Method for analyzing nucleic acids and the matrix used therefor.

Abstract

In the present invention, a nucleic acid included in a sample is analyzed by a matrix-assisted laser desorption / ionization mass spectrometer using a mixed matrix that includes 3-hydroxypicolinic acid and 2,4-dihydroxyacetophenone, or a mixed matrix that includes 3-hydroxypicolinic acid and 2,4,6-trihydroxyacetophenone monohydrate. It is thereby possible to detect [M+H]+ or [M-H]- of the nucleic acid and a fragment ion generated by desorption of said ion with high sensitivity.

Description

[Technical Field]

[0001] This invention relates to a method for analyzing nucleic acids using mass spectrometry and a matrix used therefor. [Background technology]

[0002] Nucleic acids are biomolecules composed of nucleotides, each consisting of a base, sugar, and phosphate, linked together by phosphodiester bonds. They are classified into deoxyribonucleic acid (DNA), which contains 2-deoxyribose, and ribonucleic acid (RNA), which contains ribose, depending on the sugar used. Among these, nucleic acids composed of several to several dozen nucleotides are also called oligonucleotides and can be chemically synthesized. For this reason, research into applying oligonucleotides as nucleic acid drugs has been actively conducted in recent years, increasing the importance of analyzing nucleic acids.

[0003] Matrix-assisted laser desorption / ionization (MALDI) is generally known as a gentle ionization method that can ionize non-volatile compounds with minimal degradation. Mass spectrometers utilizing this method (MALDI-MS) are widely used to obtain molecular weight information of biomacromolecules such as peptides, proteins, and glycans. However, in nucleic acid analysis using MALDI-MS, nucleic acids are easily degraded during ionization, especially those with larger molecular weights, making them susceptible to degradation of protonated molecules [M+H]. + or deprotonated molecule [MH] - (where M is a molecule and H is a hydrogen atom) is difficult to form. Also, [M+H] + or [MH] - Separately, alkali metal ion adducts of nucleic acids are easily formed. As a result, [M+H] + ya [MH] - There is a problem in that the peak intensity or signal-to-noise ratio decreases, leading to a reduction in analytical sensitivity. Therefore, various nucleic acid analysis methods that can detect these ions with high sensitivity are being investigated.

[0004] For example, in Non-Patent Document 1, in the molecular weight analysis of nucleic acids by a time-of-flight (TOF) mass spectrometer (MALDI-TOFMS) having a MALDI ion source, by adding ammonium citrate to the matrix, the peak intensity of the alkali metal ion adduct decreases, and the [M+H] of nucleic acids with a molecular weight of about 6000 + is described to have been detected with a higher S / N than before the addition of ammonium citrate. In Non-Patent Document 2, in the molecular weight analysis of nucleic acids by MALDI-TOFMS, by adding ammonium citrate to 2,4-dihydroxyacetophenone (2,4-DHAP), which is the matrix, the [M-H] of RNA with a molecular weight of about 6800 - is described to have been detected. <00001​​​​​​​​​​​It is practiced to intentionally dissociate nucleic acids using appropriate methods, determine the assignment of the resulting diverse fragment ions by mass spectrometry, and then estimate the molecular structure of the original nucleic acid. One known method for ion dissociation in this process is in-source decay (ISD). In-source decay is a technique in which ions are dissociated at the ion source simultaneously with or immediately after ionization. As mentioned above, the MALDI method is a soft ionization method, so ions are inherently difficult to dissociate. However, it is known that ion dissociation can be promoted during ionization by, for example, increasing the laser intensity to increase the energy during ionization, or by using a special matrix.

[0007] For example, Non-Patent Literature 4 describes that in structural analysis of relatively short nucleic acids with a length of 10 nucleotides, using 6-thioguanine (TG) as the matrix resulted in the detection of ISD fragment ions (ISD fragment ions) centered on w-series ions, particularly in negative ion mode. Non-Patent Literature 5 describes that in structural analysis of relatively short nucleic acids with a length of 10 nucleotides, using 1,5-diaminonaphthalene (1,5-DAN) as the matrix resulted in the detection of multiple ISD fragment ions, including aB, d, y, and w-series ions, particularly in negative ion mode. Non-Patent Literature 2 describes that in structural analysis of relatively long nucleic acids with a length of 20 nucleotides, using 2,4-dihydroxyacetophenone (2,4-DHAP) as the matrix resulted in the detection of ISD fragment ions centered on w-series ions, particularly in negative ion mode.

[0008] Furthermore, Non-Patent Literature 6 describes how, in structural analysis of synthetic oligonucleotides using MALDI-TOFMS, a large number of y-series ISD fragment ions were detected by using a mixed matrix containing anthranilic acid (AA) and 3-hydroxypicolinic acid (3-HPA). The same document also describes how, in structural analysis of synthetic oligonucleotides using MALDI-MS (IR-MALDI) with an infrared (IR) laser, y-series ISD fragment ions were detected by using a mixed matrix containing 2,5-dihydroxybenzoic acid (DHB) and anthranilic acid (AA) and nicotinic acid (NA). [Prior art documents] [Non-patent literature]

[0009] [Non-Patent Document 1] Yoshinao Wada, "Fundamentals of Genome Analysis by MS—Focusing on SNPs—", Journal of the Mass Spectrometry Society of Japan, 2003, Vol. 51, No. 2, pp. 368-373. [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 [Non-Patent Document 4] Satoshi Kimura, and 1 other, "Effect of oligonucleotide structural difference on matrix-assisted laser desorption / ionization in-source decay in comparison with collision-induced dissociation fragmentation", (USA), Rapid Communications in Mass Spectrometry, 2020, 34, e8819 [Non-Patent Document 5] 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 6] M. Sebela, "Analysis and sequencing of nucleic acids by matrix-assisted laser desorption / ionization mass spectrometry", Spectroscopy Europe, 2016, 28, 5, pp.11-15 [Non-Patent Document 7] Scott A. McLuckey, and 2 others, “Tandem mass spectrometry of small, multiply charged oligonucleotides”, Journal of the American Society for Mass Spectrometry, (USA), 1992, 3, pp. 60-70 [Overview of the project] [Problems that the invention aims to solve]

[0010] Regarding the molecular weight analysis of nucleic acids, the methods described in Non-Patent Documents 1 and 2 use matrix additives to analyze the [M+H] of relatively high-mass nucleic acids. + ya [MH] - Although it has been detected, its sensitivity is still insufficient and there is room for improvement. Furthermore, the matrix and analytical method described in Non-Patent Document 3 contain relatively high mass nucleic acids [MH] - Although it has been detected with high sensitivity, its versatility is limited, and further investigation is desired. Also, generally speaking, there are only a limited number of matrices that have been reported to be usable for nucleic acid analysis. Therefore, the [M+H] of nucleic acids + or [MH] - A new matrix is ​​desired that can detect these with high sensitivity and more effectively.

[0011] Regarding the structural analysis of nucleic acids, Non-Patent Documents 2 and 4-6 describe the detection of ISD fragment ions and subsequent sequencing of nucleic acids by using matrices with properties that facilitate ISD formation. However, these methods have drawbacks, such as low detection sensitivity, a bias in the number of detected ion species and high-sensitivity peaks to a portion of the sequence (or mass range), and the complexity of the analysis due to the detection of multiple ion species. Furthermore, decreased sensitivity leads to decreased resolution, making the analysis difficult. One reason for the low sensitivity of ISD fragment ions is the precursor ion [M+H] that generates the ISD fragment ions. + or [MH] - However, it is possible that the detection is not sufficiently sensitive. Furthermore, if ISD fragment ions can be detected with sufficient sensitivity, resolution for analysis, a sufficient number of peaks for analysis, and with as uniform an ion species as possible, nucleic acid structural analysis will become easier. However, the number of matrices reported to be usable for nucleic acid structural analysis (i.e., matrices for ISD) is limited, and new matrices are desired.

[0012] This invention was made in view of the above-mentioned problems, and concerns nucleic acids [M+H] + or [MH] - The objective is to provide an analytical method and matrix for use therein that can detect fragment ions generated by the dissociation of these ions with high sensitivity, and enable easy and reliable molecular weight analysis and structural analysis of nucleic acids. [Means for solving the problem]

[0013] The nucleic acid analysis method according to the present invention, which was developed to solve the above problems, This method involves analyzing nucleic acids contained in a sample using a matrix-assisted laser desorption / ionization mass spectrometer, employing a mixed matrix containing 3-hydroxypicolinic acid and 2,4-dihydroxyacetophenone, or a mixed matrix containing 3-hydroxypicolinic acid and 2,4,6-trihydroxyacetophenone monohydrate.

[0014] Furthermore, the matrix for nucleic acid matrix-assisted laser desorption / ionization mass spectrometry according to the present invention, which was developed to solve the above problems, contains 3-hydroxypicolinic acid and 2,4-dihydroxyacetophenone, or contains 3-hydroxypicolinic acid and 2,4,6-trihydroxyacetophenone monohydrate. [Effects of the Invention]

[0015] According to the nucleic acid analysis method and matrix used therein according to the present invention, the [M+H] of nucleic acids + or [MH] - Since a sufficient amount is generated, these can be detected with high sensitivity. Furthermore, according to the analytical method and matrix used therein, the [M+H] of nucleic acids + or [MH] - When a sufficient amount is generated, and under modified analysis conditions changed for structural analysis, [M+H] + or [MH] - Since a sufficient amount of fragment ions are generated by dissociation, fragment ions can be detected with high sensitivity. As a result, molecular weight analysis and structural analysis of nucleic acids can be performed easily and reliably. [Brief explanation of the drawing]

[0016] [Figure 1] A schematic diagram showing an example of a mass spectrometer (MALDI-ITMS) used in a nucleic acid analysis method according to one embodiment of the present invention. [Figure 2] A flowchart showing the structural analysis procedure in a nucleic acid analysis method according to one embodiment of the present invention. [Figure 3] This figure shows the mass spectra of standard nucleic acid A when various matrices are used in Reference Example 1. [Figure 4] This figure shows the mass spectra of standard nucleic acid A when various matrices (mixed matrices) are used in Reference Example 1. [Figure 5]This figure shows the mass spectrum (ISD spectrum) of fragment ions derived from standard nucleic acid A when 2,4-DHAP is used as the matrix in Reference Example 2. [Figure 6] A figure showing the mass spectra of mipomersene using various matrices in Example 1, and a table showing the detection status of [M+H]+. [Figure 7] A figure showing the mass spectra of mipomercene when using the mixed matrix 3-HPA+2,4-DHAP mixed at each mixing ratio in Example 2, and a table showing the detection status of [M+H]+. [Figure 8] This figure shows the mass spectra of mipomersen when using each matrix in Example 3-1. [Figure 9] This figure shows the mass spectra of mipomersen when using each matrix in Example 3-2. [Figure 10] A figure showing the mass spectra of mipomercene when a mixed matrix 3-HPA+2,4-DHAP containing ACD of various concentrations was used in Example 4, and a table showing the detection status of [M+H]+. [Figure 11] This figure shows the mass spectrum (ISD spectrum) of fragment ions derived from mipomercene when using the mixed matrix 3-HPA+2,4-DHAP, obtained by ISD measurement under the mass-to-charge ratio range condition of mode 3 (m / z 2000~18000) in Example 5. [Figure 12] This figure shows the mass spectrum (ISD spectrum) of fragment ions derived from mipomercene when using a mixed matrix 3-HPA+2,4-DHAP, obtained by ISD measurement under the mass-to-charge ratio range conditions of mode 2 (m / z 650~5000) in Example 5. [Figure 13] The ISD spectra of mipomercene obtained by ISD measurements under the mass-to-charge ratio range condition of mode 3 (m / z 2000~18000) in Example 5 are shown below, specifically (a) the mixed matrix 3-HPA+2,4-DHAP and (b) the ISD spectra of mipomercene using 2,4-DHAP. [Figure 14]A figure showing the mass spectra of patissirane (a 1:1 mixture of sense and antisense) using various matrices in Example 6, and a table showing the detection status of [M+H]+. [Figure 15] A figure showing the mass spectra of patissirane (a 1:1 mixture of sense and antisense) using various matrices in Example 6, and a table showing the detection status of [M+H]+. [Figure 16] A figure showing the mass spectra of patissirane (a 1:1 mixture of sense and antisense) using various matrices in Example 6, and a table showing the detection status of [M+H]+. [Figure 17] Figure showing the mass spectra of patissirane (a 1:1 mixture of sense and antisense) when various matrix solutions containing 40 mM, 70 mM, or 100 mM ACD and 50% or 70% ACN were used in Example 7, and a table showing the detection status of [M+H]+. [Modes for carrying out the invention]

[0017] The following describes one embodiment of the nucleic acid analysis method according to the present invention.

[0018] <Nucleic acid> The nucleic acids analyzed in this embodiment include not only nucleic acids but also nucleic acid-related substances such as modified nucleic acids, nucleic acid derivatives, and nucleic acid pharmaceuticals. Hereinafter, nucleic acids and nucleic acid-related substances will be collectively referred to simply as nucleic acids. The degree of polymerization (base length) of nucleic acids is not particularly limited, but it is preferable that they be oligonucleotides polymerized from several to several tens of nucleotides. In the analytical method according to this embodiment, the analytical sensitivity of nucleic acids with large molecular weights is particularly improved, so it is more preferable that the nucleic acid has a molecular weight of 3000 or more, and more preferably a molecular weight of 6000 or more. Furthermore, nucleic acids may be natural products obtained from living organisms or processed products thereof, or they may be chemically synthesized artificial nucleic acids.

[0019] <Mixed Matrix> The mixed matrix used in this embodiment is either one containing 3-hydroxypicolinic acid (3-HPA) and 2,4-dihydroxyacetophenone (2,4-DHAP) (hereinafter referred to as 3-HPA+2,4-DHAP) or one containing 3-hydroxypicolinic acid (3-HPA) and 2,4,6-trihydroxyacetophenone monohydrate (2',4',6'-trihydroxyacetophenone monohydrate: THAP) (hereinafter referred to as 3-HPA+THAP). When the nucleic acid to be analyzed is DNA, a mixed matrix containing 3-hydroxypicolinic acid (3-HPA) and 2,4-dihydroxyacetophenone (2,4-DHAP) is preferred, and when the nucleic acid to be analyzed is RNA, a mixed matrix containing 3-hydroxypicolinic acid (3-HPA) and 2,4,6-trihydroxyacetophenone monohydrate (THAP) is preferred. The reason why the optimal mixed matrix differs depending on whether the target of analysis is DNA or RNA is thought to be because DNA and RNA are composed of different types of sugars, resulting in different affinities with the matrix.

[0020] The mixing ratio of the mixed matrix is ​​not particularly limited, but if the mixed matrix contains 3-HPA and 2,4-DHAP, the [M+H] ratio of nucleic acids + or [MH] - From the viewpoint of enabling highly sensitive detection of fragment ions generated by the dissociation of the ions, a 3-HPA:2,4-DHAP ratio of 10:1 to 1:5 is preferred, 7:1 to 1:5 is more preferred, and 5:1 to 1:1 is particularly preferred. If the mixed matrix contains 3-HPA and THAP, from a similar viewpoint, a 3-HPA:THAP ratio of 1:1 to 1:5 is preferred, and 1:1 to 1:3 is more preferred.

[0021] The mixed matrix may further contain ammonium citrate dibasic (ACD) as a matrix additive. While several types of ammonium salts of citrate exist depending on the number of ammonium ions bound to the citrate ion, the salt preferably used in this embodiment is one in which two ammonium ions are bound to one citrate ion. The concentration of the matrix additive in the mixed matrix is ​​determined by the [M+H] ratio of the nucleic acid. + or [MH] - From the viewpoint of being able to detect with high sensitivity the fragment ions generated when the ions dissociate, a concentration of 10 to 100 mM is preferred, 30 to 90 mM is more preferred, and 40 to 85 mM is even more preferred. In this specification, the numerical range from the lower limit to the upper limit is indicated using the symbol "~" as "(lower limit)~(upper limit)", but the numerical range indicated in this way includes the lower limit and the upper limit themselves.

[0022] The method for preparing the analytical sample involves mixing a nucleic acid-containing sample with a mixed matrix, and then drying the resulting mixed solution on a sample plate of a mass spectrometer. The mixed solution may be prepared in advance and dropped onto the sample plate for drying, or it may be prepared on the sample plate and dried thereafter.

[0023] <Mass spectrometer> The mass spectrometer used in this embodiment is a mass spectrometer (MALDI-MS) having an ion source based on the MALDI method. Examples of MALDI-MS include time-of-flight MALDI-TOFMS and ion-trap MALDI-ITMS. The ion-trap MALDI-MS referred to here has an ion trap for capturing ions. Specifically, it includes a mass spectrometer that utilizes the mass separation function of the ion trap itself to discharge ions trapped in the ion trap in order of increasing mass-to-charge ratio (m / z), and detects the ions with a detector placed outside the ion trap. It also includes a mass spectrometer that separates the ions discharged simultaneously from the ion trap according to their mass-to-charge ratio in a mass separation unit placed outside the ion trap, such as a time-of-flight mass separation unit, and detects the ions with a detector also placed outside the ion trap.

[0024] <Structural analysis of nucleic acids> In the analytical method of this embodiment, the [M+H] of nucleic acids is determined by mass spectrometry of a sample containing nucleic acids using the mixed matrix described above. + or [MH] - The method may include a data acquisition step of acquiring mass spectral data of multiple fragment ions generated by dissociation, and a data analysis step of extracting peaks of multiple fragment ions derived from nucleic acids from the mass spectral data acquired in the data acquisition step, and determining the structure of the nucleic acid based on the mass information of the peaks. This allows for the structural analysis of nucleic acids. Below, a method for performing structural analysis will be described assuming that the mass spectrometer is an ion trap type MALDI-ITMS.

[0025] (Ion trap mass spectrometer) An ion trap type mass spectrometer comprises an ion source 1 that ionizes a sample containing the analyte, an ion trap 2 (ion trapping unit in this invention) that temporarily captures ions with a predetermined mass-to-charge ratio from the ions generated by the ion source 1 using a high-frequency electric field and separates the captured ions according to their mass-to-charge ratio (m / z), and a detection unit 3 that detects the separated ions.

[0026] Ion source 1 is an ion source utilizing the MALDI method and includes a laser irradiation unit 11 for irradiating the sample with laser light and a sample stage 12 for placing a sample plate S on which the sample is placed. Ion trap 2 is a quadrupole type ion trap including an annular ring electrode 21 and a pair of end cap electrodes 22 and 23 arranged opposite each other on either side of the ring electrode 21. An ion inlet hole 22a is formed in the inlet end cap electrode 22, and an ion outlet hole 23a is formed in the outlet end cap electrode 23. Detector 3 includes a conversion dynode 31 that converts ions into electrons and a detector (secondary electron multiplier tube) 32 that multiplies and detects electrons arriving from the conversion dynode 31.

[0027] A predetermined voltage is applied to the ring electrode 21 and the end cap electrodes 22 and 23 of the ion trap 2. The high-frequency electric field formed by this voltage traps ions in the internal space surrounded by the ring electrode 21 and the end cap electrodes 22 and 23, and also discharges ions from the internal space through the ion outlet 23a.

[0028] By changing the time (hereinafter referred to as the delay time) between irradiating the sample with laser light from ion source 1 and applying the capture voltage to the ring electrode 21 of ion trap 2 to capture ions, the range of mass-to-charge ratios of ions preferentially captured by the ion trap can be controlled. Increasing the delay time allows for more reliable capture of higher-mass ions, while shortening the delay time allows for more reliable capture of lower-mass ions compared to other ions.

[0029] The predetermined voltage applied to the ring electrode 21 and the end cap electrodes 22 and 23 may be a sinusoidal high-frequency voltage, or a rectangular wave voltage generated by rapidly switching between two different voltages. In a digital ion trap that utilizes an electric field generated by a rectangular wave voltage, the range of mass-to-charge ratios of trappable ions is controlled by changing the frequency while keeping the amplitude (voltage value) of the rectangular wave voltage constant, or by changing the duty cycle, which is the ratio of the switching intervals of the rectangular wave voltage.

[0030] Next, we will explain the procedure for performing nucleic acid structure analysis, referring to the flowchart in Figure 2.

[0031] (Structural analysis method) This section describes an example using MALDI-ITMS, which has a digital ion trap, as the mass spectrometer.

[0032] [Step 1: Obtain standard analytical conditions for MS measurement] First, the protonation molecule of nucleic acid [M+H] + or deprotonated molecule [MH] - The standard analytical conditions for performing a measurement to detect (hereinafter referred to as MS measurement) are obtained. The MS measurement corresponds to the first measurement in the present invention. The MS measurement is [M+H] + or [MH] - In order to use measurement conditions that allow for the detection of ions with the highest possible sensitivity and resolution, the measurement does not involve ion dissociation (or if ion dissociation occurs, it is very slight). The mass spectrometer has various setting items that can be appropriately set according to the type of analyte and the purpose of the analysis. In the structural analysis method of this embodiment, the ion amount setting item related to the amount of ions generated in the ion source 1, the mass-to-charge ratio range setting item related to the mass-to-charge ratio of ions captured in the ion trap 2, and the signal intensity setting item related to the signal intensity of ions in the detection unit 3 are used in the MS measurement of the analyte [M+H] + or [MH] - Obtain standard analytical conditions for detection.

[0033] Standard analytical conditions refer to the [M+H] of the nucleic acid being analyzed. + or [MH] - These are representative conditions under which detection is possible. A standard method for obtaining analytical conditions is, for example, to perform MS measurement using the default values ​​of various pre-set settings in a mass spectrometer, and then determine the [M+H] of the nucleic acid, which is the analyte. + or [MH] - If [M+H] is detected, its default value may be used as the standard analysis condition. In that case, reading the default value from a memory unit where the default value is pre-stored is equivalent to obtaining the standard analysis condition. Alternatively, for higher sensitivity and higher resolution nucleic acid [M+H] + or [MH] - You may perform MS measurements by changing the settings of various items relative to their default values ​​so that the ion can be detected, and then determine the value at which the ion is detected (threshold value, etc.).

[0034] Ion quantity setting items include the laser intensity (laser power) irradiated by the laser irradiation unit 11, mass-to-charge ratio range setting items include the RFdelay value corresponding to the delay time, and signal intensity setting items include the voltage applied to the conversion dynode 31 and the voltage applied to the detector (secondary electron multiplier tube) 32.

[0035] [Step 2: Setting up modified analysis conditions by changing the standard analysis conditions] Next, modified analysis conditions are set by changing at least one of the standard analysis conditions obtained in Step 1: the ion quantity setting item, the mass-to-charge ratio range setting item, and the signal intensity setting item. In other words, it is also possible to change only one of the ion quantity setting item, the mass-to-charge ratio range setting item, and the remaining setting items are left unchanged. In this case, compared to when MS measurement is performed under standard analysis conditions, the ion quantity setting item is changed so that the amount of ions generated by ion source 1 increases, the mass-to-charge ratio range setting item is changed so that ions on the lower mass side are preferentially captured in ion trap 2, and the signal intensity setting item is changed so that the signal intensity of ions in detection unit 3 increases.

[0036] Specifically, when changing the laser light intensity, which is an ion quantity setting item, it is preferable to set it higher than the standard analysis conditions, for example, it is more preferable to set it 1 to 40% higher than the value of the standard analysis conditions, and even more preferable to set it 1 to 30% higher. When changing the RFdelay value, which is a mass-to-charge ratio range setting item, it is preferable to set it lower than the standard analysis conditions, for example, it is more preferable to set it 5 to 30% lower than the value of the standard analysis conditions, and even more preferable to set it 10 to 20% lower (so that the delay time is 1 to 3 μs). When changing the voltage applied to the conversion dynode 31, which is a signal intensity setting item, it is preferable to set it higher than the standard analysis conditions, for example, it is preferable to set it 5 to 30% higher, and even more preferable to set it 10 to 30% higher. Similarly, when changing the voltage applied to the detector (secondary electron multiplier tube) 32, which is a signal intensity setting item, it is preferable to set it higher than the standard analysis conditions, for example, it is preferable to set it 5 to 50% higher, and even more preferable to set it 10 to 50% higher.

[0037] [Step 3: Acquisition of mass spectral data by ISD measurement using modified analysis conditions] Using the modified analytical conditions described above, measurements are performed to detect nucleic acid-derived fragment ions (decomposition products). In this embodiment, the mechanism of generation of fragment ions derived from the analyte (nucleic acid) has not yet been proven. Therefore, in this specification, the dissociation that occurs simultaneously with or immediately after ionization in the ion source of a MALDI ion trap mass spectrometer, and the dissociation of ions that occurs in the instrument thereafter, are referred to as in-source decomposition (ISD), and the measurement for detecting nucleic acid-derived fragment ions dissociated by ISD is referred to as ISD measurement. ISD measurement corresponds to the second measurement in the present invention. By performing ISD measurement using the modified analytical conditions described above, the [M+H] of nucleic acids can be detected. + or [MH] - Mass spectral data of multiple fragment ions produced by the dissociation of a precursor ion can be obtained, especially [M+H]. + or [MH] - Fragment ions in the low-mass region, far from the m / z frequency, can be detected with high sensitivity.

[0038] In step 3, the maximum value of a predetermined mass-to-charge ratio of ions captured by ion trap 2 is determined to be the [M+H] of the nucleic acid, which is the analyte. + or [MH] - ISD measurement may be performed using conditions in which the predetermined mass-charge ratio range is set so that it is smaller than the value of the mass-charge ratio. In this case, the maximum value of the predetermined mass-charge ratio of ions captured by the ion trap 2 is the [M+H] of the analyte. + or [MH] - It is preferable to set the value to be about 0.5 to 40% smaller than the mass-to-charge ratio, and more preferably about 0.5 to 20% smaller.

[0039] One method for changing the predetermined mass-to-charge ratio range is to switch the measurement mode, which is pre-installed in the instrument, from a measurement mode with a target mass range of m / z 2000~18000 to a measurement mode with a target mass range of m / z 650~5000 for an analyte with a molecular weight of approximately 6000, thereby switching to a measurement mode that does not include molecular weights of 6000. The target mass range of the measurement mode is determined according to the frequency of the high-frequency voltage applied to ion trap 2. Specifically, since the amount of ions captured by the ion trap is limited, the mass range to be measured is determined mainly by adjusting the frequency of the high-frequency voltage and setting the Low Mass Cut-Off (LMCO). When the frequency of the high-frequency voltage is increased, the LMCO is set to be small, and the mass range to be measured is set to the low-mass side. On the other hand, when the frequency of the high-frequency voltage is decreased, the LMCO is set to be large, and the mass range to be measured is set to the high-mass side. In other words, the predetermined mass-to-charge ratio range is changed by changing the frequency of the high-frequency voltage applied to ion trap 2. Another method for changing the predetermined mass-to-charge ratio range is to change the value of the duty cycle setting, which is the ratio of the switching intervals of the rectangular wave voltage pre-installed in the instrument, from, for example, the standard value of 50:50 to 52:48 for analytes with a molecular weight of approximately 6000. This method excludes ions with a m / z of 5500 or higher. As a result, mass spectral data is obtained in which fragment ions with a mass-to-charge ratio close to that of the precursor ion are excluded. Consequently, the detection sensitivity of fragment ions in the low-mass range, which are far from the precursor ion's m / z, is improved.

[0040] In step 3, ISD measurements may be performed both with and without the specified mass-to-charge ratio range, as described above, to obtain mass spectral data from each measurement.

[0041] Furthermore, the maximum value of the predetermined mass-to-charge ratio of ions captured by ion trap 2 is the [M+H] of the nucleic acid, which is the analyte. + or [MH]- In addition to using conditions in which the predetermined mass-to-charge ratio range is set to be smaller than the value of the mass-to-charge ratio, ISD measurement may also be performed using conditions in which the voltage applied to the sample stage 12 of the ion source 1 is changed to a larger value than the standard analytical conditions obtained in step 2. It is more preferable to set the voltage applied to the sample stage to be 4 to 8 times higher than the standard analytical conditions, and even more preferable to set it to be 4 to 5 times higher.

[0042] [Step 4: Analysis of Mass Spectral Data] From the mass spectral data obtained in step 3, peaks corresponding to various fragment ions are extracted, and the assignment of each fragment ion is determined based on the mass information indicated by these peaks. Combining these results, at least a portion of the original nucleic acid structure is determined. The determination of the structure includes sequence analysis and identifying the type of chemical modification or the site where the chemical modification is performed through sequence analysis. Database searches or de novo sequencing may be used for structure determination.

[0043] In Step 3, if mass spectral data is obtained both with and without a predetermined mass-to-charge ratio range, the assignment results of various fragment ions obtained from both sets of data may be combined for analysis. This allows for more reliable structural analysis of the nucleic acid, which is the analyte.

[0044] The analytical method according to the present invention will be described below with reference to examples, but these are merely illustrative examples and the present invention is not limited thereto. [Examples]

[0045] (Reference example 1) First, a matrix was selected to be used as a comparative example for the present invention.

[0046] <1. Preparation of sample solution> As a sample solution, a 10 pmol / μL aqueous solution of standard nucleic acid A (5'-TGTGCGTGTGTAGTGTGTCT-3': Sequence ID No. 1, 20 base pairs, MW 6201.1, synthesized by a requester) was prepared.

[0047] <2. Preparation of the matrix solution> As matrix solutions, 40 mg / mL 50% acetonitrile (ACN) aqueous solutions of 3-hydroxypicolinic acid (3-HPA), 2,4-dihydroxyacetophenone (2,4-DHAP), 2,4,6-trihydroxyacetophenone monohydrate (THAP), and 1,5-diaminonaphthalene (1,5-DAN) were prepared, each containing 70 mM diammonium hydrogen citrate (ACD) as a matrix additive. As a mixed matrix solution, a 0.02 mmol / 50 μL 50% ACN aqueous solution of anthranilic acid (AA) containing 70 mM ACD and a 0.01 mmol / 50 μL 50% ACN aqueous solution of nicotinic acid (NA) containing 70 mM ACD were prepared, and these were mixed in a 1:1 (v / v) (2:1 (mol / mol)) ratio to prepare a mixed matrix (AA+NA) solution. A 50 mg / mL 50% ACN aqueous solution of anthranilic acid (AA) and 3-hydroxypicolinic acid (3-HPA) containing 70 mM ACD was prepared, and these were mixed in a 1:1 (v / v) ratio to prepare a mixed matrix (AA+3-HPA) solution.

[0048] <3. Preparation of Samples for Analysis> The sample solution prepared in step 1 and the matrix solution or mixed matrix solution prepared in step 2 were mixed in a 1:1 (v / v) ratio. 1 μL of the resulting mixture was dropped onto a sample plate (SUS plate) and dried.

[0049] <4.Mass spectrometry> For mass spectrometry, a MALDI digital ion trap mass spectrometer (MALDI-DITMS, manufactured by Shimadzu Corporation, product name: MALDImini-1) was used. The sample plate containing the analytical sample prepared in step 3 was inserted into the MALDI-DITMS, and MS measurement was performed using the raster function in positive mode.

[0050] <Result> Figure 3 shows the mass spectra of standard nucleic acid A when (a) 3-HPA, (b) 2,4-DHAP, (c) THAP, and (d) 1,5-DAN are used as matrices. The arrows in the figure indicate [M+H] + This shows the detection status. [M+H] + If no peak is detected, it will be indicated as ND (not detected).

[0051] From Figure 3, when (a) 3-HPA, (b) 2,4-DHAP, and (c) THAP are used as the matrix, [M+H] + A peak was detected, but (d) when using 1,5-DAN, [M+H] + No peak was detected. 1,5-DAN has been reported as a matrix used to analyze relatively short nucleic acids of about 10 nucleotides (10mers), and it is thought that standard nucleic acid A, which is 18 nucleotides (18mers), could not be ionized by 1,5-DAN.

[0052] Figure 4 shows the mass spectra of standard nucleic acid A when (a) AA+NA and (b) AA+3-HPA are used as the mixed matrix. A magnified view of the mass spectrum is also shown for (a). From Figure 4, when (a) AA+NA is used, [M+H] + A peak was detected, along with many base-eliminating ions and alkali metal ion adducts. Also, [M+H] + The detection sensitivity was lower when using (a) 3-HPA, (b) 2,4-DHAP, and (c) THAP in Figure 3. (b) When using AA+3-HPA, the detection sensitivity of standard nucleic acid A [M+H] +No peak was detected. (a) AA+NA is a mixed matrix reported as a highly sensitive analysis matrix for high-mass nucleic acids, but at least under the conditions of this study, its sensitivity was lower than that of 3-HPA, 2,4-DHAP, and THAP. Also, (b) AA+3-HPA is reported as a matrix for ISD of nucleic acids, but at least under the conditions of this study, ionization of nucleic acid molecules itself was difficult.

[0053] Based on these results, 3-HPA, 2,4-DHAP, and THAP were selected as the comparison groups for the matrix.

[0054] (Reference example 2) Next, nucleic acid ISD measurements were performed using 2,4-DHAP as the matrix.

[0055] <1. Preparation of sample solution> As a sample solution, a 10 pmol / μL aqueous solution of standard nucleic acid A was prepared.

[0056] <2. Preparation of the matrix solution> As a matrix solution, a 40 mg / mL 50% ACN aqueous solution of 2,4-dihydroxyacetophenone (2,4-DHAP) was prepared, containing 70 mM ACD as a matrix additive.

[0057] <3. Preparation of Samples for Analysis> The sample solution prepared in step 1 and the matrix solution prepared in step 2 were mixed in a 1:1 (v / v) ratio. 1 μL of the resulting mixture was dropped onto a sample plate (SUS plate) and dried.

[0058] <4.Mass spectrometry> For mass spectrometry, a MALDI digital ion trap mass spectrometer (MALDI-DITMS, manufactured by Shimadzu Corporation, product name: MALDImini-1) was used. The sample plate containing the analytical sample prepared in step 3 was inserted into the MALDI-DITMS, and ISD measurement was performed using the raster function in positive mode. The analytical conditions for ISD measurement are as follows. The measurement mode indicates the range of mass-to-charge ratio of the ions to be measured, specifically defining the range of mass-to-charge ratio of the ions trapped in the ion trap, and this range is shown in parentheses. • Measurement mode: mode3 (m / z 2000~18000) Detector voltage (DV-1): 2000 Conversion voltage (DV-2): 8000 • RF delay value (RF): 17 • Laser power (LP): 75 • Sample stage voltage (SV): 5 Duty cycle: 50:50

[0059] <5. Data Analysis> The mass spectral data (ISD spectra) of fragment ions obtained by the ISD measurement in step 4 were analyzed, and the assignment of the main peaks was determined.

[0060] <Result> Figure 5 shows the ISD spectrum of standard nucleic acid A obtained by ISD measurement using 2,4-DHAP as the matrix. In Figure 5, each peak in the mass spectrum is labeled with the name of the corresponding oligonucleotide fragment ion species (a general name proposed in Non-Patent Literature 7). This name represents each ion species as a fragment ion series according to the nucleic acid dissociation pattern naming convention. In this naming convention, a fragment ion containing the 5' end is a n , b n , c n d n It is written as, and the fragment ion containing the 3' end in the opposite direction is x m , y m , z m , wm This is how it is written. The subscripts n and m indicate the number of constituent units (by definition, the number of bases) from the corresponding end to the dissociation site. In addition, B in the identifier represents a base of nucleic acid; for example, b13-B(A) in the same figure indicates an ion obtained by removing the base adenine (A) from the b13 ion. The same applies to the following figures.

[0061] When 2,4-DHAP was used as the matrix, the resulting ISD spectrum showed many peaks where bases had been eliminated, and was complex due to the coexistence of multiple fragment ion species. MALDI-ITMS, like the one used in this measurement, is known to be more prone to fragmentation than conventional MALDI-TOFMS due to its instrument structure characteristics. Since it is not easy to analyze the base sequence from such a mass spectrum, a simpler ISD spectrum with suppressed base elimination and a certain degree of ion species uniformity is desired to expedite the analysis.

[0062] The following describes the MS measurement of mipomersen, known as one of the nucleic acid pharmaceuticals, using the analytical method according to the present invention.

[0063] (Example 1) <1. Preparation of sample solution> As a sample solution, a 20 pmol / μL aqueous solution of mipomersen (single-stranded DNA, 5'-MG-MC-MC-MU-MC-dA-dG-dT-dC-dT-dG-dC-dT-dT-dC-MG-MC-MA-MC-MC-3' (where M represents 2'-O-(2-methoxyethyl)nucleoside and d represents 2'-deoxynucleoside. The carbon at position 5 of cytosine and uracil is substituted with a methyl group, and all phosphodiester bonds between nucleotides are substituted with phosphorothioate bonds): SEQ ID NO: 2, 20 nucleotides long, MW 7177, sample after desalting and purification of synthetic nucleic acids for research and development) was prepared.

[0064] <2. Preparation of the matrix solution> As a matrix solution, a 40 mM concentration of ACD was used to prepare a 50% ACN aqueous solution (3-HPA solution) containing 3-hydroxypicolinic acid (3-HPA) at a concentration of 40 mg / mL, along with a matrix additive. A 40 mg / mL 50% ACN aqueous solution (THAP solution) of 2,4,6-trihydroxyacetophenone monohydrate (THAP) was prepared, containing 40 mM ACD as a matrix additive. A 40 mg / mL 50% ACN aqueous solution (2,4-DHAP solution) containing 70 mM ACD was prepared. As a mixed matrix solution, a mixed matrix (3-HPA + 2,4-DHAP) solution was prepared by mixing the above 3-HPA solution and 2,4-DHAP solution in a 1:1 (v / v) ratio. Similarly, a mixed matrix (3-HPA + THAP) solution was prepared by mixing the above 3-HPA solution and THAP solution in a 1:1 (v / v) ratio.

[0065] <3. Preparation of Samples for Analysis> The sample solution prepared in step 1, the matrix solution prepared in step 2, and the mixed matrix solution were mixed in a 1:1 (v / v) ratio. 1 μL of the resulting mixture was dropped onto a sample plate (SUS plate) and dried.

[0066] <4.Mass spectrometry> For mass spectrometry, a MALDI digital ion trap mass spectrometer (MALDI-DITMS, manufactured by Shimadzu Corporation, product name: MALDImini-1) was used. The sample plate containing the analytical sample prepared in step 3 was inserted into the MALDI-DITMS, and MS measurement was performed using the raster function in positive mode.

[0067] <Result> Figure 6 shows the mass spectra of mipomersen when 3-HPA, 2,4-DHAP, and THAP are used as matrices, and 3-HPA+2,4-DHAP and 3-HPA+THAP are used as mixed matrices (from left to right: 3-HPA, 2,4-DHAP, THAP, 3-HPA+2,4-DHAP, 3-HPA+THAP). The top panel (a) is an overall view showing the region m / z 2000-15000, the middle panel (b) is a magnified view showing the region m / z 6000-8000, and the bottom panel (c) is a magnified view showing the region m / z 7100-7300. The arrows in the figures indicate [M+H] + The detection status is shown. Furthermore, below the mass spectrum, the [M+H] values ​​for each matrix are shown. + A table summarizing the details of the detection status (sensitivity (mV, S / N), resolution (R), base elimination status, adduct detection status) and the laser power used during measurement is shown. The base elimination status was determined from the mass spectrum in (b), and the adduct detection status was determined from the mass spectrum in (c).

[0068] As shown in Figure 6, when using the mixed matrix 3-HPA+2,4-DHAP, the most sensitive and sufficiently resolving conditions were observed, with the elimination of bases and the formation of alkali metal adducts suppressed, resulting in [M+H] + This was detected. This confirmed that the mixed matrix 3-HPA+2,4-DHAP is particularly effective for MS measurement of nucleic acids (DNA) with a length of 20 nucleotides or more.

[0069] (Example 2) Mass spectrometry was performed in the same manner as in Example 1, except for the method of preparing the matrix solution, and the mixing ratio of the mixed matrix 3-HPA + 2,4-DHAP was investigated. <2. Preparation of the matrix solution> A 40 mg / mL 50% ACN aqueous solution (3-HPA solution) containing 70 mM ACD as a matrix additive was prepared. A 40 mg / mL 50% ACN aqueous solution (2,4-DHAP solution) containing 70 mM ACD as a matrix additive was prepared. The prepared 3-HPA solution and 2,4-DHAP solution were mixed at the desired mixing ratio (v / v) to create a mixed matrix 3-HPA + 2,4-DHAP solution (mixing ratios (1:0), (10:1), (7:1), (5:1), (3:1), (2:1), (1:1), (1:2), (1:3), (1:5), (1:7), (1:10), (0:1)). In this case, the mixing ratio (1:0) indicates the case where 3-HPA is used alone, and the mixing ratio (0:1) indicates the case where 2,4-DHAP is used alone.

[0070] <Result> Figure 7 shows the mass spectra of mipomersene when using 3-HPA+2,4-DHAP as the mixed matrix (from left to right, in the following mixing ratios: (1:0), (10:1), (7:1), (5:1), (3:1), (2:1), (1:1), (1:2), (1:3), (1:5), (1:7), (1:10), (0:1), and the [M+H] spectra for each mixing matrix. + A table summarizing the details of the detection status (sensitivity (mV, S / N), resolution (R), base elimination status, adduct detection status, and well-to-well variability) is shown.

[0071] When comparing the use of 3-HPA + 2,4-DHAP (mixing ratio (1:0), (0:1)), i.e., 3-HPA or 2,4-DHAP alone, with the use of a mixed matrix 3-HPA + 2,4-DHAP prepared with mixing ratios (10:1) to (1:5), [M+H] + The detection sensitivity was improved. Furthermore, when using a mixed matrix 3-HPA+2,4-DHAP prepared with a mixing ratio of (7:1) to (1:5), in addition to improved detection sensitivity, base elimination was suppressed. Considering reproducibility, such as variations between wells, when using a mixed matrix 3-HPA+2,4-DHAP prepared with a mixing ratio of (5:1) to (1:1), high sensitivity, high resolution, and suppression of base elimination and alkali metal ion adduct formation were achieved [M+H]. + It was confirmed that it can be detected with high reproducibility.

[0072] (Examples 3-1 and 3-2) Mass spectrometry was performed in the same manner as in Example 1, except for a different method of preparing the matrix solution, to investigate the effects of 2,4-DHAP isomers. (Example 3-1) <2. Preparation of the matrix solution> A 40 mg / mL 50% ACN aqueous solution (3-HPA solution) of 3-hydroxypicolinic acid (3-HPA) was prepared, containing 40 mM ACD as a matrix additive. A 40 mg / mL 50% ACN aqueous solution (2,4-DHAP solution) of 2,4-dihydroxyacetophenone (2,4-DHAP) was prepared, containing 70 mM ACD as a matrix additive. A 40 mg / mL 50% ACN aqueous solution (2,5-DHAP solution) containing 70 mM ACD as a matrix additive was prepared, along with 2,5-DHAP, a positional isomer of 2,4-DHAP. The prepared 3-HPA solution and 2,4-DHAP solution, and the 3-HPA solution and 2,5-DHAP solution were mixed in a 1:1 (v / v) ratio to create mixed matrix solutions (3-HPA + 2,4-DHAP (1:1), 3-HPA + 2,5-DHAP (1:1)).

[0073] <Result> Figure 8 shows the mass spectra of mipomercene using mixed matrices 3-HPA+2,4-DHAP(1:1) and 3-HPA+2,5-DHAP(1:1). The arrows in the figure indicate [M+H] + This shows the detection status. The numbers in the figure represent [M+H]. + The peak intensity (mV) is shown. Compared to using 3-HPA+2,4-DHAP(1:1), the peak intensity when using 3-HPA+2,5-DHAP(1:1) is [M+H] + The sensitivity was low.

[0074] (Example 3-2) <2. Preparation of the matrix solution> A 40 mg / mL 50% ACN aqueous solution (3-HPA solution) containing 70 mM ACD as a matrix additive was prepared. A 40 mg / mL 50% ACN aqueous solution (2,4-DHAP solution) of 2,4-dihydroxyacetophenone (2,4-DHAP) was prepared, containing 70 mM ACD as a matrix additive. A 40 mg / mL 50% ACN aqueous solution (2,6-DHAP solution) of 2,6-DHAP, a positional isomer of 2,4-DHAP, was prepared, containing 70 mM ACD as a matrix additive. The prepared 3-HPA solution and 2,4-DHAP solution, and the 3-HPA solution and 2,6-DHAP solution were mixed in a 1:1 (v / v) ratio to create mixed matrix solutions (3-HPA + 2,4-DHAP (1:1), 3-HPA + 2,6-DHAP (1:1)).

[0075] <Result> Figure 9 shows the mass spectra of mipomercene when using the mixed matrices 3-HPA+2,4-DHAP(1:1) and 3-HPA+2,6-DHAP(1:1). When using 3-HPA+2,4-DHAP(1:1), the [M+H] of mipomercene is observed. + Although it was detected with good sensitivity, ionization of nucleic acid molecules itself was difficult when using 3-HPA + 2,6-DHAP (1:1).

[0076] (Example 4) Mass spectrometry was performed in the same manner as in Example 1, except for a different method of preparing the matrix solution, to investigate the concentration of diammonium hydrogen citrate (ACD), a matrix additive.

[0077] <2. Preparation of the matrix solution> 40 mg / mL 50% ACN aqueous solutions (3-HPA solutions) containing 3-hydroxypicolinic acid (3-HPA) as a matrix additive were prepared, each containing ACD at concentrations of 40, 70, and 100 mM. 40 mg / mL 50% ACN aqueous solutions (2,4-DHAP solutions) of 2,4-dihydroxyacetophenone (2,4-DHAP) were prepared, each containing ACD at concentrations of 40, 70, and 100 mM as a matrix additive. Mixed matrix solutions (3-HPA + 2,4-DHAP (40, 55, 70, 85, 100 mM ACD)) were prepared by mixing 3-HPA (40 mM ACD) solution and 2,4-DHAP (40 mM ACD) solution in a 1:1 (v / v) ratio. Specifically, a mixed matrix solution (3-HPA + 2,4-DHAP) containing 40 mM ACD was prepared by mixing a 3-HPA (40 mM ACD) solution and a 2,4-DHAP (40 mM ACD) solution in a 1:1 (v / v) ratio. Similarly, 3-HPA (40mM ACD) solution and 2,4-DHAP (70mM ACD) solution, 3-HPA (70mM ACD) solution and 2,4-DHAP (70mM ACD) solution, 3-HPA (70mM ACD) solution and 2,4-DHAP (100mM ACD) solution, and 3-HPA (100mM ACD) solution and 2,4-DHAP (100mM ACD) solution were mixed in a 1:1 (v / v) ratio to prepare mixed matrix (3-HPA + 2,4-DHAP) solutions containing 55, 70, 85, and 100mM ACD, respectively.

[0078] <Result> Figure 10 shows the mass spectra of mipomercene using the mixed matrix 3-HPA+2,4-DHAP at each ACD concentration (40, 55, 70, 85, 100 mM), and the [M+H] spectrum for each mixed matrix. + A table summarizing the detection status (sensitivity (mV, S / N), resolution (R), adduct detection status, and base elimination status) is shown.

[0079] Regardless of the ACD concentration of the mixed matrix used, [M+H] was obtained with good sensitivity, high resolution, and with suppressed base elimination and alkali metal ion adduct formation. + It was detected. At an ACD concentration of 100 mM, the detection sensitivity decreased slightly, but it was confirmed that a similarly good mass spectrum could be obtained at ACD concentrations of 40-85 mM.

[0080] (Example 5) Next, ISD measurement of mipomersen was performed using the analytical method according to the present invention.

[0081] <1. Preparation of sample solution> A 20 pmol / μL aqueous solution of mipomersen was prepared as the sample solution.

[0082] <2. Preparation of the matrix solution> A 40 mg / mL 50% ACN aqueous solution (3-HPA solution) of 3-hydroxypicolinic acid (3-HPA) was prepared, containing 40 mM ACD as a matrix additive. A 40 mg / mL 50% ACN aqueous solution (2,4-DHAP solution) of 2,4-dihydroxyacetophenone (2,4-DHAP) was prepared, containing 70 mM ACD as a matrix additive. The prepared 3-HPA solution and 2,4-DHAP solution were mixed in a 1:1 (v / v) ratio to create a mixed matrix solution (3-HPA + 2,4-DHAP (1:1)).

[0083] <3. Preparation of Samples for Analysis> The sample solution prepared in step 1 and the mixed matrix solution prepared in step 2 were mixed in a 1:1 (v / v) ratio. 1 μL of the resulting mixture was dropped onto a sample plate (SUS plate) and dried.

[0084] <4.Mass spectrometry> For mass spectrometry, a MALDI digital ion trap mass spectrometer (MALDI-DITMS, manufactured by Shimadzu Corporation, product name: MALDImini-1) was used. The sample plate containing the analytical sample prepared in step 3 was inserted into the MALDI-DITMS, and ISD measurement was performed in positive mode using the raster function. The analytical conditions for ISD measurement were as follows. The measurement mode indicates the range of mass-to-charge ratio of the ions to be measured, specifically defining the range of mass-to-charge ratio of the ions trapped in the ion trap, and this range is shown in parentheses. The optimal laser power value was used for each setting. • Measurement modes: mode3 (m / z 2000~18000), mode2 (m / z 650~5000) Detector voltage (DV-1): 2000 Conversion voltage (DV-2): 8000 • RF delay value (RF): 17 • Sample stage voltage (SV): 5 Duty cycle: 50:50

[0085] <5. Data Analysis> The mass spectral data (ISD spectra) of fragment ions obtained by the ISD measurement in step 4 were analyzed, and the main peaks were assigned to those values.

[0086] <Result> Figure 11 shows the ISD spectrum and assignment of the main peaks of mypomercene obtained by ISD measurement under mode 3 mass-to-charge ratio range conditions using the mixed matrix 3-HPA+2,4-DHAP. Figure 12 shows the ISD spectrum and assignment of the main peaks of mypomercene obtained by ISD measurement under mode 2 mass-to-charge ratio range conditions using the mixed matrix 3-HPA+2,4-DHAP. In Figures 11 and 12, simple ISD spectra were obtained in which a large number of ISD fragment ions, mainly a / w ions, were preferentially detected. As a result, the assignment of ISD fragment ions was made easier, and analysis could be performed quickly. Furthermore, by combining the analysis results of Figures 11 and 12, the entire sequence of mypomercene could be analyzed more reliably.

[0087] Figure 13 shows the ISD spectra of mipomercene obtained by ISD measurements under mode 3 mass-to-charge ratio range conditions, using (a) a mixed matrix 3-HPA+2,4-DHAP and (b) 2,4-DHAP. The dashed lines in the figure indicate peaks that were detected in both matrix cases and assigned to Figure 11. The sensitivity of ISD fragment ions when using the mixed matrix 3-HPA+2,4-DHAP was higher than when using the conventional ISD matrix 2,4-DHAP, and a / w ions were observed more frequently and preferentially. This confirms that using the mixed matrix 3-HPA+2,4-DHAP yields a simple ISD spectrum that facilitates structural analysis.

[0088] These results were obtained because, as shown in Figure 6 of Example 1, when MS measurements were performed using the mixed matrix 3-HPA+2,4-DHAP, the elimination of bases and the formation of alkali metal ion adducts were suppressed, resulting in [M+H] + This is thought to be related to the detection of [M+H]. ISD measurement is [M+H] + or [MH] - This method involves generating a precursor ion, and then mass spectrometry analyzing the fragment ions produced when the precursor ion disintegrates. It provides sufficient sensitivity for analyzing nucleic acid [M+H] in MS measurements using a mixed matrix 3-HPA+2,4-DHAP. + Since [M+H] was detected, it was also found in ISD measurements using the same mixed matrix. + It is thought that a sufficient amount of precursor ions were generated. As a result, when the mixed matrix has properties that make it easy to generate ISD, as in this case, a sufficient amount of fragment ions are also generated from the precursor ions by ISD, which is thought to have led to the sensitive detection of these fragment ions. Furthermore, the properties that make it difficult to form adducts during ionization and that do not cause dissociation of unstable sites such as base elimination are thought to have led to the generation of simpler ISD fragment species.

[0089] (Example 6) Next, MS measurement was performed on patisiran, known as one of the nucleic acid pharmaceuticals, using the analytical method according to the present invention.

[0090] <1. Preparation of sample solution> As the sample solution, patissirane (originally a double-stranded RNA consisting of sense and antisense, but in this example a 1:1 (mol / mol) mixture of sense and antisense was used. Sense 5'-G-Um-AA-Cm-Cm-AAGAG-Um-A-Um-Um-Cm-Cm-A-Um-dT-dT-3' (dT represents thymidine deoxyribonucleotide, Cm represents 2'-O-methylcytidine, Um represents 2'-O-methyluridine): Sequence ID 3, 21 nucleotides long (core sequence 19 nucleotides long, with a 2-nucleotide DNA overhang (dTdT) at the 3' end)), MW 6764, antisense 5'-AUGGAA-Um-ACUCUUGGU-Um-AC-dT-dT-3' (dT represents thymidine deoxyribonucleotide A 20 pmol / μL aqueous solution was prepared of 2'-O-methyluridine (SEQ ID NO: 4, 21 nucleotides long (core sequence 19 nucleotides long, with a 2-nucleotide DNA overhang (dTdT) at the 3' end), MW 6660, a sample after desalting and purification of synthetic nucleic acid for research and development).

[0091] <2. Preparation of the matrix solution> A 40 mg / mL 50% ACN aqueous solution of 3-hydroxypicolinic acid (3-HPA) containing 40 mM ACD as a matrix additive (3-HPA-1 solution) was prepared. A 40 mg / mL 50% ACN aqueous solution of 3-HPA containing 70 mM ACD as a matrix additive (3-HPA-2 solution) was prepared. A 40 mg / mL 50% ACN aqueous solution (2,4-DHAP solution) containing 70 mM ACD as a matrix additive was prepared. A 40 mg / mL 50% ACN aqueous solution (THAP-1 solution) of 2,4,6-trihydroxyacetophenone monohydrate (THAP) was prepared, containing 40 mM ACD as a matrix additive. A 40 mg / mL 50% ACN aqueous solution of THAP (THAP-2 solution) was prepared, containing 70 mM ACD as a matrix additive.

[0092] The prepared 3-HPA-1 solution and 2,4-DHAP solution were mixed in ratios of (1:1), (3:1), and (1:3) (v / v) to create various mixed matrix (3-HPA-1 + 2,4-DHAP) solutions ((1:1 ratio, 55 mM ACD), (3:1 ratio, 48 mM ACD), (1:3 ratio, 63 mM ACD)). The prepared 3-HPA-2 solution and 2,4-DHAP solution were mixed in ratios of (1:1), (3:1), and (1:3) (v / v) to create various mixed matrix (3-HPA-2 + 2,4-DHAP) solutions ((mixing ratio 1:1, 70mM ACD), (mixing ratio 3:1, 70mM ACD), (mixing ratio 1:3, 70mM ACD)). The prepared 3-HPA-1 solution and THAP-1 solution were mixed in mixing ratios of (1:1), (3:1), and (1:3) (v / v) to create various mixed matrix (3-HPA-1 + THAP-1) solutions ((mixing ratio 1:1, 40mM ACD), (mixing ratio 3:1, 40mM ACD), (mixing ratio 1:3, 40mM ACD)). The prepared 3-HPA-2 solution and THAP-2 solution were mixed at mixing ratios of (1:1), (3:1), and (1:3) (v / v) to create various mixed matrix (3-HPA-2 + THAP-2) solutions ((mixing ratio 1:1, 70mM ACD), (mixing ratio 3:1, 70mM ACD), (mixing ratio 1:3, 70mM ACD)).

[0093] <3. Preparation of Samples for Analysis> The sample solution prepared in step 1 and the mixed matrix solution prepared in step 2 were mixed in a 1:1 (v / v) ratio. 1 μL of the resulting mixture was dropped onto a sample plate (SUS plate) and dried.

[0094] <4.Mass spectrometry> For mass spectrometry, a MALDI digital ion trap type mass spectrometer (MALDI-DITMS, manufactured by Shimadzu Corporation, trade name: MALDImini-1) was used. The sample plate on which the analysis sample prepared in 3. was placed was inserted into the MALDI-DITMS, and MS measurement was performed in positive mode using the raster function.

[0095] <Results> Figs. 14, 15, and 16 show the mass spectra of patisiran and its enlarged views when using various matrix solutions, and the [M+H] of two types of patisiran samples (sense and antisense). + + Table 1 summarizes the details of the detection status of [M+H] (detection status of two types of samples, sensitivity (mV, S / N), resolution (R), adduct detection status, base elimination status) for two types of patisiran samples (sense and antisense). For sensitivity and resolution, representative numerical values for the [M+H] peak of the sense of the two types of samples are shown. Note that Figs. 15 and 16 are data obtained on the same day, and Fig. 14 is data obtained on a different day from them. +

[0096] From Figs. 14 to 16, in the mixed matrix containing 3-HPA and THAP at a mixing ratio of 1:1 or 1:3, especially the one with a mixing ratio of 1:1, for both the sense and antisense of patisiran, [M+H] was detected with good sensitivity, high resolution, and suppression of base elimination and generation of alkali metal ion adducts (Figs. 16(d), (e), (h), (i)). From this, it was confirmed that the mixed matrix 3-HPA+THAP is particularly effective for MS measurement of relatively high-mass nucleic acids (RNA) with a length of 21 bases. As shown in Fig. 6 of Example 1, in the case of mypochelsen where the nucleic acid sample is DNA, the mixed matrix containing 3-HPA and 2,4-DHAP was most suitable for the detection of [M+H]. It is considered that the reason why the types of suitable mixed matrices are different due to the difference in the type of nucleic acid is that the affinity with the matrix is different due to the difference in the sugar constituting DNA and RNA. + + + Thus, it is considered that the reason why the types of suitable mixed matrices are different due to the difference in the type of nucleic acid is that the affinity with the matrix is different due to the difference in the sugar constituting DNA and RNA.

[0097] (Example 7) Next, mass spectrometry was performed in the same manner as in Example 6, except for the method of preparing the matrix solution, to investigate the concentrations of diammonium hydrogen citrate (ACD) and acetonitrile (ACN), which are matrix additives.

[0098] <2. Preparation of the matrix solution> 40 mg / mL 50% ACN aqueous solutions of 3-hydroxypicolinic acid (3-HPA) were prepared, each containing ACD at concentrations of 40, 70, and 100 mM as a matrix additive (3-HPA-1 solution, 3-HPA-2 solution, and 3-HPA-3 solution, in order of increasing ACD concentration). 40 mg / mL 50% ACN aqueous solutions of 2,4,6-trihydroxyacetophenone (THAP) were prepared, each containing ACD at concentrations of 40, 70, and 100 mM as a matrix additive (THAP-1 solution, THAP-2 solution, and THAP-3 solution, in order of increasing ACD concentration). 40 mg / mL 70% ACN aqueous solutions of 3-HPA were prepared, each containing ACD at concentrations of 40, 70, and 100 mM as a matrix additive (3-HPA-4 solution, 3-HPA-5 solution, and 3-HPA-6 solution, in order of increasing ACD concentration). THAP was prepared in 40 mg / mL 70% ACN aqueous solutions containing ACD at concentrations of 40, 70, and 100 mM as matrix additives (THAP-4 solution, THAP-5 solution, and THAP-6 solution, in order of increasing ACD concentration).

[0099] The prepared 3-HPA-1~6 solutions and THAP-1~6 solutions were mixed in a 1:1 (v / v) ratio to create the following mixed matrices: 3-HPA-1+THAP-1 solution (40mM ACD, 50% ACN / Water), 3-HPA-2+THAP-2 solution (70mM ACD, 50% ACN / Water), 3-HPA-3+THAP-3 solution (100mM ACD, 50% ACN / Water), 3-HPA-4+THAP-4 solution (40mM ACD, 70% ACN / Water), 3-HPA-5+THAP-5 solution (70mM ACD, 70% ACN / Water), and 3-HPA-6+THAP-6 solution (100mM ACD, 70% ACN / Water).

[0100] <Result> Figure 17 shows the mass spectra of patissirane using various matrices, their magnified views, and the [M+H] of two patissirane samples (sense and antisense). + A table summarizing the detection status of the two samples (detection status of the two samples, sensitivity (mV, S / N), resolution (R), adduct detection status, and base elimination status) is shown. For sensitivity and resolution, the [M+H] of the sense sample is used as a representative example. + The values ​​relative to the peak were shown.

[0101] As shown in Figure 17, when using a mixed matrix of 3-HPA and THAP (mixing ratio 1:1), regardless of whether the ACD concentration was 40-100 mM or the solvent acetonitrile concentration was 50-70%, both the sense and antisense forms of patissirane were sensitive, high-resolution, and the elimination of bases and the formation of alkali metal ion adducts were suppressed [M+H]. + It was confirmed that [M+H] could be detected. In particular, the best [M+H] was obtained when using the 3-HPA-2+THAP-2 solution (70mM ACD, 50% ACN / Water). + A peak was obtained (Figure 17(d)).

[0102] [Pattern] It will be obvious to those skilled in the art that the exemplary embodiments described above are specific examples of the following embodiments.

[0103] (Item 1) The nucleic acid analysis method according to one aspect of the present invention uses a mixed matrix containing 3-hydroxypicolinic acid and 2,4-dihydroxyacetophenone, or a mixed matrix containing 3-hydroxypicolinic acid and 2,4,6-trihydroxyacetophenone monohydrate, and analyzes the nucleic acid contained in a sample by a matrix-assisted laser desorption ionization mass spectrometer.

[0104]

[0105] Thereby, [M+H] + or [M-H] - of the nucleic acid, and the fragment ions generated by dissociation of the ions can be detected with high sensitivity. (Item 2) The nucleic acid analysis method according to Item 1 is when the mixed matrix is a mixed matrix containing 3-hydroxypicolinic acid and 2,4-dihydroxyacetophenone, the mixing ratio of 3-hydroxypicolinic acid and 2,4-dihydroxyacetophenone may be 10:1 to 1:5.

[0106] Thereby, [M+H] + or [M-H] - of the nucleic acid, particularly DNA, and the fragment ions generated by dissociation of the ions can be detected with higher sensitivity.

[0107] (Item 3) The nucleic acid analysis method according to Item 1 is when the mixed matrix is a mixed matrix containing 3-hydroxypicolinic acid and 2,4,6-trihydroxyacetophenone monohydrate, the mixing ratio of 3-hydroxypicolinic acid and 2,4,6-trihydroxyacetophenone monohydrate may be 1:1 to 1:3.

[0108] Thereby, [M+H] + or [M-H] -This allows for more sensitive detection of fragment ions generated by the dissociation of the ion.

[0109] (Section 4) The nucleic acid analysis method described in any of paragraphs 1 to 3 is: The aforementioned mixed matrix may further contain diammonium hydrogen citrate as a matrix additive.

[0110] This results in the [M+H] of nucleic acids + or [MH] - This allows for more sensitive detection of fragment ions generated by the dissociation of the ion.

[0111] (Section 5) The nucleic acid analysis method described in any of paragraphs 1 to 4 is: By mass spectrometry using the aforementioned mixed matrix to obtain the [M+H] of the nucleic acid + or [MH] - A data acquisition process to obtain mass spectral data of multiple fragment ions generated by dissociation, The system may also include a data analysis step of extracting peaks of multiple fragment ions derived from the nucleic acid from the mass spectral data and determining the structure of the nucleic acid based on the mass information of the peaks.

[0112] This allows for the structural analysis of nucleic acids.

[0113] (Section 6) The nucleic acid analysis method described in paragraph 5 is: The matrix-assisted laser desorption / ionization mass spectrometer is an ion trap type matrix-assisted laser desorption / ionization mass spectrometer comprising: an ion source for matrix-assisted laser desorption / ionization; an ion trapping unit for separating and trapping ions with a predetermined mass-to-charge ratio from the ions generated by the ion source; and a detection unit for detecting the ions trapped by the ion trapping unit, Using the matrix-assisted laser desorption / ionization mass spectrometer, the [M+H] of the nucleic acids contained in the sample was analyzed. + or [MH] - The process includes an analysis condition acquisition step to acquire standard analysis conditions for the ion amount setting item related to the amount of ions generated in the ion source, the mass-to-charge ratio range setting item related to the mass-to-charge ratio range of ions captured in the ion capture unit, and the signal intensity setting item related to the signal intensity of ions in the detection unit, when performing a first measurement to detect the ion. The data acquisition step is a modified analysis condition in which at least one of the standard analysis conditions acquired in the analysis condition acquisition step is changed, specifically the ion amount setting item, the mass-to-charge ratio range setting item, and the signal intensity setting item, and the nucleic acid [M+H] + or [MH] - The method may also involve performing a second measurement to detect multiple fragment ions generated by the dissociation of the material, thereby obtaining mass spectral data of the multiple fragment ions.

[0114] This allows for the sensitive detection of fragment ions necessary for structural analysis across a relatively wide mass range, from low to high mass, enabling more reliable structural analysis of nucleic acids.

[0115] (Section 7) The nucleic acid analysis method described in paragraph 6 is: The modified analysis conditions are those in which the analysis conditions for the ion amount setting item are changed so that the amount of ions generated by the ion source increases compared to when the first measurement of the sample was performed under the standard analysis conditions, or the [M+H] of the nucleic acid. + or [MH] - The analysis conditions for the mass-charge ratio range setting item may be modified so that ions with lower masses than the mass-charge ratio are preferentially captured, or the analysis conditions for the signal intensity setting item may be modified so that the signal intensity of the ions in the detection unit increases.

[0116] This allows for the sensitive detection of fragment ions with low mass-to-charge ratios, which are necessary for structural analysis, enabling more reliable structural analysis of nucleic acids.

[0117] (Section 8) The nucleic acid analysis method described in paragraph 6 or 7 is: The mass-to-charge ratio range setting item is the time from when the ion source is irradiated with a laser until when a capture voltage is applied to the ion trapping unit to trap ions. The aforementioned modified analysis condition may be one in which the time is changed to a value shorter than that of the standard analysis condition. This allows for the sensitive detection of fragment ions with low mass-to-charge ratios, which are necessary for structural analysis, enabling more reliable structural analysis of nucleic acids.

[0118] (Section 9) The nucleic acid analysis method described in paragraph 8 is: The modified analysis conditions may be those in which the time is set to a value 1 to 3 microseconds shorter than that of the standard analysis conditions. This allows for more sensitive detection of fragment ions with a low mass-to-charge ratio, which are necessary for structural analysis, and enables more reliable structural analysis of nucleic acids.

[0119] (Section 10) The nucleic acid analysis method described in any of paragraphs 1 to 9 is: The matrix-assisted laser desorption / ionization mass spectrometer may be of the digital ion trap type. This results in the [M+H] of nucleic acids + or [MH] - This allows for more sensitive detection of fragment ions generated by the dissociation of the ion.

[0120] (Section 11) A matrix for nucleic acid matrix-assisted laser desorption / ionization mass spectrometry according to one aspect of the present invention is: It contains 3-hydroxypicolinic acid and 2,4-dihydroxyacetophenone, or it contains 3-hydroxypicolinic acid and 2,4,6-trihydroxyacetophenone monohydrate. This results in the [M+H] of nucleic acids + or [MH] - Furthermore, the fragment ions generated by the dissociation of the ions can be detected with high sensitivity. [Explanation of Symbols]

[0121] 1…Ion source 2…Ion trap 3...Detection unit 11…Laser irradiation area 12…Sample Stage 21... Ring electrode 22, 23… End cap electrodes 31...Conversion Dynode 32…Detector (Secondary electron multiplier tube)

Claims

1. A method for analyzing nucleic acids, comprising using a matrix-assisted laser desorption / ionization mass spectrometer to analyze nucleic acids contained in a sample using a mixed matrix comprising 3-hydroxypicolinic acid and 2,4-dihydroxyacetophenone as matrix materials, or a mixed matrix comprising 3-hydroxypicolinic acid and 2,4,6-trihydroxyacetophenone monohydrate as matrix materials.

2. The method for analyzing nucleic acids according to claim 1, wherein the mixed matrix is ​​a mixed matrix containing 3-hydroxypicolinic acid and 2,4-dihydroxyacetophenone, and the mixing ratio of 3-hydroxypicolinic acid to 2,4-dihydroxyacetophenone is 10:1 to 1:

5.

3. The method for analyzing nucleic acids according to claim 1, wherein the mixed matrix is ​​a mixed matrix containing 3-hydroxypicolinic acid and 2,4,6-trihydroxyacetophenone monohydrate, and the mixing ratio of 3-hydroxypicolinic acid to 2,4,6-trihydroxyacetophenone monohydrate is 1:1 to 1:

3.

4. The method for analyzing nucleic acids according to claim 1, wherein the mixed matrix further comprises diammonium hydrogen citrate as a matrix additive.

5. By performing mass spectrometry on the sample containing the nucleic acid using the aforementioned mixed matrix, the [M+H] of the nucleic acid can be determined. ＋ or [M-H] － A data acquisition process to obtain mass spectral data of multiple fragment ions generated by dissociation, A data analysis step of extracting peaks of multiple fragment ions derived from the nucleic acid from the mass spectral data and determining the structure of the nucleic acid based on the mass information of the peaks, A method for analyzing nucleic acids according to claim 1, comprising:

6. The matrix-assisted laser desorption / ionization mass spectrometer is an ion trap type matrix-assisted laser desorption / ionization mass spectrometer comprising: an ion source for the matrix-assisted laser desorption / ionization method; an ion trapping unit for separating and trapping ions with a predetermined mass-to-charge ratio from the ions generated by the ion source; and a detection unit for detecting the ions trapped by the ion trapping unit, Using the matrix-assisted laser desorption / ionization mass spectrometer, the [M+H] of the nucleic acids contained in the sample is obtained. ＋ or [M-H] － The process includes an analysis condition acquisition step to acquire standard analysis conditions for the ion amount setting item related to the amount of ions generated in the ion source, the mass-to-charge ratio range setting item related to the mass-to-charge ratio range of ions captured in the ion capture unit, and the signal intensity setting item related to the signal intensity of ions in the detection unit, when performing a first measurement to detect the ion. The data acquisition step is a modified analysis condition obtained by changing at least one of the standard analysis conditions acquired in the analysis condition acquisition step, namely the ion amount setting item, the mass-to-charge ratio range setting item, and the signal intensity setting item, and the nucleic acid [M+H] ＋ or [M-H] － A method for analyzing nucleic acids according to claim 5, comprising performing a second measurement to detect multiple fragment ions generated by dissociation, thereby obtaining mass spectral data of the multiple fragment ions.

7. The modified analysis conditions are those in which the analysis conditions for the ion amount setting item are changed so that the amount of ions generated by the ion source increases compared to when the first measurement of the sample was performed under the standard analysis conditions, or the nucleic acid [M+H] ＋ or [M-H] － The nucleic acid analysis method according to claim 6, wherein the analysis conditions of the mass-charge ratio range setting item are modified so that ions with a lower mass than the mass-charge ratio are preferentially captured, or the analysis conditions of the signal intensity setting item are modified so that the signal intensity of the ions in the detection unit is increased.

8. The mass-to-charge ratio range setting item is the time from when the ion source is irradiated with a laser until when a capture voltage is applied to the ion trapping unit to trap ions. The nucleic acid analysis method according to claim 6, wherein the modified analysis condition is one in which the time is changed to a value shorter than that of the standard analysis condition.

9. The nucleic acid analysis method according to claim 8, wherein the modified analysis condition is set to a value that is 1 to 3 μs shorter than the standard analysis condition.

10. The method for analyzing nucleic acids according to claim 1, wherein the matrix-assisted laser desorption / ionization mass spectrometer is of the digital ion trap type.

11. A matrix for matrix-assisted laser desorption / ionization mass spectrometry of nucleic acids, comprising 3-hydroxypicolinic acid and 2,4-dihydroxyacetophenone as matrix materials, or comprising 3-hydroxypicolinic acid and 2,4,6-trihydroxyacetophenone monohydrate as matrix materials.

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