Method for analyzing nucleic acid and matrix used therein

Abstract

Nucleic acids contained in a sample are analyzed using a matrix-assisted laser desorption ionization mass spectrometry device using 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. As a result, [M + H] + or [M-H]-in a nucleic acid and fragment ions generated by dissociation of the ions can be detected with high sensitivity.

Description

Technical Field

[0001] The present invention relates to a method for analyzing nucleic acid using mass spectrometry, and a substrate used therein. Background Art

[0002] Nucleic acid is a biopolymer composed of nucleotides composed of bases, sugars and phosphates connected by phosphodiester bonds. Depending on the sugar, it can be divided into deoxyribonucleic acid (DNA) with 2-deoxyribose and ribonucleic acid (RNA) with ribose. Among them, nucleic acids composed of several to dozens of nucleotides are also called oligonucleotides, which can be chemically synthesized. Therefore, in recent years, research on the application of oligonucleotides as nucleic acid drugs has been very active, and the importance of analyzing nucleic acids has increased.

[0003] The matrix-assisted laser desorption / ionization (MALDI) method is generally considered to be a soft ionization method that can ionize non-volatile compounds without much decomposition. Mass spectrometry devices (MALDI-MS) using it are widely used to obtain molecular weight information of biopolymers such as peptides, proteins, and sugar chains. However, when using MALDI-MS to analyze nucleic acids, nucleic acids are easily decomposed during ionization, especially the larger the molecular weight of nucleic acids, the easier it is to decompose, so it is not easy to produce protonated molecules [M+H] + or deprotonated molecule [MH] - (M is a molecule, H is a hydrogen atom). In addition, it is easy to generate a different + or [MH] - As a result, there is an alkali metal ion adduct of nucleic acid. + and [MH] - The peak intensity or S / N ratio of these ions decreases, which results in a decrease in analytical sensitivity. Therefore, various nucleic acid analysis methods that can detect these ions with high sensitivity have been studied.

[0004] For example, Non-Patent Document 1 describes that in a nucleic acid molecular weight analysis using 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 a nucleic acid with a molecular weight of about 6000 is detected at a higher S / N than before the addition of ammonium citrate. +Non-patent document 2 states that in the analysis of nucleic acid molecular weight using MALDI-TOFMS, by adding ammonium citrate to 2,4-dihydroxyacetophenone (2,4-DHAP) as a matrix, [MH] of RNA with a molecular weight of about 6800 was detected. - .

[0005] In addition, Non-Patent Document 3 states that in the analysis of nucleic acid molecular weight using MALDI-TOFMS, by using a mixed matrix of anthranilic acid (AA) and nicotinic acid (NA), it is possible to detect [MH] of DNA (21 bases in length) with a molecular weight of about 6000 with high sensitivity. - .

[0006] On the other hand, in the structural analysis of nucleic acids, the following operation is performed: using MALDI-MS, for [M+H] generated by ionization of nucleic acids by an appropriate method, + or [MH] - The various fragment ions generated by intentional dissociation are subjected to mass spectrometry analysis to determine their attribution and infer the molecular structure of the original nucleic acid. As one of the ion dissociation methods at this time, in-source decay (ISD) is known. In-source decay is a method of dissociating ions in the ion source at the same time as ionization or just after ionization. As mentioned above, the MALDI method is a soft ionization method, and it is not easy to dissociate ions. It is known that the dissociation of ions can be promoted during ionization by, for example, increasing the laser intensity, increasing the energy during ionization, or using a special matrix.

[0007] For example, non-patent document 4 records that in the structural analysis of a nucleic acid with a relatively short chain length of 10 bases, by using 6-thioguanine (TG) as a matrix, especially in the negative ion mode, ISD-based fragment ions (ISD fragment ions) mainly composed of w series ions were detected. Non-patent document 5 records that in the structural analysis of a nucleic acid with a relatively short chain length of 10 bases, by using 1,5-diaminonaphthalene (1,5-diaminonaphthalene: 1,5-DAN) as a matrix, especially in the negative ion mode, a variety of ISD fragment ions such as aB, d, y, and w series ions were detected. Non-patent document 2 records that in the structural analysis of a nucleic acid with a relatively long chain length of 20 bases, by using 2,4-dihydroxyacetophenone (2,4-DHAP) as a matrix, especially in the negative ion mode, ISD fragment ions mainly composed of w series ions were detected.

[0008] In addition, non-patent document 6 records that in the structural analysis of synthetic oligonucleotides using MALDI-TOFMS, a mixed matrix containing anthranilic acid (AA) and 3-hydroxypicolinic acid (3-HPA) was used, and many ISD fragment ions of y series ions were detected. In addition, the document records that in the structural analysis of synthetic oligonucleotides using MALDI-MS (IR-MALDI) using IR (infrared) laser, a mixed matrix containing 2,5-dihydroxybenzoic acid (2,5-dihydroxybenzoic acid; DHB) and anthranilic acid (AA) and nicotinic acid (NA) was used, and ISD fragment ions of y series ions were detected.

[0009] Prior art literature

[0010] Non-patent literature

[0011] Non-patent document 1: Yoshinao Wada, "Basics of MS Spectrometry Analysis - SNP Center Analysis", Journal of the Mass Spectrometry Society of Japan, 2003, Vol.51, No.2, pp.368-373

[0012] Non-patent document 2: H. Shimizu et al., "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

[0013] 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

[0014] Non-patent document 4: Satoshi Kimura et al., "Effect of oligonucleotide structuraldifference on matrix-assisted laser desorption / ionization in-source decay incomparison with collision-induced dissociation fragmentation", (USA), RapidCommunications in Mass Spectrometry, 2020, 34, e8819

[0015] Non-patent document 5: Nathan A. Hagan et al., "Enhanced In-Source Fragmentation in MALDI-TOF-MS of Oligonucleotides Using 1,5-Diaminonapthalene", Journal of the American Society for Mass Spectrometry, (USA), 2012, 23, pp. 773-777

[0016] Non-patent document 6: M. Sebela, "Analysis and sequencing of nucleic acids by matrix-assisted laser desorption / ionisation mass spectrometry", SpectroscopyEurope, 2016, 28, 5, pp. 11-15

[0017] Non-patent document 7: Scott A.McLuckey et al., “Tandem mass spectrometry of small, multiply charged oligonucleotides”, Journal of the American Society for Mass Spectrometry, (USA), 1992, 3, pp. 60-70 Summary of the invention

[0018] Problem that the invention aims to solve

[0019] Regarding the molecular weight analysis of nucleic acids, the methods described in Non-Patent Documents 1 and 2 use a matrix additive to detect the [M+H]+ , [MH] - However, its sensitivity is still insufficient and there is room for improvement. In addition, the matrix and analysis method described in Non-Patent Document 3 can detect relatively high-mass nucleic acid [MH] with high sensitivity. - , but the versatility is limited, and further research is expected. In addition, generally speaking, the reported matrices that can be used for nucleic acid analysis are limited. Therefore, it is expected that [M+H] + or [MH] - of new matrix.

[0020] Regarding the structural analysis of nucleic acids, non-patent documents 2 and 4 to 6 use a matrix with the property of easily generating ISD to examine ISD fragment ions and analyze nucleic acid sequences. However, they have the following problems: either the detection sensitivity is low; or the number of detected ion species and high-sensitivity peaks are biased towards some sequences (or mass ranges); or the analysis becomes complicated by detecting multiple ion species. Moreover, the decrease in sensitivity will cause a decrease in resolution, making the analysis difficult.

[0021] One of the reasons why the sensitivity of ISD fragment ions is considered to be low is that the precursor ions for generating ISD fragment ions, namely [M+H] + or [MH] - In addition, regarding ISD fragment ions, if sufficient peaks are detected with sufficient resolution for analysis and the ion species are as uniform as possible based on sensitivity, the structural analysis of nucleic acids will become easier. However, the reported matrix that can be used for the structural analysis of nucleic acids (i.e., the matrix for ISD) is limited, and a new matrix is ​​needed.

[0022] The present invention has been made in view of the above problems, and an object of the present invention is to provide a [M+H] + or [MH] - and fragment ions generated by dissociation of these ions, an analysis method capable of easily and reliably performing molecular weight analysis and structural analysis of nucleic acids, and a matrix used therein.

[0023] Solutions for solving problems

[0024] The nucleic acid analysis method of the present invention made in order to solve the above-mentioned problems is as follows:

[0025] The nucleic acid contained in the sample is analyzed by a matrix-assisted laser desorption ionization mass spectrometer using 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.

[0026] In order to solve the above problems, the matrix for MALDI-MS analysis of nucleic acid of the present invention is a matrix containing 3-hydroxypicolinic acid and 2,4-dihydroxyacetophenone, or containing 3-hydroxypicolinic acid and 2,4,6-trihydroxyacetophenone monohydrate.

[0027] Effects of the Invention

[0028] According to the nucleic acid analysis method of the present invention and the substrate used therein, a sufficient amount of [M+H] + or [MH] - , so these can be detected with high sensitivity. In addition, according to the analysis method of the present invention and the matrix used therein, a sufficient amount of [M+H] + or [MH] - , and sufficient amounts of [M+H] + or [MH] - Since the fragment ions generated by dissociation can be detected with high sensitivity, the molecular weight analysis and structural analysis of nucleic acid can be easily and reliably performed. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] Figure 1 This is a schematic diagram showing an example of a mass spectrometer (MALDI-ITMS) used in a nucleic acid analysis method according to an embodiment of the present invention.

[0030] Figure 2 This is a flowchart showing the structural analysis steps in the nucleic acid analysis method according to one embodiment of the present invention.

[0031] Figure 3 : This is a diagram showing the mass spectrum of the standard nucleic acid A in Reference Example 1 when various substrates are used.

[0032] Figure 4 : This is a diagram showing the mass spectrum of the standard nucleic acid A when various matrices (mixed matrices) are used in Reference Example 1.

[0033] Figure 5 This is a diagram showing the mass spectrum (ISD spectrum) of fragment ions derived from the standard nucleic acid A when 2,4-DHAP is used as a matrix in Reference Example 2.

[0034] Figure 6 The mass spectra of Mipomersen when various matrices were used in Example 1 and the mass spectra of [M+H] + The table of detection status.

[0035] Figure 7The graphs showing the mass spectrum of mipomersen when using the mixed matrix 3-HPA + 2,4-DHAP mixed at various mixing ratios in Example 2 and [M+H] + The table of detection status.

[0036] Figure 8 The diagram shows the mass spectrum of mipomersen when each matrix was used in Example 3-1.

[0037] Fig. 9 The diagram shows the mass spectrum of mipomersen when each matrix was used in Example 3-2.

[0038] Fig.10 The figure shows the mass spectrum of mipomersen when using the mixed matrix 3-HPA+2,4-DHAP containing various concentrations of ACD in Example 4 and the [M+H] + The table of detection status.

[0039] Fig.11 This is a diagram showing the mass spectrum (ISD spectrum) of fragment ions derived from mipomersen obtained in the ISD measurement under the mass-to-charge ratio range conditions of mode 3 (m / z 2000 to 18000) in Example 5 when a mixed matrix of 3-HPA+2,4-DHAP was used.

[0040] Fig.12 This is a diagram showing the mass spectrum (ISD spectrum) of fragment ions derived from mipomersen obtained in the ISD measurement under the mass-to-charge ratio range conditions of mode 2 (m / z 650 to 5000) in Example 5 when a mixed matrix of 3-HPA+2,4-DHAP was used.

[0041] Fig.13 The ISD spectra of mipomersen obtained in the ISD measurement under the mass-to-charge ratio range conditions of mode 3 (m / z 2000 to 18000) in Example 5 when (a) a mixed matrix 3-HPA+2,4-DHAP and (b) 2,4-DHAP were used are shown.

[0042] Fig.14 The graphs showing the mass spectra of Patisiran (a 1:1 mixture of sense strand and antisense strand) when various matrices were used in Example 6 and [M+H] + The table of detection status.

[0043] Fig.15 The graphs showing the mass spectra of patisilan (a 1:1 mixture of sense strand and antisense strand) when various matrices were used in Example 6 and [M+H] + The table of detection status.

[0044] Fig.16 The graphs showing the mass spectra of patisilan (a 1:1 mixture of sense strand and antisense strand) when various matrices were used in Example 6 and [M+H] + The table of detection status.

[0045] Fig.17 A graph showing the mass spectrum of patisilan (a 1:1 mixture of sense and antisense strands) 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 graph showing [M+H] + The table of detection status. DETAILED DESCRIPTION

[0046] One embodiment of the nucleic acid analysis method of the present invention is described below.

[0047] <Nucleic Acid>

[0048] The nucleic acid as the analysis object of the present embodiment includes, in addition to nucleic acid, nucleic acid-related substances such as modified nucleic acid, nucleic acid derivatives, and nucleic acid drugs. Nucleic acid and nucleic acid-related substances are collectively referred to as nucleic acid below. The degree of polymerization (base length) of nucleic acid is not particularly limited, and is preferably an oligonucleotide formed by polymerization of several to tens of nucleotides. The analysis method of the present embodiment can especially improve the analytical sensitivity of nucleic acids with large molecular weight, so it is more preferred that the molecular weight is more than 3000 nucleic acids, and especially more preferably the molecular weight is more than 6000 nucleic acids. In addition, nucleic acid can be a natural product obtained from a living being or a processed product thereof, or it can be a chemically synthesized artificial nucleic acid.

[0049] <Mixed matrix>

[0050] The mixed matrix used in the present embodiment is a matrix containing 3-hydroxypicolinic acid (3-HPA) and 2,4-dihydroxyacetophenone (2,4-DHAP) (hereinafter referred to as 3-HPA+2,4-DHAP), or a matrix 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. It is considered that the reason why the preferred mixed matrix differs depending on whether the analysis target is DNA or RNA is that the types of constituent sugars in DNA and RNA are different, and therefore the affinity for the matrix is ​​also different.

[0051] The mixing ratio of the mixed matrix is ​​not particularly limited. When the mixed matrix contains 3-HPA and 2,4-DHAP, the [M+H] + or [MH] - , and the fragment ions generated by the dissociation of the ions, the ratio of 3-HPA:2,4-DHAP is preferably 10:1 to 1:5, more preferably 7:1 to 1:5, and particularly preferably 5:1 to 1:1. When the mixed matrix contains 3-HPA and THAP, from the same viewpoint, the ratio of 3-HPA:THAP is preferably 1:1 to 1:5, more preferably 1:1 to 1:3.

[0052] The mixed matrix may further include ammonium citrate dibasic (ACD) as a matrix additive. There are several types of ammonium salts of citric acid depending on the number of ammonium ions bound to the citrate ion. In this embodiment, a salt in which two ammonium ions are bound to one citrate ion is preferably used. The concentration of the matrix additive in the mixed matrix is ​​determined from the [M+H] + or [MH] - , and the fragment ions generated by the dissociation of the ions, preferably 10 to 100 mM, more preferably 30 to 90 mM, and further preferably 40 to 85 mM. It should be noted that in this specification, "(lower limit) to (upper limit)" and the symbol "-" are used to represent the numerical range from the lower limit to the upper limit, and the numerical range represented in this way includes the lower limit itself and the upper limit itself.

[0053] As a method for preparing a sample for analysis, a mixed solution prepared by mixing a sample containing nucleic acid and a mixed matrix is ​​dried on a sample plate of a mass spectrometer. The mixed solution may be prepared in advance and then dripped onto the sample plate for drying, or the mixed solution may be prepared on the sample plate and then directly dried.

[0054] <Mass spectrometer>

[0055] The mass spectrometer used in this embodiment is a mass spectrometer (MALDI-MS) having an ion source based on the MALDI method. As MALDI-MS, for example, time-of-flight type MALDI-TOFMS and ion trap type MALDI-ITMS are included. The ion trap type MALDI-MS mentioned here is a device having an ion trap for capturing ions. Specifically, it includes the following mass spectrometer: a mass spectrometer that utilizes the mass separation function of the ion trap itself to discharge the ions captured in the ion trap in the order of mass-to-charge ratio (m / z) from small to large, and detects the ions with a detector configured outside the ion trap. In addition, it also includes, for example, the following mass spectrometer: a mass separation unit such as a time-of-flight type mass separation unit configured outside the ion trap separates the ions discharged together from the ion trap according to the mass-to-charge ratio, and detects them with a detector also configured outside the ion trap.

[0056] <Structural analysis of nucleic acids>

[0057] The analysis method of this embodiment may include the following steps: a data acquisition step of performing mass spectrometry analysis on a sample containing nucleic acid using the mixed matrix, thereby acquiring [M+H] + or [MH] - The method comprises the following steps: extracting peaks of multiple fragment ions derived from nucleic acid from the mass spectral data obtained in the data acquisition step, and determining the structure of the nucleic acid based on the mass information of the peaks. Thus, the structure analysis of nucleic acid can be performed. The method of performing the structure analysis is described below for the case where the mass spectrometer is an ion trap type MALDI-ITMS.

[0058] (Ion trap mass spectrometer)

[0059] The ion trap mass spectrometer comprises: an ion source 1 for ionizing a sample containing an analyte; an ion trap 2 for temporarily capturing ions of a specified mass-to-charge ratio among ions generated in the ion source 1 using a high-frequency electric field, and separating the captured ions according to the mass-to-charge ratio (m / z) (the ion capturing unit in the present invention); and a detection unit 3 for detecting the separated ions.

[0060] The ion source 1 is an ion source using the MALDI method, and includes a laser irradiation unit 11 for irradiating a sample with a laser beam, and a sample stage 12 for placing a sample plate S carrying the sample. The 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 to each other with the ring electrode 21 sandwiched therebetween. The inlet-side end cap electrode 22 is formed with an ion entrance hole 22a, and the outlet-side end cap electrode 23 is formed with an ion exit hole 23a. The detector 3 includes a conversion dynode 31 for converting ions into electrons, and a detector (secondary electron multiplier) 32 for multiplying and detecting the electrons coming from the conversion dynode 31.

[0061] Predetermined voltages are applied to the ring electrode 21 and the end cap electrodes 22 and 23 of the ion trap 2. The high frequency electric field thus formed can trap ions in the internal space surrounded by the ring electrode 21 and the end cap electrodes 22 and 23 or discharge ions from the internal space through the ion exit hole 23a.

[0062] In the ion source 1, the mass-to-charge ratio range of ions preferentially captured by the ion trap is controlled by changing the time (hereinafter referred to as the delay time) from the time when the sample is irradiated with the laser beam to the time when the trapping voltage for trapping ions is applied to the ring electrode 21 of the ion trap 2. By extending the delay time, ions on the high-mass side can be reliably captured compared with other ions, and by shortening the delay time, ions on the low-mass side can be reliably captured compared with other ions.

[0063] The prescribed voltage applied to the ring electrode 21 and the end cap electrodes 22 and 23 may be a high-frequency voltage in the form of a sine wave, or may be a rectangular wave voltage generated by switching between two different voltages at high speed. In the case of a digital ion trap using an electric field generated by a rectangular wave voltage, the mass-to-charge ratio range of ions that can be captured is controlled by changing the frequency while maintaining the amplitude (voltage value) of the rectangular wave voltage or by changing the duty cycle, which is the switching interval ratio of the rectangular wave voltage.

[0064] Then, refer to Figure 2 The flowchart of FIG. 1 illustrates the steps for performing structural analysis of nucleic acids.

[0065] (Structural analysis method)

[0066] Here, an example in which MALDI-ITMS having a digital ion trap is used as a mass spectrometry apparatus will be described.

[0067] [Step 1: Acquisition of standard analysis conditions for MS measurement]

[0068] First, obtain the protonated molecules [M+H] used to detect nucleic acids + or deprotonated molecule [MH]- The standard analytical conditions for the determination of [M+H] (hereinafter referred to as MS determination) are as follows. The MS determination is equivalent to the first determination in the present invention. In the MS determination, a method for detecting [M+H] with as high sensitivity and high resolution as possible is used. + or [MH] - The measurement conditions are such that the measurement is not accompanied by ion dissociation (or even if ion dissociation occurs, it is weak). In the mass spectrometer, there are various setting items that can be appropriately set according to the type of the analysis object, the purpose of analysis, etc. In the structure analysis method of this embodiment, the ion amount setting items related to the ion amount generated in the ion source 1, the mass-to-charge ratio range setting items related to the mass-to-charge ratio of the ions captured in the ion trap 2, and the signal intensity setting items related to the signal intensity of the ions in the detection unit 3 are obtained for detecting the [M+H] of the analysis object in the MS measurement. + or [MH] - standard analysis conditions.

[0069] Standard analysis conditions refer to the following conditions: [M+H] + or [MH] - As a method for obtaining standard analysis conditions, for example, MS measurement can be performed using the default values ​​of various setting items pre-set in the mass spectrometer. If the result is that [M+H] + or [MH] - , the default value is used as the standard analysis condition. In this case, the operation of reading the default value from a storage unit or the like in which the default value is pre-stored is equivalent to the operation of obtaining the standard analysis condition. Alternatively, in order to detect the [M+H] + or [MH] - , it is possible to change the setting values ​​of various setting items relative to the default values, perform MS measurement, and determine the value (threshold value, etc.) that can detect the ion.

[0070] Items for setting the ion quantity include the laser intensity (laser power) irradiated by the laser irradiation unit 11, items for setting the mass-to-charge ratio range include the RF delay (RFdelay) value corresponding to the delay time, and items for setting the signal intensity include the voltage applied to the conversion dynode 31 and the voltage applied to the detector (secondary electron multiplier tube) 32.

[0071] [Step 2: Changing the standard analysis conditions to change the analysis conditions]

[0072] Then, the changed analysis conditions are set to change at least one of the ion amount setting item, mass-to-charge ratio range setting item, and signal intensity setting item in the standard analysis conditions obtained in step 1. That is, the following situation may be included: only one of the ion amount setting item, mass-to-charge ratio range setting item, and signal intensity setting item is changed, and the other setting items remain unchanged. At this time, compared with the case of performing MS measurement using standard analysis conditions, the ion amount setting item is changed in a manner that the amount of ions generated in the ion source 1 is increased, the mass-to-charge ratio range setting item is changed in a manner that the ions on the low mass side are preferentially captured in the ion trap 2, and the signal intensity setting item is changed in a manner that the signal intensity of ions in the detection unit 3 is increased.

[0073] Specifically, when changing the intensity of the laser beam as an ion quantity setting item, it is preferably set to be high relative to the standard analysis conditions, for example, it is more preferably set to be about 1 to 40% higher than the value of the standard analysis conditions, and it is further preferably set to be about 1 to 30% higher. When changing the RF delay value as a mass-to-charge ratio range setting item, it is preferably set to be low relative to the standard analysis conditions, for example, it is more preferably set to be 5 to 30% lower than the value of the standard analysis conditions, and it is further preferably set to be 10 to 20% lower (so that the delay time is 1 to 3 μ seconds). When changing the applied voltage to the conversion dynode 31 as a signal intensity setting item, it is preferably set to be high relative to the standard analysis conditions, for example, it is more preferably set to be about 5 to 30% higher, and it is further preferably set to be about 10 to 30% higher. Similarly, when changing the applied voltage to the detector (secondary electron multiplier tube) 32 as a signal intensity setting item, it is preferably set to be high relative to the standard analysis conditions, for example, it is preferably set to be about 5 to 50% higher, and it is further preferably set to be about 10 to 50% higher.

[0074] [Step 3: Acquisition of mass spectrum data by ISD measurement using changed analysis conditions]

[0075] The above-mentioned changed analysis conditions are used to perform a measurement for detecting fragment ions (decomposition products) derived from nucleic acids. In the present embodiment, the generation mechanism of fragment ions derived from the object of analysis (nucleic acid) has not been confirmed. Therefore, in this specification, the dissociation that occurs simultaneously with or just after ionization in the ion source of the MALDI ion trap mass spectrometer, as well as the ion dissociation that occurs thereafter in the device are all referred to as in-source fragmentation (ISD), and the measurement for detecting fragment ions derived from nucleic acids that are dissociated by ISD is referred to as ISD measurement. The ISD measurement is equivalent to the second measurement in the present invention. By performing the ISD measurement using the above-mentioned changed analysis conditions, the [M+H] of the nucleic acid can be obtained. + or [MH] -(precursor ion) dissociation to generate multiple fragment ions. In particular, it can detect with good sensitivity the fragments that deviate from [M+H] + or [MH] - The m / z fragments are on the low mass side of the domain.

[0076] In step 3, the maximum value of the predetermined mass-to-charge ratio of the ions captured in the ion trap 2 may be smaller than [M+H] of the nucleic acid to be analyzed. + or [MH] - The ISD measurement is performed by setting the range of the predetermined mass-to-charge ratio in the form of a mass-to-charge ratio of 1. + or [MH] - The value of the mass-to-charge ratio is set to be, for example, about 0.5 to 40% smaller, and more preferably about 0.5 to 20% smaller.

[0077] As a method of changing the range of the specified mass-to-charge ratio, there is, for example, the following method, etc.: the measurement mode pre-integrated in the device for the analysis object with a molecular weight of about 6000 is switched from a measurement mode with a target mass range of m / z 2000 to 18000 to a measurement mode with a target mass range of m / z 650 to 5000, thereby switching to a measurement mode that does not include a molecular weight of 6000 as an object. The target mass range of the measurement mode can be determined according to the frequency of the high-frequency voltage applied to the ion trap 2. Specifically, the amount of ions captured in the ion trap is limited, so the mass range to be measured is mainly determined by adjusting the frequency of the high-frequency voltage and setting a cutoff value (LowMass Cut-Off: LMCO) on the low mass side. If 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, if the frequency of the high-frequency voltage is reduced, the LMCO is set to be large and the mass range to be measured is set to the high mass side. That is, the range of the specified mass-to-charge ratio is changed by changing the frequency of the high-frequency voltage applied to the ion trap 2. In addition, as a method of changing the range of the specified mass-to-charge ratio, there is a method such as the following: for an analyte with a molecular weight of about 6000, the ratio of the switching interval of the rectangular wave voltage pre-integrated in the device, that is, the value of the setting item of the duty ratio, for example, is changed from a standard value of 50:50 to a value of 52:48, thereby excluding ions with m / z 5500 or more. By doing so, mass spectrum data can be obtained that excludes fragment ions having a mass-to-charge ratio close to the m / z of the precursor ion. As a result, the detection sensitivity of fragment ions in the low-mass region that deviates from the m / z of the precursor ion is improved.

[0078] In step 3, the ISD measurement can be performed in two ways: using and not using the conditions in which the range of the mass-to-charge ratio is set as described above, thereby acquiring mass spectrum data obtained in each measurement.

[0079] In addition, the maximum value of the predetermined mass-to-charge ratio of the ions captured in the ion trap 2 may be smaller than the [M+H] value of the nucleic acid to be analyzed. + or [MH] - On the basis of the condition formed by setting the range of the prescribed mass-to-charge ratio in the manner of the mass-to-charge ratio value, the standard analysis condition for the applied voltage to the sample stage 12 of the ion source 1 is further obtained in step 2, and the ISD measurement is performed using the condition formed by changing the applied voltage to the sample stage 12 to a larger value than the analysis condition. The applied voltage to the sample stage is more preferably set to be about 4 to 8 times higher than the standard analysis condition, and further preferably set to be about 4 to 5 times higher.

[0080] [Step 4: Analysis of mass spectrometry data]

[0081] Peaks corresponding to various fragment ions are extracted from the mass spectrometry data obtained in step 3, and the attribution of various fragment ions is determined based on the mass information shown in the peaks, and at least a part of the structure of the original nucleic acid is determined based on the results. The determination of the structure includes sequence analysis and determining the type of chemical modification or the site of the chemical modification through sequence analysis. The determination of the structure can be performed by database retrieval and de novo sequencing.

[0082] In step 3, when mass spectrometry data is acquired under the conditions of using and not using the conditions of setting the range of the specified mass-to-charge ratio, the attribution results of various fragment ions obtained in these two cases can be combined and analyzed. By doing so, the structural analysis of nucleic acids as the object of analysis can be performed more reliably.

[0083] The analysis method of the present invention will be described below by way of examples, which are merely illustrative and the present invention is not limited thereto.

[0084] Example

[0085] (Reference Example 1)

[0086] First, a substrate to be used as a comparative object of the present invention is selected.

[0087] <1. Preparation of sample solution>

[0088] As a sample solution, a 10 pmol / μL aqueous solution of a standard nucleic acid A (5′-TGTGCGTGTGTAGTGTGTCT-3′: sequence number 1, 20 bases in length, MW 6201.1, commissioned synthesis product) was prepared.

[0089] <2. Preparation of matrix solution>

[0090] As a matrix solution, 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 respectively, containing 70 mM diammonium hydrogen citrate (ACD) as a matrix additive.

[0091] 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 they were mixed at a ratio of 1:1 (v / v) (2:1 (mol / mol)) 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 they were mixed at a ratio of 1:1 (v / v) to prepare a mixed matrix (AA+3-HPA) solution.

[0092] <3. Preparation of analytical samples>

[0093] The sample solution prepared in 1. and the matrix solution or mixed matrix solution prepared in 2. were mixed at 1:1 (v / v), and 1 μL of the obtained mixed solution was dropped onto a sample plate (SUS plate) and dried.

[0094] <4. Mass spectrometry analysis>

[0095] For mass spectrometry, a MALDI digital ion trap mass spectrometer (MALDI-DITMS, manufactured by Shimadzu Corporation, trade name: MALDImini-1) was used. The sample plate carrying the analytical sample prepared in 3. was inserted into the MALDI-DITMS, and MS measurement was performed in the positive mode using the grating function.

[0096] <Results>

[0097] Figure 3 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 are shown. The arrows in the figure indicate [M+H] + Detection of [M+H] was not detected. + The peak time is expressed as ND (not detected).

[0098] Depend on Figure 3 , [M+H] was detected when (a) 3-HPA, (b) 2,4-DHAP, and (c) THAP were used as matrices. + The peak of [M+H] was not detected when (d) 1,5-DAN was used. + 1,5-DAN is reported as a matrix for analyzing relatively short nucleic acids of about 10 bases in length (10mer), and it is believed that the standard nucleic acid A of 18 bases in length (18mer) is not ionized in the case of 1,5-DAN.

[0099] Figure 4 The mass spectra of standard nucleic acid A when (a) AA+NA and (b) AA+3-HPA are used as mixed matrices are shown. (a) also shows an enlarged view of the mass spectrum. Figure 4 , [M+H] was detected when (a) AA+NA was used + peaks, but also detected a variety of base ions and alkali metal ion adducts. In addition, [M+H] + The detection sensitivity is lower than Figure 3 When (a) 3-HPA, (b) 2,4-DHAP, and (c) THAP were used. When (b) AA+3-HPA was used, [M+H] of standard nucleic acid A was not detected. + (a) AA+NA is a mixed matrix reported as a matrix for high-sensitivity analysis of high-quality nucleic acids, but at least under the conditions of this time, the sensitivity is lower than 3-HPA, 2,4-DHAP, and THAP. In addition, (b) AA+3-HPA is reported as a matrix for ISD of nucleic acids, but at least under the conditions of this time, the ionization of nucleic acid molecules itself is difficult.

[0100] Based on the above results, 3-HPA, 2,4-DHAP, and THAP were selected as substrates for comparison.

[0101] (Reference Example 2)

[0102] Then, ISD assay of nucleic acids was performed using 2,4-DHAP as a matrix.

[0103] <1. Preparation of sample solution>

[0104] As a sample solution, a 10 pmol / μL aqueous solution of standard nucleic acid A was prepared.

[0105] <2. Preparation of matrix solution>

[0106] As a matrix solution, a 40 mg / mL 50% ACN aqueous solution of 2,4-dihydroxyacetophenone (2,4-DHAP) containing 70 mM ACD as a matrix additive was prepared.

[0107] <3. Preparation of analytical samples>

[0108] The sample solution prepared in 1. and the matrix solution prepared in 2. were mixed at 1:1 (v / v), and 1 μL of the obtained mixed solution was dropped onto a sample plate (SUS plate) and dried.

[0109] <4. Mass spectrometry analysis>

[0110] In the mass spectrometry analysis, a MALDI digital ion trap mass spectrometer (MALDI-DITMS, manufactured by Shimadzu Corporation, trade name: MALDImini-1) is used. The sample plate carrying the analytical sample prepared in 3. is inserted into the MALDI-DITMS, and the ISD measurement is performed using the grating function in the positive mode. The analysis conditions when performing the ISD measurement are as follows. The measurement mode shows the range of the mass-to-charge ratio of the ions to be measured, specifically, the range of the mass-to-charge ratio of the ions captured in the ion trap is specified, and the range is shown in brackets.

[0111] ·Measurement mode: Mode 3 (m / z 2000~18000)

[0112] Detector voltage (DV-1): 2000

[0113] Conversion voltage (DV-2): 8000

[0114] RF delay value (RF): 17

[0115] Laser power (LP): 75

[0116] Sample stage voltage (SV): 5

[0117] Duty cycle: 50:50

[0118] <5. Data Analysis>

[0119] The attribution of the main peak was determined for the mass spectrum data (ISD spectrum) of the fragment ions obtained by the ISD measurement in 4.

[0120] <Results>

[0121] Figure 5 The ISD spectrum of the standard nucleic acid A obtained by performing ISD measurement using 2,4-DHAP as a matrix is ​​shown. Figure 5In the present invention, the name of the corresponding oligonucleotide fragment ion species is marked on each peak in the mass spectrum (the general name suggested in non-patent document 7). The name is used to represent each ion species using a fragment ion series that conforms to the naming rules of the nucleic acid dissociation mode. In this naming rule, the fragment ion containing the 5' end is recorded as a n , b n 、c n ,d n , the fragment ion containing the 3' end in the opposite direction is denoted as x m ,y m 、z m 、w m It should be noted that the subscripts n and m represent the number of constituent units from the corresponding end to the dissociation site (defined by the number of bases). In addition, B in the identifier represents the base of the nucleic acid, for example, b13-B(A) in the figure represents the ion formed by the detachment of adenine base (A) from b13 ion. The same is true in the following figures.

[0122] When 2,4-DHAP is used as a matrix, the obtained ISD spectrum shows peaks of multiple bases detaching, and multiple fragment ion species are mixed and complicated. It is known that the MALDI-ITMS used in this measurement is more prone to fragmentation due to its device structure characteristics compared to the previous MALDI-TOFMS. It is difficult to perform base sequence analysis using such a mass spectrum, so in order to make the analysis faster, it is expected to suppress base detachment and obtain a simpler ISD spectrum with ion species that are unified to a certain extent.

[0123] Next, mipomersen, which is known as one of the nucleic acid drugs, was subjected to MS measurement using the analytical method of the present invention.

[0124] (Example 1)

[0125] <1. Preparation of sample solution>

[0126] 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-dG-dC-dT-dT-dC-MG-MC-MA-MC-MC-3' (M represents 2'-O-(2-methoxyethyl) nucleoside, d represents 2'-deoxynucleoside. The carbon at the 5-position of cytosine and uracil is substituted with a methyl group, and the phosphodiester bonds between all nucleotides are replaced with phosphorothioate bonds.): sequence number 2, 20 bases in length, MW 7177, a sample after desalting and purification of a synthetic nucleic acid for research and development) was prepared.

[0127] <2. Preparation of matrix solution>

[0128] As a matrix solution, a 40 mg / mL 50% ACN aqueous solution (3-HPA solution) of 3-hydroxypicolinic acid (3-HPA) containing 40 mM ACD as a matrix additive was prepared.

[0129] A 40 mg / mL 50% ACN aqueous solution (THAP solution) of 2,4,6-trihydroxyacetophenone monohydrate (THAP) containing 40 mM ACD as a matrix additive was prepared.

[0130] A 40 mg / mL 50% ACN aqueous solution (2,4-DHAP solution) of 2,4-dihydroxyacetophenone (2,4-DHAP) containing 70 mM ACD was prepared.

[0131] 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 at a ratio of 1:1 (v / v). Similarly, a mixed matrix (3-HPA+THAP) solution was prepared by mixing the above 3-HPA solution and THAP solution at a ratio of 1:1 (v / v).

[0132] <3. Preparation of analytical samples>

[0133] The sample solution prepared in 1. and the matrix solution and mixed matrix solution prepared in 2. were mixed at 1:1 (v / v), and 1 μL of the obtained mixed solution was dropped onto a sample plate (SUS plate) and dried.

[0134] <4. Mass spectrometry analysis>

[0135] For mass spectrometry, a MALDI digital ion trap mass spectrometer (MALDI-DITMS, manufactured by Shimadzu Corporation, trade name: MALDImini-1) was used. The sample plate carrying the analytical sample prepared in 3. was inserted into the MALDI-DITMS, and MS measurement was performed in the positive mode using the grating function.

[0136] <Results>

[0137] Figure 6The 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 are shown (3-HPA, 2,4-DHAP, THAP, 3-HPA+2,4-DHAP, and 3-HPA+THAP are shown in order from the left). The upper section (a) is an overall view showing the region of m / z 2000 to 15000, the middle section (b) is an enlarged view showing the region of m / z 6000 to 8000, and the lower section (c) is an enlarged view showing the region of m / z 7100 to 7300. The arrows in the figure indicate [M+H] + Furthermore, the [M+H] values ​​when using each matrix are summarized below the mass spectrum. + Table of the detection details (sensitivity (mV, S / N), resolution (R), base detachment, adduct detection) and laser power during measurement. The base detachment is determined based on the mass spectrum of (b), and the adduct detection is determined based on the mass spectrum of (c).

[0138] Depend on Figure 6 When the mixed matrix 3-HPA+2,4-DHAP was used, [M+H] was detected with the highest sensitivity and sufficient resolution in a state where the detachment of the base and the formation of alkali metal adducts were suppressed. + Thus, it was 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 bases or more.

[0139] (Example 2)

[0140] Mass spectrometry was performed in the same manner as in Example 1, except that the method for preparing the matrix solution was different, to examine the mixing ratio of the mixed matrix 3-HPA+2,4-DHAP.

[0141] <2. Preparation of matrix solution>

[0142] A 40 mg / mL 50% ACN aqueous solution (3-HPA solution) of 3-hydroxypicolinic acid (3-HPA) containing 70 mM ACD as a matrix additive was prepared.

[0143] A 40 mg / mL 50% ACN aqueous solution (2,4-DHAP solution) of 2,4-dihydroxyacetophenone (2,4-DHAP) containing 70 mM ACD as a matrix additive was prepared.

[0144] The prepared 3-HPA solution and 2,4-DHAP solution were mixed at a desired mixing ratio (v / v) to prepare a mixed matrix 3-HPA+2,4-DHAP (mixing ratio (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)) solution. In this case, the mixing ratio (1:0) indicates the case of using 3-HPA alone, and the mixing ratio (0:1) indicates the case of using 2,4-DHAP alone.

[0145] <Results>

[0146] Figure 7 The mass spectra of mipomersen when 3-HPA+2,4-DHAP (mixing ratios from the left: (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) are shown, and the [M+H] + A table showing the details of the detection conditions (sensitivity (mV, S / N), resolution (R), base detachment conditions, adduct detection conditions, and deviation between wells).

[0147] Compared with 3-HPA+2,4-DHAP (mixing ratio (1:0), (0:1)), that is, when 3-HPA or 2,4-DHAP was used alone, when a mixed matrix 3-HPA+2,4-DHAP prepared at a mixing ratio of (10:1) to (1:5) was used, [M+H] + The detection sensitivity of [M+H] was improved. In addition, when the mixed matrix 3-HPA+2,4-DHAP prepared at a mixing ratio of (7:1) to (1:5) was used, the detection sensitivity was improved and the detachment of the base was suppressed. Considering the reproducibility of the deviation between the wells, etc., it was confirmed that when the mixed matrix 3-HPA+2,4-DHAP prepared at a mixing ratio of (5:1) to (1:1) was used, it was possible to detect [M+H] with high sensitivity and high resolution and with good reproducibility while suppressing the detachment of the base and the formation of alkali metal ion adducts. + .

[0148] (Example 3-1, Example 3-2)

[0149] Mass spectrometry was performed in the same manner as in Example 1, except that the method for preparing the substrate solution was different, to examine the effect of the isomers of 2,4-DHAP.

[0150] (Example 3-1)

[0151] <2. Preparation of matrix solution>

[0152] A 40 mg / mL 50% ACN aqueous solution (3-HPA solution) of 3-hydroxypicolinic acid (3-HPA) containing 40 mM ACD as a matrix additive was prepared.

[0153] A 40 mg / mL 50% ACN aqueous solution (2,4-DHAP solution) of 2,4-dihydroxyacetophenone (2,4-DHAP) containing 70 mM ACD as a matrix additive was prepared.

[0154] A 40 mg / mL 50% ACN aqueous solution of 2,5-DHAP (2,5-DHAP solution), which is a positional isomer of 2,4-DHAP, was prepared containing 70 mM ACD as a matrix additive.

[0155] The prepared 3-HPA solution and 2,4-DHAP solution, and 3-HPA solution and 2,5-DHAP solution were mixed at a ratio of 1:1 (v / v) to prepare mixed matrix (3-HPA+2,4-DHAP (1:1), 3-HPA+2,5-DHAP (1:1)) solutions.

[0156] <Results>

[0157] Figure 8 The mass spectra of mipomersen when using the mixed matrix 3-HPA+2,4-DHAP (1:1) and 3-HPA+2,5-DHAP (1:1) are shown. The arrows in the figure indicate [M+H] + The values ​​in the figure represent [M+H] + Compared with the case of using 3-HPA+2,4-DHAP (1:1), the peak intensity (mV) of [M+H] + The sensitivity is low.

[0158] (Example 3-2)

[0159] <2. Preparation of matrix solution>

[0160] A 40 mg / mL 50% ACN aqueous solution (3-HPA solution) of 3-hydroxypicolinic acid (3-HPA) containing 70 mM ACD as a matrix additive was prepared.

[0161] A 40 mg / mL 50% ACN aqueous solution (2,4-DHAP solution) of 2,4-dihydroxyacetophenone (2,4-DHAP) containing 70 mM ACD as a matrix additive was prepared.

[0162] A 40 mg / mL 50% ACN aqueous solution of 2,6-DHAP (2,6-DHAP solution), which is a positional isomer of 2,4-DHAP, was prepared containing ACD at a concentration of 70 mM as a matrix additive.

[0163] The prepared 3-HPA solution and 2,4-DHAP solution, and 3-HPA solution and 2,6-DHAP solution were mixed at a ratio of 1:1 (v / v) to prepare mixed matrix (3-HPA+2,4-DHAP (1:1), 3-HPA+2,6-DHAP (1:1)) solutions.

[0164] <Results>

[0165] Fig. 9 The mass spectra of mipomersen when using the mixed matrix 3-HPA + 2,4-DHAP (1:1) and 3-HPA + 2,6-DHAP (1:1) are shown. When using 3-HPA + 2,4-DHAP (1:1), the [M+H] + However, when using 3-HPA+2,6-DHAP (1:1), the ionization of nucleic acid molecules itself is more difficult.

[0166] (Example 4)

[0167] Mass spectrometry was performed in the same manner as in Example 1, except that the method for preparing the matrix solution was different, to examine the concentration of diammonium hydrogen citrate (ACD) as a matrix additive.

[0168] <2. Preparation of matrix solution>

[0169] A 40 mg / mL 50% ACN aqueous solution (3-HPA solution) of 3-hydroxypicolinic acid (3-HPA) containing 40, 70, and 100 mM ACD as a matrix additive was prepared.

[0170] A 40 mg / mL 50% ACN aqueous solution (2,4-DHAP solution) of 2,4-dihydroxyacetophenone (2,4-DHAP) containing 40, 70, and 100 mM ACD as a matrix additive was prepared.

[0171] The 3-HPA solution and 2,4-DHAP solution containing various concentrations of ACD were mixed at 1:1 (v / v) to prepare a mixed matrix (3-HPA + 2,4-DHAP (40, 55, 70, 85, 100 mMACD)) solution. Specifically, the 3-HPA (40 mMACD) solution and the 2,4-DHAP (40 mMACD) solution were mixed at 1:1 (v / v) to prepare a mixed matrix (3-HPA + 2,4-DHAP) solution containing 40 mM ACD. Similarly, 3-HPA (40mMACD) solution and 2,4-DHAP (70mMACD) solution, 3-HPA (70mMACD) solution and 2,4-DHAP (70mMACD) solution, 3-HPA (70mM ACD) solution and 2,4-DHAP (100mMACD) solution, and 3-HPA (100mMACD) solution and 2,4-DHAP (100mMACD) solution were mixed in a ratio of 1:1 (v / v) to prepare mixed matrix (3-HPA+2,4-DHAP) solutions containing 55, 70, 85, and 100mM ACD.

[0172] <Results>

[0173] Fig.10 The mass spectra of mipomersen when using the mixed matrix 3-HPA+2,4-DHAP at various ACD concentrations (40, 55, 70, 85, 100 mM) and the [M+H] + The table shows the detection conditions (sensitivity (mV, S / N), resolution (R), adduct detection conditions, base separation conditions).

[0174] When using mixed matrices of any ACD concentration, [M+H] was detected with good sensitivity and high resolution, and the detachment of bases and the formation of alkali metal ion adducts were suppressed. + The detection sensitivity slightly decreased when the ACD concentration was 100 mM, but it was confirmed that a similarly good mass spectrum could be obtained when the ACD concentration was 40 to 85 mM.

[0175] (Example 5)

[0176] Then, the ISD of mipomersen was measured using the analytical method of the present invention.

[0177] <1. Preparation of sample solution>

[0178] As a sample solution, a 20 pmol / μL aqueous solution of mipomersen was prepared.

[0179] <2. Preparation of matrix solution>

[0180] A 40 mg / mL 50% ACN aqueous solution (3-HPA solution) of 3-hydroxypicolinic acid (3-HPA) containing 40 mM ACD as a matrix additive was prepared.

[0181] A 40 mg / mL 50% ACN aqueous solution (2,4-DHAP solution) of 2,4-dihydroxyacetophenone (2,4-DHAP) containing 70 mM ACD as a matrix additive was prepared.

[0182] The prepared 3-HPA solution and 2,4-DHAP solution were mixed at 1:1 (v / v) to prepare a mixed matrix (3-HPA+2,4-DHAP (1:1)) solution.

[0183] <3. Preparation of analytical samples>

[0184] The sample solution prepared in 1. and the mixed matrix solution prepared in 2. were mixed at 1:1 (v / v), and 1 μL of the obtained mixed solution was dropped onto a sample plate (SUS plate) and dried.

[0185] <4. Mass spectrometry analysis>

[0186] In the mass spectrometry analysis, a MALDI digital ion trap mass spectrometer (MALDI-DITMS, manufactured by Shimadzu Corporation, trade name: MALDImini-1) is used. The sample plate carrying the analytical sample prepared in 3. is inserted into the MALDI-DITMS, and the ISD measurement is performed using the grating function in the positive mode. The analysis conditions when performing the ISD measurement are as follows. The measurement mode shows the range of the mass-to-charge ratio of the ions to be measured, specifically, the range of the mass-to-charge ratio of the ions captured in the ion trap is specified, and the range is shown in brackets. The laser power uses the optimal value.

[0187] · Measurement mode: Mode 3 (m / z 2000-18000), Mode 2 (m / z 650-5000)

[0188] Detector voltage (DV-1): 2000

[0189] Conversion voltage (DV-2): 8000

[0190] RF delay value (RF): 17

[0191] Sample stage voltage (SV): 5

[0192] Duty cycle: 50:50

[0193] <5. Data Analysis>

[0194] The attribution of the main peak was determined for the mass spectrum data (ISD spectrum) of the fragment ions obtained by the ISD measurement in 4.

[0195] <Results>

[0196] Fig.11 The ISD spectrum of mipomersen obtained in the ISD measurement under the mass-to-charge ratio range conditions of mode 3 and using the mixed matrix 3-HPA+2,4-DHAP and the attribution of the main peaks are shown. Fig.12 The ISD spectrum of mipomersen obtained in the ISD measurement under the mass-to-charge ratio range conditions of mode 2 and using the mixed matrix 3-HPA+2,4-DHAP and the attribution of the main peaks are shown. Fig.11 and Fig.12 In this study, a concise ISD spectrum was obtained in which multiple ISD fragment ions, mainly a / w ions, were preferentially detected. As a result, the attribution of ISD fragment ions became easy to determine, and analysis could be performed quickly. In addition, by integrating Fig.11 and Fig.12 The analysis results can more reliably analyze the entire sequence of mipomersen.

[0197] Fig.13 The ISD spectra of mipomersen obtained in the ISD measurement under the mass-to-charge ratio range conditions of mode 3 when (a) the mixed matrix 3-HPA+2,4-DHAP and (b) 2,4-DHAP were used. The dotted line in the figure indicates that both matrices were detected and Fig.11 The sensitivity of ISD fragment ions when using the mixed matrix 3-HPA + 2,4-DHAP is higher than when using 2,4-DHAP, which is a conventional ISD matrix, and a / w ions are preferentially and more frequently observed. This confirms that by using the mixed matrix 3-HPA + 2,4-DHAP, a simple ISD spectrum that can be easily analyzed can be obtained.

[0198] It can be considered that obtaining such a result is related to the following: Figure 6 As shown in the results, when the mixed matrix 3-HPA+2,4-DHAP was used for MS measurement, [M+H] was detected with good sensitivity and high resolution while suppressing the detachment of bases and the formation of alkali metal ion adducts. + The ISD measurement is based on the generation of [M+H] + or [MH] - The method of using a mixed matrix of 3-HPA+2,4-DHAP as a precursor ion and then performing mass spectrometry analysis on the fragment ions generated by fragmentation of the precursor ion. It is believed that the [M+H] +Therefore, the precursor ions [M+H] were generated in sufficient amounts in the ISD measurement using the same mixed matrix. + As a result, when the mixed matrix has the characteristic of being easy to generate ISD as in this case, a sufficient amount of fragment ions generated from the precursor ions are generated through ISD, which allows the fragment ions to be detected with good sensitivity. In addition, it is believed that the characteristics of not easily generating adducts during ionization and not generating dissociation of unstable sites such as base detachment lead to the generation of simpler ISD fragment species.

[0199] (Example 6)

[0200] Then, the MS measurement of Patisiran, which is known as one of the nucleic acid drugs, was performed using the analytical method of the present invention.

[0201] <1. Preparation of sample solution>

[0202] As a sample solution, Patisiran (originally double-stranded RNA consisting of a sense strand and an antisense strand, and in this example, a 1:1 (mol / mol) mixture of the sense strand and the antisense strand was used) was prepared.

[0203] Sense strand 5'-G-Um-AA-Cm-Cm-AAGAG-Um-A-Um-Um-Cm-Cm-A-Um-dT-dT-3'

[0204] (dT represents thymidine deoxyribonucleotide, Cm represents 2'-O-methylcytidine, and Um represents 2'-O-methyluridine): SEQ ID NO. 3, 21 bases in length (core sequence 19 bases in length, 3'-end DNA overhang with 2 bases (dTdT)). ), MW 6764, antisense strand 5'-AUGGAA-Um-ACUCUUGGU-Um-AC-dT-dT-3' (dT represents thymidine deoxyribonucleotide, and Um represents 2'-O-methyluridine): SEQ ID NO. 4, 21 bases in length (core sequence 19 bases in length, 3'-end DNA overhang with 2 bases (dTdT). ), MW 6660, sample after desalting and purification of synthetic nucleic acid for research and development. ) in a 20 pmol / μL aqueous solution.

[0205] <2. Preparation of matrix solution>

[0206] 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.

[0207] A 40 mg / mL 50% ACN aqueous solution (2,4-DHAP solution) of 2,4-dihydroxyacetophenone (2,4-DHAP) containing 70 mM ACD as a matrix additive was prepared.

[0208] A 40 mg / mL 50% ACN aqueous solution (THAP-1 solution) of 2,4,6-trihydroxyacetophenone monohydrate (THAP) containing 40 mM ACD as a matrix additive was prepared.

[0209] A 40 mg / mL 50% ACN aqueous solution of THAP (THAP-2 solution) containing 70 mM ACD as a matrix additive was prepared.

[0210] The prepared 3-HPA-1 solution and 2,4-DHAP solution were mixed at a mixing ratio of (1:1), (3:1), and (1:3) (v / v) to prepare various mixed matrix (3-HPA-1+2,4-DHAP) solutions ((mixing ratio 1:1, 55 mMACD), (mixing ratio 3:1, 48 mMACD), (mixing ratio 1:3, 63 mMACD)).

[0211] The prepared 3-HPA-2 solution and 2,4-DHAP solution were mixed at a mixing ratio of (1:1), (3:1), and (1:3) (v / v) to prepare various mixed matrix (3-HPA-2+2,4-DHAP) solutions ((mixing ratio 1:1, 70 mMACD), (mixing ratio 3:1, 70 mMACD), (mixing ratio 1:3, 70 mMACD)).

[0212] The prepared 3-HPA-1 solution and THAP-1 solution were mixed at a mixing ratio of (1:1), (3:1), and (1:3) (v / v) to prepare various mixed matrix (3-HPA-1+THAP-1) solutions ((mixing ratio 1:1, 40 mMACD), (mixing ratio 3:1, 40 mMACD), (mixing ratio 1:3, 40 mMACD)).

[0213] The prepared 3-HPA-2 solution and THAP-2 solution were mixed at a mixing ratio of (1:1), (3:1), and (1:3) (v / v) to prepare various mixed matrix (3-HPA-2+THAP-2) solutions ((mixing ratio 1:1, 70 mMACD), (mixing ratio 3:1, 70 mMACD), (mixing ratio 1:3, 70 mMACD)).

[0214] <3. Preparation of analytical samples>

[0215] The sample solution prepared in 1. and the mixed matrix solution prepared in 2. were mixed at 1:1 (v / v), and 1 μL of the obtained mixed solution was dropped onto a sample plate (SUS plate) and dried.

[0216] <4. Mass spectrometry analysis>

[0217] For mass spectrometry, a MALDI digital ion trap mass spectrometer (MALDI-DITMS, manufactured by Shimadzu Corporation, trade name: MALDImini-1) was used. The sample plate carrying the analytical sample prepared in 3. was inserted into the MALDI-DITMS, and MS measurement was performed in the positive mode using the grating function.

[0218] <Results>

[0219] Fig.14 , 15 16 shows the mass spectra of Patisiran when using various matrix solutions and their enlarged images, and summarizes the [M+H] + The following table shows the details of the detection of the two samples (detection of the two samples, sensitivity (mV, S / N), resolution (R), adduct detection, base separation). Regarding sensitivity and resolution, the [M+H] + The peak value. It should be noted that Fig.15 and Fig.16 For data obtained on the same day, Fig.14 For data obtained on a different date than these.

[0220] Depend on Figures 14 to 16 It was confirmed that the mixed matrix containing 3-HPA and THAP at a mixing ratio of 1:1 or 1:3, especially the mixed ratio of 1:1, had good sensitivity and high resolution. In addition, [M+H] was detected for both the sense and antisense strands of Patisilan while suppressing the detachment of bases and the formation of alkali metal ion adducts. + ( Fig.16(d), (e), (h), (i)). It was confirmed that the mixed matrix 3-HPA+THAP is particularly effective for MS determination of 21 bases long nucleic acid (RNA) with relatively high mass. Figure 6 As shown in the figure, when the nucleic acid sample is mipomersen as DNA, the mixed matrix containing 3-HPA and 2,4-DHAP is most suitable for [M+H] + Therefore, the preferred mixed matrix type varies depending on the type of nucleic acid, and it is believed that the difference in the constituent sugars of DNA and RNA leads to different affinities with the matrix.

[0221] (Example 7)

[0222] Then, mass spectrometry was performed in the same manner as in Example 6, except that the method for preparing the matrix solution was different, to examine the concentrations of diammonium hydrogen citrate (ACD) and acetonitrile (ACN) as matrix additives.

[0223] <2. Preparation of matrix solution>

[0224] A 40 mg / mL 50% ACN aqueous solution of 3-hydroxypicolinic acid (3-HPA) containing 40, 70, and 100 mM ACD as a matrix additive was prepared (the ACD concentrations from small to large were 3-HPA-1 solution, 3-HPA-2 solution, and 3-HPA-3 solution).

[0225] A 40 mg / mL 50% ACN aqueous solution of 2,4,6-trihydroxyacetophenone (THAP) containing 40, 70, and 100 mM ACD as a matrix additive was prepared (the ACD concentrations in descending order are THAP-1 solution, THAP-2 solution, and THAP-3 solution).

[0226] A 40 mg / mL 70% ACN aqueous solution of 3-HPA containing 40, 70, and 100 mM ACD as a matrix additive was prepared (the ACD concentrations in descending order are 3-HPA-4 solution, 3-HPA-5 solution, and 3-HPA-6 solution).

[0227] A 40 mg / mL 70% ACN aqueous solution of THAP containing 40, 70, and 100 mM ACD as a matrix additive was prepared (the ACD concentrations were in descending order: THAP-4 solution, THAP-5 solution, and THAP-6 solution).

[0228] The prepared 3-HPA-1~6 solutions and THAP-1~6 solutions were mixed in a mixing ratio of 1:1 (v / v) to prepare mixed matrix 3-HPA-1+THAP-1 solution (40mMACD, 50% ACN / Water), 3-HPA-2+THAP-2 solution (70mMACD, 50% ACN / Water), 3-HPA-3+THAP-3 solution (100mMACD, 50% ACN / Water), 3-HPA-4+THAP-4 solution (40mMACD, 70% ACN / Water), 3-HPA-5+THAP-5 solution (70mMACD, 70% ACN / Water), and 3-HPA-6+THAP-6 solution (100mMACD, 70% ACN / Water), respectively.

[0229] <Results>

[0230] Fig.17 The mass spectra of Patisiran when using various matrices and their enlarged images are shown, as well as the sum of [M+H] of two Patisiran samples (sense strand and antisense strand). + The following table shows the details of the detection of the two samples (detection of the two samples, sensitivity (mV, S / N), resolution (R), adduct detection, base separation). For sensitivity and resolution, the [M+H] + The value of the peak.

[0231] Depend on Fig.17 When a mixed matrix of 3-HPA and THAP (mixing ratio 1:1) was used, the ACD concentration was 40-100 mM and the solvent acetonitrile concentration was 50-70%, and the sensitivity was good and the high resolution was high. In addition, the sense and antisense strands of Patisilan were detected while suppressing the detachment of bases and the formation of alkali metal ion adducts. [M+H] + Among them, the best [M+H] + peak( Fig.17 (d)).

[0232] [Way]

[0233] It will be apparent to those skilled in the art that the above-described exemplary embodiments are specific examples of the following aspects.

[0234] (Item 1)

[0235] A method for analyzing nucleic acid according to one embodiment of the present invention is:

[0236] The nucleic acid contained in the sample is analyzed by a matrix-assisted laser desorption ionization mass spectrometer using 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.

[0237] Thus, the [M+H] of nucleic acid can be detected with high sensitivity. + or [MH] - , and fragment ions generated by the dissociation of the ion.

[0238] (Item 2)

[0239] The method for analyzing nucleic acid according to item 1, wherein

[0240] When the mixed matrix is ​​a mixed matrix containing 3-hydroxypicolinic acid and 2,4-dihydroxyacetophenone, the mixing ratio of 3-hydroxypicolinic acid to 2,4-dihydroxyacetophenone may be 10:1 to 1:5.

[0241] This makes it possible to detect nucleic acids, especially DNA, with higher sensitivity. + or [MH] - , and fragment ions generated by the dissociation of the ion.

[0242] (Item 3)

[0243] The method for analyzing nucleic acid according to item 1, wherein

[0244] When the mixed base comprises 3-hydroxypicolinic acid and 2,4,6-trihydroxyacetophenone monohydrate, the mixing ratio of 3-hydroxypicolinic acid to 2,4,6-trihydroxyacetophenone monohydrate may be 1:1 to 1:3.

[0245] This makes it possible to detect nucleic acids, especially RNA, with higher sensitivity. + or [MH] - , and fragment ions generated by the dissociation of the ion.

[0246] (Item 4)

[0247] The method for analyzing nucleic acid according to any one of items 1 to 3, wherein

[0248] The mixed matrix may further comprise diammonium hydrogen citrate as a matrix additive.

[0249] This makes it possible to detect [M+H] of nucleic acid with higher sensitivity. + or [MH] - , and fragment ions generated by the dissociation of the ion.

[0250] (Item 5)

[0251] The method for analyzing nucleic acid according to any one of items 1 to 4 may include the following steps:

[0252] In a data acquisition step, a mass spectrometry analysis is performed on a sample containing the nucleic acid using the mixed matrix to acquire [M+H] + or [MH] - mass spectral data of the plurality of fragment ions generated by dissociation; and

[0253] The data analysis step extracts peaks of a plurality of fragment ions derived from the nucleic acid from the mass spectrum data, and determines the structure of the nucleic acid based on mass information of the peaks.

[0254] This enables structural analysis of nucleic acids.

[0255] (Item 6)

[0256] The method for analyzing nucleic acid according to item 5, wherein

[0257] The matrix-assisted laser desorption ionization mass spectrometry device is an ion trap type matrix-assisted laser desorption ionization mass spectrometry device, which comprises: an ion source based on a matrix-assisted laser desorption ionization method, an ion capture unit for separating and capturing ions of a predetermined mass-to-charge ratio from ions generated in the ion source, and a detection unit for detecting ions captured by the ion capture unit.

[0258] The nucleic acid analysis method comprises: an analysis condition acquisition step of acquiring [M+H] for detecting the nucleic acid contained in the sample using the matrix-assisted laser desorption ionization mass spectrometry apparatus; + or [MH] - standard analysis conditions for the ion quantity setting item related to the ion quantity generated in the above-mentioned ion source, the mass-to-charge ratio range setting item related to the mass-to-charge ratio range of the ions captured in the above-mentioned ion capturing section, and the signal intensity setting item related to the ion signal intensity in the above-mentioned detection section during the first measurement,

[0259] The data acquisition step may be a step of performing the [M+H] analysis for detecting the nucleic acid under the changed analysis conditions obtained by changing at least one of the ion amount setting item, the mass-to-charge ratio range setting item, and the signal intensity setting item in the standard analysis conditions acquired in the analysis condition acquisition step. + or [MH] - The second measurement of the plurality of fragment ions generated by dissociation is performed to obtain mass spectrum data of the plurality of fragment ions.

[0260] This makes it possible to detect fragment ions required for structural analysis with high sensitivity in a wide mass range from a low mass range to a high mass range, and to more reliably perform structural analysis of nucleic acids.

[0261] (Item 7)

[0262] The method for analyzing nucleic acid according to item 6, wherein

[0263] The changed analysis conditions may be: the analysis conditions of the ion quantity setting item are changed so that the amount of ions generated in the ion source increases compared to the first measurement of the sample under the standard analysis conditions, so that the [M+H] + or [MH] - The conditions obtained by changing the analysis conditions of the above-mentioned mass-to-charge ratio range setting item in a manner that increases the mass-to-charge ratio of ions on the lower mass side, or the conditions obtained by changing the analysis conditions of the above-mentioned signal intensity setting item in a manner that increases the signal intensity of ions in the above-mentioned detection unit.

[0264] This makes it possible to detect with high sensitivity the fragment ions on the low mass-to-charge ratio side among the fragment ions required for structural analysis, and to more reliably perform structural analysis of nucleic acids.

[0265] (Item 8)

[0266] The method for analyzing nucleic acid according to item 6 or 7, wherein

[0267] The mass-to-charge ratio range setting item is the time from the ion source irradiating the laser to the ion trapping unit applying the trapping voltage for trapping ions.

[0268] The changed analysis conditions may be conditions in which the time is changed to a value shorter than the standard analysis conditions.

[0269] This makes it possible to detect with high sensitivity the fragment ions on the low mass-to-charge ratio side among the fragment ions required for structural analysis, and to more reliably perform structural analysis of nucleic acids.

[0270] (Item 9)

[0271] The method for analyzing nucleic acid according to item 8, wherein

[0272] The changed analysis condition may be a condition in which the time is set to a value shorter than the standard analysis condition by 1 to 3 μsec.

[0273] This makes it possible to detect with high sensitivity the fragment ions on the low mass-to-charge ratio side among the fragment ions required for structural analysis, and to more reliably perform structural analysis of nucleic acids.

[0274] (Item 10)

[0275] The method for analyzing nucleic acid according to any one of items 1 to 9, wherein

[0276] The matrix-assisted laser desorption ionization mass spectrometry device may be a digital ion trap type.

[0277] This makes it possible to detect [M+H] of nucleic acid with higher sensitivity. + or [MH] - , and fragment ions generated by the dissociation of the ion.

[0278] (Item 11)

[0279] The matrix for matrix-assisted laser desorption ionization mass spectrometry analysis of nucleic acid according to one embodiment of the present invention is:

[0280] A matrix comprising 3-hydroxypicolinic acid and 2,4-dihydroxyacetophenone or a matrix comprising 3-hydroxypicolinic acid and 2,4,6-trihydroxyacetophenone monohydrate.

[0281] Thus, the [M+H] of nucleic acid can be detected with high sensitivity. + or [MH] - , and fragment ions generated by the dissociation of the ion.

[0282] Description of Reference Numerals

[0283] 1…Ion source

[0284] 2…Ion trap

[0285] 3…Detection Department

[0286] 11…Laser irradiation part

[0287] 12…Sample stage

[0288] 21…Ring electrode

[0289] 22, 23…End cap electrode

[0290] 31…Conversion multiplier

[0291] 32…Detector (Secondary Electron Multiplier Tube)

Claims

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

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

5.

3. The method for analyzing nucleic acid according to claim 1, wherein: When the mixed matrix is ​​a mixed matrix comprising 3-hydroxypicolinic acid and 2,4,6-trihydroxyacetophenone monohydrate, 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 acid according to claim 1, wherein The mixed matrix also contains diammonium hydrogen citrate as a matrix additive.

5. The method for analyzing nucleic acid according to claim 1, comprising the following steps: A data acquisition step of performing mass spectrometry analysis on a sample containing the nucleic acid using the mixed matrix to acquire [M+H] + or [MH] - mass spectral data of the plurality of fragment ions generated by dissociation; and The data analysis step extracts peaks of a plurality of fragment ions derived from the nucleic acid from the mass spectrum data, and determines the structure of the nucleic acid based on mass information of the peaks.

6. The method for analyzing nucleic acid according to claim 5, wherein: The matrix-assisted laser desorption ionization mass spectrometry device is an ion trap type matrix-assisted laser desorption ionization mass spectrometry device, which comprises: an ion source based on a matrix-assisted laser desorption ionization method, an ion capture unit for separating and capturing ions of a predetermined mass-to-charge ratio from ions generated by the ion source, and a detection unit for detecting ions captured by the ion capture unit. The nucleic acid analysis method comprises: an analysis condition acquisition step of acquiring [M+H] for detecting the nucleic acid contained in a sample using the matrix-assisted laser desorption ionization mass spectrometry device; + or [MH] - standard analysis conditions for the ion quantity setting item related to the ion quantity generated in the ion source, the mass-to-charge ratio range setting item related to the mass-to-charge ratio range of the ions captured in the ion capturing section, and the signal intensity setting item related to the ion signal intensity in the detection section during the first measurement, The data acquisition step is a step of performing a [M+H] analysis for detecting the nucleic acid under the changed analysis conditions obtained by changing at least one of the ion amount setting item, the mass-to-charge ratio range setting item, and the signal intensity setting item in the standard analysis conditions acquired in the analysis condition acquisition step. + or [MH] - The plurality of fragment ions generated by dissociation are subjected to a second measurement to obtain mass spectrum data of the plurality of fragment ions.

7. The method for analyzing nucleic acid according to claim 6, wherein: The changed analysis conditions are conditions obtained by changing the analysis conditions of the ion quantity setting item so as to increase the amount of ions generated in the ion source compared to when the first measurement of the sample is performed under the standard analysis conditions, and by preferentially capturing [M+H] + or [MH] - The analysis conditions of the mass-to-charge ratio range setting item are changed in a manner that increases the mass-to-charge ratio of ions on the lower mass side, or the analysis conditions of the signal intensity setting item are changed in a manner that increases the signal intensity of ions in the detection unit.

8. The method for analyzing nucleic acid according to claim 6, wherein: The mass-to-charge ratio range setting item is the time from the ion source irradiating the laser to the ion trapping unit applying a trapping voltage for trapping ions. The changed analysis conditions are conditions in which the time is changed to a value shorter than the standard analysis conditions.

9. The method for analyzing nucleic acid according to claim 8, wherein: The changed analysis conditions are conditions in which the time is set to a value shorter than the standard analysis conditions by 1 to 3 μsec.

10. The method for analyzing nucleic acid according to claim 1, wherein: The matrix-assisted laser desorption ionization mass spectrometry analysis device is of a digital ion trap type.

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

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