Nucleic acid structural analysis method

Abstract

This nucleic-acid structural analysis method utilizes an ion trap mass spectrometer having an ion source based on MALDI, the method including: an ionization step for ionizing with the ion source a nucleic acid contained in a sample; an ionic dissociation step for dissociating a protonated molecule or a deprotonated molecule of the nucleic acid, generated in the ionization step, by collision-induced dissociation inside the ion trap in the mass spectrometer, to generate a plurality of fragment ions; a mass analysis step for carrying out mass analysis on the plurality of fragment ions generated in the ionic dissociation step, to acquire mass information about the plurality of fragment ions; and a structure-determining step for determining at least a part of the structure of the nucleic acid, on the basis of the mass information about the plurality of fragment ions acquired in the mass analysis step. This can provide a novel nucleic-acid structural analysis method by which fragment ions can be detected with advanced sensitivity.

Description

[Technical Field]

[0001] The present invention relates to a method for analyzing the structure of nucleic acids using mass spectrometry. [Background technology]

[0002] Nucleic acids are biopolymers in which nucleotides, each consisting of a base, sugar, and phosphate, are linked by phosphodiester bonds, and are classified as deoxyribonucleic acid (DNA) or ribonucleic acid (RNA) depending on the sugar. Among these, nucleic acids, also known as oligonucleotides, which are polymerized from several to around 20 nucleotides, can be chemically synthesized, and in recent years, active research has been conducted into the application of oligonucleotides as nucleic acid medicines.

[0003] A method using mass spectrometry is known as a method for structural analysis of nucleic acids and oligonucleotides, i.e., a method for identifying the bonding order (base sequence) of nucleotides constituting DNA or RNA, the type of chemical modification, or the site of said chemical modification. In this method, the nucleic acid to be analyzed is intentionally fragmented, and the various partial structures generated are analyzed by mass spectrometry.

[0004] For example, in the method described in Non-Patent Document 1, MS / MS analysis (MS ) is performed using a tandem time-of-flight (TOF) mass spectrometer (MALDI-TOF / TOF-MS) having an ion source based on the matrix-assisted laser desorption / ionization (MALDI) method. 2 By performing this analysis, the structure of nucleic acids with a molecular weight of about 1200, which are composed of four nucleotides, is analyzed. Specifically, first, in the mass separation section at the front, the protonated molecule ([M+H] +) is selected as a precursor ion, and then in the subsequent collision chamber, the precursor ion is dissociated by collision-induced dissociation (CID) to generate various fragment ions (also called product ions).Then, in the subsequent mass separation section, the various fragment ions are separated, and structural analysis is performed based on the mass information of the fragment ions obtained by detecting them.

[0005] Furthermore, since the MALDI method is generally a soft ionization method, ions are difficult to dissociate. However, it is known that dissociation of ions during ionization can be promoted, for example, by increasing the intensity of the laser light to increase the energy during ionization or by using a special matrix. This technique of dissociating ions simultaneously with or immediately after ionization is called in-source decay (ISD). In the methods described in Non-Patent Documents 2 to 4, a time-of-flight mass spectrometer (MALDI-TOF-MS) equipped with a MALDI ion source is used to perform mass analysis of various fragment ions of nucleic acids generated by in-source decay, and structural analysis of nucleic acids is performed based on the mass information of the fragment ions obtained thereby. [Prior art documents] [Non-patent literature]

[0006] [Non-Patent Document 1] Thomas E. Andersen, and 2 others, "RNA Fragmentation in MALDI Mass Spectrometry Studied by H / D-Exchange: Mechanisms of General Applicability to Nucleic Acids", Journal of the American Society for Mass Spectrometry, (USA), 2006, 17, pp.1353-1368 [Non-patent document 2] Nathan A. Hagan, 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 3] Hisao 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 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] 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 Summary of the Invention [Problem to be solved by the invention]

[0007] To perform structural analysis by MS / MS analysis using collision-induced dissociation as in Non-Patent Document 1, a protonated molecule ([M+H] + ) or deprotonated molecule ([MH] - ) (M is a molecule, H is a hydrogen atom) must be introduced into the collision cell in sufficient quantities. However, in the mass spectrometry of nucleic acids, nucleic acids tend to decompose during ionization, especially the larger the molecular weight of the nucleic acid, so [M+H] + or [MH] - It is difficult to generate [M+H]. + or [MH] - In addition, alkali metal ion adducts of nucleic acids are easily formed, so [M+H] + or [MH] - Therefore, it is difficult to introduce a sufficient amount of molecular weight-related ions into the collision cell, which can make structural analysis of nucleic acids by MS / MS analysis difficult. Furthermore, in structural analysis using in-source decay as in Non-Patent Documents 2 to 4, there is a problem that the resolution and sensitivity of some of the detected fragment ions tend to be low.

[0008] The present invention has been made in view of the above-mentioned problems, and has as its object to provide a novel method for analyzing the structure of nucleic acids, which is capable of detecting fragment ions with high sensitivity. [Means for solving the problem]

[0009] The method for analyzing the structure of nucleic acid according to the present invention, which has been achieved to solve the above problems, comprises: A method for analyzing the structure of nucleic acids using an ion trap mass spectrometer having an ion source based on matrix-assisted laser desorption / ionization, comprising: an ionization step of ionizing nucleic acids contained in a sample using the ion source; an ion dissociation step of dissociating the protonated or deprotonated molecules of the nucleic acid produced in the ionization step by collision-induced dissociation inside an ion trap of the mass spectrometer to produce a plurality of fragment ions; a mass analysis step of performing mass analysis on the plurality of fragment ions generated by the ion dissociation step to obtain mass information of the plurality of fragment ions; a structure determination step of determining at least a part of the structure of the nucleic acid based on mass information of the plurality of fragment ions obtained by the mass analysis step; It has the following characteristics. [Effects of the Invention]

[0010] As a result of extensive research, the inventors of the present invention have found that when mass spectrometry of nucleic acids is performed using an ion trap mass spectrometer equipped with an ion source based on the MALDI method, the influence of the formation of alkali metal ion adducts is suppressed, and as a result, the [M+H] of nucleic acids is reduced. + or [MH] - Based on this finding, we have discovered that the above-mentioned mass spectrometer can be used to detect nucleic acids by collision-induced dissociation. 2 Analysis revealed that [M+H] + or [MH] - It was also found that the fragment ions generated from the ion beam can be detected with high sensitivity.

[0011] The collision-induced dissociation performed by the ion trap mass spectrometer used in the present invention involves a relatively low kinetic energy of approximately 1000 eV (1 keV) or less (several to several hundred eV) during collisions between the collision gas and the target ions, and is therefore called low-energy collision-induced dissociation (LE-CID). In LE-CID, multiple low-energy collisions cause the collision energy to accumulate as vibrational energy within the sample molecules, inducing ion dissociation. The accumulated energy is redistributed based on the molecular structure, resulting in ion dissociation beyond the critical point of the bonded state. On the other hand, in high-energy collision-induced dissociation (HE-CID), where the collision energy is 1000 eV or more, most fragment ions are generated in a single collision, and the ions dissociate primarily through simple fragmentation at the collision site. Thus, the ion dissociation mechanisms differ between LE-CID and HE-CID, and the resulting fragment ions are also different.

[0012] According to the method for analyzing the structure of nucleic acid of the present invention, fragment ions can be detected with high sensitivity, and a novel method for analyzing the structure of nucleic acid that utilizes an ion dissociation method different from conventional methods can be provided. [Brief explanation of the drawings]

[0013] [Figure 1] FIG. 1 shows the mass spectrum of standard nucleic acid A obtained by MS analysis in Example 1. [Figure 2] FIG. 1 shows the product ion spectrum of standard nucleic acid A obtained by MS2 analysis in Example 1. DETAILED DESCRIPTION OF THE INVENTION

[0014] Hereinafter, one embodiment of the method for analyzing the structure of nucleic acid according to the present invention will be described.

[0015] (nucleic acid) The number of nucleotides constituting the nucleic acid to be analyzed in this embodiment is not particularly limited, but oligonucleotides in which several to several tens of nucleotides are linked are preferred. Of these, oligonucleotides in which approximately 2 to 20 nucleotides are linked are preferred. Furthermore, the nucleic acid may be a natural product obtained from an organism or a processed product thereof, or may be a chemically synthesized artificial nucleic acid.

[0016] (Mass spectrometer) In this embodiment, an ion trap type mass spectrometer having an ion source based on the MALDI method is used. Specifically, the mass spectrometer is equipped with an ion source based on the MALDI method and an ion trap that holds ions therein and has the functions of separating ions according to their mass-to-charge ratio and dissociating ions by collision-induced dissociation. This apparatus not only performs analysis without ion dissociation (hereinafter referred to as MS analysis), but also MS analysis, which repeats ion selection and dissociation one or more times. n Analysis (n is an integer of 2 or more) can be performed.

[0017] The term "ion trap mass spectrometer" as used herein refers to a mass spectrometer that has an ion trap for trapping ions generated in an ion source. Specifically, it includes a mass spectrometer that uses the mass separation function of the ion trap itself to eject ions trapped in the ion trap in ascending order of mass-to-charge ratio (m / z) and detects the ions with a detector located outside the ion trap. It also includes a mass spectrometer that separates ions simultaneously ejected from the ion trap according to their mass-to-charge ratio in a mass separator located outside the ion trap, such as a time-of-flight mass analyzer, and detects the ions with a detector also located outside the ion trap.

[0018] The type of ion trap is not particularly limited. In the case of an RF trap that uses a radio frequency (RF) electric field to trap and eject ions, it may be an ion trap that traps ions using an electric field generated by applying a sinusoidal radio frequency voltage to a ring electrode, or a digital ion trap that traps ions using an electric field generated by applying a square wave voltage to a ring electrode, which is generated by switching two different voltages at high speed. In a digital ion trap, the m / z range of ions that can be trapped is controlled by changing the frequency while maintaining the amplitude (voltage value) of the square wave voltage constant. A digital ion trap is preferably used as the ion trap.

[0019] The method for analyzing the structure of nucleic acid of this embodiment includes an ionization step of ionizing nucleic acid contained in a sample using an ion source based on the MALDI method; an ion dissociation step of dissociating protonated or deprotonated molecules of nucleic acid produced in the ionization step by collision-induced dissociation inside an ion trap; a mass analysis step of performing mass analysis on multiple fragment ions derived from the nucleic acid produced in the ion dissociation step; and a structure determination step of determining the structure of the nucleic acid based on mass information of the multiple fragment ions obtained in the mass analysis step.

[0020] (Ionization process) In the ionization step, an analytical sample containing nucleic acids and a matrix substance is irradiated with laser light in an ion source for the MALDI method, thereby ionizing the nucleic acids together with the matrix substance.

[0021] The matrix substance can be selected appropriately depending on the type of nucleic acid to be analyzed, such as 3-hydroxypicolinic acid (3-HPA), 2,4-dihydroxyacetophenone (2,4-DHAP), 2,5-dihydroxybenzoic acid (DHB), 2',4',6'-trihydroxyacetophenone monohydrate (THAP), 6-aza-2-thiothymine (ATT), 3-aminopyrazine-2-carboxylic acid (APCA), anthranilic acid (AA), and nicotinic acid (NA). Among these, 3-HPA, 2,4-DHAP, and THAP are preferably used, with 3-HPA and 2,4-DHAP being more preferred, and 3-HPA being particularly preferred. Mixed matrices containing two or more matrix substances may also be used, with mixed matrices of 3-HPA and 2,4-DHAP, 3-HPA and THAP, and 2,4-DHAP and THAP being particularly preferred.

[0022] The analytical sample is prepared by preparing a mixed solution of a nucleic acid-containing sample and a matrix substance, and then drying the mixed solution on a sample plate of a mass spectrometer. The mixed solution may be prepared in advance and then dropped onto the sample plate and dried, or the mixed solution may be prepared on the sample plate and then dried as is.

[0023] The analytical sample may further contain a matrix additive. Diammonium hydrogen citrate (ammonium citrate dibasic: ACD) can be used as the 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.

[0024] When the analytical sample contains a matrix additive, the order in which the nucleic acid-containing sample, matrix substance, and matrix additive are mixed is not particularly limited. However, it is preferable to prepare a matrix / additive mixed solution containing the matrix substance and matrix additive in advance and then mix the nucleic acid-containing sample solution with the matrix / additive mixed solution to prepare the analytical sample. In this case, the analytical sample may be prepared by premixing the sample solution and the matrix / additive mixed solution, dropping the mixed solution onto a sample plate and drying it, or by dropping the sample solution and the matrix / additive mixed solution onto the sample plate separately, mixing them on the sample plate, and drying them. The mixing ratio of the sample solution and the matrix / additive mixed solution is not particularly limited. Preparing the matrix / additive mixed solution in advance facilitates the preparation of the analytical sample. The concentration of the matrix additive in the matrix / additive mixed solution is preferably 10 to 100 mM, more preferably 30 to 70 mM, to generate sufficient amounts of ions related to the molecular weight of nucleic acids. In this specification, a numerical range from a lower limit value to an upper limit value is expressed using the symbol "to" as "(lower limit value) to (upper limit value)", but the numerical range expressed in this manner includes the lower limit value itself and the upper limit value itself.

[0025] (Ion dissociation process) In the ion dissociation step, all ions generated in the ionization step are first trapped in the ion trap, and the protonated molecules ([M+H] + ) or deprotonated molecule ([MH] -Ions other than the protonated molecule are ejected from the ion trap, and the protonated or deprotonated molecule is selected as the precursor ion. Next, an inert gas such as argon is introduced into the ion trap, and the protonated or deprotonated molecule is dissociated by collision-induced dissociation. This generates various fragment ions (product ions) derived from nucleic acids.

[0026] The collision-induced dissociation in the ion trap mass spectrometer used in this embodiment corresponds to low-energy collision-induced dissociation (LE-CID). In LE-CID, multiple collisions cause collision energy to accumulate as vibrational energy within sample molecules, inducing ion dissociation. The accumulated energy is redistributed in accordance with the molecular structure, causing ions to dissociate from points beyond the limit of the molecular bonding state. On the other hand, the collision-induced dissociation in a time-of-flight mass spectrometer corresponds to high-energy collision-induced dissociation (HE-CID), in which ions simply dissociate at the point of collision. Thus, the type of CID (LE-CID or HE-CID) differs depending on the mass spectrometer used, and LE-CID differs from HE-CID in the way ions dissociate.

[0027] (Mass spectrometry process) In the mass spectrometry step, various fragment ions derived from nucleic acids generated in the ion dissociation step are subjected to mass spectrometry (MS 2 In the case where the mass spectrometer used in this embodiment utilizes the mass separation function of the ion trap itself, mass analysis is performed by ejecting fragment ions from the ion trap in ascending order of mass-to-charge ratio, and detecting the ejected ions with a detector located outside the ion trap. In the case where the mass spectrometer utilizes the mass separation function of a mass separation section other than an ion trap, mass analysis is performed by introducing various fragment ions ejected simultaneously from the ion trap into a mass separator located outside the ion trap, where the ions are separated according to their mass-to-charge ratio, and detecting the separated fragment ions with a detector also located outside. 2By performing this analysis, mass spectra (product ion spectra) of various fragment ions can be obtained. As mentioned above, the way ions dissociate differs between LE-CID and HE-CID, so MS using LE-CID with an ion trap mass spectrometer is 2 The analysis was performed using MS with HE-CID. 2 A different product ion spectrum can be obtained from the analysis.

[0028] (Structure determination process) In the structure determination step, peaks corresponding to various fragment ions are extracted from the mass spectrum obtained in the mass analysis step, and the various fragment ions are assigned based on the mass-to-charge ratio (mass information) indicated by the peaks. The results are combined to determine at least a portion of the structure of the original nucleic acid. Structure determination includes sequence analysis and identifying the type of chemical modification or the site where the chemical modification has been applied by sequence analysis. Structure determination may also be performed using database search or de novo sequencing.

[0029] The method for analyzing the structure of nucleic acid according to the present invention will be explained below with reference to examples, but these are merely examples and the present invention is not limited thereto. [Example]

[0030] <1. Preparation of sample solution> As a sample solution, a 100 pmol / μL aqueous solution of standard nucleic acid A (5′-CAATGTGC-3′: MW 2409.6) was prepared.

[0031] 2. Preparation of matrix / additive mixture solution A 40 mg / mL aqueous solution of 3-hydroxypicolinic acid (3-HPA) in 50% acetonitrile (ACN) containing 40 mM diammonium hydrogen citrate (ACD) as a matrix additive was prepared as a matrix-additive mixed solution.

[0032] 3. Preparation of analytical samples The sample solution prepared in 1. and the matrix / additive mixed solution prepared in 2. were mixed at a 1:1 (v / v) ratio, and 1 μL of the resulting mixture was dropped onto a sample plate (SUS plate) and dried.

[0033] <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 analytical sample prepared in 3. was inserted into the MALDI-DITMS, and MS analysis and MS 2 The analysis was carried out.

[0034] <5.Results> Figure 1 shows the mass spectrum obtained by MS analysis. The arrows in the figure indicate the protonated molecules ([M+H] + ) detection status. From Figure 1, [M+H] + was confirmed to be detected with high sensitivity.

[0035] Figure 2 shows [M+H] + MS 2 The product ion spectrum obtained by the analysis is shown in Figure 2. In Figure 2, each peak in the mass spectrum is given the name of the corresponding fragment ion species of the oligonucleotide (the general name proposed in Non-Patent Document 5). This name represents the nuclear ion species as a fragment ion series according to the nomenclature of dissociation patterns of nucleic acids. In this nomenclature, fragment ions containing the 5' end are named as a n , b n , c n , d n and the fragment ion containing the 3' end in the opposite direction is denoted x m , y m , z m , w mThe subscripts n and m indicate the number of constituent units (by definition, the number of bases) from the corresponding end to the dissociation site. As shown in Figure 2, the peaks of various fragment ions were detected with high sensitivity, and sufficient types of fragment ions could be assigned to perform structural analysis based on the w-series ions, enabling the analysis of the entire base sequence of standard nucleic acid A.

[0036] [Aspect] It will be appreciated by those skilled in the art that the exemplary embodiments described above are examples of the following aspects.

[0037] (Section 1) A method for analyzing the structure of nucleic acid according to one aspect of the present invention comprises: A method for analyzing the structure of nucleic acids using an ion trap mass spectrometer having an ion source based on matrix-assisted laser desorption / ionization, comprising: an ionization step of ionizing nucleic acids contained in a sample using the ion source; an ion dissociation step of dissociating the protonated or deprotonated molecules of the nucleic acid produced in the ionization step by collision-induced dissociation inside an ion trap of the mass spectrometer to produce a plurality of fragment ions; a mass analysis step of performing mass analysis on the plurality of fragment ions generated by the ion dissociation step to obtain mass information of the plurality of fragment ions; a structure determination step of determining at least a part of the structure of the nucleic acid based on mass information of the plurality of fragment ions obtained by the mass analysis step; It has the following characteristics.

[0038] This makes it possible to provide a novel method for analyzing the structure of nucleic acids, which allows for the sensitive detection of fragment ions.

[0039] (Section 2) The method for analyzing the structure of nucleic acid according to item 1 includes: The ionization step may be carried out by irradiating a laser beam onto an analytical sample containing the nucleic acid, a matrix substance, and a matrix additive which is diammonium hydrogen citrate.

[0040] This allows for the generation of more protonated or deprotonated nucleic acid molecules, enabling fragment ions to be detected with higher sensitivity.

[0041] (Section 3) The method for analyzing the structure of nucleic acid according to item 2 includes the steps of: The matrix material may be 3-hydroxypicolinic acid.

[0042] This allows for the generation of more protonated or deprotonated nucleic acid molecules, enabling fragment ions to be detected with higher sensitivity.

[0043] (Section 4) The method for analyzing the structure of nucleic acid according to item 2 or 3 includes: When a matrix-additive mixed solution containing the matrix substance and the matrix additive is prepared, the concentration of the matrix additive in the matrix-additive mixed solution may be 10 to 100 mM.

[0044] This allows for the generation of more protonated or deprotonated nucleic acid molecules, enabling fragment ions to be detected with higher sensitivity.

[0045] (Section 5) The method for analyzing the structure of nucleic acid according to any one of items 2 to 4 includes the steps of: A matrix-additive mixed solution containing the matrix material and the matrix additive is prepared in advance, The analytical sample may be prepared by mixing the sample with the matrix / additive mixed solution.

[0046] This allows the sample for analysis to be easily prepared.

[0047] (Section 6) The method for analyzing the structure of nucleic acid according to any one of items 1 to 5 includes: The mass spectrometer may be a digital ion trap type mass spectrometer.

[0048] This allows for the generation of more protonated or deprotonated nucleic acid molecules, enabling fragment ions to be detected with higher sensitivity.

Claims

1. A method for analyzing the structure of nucleic acids using an ion trap mass spectrometer having an ion source based on matrix-assisted laser desorption / ionization, comprising: an ionization step of ionizing nucleic acids contained in a sample using the ion source; an ion dissociation step of dissociating the protonated or deprotonated molecules of the nucleic acid produced in the ionization step by collision-induced dissociation inside an ion trap of the mass spectrometer to produce a plurality of fragment ions; a mass analysis step of performing mass analysis on the plurality of fragment ions generated by the ion dissociation step to obtain mass information of the plurality of fragment ions; a structure determination step of determining at least a part of the structure of the nucleic acid based on mass information of the plurality of fragment ions obtained by the mass analysis step; A method for analyzing the structure of a nucleic acid having the formula:

2. 2. The method for analyzing the structure of nucleic acid according to claim 1, wherein the ionization step is carried out by irradiating a laser beam onto an analytical sample containing the nucleic acid, a matrix substance, and a matrix additive which is diammonium hydrogen citrate.

3. 3. The method for analyzing the structure of nucleic acid according to claim 2, wherein the matrix substance is 3-hydroxypicolinic acid.

4. 3. The method for analyzing the structure of nucleic acid according to claim 2, wherein when a matrix / additive mixed solution containing the matrix substance and the matrix additive is prepared, the concentration of the matrix additive in the matrix / additive mixed solution is 10 to 100 mM.

5. a matrix-additive mixed solution containing the matrix material and the matrix additive is prepared in advance; 3. The method for analyzing the structure of nucleic acid according to claim 2, wherein the sample for analysis is prepared by mixing the sample with the matrix / additive mixed solution.

6. 2. The method for analyzing the structure of nucleic acid according to claim 1, wherein the mass spectrometer is a digital ion trap type mass spectrometer.

7. A method for analyzing the structure of nucleic acid as described in claim 1, wherein the collision-induced dissociation involves colliding protonated or deprotonated molecules of the nucleic acid with a collision gas at a kinetic energy of 1000 eV (1 keV) or less.

8. A method for analyzing the structure of nucleic acid as described in claim 1, wherein the collision-induced dissociation involves multiple collisions in which protonated or deprotonated molecules of the nucleic acid collide with a collision gas multiple times, causing collision energy to be accumulated inside the molecule as vibrational energy, and the accumulated vibrational energy is redistributed in accordance with the structure of the molecule, resulting in dissociation of the molecule at a point where the binding state within the molecule exceeds a limit point.

9. A method for analyzing the structure of a nucleic acid as described in claim 1, wherein the nucleic acid is an oligonucleotide consisting of 2 to 20 nucleotides linked together.

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