Method for analyzing structure of sample molecule

Abstract

A method for analyzing the structure of a sample molecule according to the present invention is a method for analyzing the structure of a sample molecule using a mass spectrometer. The mass spectrometry device is provided with: an ion source based on the MALDI method; an ion capture unit for capturing ions having a predetermined mass-to-charge ratio from ions generated by the ion source; and a detection unit for detecting the ions captured by the ion capture unit. The method for analyzing the structure of a sample molecule comprises the steps of: acquiring standard analysis conditions for an ion amount setting item, a mass-to-charge ratio range setting item, and a signal strength setting item when measuring molecular weight-related ions; performing product ion measurement under a modified analysis condition obtained by modifying at least one of the ion amount setting item, the mass-to-charge range setting item, and the signal strength setting item among the standard analysis conditions, and acquiring mass spectrum data of the product ion measurement; extracting peaks corresponding to fragment ions from the mass spectrum data; and determining at least a portion of the structure of the sample molecule on the basis of the mass information of the extracted peaks. As a result, fragment ions required for structural analysis can be preferentially detected with good sensitivity.

Description

Technical Field

[0001] The present invention relates to a method for analyzing the structure of sample molecules using mass spectrometry. Background Art

[0002] In a mass spectrometer (MALDI-MS) having an ion source based on the matrix-assisted laser desorption / ionization (MALDI) method, a laser is briefly irradiated onto an analytical sample prepared by mixing the sample and an ionization assisting agent called a matrix, thereby ionizing the sample molecules in the analytical sample, and the generated ions are introduced into a mass separator to be separated and detected according to their mass-to-charge ratio (m / z).

[0003] The MALDI method is generally known as a flexible ionization method that can ionize a non-volatile compound without much decomposition. Therefore, MALDI-MS is widely used to obtain molecular weight information of biological macromolecules such as nucleic acids or nucleic acid-related substances, peptides, proteins, and sugar chains. In addition, in the structural analysis of such biological macromolecules, the molecular weight-related ions (precursor ions) generated from the sample molecules in the MALDI ion source are intentionally fragmented using an appropriate method, and the various fragment ions thus generated are subjected to mass spectrometry analysis, thereby estimating the structure of the sample molecule based on the mass information of the fragment ions.

[0004] For example, in the nucleic acid structure analysis method described in non-patent documents 1 to 3, MALDI-TOFMS is used to perform mass spectrometry analysis of various fragment ions (ISD fragments) of nucleic acids generated by in-source decay (In-Source Decay: ISD), and the base sequence information of nucleic acids is analyzed based on the mass information of the fragment ions obtained thereby. The so-called in-source decay is a method of causing ions to be fragmented in an ion source at the same time as ionization or just after ionization. Since the MALDI method is a flexible ionization method as described above, the following is known: although ions are originally difficult to fragment, for example, by increasing the intensity of the laser to increase the energy during ionization, or by using a special matrix, the fragmentation of ions is promoted during ionization. It is known that ISD fragments such as a, b, c, d / w, x, y, and z-ions are generated by specific cleavage at a phosphodiester bond (or phosphorothioate bond, etc.) site in the ISD of nucleic acid molecules.

[0005] Prior art literature

[0006] Non-patent literature

[0007] Non-patent document 1: Nathan A. Hagan, 5 others, "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

[0008] Non-patent document 2: Hisao Shimizu, 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

[0009] Non-patent document 3: Satoshi Kimura, 1 other person, "Effect of oligonucleotidestructural difference on matrix-assisted laser desorption / ionization in-source decay in comparison with collision-induced dissociationfragmentation", (USA), Rapid Communications in Mass Spectrometry, 2020, 34, e8819

[0010] Non-patent document 4: Scott A.McLuckey, and two 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

[0011] Problem that the invention aims to solve

[0012] In the methods described in non-patent literatures 1 to 3, MALDI-TOFMS is used for the mass spectrometer, and most of the ions generated by the ion source are introduced into the mass separation section, and the ions with the smallest mass are separated and detected. Therefore, when ISD is performed, fragment ions are detected in a wide range from the low-mass region to the high-mass region. In this case, there are the following problems: the peaks of the fragment ions on the low-mass region side overlap with the peaks of the cluster ions derived from the matrix, or the detection is suppressed due to the ion suppression effect, etc. In addition, in MALDI-TOFMS, since the ions on the low-mass region side are preferentially detected, it is sometimes difficult to obtain the sensitivity of the fragment ions on the high-mass side. In addition, since the mass region of the object measured at one time is wide, there is a problem that the overall detection sensitivity and resolution of the fragment ions are easily reduced, especially under the condition that the fragmentation efficiency from the precursor ions is low.

[0013] There are also the following problems: the peaks of fragment ions near the m / z value of the precursor ion overlap with peaks other than the fragment ions (in the case of nucleic acids, base desorption peaks, etc.), or excessive energy is applied to the precursor ions during fragmentation, resulting in incomplete ion separation and reduced resolution.

[0014] For example, in non-patent document 3 Figure 5 The ISD spectra of nucleic acids obtained by MALDI-TOFMS are described in Figure 5 In the ISD spectrum, the peak of the fragment ion near the m / z value of the precursor ion that has undergone cleavage in the base sequence portion near the 3' end or the 5' end overlaps with the peaks other than the fragment ions. In addition, compared with the peak of the fragment ion obtained by cleavage in the base sequence portion away from the 3' end or the 5' end (the peak of the fragment ion detected using the m / z value away from the low mass region relative to the vicinity of the m / z value of the precursor ion), the sensitivity and resolution become lower.

[0015] The present invention has been made in view of the above problems, and an object of the present invention is to provide a structural analysis method capable of detecting fragment ions required for structural analysis of sample molecules with good sensitivity in a relatively wide mass range from a low mass region to a high mass region.

[0016] Solutions for solving problems

[0017] The method for analyzing the structure of a sample molecule according to the present invention, which is accomplished in order to solve the above-mentioned problems, is a method for analyzing the structure of a sample molecule using an ion trap mass spectrometer, wherein the ion trap mass spectrometer comprises: an ion source based on a matrix-assisted laser desorption ionization (MALDI) 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, and the method for analyzing the structure of a sample molecule comprises the following steps:

[0018] a standard analysis condition acquisition step for respectively acquiring standard analysis conditions for ion quantity setting items related to the quantity of ions generated by the ion source, a mass-to-charge ratio range setting item related to the mass-to-charge ratio range of ions captured in the ion capture section, and a signal intensity setting item related to the signal intensity of ions in the detection section when the mass spectrometer is used to perform molecular weight associated ion measurement for detecting molecular weight associated ions of the sample molecules contained in the sample;

[0019] A product ion measurement data acquisition step, performing a product ion measurement for detecting a plurality of first fragment ions generated by the fragmentation of the molecular weight-related ions of the sample molecule under a changed analysis condition obtained by changing at least one of the analysis conditions of the ion quantity setting item, the mass-to-charge ratio range setting item, and the signal intensity setting item in the standard analysis condition acquired in the standard analysis condition acquisition step, and acquiring mass spectrum data of the product ion measurement;

[0020] a first fragment peak extraction step of extracting a peak corresponding to the first fragment ion from the mass spectrum data acquired in the product ion measurement data acquisition step; and

[0021] The data analysis step specifies at least a part of the structure of the sample molecule based on the mass information of the peak extracted in the first fragment peak extraction step.

[0022] Another embodiment of the present invention completed to solve the above-mentioned problem is a method for analyzing the structure of a sample molecule using an ion trap mass spectrometer, the ion trap mass spectrometer comprising: an ion source based on a matrix-assisted laser desorption ionization (MALDI) 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 method comprising the following steps:

[0023] a standard analysis condition acquisition step for respectively acquiring standard analysis conditions for ion quantity setting items related to the quantity of ions generated by the ion source, a mass-to-charge ratio range setting item related to the mass-to-charge ratio range of ions captured in the ion capture section, and a signal intensity setting item related to the signal intensity of ions in the detection section when the mass spectrometer is used to perform molecular weight associated ion measurement for detecting molecular weight associated ions of the sample molecules contained in the sample;

[0024] A product ion determination step, performing product ion determination for detecting a plurality of first fragment ions generated by the fragmentation of the molecular weight-related ions of the sample molecule under changed analysis conditions obtained by changing at least one of the analysis conditions of the ion quantity setting item, the mass-to-charge ratio range setting item, and the signal intensity setting item in the standard analysis conditions acquired in the standard analysis condition acquisition step;

[0025] An MS / MS measurement condition setting step of setting measurement conditions for performing MS / MS measurement in which at least one first fragment ion among the plurality of first fragment ions detected in the product ion measurement is set as a precursor ion;

[0026] an MS / MS measurement data acquisition step of acquiring mass spectrum data of the MS / MS measurement by performing the MS / MS measurement based on the measurement conditions set in the MS / MS measurement condition setting step;

[0027] a second fragment peak extraction step of extracting a peak corresponding to a second fragment ion generated in the MS / MS measurement from the mass spectrum data acquired in the MS / MS measurement data acquisition step; and

[0028] The data analysis step specifies a part of the structure of the sample molecule based on the mass information of the peak extracted in the second fragment peak extraction step.

[0029] In the present invention, molecular-related ions are a general term for ions that directly contribute to obtaining molecular weight information, and specifically include, for example, protonated molecules, deprotonated molecules, sodium adduct molecules, and the like.

[0030] Effects of the Invention

[0031] According to the present invention, it is possible to detect the fragment ions required for the structural analysis of the sample molecule with good sensitivity in a relatively wide mass region from the low mass region to the high mass region. As a result, it is possible to detect a sufficient number of fragment ions for the structural analysis of the sample molecule. In particular, it is possible to determine more structural information such as sequence information for the molecular structure of the sample molecule containing relatively high mass molecules such as biological macromolecules.

[0032] In addition, according to another embodiment of the present invention, the fragment ions (second fragment ions) obtained by further fragmenting the fragment ions (first fragment ions) derived from the molecular weight-related ions can be detected with good sensitivity. As a result, the structural information can be more reliably determined for the molecular structure of the sample molecule that cannot be accurately analyzed using only the mass information of the first fragment ions. BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figure 1 This is a schematic configuration diagram showing an example of a mass spectrometry device for implementing the structure analysis method involved in the present invention.

[0034] Figure 2 This is a flowchart showing the process of the structural analysis method according to one embodiment of the present invention.

[0035] Figure 3 This is a flowchart showing the process of a structural analysis method according to another embodiment of the present invention.

[0036] Figure 4 1 and 2 are diagrams showing the mass spectrum of the standard nucleic acid A when the conditions (a) to (e) in Example 1 are used.

[0037] Figure 5 3 is a diagram showing the mass spectrum of the standard nucleic acid A and the sequence analysis status of the standard nucleic acid A when the condition (e) is used in Example 1.

[0038] Figure 6 This is a diagram showing the mass spectrum of the standard nucleic acid A when conditions (b) to (d) in Example 2 are used.

[0039] Figure 7 1 and 2 are diagrams showing the mass spectrum of the standard nucleic acid A and the sequence analysis status of the standard nucleic acid A when the condition (d) is used in Example 2.

[0040] Fig. 8A This is a diagram showing the mass spectrum (m / z 2000 to 6000) of the standard nucleic acid A when the conditions (b) and (c) in Example 3 are used.

[0041] Figure 8BThis is a diagram showing the mass spectrum (m / z 3000 to 5000) of the standard nucleic acid A when the conditions (b) and (c) in Example 3 are used.

[0042] Fig. 9 1 and 2 are diagrams showing the mass spectrum of the standard nucleic acid A and the sequence analysis status of the standard nucleic acid A when the condition (c) is used in Example 3.

[0043] Fig.10 1 and 2 are diagrams showing the mass spectrum of the standard nucleic acid A and the sequence analysis status of the standard nucleic acid A when the conditions (b) and (c) in Example 3 are used.

[0044] Fig.11 The diagram shows the mass spectrum of mipomersen when conditions (a) and (b) are used in Example 4, and the sequence analysis status of mipomersen.

[0045] Fig.12 It is a diagram showing the mass spectrum of mipomersen when the measurement conditions of MS / MS measurement in Example 4 are used and the sequence analysis status of mipomersen. DETAILED DESCRIPTION

[0046] In order to solve the above problems, the inventors of the present application have attempted to use an ion trap type (IonTrap: IT) mass spectrometer (MALDI-ITMS) with a MALDI ion source and a matrix for ISD that has been confirmed to promote ion fragmentation to perform structural analysis of sample molecules. In addition, there are almost no reports of attempts to use MALDI-ITMS for ISD so far. As a result, the following was confirmed: although ion fragmentation occurs, the fragment ions obtained therefrom are limited to ions with m / z values ​​close to those of the precursor ions. It is believed that the reason for this is that the ion capture conditions based on the space charge effect of MALDI-ITMS are different in the precursor ions and the fragment ions on the low mass-to-charge ratio side obtained by more fragmentation of the precursor ions. Such a phenomenon is particularly significant when the object of analysis is a high-mass molecule.

[0047] An object of the present invention is to provide a method for preferentially detecting, with high sensitivity, fragment ions, particularly those in a low-mass region, among fragment ions required for analysis in a method for analyzing the structure of a sample molecule using MALDI-ITMS.

[0048] Hereinafter, one embodiment of the method for analyzing the structure of a sample molecule according to the present invention will be described with reference to the drawings. Figure 1 1 is a schematic configuration diagram showing an example of a mass spectrometry apparatus for implementing the structure analysis method according to the present embodiment.

[0049] <Mass spectrometer>

[0050] The mass spectrometry device used in the present embodiment is an ion trap type mass spectrometry device, comprising: an ion source 1, which ionizes a sample containing an analysis object; an ion trap 2 (the ion capturing unit in the present invention), which temporarily captures ions of a specified mass-to-charge ratio among ions generated by the ion source 1 through the action of a high-frequency electric field, and separates the captured ions according to the mass-to-charge ratio (m / z); and a detection unit 3, which detects the separated ions.

[0051] The ion source 1 is an ion source using the MALDI method, and includes a laser irradiation unit 11 for irradiating a sample with laser light, and a sample stage 12 for placing a sample plate S on which the sample is placed. The ion trap 2 is a quadrupole-type ion trap, and includes a circular ring electrode 21 and a pair of end cap electrodes 22 and 23 arranged opposite to each other across the ring electrode 21. An ion entrance hole 22a is formed in the entrance side end cap electrode 22, and an ion exit hole 23a is formed in the exit side end cap electrode 23. The detector 3 includes a conversion dynode 31 for converting ions into electrons, and a detector (secondary electron multiplier tube) 32 for multiplying the electrons from the conversion dynode 31 for detection.

[0052] Predetermined voltages are applied to the annular electrode 21 and the end cap electrodes 22, 23 of the ion trap 2. The high frequency electric field thus formed can trap ions in the inner space surrounded by the annular electrode 21 and the end cap electrodes 22, 23, or discharge ions from the inner space through the ion exit hole 23a.

[0053] The range of mass-to-charge ratios of ions preferentially captured in the ion trap is controlled by changing the time (hereinafter referred to as delay time) from irradiating the sample with laser light by the ion source 1 until the capture voltage for capturing ions is applied to the annular electrode 21 of the ion trap 2. If the delay time is extended, ions with a higher mass side than other ions can be reliably captured, and if the delay time is shortened, ions with a lower mass side than other ions can be reliably captured.

[0054] The prescribed voltage applied to the annular electrode 21 and the end cap electrodes 22 and 23 can be either a high-frequency voltage in the form of a sine wave or a rectangular wave voltage generated by switching two different voltages at high speed. In a digital ion trap using an electric field generated by a rectangular wave voltage, the range of the mass-to-charge ratio of ions that can be captured is controlled by changing the frequency while maintaining the amplitude (voltage value) of the rectangular wave voltage constant, or by changing the ratio of the switching interval of the rectangular wave voltage, i.e., the duty cycle.

[0055] The ion trap type mass spectrometer in this embodiment includes a mass spectrometer that uses the mass separation function of the ion trap to discharge the ions captured in the ion trap in the order of mass-to-charge ratio from small to large, and uses a detector arranged outside the ion trap to detect the ions, and also includes a mass spectrometer that uses a mass separation unit such as a time-of-flight mass separation unit arranged outside the ion trap to separate the ions discharged from the ion trap according to the mass-to-charge ratio, and uses a detector also arranged outside the ion trap to detect the ions. In addition, it can also be a tandem type mass spectrometer having a structure in which two mass separation units are connected in series so as to be able to perform the MS / MS measurement described later.

[0056] Next, refer to Figure 2 The process of the structural analysis method of this embodiment is described with reference to the flowchart of FIG.

[0057] <Structural analysis method>

[0058] Here, an example in which an ion trap type mass spectrometer having a digital ion trap is used as the mass spectrometer will be described.

[0059] [Step 101: Acquisition of standard analysis conditions for MS measurement]

[0060] First, molecular weight-related ions (proton addition molecules [M+H]) for detecting sample molecules as the analyte are obtained. + , deprotonated molecule [MH] - The standard analysis conditions are as follows when performing measurements (equivalent to the molecular weight-related ion measurement in the present invention. It will be referred to as MS measurement hereinafter). MS measurement is a measurement that uses measurement conditions for detecting molecular weight-related ions with as high sensitivity and resolution as possible without ion fragmentation (or, even if ion fragmentation occurs, it is very small). In a mass spectrometer, there are various setting items that can be appropriately set according to the type of analysis object or the purpose of analysis. In the structure analysis method involved in the present embodiment, standard analysis conditions are obtained for an ion quantity setting item related to the ion quantity generated by the ion source 1, a mass-to-charge ratio range setting item related to the mass-to-charge ratio range of ions captured in the ion trap 2, and a signal intensity setting item related to the signal intensity of ions in the detection unit 3.

[0061] Standard analysis conditions are representative conditions among the conditions that can detect the molecular weight-related ions of the sample molecules. As a method for obtaining standard analysis conditions, for example, if the result of performing MS measurement in a mass spectrometer using the default values ​​of various setting items set in advance is the detection of the molecular weight-related ions of the sample molecules, the default values ​​can also be set as standard analysis conditions. In this case, reading the default values ​​from a storage unit in which the default values ​​are pre-stored is equivalent to obtaining the standard analysis conditions. Alternatively, after changing the setting values ​​of various setting items relative to the default values ​​so that the molecular weight-related ions of the sample molecules are detected with higher sensitivity and higher resolution, MS measurement can be performed, and the value (threshold value, threshold value, etc.) that can detect the molecular weight-related ions of the sample molecules can be obtained.

[0062] Items for setting the ion quantity include the laser intensity (laser power) used for irradiation by the laser irradiation unit 11, items for setting the mass-to-charge ratio range include the RFdelay value corresponding to the delay time, and items for setting the signal intensity include the voltage applied to the conversion dynode 31 or the voltage applied to the detector (secondary electron multiplier tube) 32.

[0063] [Step 102: Setting of modified analysis conditions obtained by changing the standard analysis conditions]

[0064] Next, the changed analysis conditions are set 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 obtained in step 101. That is, there may be a situation where only any one of the ion amount setting item, the mass-to-charge ratio range setting item, and the signal intensity setting item is changed without changing the remaining setting items. At this time, the ion amount setting item is changed in a manner that increases the amount of ions generated by the ion source 1 compared to the case where MS measurement is performed under the standard analysis conditions, the mass-to-charge ratio range setting item is changed in a manner that preferentially captures ions on the low mass side in the ion trap 2 compared to the case where MS measurement is performed under the standard analysis conditions, and the signal intensity setting item is changed in a manner that increases the signal intensity of ions in the detection unit 3 compared to the case where MS measurement is performed under the standard analysis conditions.

[0065] Specifically, when changing the intensity of the laser as an ion quantity setting item, it is preferred to set the intensity of the laser higher relative to the standard analysis conditions, and it is more preferred to set the intensity of the laser to, for example, about 1% to 40% higher than the value of the standard analysis conditions, and it is further preferred to set the intensity of the laser to, for example, about 1% to 30% higher than the value of the standard analysis conditions. When changing the RFdelay value as a mass-to-charge ratio range setting item, it is preferred to set the RFdelay value lower relative to the standard analysis conditions, and it is more preferred to set the RFdelay value to, for example, 5% to 30% lower than the value of the standard analysis conditions, and it is further preferred to set the RFdelay value to, for example, 10% to 20% lower than the value of the standard analysis conditions (so that the delay time is 1 microsecond to 3 microseconds). When changing the applied voltage applied to the conversion dynode 31 as a signal intensity setting item, it is preferred to set the applied voltage higher relative to the standard analysis conditions, and it is, for example, preferably set to about 5% to 30% higher, and it is further preferred to set to about 10% to 30% higher. When changing the applied voltage to the detector (secondary electron multiplier tube) 32 as a signal intensity setting item, it is preferred to set the applied voltage higher than the standard analysis conditions, for example, preferably about 5% to 50% higher, and more preferably about 10% to 50% higher.

[0066] [Step 103: Acquisition of Data Obtained by ISD Measurement Using Changed Analysis Conditions]

[0067] The above-mentioned changed analysis conditions are used to perform a determination (equivalent to the product ion determination of the present application) for detecting fragment ions generated by the fragmentation of the molecular weight-related ions (precursor ions) of the sample molecules, and obtain data related to the detected ions. In the present invention, since the generation mechanism of fragment ions derived from sample molecules has not yet been confirmed, in this specification, the fragmentation that occurs in the ion source of the MALDI ion trap mass spectrometer at the same time as or just after ionization, and the fragmentation of ions that occurs in the device thereafter are collectively referred to as in-source fragmentation (ISD), and the above-mentioned determination (product ion determination) performed with ISD is referred to as ISD determination. In addition, the fragment ions generated in the above-mentioned determination will be referred to as first fragment ions or ISD fragments hereinafter. According to the ISD determination using the above-mentioned changed analysis conditions, the first fragment ions on the low-mass region side of the m / z value away from the molecular weight-related ions can be detected with good sensitivity.

[0068] In step 103, the ISD measurement may be further performed using a condition in which the range of the prescribed mass-to-charge ratio is set in such a manner that the maximum value of the prescribed mass-to-charge ratio of the ions captured in the ion trap 2 is smaller than the value of the mass-to-charge ratio of the ions associated with the molecular weight of the sample molecule. In this case, it is preferably set so that the maximum value of the prescribed mass-to-charge ratio of the ions captured in the ion trap 2 is smaller than the value of the mass-to-charge ratio of the ions associated with the molecular weight of the sample molecule by, for example, about 0.5% to 40%, and more preferably set so that it is smaller by about 0.5% to 20%.

[0069] As a method of changing the range of the predetermined mass-to-charge ratio, there is, for example, the following method: for an analyte having a molecular weight of about 6000, the measurement mode pre-programmed in the device is switched from a measurement mode having a target mass range of m / z 2000 to 18000 to a measurement mode having 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 a target. The target mass range of the measurement mode is determined by the frequency of the high-frequency voltage applied to the ion trap 2. Specifically, since the amount of ions captured in the ion trap is limited, the mass range to be measured is determined mainly by adjusting the frequency of the high-frequency voltage and setting a cutoff value (Low Mass Cut-Off: LMCO) on the low mass side. If the frequency of the high-frequency voltage is increased, the LMCO is set 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 large and the mass range to be measured is set to the high mass side. That is, the predetermined mass-to-charge ratio range is changed by changing the frequency of the high-frequency voltage applied to the ion trap 2.

[0070] In addition, as another method for changing the range of the specified mass-to-charge ratio, there is the following method: for an analyte having a molecular weight of about 6000, by changing the value of the setting item of the ratio of the switching interval of the rectangular wave voltage pre-programmed into the device, i.e., the duty cycle, from a standard value of 50:50 to a value of 52:48, for example, to exclude ions with m / z 5500 or more. In this way, mass spectrum data is obtained after the first fragment ions having a mass-to-charge ratio close to the m / z value of the precursor ion are excluded. As a result, the detection sensitivity of the first fragment ions in the low-mass region far from the m / z value of the precursor ion is improved.

[0071] In step 103, the mass spectrum data obtained in each measurement can be obtained by performing ISD measurement in both the case where the condition of setting the mass-to-charge ratio range as described above is used and the case where the condition of setting the mass-to-charge ratio range as described above is not used. Thus, the first fragment ion derived from the sample molecule can be detected from the low mass region to the high mass region.

[0072] In addition, in addition to using the condition that the range of the predetermined mass-to-charge ratio is set so that the maximum value of the predetermined mass-to-charge ratio of the ions captured in the ion trap 2 is smaller than the value of the mass-to-charge ratio of the molecular weight-related ions of the analysis object, the ISD measurement may be performed using the condition that the standard analysis condition is acquired in step 102 for the applied voltage applied to the sample stage 12 of the ion source 1 and the applied voltage applied to the sample stage 12 is changed to a larger value than the analysis condition. The applied voltage applied to the sample stage is preferably set to be about 4 to 8 times higher than the standard analysis condition, and more preferably set to be about 4 to 5 times higher.

[0073] [Step 104: Preparation of ISD spectrum]

[0074] A mass spectrum (ISD spectrum) is prepared based on the data obtained by the ISD measurement in step 103. The mass spectrum data in the present invention includes information on the mass-to-charge ratio and signal intensity of each peak in the mass spectrum. In step 104, mass spectrum data (ISD spectrum data) obtained by the ISD measurement is acquired.

[0075] [Step 105: Analysis of mass spectrum data]

[0076] Peaks corresponding to various first fragment ions are extracted from the ISD spectrum data obtained in step 104, and the attribution of various first fragment ions is determined based on the mass information represented by the peaks, and at least a part of the structure of the original sample molecule is determined by combining these results. The determination of the structure includes sequence analysis, specifying the type of chemical modification or the site where the chemical modification is applied by sequence analysis. In the determination of the structure, database retrieval and de novo sequencing can also be used.

[0077] Alternatively, in step 103, ISD measurement is performed in both cases, when the condition of setting a predetermined mass-to-charge ratio range is used and when the condition of not setting a predetermined mass-to-charge ratio range is used, and when mass spectrum data of ISD measurement in both cases are obtained in step 104, the attribution results of various first fragment ions obtained from the two cases are combined and analyzed. In this way, the structural analysis of the object to be analyzed can be performed more reliably.

[0078] <Analysis object>

[0079] The object of analysis in the present invention is, for example, a sample molecule containing a relatively high-mass molecule such as a biopolymer. Examples of the biopolymer include nucleic acids, peptides, sugar chains, proteins, and lipids.

[0080] The nucleic acid mentioned here also includes nucleic acid-related substances such as modified nucleic acids, nucleic acid derivatives, and nucleic acid pharmaceuticals. Below, nucleic acids and nucleic acid-related substances are collectively referred to as nucleic acids. When the object of analysis is nucleic acid, the degree of polymerization (base length) of the nucleic acid is not particularly limited, and is preferably an oligonucleotide formed by polymerization of several to dozens of nucleotides. In the analysis method involved in the present invention, the analytical sensitivity of nucleic acids with large molecular weight is particularly improved, so nucleic acids with a molecular weight of more than 3000, especially a molecular weight of more than 6000 are more preferred. In addition, nucleic acids can be natural products obtained from organisms or their processed products, or they can be artificially synthesized nucleic acids synthesized by chemical synthesis.

[0081] <Method for preparing analytical samples>

[0082] Here, as an example, the analyte is assumed to be nucleic acid. A mixed solution obtained by mixing a sample containing nucleic acid and a matrix substance is dripped onto a sample plate and dried to prepare a sample for analysis. In this case, the mixed solution may be prepared in advance, dripped onto the sample plate and dried, or the mixed solution may be prepared on the sample plate and directly dried.

[0083] As the substrate substance, an appropriate substance can be selected according to the type of nucleic acid. For example, 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) can be mentioned. Among them, 2,4-DHAP and THAP are preferred. In addition, a mixed matrix obtained by mixing two or more matrix substances may be used. Among them, a mixed matrix obtained by mixing 3-HPA and 2,4-DHAP, 3-HPA and THAP, and 2,4-DHAP and THAP, respectively, is preferred.

[0084] The analytical sample may also contain a matrix additive. As a matrix additive, diammonium citrate (ACD) can be used. There are several types of ammonium salts of citric acid depending on the number of ammonium ions bonded to the citrate ion, but in this embodiment, a salt having two ammonium ions bonded to one citrate ion is preferably used.

[0085] In the case where the sample for analysis contains a matrix additive, the order in which the sample containing nucleic acid, the matrix substance, and the matrix additive are mixed is not particularly limited. It is preferred to prepare a matrix and additive mixed solution containing a matrix substance and a matrix additive in advance, and mix the sample solution containing nucleic acid with the matrix and additive mixed solution to prepare the sample for analysis. In this case, the sample for analysis can be prepared by dropping a mixed solution prepared by mixing the sample solution with the matrix and additive mixed solution on a sample plate and drying it, or by dropping the sample solution and the matrix and additive mixed solution on a sample plate, respectively, and mixing and drying them on the sample plate to prepare the sample for analysis. By preparing the matrix and additive mixed solution in advance, the preparation of the sample for analysis becomes easy. From the viewpoint of generating a sufficient amount of molecular weight-related ions of nucleic acids, the concentration of the matrix additive in the matrix and additive mixed solution is preferably 10mM to 100mM, and more preferably 30mM to 70mM.

[0086] Next, another embodiment of the method for analyzing the structure of a sample molecule according to the present invention will be described with reference to the drawings. Figure 3 This is a flowchart showing the process of the structural analysis method according to the present embodiment.

[0087] [Step 201: Acquisition of standard analysis conditions for MS measurement]

[0088] [Step 202: Setting of modified analysis conditions obtained by modifying standard analysis conditions]

[0089] [Step 203: Acquisition of Data Obtained by ISD Measurement Using Changed Analysis Conditions]

[0090] [Step 204: Preparation of ISD spectrum]

[0091] The standard analysis conditions for MS measurement are obtained by the same method as steps 101 to 104 described above (step 201), and the change conditions obtained by changing the standard analysis conditions are set (step 202), and data obtained by ISD measurement using the change conditions are obtained (step 203), and an ISD spectrum is prepared based on the data to obtain ISD spectrum data (step 204).

[0092] [Measurement 205: Setting of measurement conditions for MS / MS measurement]

[0093] At least one first fragment ion among the plurality of first fragment ions detected in the ISD measurement of step 203 is selected as a precursor ion based on the ISD spectrum data acquired in step 204, and measurement conditions for performing MS / MS measurement of the precursor ion are set. MS / MS measurement is generally known as the following measurement method: in a mass separation section at a previous stage, ions with a specific mass-to-charge ratio are selected from ions generated by sample molecules as precursor ions, the precursor ions are fragmented by collision induced dissociation (CID) in a subsequent collision chamber to generate various fragment ions (product ions), and the fragment ions (product ions) are separated in a mass separation section at a subsequent stage. In the MS / MS measurement of the ion trap type mass spectrometer used in the present embodiment, these ions are screened as precursor ions by leaving only ions in a specific mass range (with a specific mass-to-charge ratio) inside the ion trap 2. The precursor ions are then fragmented by CID with a gas such as argon introduced into the ion trap 2 to generate product ions containing various fragment ions. Thereafter, a mass scan is performed to discharge the ions in order from the smallest mass-to-charge ratio to the largest.

[0094] As precursor ion, preferably select the first fragment ion that is detected with relatively high sensitivity and does not overlap with other peaks as far as possible in the ISD spectrum data acquired by step 204. If this first fragment ion is set as precursor ion, multiple fragment ions (hereinafter, the fragment ion produced in the MS / MS determination is also referred to as the second fragment ion) produced in the MS / MS determination later are detected with good sensitivity. In addition, preferably select the first fragment ion in the low-quality region of the m / z value away from the molecular weight associated ion as precursor ion. If this first fragment ion is set as precursor ion, the mass spectrum obtained by MS / MS determination will not become complicated, and data analysis becomes easy. The m / z value of the first fragment selected as precursor ion is preferably below m / z 5000, more preferably below m / z 3000, and further preferably below m / z 2000.

[0095] [Step 206: Acquisition of Data Obtained by MS / MS Measurement]

[0096] MS / MS measurement is performed based on the measurement conditions set in step 205, and data related to the detected peaks are acquired.

[0097] [Step 207: Preparation of MS / MS spectrum]

[0098] A mass spectrum (MS / MS spectrum) is produced based on the data obtained in step 206 to acquire mass spectrum data (MS / MS spectrum data) obtained by MS / MS measurement.

[0099] [Step 208: Analysis of mass spectrum data]

[0100] Similar to step 105, the peaks corresponding to the various first fragment ions are extracted from the ISD spectrum data obtained in step 204, and the attribution of the various first fragment ions is determined according to the mass information represented by the peaks, and at least a part of the structure of the original sample molecule is determined by combining these results. In addition, the peaks corresponding to the various second fragment ions are extracted from the MS / MS spectrum data obtained in step 207, and the attribution of the various second fragment ions is determined according to the mass information represented by the peaks, and a part of the structure of the original sample molecule is determined by combining these results. By combining the peak attribution results of the ISD measurement with the peak attribution results of the MS / MS measurement, more information related to the structure of the sample molecule can be obtained, and the accuracy of the structural analysis is improved. According to the peak attribution results of the MS / MS measurement, it is easier to obtain information on the terminal structure of the sample molecule, which is difficult to obtain structural information due to the sensitivity of the ISD fragments being easily reduced in the case of only ISD measurement. In particular, when the sample molecule is a nucleic acid, the structure (base sequence, modification information, etc.) near the 3' end or 5' end of the nucleic acid can be determined according to the peak attribution results of the MS / MS measurement.

[0101] In the present embodiment, in step 208, the attribution of the first fragment ion and the second fragment ion is determined based on the ISD spectrum data obtained in step 204 and the MS / MS spectrum data obtained in step 207, respectively, but the attribution of the second fragment ion may be determined based only on the MS / MS spectrum data obtained in step 207. In this case, if the first fragment ion generated by the ISD measurement can be detected in step 203 and the m / z value of the first fragment ion can be obtained, step 204 (production of the ISD spectrum, acquisition of the ISD spectrum data) may not be performed, and in step 208, the attribution results of the various second fragment ions are combined to determine only a portion of the structure of the original sample molecule.

[0102] The structural analysis method according to the present invention will be described below by way of examples. However, these are merely illustrative and the present invention is not limited thereto.

[0103] Example 1

[0104] <1. Preparation of sample solution>

[0105] As a sample solution, a 10 pmol / μL aqueous solution of a standard nucleic acid A (5′-TGTGCGTGTGTAGTGTGTCT-3′: MW 6201.1, DNA, sequence number 1, synthesis request product) was prepared.

[0106] <2. Preparation of substrate solution>

[0107] As a matrix solution, a 50% acetonitrile (ACN) aqueous solution containing 40 mg / mL of 2,4-dihydroxyacetophenone (2,4-DHAP) and 70 mM diammonium hydrogen citrate (ACD) as a matrix additive was prepared.

[0108] <3. Preparation of Samples for Analysis>

[0109] The sample solution prepared in 1. and the matrix solution prepared in 2. were mixed at a ratio of 1:1 (v / v), and 1 μL of the obtained mixed solution was dropped onto a sample plate (SUS plate) and dried.

[0110] <4. Mass spectrometry analysis>

[0111] A MALDI digital ion trap mass spectrometer (MALDI-DITMS, manufactured by Shimadzu Corporation, trade name: MALDImini-1) was used for mass spectrometry. The sample plate on which the analytical sample prepared in 3. was placed was inserted into the MALDI-DITMS, and MS and ISD measurements were performed using the raster function in the positive mode.

[0112] <4-1. MS measurement>

[0113] As conditions for performing MS measurement (various setting values ​​of the mass spectrometer), the values ​​in the following Table 1 were used. The measurement mode is a mode that indicates the range of the mass-to-charge ratio of the ions to be measured, and more specifically, is a mode for defining the range of the mass-to-charge ratio of the ions captured in the ion trap, and the range is shown in parentheses. The same applies to the following tables.

[0114] [Table 1]

[0115]

[0116] <4-2. ISD measurement>

[0117] As conditions for performing ISD measurement (various setting values ​​of the mass spectrometer), the values ​​in the following Table 2 were used.

[0118] [Table 2]

[0119]

[0120] <5. Structural analysis>

[0121] The base sequence of the standard nucleic acid A was analyzed based on the mass spectrum data of the obtained fragment ions.

[0122] <Results>

[0123] exist Figure 4 The mass spectra of the standard nucleic acid A obtained by MS measurement and ISD measurement under various conditions are shown in the figure (the mass spectra when conditions (a), (b), (c), (d), and (e) are used in order from the top). In the mass spectra of each condition, the left side is the mass spectrum showing the entire mass-to-charge ratio (m / z 3300 to 6300) measured, and the right side is the mass spectrum after a part of it (m / z 4400 to 5200) is magnified. In addition, the arrows in the figure show the molecular weight-related ions ([M+H] + The numerical values ​​in the figure represent the peak intensity (mV) of the strongest ion in each mass spectrum.

[0124] according to Figure 4 It was confirmed that the protonated molecules [M+H] of the standard nucleic acid A were detected with high sensitivity in the mass spectrum of m / z 3300 to 6300 under condition (a). + At the same time, under condition (a), although several peaks containing fragment ions of base desorption ions were detected, the number of fragment ions was small and the detection sensitivity was low ( Figure 4 (a)).

[0125] In contrast, it was confirmed that under conditions (b) to (e) obtained by gradually changing the detector voltage, conversion dynode voltage, RF delay value, and laser power, the detection sensitivity of the fragment ion peaks was improved and the number of detected peaks increased ( Figure 4 In particular, when the measurement under condition (a) is compared with the measurement under condition (e), the sensitivity of the fragment ion peaks detected on the low mass-to-charge ratio side is improved, and the number of detected peaks increases significantly.

[0126] exist Figure 5 The ISD spectrum of the fragment ions obtained under condition (e) and the analysis of the base sequence of the nucleic acid are shown in FIG. Figure 5 , most of the sequence of the standard nucleic acid A (90% of the entire sequence excluding the sequence near the center) can be determined.

[0127] The above results are believed to be caused by the following: by changing the laser power to a value higher than the value in the usual MS measurement, the amount of ions generated in the ion source increases, and by changing the detector voltage and the conversion dynode voltage to values ​​higher than the values ​​in the usual MS measurement, each ion signal is amplified. In addition, it is believed that by changing the RFdelay value to a value lower than the value in the usual MS measurement, fragment ions on the side of the lower mass-to-charge ratio compared with the precursor ion can be preferentially captured, and as a result, the detection sensitivity of the fragment ions can be improved. Based on the above, it was confirmed that ISD analysis using MALDI-ITMS can be performed, and most of the sequence can be determined for a standard nucleic acid (MW 6201) with a relatively high mass.

[0128] Example 2

[0129] Mass spectrometry analysis and structural analysis were performed in the same manner as in Example 1 except that the measurement conditions for MS measurement and ISD measurement were different.

[0130] <4-1. MS measurement>

[0131] As conditions for MS measurement (various setting values ​​of the mass spectrometer), the values ​​in Table 3 below were used.

[0132] [Table 3]

[0133]

[0134] <4-2. ISD measurement>

[0135] As conditions for performing ISD measurement (various setting values ​​of the mass spectrometer), the values ​​in the following Table 4 were used.

[0136] [Table 4]

[0137]

[0138] <Results>

[0139] First, under condition (a), although not shown in the figure, it was confirmed that the protonated molecules [M+H] of the standard nucleic acid A + Detected with high sensitivity.

[0140] Then, in Figure 6 The ISD spectra of the standard nucleic acid A obtained by ISD measurement under various conditions are shown in FIG. 1 (ISD spectra when conditions (b), (c), and (d) are used in order from the bottom). When the measurement is performed using condition (b) obtained by changing the detector voltage, conversion dynode voltage, RF delay value, and laser power relative to condition (a), it is easy to detect fragment ions on the high mass region side close to the precursor ion ( Figure 6 On the other hand, when the measurement is performed under conditions (c) and (d) in which the measurement mode is changed in addition to the above changes so that the maximum value of the mass-to-charge ratio range of the ions captured in the ion trap is smaller than the mass-to-charge ratio of the precursor ions, fragment ions are detected even on the low-mass region side away from the precursor ions ( Figure 6 In particular, under condition (d) where the sample stage voltage is set higher than condition (a), it is confirmed that many fragment ions on the low-mass region side are detected with relatively high sensitivity ( Figure 6 In addition, regarding conditions (b) to (d), in order to more easily understand the influence of the measurement mode and the sample stage voltage, a comparative evaluation was performed by making the other analysis conditions the same (Table 4).

[0141] exist Figure 5 The ISD spectrum of the standard nucleic acid A and the sequence and attribution of the standard nucleic acid A are shown in Table 4 (condition (b)) when the ISD measurement is performed under the condition in which the laser power is changed to 65 (i.e., under the condition in Table 2 (e)). At least in this experiment, under the condition in Table 4 (b) when the laser power is changed to 75 (conditions in Table 4 (b), Figure 6 (b)) and when the laser power is changed to 65 (condition (e) of Table 2, Figure 5 ), the obtained ISD spectrum is almost unchanged. Therefore, using Figure 5 The following is to explain the attribution of the ISD spectrum under condition (b) of Table 4. Figure 5 , many fragment ions can be assigned, and about 90% of the base sequence can be determined. However, due to the low sensitivity of the fragment ions on the low-mass region side, it is not possible to analyze the entire sequence.

[0142] exist Figure 7 The case where ISD measurement was performed under condition (d) is shown in FIG. Figure 6 The ISD spectrum of the standard nucleic acid A under the condition of (d) and the sequence and attribution of the standard nucleic acid A. Figure 7 In Figure 5 Many fragment ions in the low-mass region of the peak could not be observed due to low detection sensitivity, and about 60% of the base sequence was determined. However, since fragment ions could not be detected in the high-mass region near the precursor ion of the standard nucleic acid A, the entire sequence could not be analyzed.

[0143] Finally, by Figure 5 The analytical results obtained from the mass spectrometry data and based on Figure 7By combining the analysis results obtained from the mass spectrometry data, the entire sequence of the standard nucleic acid with a relatively high mass (MW 6201) can be analyzed.

[0144] Example 3

[0145] Mass spectrometry analysis and structural analysis were performed in the same manner as in Example 1 except that the measurement conditions for MS measurement and ISD measurement were different.

[0146] <4-1. MS measurement>

[0147] As conditions for MS measurement (various setting values ​​of the mass spectrometer), the values ​​in the following Table 5 were used.

[0148] [Table 5]

[0149]

[0150] <4-2. ISD measurement>

[0151] As conditions for performing ISD measurement (various setting values ​​of the mass spectrometer), the values ​​in the following Table 6 were used.

[0152] [Table 6]

[0153]

[0154] <Results>

[0155] First, under condition (a), although not shown in the figure, it was confirmed that the protonated molecules [M+H] of the standard nucleic acid A + Detected with high sensitivity.

[0156] Then, in Fig. 8A , 8B The ISD spectra of the standard nucleic acid A obtained by the ISD measurement under various conditions are shown in FIG. 1 (the ISD spectra when the conditions (b) and (c) are used in order from the following). In the ISD spectra under various conditions, Fig. 8A is a graph showing the entire mass-to-charge ratio (m / z 2000 to 6000) measured, Figure 8B This is an enlarged view of a part (m / z 3000 to 5000).

[0157] When the measurement is performed using condition (b) obtained by changing the detector voltage, conversion dynode voltage, RF delay value, and laser power relative to condition (a), it is easy to detect fragment ions ( Fig. 8AOn the other hand, when the measurement is performed using the set condition (c) in which the duty ratio related to the high-frequency voltage applied to the ion trap is changed in addition to the above-mentioned change so that the maximum value of the mass-to-charge ratio range of the ions captured in the ion trap is smaller than the mass-to-charge ratio of the precursor ions, the ions near the precursor ions are excluded ( Fig. 8A (c-2)), it is easy to detect fragment ions in the low mass region far from the m / z value of the precursor ion ( Fig. 8A (c-2), Figure 8B As a result, the peak of the fragment ion on the low-mass region side is detected with higher intensity by changing the duty ratio.

[0158] exist Fig. 9 The ISD spectrum of the standard nucleic acid A when the ISD measurement is performed under condition (c) and the sequence and attribution of the standard nucleic acid A are shown in FIG. Fig. 9 If ions with low peak intensity are also included, many fragment ions can be assigned and the entire sequence can be analyzed. However, under this condition, the intensity of fragment ions near the precursor ion is low.

[0159] exist Fig.10 The ISD spectra of standard nucleic acid A when ISD measurement was performed under conditions (b) and (c) are shown (the upper part is condition (b), the lower part is condition (c)), as well as the sequence of standard nucleic acid A and its attribution status. In the mass spectrum when condition (c) is used, the entire sequence of the nucleic acid can be analyzed, but the peak intensity of ions near the precursor ions is low. On the other hand, in the mass spectrum when condition (b) is used, fragment ions near the precursor are observed with sufficient intensity. By combining the analysis results obtained from the mass spectral data of conditions (b) and (c), the entire sequence of the standard nucleic acid with a relatively high mass (MW 6201) can be analyzed more reliably.

[0160] Example 4

[0161] <1. Preparation of sample solution>

[0162] As a sample solution, a 20 pmol / μL aqueous solution of mipomersen (a desalted and purified sample of synthetic nucleic acid for research and development. DNA: total length 20 bases, MW 7177) (5'-MG-MC-MC-MU-MC-dA-dG-dT-dC-dT-dG-dC-dT-dT-dC-MG-MC-MA-MC-MC-3', M is 2'-O-(2-methoxyethyl)nucleoside, d is 2'-deoxynucleoside. The carbon at the 5-position of cytosine and uracil is replaced by a methyl group, and all the phosphodiester bonds between nucleotides are replaced by phosphorothioate bonds.: sequence number 2) was prepared.

[0163] <2. Preparation of substrate solution>

[0164] 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, and a 40 mg / mL 50% ACN aqueous solution (2,4-DHAP solution) of 2,4-DHAP containing 70 mM ACD as a matrix additive were prepared, and a mixed matrix (3-HPA+2,4-DHAP, 1:1) solution was prepared by mixing the 3-HPA solution and the 2,4-DHAP solution in a ratio of 1:1 (v / v).

[0165] <3. Preparation of Samples for Analysis>

[0166] The sample solution prepared in 1. and the mixed matrix solution prepared in 2. were mixed at a ratio of 1:1 (v / v), and 1 μL of the obtained mixed solution was dropped onto a sample plate (SUS plate) and dried.

[0167] <4. Mass spectrometry analysis>

[0168] <4-1. ISD measurement>

[0169] The sample plate of 3. was inserted into MALDI-DITMS (manufactured by Shimadzu Corporation, trade name: MALDImini-1), and ISD measurement was performed using the grating function in the positive mode. As the conditions for performing the ISD measurement (various setting values ​​of the mass spectrometer), the values ​​in the following Table 7 were used. Regarding the laser power, the optimal value was used under each condition.

[0170] [Table 7]

[0171]

[0172] <4-2. MS / MS measurement>

[0173] In the ISD spectrum obtained by the ISD measurement in 4-1., MS / MS measurement (low energy CID) was performed using measurement conditions in which an ISD fragment on the relatively low mass side (w5-ion in this case) was used as a precursor ion.

[0174] <5. Structural analysis>

[0175] The base sequence of mipomersen was analyzed by assigning the main fragment ion peaks of the ISD spectrum obtained by the ISD measurement in 4-1. and the MS / MS spectrum obtained by the MS / MS measurement in 4-2.

[0176] <Results>

[0177] exist Fig.11 The ISD spectrum of mipomersen obtained using the mixed matrix 3-HPA+2,4-DHAP and the attribution of the main peaks are shown in FIG. Fig.11 (a) is the ISD spectrum obtained by performing ISD measurement using the condition (a) of Table 7, Fig.11 (b) is the ISD spectrum obtained by performing ISD measurement using the condition (b) of Table 7, Fig.11 The portion indicated by the bar x2 in (b) has a peak intensity twice as high as that of the other portions. Fig.11 In the ISD spectrum, the name of the corresponding oligonucleotide fragment ion species (the general name proposed in non-patent document 4) is annotated for each peak. The name is the name of each ion species using the fragment ion sequence according to the naming rules of the nucleic acid fragmentation pattern, and the fragment ion containing the 5' end is recorded as a n 、b n 、c n d n , fragment ions containing the 3' terminus in the opposite direction are recorded as x m ,y m 、z m 、w m In addition, the subscripts n and m represent the number of constituent units from the corresponding end to the cleavage site (by definition, the number of bases). In addition, the B in the name of the fragment ion species represents the base of the nucleic acid, for example, Fig.11 a in (b) 16 -B(G) indicates that the base guanine (G) is from a 16 Ions obtained by ion desorption. The same is true in the following figures.

[0178] Fig.11 The portion surrounded by a box on the right end of (b) represents the molecular weight-related ion [M+H] +The spectrum near the m / z value of (precursor ion). In this region, it was confirmed that the base desorption peak of the precursor ion was particularly detected with high sensitivity, and the detection of the ISD fragment peak was hindered because the base desorption peak overlapped with the ISD fragment peak.

[0179] exist Fig.12 It will be shown in Fig.11 The w5-ion detected in the ISD spectrum is set as the MS / MS spectrum of the precursor ion and the main peak attribution status. Fig.11 Compared with the ISD spectrum and peak attribution of Fig.12 In the present invention, multiple second fragment ions containing terminal sequence information are generated with sufficient intensity and resolution, which can more reliably analyze the terminal sequence. Fig.12 In the MS / MS spectrum of Fig.11 The overlap of the product ion peak near the precursor ion such as the base desorption peak observed in the ISD spectrum of (b) and the peak of the second fragment ion.

[0180] As a whole, from Fig.11 After obtaining as much base sequence information as possible from the ISD spectrum, an MS / MS measurement is performed using the ISD fragments of the low-mass region that are relatively far from the m / z value of the molecular weight-related ion as precursor ions, thereby clearly detecting the fragment ion peaks near the m / z value of the molecular weight-related ion that is likely to hinder peak detection in the ISD measurement and analyzing the base sequence at the end, thereby enabling a more reliable full base sequence analysis. That is, by combining ISD measurement and MS / MS measurement, a more reliable base sequence analysis can be performed.

[0181] The method of combining ISD measurement and MS / MS measurement to perform structural analysis can be used basically regardless of the object of analysis. In addition, it can be improved to enable more reliable terminal sequence analysis, which is a subject of ISD analysis of MALDI-MS. It is believed that this method is particularly effective when using MALDI-IT-TOFMS, which is prone to fragmentation, to measure unstable nucleic acids that are prone to cleavage such as base desorption.

[0182] [Way]

[0183] It will be clear to those skilled in the art that the above-described exemplary embodiments are specific examples of the following aspects.

[0184] (First Item)

[0185] A method for analyzing the structure of a sample molecule according to one embodiment of the present invention is a method for analyzing the structure of a sample molecule using an ion trap mass spectrometer, wherein the ion trap mass spectrometer comprises: an ion source based on a matrix-assisted laser desorption ionization (MALDI) 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, and the method for analyzing the structure of a sample molecule comprises the following steps:

[0186] a standard analysis condition acquisition step for respectively acquiring standard analysis conditions for ion quantity setting items related to the quantity of ions generated by the ion source, a mass-to-charge ratio range setting item related to the mass-to-charge ratio range of ions captured in the ion capture section, and a signal intensity setting item related to the signal intensity of ions in the detection section when the mass spectrometer is used to perform molecular weight associated ion measurement for detecting molecular weight associated ions of the sample molecules contained in the sample;

[0187] A product ion measurement data acquisition step, performing a product ion measurement for detecting a plurality of first fragment ions generated by the fragmentation of the molecular weight-related ions of the sample molecule under a changed analysis condition obtained by changing at least one of the analysis conditions of the ion quantity setting item, the mass-to-charge ratio range setting item, and the signal intensity setting item in the standard analysis condition acquired in the standard analysis condition acquisition step, and acquiring mass spectrum data of the product ion measurement;

[0188] a first fragment peak extraction step of extracting a peak corresponding to the first fragment ion from the mass spectrum data acquired in the product ion measurement data acquisition step; and

[0189] The data analysis step specifies at least a part of the structure of the sample molecule based on the mass information of the peak extracted in the first fragment peak extraction step.

[0190] This makes it possible to detect fragment ions required for structural analysis with high sensitivity in a relatively wide mass range from a low-mass region to a high-mass region.

[0191] (Second Item)

[0192] In the method for analyzing the structure of a sample molecule according to the first item, it is also possible that:

[0193] The mass spectrometer is a device capable of performing MS / MS measurement,

[0194] The method for analyzing the structure of the sample molecule further comprises the following steps:

[0195] An MS / MS measurement condition setting step of setting measurement conditions for performing MS / MS measurement in which at least one first fragment ion among the plurality of first fragment ions detected in the product ion measurement is set as a precursor ion;

[0196] An MS / MS measurement data acquisition step of performing the MS / MS measurement based on the measurement conditions set in the MS / MS measurement condition setting step to acquire mass spectrum data of the MS / MS measurement; and

[0197] a second fragment peak extraction step of extracting a peak corresponding to a second fragment ion generated in the MS / MS measurement from the mass spectrum data acquired in the MS / MS measurement data acquisition step;

[0198] In the data analysis step, at least a part of the structure of the sample molecule is determined based on the mass information of the peak extracted in the first fragment peak extraction step and the mass information of the peak extracted in the second fragment peak extraction step.

[0199] Thus, not only the first fragment ion derived from the molecular weight-related ion can be detected with good sensitivity, but also the second fragment ion obtained by further fragmenting the first fragment ion can be detected with good sensitivity. As a result, for sample molecules whose overall molecular structure cannot be accurately analyzed using only the mass information of the first fragment ion, the structural information can be more reliably determined based on the mass information of the first fragment ion and the second fragment ion.

[0200] (Third Item)

[0201] A method for analyzing the structure of a sample molecule according to another embodiment of the present invention is a method for analyzing the structure of a sample molecule using an ion trap mass spectrometer, wherein the ion trap mass spectrometer comprises: an ion source based on a matrix-assisted laser desorption ionization (MALDI) 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, and the method for analyzing the structure of a sample molecule comprises the following steps:

[0202] a standard analysis condition acquisition step for respectively acquiring standard analysis conditions for ion quantity setting items related to the quantity of ions generated by the ion source, a mass-to-charge ratio range setting item related to the mass-to-charge ratio range of ions captured in the ion capture section, and a signal intensity setting item related to the signal intensity of ions in the detection section when the mass spectrometer is used to perform molecular weight associated ion measurement for detecting molecular weight associated ions of the sample molecules contained in the sample;

[0203] A product ion determination step, performing product ion determination for detecting a plurality of first fragment ions generated by the fragmentation of the molecular weight-related ions of the sample molecule under changed analysis conditions obtained by changing at least one of the analysis conditions of the ion quantity setting item, the mass-to-charge ratio range setting item, and the signal intensity setting item in the standard analysis conditions acquired in the standard analysis condition acquisition step;

[0204] An MS / MS measurement condition setting step of setting measurement conditions for performing MS / MS measurement in which at least one first fragment ion among the plurality of first fragment ions detected in the product ion measurement is set as a precursor ion;

[0205] an MS / MS measurement data acquisition step of acquiring mass spectrum data of the MS / MS measurement by performing the MS / MS measurement based on the measurement conditions set in the MS / MS measurement condition setting step;

[0206] a second fragment peak extraction step of extracting a peak corresponding to a second fragment ion generated in the MS / MS measurement from the mass spectrum data acquired in the MS / MS measurement data acquisition step; and

[0207] The data analysis step specifies a part of the structure of the sample molecule based on the mass information of the peak extracted in the second fragment peak extraction step.

[0208] Thus, the second fragment ions obtained by further fragmenting the first fragment ions derived from the molecular weight-related ions can be detected with high sensitivity. As a result, the structural information of the molecular structure of the sample molecule that cannot be accurately analyzed using only the mass information of the first fragment ions can be determined more reliably.

[0209] (4th item)

[0210] The method for analyzing the structure of a sample molecule according to any one of the first to third items may be:

[0211] The changed analysis conditions are conditions obtained by changing the analysis conditions of the ion amount setting item in a manner that increases the amount of ions generated by the ion source compared to when the molecular weight associated ions of the sample are measured under the standard analysis conditions, or conditions obtained by changing the analysis conditions of the mass-to-charge ratio range setting item in a manner that preferentially captures ions on the lower mass side than the mass-to-charge ratio of the molecular weight associated ions of the sample molecules, or conditions obtained by changing the analysis conditions of the signal intensity setting item in a manner that increases the signal intensity of the ions in the detection unit.

[0212] This makes it possible to detect, with high sensitivity, fragment ions, particularly those at a low mass-to-charge ratio, among the fragment ions required for analysis.

[0213] (Fifth Item)

[0214] The method for analyzing the structure of a sample molecule according to any one of the first to fourth items may be:

[0215] The changed analysis conditions are obtained by changing the analysis conditions of the ion amount setting item so as to increase the amount of ions generated by the ion source, and changing the analysis conditions of the signal intensity setting item so as to increase the signal intensity of ions in the detection unit.

[0216] This makes it possible to detect, among the fragment ions required for analysis, particularly the fragment ions at the low mass-to-charge ratio side with higher sensitivity.

[0217] (Sixth Item)

[0218] The method for analyzing the structure of a sample molecule according to any one of the first to fifth items may be:

[0219] The ion quantity setting item is the intensity of the laser irradiated by the ion source.

[0220] The modified analysis conditions are conditions obtained by changing the laser intensity to a value greater than the laser intensity of the standard analysis conditions.

[0221] This makes it possible to detect, among the fragment ions required for analysis, particularly the fragment ions at the low mass-to-charge ratio side with higher sensitivity.

[0222] (Item 7)

[0223] The method for analyzing the structure of a sample molecule according to any one of the first to sixth items may be:

[0224] The mass-to-charge ratio range setting item is the time from irradiation of laser light by the ion source to application of a trapping voltage for trapping ions to the ion trapping unit.

[0225] The modified analysis condition is a condition obtained by changing the time to a value shorter than the time of the standard analysis condition.

[0226] This makes it possible to detect, among the fragment ions required for analysis, particularly the fragment ions at the low mass-to-charge ratio side with higher sensitivity.

[0227] (Item 8)

[0228] According to the method for analyzing the structure of the sample molecule described in item 7, it is also possible to:

[0229] The modified analysis condition is a condition in which the time is set to a value shorter than the time in the standard analysis condition by 1 microsecond to 3 microseconds.

[0230] This makes it possible to detect, among the fragment ions required for analysis, particularly the fragment ions at the low mass-to-charge ratio side with higher sensitivity.

[0231] (Item 9)

[0232] The method for analyzing the structure of a sample molecule according to any one of the first to eighth items may be:

[0233] The detection unit includes a conversion dynode and a detector.

[0234] The signal intensity setting items are the applied voltage applied to the conversion dynode and the applied voltage applied to the detector,

[0235] The changed analysis condition is a condition obtained by changing at least one of the voltage applied to the conversion dynode and the voltage applied to the detector to a value larger than the standard analysis condition.

[0236] This makes it possible to detect, among the fragment ions required for analysis, particularly the fragment ions at the low mass-to-charge ratio side with higher sensitivity.

[0237] (Item 10)

[0238] The method for analyzing the structure of a sample molecule according to any one of the first to ninth items may be:

[0239] The product ion measurement is performed by setting the predetermined mass-to-charge ratio range so that the maximum value of the predetermined mass-to-charge ratio of the ions captured by the ion capturing unit is smaller than the mass-to-charge ratio value of the molecular weight-related ions of the sample molecule.

[0240] This makes it possible to detect, among the fragment ions required for analysis, particularly the fragment ions at the low mass-to-charge ratio side with higher sensitivity.

[0241] (Item 11)

[0242] According to the method for analyzing the structure of a sample molecule as recited in the tenth item, it is also possible that:

[0243] The frequency of the trapping voltage in the ion trapping unit is set so as to set the predetermined mass-to-charge ratio range.

[0244] This makes it possible to detect, among the fragment ions required for analysis, particularly the fragment ions at the low mass-to-charge ratio side with higher sensitivity.

[0245] (Item 12)

[0246] According to the method for analyzing the structure of a sample molecule as recited in the tenth item, it is also possible that:

[0247] The mass spectrometer is a digital ion trap mass spectrometer.

[0248] The duty ratio of the trapping voltage in the ion trapping section is set so as to set the predetermined mass-to-charge ratio range.

[0249] This makes it possible to detect, among the fragment ions required for analysis, particularly the fragment ions at the low mass-to-charge ratio side with higher sensitivity.

[0250] (Item 13)

[0251] The method for analyzing the structure of a sample molecule according to any one of items 10 to 12 may be:

[0252] The mass spectrometer comprises a sample stage for placing a sample plate on which the sample is placed.

[0253] In the standard analysis condition acquisition step, a standard analysis condition for a voltage applied to the sample stage when performing the molecular weight-related ion measurement is further acquired.

[0254] The product ion measurement is performed under the condition that the voltage applied to the sample stage is changed to a value larger than the voltage applied under the standard analysis condition.

[0255] This makes it possible to detect, among the fragment ions required for analysis, particularly the fragment ions at the low mass-to-charge ratio side with higher sensitivity.

[0256] (Item 14)

[0257] The method for analyzing the structure of a sample molecule according to any one of the first, second, and fourth to thirteenth items may be:

[0258] The mass spectrometer is a digital ion trap mass spectrometer.

[0259] In the product ion measurement data acquisition process, at least one of the frequency of the capture voltage and the duty cycle of the capture voltage in the ion capture section is changed in such a way that the maximum value of the specified mass-to-charge ratio of the ions captured by the ion capture section is smaller than the value of the mass-to-charge ratio of the molecular weight-related ions of the sample molecule, or the product ion measurement is performed without changing at least one of the frequency of the capture voltage and the duty cycle of the capture voltage, so as to respectively acquire mass spectrum data of the product ion measurement,

[0260] In the first fragment peak extraction step, peaks corresponding to the plurality of first fragment ions are extracted from both the mass spectrometry data acquired by performing the product ion measurement in the product ion measurement data acquisition step by changing at least one of the frequency of the capture voltage and the duty cycle of the capture voltage, and the mass spectrometry data acquired by performing the product ion measurement without changing at least one of the frequency of the capture voltage and the duty cycle of the capture voltage,

[0261] In the data analysis step, at least a part of the structure of the sample molecule is determined based on the mass information of the peak extracted in the first fragment peak extraction step.

[0262] This makes it possible to more reliably analyze the structure of the sample molecules.

[0263] (Item 15)

[0264] The method for analyzing the structure of a sample molecule according to any one of the first to fourteenth items may be:

[0265] The sample molecule is a nucleic acid or a nucleic acid-related substance.

[0266] (Item 16)

[0267] According to the method for analyzing the structure of the sample molecule described in the second item, it is also possible to:

[0268] The sample molecule is a nucleic acid or a nucleic acid-related substance, and a part of the structure of the sample molecule is a structure of a terminal portion of the nucleic acid or the nucleic acid-related substance.

[0269] (Item 17)

[0270] According to the method for analyzing the structure of the sample molecule described in the third item, it is also possible to:

[0271] The sample molecule is a nucleic acid or a nucleic acid-related substance, and a part of the structure of the sample molecule is a structure of a terminal portion of the nucleic acid or the nucleic acid-related substance.

[0272] Description of Reference Numerals

[0273] 1: ion source; 2: ion trap; 3: detection unit; 11: laser irradiation unit; 12: sample stage; 21: annular electrode; 22, 23: end cap electrodes; 31: conversion dynode; 32: detector (secondary electron multiplier tube).

Claims

1. A method for analyzing the structure of a sample molecule, which is a method for analyzing the structure of a sample molecule using an ion trap mass spectrometer, wherein the ion trap mass spectrometer comprises: an ion source based on a matrix-assisted laser desorption ionization method (MALDI 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, wherein the method for analyzing the structure of a sample molecule comprises the following steps: a standard analysis condition acquisition step for respectively acquiring standard analysis conditions for ion quantity setting items related to the quantity of ions generated by the ion source, a mass-to-charge ratio range setting item related to the mass-to-charge ratio range of ions captured in the ion capture section, and a signal intensity setting item related to the signal intensity of ions in the detection section when the mass spectrometer is used to perform molecular weight associated ion measurement for detecting molecular weight associated ions of the sample molecules contained in the sample; A product ion measurement data acquisition step, performing a product ion measurement for detecting a plurality of first fragment ions generated by the fragmentation of the molecular weight-related ions of the sample molecule under a changed analysis condition obtained by changing at least one of the analysis conditions of the ion quantity setting item, the mass-to-charge ratio range setting item, and the signal intensity setting item in the standard analysis condition acquired in the standard analysis condition acquisition step, and acquiring mass spectrum data of the product ion measurement; a first fragment peak extraction step of extracting a peak corresponding to the first fragment ion from the mass spectrum data acquired in the product ion measurement data acquisition step; and The data analysis step specifies at least a part of the structure of the sample molecule based on the mass information of the peak extracted in the first fragment peak extraction step.

2. The method for analyzing the structure of a sample molecule according to claim 1, wherein: The mass spectrometer is a device capable of performing MS / MS measurement, The method for analyzing the structure of the sample molecule further comprises the following steps: An MS / MS measurement condition setting step of setting measurement conditions for performing MS / MS measurement in which at least one first fragment ion among the plurality of first fragment ions detected in the product ion measurement is set as a precursor ion; An MS / MS measurement data acquisition step of performing the MS / MS measurement based on the measurement conditions set in the MS / MS measurement condition setting step to acquire mass spectrum data of the MS / MS measurement; as well as a second fragment peak extraction step of extracting a peak corresponding to a second fragment ion generated in the MS / MS measurement from the mass spectrum data acquired in the MS / MS measurement data acquisition step; In the data analysis step, at least a part of the structure of the sample molecule is determined based on the mass information of the peak extracted in the first fragment peak extraction step and the mass information of the peak extracted in the second fragment peak extraction step.

3. A method for analyzing the structure of a sample molecule, which is a method for analyzing the structure of a sample molecule using an ion trap mass spectrometer, wherein the ion trap mass spectrometer comprises: an ion source based on a matrix-assisted laser desorption ionization method (MALDI 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, wherein the method for analyzing the structure of a sample molecule comprises the following steps: a standard analysis condition acquisition step for respectively acquiring standard analysis conditions for ion quantity setting items related to the quantity of ions generated by the ion source, a mass-to-charge ratio range setting item related to the mass-to-charge ratio range of ions captured in the ion capture section, and a signal intensity setting item related to the signal intensity of ions in the detection section when the mass spectrometer is used to perform molecular weight associated ion measurement for detecting molecular weight associated ions of the sample molecules contained in the sample; A product ion determination step, performing product ion determination for detecting a plurality of first fragment ions generated by the fragmentation of the molecular weight-related ions of the sample molecule under changed analysis conditions obtained by changing at least one of the analysis conditions of the ion quantity setting item, the mass-to-charge ratio range setting item, and the signal intensity setting item in the standard analysis conditions acquired in the standard analysis condition acquisition step; An MS / MS measurement condition setting step of setting measurement conditions for performing MS / MS measurement in which at least one first fragment ion among the plurality of first fragment ions detected in the product ion measurement is set as a precursor ion; an MS / MS measurement data acquisition step of acquiring mass spectrum data of the MS / MS measurement by performing the MS / MS measurement based on the measurement conditions set in the MS / MS measurement condition setting step; a second fragment peak extraction step of extracting a peak corresponding to a second fragment ion generated in the MS / MS measurement from the mass spectrum data acquired in the MS / MS measurement data acquisition step; and The data analysis step specifies a part of the structure of the sample molecule based on the mass information of the peak extracted in the second fragment peak extraction step.

4. The method for analyzing the structure of a sample molecule according to claim 1, wherein: The changed analysis conditions are conditions obtained by changing the analysis conditions of the ion amount setting item in a manner that increases the amount of ions generated by the ion source compared to when the molecular weight associated ions of the sample are measured under the standard analysis conditions, or conditions obtained by changing the analysis conditions of the mass-to-charge ratio range setting item in a manner that preferentially captures ions on the lower mass side than the mass-to-charge ratio of the molecular weight associated ions of the sample molecules, or conditions obtained by changing the analysis conditions of the signal intensity setting item in a manner that increases the signal intensity of the ions in the detection unit.

5. The method for analyzing the structure of a sample molecule according to claim 1, wherein: The changed analysis conditions are obtained by changing the analysis conditions of the ion amount setting item so as to increase the amount of ions generated by the ion source, and changing the analysis conditions of the signal intensity setting item so as to increase the signal intensity of ions in the detection unit.

6. The method for analyzing the structure of a sample molecule according to claim 1, wherein: The ion quantity setting item is the intensity of the laser irradiated by the ion source. The modified analysis conditions are conditions obtained by changing the laser intensity to a value greater than the laser intensity of the standard analysis conditions.

7. The method for analyzing the structure of a sample molecule according to claim 1, wherein: The mass-to-charge ratio range setting item is the time from irradiation of laser light by the ion source to application of a trapping voltage for trapping ions to the ion trapping unit. The modified analysis condition is a condition obtained by changing the time to a value shorter than the time of the standard analysis condition.

8. The method for analyzing the structure of a sample molecule according to claim 7, wherein: The modified analysis condition is a condition in which the time is set to a value shorter than the time in the standard analysis condition by 1 microsecond to 3 microseconds.

9. The method for analyzing the structure of a sample molecule according to claim 1, wherein: The detection unit includes a conversion dynode and a detector. The signal intensity setting items are the applied voltage applied to the conversion dynode and the applied voltage applied to the detector, The changed analysis condition is a condition obtained by changing at least one of the voltage applied to the conversion dynode and the voltage applied to the detector to a value larger than the standard analysis condition.

10. The method for analyzing the structure of a sample molecule according to claim 1, wherein: The product ion measurement is performed by setting the predetermined mass-to-charge ratio range so that the maximum value of the predetermined mass-to-charge ratio of the ions captured by the ion capturing unit is smaller than the mass-to-charge ratio value of the molecular weight-related ions of the sample molecule.

11. The method for analyzing the structure of a sample molecule according to claim 10, wherein: The frequency of the trapping voltage in the ion trapping unit is set so as to set the predetermined mass-to-charge ratio range.

12. The method for analyzing the structure of a sample molecule according to claim 10, wherein: The mass spectrometer is a digital ion trap mass spectrometer. The duty ratio of the trapping voltage in the ion trapping section is set so as to set the predetermined mass-to-charge ratio range.

13. The method for analyzing the structure of a sample molecule according to claim 10, wherein: The mass spectrometer comprises a sample stage for placing a sample plate on which the sample is placed. In the standard analysis condition acquisition step, a standard analysis condition for a voltage applied to the sample stage when performing the molecular weight-related ion measurement is further acquired. The product ion measurement is performed under the condition that the voltage applied to the sample stage is changed to a value larger than the voltage applied under the standard analysis condition.

14. The method for analyzing the structure of a sample molecule according to claim 1, wherein: The mass spectrometer is a digital ion trap mass spectrometer. In the product ion measurement data acquisition process, at least one of the frequency of the capture voltage and the duty cycle of the capture voltage in the ion capture section is changed in such a way that the maximum value of the specified mass-to-charge ratio of the ions captured by the ion capture section is smaller than the value of the mass-to-charge ratio of the molecular weight-related ions of the sample molecule, or the product ion measurement is performed without changing at least one of the frequency of the capture voltage and the duty cycle of the capture voltage, so as to respectively acquire mass spectrum data of the product ion measurement, In the first fragment peak extraction step, peaks corresponding to the plurality of first fragment ions are extracted from both the mass spectrometry data acquired by performing the product ion measurement in the product ion measurement data acquisition step by changing at least one of the frequency of the capture voltage and the duty cycle of the capture voltage, and the mass spectrometry data acquired by performing the product ion measurement without changing at least one of the frequency of the capture voltage and the duty cycle of the capture voltage, In the data analysis step, at least a part of the structure of the sample molecule is determined based on the mass information of the peak extracted in the first fragment peak extraction step.

15. The method for analyzing the structure of a sample molecule according to claim 1, wherein: The sample molecule is a nucleic acid or a nucleic acid-related substance.

16. The method for analyzing the structure of a sample molecule according to claim 2, wherein: The sample molecule is a nucleic acid or a nucleic acid-related substance, and a part of the structure of the sample molecule is a structure of a terminal portion of the nucleic acid or the nucleic acid-related substance.

17. The method for analyzing the structure of a sample molecule according to claim 3, wherein: The sample molecule is a nucleic acid or a nucleic acid-related substance, and a part of the structure of the sample molecule is a structure of a terminal portion of the nucleic acid or the nucleic acid-related substance.

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