on the well. Alternatively, a matrix solution and a sample solution are dropped into each well, mixed on the well, and dried to form a sample / matrix mixed crystal. In either case, the sample / matrix mixed crystal is subjected to MALDI mass spectrometry as an analytical sample. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Nathan A. Hagan, and 5 others, "Enhanced In-Source Fragmentationin MALDI-TOF-MS of Oligonucleotides Using 1,5-Diaminonapthalene", Journal of the American Society for Mass Spectrometry, (USA), 2012, 23, pp.773-777 [Non-Patent Document 2] H. Shimizu, and 6 others, "Application of high-resolution ESI and MALDI mass spectrometry to metabolite profiling of small interfering RNA duplex", Journal of Mass Spectrometry, (USA), 2012, 47, pp. 1015-1022 [Non-Patent Document 3] Li-Kang Zhang, et al., "Matrix-assisted laser desorption / ionization mass spectrometry methods for oligodeoxynucleotides: Improvements in matrix, detection limits, quantification, and sequencing", Journal of the American Society for Mass Spectrometry, (USA), 2000, 11, pp. 854-865 Summary of the Invention [Problem to be solved by the invention]

[0006] Conventionally, when an analytical sample is prepared using a matrix known as a matrix for MALDI mass spectrometry of nucleic acids, sample / matrix mixed crystals are often formed non-uniformly on the well. Therefore, even if a laser beam is irradiated onto the well to obtain molecular weight-related ions of the nucleic acid to be analyzed, a sufficient amount of molecular weight-related ions cannot be generated depending on the irradiation position. If the laser beam can be irradiated onto a location on the well where the sample / matrix mixed crystals are formed and where the ions of the sample molecules to be analyzed are relatively easy to detect (called a sweet spot), a good molecular weight-related ion peak can be detected with sufficient sensitivity. However, if the operator is inexperienced, it is difficult to find such a location in a short time, and the analysis takes a long time.

[0007] By irradiating the sample / matrix mixed crystal formed on the well with a laser using the raster scan function, mass spectrometry of the analyte contained in the sample / matrix mixed crystal can be performed more easily and quickly. However, if the sample / matrix mixed crystal is formed unevenly on the well and the crystal sites on the well vary widely, or if the sample / matrix mixed crystal is uneven and the distribution (position) of the sample in the mixed crystal varies widely, the amount of ions of the analyte generated is likely to vary depending on the irradiation position. For example, depending on the irradiation position, it is likely that excessive or insufficient molecular weight-related ions of the analyte are generated.

[0008] Furthermore, compared to DNA, there are fewer types of matrices reported for RNA that can be used in MALDI mass spectrometry, and the above problems are easily caused by conventional matrices reported for RNA analysis. Therefore, a highly sensitive and rapid analysis method for RNA has been desired.

[0009] An object of the present invention is to provide a method for easily and quickly detecting molecular weight-related ions with high sensitivity when analyzing RNA using MALDI mass spectrometry. [Means for solving the problem]

[0010] The method for analyzing RNA according to the present invention, which has been devised to solve the above problems, comprises: A mixed matrix containing 2,4-dihydroxyacetophenone (DHAP) and 2,4,6-trihydroxyacetophenone monohydrate (THAP) is used as the matrix, and RNA contained in the sample is analyzed by a matrix-assisted laser desorption / ionization mass spectrometer.

[0011] In addition, the matrix for matrix-assisted laser desorption / ionization mass spectrometry of RNA according to the present invention, which has been made to solve the above problems, contains 2,4-dihydroxyacetophenone (DHAP) and 2,4,6-trihydroxyacetophenone monohydrate (THAP). Effect of the Invention

[0012] According to the method for analyzing RNA of the present invention, when analyzing RNA contained in a sample using a matrix-assisted laser desorption / ionization mass spectrometry device, a relatively uniform sample / matrix mixed crystal can be created, and molecular weight-related ions can be detected easily and quickly with high sensitivity. In addition, by preparing an analytical sample for a sample containing RNA using the matrix for RNA matrix-assisted laser desorption / ionization mass spectrometry of the present invention, when performing matrix-assisted laser desorption / ionization mass spectrometry, a relatively uniform sample / matrix mixed crystal can be created, and molecular weight-related ions can be detected easily and quickly with high sensitivity. Therefore, RNA in a sample can be well analyzed. [Brief description of the drawings]

[0013] [Figure 1] FIG. 1 shows mass spectra of Patisiran (sense strand) when various matrices are used in Example 1, and a table showing the detection status of molecular weight-related ions. [Diagram 2] FIG. 1 shows mass spectra of Patisiran (sense strand) when various matrices are used in Example 2, and a table showing the detection status of molecular weight-related ions. [Diagram 3] FIG. 1 shows the mass spectrum of Patisiran (antisense strand) when various matrices are used in Example 3, and a table showing the detection status of molecular weight-related ions. [Figure 4] FIG. 1 shows the mass spectrum of Patisiran (sense strand) when various matrices are used in Example 4, and a table showing the detection status of molecular weight-related ions. [Diagram 5]FIG. 1 shows mass spectra of mipomersen when various matrices are used in Reference Example 1, and a table showing the detection status of molecular weight-related ions. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] Hereinafter, one embodiment of the RNA analysis method according to the present invention will be described.

[0015] (Substances to be analyzed) The RNA, which is the substance to be analyzed in this embodiment, may be a natural product obtained from an organism or a processed product thereof, or may be a chemically synthesized artificial nucleic acid. In addition, RNA includes RNA-related substances such as modified RNA, RNA derivatives, and RNA medicines. The degree of polymerization (base length) of RNA is not particularly limited, but it is preferably an oligonucleotide in which several to several tens of nucleotides are polymerized. In the analysis method of this embodiment, the molecular weight of RNA is preferably 3000 or more, and more preferably 6000 or more, from the viewpoint of being able to analyze high molecular weight RNA with good sensitivity.

[0016] (Preparation of Analytical Samples) The preparation of the analytical sample is carried out by mixing a sample containing RNA as an analyte with 2,4-dihydroxyacetophenone (hereinafter referred to as DHAP) and 2,4,6-trihydroxyacetophenone monohydrate (hereinafter referred to as THAP) as a matrix. Specifically, for example, a sample solution and a mixed matrix solution containing DHAP and THAP are prepared and mixed in advance, or a sample solution, a matrix solution containing DHAP, and a matrix solution containing THAP are prepared and mixed in advance to prepare a sample / mixed matrix mixed solution, which is dropped onto a well and dried (this method is called the pre-mix method). The sample solution and the mixed matrix solution may be dropped separately onto the well, mixed, and dried, or the sample solution, the matrix solution containing DHAP, and the matrix solution containing THAP may be dropped separately onto the well, mixed, and dried (this method is called the on-target method). In this manner, a sample / matrix mixed crystal (corresponding to the sample / matrix mixture of the present invention), which is a mixed crystal of the analyte and the matrix, is formed on the well.

[0017] The mixing ratio of DHAP to THAP in the mixed matrix (or in the mixed matrix solution) is preferably 30:1 to 10:1, and more preferably 30:1 to 15:1, from the viewpoint of improving the uniformity of the mixed crystals and enabling RNA to be analyzed with high sensitivity.

[0018] The matrix or matrix solution may further contain diammonium hydrogen citrate as a matrix additive. There are several types of ammonium salts of citrate depending on the number of ammonium ions bound to the citrate ion, but the salt preferably used in this embodiment is a salt in which two ammonium ions are bound to one citrate ion. The concentration of diammonium hydrogen citrate in the matrix solution is preferably 20 to 100 mM from the viewpoint of being able to detect the molecular weight related ions of RNA with good sensitivity. The solvent of the matrix solution is not particularly limited, and a solvent generally used as a solvent for matrix solutions can be used. For example, an aqueous solution containing 20 to 80% of an organic solvent such as acetonitrile, methanol, or ethanol can be used. Among them, it is preferable to use an aqueous solution containing 50 to 70% acetonitrile, and it is particularly preferable to use an aqueous solution containing 50% acetonitrile. The matrix additive may be added to a matrix solution containing DHAP or THAP, or may be added to a mixed matrix solution containing DHAP and THAP.

[0019] (Mass spectrometry) In the analysis method of the present embodiment, a mass spectrometer (MALDI-MS) having an ion source based on the MALDI method is used. Examples of MALDI-MS include MALDI-TOFMS of a time-of-flight (TOF) type and MALDI-ITMS of an ion trap (IT) type. The ion trap type MALDI-ITMS has an ion trap for trapping ions, and includes mass spectrometers that use the mass separation function of the ion trap itself to eject ions captured in the ion trap in ascending order of mass-to-charge ratio (m / z) and detect the ions with a detector arranged outside the ion trap, as well as mass spectrometers that separate ions ejected all at once from the ion trap according to their mass-to-charge ratios in a mass separator arranged outside the ion trap, such as a time-of-flight mass separator, and detect the ions with a detector also arranged outside the ion trap.

[0020] The ion trap of the MALDI-ITMS may be an RF trap that uses a radio frequency (RF) electric field to capture and eject ions, and may be an ion trap that uses an electric field generated by applying a sinusoidal radio frequency voltage to a ring electrode to capture ions, or a digital ion trap that uses an electric field generated by applying a square wave voltage to a ring electrode, which is generated by switching between two different voltages at high speed. In the digital ion trap, the m / z range of ions that can be captured is controlled by changing the frequency while keeping the amplitude (voltage value) of the square wave voltage constant. When the frequency is scanned to the low frequency side, the trapped ions are ejected in ascending order of m / z, and a mass spectrum is obtained by detecting them with a detector. It is preferable to use a digital ion trap type MALDI-ITMS as the mass spectrometer.

[0021] The mass spectrometer may be equipped with a function of measurement by raster scanning. Measurement by raster scanning is a measurement technique in which measurement data is obtained by performing a specified number of laser irradiations at each of a number of measurement points previously set at different positions on a well, and the final measurement data is derived by integrating all of the measurement data. Since the laser irradiation position is mechanically moved, the influence of the variation in the measurement results due to the difference in the laser irradiation position can be reduced in the integrated data. Measurement by raster scanning does not require manual movement of the irradiation position, so that easy and quick measurement is possible. In addition, since measurement by raster scanning is an automatic measurement function that does not involve the intervention of an artificial selection operation of the measurement position, it is possible to obtain more objective and highly reproducible data.

[0022] The setting conditions for raster scanning are not particularly limited, but can be adjusted appropriately depending on the well diameter of the sample plate of the device to be used, the laser diameter, etc. It is preferable to irradiate the wells of the sample plate and the sample / matrix mixture on the wells with the laser a sufficient number of times for peak detection so that the laser irradiation positions do not overlap as much as possible. Specific setting values ​​for the setting conditions for raster scanning are, for example, 25 points per well (indicating that the number of laser irradiation positions on the well is 25 points per well), 4 shots / point (indicating that the number of laser irradiations per laser irradiation position is 4 times), spacing: 0.2 mm (indicating that the interval between the laser irradiation positions is 0.2 mm), size: 0.8 mm (indicating that when performing the above 25-point laser irradiation, the scanning is performed by scanning 0.8 mm in the horizontal direction and then turning back. In other words, it indicates the length of the horizontal axis in the square-shaped laser irradiation range), etc., and a square range of 5 x 5 = 25 points automatically calculated based on them is raster scanned.

[0023] The RNA analysis method according to the present invention will be described below with reference to several examples, but these are merely illustrative and the present invention is not limited to these. EXAMPLES

[0024] <1. Preparation of sample solution> As a sample solution, a 20 pmol / μL aqueous solution of Patisiran (a sample obtained after desalting and purification of synthetic nucleic acid for research and development. Originally, it is a double-stranded RNA consisting of sense and antisense, but only the sense was used in this example. Sense: total length is 21 bases, core sequence is 19 bases, and has a 2-base DNA overhang (dTdT) at the 3' end, MW 6764) (5'-G-Um-AA-Cm-Cm-AAGAG-Um-A-Um-Um-Cm-Cm-A-Um-dT-dT-3', dT is thymidine deoxyribonucleotide, Cm is 2'-O-methylcytidine, and Um is 2'-O-methyluridine.: Sequence No. 1) was prepared.

[0025] 2. Preparation of matrix solution As matrix solutions, 40 mg / mL 50% acetonitrile solution of 2,4-dihydroxyacetophenone (DHAP) containing 70 mM diammonium hydrogen citrate as a matrix additive (DHAP solution) and 40 mg / mL 50% acetonitrile solution of 2,4,6-trihydroxyacetophenone monohydrate (THAP) containing 70 mM diammonium hydrogen citrate as a matrix additive (THAP solution) were prepared. The prepared DHAP solution and THAP solution were mixed at a mixing ratio of 30:1, 20:1, 15:1, 10:1, 5:1, 3:1, 1:1, 1:3, 1:5, and 1:10 (v / v) to prepare each mixed matrix solution.

[0026] 3. Preparation of analytical samples The sample solution of 1. and the matrix solution of 2. were mixed at 1:1 (v / v), and 1 μL of the resulting mixed solution was dropped onto the well of a sample plate (SUS plate). After dropping, the sample plate was placed in an ultra-low humidity dry box (product name: McDry, manufactured by ERC Co., Ltd.) and dried.

[0027] <4.Mass spectrometry> For mass spectrometry, a MALDI digital ion trap mass spectrometer (MALDI-DITMS, product name: MALDImini-1, manufactured by Shimadzu Corporation) was used. The sample plate in 3 was inserted into the MALDI-DITMS, and measurements were performed in positive mode using the raster scan function. The raster scan settings were 4 shots / point and 25 points. The optimal laser power for each matrix was used. The laser power depends on the matrix, and for peak detection, higher laser power is required for THAP than for DHAP. The optimal laser power for each matrix was set while checking the peak detection situation. Specifically, conditions with relatively high sensitivity and resolution were searched for. The mixed matrix had the lowest optimal laser power.

[0028] <Result> The mass spectrum and [M+H] of Patisiran sense when DHAP, THAP, and a mixed matrix containing DHAP and THAP at various mixing ratios (DHAP+THAP) were used as the matrix. + The peak detection status (sensitivity, resolution, base removal status, adduct detection status, and uniformity of peak detection by raster scan measurement) is summarized in a table in Figure 1. Note that a mixing ratio of 1:0 indicates the case where DHAP was used alone, and a mixing ratio of 0:1 indicates the case where THAP was used alone.

[0029] In the base desorption situation, the base desorption occurs during the ionization process, forming molecular weight related ions ([M+H] + The likelihood of this occurring varies depending on the characteristics of each matrix. For example, matrices that perform soft ionization are less likely to cause base desorption, while matrices that perform hard ionization are more likely to cause base desorption.

[0030] Although detailed analysis of the adducts in the adduct detection situation has not been performed, it is estimated from the m / z values ​​that the adducts are oxidized ([M+O] + etc.), alkali metal ion adducts ([M+Na] + , [M+O+Na] + , [M+2Na] + etc.), base adducts ([M+B+H] + etc.), matrix ion adducts ([M+m+H] + Adducts that are not originally present in the sample but are generated by the influence of some impurities may have been generated during the preparation or ionization process, and the likelihood of their generation varies depending on the characteristics of each matrix. It is also possible that the affinity of the matrix with the sample or alkali metal ions is related, but the details are not known.

[0031] If base elimination or adduct detection occurs easily, the nucleic acid molecule to be analyzed will be converted into molecular weight related ions ([M+H] +) and are ionized as multiple ion species and are detected dispersedly in multiple peaks, resulting in a decrease in sensitivity for molecular weight-related ions. Furthermore, because multiple ion peaks are detected, data analysis becomes complicated. Therefore, a matrix that can further suppress base elimination and the detection of adduct peaks is desired.

[0032] The mass spectrum in Figure 1 is the final mass spectrum obtained in the raster scan measurement (the mass spectrum obtained by integrating all the mass spectrum data at each measurement point). The sensitivity, resolution, base desorption status, and adduct detection status in the table are based on the mass spectrum data in Figure 1.

[0033] In this example, the base removal state is [M+H] + The S / N ratio of the representative base desorption ion peaks generated by base desorption to the S / N ratio of the peak was used for evaluation (%) (specific S / N values ​​are shown in parentheses in the table). The detection status of adducts was also evaluated based on the [M+H] + The evaluation was based on the ratio (%) of the S / N value of a representative adduct peak to the S / N value of the peak (specific S / N values ​​are shown in parentheses in the table).

[0034] The uniformity of peak detection by raster scanning is [M+H] at a sensitivity of S / N>2. + Number of measurement points where peaks were detected, [M+H] at a sensitivity of S / N>5 + The number of measurement points where the peak was detected, and [M+H] at a sensitivity of S / N>10 + The number of measurement points at which peaks were detected was evaluated as a percentage of the total number of measurement points during raster scanning (25 in this example) (the specific numbers of measurement points are shown in parentheses in the table).

[0035] As shown in Figure 1, when using a mixed matrix (DHAP+THAP) in which DHAP and THAP were mixed at a mixing ratio of 30:1 to 10:1, the molecular weight-related ions [M+H] of RNA +was detected with high sensitivity. In addition, when DHAP was used alone, [M+H] was detected with high sensitivity, for example, at a S / N>10. + The measurement points where [M+H] was detected were about 60% of the measurement points measured by raster scanning (Figure 1(a)). When THAP was used alone, [M+H] was detected at a sensitivity of S / N>10. + The measurement points where [M+H] was detected were about 20% of the measurement points measured by raster scanning (Figure 1(l)). However, when a mixed matrix (DHAP+THAP, mixing ratio 30:1 to 10:1) was used, [M+H] was detected at a sensitivity of S / N>10 at more than 90% of the measurement points measured by raster scanning. + The resolution was comparable in all matrices, and the detection of base desorption and adduct peaks was similarly suppressed in all matrices (Figure 1(b)-(e)).

[0036] From the above, it was confirmed that when a mixed matrix (DHAP + THAP, mixing ratio 30:1 to 10:1) was used, the detection sensitivity of RNA molecular weight-related ions was improved compared to the conventional method using DHAP or THAP alone, and the uniformity in measurements by raster scanning was improved. EXAMPLES

[0037] In <2. Preparation of matrix solution>, DHAP solution and THAP solution were mixed at a mixing ratio of 100:1, 50:1, 30:1, 20:1, 15:1, 10:1, 5:1, and 1:1 (v / v) to prepare each mixed matrix solution, and mass spectrometry of RNA (Patisiran sense) was performed in the same manner as in Example 1.

[0038] <Result> The mass spectrum and [M+H] of Patisiran sense when DHAP, THAP, and a mixed matrix containing DHAP and THAP at various mixing ratios (DHAP+THAP) were used as the matrix. +The peak detection conditions (sensitivity, resolution, base removal condition, adduct detection condition, and uniformity of peak detection by raster scan measurement) of Example 2 are summarized in a table in Figure 2. Note that the measurement in Example 2 was carried out on a day different from that in Example 1.

[0039] FIG. 2 also confirmed that when a mixed matrix (DHAP+THAP, mixing ratio 30:1 to 10:1) was used, the sensitivity and uniformity were improved compared to DHAP and THAP. EXAMPLES

[0040] Mass spectrometry of RNA (antisense of Patisiran) was performed in the same manner as in Example 1, except for <1. Preparation of sample solution> and <2. Preparation of matrix solution>.

[0041] <1. Preparation of sample solution> As a sample solution, a 20 pmol / μL aqueous solution of Patisiran (a sample obtained after desalting and purification of synthetic nucleic acid for research and development. Originally, it is a double-stranded RNA consisting of sense and antisense, but in this example, only the antisense was used. Antisense: total length is 21 bases, core sequence is 19 bases, has 2 bases of DNA overhang (dTdT) at the 3' end, MW 6660) (5'-AUGGAA-Um-ACUCUUGGU-Um-AC-dT-dT-3', dT is thymidine deoxyribonucleotide, Um is 2'-O-methyluridine.: sequence number 2) was prepared.

[0042] 2. Preparation of matrix solution As the matrix solution, a DHAP solution and a THAP solution were prepared in the same manner as in Example 1. The prepared DHAP solution and the THAP solution were mixed at a mixing ratio of 30:1 and 20:1 (v / v) to prepare each mixed matrix solution. In addition, as the matrix solution, a 40 mg / mL 50% acetonitrile aqueous solution of 3-hydroxypicolinic acid (3-HPA) containing 70 mM diammonium hydrogen citrate as a matrix additive (3-HPA solution) was prepared.

[0043] <Result> The mass spectra and [M+H] of the antisense of Patisiran when DHAP, THAP, a mixed matrix containing DHAP and THAP at various mixing ratios (DHAP+THAP), and 3-HPA were used as matrices. + Figure 3 shows a table summarizing the peak detection status (sensitivity, resolution, base removal status, adduct detection status, and uniformity of peak detection by raster scan measurement).

[0044] As shown in Figure 3, when the mixed matrix (DHAP+THAP, mixing ratio 30:1, 20:1) was used, the [M+H] + In addition, when the mixed matrix was used, [M+H] was detected with high sensitivity at S / N>10 in more than 90% of the measurement points in the raster scan. + Furthermore, when the mixed matrix was used, the detection of base elimination and adduct peaks was suppressed to a relatively low level, and the resolution was as high as that of the DHAP, THAP, and 3-HPA matrices.

[0045] From the above, when the mixed matrix (DHAP + THAP, mixing ratio 30:1, 20:1) was used, the RNA measured in Example 1 was not only for the sense but also for the antisense of Patisiran, and the [M + H] was obtained with high sensitivity, high uniformity, sufficient resolution, base removal status, and adduct detection status. + It was confirmed that it is possible to detect EXAMPLES

[0046] Mass spectrometry of RNA (sense of patisiran) was performed in the same manner as in Example 1, except for <2. Preparation of matrix solution>.

[0047] 2. Preparation of matrix solution As the matrix solutions, DHAP solution and THAP solution were prepared in the same manner as in Example 1. The prepared DHAP solution and THAP solution were mixed in a mixing ratio of 30:1 and 20:1 (v / v) to prepare each mixed matrix solution. In addition, 3-HPA solution was prepared in the same manner as in Example 3, and the prepared 3-HPA solution and THAP solution were mixed in a mixing ratio of 1:1 and 1:3 (v / v) to prepare each mixed matrix solution.

[0048] <Result> The mass spectra and [M+H] of Patisiran sense when DHAP, a mixed matrix containing DHAP and THAP at various mixing ratios (DHAP+THAP), and a mixed matrix containing 3-HPA and THAP at various mixing ratios (3-HPA+THAP) were used as the matrix. + Figure 4 shows a table summarizing the peak detection status (sensitivity, resolution, base removal status, adduct detection status, and uniformity of peak detection by raster scan measurement).

[0049] As shown in Figure 4, when comparing DHAP+THAP and 3-HPA+THAP, the use of DHAP+THAP resulted in higher sensitivity, higher uniformity, and a higher [M+H] + The resolution, base removal, and adduct detection were comparable. From the above, it was confirmed that the combination of DHAP and THAP is particularly effective in improving the sensitivity and uniformity in RNA analysis. Reference Example 1

[0050] DNA mass spectrometry was performed in the same manner as in Example 1, except for <1. Preparation of sample solution> and <2. Preparation of matrix solution>.

[0051] <1. Preparation of sample solution> As a sample solution, a 20 pmol / μL aqueous solution of mipomersen (a desalted and purified synthetic nucleic acid for research and development. DNA: total length 20 bases, MW 7177) (5'-MG-MC-MC-MU-MC-dA-dG-dT-dC-dT-dG-dC-dT-dT-dC-MG-MC-MA-MC-MC-3', M stands for 2'-O-(2-methoxyethyl)nucleoside, d stands for 2'-deoxynucleoside. The carbon at the 5th position of cytosine and uracil is replaced with a methyl group, and all phosphodiester bonds between nucleotides are replaced with phosphorothioate bonds.: SEQ ID NO: 3) was prepared.

[0052] 2. Preparation of matrix solution As matrix solutions, DHAP solutions and THAP solutions were prepared in the same manner as in Example 1. The prepared DHAP solutions and THAP solutions were mixed at mixing ratios of 50:1, 30:1, 20:1, 10:1, 5:1, 1:1, 1:5, 1:10, and 1:30 (v / v) to prepare each mixed matrix solution.

[0053] <Result> Mass spectra of mipomersen and [M+H] for DHAP, THAP, and a mixed matrix containing DHAP and THAP at various mixing ratios (DHAP+THAP) were used as matrices. + Figure 5 shows a table summarizing the peak detection status (sensitivity, resolution, base removal status, adduct detection status, and uniformity of peak detection by raster scan measurement).

[0054] As shown in Figure 5, the effect of improving the sensitivity and uniformity of the mixed matrix (DHAP + THAP) was not confirmed for mipomersen, which is DNA. From the above, it was found that DHAP + THAP is an effective matrix, especially for RNA analysis.

[0055] [Aspects] It will be apparent to those skilled in the art that the above-described exemplary embodiments are illustrative of the following aspects.

[0056] (Item 1) A method for analyzing RNA according to one aspect of the present invention comprises: A mixed matrix containing 2,4-dihydroxyacetophenone (DHAP) and 2,4,6-trihydroxyacetophenone monohydrate (THAP) is used as the matrix, and RNA contained in the sample is analyzed by a matrix-assisted laser desorption / ionization mass spectrometer.

[0057] By using a mixed matrix containing 2,4-dihydroxyacetophenone and 2,4,6-trihydroxyacetophenone monohydrate, the uniformity of the sample / mixed matrix mixed crystals formed as an analytical sample and consisting of an RNA-containing sample and a matrix, and the uniformity of the distribution of the RNA sample in the mixed crystals are improved. Therefore, when analyzing RNA contained in a sample using a matrix-assisted laser desorption / ionization mass spectrometer, an appropriate amount of molecular weight-related ions of the analysis target sample can be generated regardless of the laser irradiation position, and RNA can be analyzed relatively uniformly, and therefore easily and quickly. In addition, the molecular weight-related ions of RNA can be detected with relatively high sensitivity. Furthermore, the molecular weight-related ions can be detected with sufficient resolution while suppressing the detection of base desorption and adduct peaks.

[0058] (2) The method for analyzing RNA according to (1), The mixed matrix may have a mixing ratio of DHAP and THAP of 30:1 to 10:1.

[0059] This allows RNA to be analyzed with higher sensitivity. Furthermore, it is possible to generate a more uniform and appropriate amount of molecular weight-related ions of the RNA sample. Therefore, RNA can be analyzed easily, quickly, and satisfactorily.

[0060] (Item 3) The method for analyzing RNA according to item 1, The mixed matrix may use diammonium hydrogen citrate as a matrix additive, and may be used to analyze RNA contained in the sample.

[0061] Diammonium hydrogen citrate contributes to improving the sensitivity, especially for high mass molecules, which allows for more sensitive analysis of RNA.

[0062] (Item 4) In the method for analyzing RNA according to items 1 to 3, A sample solution containing RNA, a matrix solution containing DHAP, and a matrix solution containing THAP may be mixed to prepare a sample / mixed-matrix mixed solution, or a sample solution containing RNA and a mixed matrix solution containing DHAP and THAP may be mixed to prepare a sample / mixed-matrix mixed solution, and the sample / mixed-matrix mixed solution prepared by dropping the sample / mixed-matrix mixed solution onto a sample plate may be measured by the matrix-assisted laser desorption / ionization mass spectrometry device.

[0063] By premixing a sample solution containing RNA, a matrix solution containing DHAP, and a matrix solution containing THAP to prepare a sample / mixed matrix mixed solution, or premixing a sample solution containing RNA with a mixed matrix solution containing DHAP and THAP to prepare a sample / mixed matrix mixed solution, the uniformity of the mixed crystal (sample / mixed matrix mixture) of the sample containing RNA and the mixed matrix formed as an analytical sample on a sample plate and the uniformity of the distribution of the RNA sample in the mixed crystal are further improved. Therefore, when analyzing RNA contained in a sample using a matrix-assisted laser desorption / ionization mass spectrometer, an appropriate amount of molecular weight-related ions of the RNA sample can be generated regardless of the laser irradiation position, and RNA can be analyzed relatively uniformly, easily, quickly, and with high sensitivity.

[0064] (5) The method for analyzing RNA according to any one of (1) to (3), The RNA contained in the sample may be analyzed by measuring a sample / mixed matrix mixture prepared by mixing a sample solution containing RNA with a mixed matrix solution containing DHAP and THAP using the matrix-assisted laser desorption / ionization mass spectrometry device.

[0065] By preparing the sample / mixed matrix mixture after preparing the sample solution and the mixed matrix solution, the matrix in the mixed matrix, and the mixed matrix and RNA are more likely to be mixed uniformly, and the uniformity of the sample / mixed matrix mixture formed and the uniformity of the distribution of the RNA sample in the mixture are improved, thereby allowing the RNA molecules to be detected relatively uniformly, and therefore easily, quickly, and with high sensitivity.

[0066] (Item 6) In the method for analyzing RNA according to items 1 to 3, The method may involve preparing a sample solution containing RNA, a matrix solution containing either DHAP or THAP, and a matrix solution containing the other of DHAP and THAP, and measuring sample / mixed matrix mixtures prepared from these sample solutions and each matrix solution using the matrix-assisted laser desorption / ionization mass spectrometry apparatus, thereby analyzing the RNA contained in the sample.

[0067] By preparing the sample / mixed matrix mixture after adjusting the sample solution and the matrix solution, the matrix in the mixed matrix, and the mixed matrix and RNA are easily mixed uniformly, and the uniformity of the sample / mixed matrix mixture and the uniformity of the distribution of the RNA sample in the mixture are improved, so that the RNA molecules can be detected relatively uniformly, and therefore easily, quickly, and with high sensitivity.

[0068] (Item 7) In the method for analyzing RNA according to items 1 to 3, The matrix-assisted laser desorption / ionization mass spectrometer may be of a digital ion trap type.

[0069] In digital ion trap mass spectrometers, fragmentation is more likely to occur than in time-of-flight mass spectrometers, and sensitivity and resolution are more likely to be affected by the amount of ions. This makes it easier to obtain the effects of the present invention, and allows for more effective analysis of RNA.

[0070] (Item 8) A matrix for matrix-assisted laser desorption / ionization mass spectrometry of RNA according to one aspect of the present invention comprises: It contains 2,4-dihydroxyacetophenone (DHAP) and 2,4,6-trihydroxyacetophenone monohydrate (THAP).

[0071] As a result, when analyzing RNA contained in a sample using a matrix-assisted laser desorption / ionization mass spectrometer, molecular weight-related ions can be generated in a relatively uniform and appropriate amount and detected with higher sensitivity, enabling good analysis of RNA.

Claims

1. A method for analyzing RNA, which uses a mixed matrix containing 2,4-dihydroxyacetophenone (DHAP) and 2,4,6-trihydroxyacetophenone monohydrate (THAP) as a matrix, and analyzes RNA contained in a sample using a matrix-assisted laser desorption / ionization mass spectrometer.

2. The mixing ratio of DHAP to THAP in the mixed matrix is ​​30:1 to 10:1; The method for analyzing RNA according to claim 1.

3. Using diammonium hydrogen citrate as a matrix additive in the mixed matrix, RNA contained in the sample is analyzed. The method for analyzing RNA according to claim 1.

4. The method for analyzing RNA according to any one of claims 1 to 3, wherein a sample / mixed matrix solution is prepared by mixing a sample solution containing RNA, a matrix solution containing DHAP, and a matrix solution containing THAP, or a sample solution containing RNA and a mixed matrix solution containing DHAP and THAP to prepare a sample / mixed matrix solution, and the sample / mixed matrix solution is then dropped onto a sample plate and dried, thereby preparing a sample / mixed matrix mixture, which is then measured by the matrix-assisted laser desorption / ionization mass spectrometer.

5. The method for analyzing RNA according to any one of claims 1 to 3, wherein a sample solution containing RNA and a mixed matrix solution containing DHAP and THAP are each dropped onto a sample plate to prepare a sample / mixed matrix mixed solution on the sample plate, and the sample / mixed matrix solution is dried to prepare a sample / mixed matrix mixture, which is then measured by the matrix-assisted laser desorption / ionization mass spectrometer.

6. a sample solution containing RNA, a matrix solution containing DHAP, and a matrix solution containing THAP are each dropped onto a sample plate to prepare a sample / mixed matrix mixed solution on the sample plate, and the sample / mixed matrix mixed solution is dried to prepare a sample / mixed matrix mixture, which is then measured using the matrix-assisted laser desorption / ionization mass spectrometer; The method for analyzing RNA according to any one of claims 1 to 3.

7. The method for analyzing RNA according to any one of claims 1 to 3, wherein the matrix-assisted laser desorption / ionization mass spectrometer is a digital ion trap type.

8. A matrix for matrix-assisted laser desorption / ionization mass spectrometry of RNA, comprising 2,4-dihydroxyacetophenone (DHAP) and 2,4,6-trihydroxyacetophenone monohydrate (THAP).

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