Method for structural analysis of sample molecule

Abstract

This method for structural analysis of a sample molecule is a method for analyzing the structure of a sample molecule using a mass spectrometer provided with a MALDI-type ion source, an ion-capturing unit for capturing ions having a prescribed mass-to-charge ratio from among the ions produced by the ion source, and a detection unit for detecting the ions captured by the ion-capturing unit, the method having: a step for acquiring standard analysis conditions for an ion amount setting item, a mass-to-charge ratio range setting item, and a signal intensity setting item for performing a molecular weight-related ion measurement; a step for performing a product ion measurement under a modified analysis condition in which an analysis condition for at least one of the ion amount setting item, the mass-to-charge ratio range setting item, and the signal intensity setting item from among the standard analysis conditions has been modified, and acquiring mass spectral data for the product ion measurement; a step for extracting a peak corresponding to fragment ions from the mass spectral data; and a step for determining at least a part of the structure of the sample molecule on the basis of mass information for the extracted peak. It is thereby possible to detect, at a high sensitivity and in a prioritized manner, fragment ions necessary for structural analysis.

Description

Method for structural analysis of sample molecules

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

[0002] In a mass spectrometer (MALDI-MS) with an ion source using the matrix-assisted laser desorption / ionization (MALDI) method, an analytical sample is prepared by mixing the sample with an ionization aid called a matrix, and laser light is irradiated onto the analytical sample for a short period of time to ionize the sample molecules in the analytical sample. The generated ions are then introduced into a mass separator where they are separated and detected according to their mass-to-charge ratio (m / z).

[0003] MALDI is generally known as a soft ionization method capable of ionizing refractory compounds without significant decomposition. Therefore, MALDI-MS is widely used to obtain molecular weight information for biopolymers such as nucleic acids, nucleic acid-related substances, peptides, proteins, and sugar chains. Furthermore, in structural analysis of such biopolymers, molecular weight-related ions (precursor ions) generated from sample molecules in a MALDI ion source are intentionally dissociated by an appropriate method, and the resulting various fragment ions are subjected to mass analysis, allowing the structure of the sample molecule to be inferred based on the mass information of the fragment ions.

[0004] For example, in the nucleic acid structural analysis methods described in Non-Patent Documents 1 to 3, mass analysis is performed using a MALDI-TOFMS to analyze various fragment ions (ISD fragments) of nucleic acids generated by in-source decay (ISD), and the base sequence information of the nucleic acid is analyzed based on the mass information of the fragment ions obtained thereby. In-source decay is a technique in which ions are dissociated in the ion source simultaneously with or immediately after ionization. As described above, MALDI is a soft ionization method, and ions are inherently difficult to dissociate. However, it is known that ion dissociation during ionization can be promoted, for example, by increasing the intensity of the laser light to increase the energy during ionization or by using a special matrix. In the ISD of nucleic acid molecules, 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 single site of a phosphodiester bond (or phosphorothioate bond, etc.).

[0005] Nathan A. Hagan, et al., "Enhanced In-Source Fragmentation in MALDI-TOF-MS of Oligonucleotides Using 1,5-Diaminonapthalene", Journal of the American Society for Mass Spectrometry, (USA), 2012, 23, pp.773-777 Hisao Shimizu, et al., "Application of high-resolution ESI and MALDI mass spectrometry to metabolite profiling of small interfering RNA duplex", Journal of Mass Spectrometry, (USA), 2012, 47, pp.1015?1022 Satoshi Kimura, et al., "Effect of oligonucleotide structural difference on matrix-assisted laser desorption / ionization in-source decay in comparison with collision-induced dissociation fragmentation", (USA), Rapid Communications in Mass Spectrometry, 2020, 34, e8819 Scott A. McLuckey, et al., “Tandem mass spectrometry of small, multiply charged oligonucleotides”, Journal of the American Society for Mass Spectrometry, (USA), 1992, 3, pp. 60-70

[0006] In the methods described in Non-Patent Documents 1 to 3, a MALDI-TOFMS is used as a mass spectrometer, and most of the ions generated in the ion source are introduced into a mass separation section, where they are separated and detected, starting with those with the lowest mass. Therefore, when ISD is performed, fragment ions are detected over a wide range, from low to high mass. In this case, there are problems such as the peaks of fragment ions in the low mass range overlapping with peaks of cluster ions derived from the matrix, or detection being suppressed due to ion suppression effects, etc. In addition, since ions in the low mass range are preferentially detected in MALDI-TOFMS, it may be difficult to obtain sensitivity for fragment ions in the high mass range. Furthermore, since the target mass range for a single measurement is wide, there is a problem that the detection sensitivity and resolution of the entire fragment ions are likely to decrease, especially under conditions where the fragmentation efficiency from precursor ions is low.

[0007] Furthermore, there are problems such as the peak of a fragment ion near the m / z value of the precursor ion overlapping with a peak other than the fragment ion (such as a base desorption peak in the case of nucleic acids), or the application of excessive energy to the precursor ion during fragmentation resulting in fragment ions that cannot be completely separated, resulting in a decrease in resolution.

[0008] For example, Figure 5 of Non-Patent Document 3 shows an ISD spectrum of nucleic acid by MALDI-TOFMS. In the ISD spectrum of Figure 5, the peaks of fragment ions near the m / z value of precursor ions dissociated in the base sequence portion near the 3' or 5' end overlap with peaks other than fragment ions, and the sensitivity and resolution are lower than the peaks of fragment ions dissociated in the base sequence portion away from the 3' or 5' end (peaks of fragment ions detected at m / z values ​​away from the vicinity of the m / z value of the precursor ion toward the low mass region).

[0009] The present invention has been made in view of the above-mentioned problems, and aims to provide a structural analysis method that can detect fragment ions necessary for structural analysis of sample molecules with high sensitivity over a relatively wide mass range from low to high mass.

[0010] The method for analyzing the structure of a sample molecule according to the present invention, which has been achieved to solve the above-mentioned problems, is a method for analyzing the structure of a sample molecule using an ion trap mass spectrometer equipped with an ion source employing a matrix-assisted laser desorption / ionization (MALDI) method, an ion trapping unit for separating and trapping ions of a predetermined mass-to-charge ratio from among ions generated by the ion source, and a detection unit for detecting the ions trapped by the ion trapping unit, and includes a standard analysis condition acquisition step of acquiring standard analysis conditions for an ion amount setting item relating to the amount of ions generated by the ion source, a mass-to-charge ratio range setting item relating to the mass-to-charge ratio range of ions trapped by the ion trapping unit, and a signal intensity setting item relating to the signal intensity of ions in the detection unit, when performing molecular weight-related ion measurement to detect molecular weight-related ions of the sample molecule contained in a sample using the mass spectrometer; The method comprises: a product ion measurement data acquisition step of performing product ion measurement to detect a plurality of first fragment ions generated by dissociation of molecular weight-related ions of the sample molecule under modified analysis conditions obtained by changing at least one of the analysis conditions for the ion amount setting item, the mass-to-charge ratio range setting item, and the signal intensity setting item among the standard analysis conditions 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 peaks corresponding to the first fragment ions from the mass spectrum data acquired in the product ion measurement data acquisition step; and a data analysis step of determining at least a part of the structure of the sample molecule based on mass information of the peaks extracted in the first fragment peak extraction step.

[0011] Another aspect of the present invention, which has been made to solve the above-mentioned problems, is a method for analyzing the structure of a sample molecule using an ion trap mass spectrometer equipped with an ion source employing a matrix-assisted laser desorption / ionization (MALDI) method, an ion trapping section for separating and trapping ions of a predetermined mass-to-charge ratio from among ions generated by the ion source, and a detection section for detecting the ions trapped by the ion trapping section, the method comprising: a standard analysis condition acquisition step for acquiring standard analysis conditions, when performing molecular weight-related ion measurement to detect molecular weight-related ions of the sample molecule contained in a sample using the mass spectrometer, including an ion amount setting item relating to the amount of ions generated by the ion source, a mass-to-charge ratio range setting item relating to the mass-to-charge ratio range of ions trapped by the ion trapping section, and a signal intensity setting item relating to the signal intensity of ions in the detection section; and a product ion measurement step for performing product ion measurement to detect a plurality of first fragment ions generated by dissociation of the molecular weight-related ions of the sample molecule under modified analysis conditions obtained by changing at least one of the ion amount setting item, the mass-to-charge ratio range setting item, and the signal intensity setting item from the standard analysis conditions acquired in the standard analysis condition acquisition step. The method comprises an MS / MS measurement condition setting step of setting measurement conditions for performing MS / MS measurement using at least one of the plurality of first fragment ions detected in the product ion measurement as a precursor ion; an MS / MS measurement data acquisition step of acquiring mass spectral 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 peaks corresponding to second fragment ions generated in the MS / MS measurement from the mass spectral data acquired in the MS / MS measurement data acquisition step; and a data analysis step of determining a part of the structure of the sample molecule based on mass information of the peak extracted in the second fragment peak extraction step.

[0012] In the present invention, the term "molecular weight-related ions" refers to a general term for ions that are directly useful for obtaining molecular weight information, and specifically includes, for example, protonated molecules, deprotonated molecules, and sodium ion adduct molecules.

[0013] According to the present invention, fragment ions necessary for structural analysis of sample molecules can be detected with high sensitivity over a relatively wide mass range, from low to high mass. As a result, a sufficient number of fragment ions can be detected to perform structural analysis of the sample molecules. In particular, more structural information, such as sequence information, can be determined for the molecular structures of sample molecules containing relatively high-mass molecules such as biopolymers. Furthermore, according to another aspect of the present invention, fragment ions (second fragment ions) obtained by further dissociating fragment ions (first fragment ions) derived from molecular-related ions can be detected with high sensitivity. As a result, structural information can be more reliably determined for molecular structure portions of sample molecules that cannot be analyzed accurately based on mass information of the first fragment ions alone.

[0014] 1 is a schematic diagram showing an example of a mass spectrometer used to carry out the structural analysis method of the present invention. 2 is a flowchart showing the procedure of a structural analysis method according to one embodiment of the present invention. 3 is a flowchart showing the procedure of a structural analysis method according to another embodiment of the present invention. 4 is a diagram showing the mass spectrum of standard nucleic acid A when conditions (a) to (e) are used in Example 1. 5 is a diagram showing the mass spectrum of standard nucleic acid A and the sequence analysis status of standard nucleic acid A when condition (e) is used in Example 1. 6 is a diagram showing the mass spectrum of standard nucleic acid A when conditions (b) to (d) are used in Example 2. 7 is a diagram showing the mass spectrum of standard nucleic acid A and the sequence analysis status of standard nucleic acid A when condition (d) is used in Example 2. 8 is a diagram showing the mass spectrum (m / z 2000-6000) of standard nucleic acid A when conditions (b) and (c) are used in Example 3. 9 is a diagram showing the mass spectrum (m / z 3000-5000) of standard nucleic acid A when conditions (b) and (c) are used in Example 3. A figure showing the mass spectrum of standard nucleic acid A and the sequence analysis status of standard nucleic acid A when condition (c) is used in Example 3. A figure showing the mass spectrum of standard nucleic acid A and the sequence analysis status of standard nucleic acid A when conditions (b) and (c) are used in Example 3. A figure showing the mass spectrum of mipomersen and the sequence analysis status of mipomersen when conditions (a) and (b) are used in Example 4. A figure showing the mass spectrum of mipomersen and the sequence analysis status of mipomersen when measurement conditions for MS / MS measurement are used in Example 4.

[0015] To address the above-mentioned issues, the present inventors attempted to perform structural analysis of sample molecules using an ion trap (IT) mass spectrometer (MALDI-ITMS) with a MALDI ion source and an ISD matrix that has been shown to promote ion dissociation. However, there have been few reports of ISD using MALDI-ITMS. As a result, it was confirmed that, although ion dissociation occurs, the resulting fragment ions are limited to m / z values ​​close to those of the precursor ion. This is thought to be due to the difference in ion capture conditions caused by the space charge effect of MALDI-ITMS between precursor ions and low-mass-to-charge fragment ions obtained by more dissociation from the precursor ion. This phenomenon was particularly pronounced when the analyte was a high-mass molecule.

[0016] The present invention aims to provide a method for structural analysis of sample molecules using MALDI-ITMS, which is capable of detecting, with high sensitivity and priority, fragment ions particularly in the low mass region among the fragment ions required for analysis.

[0017] An embodiment of the method for structural analysis of a sample molecule according to the present invention will now be described with reference to the drawings. Fig. 1 is a schematic diagram showing an example of a mass spectrometer used to carry out the method for structural analysis according to the present invention.

[0018] <Mass Spectrometer> The mass spectrometer used in this embodiment is an ion trap mass spectrometer, and includes an ion source 1 that ionizes a sample containing an analyte, an ion trap 2 (the ion trapping unit of the present invention) that temporarily traps ions of a predetermined mass-to-charge ratio among the ions generated by the ion source 1 using the action of a radio frequency electric field and separates the trapped ions according to their mass-to-charge ratio (m / z), and a detection unit 3 that detects the separated ions.

[0019] The ion source 1 is an ion source that utilizes the MALDI method and includes a laser irradiation unit 11 that irradiates a sample with laser light and a sample stage 12 for placing a sample plate S on which a sample is placed. The ion trap 2 is a quadrupole ion trap that includes a circular ring electrode 21 and a pair of end cap electrodes 22, 23 that are arranged opposite each other across the ring electrode 21. An ion entrance aperture 22a is formed in the entrance end cap electrode 22, and an ion exit aperture 23a is formed in the exit end cap electrode 23. The detector 3 includes a conversion dynode 31 that converts ions into electrons, and a detector (secondary electron multiplier) 32 that multiplies and detects the electrons arriving from the conversion dynode 31.

[0020] A predetermined voltage is applied to the ring electrode 21 and the end cap electrodes 22, 23 of the ion trap 2. The radio frequency electric field thus formed can trap ions in the internal space surrounded by the ring electrode 21 and the end cap electrodes 22, 23, and eject ions from the internal space through the ion exit hole 23 a.

[0021] The range of mass-to-charge ratios of ions that are preferentially trapped in the ion trap can be controlled by changing the time (hereinafter referred to as the delay time) between when the sample is irradiated with laser light from the ion source 1 and when a trapping voltage for trapping ions is applied to the ring electrode 21 of the ion trap 2. By increasing the delay time, ions on the higher mass side can be trapped more reliably than other ions, while by decreasing the delay time, ions on the lower mass side can be trapped more reliably than other ions.

[0022] The predetermined voltage applied to the ring electrode 21 and the end cap electrodes 22, 23 may be a sinusoidal high-frequency voltage or a square-wave voltage generated by rapidly switching between two different voltages. In a digital ion trap that utilizes the electric field generated by the square-wave voltage, the range of mass-to-charge ratios of ions that can be trapped is controlled by changing the frequency while maintaining the amplitude (voltage value) of the square-wave voltage constant, or by changing the duty ratio, which is the ratio of the switching intervals of the square-wave voltage.

[0023] The ion trap mass spectrometer in this embodiment includes not only a mass spectrometer that uses the mass separation function of the ion trap itself to eject ions trapped in the ion trap in ascending order of mass-to-charge ratio and detects the ions with a detector located outside the ion trap, but also a mass spectrometer that separates ions ejected all at once from the ion trap according to their mass-to-charge ratio with a mass separator located outside the ion trap, such as a time-of-flight mass separator, and detects the ions with a detector also located outside the ion trap. Furthermore, the ion trap mass spectrometer may be a tandem mass spectrometer having two mass separators connected in series to enable MS / MS measurements, as described below.

[0024] Next, the procedure of the structural analysis method of this embodiment will be described with reference to the flowchart of FIG.

[0025] <Structural Analysis Method> Here, an example will be described in which an ion trap mass spectrometer having a digital ion trap is used as the mass spectrometer.

[0026] [Step 101: Obtaining standard analysis conditions for MS measurement] First, the molecular weight-related ions (protonated molecules [M+H]) of the sample molecules to be analyzed are obtained. ＋ , deprotonated molecule [M-H] － Standard analytical conditions are acquired for a measurement (corresponding to molecular-weight-related ion measurement in the present invention; hereinafter referred to as MS measurement) for detecting molecular-weight-related ions, etc. The MS measurement uses measurement conditions for detecting molecular-weight-related ions with as high a sensitivity and resolution as possible, and therefore does not involve ion dissociation (or, even if ion dissociation occurs, it occurs only to a very small extent). Mass spectrometers have various setting items that can be appropriately set depending on the type of analyte and the purpose of analysis, and in the structural analysis method according to this embodiment, standard analytical conditions are acquired for an ion amount setting item relating to the amount of ions generated by the ion source 1, a mass-to-charge ratio range setting item relating to the mass-to-charge ratio range of ions trapped in the ion trap 2, and a signal intensity setting item relating to the signal intensity of ions in the detection unit 3.

[0027] Standard analytical conditions are representative conditions among conditions under which molecular weight-related ions of sample molecules can be detected. As a method for acquiring standard analytical conditions, for example, if an MS measurement is performed using default values ​​for various setting items pre-set in a mass spectrometer and molecular weight-related ions of sample molecules are detected as a result, the default values ​​may be used as the standard analytical conditions. In this case, acquiring the standard analytical conditions involves reading the default values ​​from a memory unit or the like in which the default values ​​are pre-stored. Alternatively, the setting values ​​of various setting items may be changed from the default values ​​so that molecular weight-related ions of sample molecules are detected with higher sensitivity and higher resolution, and then an MS measurement may be performed to determine the value (threshold value, etc.) at which molecular weight-related ions of sample molecules are detected.

[0028] The ion quantity setting item is the laser intensity (laser power) emitted by the laser irradiation unit 11, etc., the mass-to-charge ratio range setting item is the RF delay value corresponding to the delay time, etc., and the signal intensity setting item is the voltage applied to the conversion dynode 31 and the voltage applied to the detector (secondary electron multiplier) 32, etc.

[0029] [Step 102: Setting Modified Analysis Conditions by Changing the Standard Analysis Conditions] Next, modified analysis conditions are set by changing at least one of the ion quantity setting item, mass-to-charge ratio range setting item, and signal intensity setting item from the standard analysis conditions obtained in step 101. That is, only one of the ion quantity setting item, mass-to-charge ratio range setting item, and signal intensity setting item may be changed, with the remaining settings remaining unchanged. In this case, the ion quantity setting item is changed to increase the amount of ions generated by the ion source 1, the mass-to-charge ratio range setting item is changed to preferentially capture low-mass ions in the ion trap 2, and the signal intensity setting item is changed to increase the signal intensity of ions in the detection unit 3, compared to when MS measurement is performed under the standard analysis conditions.

[0030] Specifically, when changing the laser light intensity, which is an ion quantity setting item, it is preferable to set it higher than the standard analysis conditions, for example, about 1 to 40% higher than the value of the standard analysis conditions, more preferably about 1 to 30% higher. When changing the RF delay value, which is an ion mass-to-charge ratio range setting item, it is preferable to set it lower than the standard analysis conditions, for example, more preferably about 5 to 30% lower than the value of the standard analysis conditions, more preferably about 10 to 20% lower (so that the delay time is 1 to 3 μsec). When changing the voltage applied to the conversion dynode 31, which is a signal intensity setting item, it is preferable to set it higher than the standard analysis conditions, for example, about 5 to 30% higher, more preferably about 10 to 30% higher. When changing the voltage applied to the detector (secondary electron multiplier) 32, which is also a signal intensity setting item, it is preferable to set it higher than the standard analysis conditions, for example, about 5 to 50% higher, more preferably about 10 to 50% higher.

[0031] [Step 103: Acquiring Data by In-Source Decay Measurement Using Modified Analysis Conditions] Using the modified analysis conditions, a measurement (corresponding to product ion measurement in the present application) is performed to detect fragment ions generated by dissociation of molecular-related ions (precursor ions) of sample molecules, and data on the detected ions is acquired. In the present invention, since the mechanism for generating fragment ions derived from sample molecules has not yet been proven, in this specification, the dissociation that occurs simultaneously with or immediately after ionization in the ion source of a MALDI ion trap mass spectrometer, as well as the subsequent dissociation of ions generated within the instrument, are generally referred to as in-source decay (ISD), and the above measurement involving ISD (product ion measurement) is referred to as ISD measurement. In addition, hereinafter, the fragment ions generated in the above measurement are referred to as first fragment ions or ISD fragments. ISD measurement using the modified analysis conditions enables high-sensitivity detection of first fragment ions, particularly those in the low-mass region away from the m / z value of the molecular-related ions.

[0032] In step 103, the ISD measurement may be performed under conditions in which a predetermined range of mass-to-charge ratios is set so that the maximum value of the predetermined mass-to-charge ratio of ions trapped in the ion trap 2 is smaller than the value of the mass-to-charge ratio of ions related to the molecular weight of the sample molecule. In this case, the maximum value of the predetermined mass-to-charge ratio of ions trapped in the ion trap 2 is preferably set to be smaller than the value of the mass-to-charge ratio of ions related to the molecular weight of the sample molecule by, for example, 0.5 to 40%, and more preferably by 0.5 to 20%.

[0033] One method for changing the predetermined mass-to-charge ratio range is to switch the measurement mode pre-installed in the device from a target mass range of m / z 2000 to 18000 to a target mass range of m / z 650 to 5000 for an analyte with a molecular weight of approximately 6000, thereby switching the measurement mode to one that does not include the target mass of 6000. The target mass range of the measurement mode is determined by the frequency of the radio-frequency voltage applied to the ion trap 2. Specifically, because the amount of ions trapped in the ion trap is limited, the mass range to be measured is determined primarily by adjusting the frequency of the radio-frequency voltage and setting the low-mass cutoff (LMCO). Increasing the frequency of the radio-frequency voltage reduces the LMCO, and the mass range to be measured is shifted to the lower mass side. Conversely, decreasing the frequency of the radio-frequency voltage increases the LMCO, and the mass range to be measured is shifted to the higher mass side. In other words, the predetermined mass-to-charge ratio range can be changed by changing the frequency of the radio-frequency voltage applied to the ion trap 2.

[0034] Another method for changing the predetermined mass-to-charge ratio range is to exclude ions with an m / z of 5500 or greater by changing the duty ratio setting, which is the ratio of the switching intervals of the square-wave voltages pre-installed in the instrument, for an analyte with a molecular weight of approximately 6000, from the standard value of 50:50 to a value of 52:48. This method obtains mass spectrum data that excludes first fragment ions with mass-to-charge ratios close to the m / z value of the precursor ion. As a result, the detection sensitivity of first fragment ions in the low mass range away from the m / z value of the precursor ion is improved.

[0035] In step 103, mass spectrum data obtained by performing ISD measurements both under the condition of setting a predetermined range of mass-to-charge ratios as described above and under the condition of not using the same may be acquired, thereby making it possible to detect first fragment ions derived from sample molecules over a range from low to high mass.

[0036] Furthermore, in addition to using conditions that set a predetermined range of mass-to-charge ratios so that the maximum value of the predetermined mass-to-charge ratio of ions trapped in the ion trap 2 is smaller than the value of the mass-to-charge ratio of ions related to the molecular weight of the analyte, ISD measurement may also be performed using conditions in which the voltage applied to the sample stage 12 of the ion source 1 is changed to a value higher than the standard analysis conditions obtained in step 102. The voltage applied to the sample stage is preferably set 4 to 8 times higher, and more preferably 4 to 5 times higher, than the standard analysis conditions.

[0037] [Step 104: Creation of In-Situ Dispersion (ISD) Spectrum] A mass spectrum (ISD spectrum) is created 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 of each peak in the mass spectrum and its signal intensity, and mass spectrum data (ISD spectrum data) from the ISD measurement is acquired in step 104.

[0038] [Step 105: Analysis of Mass Spectral Data] Peaks corresponding to various first fragment ions are extracted from the ISD spectral data acquired in step 104, and the assignments of the various first fragment ions are determined based on the mass information indicated by the peaks. These results are combined to determine at least a portion of the structure of the original sample molecule. The structure determination includes sequence analysis and identifying the type of chemical modification or the site where the chemical modification has been applied through sequence analysis. Database search or de novo sequencing may be used to determine the structure.

[0039] In step 103, ISD measurements are performed both with and without conditions for setting a predetermined range of mass-to-charge ratios, and mass spectrum data from both ISD measurements is acquired in step 104. Analysis may be performed by combining the assignment results of various first fragment ions obtained from both of these measurements. This allows for more reliable structural analysis of the analyte.

[0040] <Analyte> The analyte in the present invention is, for example, a sample molecule containing relatively high-mass molecules such as biopolymers, etc. Examples of biopolymers include nucleic acids, peptides, sugar chains, proteins, and lipids.

[0041] The term "nucleic acid" as used herein also includes nucleic acid-related substances such as modified nucleic acids, nucleic acid derivatives, and nucleic acid pharmaceuticals. Hereinafter, nucleic acids and nucleic acid-related substances will be collectively referred to simply as "nucleic acids." When the analyte is a nucleic acid, the degree of polymerization (base length) of the nucleic acid is not particularly limited, but an oligonucleotide composed of several to several tens of nucleotides is preferred. In the analytical method of the present invention, the analytical sensitivity of nucleic acids with large molecular weights is particularly improved, and therefore nucleic acids with a molecular weight of 3000 or more, and more preferably 6000 or more, are preferred. Furthermore, nucleic acids may be natural products obtained from living organisms or processed products thereof, or may be chemically synthesized artificial nucleic acids.

[0042] <Method of Preparing an Analysis Sample> In this example, the analyte is assumed to be a nucleic acid. The analysis sample is prepared by dropping a mixed solution of a nucleic acid-containing sample and a matrix substance onto a sample plate and drying it. In this case, the mixed solution may be prepared in advance, dropped onto the sample plate, and dried, or the mixed solution may be prepared on the sample plate and dried as is.

[0043] The matrix material can be selected appropriately depending on the type of nucleic acid. Examples include 3-hydroxypicolinic acid (3-HPA), 2,4-dihydroxyacetophenone (2,4-DHAP), 2,5-dihydroxybenzoic acid (DHB), 2',4',6'-trihydroxyacetophenone monohydrate (THAP), 6-aza-2-thiothymine (ATT), 3-aminopyrazine-2-carboxylic acid (APCA), anthranilic acid (AA), and nicotinic acid (NA). Among these, 2,4-DHAP and THAP are preferred. A mixed matrix obtained by mixing two or more matrix substances may also be used, and among these, mixed matrices obtained by mixing 3-HPA and 2,4-DHAP, 3-HPA and THAP, and 2,4-DHAP and THAP are preferred.

[0044] The analytical sample may further contain a matrix additive. Diammonium hydrogen citrate (ammonium citrate dibasic: ACD) can be used as the matrix additive. There are several types of ammonium salts of citric acid, depending on the number of ammonium ions bound to the citrate ion. In this embodiment, however, a salt in which two ammonium ions are bound to one citrate ion is preferably used.

[0045] When the analytical sample contains a matrix additive, the order in which the nucleic acid-containing sample, matrix substance, and matrix additive are mixed is not particularly limited. However, it is preferable to prepare a matrix / additive mixed solution containing the matrix substance and matrix additive in advance, and then mix the nucleic acid-containing sample solution with the matrix / additive mixed solution to prepare the analytical sample. In this case, the analytical sample may be prepared by dropping a premixed solution of the sample solution and the matrix / additive mixed solution onto a sample plate and drying it. Alternatively, the analytical sample may be prepared by dropping the sample solution and the matrix / additive mixed solution onto a sample plate, mixing them on the sample plate, and drying them. Preparing the matrix / additive mixed solution in advance facilitates the preparation of the analytical sample. The concentration of the matrix additive in the matrix / additive mixed solution is preferably 10 to 100 mM, more preferably 30 to 70 mM, from the viewpoint of generating a sufficient amount of ions related to the molecular weight of the nucleic acid.

[0046] 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. Fig. 3 is a flow chart showing the steps of the method for analyzing the structure of this embodiment.

[0047] [Step 201: Obtaining standard analysis conditions when performing MS measurement] [Step 202: Setting modified analysis conditions by modifying the standard analysis conditions] [Step 203: Obtaining data by ISD measurement using modified analysis conditions] [Step 204: Creating an ISD spectrum] Using a method similar to steps 101 to 104 described above, standard analysis conditions when performing MS measurement are obtained (step 201), modified conditions by modifying the standard analysis conditions are set (step 202), data is obtained by ISD measurement using the modified conditions (step 203), an ISD spectrum is created based on the data, and ISD spectrum data is obtained (step 204).

[0048] [Step 205: Setting Measurement Conditions for MS / MS Measurement] Based on the ISD spectrum data acquired in step 204, at least one of the multiple first fragment ions detected in the ISD measurement in step 203 is selected as a precursor ion, and measurement conditions for performing MS / MS measurement of the precursor ion are set. MS / MS measurement is generally known as a measurement technique in which an ion having a specific mass-to-charge ratio is selected as a precursor ion from ions generated from sample molecules in a mass separation unit in a preceding stage, the precursor ion is dissociated by collision induced dissociation (CID) in a subsequent collision cell to generate various fragment ions (product ions), and the fragment ions (product ions) are separated in a mass separation unit in a subsequent stage. In MS / MS measurements in the ion trap mass spectrometer used in this embodiment, only ions within a specific mass range (having a specific mass-to-charge ratio) are left inside the ion trap 2, and these ions are selected as precursor ions. Subsequently, the precursor ions are dissociated by CID with a gas such as argon introduced into the ion trap 2 to generate product ions including various fragment ions, and then a mass scan is performed to eject the ions in ascending order of mass-to-charge ratio.

[0049] As the precursor ion, it is preferable to select a first fragment ion that is detected with relatively high sensitivity in the ISD spectrum data acquired in step 204 and that overlaps as little as possible with other peaks. Using such a first fragment ion as the precursor ion allows multiple fragment ions generated in the subsequent MS / MS measurement (hereinafter, the fragment ions generated in the MS / MS measurement will also be referred to as second fragment ions) to be detected with high sensitivity. Furthermore, it is preferable to select a first fragment ion in a low mass range away from the m / z value of the molecular weight-related ions as the precursor ion. Using such a first fragment ion as the precursor ion prevents the mass spectrum obtained by the MS / MS measurement from becoming complex, facilitating data analysis. The m / z value of the first fragment selected as the precursor ion is preferably m / z 5000 or less, more preferably m / z 3000 or less, and even more preferably m / z 2000 or less.

[0050] [Step 206: Acquiring Data by MS / MS Measurement] MS / MS measurement is performed based on the measurement conditions set in step 205, and data on the detected peaks is acquired.

[0051] [Step 207: Creating an MS / MS Spectrum] A mass spectrum (MS / MS spectrum) is created based on the data obtained in step 206, and mass spectrum data (MS / MS spectrum data) from the MS / MS measurement is obtained.

[0052] [Step 208: Analysis of Mass Spectral Data] As in step 105, peaks corresponding to various first fragment ions are extracted from the ISD spectrum data acquired in step 204, and the assignments of the various first fragment ions are determined based on the mass information indicated by the peaks. These results are combined to determine at least a portion of the structure of the original sample molecule. Furthermore, peaks corresponding to various second fragment ions are extracted from the MS / MS spectrum data acquired in step 207, and the assignments of the various second fragment ions are determined based on the mass information indicated by the peaks. These results are combined to determine a portion of the structure of the original sample molecule. By combining the peak assignment results of the ISD measurement and the MS / MS measurement, more information about the structure of the sample molecule can be obtained, improving the accuracy of structural analysis. The peak assignment results of the MS / MS measurement make it easier to obtain information about the terminal structure of the sample molecule, which is difficult to obtain from ISD measurement alone due to the low sensitivity of ISD fragments. 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 from the peak assignment results of MS / MS measurement.

[0053] In this embodiment, in step 208, the assignments of the first fragment ions and the second fragment ions are respectively determined based on the ISD spectrum data acquired in step 204 and the MS / MS spectrum data acquired in step 207, but it is also possible to simply determine the assignment of the second fragment ions based on the MS / MS spectrum data acquired in step 207. In that case, if the first fragment ions generated by the ISD measurement in step 203 are detected and the m / z values ​​of the first fragment ions can be obtained, step 204 (creation of the ISD spectrum, acquisition of the ISD spectrum data) does not need to be performed, and in step 208, the assignment results of various second fragment ions are combined to determine only part of the structure of the original sample molecule.

[0054] The structural analysis method according to the present invention will be described below with reference to examples, but these are merely examples and the present invention is not limited to these.

[0055] <1. Preparation of sample solution> As a sample solution, a 10 pmol / μL aqueous solution of standard nucleic acid A (5′-TGTGCGTGTGTAGTGTGTCT-3′: MW 6201.1, DNA, SEQ ID NO: 1, synthesized on request) was prepared.

[0056] 2. Preparation of Matrix Solution As a matrix solution, a 40 mg / mL aqueous solution of 2,4-dihydroxyacetophenone (2,4-DHAP) in 50% acetonitrile (ACN) containing 70 mM diammonium hydrogen citrate (ACD) as a matrix additive was prepared.

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

[0058] <4. Mass Analysis> A MALDI digital ion trap mass spectrometer (MALDI-DITMS, manufactured by Shimadzu Corporation, product name: MALDImini-1) was used for mass analysis. The sample plate carrying the analytical sample prepared in 3. was inserted into the MALDI-DITMS, and MS measurement and ISD measurement were performed using the raster function in positive mode.

[0059] <4-1. MS Measurement> The values ​​in Table 1 below were used as the conditions (various settings of the mass spectrometer) for MS measurement. The measurement mode indicates the range of mass-to-charge ratios of the ions to be measured, and more specifically, specifies the range of mass-to-charge ratios of ions to be trapped in the ion trap, and this range is shown in parentheses. This also applies to the following tables.

[0060]

[0061] <4-2. ISD Measurement> The values ​​in Table 2 below were used as the conditions (various settings of the mass spectrometer) for ISD measurement.

[0062]

[0063] <5. Structural Analysis> The base sequence of standard nucleic acid A was analyzed from the mass spectrum data of the obtained fragment ions.

[0064] <Results> Figure 4 shows the mass spectra of standard nucleic acid A obtained by MS measurement and ISD measurement under each condition (mass spectra under conditions (a), (b), (c), (d), and (e) from top to bottom). Of the mass spectra under each condition, the left side shows the entire measured mass-to-charge ratio (m / z 3300-6300), and the right side shows an enlarged portion of that (m / z 4400-5200). The arrows in the figure indicate molecular weight-related ions ([M+H] ＋ , precursor ions). The values ​​in the figure indicate the peak intensity (mV) of the most intense ion in each mass spectrum.

[0065] As shown in FIG. 4, in the mass spectrum of m / z 3300 to 6300 under condition (a), the protonated molecule [M+H] of standard nucleic acid A ＋ At the same time, under condition (a), several fragment ion peaks including base-release ions were detected, but the number of fragment ions was small and the detection sensitivity was low (Figure 4(a)).

[0066] In contrast, under conditions (b) to (e), in which the detector voltage, conversion dynode voltage, RF delay value, and laser power were changed little by little, it was confirmed that the detection sensitivity of fragment ion peaks improved and the number of detected peaks increased (FIGS. 4(b) to (e)). In particular, when comparing measurements performed under condition (a) with measurements performed under condition (e), the sensitivity of fragment ion peaks detected on the low mass-to-charge ratio side improved and the number of detected peaks increased significantly.

[0067] The ISD spectrum of the fragment ions obtained under condition (e) and the status of the nucleic acid base sequence analysis are shown in Figure 5. As can be seen from Figure 5, most of the sequence of standard nucleic acid A (90% of the entire sequence, excluding the sequence near the center) was determined.

[0068] The above results are believed to be due to the fact that changing the laser power to a value higher than that used in normal MS measurements increases the amount of ions generated in the ion source, and changing the detector voltage and conversion dynode voltage to values ​​higher than those used in normal MS measurements amplifies the individual ion signals. Furthermore, changing the RF delay value to a value lower than that used in normal MS measurements preferentially captures fragment ions with lower mass-to-charge ratios than the precursor ion, resulting in improved detection sensitivity for fragment ions. From the above, it was confirmed that ISD analysis using MALDI-ITMS is possible, and that most of the sequence can be determined even for a relatively high-mass standard nucleic acid (MW 6201).

[0069] Mass spectrometry and structural analysis were performed in the same manner as in Example 1, except that the measurement conditions for the MS measurement and the ISD measurement were different.

[0070] <4-1. MS Measurement> The values ​​in Table 3 below were used as conditions (various settings of the mass spectrometer) for MS measurement.

[0071]

[0072] <4-2. ISD Measurement> The values ​​in Table 4 below were used as the conditions (various setting values ​​of the mass spectrometer) for ISD measurement.

[0073]

[0074] <Results> First, under condition (a), although not shown, the protonated molecule [M+H] of standard nucleic acid A ＋ was confirmed to be detected with high sensitivity.

[0075] Next, Figure 6 shows the ISD spectra of standard nucleic acid A obtained by ISD measurement under each condition (ISD spectra under conditions (b), (c), and (d) from bottom to top). When measurement was performed under condition (b), which changed the detector voltage, conversion dynode voltage, RF delay value, and laser power compared to condition (a), fragment ions were more likely to be detected in the high-mass region close to the precursor ion (Figure 6(b)). On the other hand, when measurement was performed under conditions (c) and (d), which in addition to the above changes, changed the measurement mode so that the maximum value of the mass-to-charge ratio range of ions trapped in the ion trap was smaller than the mass-to-charge ratio of the precursor ion, fragment ions were also detected in the low-mass region farther from the precursor ion (Figures 6(c) and (d)). In particular, under condition (d), in which the sample stage voltage was set higher than that under condition (a), it was confirmed that a large number of fragment ions in the low-mass region were detected with relatively high sensitivity (Figure 6(d)). In addition, in order to make it easier to understand the influence of the measurement mode and sample stage voltage, the comparative evaluation was carried out under conditions (b) to (d) with the other analysis conditions kept constant (Table 4).

[0076] FIG. 5 shows the ISD spectrum of standard nucleic acid A and the sequence and its assignment status when ISD measurement was performed under the conditions of condition (b) in Table 4, except that the laser power was changed to 65 (i.e., condition (e) in Table 2). At least in this experiment, the ISD spectrum obtained under condition (b) in Table 4 was almost the same when the laser power was set to 75 (condition (b) in Table 4, FIG. 6(b)) and when it was set to 65 (condition (e) in Table 2, FIG. 5). Therefore, the assignment status of the ISD spectrum under condition (b) in Table 4 will be explained using FIG. 5. As shown in FIG. 5, a large number of fragment ions could be assigned, and approximately 90% of the base sequence could be determined. However, due to the low sensitivity of fragment ions in the low mass region, it was not possible to analyze the entire sequence.

[0077] Figure 7 shows the ISD spectrum of standard nucleic acid A when ISD measurement was performed under condition (d) (conditions of Figure 6(d)), as well as the sequence of standard nucleic acid A and its assignment status. In Figure 7, many fragment ions were detected, including fragment ions in the low mass region where peaks could not be observed in Figure 5 due to low detection sensitivity, and it was possible to determine about 60% of the base sequence. However, since fragment ions could not be detected in the high mass region near the precursor ion of standard nucleic acid A, it was not possible to analyze the entire sequence.

[0078] Finally, by combining the analysis results obtained from the mass spectrum data of FIG. 5 and the analysis results obtained from the mass spectrum data of FIG. 7, the entire sequence of the standard nucleic acid with a relatively high mass (MW 6201) could be analyzed.

[0079] Mass spectrometry and structural analysis were performed in the same manner as in Example 1, except that the measurement conditions for the MS measurement and the ISD measurement were different.

[0080] <4-1. MS Measurement> The values ​​in Table 5 below were used as conditions (various settings of the mass spectrometer) for MS measurement.

[0081]

[0082] <4-2. ISD Measurement> The values ​​in Table 6 below were used as the conditions (various setting values ​​of the mass spectrometer) for ISD measurement.

[0083]

[0084] <Results> First, under condition (a), although not shown, the protonated molecule [M+H] of standard nucleic acid A ＋ was confirmed to be detected with high sensitivity.

[0085] Next, Figures 8A and 8B show the ISD spectra of standard nucleic acid A obtained by ISD measurement under each condition (from bottom to top, ISD spectra under conditions (b) and (c)). Of the ISD spectra under each condition, Figure 8A shows the entire measured mass-to-charge ratio (m / z 2000-6000), and Figure 8B shows an enlarged portion of that spectrum (m / z 3000-5000).

[0086] When measurements were performed under condition (b), which changed the detector voltage, conversion dynode voltage, RF delay value, and laser power compared to condition (a), fragment ions were more easily detected in the high-mass region near the precursor ion (Figure 8A(b-2)). On the other hand, when measurements were performed under condition (c), which changed the duty ratio of the radio-frequency voltage applied to the ion trapping section so that the maximum value of the mass-to-charge ratio range of ions trapped in the ion trap was smaller than the mass-to-charge ratio of the precursor ion, ions near the precursor ion were excluded (Figure 8A(c-2)), and fragment ions in the low-mass region farther from the m / z value of the precursor ion were more easily detected (Figures 8A(c-2) and 8B(c-1)). As a result, the change in duty ratio resulted in higher intensity peaks of fragment ions in the low-mass region.

[0087] Figure 9 shows the ISD spectrum of standard nucleic acid A, the sequence of standard nucleic acid A, and its assignment status when ISD measurement was performed under condition (c). As can be seen from Figure 9, including ions with low peak intensity, a large number of fragment ions could be assigned, and the entire sequence could be analyzed. However, under these conditions, the intensity of fragment ions near the precursor ion was low.

[0088] FIG. 10 shows the ISD spectra of standard nucleic acid A when ISD measurements were performed under conditions (b) and (c) (the upper row shows condition (b) and the lower row shows condition (c)), as well as the sequence of standard nucleic acid A and its assignment status. In the mass spectrum obtained under condition (c), the entire sequence of the nucleic acid could be analyzed, but the peak intensity of ions near the precursor ion was low. On the other hand, in the mass spectrum obtained under condition (b), fragment ions near the precursor were observed with sufficient intensity. By combining the analysis results obtained from the mass spectral data under conditions (b) and (c), the entire sequence of a standard nucleic acid with a relatively high mass (MW 6201) could be analyzed more reliably.

[0089] <1. Preparation of 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 represents 2'-O-(2-methoxyethyl)nucleoside, d represents 2'-deoxynucleoside; the carbon atoms at the 5-positions of cytosine and uracil have been substituted with methyl groups, and all internucleotide phosphodiester bonds have been substituted with phosphorothioate bonds; SEQ ID NO: 2) was prepared as a sample solution.

[0090] <2. Preparation of matrix solution> A 40 mg / mL 50% ACN aqueous solution of 3-hydroxypicolinic acid (3-HPA) containing 40 mM ACD as a matrix additive (3-HPA solution) and a 40 mg / mL 50% ACN aqueous solution of 2,4-DHAP containing 70 mM ACD as a matrix additive (2,4-DHAP solution) were prepared, and the 3-HPA solution and the 2,4-DHAP solution were mixed at a 1:1 (v / v) ratio to prepare a mixed matrix (3-HPA + 2,4-DHAP, 1:1) solution.

[0091] <3. Preparation of analytical sample> The sample solution prepared in 1. and the mixed matrix solution prepared in 2. were mixed at a 1:1 (v / v) ratio, and 1 μL of the resulting mixture was dropped onto a sample plate (SUS plate) and dried.

[0092] <4. Mass Analysis> <4-1. Indirect Dispersion Measurement> The sample plate from 3. was inserted into a MALDI-DITMS (Shimadzu Corporation, product name: MALDImini-1), and Indirect Dispersion measurement was performed using the raster function in positive mode. The conditions for Indirect Dispersion measurement (various settings of the mass spectrometer) used were the values ​​in Table 7 below. The optimal laser power value for each condition was used.

[0093]

[0094] <4-2. MS / MS Measurement> In the ISD spectrum obtained by the ISD measurement in 4-1, the ISD fragment on the relatively low mass side (in this case, w ５ MS / MS measurement (low energy CID) was carried out under measurement conditions in which 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 3

[0095] <5. Structural analysis> 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 were assigned, and the base sequence of mipomersen was analyzed.

[0096] <Results> Figure 11 shows the ISD spectrum of mipomersen obtained using the mixed matrix 3-HPA + 2,4-DHAP and the assignment of the main peaks. Figure 11(a) is the ISD spectrum obtained by performing ISD measurement using condition (a) in Table 7, and Figure 11(b) is the ISD spectrum obtained by performing ISD measurement using condition (b) in Table 7. In the part indicated by the bar labeled x2 in Figure 11(b), the peak intensity is doubled compared to the other parts. In the ISD spectrum of Figure 11, each peak is given the name of the fragment ion species of the corresponding oligonucleotide (the general name proposed in Non-Patent Document 4). This name represents each ion species as a fragment ion series according to the nomenclature of nucleic acid dissociation patterns, and fragment ions containing the 5' end are named a ｎ , b ｎ , c ｎ , d ｎ and the fragment ion containing the 3' end in the opposite direction is represented as x ｍ , y ｍ , z ｍ , w ｍ The subscripts n and m indicate the number of constituent units from the corresponding end to the dissociation site (by definition, the number of bases). Also, B in the name of the fragment ion species represents the base of the nucleic acid, for example, a １６ -B(G) is a １６ This shows that the base guanine (G) has been eliminated from the ion. This also applies to the following figures.

[0097] The area enclosed by a box on the right side of Figure 11(b) is the molecular weight-related ion [M+H]. ＋ The spectrum around the m / z value of (precursor ion) is shown. In this region, base desorption peaks of the precursor ion are particularly detected with high sensitivity, and it was confirmed that the detection of the ISD fragment peaks is hindered by the overlap of the base desorption peaks and the ISD fragment peaks.

[0098] FIG. 12 shows the w detected in the ISD spectrum of FIG. ５ The MS / MS spectrum and main peak assignments for the α-ion precursor ion are shown in Figure 11. Compared to the ISD spectrum and peak assignments in Figure 12, multiple types of second fragment ions containing terminal sequence information were generated with sufficient intensity and resolution, allowing for more reliable analysis of the terminal sequence. Furthermore, the MS / MS spectrum in Figure 12 did not show any overlap between product ion peaks near the precursor ion, such as base desorption peaks, and second fragment ion peaks, as seen in the ISD spectrum in Figure 11(b).

[0099] Overall, after obtaining as much base sequence information as possible from the ISD spectrum in Figure 11, MS / MS measurements were performed using ISD fragments in the low mass range relatively far from the m / z values ​​of the molecular weight-related ions as precursor ions. This enabled clear detection of fragment ion peaks near the m / z values ​​of the molecular weight-related ions, which are prone to peak detection being hindered by ISD measurements, and analysis of the terminal base sequences, enabling more reliable analysis of the entire base sequence. In other words, the combination of ISD and MS / MS measurements enabled more reliable base sequence analysis.

[0100] This method of structural analysis, combining ISD and MS / MS measurements, can basically be used for any analyte. Furthermore, it can be improved to more reliably perform terminal sequence analysis, which is a challenge in MALDI-MS ISD analysis. This method is considered to be particularly effective for unstable nucleic acids that are prone to cleavage, such as base elimination, and for measurements using MALDI-IT-TOFMS, which are prone to fragmentation.

[0101] Aspects It will be apparent to those skilled in the art that the exemplary embodiments described above are examples of the following aspects.

[0102] (Item 1) A method for analyzing the structure of a sample molecule according to one aspect of the present invention is a method for analyzing the structure of a sample molecule using an ion trap mass spectrometer equipped with an ion source employing a matrix-assisted laser desorption / ionization (MALDI) method, an ion trapping unit for separating and trapping ions of a predetermined mass-to-charge ratio from among ions generated by the ion source, and a detection unit for detecting the ions trapped by the ion trapping unit, the method comprising: a standard analysis condition acquisition step of acquiring standard analysis conditions for an ion amount setting item relating to the amount of ions generated by the ion source, a mass-to-charge ratio range setting item relating to the mass-to-charge ratio range of ions trapped by the ion trapping unit, and a signal intensity setting item relating to the signal intensity of ions in the detection unit when performing molecular weight-related ion measurement to detect molecular weight-related ions of the sample molecule contained in a sample using the mass spectrometer; The method comprises: a product ion measurement data acquisition step of performing product ion measurement to detect a plurality of first fragment ions generated by dissociation of molecular weight-related ions of the sample molecule under modified analysis conditions obtained by changing at least one of the analysis conditions, namely, the ion amount setting item, the mass-to-charge ratio range setting item, and the signal intensity setting item, out of the standard analysis conditions acquired in the standard analysis condition acquisition step; a first fragment peak extraction step of extracting peaks corresponding to the first fragment ions from the mass spectrum data acquired in the product ion measurement data acquisition step; and a data analysis step of determining at least a part of the structure of the sample molecule based on mass information of the peaks extracted in the first fragment peak extraction step.

[0103] This allows fragment ions necessary for structural analysis to be detected with good sensitivity over a relatively wide mass range from low to high mass.

[0104] (Item 2) The method for analyzing the structure of a sample molecule described in Item 1, wherein the mass spectrometer is an apparatus capable of MS / MS measurement, further comprising: an MS / MS measurement condition setting step of setting measurement conditions for executing MS / MS measurement in which at least one of the plurality of first fragment ions detected in the product ion measurement is used 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 and acquiring mass spectral data of the MS / MS measurement; and a second fragment peak extraction step of extracting peaks corresponding to second fragment ions generated in the MS / MS measurement from the mass spectral data acquired in the MS / MS measurement data acquisition step, wherein the data analysis step may determine at least a part of the structure of the sample molecule based on mass information of the peak extracted in the first fragment peak extraction step and mass information of the peak extracted in the second fragment peak extraction step.

[0105] This makes it possible to detect with high sensitivity not only first fragment ions derived from molecular weight-related ions but also second fragment ions obtained by further dissociating the first fragment ions. As a result, for sample molecules whose entire molecular structure cannot be analyzed with high accuracy based on the mass information of the first fragment ions alone, structural information can be determined more reliably based on the mass information of the first fragment ions and the second fragment ions.

[0106] (Item 3) A method for analyzing the structure of a sample molecule according to another aspect of the present invention is a method for analyzing the structure of a sample molecule using an ion trap mass spectrometer equipped with an ion source employing a matrix-assisted laser desorption / ionization (MALDI) method, an ion trapping unit for separating and trapping ions of a predetermined mass-to-charge ratio from among ions generated by the ion source, and a detection unit for detecting the ions trapped by the ion trapping unit, the method comprising: a standard analysis condition acquisition step for acquiring standard analysis conditions, when performing molecular weight-related ion measurement to detect molecular weight-related ions of the sample molecule contained in a sample using the mass spectrometer, including an ion amount setting item related to the amount 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 trapped by the ion trapping unit, and a signal intensity setting item related to the signal intensity of ions in the detection unit; and a product ion measurement step for performing product ion measurement to detect a plurality of first fragment ions generated by dissociation of the molecular weight-related ions of the sample molecule under modified analysis conditions obtained by changing at least one of the ion amount setting item, the mass-to-charge ratio range setting item, and the signal intensity setting item among the standard analysis conditions acquired in the standard analysis condition acquisition step. The method comprises an MS / MS measurement condition setting step of setting measurement conditions for performing MS / MS measurement using at least one of the plurality of first fragment ions detected in the product ion measurement as a precursor ion; an MS / MS measurement data acquisition step of acquiring mass spectral 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 peaks corresponding to second fragment ions generated in the MS / MS measurement from the mass spectral data acquired in the MS / MS measurement data acquisition step; and a data analysis step of determining a part of the structure of the sample molecule based on mass information of the peak extracted in the second fragment peak extraction step.

[0107] This allows for the sensitive detection of second fragment ions, which are generated by further dissociating the first fragment ions derived from molecular-related ions, and as a result, it is possible to more reliably determine structural information about the molecular structure of a sample molecule that cannot be analyzed accurately based on the mass information of the first fragment ions alone.

[0108] (4) In the method for analyzing the structure of a sample molecule described in any one of paragraphs 1 to 3, the modified analysis conditions may be modified so that the amount of ions generated in the ion source is increased compared to when the molecular weight-related ions of the sample are measured under the standard analysis conditions, or the analysis conditions for the mass-to-charge ratio range setting item are modified so that ions lower in mass than the mass-to-charge ratio of the molecular weight-related ions of the sample molecule are preferentially captured, or the analysis conditions for the signal intensity setting item are modified so that the signal intensity of ions in the detection unit is increased.

[0109] This makes it possible to detect fragment ions, particularly those on the low mass-to-charge ratio side, among the fragment ions required for analysis with high sensitivity.

[0110] (Item 5) In the method for analyzing the structure of a sample molecule described in any one of Items 1 to 4, the changed analysis conditions may be such that the analysis conditions for the ion amount setting item are changed so that the amount of ions generated in the ion source is increased, and the analysis conditions for the signal intensity setting item are changed so that the signal intensity of the ions in the detection unit is increased.

[0111] This allows fragment ions necessary for analysis, particularly those on the low mass-to-charge ratio side, to be detected with higher sensitivity.

[0112] (Item 6) In the method for analyzing the structure of a sample molecule described in any one of Items 1 to 5, the ion amount setting item may be the intensity of a laser irradiated by the ion source, and the modified analysis conditions may be such that the laser intensity is changed to a value greater than that of the standard analysis conditions.

[0113] This allows fragment ions necessary for analysis, particularly those on the low mass-to-charge ratio side, to be detected with higher sensitivity.

[0114] (Item 7) In the method for analyzing the structure of a sample molecule described in any one of Items 1 to 6, the mass-to-charge ratio range setting item may be a time from when a laser is irradiated from the ion source to when a capture voltage for capturing ions is applied to the ion capture section, and the modified analysis conditions may be a time that is changed to a value shorter than that of the standard analysis conditions.

[0115] This allows fragment ions necessary for analysis, particularly those on the low mass-to-charge ratio side, to be detected with higher sensitivity.

[0116] (Item 8) In the method for analyzing the structure of a sample molecule according to item 7, the modified analysis conditions may be such that the time is set to a value that is 1 to 3 μsec shorter than that of the standard analysis conditions.

[0117] This allows fragment ions necessary for analysis, particularly those on the low mass-to-charge ratio side, to be detected with even greater sensitivity.

[0118] (Item 9) In the method for analyzing the structure of a sample molecule described in any one of Items 1 to 8, the detection unit may comprise a conversion dynode and a detector, the signal intensity setting items may be a voltage applied to the conversion dynode and a voltage applied to the detector, and the modified analysis conditions may be such that at least one of the voltage applied to the conversion dynode and the voltage applied to the detector is changed to a value greater than that of the standard analysis conditions.

[0119] This allows fragment ions necessary for analysis, particularly those on the low mass-to-charge ratio side, to be detected with higher sensitivity.

[0120] (Item 10) In the method for analyzing the structure of a sample molecule described in any one of Items 1 to 9, the product ion measurement may be performed by setting a range of the predetermined mass-to-charge ratio so that the maximum value of the predetermined mass-to-charge ratio of ions captured by the ion trapping unit is smaller than the value of the mass-to-charge ratio of ions related to the molecular weight of the sample molecule.

[0121] This allows fragment ions necessary for analysis, particularly those on the low mass-to-charge ratio side, to be detected with high sensitivity.

[0122] (Item 11) In the method for analyzing the structure of a sample molecule according to item 10, the predetermined range of mass-to-charge ratios may be set by setting a frequency of the trapping voltage in the ion trapping unit.

[0123] This allows fragment ions necessary for analysis, particularly those on the low mass-to-charge ratio side, to be detected with high sensitivity.

[0124] (Item 12) In the method for analyzing the structure of a sample molecule described in Item 10, the mass spectrometer may be a digital ion trap mass spectrometer, and the predetermined mass-to-charge ratio range may be set by setting a duty ratio of the trapping voltage in the ion trapping section.

[0125] This allows fragment ions necessary for analysis, particularly those on the low mass-to-charge ratio side, to be detected with higher sensitivity.

[0126] (Item 13) In the method for analyzing the structure of a sample molecule described in any one of Items 10 to 12, the mass spectrometer may include a sample stage for placing a sample plate on which the sample is placed, the standard analysis condition acquisition step may further acquire standard analysis conditions for a voltage applied to the sample stage when measuring the molecular weight-related ions, and the product ion measurement may be performed under conditions in which the voltage applied to the sample stage is changed to a value greater than the standard analysis conditions.

[0127] This allows fragment ions necessary for analysis, particularly those on the low mass-to-charge ratio side, to be detected with even greater sensitivity.

[0128] (Item 14) In the method for analyzing the structure of a sample molecule according to any one of Items 1, 2, and 4 to 13, the mass spectrometer is a digital ion trap type mass spectrometer, and the product ion measurement data acquisition step further comprises performing the product ion measurement and acquiring mass spectrum data of the product ion measurement by changing at least one of the frequency of the trapping voltage and the duty ratio of the trapping voltage in the ion trapping unit, or without changing at least one of the frequency of the trapping voltage and the duty ratio of the trapping voltage, so that the maximum value of the predetermined mass-to-charge ratio of ions trapped by the ion trapping unit is smaller than the value of the mass-to-charge ratio of ions related to the molecular weight of the sample molecule, and the first fragment peak extraction step extracts peaks corresponding to the plurality of first fragment ions from both the mass spectrum data obtained by performing the product ion measurement in the product ion measurement data acquisition step while changing at least one of the frequency of the trapping voltage and the duty ratio of the trapping voltage, and the mass spectrum data obtained by performing the product ion measurement without changing at least one of the frequency of the trapping voltage and the duty ratio of the trapping voltage, The data analysis step may determine at least a part of the structure of the sample molecule based on mass information of the peak extracted in the first fragment peak extraction step, thereby enabling more reliable analysis of the structure of the sample molecule.

[0129] (15) In the method for analyzing the structure of a sample molecule according to any one of items 1 to 14, the sample molecule may be a nucleic acid or a nucleic acid-related substance.

[0130] (Item 16) In the method for analyzing the structure of a sample molecule according to Item 2, the sample molecule may be a nucleic acid or a nucleic acid-related substance, and a part of the structure of the sample molecule may be a structure of a terminal portion of the nucleic acid or nucleic acid-related substance.

[0131] (Item 17) In the method for analyzing the structure of a sample molecule according to item 3, the sample molecule may be a nucleic acid or a nucleic acid-related substance, and a part of the structure of the sample molecule may be a structure of a terminal portion of the nucleic acid or nucleic acid-related substance.

[0132] REFERENCE SIGNS LIST 1 ion source 2 ion trap 3 detection unit 11 laser irradiation unit 12 sample stage 21 ring electrode 22, 23 end cap electrodes 31 conversion dynode 32 detector (secondary electron multiplier)

Claims

1. A method for analyzing a structure of a sample molecule using an ion trap mass spectrometer including an ion source using a matrix assisted laser desorption / ionization (MALDI) method, an ion trapping section for separating and trapping ions having a predetermined mass-to-charge ratio from among ions generated by the ion source, and a detection section for detecting the ions trapped by the ion trapping section, comprising: a standard analysis condition acquisition step of acquiring standard analysis conditions for an ion amount setting item relating to the amount of ions generated in the ion source, a mass-to-charge ratio range setting item relating to the mass-to-charge ratio range of ions trapped in the ion trapping unit, and a signal intensity setting item relating to the signal intensity of ions in the detection unit, when performing molecular weight-related ion measurement to detect molecular weight-related ions of the sample molecules contained in a sample using the mass spectrometer; an MS measurement data acquisition step of performing an MS measurement for detecting a plurality of first fragment ions generated by dissociation of 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 amount setting item, the mass-to-charge ratio range setting item, and the signal intensity setting item among the standard analysis conditions acquired in the standard analysis condition acquisition step, and acquiring mass spectrum data of the MS 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 MS measurement data acquisition step; a data analysis step of determining at least a part of the structure of the sample molecule based on mass information of the peak extracted in the first fragment peak extraction step; The method for analyzing the structure of a sample molecule comprises:

2. 2. The method for analyzing a structure of a sample molecule according to claim 1, The mass spectrometer is capable of MS / MS measurement, moreover, an MS / MS measurement condition setting step of setting measurement conditions for performing an MS / MS measurement in which at least one of the plurality of first fragment ions detected in the MS measurement is used 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 and acquiring mass spectrum data of the MS / MS measurement; 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; having In the data analysis step, at least a part of the structure of the sample molecule is determined based on mass information of the peak extracted in the first fragment peak extraction step and mass information of the peak extracted in the second fragment peak extraction step. A method for analyzing the structure of sample molecules.

3. A method for analyzing a structure of a sample molecule using an ion trap mass spectrometer including an ion source using a matrix assisted laser desorption / ionization (MALDI) method, an ion trapping section for separating and trapping ions having a predetermined mass-to-charge ratio from among ions generated by the ion source, and a detection section for detecting the ions trapped by the ion trapping section, comprising: a standard analysis condition acquisition step of acquiring standard analysis conditions for an ion amount setting item relating to the amount of ions generated in the ion source, a mass-to-charge ratio range setting item relating to the mass-to-charge ratio range of ions trapped in the ion trapping unit, and a signal intensity setting item relating to the signal intensity of ions in the detection unit, when performing molecular weight-related ion measurement to detect molecular weight-related ions of the sample molecules contained in a sample using the mass spectrometer; an MS measurement step of performing an MS measurement to detect a plurality of first fragment ions generated by dissociation of 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 amount setting item, the mass-to-charge ratio range setting item, and the signal intensity setting item among 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 an MS / MS measurement in which at least one of the plurality of first fragment ions detected in the MS measurement is used 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; a data analysis step of determining a part of the structure of the sample molecule based on mass information of the peak extracted in the second fragment peak extraction step; The method for analyzing the structure of a sample molecule comprises:

4. 2. The method for analyzing a structure of a sample molecule according to claim 1, wherein the changed analysis conditions are analysis conditions for the ion amount setting item changed so that the amount of ions generated in the ion source is increased compared to when the molecular weight-related ions of the sample molecule are measured under the standard analysis conditions, analysis conditions for the mass-to-charge ratio range setting item changed so that ions on the lower mass side than the mass-to-charge ratio of the molecular weight-related ions of the sample molecule are preferentially captured, or analysis conditions for the signal intensity setting item changed so that the signal intensity of ions in the detection unit is increased.

5. 2. The structural analysis method 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 in the ion source, and by changing the analysis conditions of the signal intensity setting item so as to increase the signal intensity of the ions in the detection unit.

6. The ion amount setting item is a laser intensity irradiated from the ion source, 2. The method for analyzing a structure of a sample molecule according to claim 1, wherein the modified analysis conditions are such that the laser intensity is changed to a value greater than that of the standard analysis conditions.

7. the mass-to-charge ratio range setting item is a time from when a laser is irradiated from the ion source to when a trapping voltage for trapping ions is applied to the ion trapping unit, 2. The method for analyzing a structure of a sample molecule according to claim 1, wherein the modified analytical conditions are such that the time is changed to a value shorter than that of the standard analytical conditions.

8. 8. The method for analyzing a structure of a sample molecule according to claim 7, wherein the modified analysis conditions set the time to a value that is 1 to 3 μsec shorter than that of the standard analysis conditions.

9. the detection unit includes a conversion dynode and a detector, the signal intensity setting items are a voltage applied to the conversion dynode and a voltage applied to the detector, 2. The method for analyzing a structure of a sample molecule according to claim 1, wherein the modified analysis conditions are such that at least one of the voltage applied to the conversion dynode and the voltage applied to the detector is changed to a value greater than that of the standard analysis conditions.

10. 2. The method for analyzing a structure of a sample molecule according to claim 1, wherein the MS measurement is performed by setting a range of the predetermined mass-to-charge ratio such that a maximum value of the predetermined mass-to-charge ratio of ions trapped by the ion trapping unit is smaller than a value of a mass-to-charge ratio of an ion related to a molecular weight of the sample molecule.

11. 11. The method for analyzing a structure of a sample molecule according to claim 10, further comprising the step of: setting a frequency of a trapping voltage for trapping ions in the ion trapping section to set the predetermined range of mass-to-charge ratios.

12. The mass spectrometer is a digital ion trap type mass spectrometer, 11. The method for analyzing a structure of a sample molecule according to claim 10, further comprising the step of: setting a duty ratio of a trapping voltage for trapping ions in the ion trapping portion to set the predetermined range of mass-to-charge ratios.

13. the mass spectrometer includes a sample stage for mounting a sample plate on which samples are placed; The standard analysis condition acquisition step further includes acquiring standard analysis conditions for a voltage applied to the sample stage when the molecular weight-related ion measurement is performed; 11. The method for analyzing a structure of a sample molecule according to claim 10, wherein the MS measurement is performed under conditions in which a voltage applied to the sample stage is changed to a value greater than the standard analysis conditions.

14. The mass spectrometer is a digital ion trap type mass spectrometer, the MS measurement data acquisition step further comprises performing the MS measurement while changing at least one of a frequency of a trapping voltage for trapping ions in the ion trapping section and a duty ratio of the trapping voltage so that the maximum value of the predetermined mass-to-charge ratio of the ions trapped in the ion trapping section is smaller than a mass-to-charge ratio value of an ion related to a molecular weight of the sample molecule, or without changing at least one of the frequency of the trapping voltage and the duty ratio of the trapping voltage, and acquiring mass spectrum data of the MS measurement; The first fragment peak extraction step extracts peaks corresponding to the first fragment ions from both mass spectrum data acquired by performing the MS measurement while changing at least one of a frequency of the trapping voltage and a duty ratio of the trapping voltage in the MS measurement data acquisition step and mass spectrum data acquired by performing the MS measurement without changing at least one of a frequency of the trapping voltage and a duty ratio of the trapping voltage in the MS measurement data acquisition step; 2. The method for analyzing a structure of a sample molecule according to claim 1, wherein the data analyzing step determines at least a part of the structure of the sample molecule based on mass information of the peak extracted in the first fragment peak extracting 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. 3. 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. 4. 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.