Method for analyzing the structure of nucleic acids and apparatus for analyzing the structure of nucleic acids

Abstract

Provided is a nucleic acid structure analysis method comprising: a HAD-MSn analysis step (101) of dissociating ions derived from a test nucleic acid by introducing hydrogen radicals into a space containing the ions, and performing mass spectrometry of a plurality of thus generated fragment ions to collect information on the m / z of the plurality of fragment ions; and a structure estimation step (103) of estimating the structure of the test nucleic acid on the basis of the information on the m / z of the plurality of fragment ions obtained in the HAD-MSn analysis step. The present invention thereby makes it possible to handle precursor ions of various electric charges and facilitate structure estimation of nucleic acids based on mass spectra.

Description

[Technical Field]

[0001] This invention relates to a method for analyzing the structure of nucleic acids and an apparatus for analyzing the structure of nucleic acids. [Background technology]

[0002] In recent years, mass spectrometry has become widely used as one method for analyzing the structure of nucleic acids, that is, for determining the base sequence of DNA or RNA, or for identifying the type of chemical modification or the site where such modification has occurred. Such analyses utilize mass spectrometry (MS), which involves n-1 ion cleavage. n Using a mass spectrometer capable of performing analysis (where n is an integer greater than or equal to 2), MS is applied to the nucleic acid to be analyzed. n The analysis is performed. Specifically, ions generated from the target nucleic acid (test nucleic acid) are dissociated using an appropriate dissociation method to produce various fragment ions (also called product ions) in which the original ion (called the precursor ion) is fragmented. These fragment ions are separated and detected according to their m / z (mass-to-charge ratio), and a mass spectrum is created showing the m / z and detection intensity of each fragment ion.

[0003] MS of nucleic acids as described above n Known methods for dissociating precursor ions in analysis include collision-induced dissociation (CID), which promotes dissociation by colliding the precursor ion with a gas (usually an inert gas); infrared multiphoton dissociation (IRMPD), which promotes dissociation of the precursor ion by increasing its internal energy with an infrared laser; electron transfer dissociation (ETD), which collides the precursor ion with a negative ion; and electron capture dissociation (ECD), which irradiates the precursor ion with electrons. [Prior art documents] [Patent Documents]

[0004] [Patent Document 1] Japanese Unexamined Patent Application Publication No. 2016-223777 [Patent Document 2] International Publication No. 2015 / 133259 [Non-Patent Document]

[0005] [Non-Patent Document 1] Stefan Schurch, Mass Spectrometry Reviews, 2016, Volume35, Issue 4, pp.483-523 [Summary of the Invention] [Problems to be Solved by the Invention]

[0006] When a nucleic acid-derived precursor ion is dissociated by the CID method or the IRMPD method, fragment ions (base-eliminated ions) from which a nucleobase (hereinafter sometimes simply referred to as a base) has been eliminated may occur (for example, see Non-Patent Document 1), or internal fragment ions generated by dissociation of the sugar-phosphate backbone of the precursor ion at multiple sites, or neutral loss ions generated by removing a species having no charge from the precursor ion are likely to occur. These base-eliminated ions, internal fragment ions, or neutral loss ions do not contribute to the estimation of the base sequence. Therefore, when the CID method or the IRMPD method is used, there is a problem that it is difficult to analyze the mass spectrum and estimate the base sequence of the nucleic acid.

[0007] On the other hand, the ETD or ECD method is less likely to produce the base-desorbed ions, internal fragment ions, or neutral loss ions mentioned above. However, the ETD and ECD methods are generally only effective for positive ions, and it is difficult to dissociate negative ions. There is a method called negative ETD (NETD) that can dissociate negative ions, but even this method has the limitation that it cannot dissociate monovalent negative ions. Furthermore, the ETD and ECD methods, by their very nature, can only dissociate multivalent ions. As is well known, the ions produced by ionization methods such as matrix-assisted laser desorption / ionization (MALDI) are mainly monovalent positive ions, so the inability to dissociate monovalent positive ions is a major limitation for analytical techniques.

[0008] The present invention has been made in view of these points, and its objective is to provide a method and apparatus for analyzing the structure of nucleic acids that can handle precursor ions of various charges and facilitate the estimation of nucleic acid structure based on mass spectrometry. [Means for solving the problem]

[0009] The nucleic acid structure analysis method according to the present invention, which was developed to solve the above problems, HAD-MS involves introducing hydrogen radicals into a space containing ions derived from the nucleic acid being tested to dissociate the ions, and then using mass spectrometry to collect information on the m / z values ​​of the resulting multiple fragment ions. n Analysis steps, The aforementioned HAD-MS n A structure estimation step in which the structure of the test nucleic acid is estimated based on the m / z information of the plurality of fragment ions obtained in the analysis step, It possesses the following characteristics.

[0010] Furthermore, the nucleic acid structure analysis apparatus according to the present invention, which was developed to solve the above problems, HAD-MS involves introducing hydrogen radicals into a space containing ions derived from the nucleic acid being tested to dissociate the ions, and then using mass spectrometry to collect information on the m / z values ​​of the resulting multiple fragment ions. n Analysis execution unit, The aforementioned HAD-MS n A structure estimation unit estimates the structure of the test nucleic acid based on the m / z information of the plurality of fragment ions obtained by the analysis execution unit, It possesses the following characteristics. [Effects of the Invention]

[0011] According to the nucleic acid structure analysis method or nucleic acid structure analysis apparatus of the present invention described above, it is possible to handle precursor ions with various charges and to easily estimate the structure of nucleic acids based on mass spectrometry. [Brief explanation of the drawing]

[0012] [Figure 1] A schematic diagram of a nucleic acid structure analysis device according to one embodiment of the present invention. [Figure 2] A flowchart showing an example of the procedure for analyzing the structure of nucleic acids using the aforementioned apparatus. [Figure 3] A figure showing the mass spectrum of fragment ions obtained by CID-MS2 analysis of a nucleic acid sample, as well as the base sequence of the nucleic acid sample and the assigned series ions. [Figure 4] A figure showing the mass spectrum of fragment ions obtained by HAD-MS2 analysis of a nucleic acid sample, as well as the base sequence of the nucleic acid sample and the assigned series ions. [Figure 5] A flowchart illustrating another example of the procedure for analyzing the structure of nucleic acids using the aforementioned apparatus. [Modes for carrying out the invention]

[0013] The inventors of this invention are diligently researching a method of dissociating ions by attaching hydrogen radicals to ions as a method of unpaired electron-induced dissociation using uncharged particles, and have already proposed a method for efficiently dissociating peptide-derived ions using hydrogen radicals (see Patent Document 1). This dissociation method can dissociate monovalent ions and negative ions with high efficiency, which could not be dissociated by unpaired electron-induced dissociation methods using charged particles such as the ETD method and the ECD method.

[0014] In the process of repeatedly conducting various experiments on the ion dissociation method by attachment of hydrogen radical particles (Hydrogen Attachment Dissociation, hereinafter referred to as "HAD") as described above, the inventors discovered that when ions derived from nucleic acids are dissociated by HAD, fragment ions that do not contribute to the determination of nucleic acid structure, such as the base-deleted ions, neutral-loss ions, and internal fragment ions described above, are less likely to be generated. Based on this finding, the inventors conceived the present invention.

[0015] Hereinafter, an embodiment of the nucleic acid structure analysis apparatus and structure analysis method according to the present invention will be described with reference to the drawings. Figure 1 is a schematic configuration diagram showing the nucleic acid structure analysis apparatus according to this embodiment.

[0016] This nucleic acid structure analysis apparatus includes a mass spectrometer, which comprises an ion source 1 for ionizing the target sample component (i.e., the nucleic acid to be tested), an ion trap 2 for capturing the ions generated by the ion source 1 using a high-frequency electric field, a time-of-flight mass separation unit 3 for separating the ions ejected from the ion trap 2 according to m / z, and an ion detector 4 for detecting the separated ions. The mass spectrometer also includes a hydrogen radical supply unit 5 for supplying hydrogen radicals into the ion trap 2 to dissociate the ions trapped in the ion trap 2, a gas supply unit 6 for supplying a predetermined gas into the ion trap 2, a trap voltage generation unit 7, and a control unit 8. The detection signal from the ion detector 4 is input to a data processing unit 9. These components—ion source 1, ion trap 2, time-of-flight mass separation unit 3, ion detector 4, hydrogen radical supply unit 5, gas supply unit 6, trap voltage generation unit 7, and control unit 8—constitute the "HAD-MS" of this invention. n This corresponds to the "Analysis Execution Unit".

[0017] The control unit 8 and the data processing unit 9 are actually computers such as personal computers. The data processing unit 9 performs analytical processing characteristic of the nucleic acid structure analysis device according to this embodiment, and includes a mass spectrum creation unit 91, a peak extraction unit 92, a structure estimation unit 93, and a display processing unit 94 as functional blocks. These functional blocks are realized by executing a predetermined program pre-installed on the computer. Furthermore, a storage unit 95 provided in the computer constituting the data processing unit 9 stores a nucleic acid database (nucleic acid DB) 96 containing information such as the base sequences of numerous known natural or artificial nucleic acids. In addition, the data processing unit 9 is connected to an input unit 97 consisting of a keyboard and a pointing device such as a mouse, and a display unit 98 consisting of a liquid crystal display or the like.

[0018] The ion source 1 is an ion source that uses an ionization method such as the MALDI method. The ion trap 2 is a three-dimensional quadrupole type ion trap including an annular ring electrode 21 and a pair of end cap electrodes 22 and 24 disposed opposite to each other with the ring electrode 21 interposed therebetween. In accordance with an instruction from the control unit 8, the trap voltage generation unit 7 applies, to each of the electrodes 21, 22, and 24, either a high-frequency voltage or a DC voltage at a predetermined timing, or a voltage obtained by synthesizing them. The time-of-flight mass separation unit 3 is linear in this example, but may be a reflectron type, a multi-turn type, etc., and instead of a time-of-flight mass separator, for example, mass separation may be performed using the ion separation function of the ion trap 2 itself, or an orbitrap or the like may be used.

[0019] The hydrogen radical supply unit 5 includes a hydrogen radical supply source 51 that stores or generates hydrogen radicals, a valve 52 whose flow rate can be adjusted, a nozzle 53 that ejects hydrogen radicals, and a skimmer 54 that has an opening on the central axis of the ejection flow from the nozzle 53 and separates a gas such as diffusing hydrogen molecules to extract a hydrogen radical flow with a small diameter.

[0020] The gas supply unit 6 includes a gas supply source 61 that stores an inert gas such as helium gas or argon gas, and a valve 62 whose flow rate can be adjusted.

[0021] The procedure of structure analysis in the nucleic acid structure analysis apparatus of the present embodiment will be described while referring to the flowchart of FIG. 2.

[0022] [Step 101: HAD-MS of the test nucleic acid n analysis] First, for a sample containing a test nucleic acid, mass spectrometry (hereinafter referred to as HAD-MS) nAn analysis (called "analysis") is performed. At this time, various ions generated from the sample containing the nucleic acid to be tested in the ion source 1 are ejected from the ion source 1 in a packet shape and introduced into the ion trap 2 through the ion introduction hole 23 formed in the inlet end cap electrode 22. The ions derived from the nucleic acid to be tested introduced into the ion trap 2 are trapped in a high-frequency electric field formed inside the ion trap 2 by the voltage applied to the ring electrode 21 from the trap voltage generation unit 7. After that, a predetermined voltage is applied to the ring electrode 21 etc. from the trap voltage generation unit 7, thereby exciting ions other than the target ions with a specific m / z and removing them from the ion trap 2. As a result, precursor ions with a specific m / z are selectively trapped inside the ion trap 2.

[0023] Subsequently, valve 62 is opened in the gas supply unit 6, and an inert gas is introduced into the ion trap 2 as a cooling gas, thereby cooling the precursor ions. As a result, the precursor ions are concentrated near the center of the ion trap 2.

[0024] In this state, the valve 52 of the hydrogen radical supply unit 5 is opened, and gas containing hydrogen radicals is ejected from the nozzle 53. The skimmer 54 located ahead of the ejected flow removes gases such as hydrogen gas, and the hydrogen radicals that pass through the opening of the skimmer 54 become a narrow beam and pass through the radical particle inlet 26 drilled in the ring electrode 21. These hydrogen radicals are then introduced into the ion trap 2 and react with precursor ions trapped in the ion trap 2. At this time, the opening of the valve 52 is set so that the flow rate of hydrogen radicals introduced into the ion trap 2 is a predetermined flow rate (for example, 4 × 10⁻¹⁰ 10The reaction rate is adjusted to be greater than or equal to [atoms / s]. The introduction time of the hydrogen radical is also set as appropriate. Through reaction with the hydrogen radical, the precursor ion undergoes unpaired electron-induced dissociation, generating fragment ions derived from the precursor ion. The various fragment ions generated by the dissociation of ions through the reaction with the hydrogen radical (i.e., HAD) are captured in the ion trap 2 and cooled.

[0025] Subsequently, at a predetermined timing, a DC high voltage is applied from the trap voltage generation unit 7 to the end cap electrodes 22 and 24. This causes the fragment ions trapped in the ion trap 2 to receive acceleration energy and are simultaneously ejected through the ion ejection port 25. In this way, fragment ions with a certain acceleration energy are introduced into the flight space of the time-of-flight mass separation unit 3 and separated according to m / z as they fly through the flight space. The ion detector 4 sequentially detects the separated fragment ions. Upon receiving the detection signal from the ion detector 4, the data processing unit 91 creates a time-of-flight spectrum with the ejection time of the fragment ions from the ion trap 2 set to time zero. Then, by converting the flight time to m / z using pre-determined mass calibration information, a mass spectrum of fragment ions is created.

[0026] The mass spectrum obtained in this way shows peaks of various fragment ions derived from the nucleic acid being tested. However, by performing ion dissociation using the HAD method, the generation of fragment ions that do not contribute to the structural estimation of the nucleic acid being tested, such as fragment ions from which nucleic acid bases have been removed, can be suppressed. This will be explained below with reference to experimental examples.

[0027] For the actual measurement, a single-stranded DNA nucleic acid sample (base sequence: 5'-GCACATTG-3', molecular weight: 2409.6) was used, and MS with ion dissociation by CID was performed on the nucleic acid sample. 2 Analysis (i.e., CID-MS) 2 Analysis) and MS with ion dissociation by HAD 2Analysis (i.e., HAD-MS) 2 The analysis was performed. The mass spectrometer used for the measurement was a MALDI digital ion trap time-of-flight mass spectrometer (MALDI-DIT-TOF MS: Shimadzu Corporation) equipped with a hydrogen radical particle irradiation mechanism, as shown in Figure 1. For the actual measurement, the nucleic acid sample was introduced into ion source 1, and trivalent negative ions [M-3H] were irradiated. 3- MS as Precursor ion 2 An analysis was performed. (Note: CID-MS) 2 In the analysis, CID gas is supplied from the gas supply unit 6 to the ion trap 2 to dissociate precursor ions, and HAD-MS is performed. 2 In the analysis, precursor ions were dissociated by supplying hydrogen radicals from the hydrogen radical supply unit 5 to the ion trap 2.

[0028] Said CID-MS 2 The results of the analysis are shown in Figure 3, HAD-MS. 2 The results of the analysis are shown in Figure 4. In Figures 3 and 4, the base sequence of the nucleic acid sample and identifiers indicating each ion species assigned from the peaks on the mass spectrum are shown above the mass spectrum of the fragment ions. In addition, in Figures 3 and 4, each peak in the mass spectrum is labeled with its m / z and an identifier representing the ion species corresponding to that peak. The identifiers represent each ion species as a fragment ion series according to the dissociation pattern naming convention for nucleic acids. In this naming convention, fragment ions containing the 5' end are a n - , b n - , c n - d n - It is written as such, and the fragment ion at the opposite 3' end is w m - , x m - , y m - , z m - It is written as follows. n and mThis indicates the number of constituent units (i.e., the number of nucleotides) from the corresponding end to the dissociation site.

[0029] The mass spectrum (CID spectrum) shown in Figure 3 reveals not only peaks of fragment ions with retained nucleic acid bases that can be attributed to a base sequence, but also peaks [XB(Y)] of fragment ions with removed nucleic acid bases that cannot be attributed to a base sequence. For example, [a5-B(A)] in the same figure. 2- is a5 2- This peak represents the elimination of one adenine (A) nucleic acid base from an ion. However, it is not possible to determine which of the two A bases in the nucleic acid sample was eliminated; therefore, this peak does not contribute to the estimation of the base sequence. Such base elimination peaks not only do not contribute to the estimation of the base sequence, but they also complicate the mass spectrum, making base sequence analysis based on the mass spectrum difficult.

[0030] In contrast, the mass spectrum (HAD spectrum) shown in Figure 4 does not show the base elimination peaks described above, indicating that the base sequence of the nucleic acid can be identified over a wider range than when using the CID spectrum. Specifically, from the fragment ion peaks on the spectrum, the sequence of three bases on the 5' side (GCA) and the sequence of two bases on the 3' side (TG) can be identified. Furthermore, from the m / z difference between fragment ion z5 and fragment ion a7, it can be identified that the combination of nucleic acid bases C, A, and T is between the three bases on the 5' side and the two bases on the 3' side. Although the HAD spectrum in this experimental example could not cover the entire base sequence of the nucleic acid sample, it is thought that the number of fragment ions can be increased and the base sequence coverage can be improved by adjusting the mass spectrometry conditions to increase the amount of precursor ions or to improve the reaction efficiency of HAD.

[0031] [Step 102: Create a peak list] In the nucleic acid structure analysis apparatus according to this embodiment, when a mass spectrum of fragment ions is created in the mass spectrum creation unit 91, the peak extraction unit 92 extracts all peaks that satisfy predetermined conditions (for example, signal intensity being above a predetermined threshold) from the mass spectrum and creates a peak list that records the m / z and intensity of each peak. Furthermore, the peak extraction unit 92 inputs the information that "the ions corresponding to each peak listed in the peak list are fragment ions that retain nucleic acid bases" to the structure estimation unit 93 along with the peak list.

[0032] [Step 103: Estimation of the structure of the nucleic acid to be tested] Upon receiving the aforementioned information and peak list, the structure estimation unit 93 estimates the base sequence of the nucleic acid to be tested by database search or de novo sequencing.

[0033] When estimating a base sequence by database search, the structure estimation unit 93 creates an m / z list, which is a list containing the base sequences of multiple fragments that can arise from each known nucleic acid and the theoretical m / z values ​​of the ions (fragment ions) corresponding to each fragment, based on the base sequences of each of the many known nucleic acids recorded in the nucleic acid database 96. The unit then compares the m / z list for each known nucleic acid with the peak list of the nucleic acid under test.

[0034] Conventional CID-MS n When analyzing a test nucleic acid, both fragment ions with detached nucleic acid bases (base-detached ions) and fragment ions with intact nucleic acid bases (base-retaining ions) are generated. Therefore, the database search required considering both types of fragments. Specifically, for each of the multiple fragments that can be generated from the known nucleic acid, it was necessary to calculate the theoretical m / z for both the case where the ion corresponding to that fragment is a base-detached ion and the case where it is a base-retaining ion. This required comparing the m / z list containing these theoretical m / z values ​​with the peak list of the test nucleic acid. This increased the computational load and could potentially lead to a higher error rate.

[0035] In contrast, the nucleic acid structure analyzer according to this embodiment uses HAD-MS to analyze the nucleic acid to be tested. n Since the analysis is performed by analysis, it can be assumed that all fragment ions generated from the nucleic acid under test are base-holding ions. Therefore, in the apparatus according to this embodiment, when performing the database search as described above, in addition to the peak list of the nucleic acid under test, the information that "the ions corresponding to each peak listed in the peak list are fragment ions that hold nucleic acid bases" is also used. That is, for each of the many known nucleic acids recorded in the nucleic acid database 96, the structure estimation unit 93 calculates the m / z for each of the multiple fragments that can be generated from the known nucleic acid and the ions corresponding to each fragment are base-holding ions, and creates an m / z list that associates the base sequence of each fragment with the theoretical m / z of the corresponding ion, and compares the m / z list for each known nucleic acid with the peak list of the nucleic acid under test. Then, for each known nucleic acid, a score is calculated that indicates the probability that the base sequence of the known nucleic acid is the same as the base sequence of the nucleic acid under test.

[0036] Specifically, the following process is performed, for example. First, for each peak included in the peak list of the nucleic acid to be tested, it is checked whether the m / z of the peak falls within a predetermined m / z range centered on the theoretical m / z of the fragment that can arise from the known sequence, thereby determining whether the peak can be assigned to that fragment. Then, once the assignment of all peaks in the peak list has been determined, the assigned peak sum value is calculated by summing the signal intensities of all the peaks that could be assigned, and the total peak sum value is calculated by summing the signal intensities of all peaks detected in the mass spectrum, and the score is calculated based on the ratio of the assigned peak sum value and the total peak sum value. This score indicates the degree of agreement between the base sequence of the known nucleic acid and the mass spectrum obtained by actually measuring the nucleic acid to be tested, and the higher the score, the better the base sequence of the known nucleic acid matches the mass spectrum obtained by actual measurement. Then, a score is calculated for each of the multiple known nucleic acids included in the nucleic acid database 96, and the base sequence of the known nucleic acid with the highest score is used as the estimated base sequence of the nucleic acid being tested.

[0037] Furthermore, when the structure estimation unit 93 performs base sequence estimation by de novo sequencing, it first estimates the composition of the nucleic acid from the m / z of its precursor ion, and derives multiple predicted base sequences (predicted sequences) from this composition. Then, for each predicted sequence, it calculates the base sequences of multiple fragments that can be generated from the nucleic acid having the predicted sequence, and the theoretical m / z of the ion corresponding to each fragment (fragment ion), and creates a mass list that associates the base sequences with the theoretical m / z. In this embodiment as well, since only the m / z of the base-holding ion needs to be calculated as the theoretical m / z, the amount of computation can be reduced. Subsequently, for each of the predicted sequences, it creates an m / z list that associates the base sequence of the fragment with the theoretical m / z of the corresponding ion, and compares these m / z lists with the peak list of the nucleic acid. In this case as well, for example, the same score as above is calculated for each predicted sequence, and the predicted sequence with the highest score is taken as the estimated base sequence of the nucleic acid.

[0038] [Step 104: Displaying Results] Once the structural estimation of the nucleic acid to be tested is complete, the display processing unit 94 displays the estimated base sequence of the nucleic acid to be tested, along with the mass spectrum of the fragment ions derived from the nucleic acid to be tested obtained in step 101, on the screen of the display unit 98.

[0039] In the above example, the structure of the nucleic acid being tested was shown as an example of estimating its base sequence. However, the structure estimation device in this embodiment may also estimate the type of chemical modifications, such as post-transcriptional modifications, if the nucleic acid being tested has such modifications. In that case, the storage unit 95 stores a modification database in addition to (or instead of) the nucleic acid database 96, which contains information on the type of chemical modification to the nucleotide, the amount of change in m / z caused by the various modifications, and, if the site within the nucleic acid receiving the modification is known, information on that site. Then, when performing a database search or de novo sequencing structure estimation using the mass list created from the mass spectrum obtained in step 101, the modification database is referred to to perform structure estimation that takes various chemical modifications into account. This makes it possible to estimate not only the base sequence of the nucleic acid being tested, but also the type of chemical modification (or the type of chemical modification and the site where the modification is applied) to the nucleic acid being tested.

[0040] Since nucleotides are composed of sugar, phosphate groups, and bases, the types of chemical modifications to be stored in the modification database can be broadly classified into modifications to sugars, modifications to phosphate groups, and modifications to bases. Here, chemical modifications include both modifications that occur in vivo, such as post-transcriptional modifications, and modifications that are artificially introduced. Examples of modifications to sugars include 2'-O-methoxyethylation (2'-MOE), 2'-O-Methylation (2'-OMe), and 2'-Fluoroation (2'-F), which are modifications at the 2' position of the sugar moiety, as well as 2',4'-BNA(LNA) modifications in which the oxygen atom at the 2' position of the sugar moiety and the carbon atom at the 4' position are bridged, O-alkylation or O-alkoxyalkylation at the 2' position of the sugar moiety, conversion from the F-form at the 2' position of the pyrimidine sugar moiety to the anhydro or arabino-form, conversion to the Abasic-form, conversion to the 2'-5' phosphodiester conjugate, and conversion to the 3'→3'(5'→5') conjugate. Modifications to the phosphate group include, for example, sulfurization (S-modification), conversion from PS to PO (substitution of a sulfur atom bonded to a phosphorus atom with an oxygen atom), trichloroacetaldehyde reaction products, conversion to C-phosphonates, ethylene phosphodiesterification, and phosphorodithioate formation. Modifications to the base include, for example, pseudouridineization, deamination, cyanoethyl addition to thymine, methylamine addition to cytosine, and isobutyl addition to guanine, as well as cytosine converters, adenine converters, guanine converters, pyrimidine converters, and thymine converters.

[0041] As described above, the experimental examples shown in Figures 3 and 4 confirmed that base elimination can be suppressed by ion dissociation using the HAD method. Bases are chemical modification groups for sugars with a molecular weight of approximately 150. Therefore, it is predicted that the elimination of modification groups with a molecular weight of about the same as (or smaller than) a base can be suppressed among the above-mentioned "modifications to sugars." Furthermore, it is thought that the elimination of modification groups can be suppressed even for "modifications to bases" if the modification does not significantly increase the molecular weight of the base. Moreover, it is predicted that the elimination of modification groups can be suppressed even for "modifications to phosphate groups" if the molecular weight of the modification group is about the same as (or smaller than) that of the base. Thus, since it is predicted that ion dissociation using the HAD method can suppress not only the elimination of bases as described above, it is thought that fragment ions useful for estimating the type and site of such chemical modifications can be generated by subjecting precursor ions derived from nucleic acids to HAD dissociation.

[0042] It should be noted that the above embodiments are merely examples of the present invention, and any modifications, additions, or alterations made as appropriate within the scope of the present invention will naturally be included within the scope of the claims. For example, in the ion trap mass spectrometer of the above embodiments, the ion trap has a three-dimensional quadrupole configuration, but a linear ion trap may also be used. Furthermore, the mass spectrometer is not limited to one equipped with an ion trap; MS n Any device capable of analysis will suffice; for example, a triple quadrupole mass spectrometer equipped with quadrupole mass filters on either side of a collision cell can be used.

[0043] Furthermore, although the nucleic acid database 96 or the modification database is stored within the data processing unit 9 as described above, the system is not limited to this, and these databases may also be stored in an external device connected via an interface (not shown) provided on the computer constituting the data processing unit 9. Alternatively, the nucleic acid database 96 or the modification database may be stored on a server on the Internet, and the computer constituting the data processing unit 9 may be connected to the Internet via the interface to utilize these databases. As for such databases, databases prepared in advance by public institutions or mass spectrometer manufacturers may be used, or, for example, a database independently constructed by the user may be used.

[0044] Furthermore, the nucleic acid structure analyzer according to this embodiment may also be configured to promote the dissociation of precursor ions by collisional activation in order to improve the dissociation efficiency of precursor ions by the HAD method. Collisional activation is a method of providing auxiliary energy from an external source by performing an operation similar to the CID method, but with an energy level low enough that the ions do not dissociate (see, for example, Patent Document 2).

[0045] When promoting dissociation by collisional activation, an inert gas is introduced into the ion trap 2 from the gas supply unit 6 for a predetermined period before or after (or both before and after) the introduction of a hydrogen radical into the ion trap 2 where precursor ions derived from the nucleic acid being tested are trapped. Simultaneously, a predetermined resonance excitation voltage is applied to the end cap electrodes 22 and 24 from the trap voltage generation unit 7, thereby exciting the precursor ions trapped in the ion trap 2 and causing them to collide with the gas. This provides energy to the precursor ions before they react with the hydrogen radical, or to the precursor ions that have reacted with the hydrogen radical but remain undissociated in the ion trap 2, thereby promoting the dissociation of these ions.

[0046] Furthermore, the nucleic acid structure analysis apparatus and nucleic acid structure analysis method according to this embodiment use mass spectrometry (HAD-MS) with ion dissociation by the HAD method as described above for the nucleic acid to be tested. n After performing the analysis (or HAD-MS) n Before performing the analysis, the nucleic acid to be tested is subjected to mass spectrometry (CID-MS) with ion dissociation by the CID method. n The structure of the nucleic acid may be estimated by performing an analysis and using the mass spectra obtained from both analyses in a complementary manner. In this case, the ion source 1, ion trap 2, time-of-flight mass separation unit 3, ion detector 4, gas supply unit 6, trap voltage generation unit 7, and control unit 8 in the nucleic acid structure analyzer according to this embodiment are the "CID-MS" of the present invention. n This corresponds to the "Analysis Execution Unit".

[0047] Figure 5 shows an example of the nucleic acid structure analysis procedure in this case. Note that in the same figure, HAD-MS of the test nucleic acid is shown. n After performing the analysis (step 201), CID-MS of the test nucleic acid. n Although analysis (step 202) is to be performed, HAD-MS n Analysis and CID-MS n The steps may be performed in the reverse order. Also, HAD-MS n In the analysis, the dissociation of precursor ions may be promoted by collisional activation as described above.

[0048] Said CID-MS n In the analysis, precursor ions with a specific m / z within ion trap 2 (HAD-MS) nThe precursor ions (with the same m / z as the precursor ions to be dissociated in the analysis) are captured, and the valve 62 of the gas supply unit 6 is opened to introduce an inert gas as a cooling gas into the ion trap 2 to cool the precursor ions. Then, a predetermined AC voltage is applied from the trap voltage generation unit 7 to the end cap electrodes 22 and 24, causing the precursor ions to resonantly excite and collide with the inert gas (at this time, the inert gas functions as a CID gas). As a result, the precursor ions undergo energy-accumulating dissociation, and fragment ions derived from the nucleic acid to be tested are generated. Subsequently, at a predetermined timing, the fragment ions in the ion trap are introduced into the flight space of the time-of-flight mass separation unit 3, separated according to their m / z, and detected by the ion detector 4.

[0049] Subsequently, the peak extraction unit 92 creates a peak list from the mass spectrum (HAD spectrum) obtained in step 201 and the mass spectrum (CID spectrum) obtained in step 202 (step 203), and the structure estimation unit 93 estimates the structure of the nucleic acid by database search or de novo sequencing using both peak lists (step 204). Then, the display processing unit 94 displays the results of the structure estimation on the display unit 98 (step 205).

[0050] In this way, by applying the HAD method, an unpaired electron induction type dissociation method, and the CID method, an energy storage type dissociation method, to precursor ions derived from the nucleic acid being tested, and collecting information on fragment ions, it is possible to improve sequence coverage (i.e., the proportion of the entire base sequence of the nucleic acid being tested in which the sequence has been identified).

[0051] [Aspect] Those skilled in the art will understand that the above-described exemplary embodiments are specific examples of the following embodiments.

[0052] (Section 1) A method for analyzing the structure of nucleic acids according to one aspect of the present invention is: HAD-MS involves introducing hydrogen radicals into a space containing ions derived from the nucleic acid being tested to dissociate the ions, and then using mass spectrometry to collect information on the m / z values ​​of the resulting multiple fragment ions. n Analysis steps, The aforementioned HAD-MS n A structure estimation step in which the structure of the test nucleic acid is estimated based on the m / z information of the plurality of fragment ions obtained in the analysis step, It possesses the following characteristics.

[0053] (Section 2) The nucleic acid structure analysis method described in Section 1 is: In the structure estimation step, the base sequence of the nucleic acid to be tested is estimated by de novo sequencing, and the de novo sequencing may be performed on the condition that each of the plurality of fragment ions possesses a chemical modification group for nucleic acid bases or nucleotides.

[0054] (Section 3) The nucleic acid structure analysis method described in Section 1 is: In the structure estimation step, the base sequence of the nucleic acid to be tested is estimated by a database search of a nucleic acid database containing the base sequences of multiple known nucleic acids, and the database search may be performed with the condition that each of the multiple fragment ions possesses a chemical modification group for a nucleic acid base or nucleotide.

[0055] (Section 4) The nucleic acid structure analysis method described in any of Sections 1 to 3 is: The aforementioned HAD-MS n In the analysis step, neutral particles may be introduced into the space at least one of the periods before or after the introduction of hydrogen radicals, and the ions derived from the nucleic acid to be tested present in the space may be excited and collided with the neutral particles to promote the dissociation of the ions.

[0056] (Section 5) The nucleic acid structure analysis method described in any of Sections 1 to 3 is: CID-MS involves introducing a neutral molecule into a space containing ions derived from the nucleic acid being tested, causing the ions to dissociate by colliding with the neutral molecule, and then collecting m / z information of the multiple fragment ions generated by mass spectrometry. n Analysis steps, It further possesses, In the structural analysis step, the HAD-MS n The m / z information of the multiple fragment ions obtained in the analysis step and the CID-MS n The structure of the nucleic acid to be tested may be estimated based on the m / z information of the multiple fragment ions obtained in the analysis step.

[0057] (Article 6) The nucleic acid structure analysis method described in Article 2 or Article 3 is: The chemical modification group for the nucleotide may be a chemical modification group for a sugar contained in the nucleotide.

[0058] (Section 7) A nucleic acid structure analysis device according to one aspect of the present invention is: HAD-MS involves introducing hydrogen radicals into a space containing ions derived from the nucleic acid being tested to dissociate the ions, and then using mass spectrometry to collect information on the m / z values ​​of the resulting multiple fragment ions. n Analysis execution unit, The aforementioned HAD-MS n A structure estimation unit estimates the structure of the test nucleic acid based on the m / z information of the plurality of fragment ions obtained by the analysis execution unit, It possesses the following characteristics.

[0059] (Section 8) The nucleic acid structure analysis apparatus described in Section 7 is: The structure estimation unit estimates the base sequence of the nucleic acid under test by de novo sequencing, and may perform the de novo sequencing on the condition that each of the plurality of fragment ions possesses a chemical modification group for nucleic acid bases or nucleotides.

[0060] (Section 9) The nucleic acid structure analysis apparatus described in Section 7 is: The structure estimation unit estimates the base sequence of the nucleic acid under test by performing a database search on a nucleic acid database containing the base sequences of a plurality of known nucleic acids, and the database search may be performed with the condition that each of the plurality of fragment ions possesses a chemical modification group for nucleic acid bases or nucleotides.

[0061] (Item 10) A nucleic acid structure analyzer described in any of items 7 to 9 is: The aforementioned HAD-MS n The analysis unit may further introduce neutral particles into the space during at least one of the periods before or after introducing hydrogen radicals into the space, and promote the dissociation of ions by exciting the ions derived from the nucleic acid to be tested that are present in the space and causing them to collide with the neutral particles.

[0062] (Item 11) A nucleic acid structure analyzer described in any of items 7 to 9 is: CID-MS involves introducing a neutral molecule into a space containing ions derived from the nucleic acid being tested, causing the ions to dissociate by colliding with the neutral molecule, and then collecting m / z information of the multiple fragment ions generated by mass spectrometry. n analysis department, It further possesses, The structural analysis department performs the HAD-MS n The m / z information of the multiple fragment ions collected by the analysis unit and the CID-MS n The structure of the nucleic acid to be tested may be estimated based on the m / z information of the multiple fragment ions collected by the analysis unit.

[0063] (Paragraph 12) The nucleic acid structure analyzer described in paragraph 8 or 9 is: The chemical modification group for the nucleotide may be a chemical modification group for a sugar contained in the nucleotide.

[0064] The nucleic acid structure analysis method described in paragraph 1 or the nucleic acid structure analysis apparatus described in paragraph 7 can accommodate precursor ions with various charges and facilitate the estimation of nucleic acid structure based on mass spectrometry.

[0065] According to the nucleic acid structure analysis method described in paragraph 2 or the nucleic acid structure analysis apparatus described in paragraph 8, the structure of the nucleic acid to be tested can be estimated by the database search without considering the case where each of the plurality of fragment ions is a base-eliminated ion or an ion from which a chemically modified group has been eliminated, thus reducing the computational load in the structure estimation.

[0066] According to the nucleic acid structure analysis method described in paragraph 3 or the nucleic acid structure analysis apparatus described in paragraph 9, the structure of the nucleic acid to be tested can be estimated by de novo sequencing without considering the case where each of the plurality of fragment ions is a base-eliminated ion or an ion from which a chemically modified group has been eliminated, thus reducing the computational load in the structure estimation.

[0067] According to the nucleic acid structure analysis method described in paragraph 4 or the nucleic acid structure analysis apparatus described in paragraph 10, HAD-MS n This method can promote the dissociation of precursor ions during analysis, thereby improving the signal-to-noise ratio (SNR) of the analysis and increasing the accuracy of structural estimation.

[0068] The nucleic acid structure analysis method described in paragraph 5 or the nucleic acid structure analysis apparatus described in paragraph 11 can improve sequence coverage in structure estimation. [Explanation of Symbols]

[0069] 1…Ion source 2…Ion trap 3…Time-of-flight mass separator 4…Ion detector 5…Hydrogen radical supply unit 6…Gas supply unit 7... Trap voltage generation unit 8... Control Unit 9…Data Processing Unit 91...Mass spectrum generation unit 92...Peak extraction section 93…Structure estimation part 96… Nucleic Acid Database

Claims

1. A HAD-MSn analysis step involves introducing hydrogen radicals into a space containing ions derived from the nucleic acid to be tested to dissociate the ions, and then collecting m / z information of the multiple fragment ions generated by mass spectrometry. A structure estimation step is performed to estimate the structure of the test nucleic acid based on the m / z information of the plurality of fragment ions obtained in the HAD-MSn analysis step, A method for analyzing the structure of nucleic acids.

2. The method for analyzing the structure of a nucleic acid according to claim 1, wherein the base sequence of the nucleic acid to be tested is estimated by de novo sequencing in the structure estimation step, and the de novo sequencing is performed on the condition that each of the plurality of fragment ions holds a chemical modification group for nucleic acid bases or nucleotides.

3. The method for analyzing the structure of a nucleic acid according to claim 1, wherein in the structure estimation step, the base sequence of the nucleic acid to be tested is estimated by a database search of a nucleic acid database containing the base sequences of a plurality of known nucleic acids, and the database search is performed with the condition that each of the plurality of fragment ions possesses a chemical modification group for a nucleic acid base or nucleotide.

4. The method for analyzing the structure of a nucleic acid according to claim 1, wherein, in the HAD-MSn analysis step, neutral particles are introduced into the space during at least one of the periods before or after the introduction of hydrogen radicals into the space, and ions derived from the nucleic acid to be tested present in the space are excited and collided with the neutral particles to promote the dissociation of the ions.

5. A CID-MSn analysis step involves introducing a neutral molecule into a space containing ions derived from the nucleic acid to be tested, causing the ions to dissociate by colliding with the neutral molecule, and collecting m / z information of the multiple fragment ions generated by mass spectrometry. It further possesses, The method for analyzing the structure of a nucleic acid according to claim 1, wherein in the structure estimation step, the structure of the nucleic acid to be tested is estimated based on the m / z information of the plurality of fragment ions obtained in the HAD-MSn analysis step and the m / z information of the plurality of fragment ions obtained in the CID-MSn analysis step.

6. The method for analyzing the structure of a nucleic acid according to claim 2, wherein the chemical modification group for the nucleotide is a chemical modification group for a sugar contained in the nucleotide.

7. A HAD-MSn analysis execution unit that dissociates ions by introducing hydrogen radicals into a space containing ions derived from the nucleic acid to be tested, and collects m / z information of the multiple fragment ions generated by mass spectrometry, A structure estimation unit estimates the structure of the test nucleic acid based on the m / z information of the plurality of fragment ions obtained by the HAD-MSn analysis execution unit, A nucleic acid structure analyzer having the following features.

8. The nucleic acid structure analysis apparatus according to claim 7, wherein the structure estimation unit estimates the base sequence of the nucleic acid to be tested by de novo sequencing, and performs the de novo sequencing on the condition that each of the plurality of fragment ions holds a chemical modification group for nucleic acid bases or nucleotides.

9. The nucleic acid structure analysis apparatus according to claim 7, wherein the structure estimation unit estimates the base sequence of the nucleic acid to be tested by performing a database search on a nucleic acid database containing the base sequences of a plurality of known nucleic acids, and the database search is performed with the condition that each of the plurality of fragment ions possesses a chemical modification group for a nucleic acid base or nucleotide.

10. The nucleic acid structure analysis apparatus according to claim 7, wherein the HAD-MSn analysis unit further introduces neutral particles into the space and promotes the dissociation of ions by exciting ions derived from the nucleic acid to be tested present in the space and causing them to collide with the neutral particles during at least one of the periods before or after introducing hydrogen radicals into the space.

11. A CID-MSn analysis unit introduces a neutral molecule into a space containing ions derived from the nucleic acid to be tested, causes the ions to dissociate by colliding with the neutral molecule, and collects m / z information of the multiple fragment ions generated by mass spectrometry. It further possesses, The nucleic acid structure analysis apparatus according to claim 7, wherein the structure estimation unit estimates the structure of the test nucleic acid based on the m / z information of the plurality of fragment ions collected by the HAD-MSn analysis unit and the m / z information of the plurality of fragment ions collected by the CID-MSn analysis unit.

12. The nucleic acid structure analysis apparatus according to claim 8, wherein the chemical modification group for the nucleotide is a chemical modification group for a sugar contained in the nucleotide.

13. The method for analyzing the structure of a nucleic acid according to claim 1, wherein the space where ions derived from the nucleic acid to be tested exist is a space in which precursor ions, which are ions having a specific m / z among the ions derived from the nucleic acid to be tested, are selectively captured.

14. The nucleic acid structure analysis apparatus according to claim 7, wherein the space where ions derived from the nucleic acid to be tested exist is a space in which precursor ions, which are ions having a specific m / z among the ions derived from the nucleic acid to be tested, are selectively captured.

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