Mass spectrometry data analysis method and mass spectrometry device

Abstract

An embodiment of the mass spectrometer according to the present invention comprises: a measurement unit (11) that acquires data constituting a fragment spectrum, by performing mass spectrometry involving fragmentation on a target compound having an array structure in which a plurality of known substances are linked; a fragment estimation unit (24) that estimates fragment ions derived from the target compound and corresponding to peaks observed in the fragment spectrum; a coverage creation unit (26) that creates an array coverage in which the estimated fragment ions are associated with the array structure of the target compound; a display unit (4) that is capable of displaying an image; an analysis result display process unit (27) that displays, on the same display screen, the array coverage and a fragment list that displays a list of the estimated fragment ions and allows reception of designation of whether each fragment ion is to be selected or not to be selected; and a designation reception unit (28) that receives the user's designation of whether each fragment ion is to be selected or not to be selected in the displayed fragment list. The coverage creation unit creates the array coverage in which only fragment ions designated to be selected are reflected, when the user designates fragment ions to be selected or not to be selected.

Description

Mass spectrometry data analysis method and mass spectrometry apparatus

[0001] The present invention relates to a method for analyzing data obtained by mass spectrometry and a mass spectrometer using the analysis method, and more particularly to a method and apparatus that are useful for structural analysis of polymeric compounds such as oligonucleic acids and peptides.

[0002] Nucleic acid drugs have been attracting attention in recent years, and research and development of nucleic acid drugs is being actively pursued both domestically and internationally. Nucleic acid drugs are chemically synthesized pharmaceuticals made from oligonucleotides (oligonucleotides), which are building blocks of DNA and RNA. The process of chemically synthesizing oligonucleotides results in the inclusion of various impurities known as related substances (hereinafter, oligonucleotide related substances are referred to as "impurities"). Therefore, identifying these impurities is an important task in the research and development of nucleic acid drugs.

[0003] In order to identify impurities present in oligonucleic acids, a method using a mass spectrometer capable of MS / MS analysis has been known. As described in Non-Patent Document 1, when ions derived from oligonucleic acids or impurities are dissociated by a method such as collision-induced dissociation (CID), fragment ions of the a, b, c, d, w, x, y, and z series, which are characteristic of their structures, are generated. Therefore, conventional systems for analyzing oligonucleic acids equipped with software described in Non-Patent Document 2 and the like detect these specific series of fragment ions in MS / MS spectra (fragment spectra) and use the detection results to estimate the base sequences of the oligonucleic acids or impurities.

[0004] In conventional oligonucleic acid analysis systems described in Non-Patent Documents 2 and 3, a sequence coverage is created and displayed in which each fragment ion corresponds to the sequence of the original compound (oligonucleic acid) based on the identification results of peaks detected in the MS / MS spectrum. The user can visually check the type (series) of fragments and their comprehensiveness using this sequence coverage, and can accurately obtain information such as base deletions and modification positions in impurities.

[0005] Scott A. McLuckey and two others, "Tandem mass spectrometry of small, multiply charged oligonucleotides," Journal of American Society Mass Spectrometry, 1992, Vol. 3, No. 1, pp. 60-70. "LabSolutions Insight Biologics Sequence Identification Software for Oligonucleotides," Shimadzu Corporation, [online], [Retrieved May 24, 2024], Internet <URL: https: / / www.an.shimadzu.co.jp / products / liquid-chromatograph-mass-spectrometry / lc-ms-software / labsolutions-insight-biologics / index.html>. Noriko Kato and one other, "LabSolutions Insight™ "Oligonucleotide Impurity Analysis Workflow Using Biologics," Shimadzu Corporation, [online], [searched May 24, 2024], Internet <URL: https: / / www.an.shimadzu.co.jp / sites / an.shimadzu.co.jp / files / pim / pim_document_file / an_jp / applications / application_note / 21751 / an_01-00595-jp.pdf>

[0006] In mass spectrometry of oligonucleic acids as described above, multiply charged ions are likely to be generated, and many multiply charged ion peaks with a wide range of valences are observed in MS / MS spectra, resulting in complex spectral patterns. Furthermore, when performing mass spectrometry on biopolymer compounds such as oligonucleic acids, peaks reflecting isotope distribution are observed, but if the signal intensity of the precursor ion is low, some or many of the isotope peaks may not be observed in the MS / MS spectrum. Therefore, when analyzing MS / MS spectra, misidentification of peaks due to incorrect estimation of valence, etc., often occurs, resulting in a problem of reduced accuracy of the displayed sequence coverage.

[0007] Furthermore, in peptide analysis such as peptide mapping, a technique is known in which, in order to analyze relatively large peptides, fragments of the peptides are observed as multiply charged ions, and the observation results are mapped to evaluate the coverage of the amino acid sequence. In this case, as in the case of the oligonucleic acid described above, there is a problem in that the coverage display is inaccurate due to misidentification of multiply charged ion peaks observed in the MS / MS spectrum.

[0008] The present invention has been made to solve these problems, and its main object is to provide a mass spectrometry data analysis method and mass spectrometer that allows a user to easily correct coverage information for base sequences and amino acid sequences using fragment information obtained from fragment spectra when analyzing the structure of polymeric compounds such as oligonucleic acids and peptides, thereby improving the accuracy of the information.

[0009] One aspect of the mass spectrometry data analysis method according to the present invention is a method for analyzing a compound having a sequence structure in which multiple known types of substances are linked together, using mass spectrometry, and includes: a fragment estimation step for estimating fragment ions derived from a target compound that correspond to peaks observed in a fragment spectrum obtained for the target compound; a coverage creation step for creating sequence coverage that associates the fragment ions estimated in the fragment estimation step with the sequence structure of the target compound; an analysis result display step for displaying, on a single display screen, a fragment list that displays a list of the fragment ions estimated in the fragment estimation step and allows for acceptance / non-acceptance of each fragment ion, and the sequence coverage; a designation receiving step for receiving, by a user, designation of acceptance / non-acceptance of each fragment ion in the displayed fragment list; and a coverage display change step for changing the sequence coverage displayed on the display unit to a sequence coverage that reflects only the fragment ions designated as accepted, in accordance with the user's designation of acceptance / non-acceptance in the designation receiving step.

[0010] a fragment ion estimation unit that estimates fragment ions derived from the target compound that correspond to peaks observed in the fragment spectrum; a coverage creation unit that creates sequence coverage that associates the fragment ions estimated by the fragment estimation unit with the sequence structure of the target compound; a display unit that can display images; an analysis result display processing unit that displays, on the same screen of the display unit, a fragment list that displays the fragment ions estimated by the fragment estimation unit and that can accept an acceptance / non-acceptance designation for each fragment ion, and the sequence coverage; and a designation receiving unit that receives a user's designation for acceptance / non-acceptance of each fragment ion in the fragment list displayed on the display unit, wherein the coverage creation unit creates sequence coverage that reflects only the fragment ions that have been designated as accepted, when the user has designated whether to accept or not accept a fragment ion via the designation receiving unit.

[0011] In the present invention, for example, if the user determines that some fragment ions estimated by automatic analysis to be derived from the target compound are likely to be invalid (misidentified), the user performs an operation to designate the fragment ions as unaccepted in the fragment list. In response to this operation, the sequence coverage displayed on the screen of the display unit is changed to a sequence coverage that includes the remaining fragment ions, excluding the fragment ions designated as unaccepted by the user. In this way, according to the present invention, when analyzing the structure of a polymer compound such as an oligonucleic acid or a peptide, the user can easily correct the coverage information of the base sequence or amino acid sequence using fragment information, thereby improving its accuracy.

[0012] 1 is a schematic diagram of an oligo-nucleic acid sequence analysis device according to one embodiment of the present invention; FIG. 2 is a flowchart showing an example of an analysis procedure in the oligo-nucleic acid sequence analysis device according to this embodiment; FIG. 3 is a schematic diagram of an analysis result screen displayed at the end of analysis processing in the oligo-nucleic acid sequence analysis device according to this embodiment; FIG. 4 is a diagram showing an example of a display of the sequence coverage display area in FIG. 3; FIG. 5 is a diagram showing an example of a display of the MS / MS spectrum display area in FIG. 3; FIG. 6 is a diagram showing an example of a display of the identification result display area in FIG. 3; FIG. 7 is a diagram showing an example of a display of the fragment result display area in FIG. 3; FIG. 8 is a diagram showing a display state when one fragment is not adopted in the fragment result table; FIG. 9 is a diagram showing a sequence coverage display when one fragment is not adopted as shown in FIG. 10; FIG. 11 is a diagram showing a part of a setting screen when setting parameters related to peak detection; FIG. 12 is an explanatory diagram of adjacent isotope detection.

[0013] [Compound to be analyzed] The compound to be analyzed in the present invention is a compound having a sequence structure in which multiple known types of substances are linked together. The "substance" referred to here is, for example, a base or an amino acid. When the substance is a base, the compound is an oligonucleic acid or a nucleic acid. On the other hand, when the substance is an amino acid, the compound is a peptide or a protein.

[0014] [Conditions for Mass Spectrometer] The mass spectrometer used in the present invention is capable of performing mass analysis of compounds in a sample, involving ion fragmentation. Fragmentation here refers to dissociating ions derived from the target compound using various dissociation techniques, such as CID, photoinduced dissociation, electron capture dissociation, electron transfer dissociation, and radical-induced dissociation, as well as the so-called in-source decay (ISD) technique, in which ions are fragmented. Therefore, the fragment spectrum referred to here includes not only MS / MS spectra obtained by MS / MS (=MS2) measurements, but also ISD spectra.

[0015] The mass spectrometer used in the present invention employs an ionization technique that generates multiply charged ions (i.e., ions with multiple valences) from compounds in a sample. Well-known examples of such ionization techniques include electrospray ionization (ESI) and similar ionization techniques (i.e., ionization techniques that utilize the electrospray phenomenon). Furthermore, matrix-assisted laser desorption / ionization (MALDI), which is generally considered to be difficult to generate multiply charged ions, can also generate multiply charged ions depending on the ionization conditions, such as increasing the laser power or using a special matrix. Therefore, the present invention is applicable not only to mass spectrometers employing ESI but also to mass spectrometers employing ionization techniques such as MALDI.

[0016] The mass analyzer used in the present invention has a mass resolution that allows isotope peaks to be separated and observed. Generally, time-of-flight mass analyzers have high mass resolution and are therefore suitable as the mass analyzer used here. For example, even with a quadrupole mass filter, it is possible to increase the mass resolution to a certain extent by slowing the scan speed. Similarly, with an ion trap mass separator, it is possible to increase the mass resolution to a level that allows isotope peaks to be separated by appropriately setting conditions such as the scan speed. For these reasons, mass analyzers using a quadrupole mass filter or an ion trap as a mass separator can also be applied to the present invention.

[0017] [Configuration of an Oligo-Nucleic Acid Sequence Analysis Device of an Embodiment] An oligo-nucleic acid sequence analysis device of an embodiment of the present invention will be described with reference to the accompanying drawings. Fig. 1 is a schematic diagram of the configuration of an oligo-nucleic acid sequence analysis device of this embodiment.

[0018] This oligo-nucleic acid sequence analyzer comprises a measurement unit 1, a data analysis unit 2, an input unit 3, and a display unit 4. The measurement unit 1 includes a liquid chromatograph unit (LC unit) 10 and a tandem mass spectrometer unit (MS / MS unit) 11. The MS / MS unit 11 is, for example, a quadrupole time-of-flight mass spectrometer equipped with an ESI source as an ion source and capable of fragmenting ions by CID in a collision cell. Specifically, the measurement unit 1 may be, for example, a Nexera manufactured by Shimadzu Corporation. TM XS inert and LCMS-9050 can be used, but it goes without saying that the MS / MS unit 11 is not limited to these.

[0019] The data analysis unit 2 includes, as functional blocks, a spectrum data storage unit 20, an analysis parameter setting unit 21, a chromatographic peak detection unit 22, a spectrum creation unit 23, a spectrum analysis unit 24, a theoretical isotope spectrum calculation unit 25, an analysis result information creation unit 26, an analysis result display processing unit 27, and an operation reception unit 28.

[0020] The data analysis unit 2 is actually a computer such as a personal computer or a workstation, and the above-mentioned functional blocks are realized by executing one or more dedicated software programs (computer programs) installed on the computer. Such computer programs can be provided to the user by being stored on a computer-readable, non-transitory recording medium such as a CD-ROM, DVD-ROM, memory card, or USB memory (dongle). Alternatively, they can be provided to the user in the form of data transfer via a communication line such as the Internet. Alternatively, they can be pre-installed on a computer that is part of the system when the user purchases the system.

[0021] The input unit 3 and the display unit 4 are user interfaces, and the input unit 3 is a keyboard and a pointing device (mouse, etc.) attached to the computer, while the display unit 4 is a monitor display attached to the computer that can display images.

[0022] [Procedure for Oligo-Nucleic Acid Sequence Analysis] Fig. 2 is a flowchart showing the analysis procedure in the oligo-nucleic acid sequence analysis device of this embodiment. The oligo-nucleic acid sequence analysis device of this embodiment comprehensively analyzes the characteristics of the target oligo-nucleic acid and its impurities contained in a sample, and the analysis is performed according to the procedure shown in Fig. 2.

[0023] First, an LC / MS measurement is performed on a sample containing an oligonucleic acid and impurities to be analyzed, using the measurement unit 1, to obtain MS spectrum and MS / MS spectrum data. Here, in order to comprehensively detect unknown impurities, the MS / MS unit 11 performs MS / MS measurement by DDA (Data Dependent Analysis) (step S1).

[0024] When a sample is introduced into the LC unit 10, the compounds in the sample (oligonucleotides and impurities) are separated in time as they pass through the LC unit 10 and then introduced into the MS / MS unit 11. The MS / MS unit 11 repeatedly performs mass analysis (MS analysis) over a predetermined mass-to-charge ratio (m / z) range. When a peak satisfying a predetermined condition (e.g., signal intensity above a predetermined threshold) is detected in the MS spectrum obtained by the MS analysis, an MS / MS analysis targeting the ions corresponding to that peak is performed following the MS analysis. Therefore, an MS spectrum and an MS / MS spectrum associated with the MS spectrum are obtained for the oligonucleotides and their impurities contained in the sample. The data constituting these spectra are sent to the data analysis unit 2 and stored in the spectrum data storage unit 20.

[0025] When performing analysis after spectral data has been acquired for the sample to be analyzed as described above, the user first sets analysis parameters (step S2). That is, when the user performs a predetermined operation on the input unit 3, the analysis parameter setting unit 21 displays a parameter setting screen in a predetermined format on the display unit 4. While viewing this setting screen, the user operates the input unit 3 to input information such as the sequence of the target oligonucleic acid. Furthermore, various search conditions related to anticipated impurities, such as differences in chain length, nucleic acid base deletion, depurination / pyrimidination, deamination, and adducts or modifications such as protective residues, can be set. Furthermore, peak detection conditions and waveform processing conditions for chromatograms and spectra (MS spectra, MS / MS spectra) can also be set here.

[0026] When the user sets the analytical parameters and issues an instruction to start the analysis, the data analysis unit 2 then initiates an analytical process based on the data stored in the spectrum data storage unit 20. First, the chromatographic peak detection unit 22 creates a total ion chromatogram (TIC) or a base peak chromatogram (BPC) using the stored MS spectrum data, or creates an LC chromatogram based on data acquired by a separately provided PDA detector or the like, and detects peaks (chromatographic peaks) in the chromatograms (step S3). The retention times at which chromatographic peaks are detected are the retention times at which the compounds in the sample, i.e., the target oligonucleic acid and impurities, elute.

[0027] Next, the spectrum creation unit 23 creates an MS spectrum and an MS / MS spectrum using the MS spectrum data and MS / MS spectrum data acquired around the retention time at which the chromatographic peak was detected. The spectrum analysis unit 24 then extracts mass peaks from the MS spectrum that are presumed to be derived from the target oligonucleic acid and impurities according to the search conditions set in step S2 (step S4). Since many multiply charged ion peaks derived from the same compound but with different valences are observed in the MS spectrum, all of these multiply charged ion peaks are also detected.

[0028] Furthermore, the spectrum analysis unit 24 detects mass peaks in the MS / MS spectrum obtained by MS / MS analysis linked to the mass peaks detected in the MS spectrum, i.e., targeting the mass peaks, and searches for fragment ions corresponding to each mass peak according to the set search conditions (step S5). Then, when fragment ions presumably derived from the same compound are determined, the oligonucleic acid and impurities are identified based on the results (step S6).

[0029] Furthermore, the theoretical isotope spectrum calculation unit 25 calculates a theoretical isotope spectrum that indicates the theoretical isotope distribution of the ion based on the chemical formula of the identified fragment ion (step S7).

[0030] When the series of analyses is completed as described above, the analysis result information creation unit 26 aggregates the fragment identification results, compound identification results, etc., and creates information for displaying the analysis results. As will be described in detail later, the analysis result information creation unit 26 creates an identification result table that is a list of identified compounds, a fragment result table that is a list of fragments corresponding to the identified compounds, sequence coverage that shows the correspondence between the base sequences of the identified compounds and each fragment ion, paired spectra that show a comparison between the measured MS / MS spectrum near the m / z value of each fragment ion and the theoretical isotope spectrum of the fragment ion, etc. (step S8).

[0031] The analysis result display processing unit 27 then creates an analysis result screen on which the information created by the analysis result information creation unit 26 is arranged, and displays the created analysis result screen on the display unit 4 (step S9). Fig. 3 is a schematic diagram of an example of the analysis result screen 50, and Figs. 4 to 7 each show an example of the display area of ​​the analysis result screen 50 shown in Fig. 3. In the example shown in Fig. 3, the analysis result screen 50 has a sequence coverage display area 51, a spectrum display area 52, an identification result display area 53, and a fragment result display area 54.

[0032] The identification result display area 53 displays an identification result table 530 showing the identification results of oligosaccharide chains and impurities, an example of which is shown in Figure 6. This identification result table 530 displays information such as retention time, mass, mass error, area (peak area in TIC), nucleic acid base sequence, and chromatogram for each identified oligosaccharide chain or impurity. In the identification result table 530, a compound (oligosaccharide chain or impurity) shown in any one row can be selected by clicking, and in Figure 6, the compound in the top row is selected (when selected, the background color of the left side of that row changes).

[0033] 7, the fragment result display area 54 displays a fragment result table 540 showing the results of all detected fragment ions corresponding to one compound selected on the identification result table 530. The fragment result table 540 displays information for each fragment ion, such as the type of fragment ion, the measured m / z value, the theoretical m / z value, the charge, the isotope position (the position of the ion peak in the isotope distribution used for identification), the intensity, and the mass error. In this fragment result table 540, a fragment shown in any one of the rows can also be selected by clicking, and in FIG. 7, the fragment (d16) in the fourth row from the top is selected (when selected, the background color of the left side of the row changes).

[0034] The sequence coverage display area 51 displays a sequence coverage 510, which is the result of associating fragment ions with the base sequence of the target oligonucleic acid, as shown in Figure 4. This sequence coverage display format has two types: branch mode and fill mode. Figure 4 shows an example of sequence coverage in branch mode, in which the detected fragment ions of each series (a, b, c, d, w, x, y, and z) are associated in a branch-like manner with each cleavage position in the base sequence. In Figure 4, bases are represented by a single capital letter (here, A, C, G, or T) surrounded by a hexagon. Also in Figure 4, the "s" surrounded by a circle indicates a thiophosphate modification, and the "d" surrounded by a square indicates a 2'-deoxy sugar modification. This branch mode sequence coverage display is useful for confirming fragment series.

[0035] On the other hand, the sequence coverage in fill mode is not shown here, but is a diagram in which long horizontal bars indicate to which range of the oligonucleic acid base sequence each detected fragment ion corresponds. The display color of the bar corresponds to the signal intensity. This sequence coverage display is useful for checking the intensity and comprehensiveness of each fragment ion. The display format of the sequence coverage can be easily switched by a predetermined operation.

[0036] The spectrum display area 52 displays a paired spectrum 20 including a measured MS / MS spectrum in a narrow m / z range near the measured m / z of one fragment ion selected in the fragment result table 540, and a theoretical isotope spectrum of that fragment ion. Specifically, as shown in an example in Fig. 5 , the paired spectrum 520 has a measured MS / MS spectrum 522 arranged on the upper side, and a theoretical isotope spectrum 523 arranged line-symmetrically below the measured MS / MS spectrum 522, sharing a horizontal axis (m / z axis) 521 with the measured MS / MS spectrum 522. That is, the measured MS / MS spectrum 522 is a spectrum whose signal intensity increases upward, while the theoretical isotope spectrum 523 is a spectrum whose signal intensity increases downward.

[0037] In this pair of spectra 520, the horizontal axis (m / z axis) 521 is shared, making it possible to check at a glance whether both mass peaks exist at the same m / z value or at very similar m / z values. Furthermore, the vertical bar representing the isotope peak with the greatest signal intensity among the isotope peaks, i.e., the most bandutant isotope peak, is surrounded by a background 524, as shown in FIG. 5. This allows the user to easily identify the most bandutant isotope peak in this pair of spectra.

[0038] Note that the measured MS / MS spectrum 522 is not an MS / MS spectrum obtained using a single peak observed in the MS spectrum as the precursor ion in the MS / MS analysis, but rather a merged (integrated) MS / MS spectrum obtained by performing multiple MS / MS analyses using multiple peaks observed in the same MS spectrum as precursor ions. In other words, this merged MS / MS spectrum reflects fragments of various valences generated by CID of multiply charged ions derived from the same compound but with different valences.

[0039] In the leftmost column of the fragment result table 540, a check box 542 is arranged for each fragment ion. In the example of FIG. 7 , the check boxes 542 for all fragment ions (including those that cannot be seen unless scrolling down) are checked. When the analysis process is completed and this analysis result screen 50 is displayed, all of the fragment ions will have check marks in the check boxes 542. The fragment ions with these check marks are reflected in the sequence coverage 510 displayed in the sequence coverage display area 51.

[0040] After the analysis parameters are set as described above, a series of analyses are performed automatically, but the analysis results are not necessarily all correct. Therefore, the user is usually required to check the analysis result screen 50 to see if the analysis is appropriate. One check item is to check whether the fragment ion identification is appropriate. For example, in the examples of Figures 3 to 7, when the information for each fragment ion is checked in the fragment result table 540, it is found that fragment ion d16 tends to differ from other fragment ions in terms of valence, isotope position, etc. However, these differences alone do not determine whether fragment ion d16 is misidentified.

[0041] Therefore, in order to check the measured MS / MS spectrum and theoretical MSMS spectrum corresponding to this fragment ion d16, the user uses the input unit 3 to click on the column for fragment ion d16 on the fragment result table 540. Then, the operation accepting unit 28 accepts this operation and displays a paired spectrum 520 corresponding to the specified fragment ion in the spectrum display area 52. However, the paired spectrum 520 shown in FIG. 5 is in a state in which fragment ion d16 has been specified in the fragment result table 540.

[0042] As described above, the paired spectrum 520 displayed in the spectrum display area 52 allows one to quickly confirm whether the theoretically observed isotope peaks are actually observed in the measured MS / MS spectrum. In the example of FIG. 5 , it is clear at a glance that no isotope peaks adjacent to the peak with the measured m / z value of 847.07681, which is the most bandgap isotope peak, are observed. Therefore, the user can determine that this fragment ion is likely not a fragment ion derived from the target oligonucleic acid or impurities. Therefore, if such a determination is made, the user unchecks the checkbox 542 in the row of fragment ion d16 in the fragment result table 540 (step S10) to essentially delete this fragment ion from the fragmentation results. Figure 8 shows the unchecked state.

[0043] The operation reception unit 28 receives an operation to uncheck the checkbox via the input unit 3, and the analysis result information creation unit 26 changes the sequence coverage to be displayed to one that does not reflect the unchecked fragment ions (step S11). As a result, the sequence coverage displayed on the display unit 4 is changed to one that does not substantially include the deleted fragment ions (step S12). The identification result table 530 includes a numerical value item called an MS / MS score calculated based on the fragment coverage. Since this is an index indicating the degree of coverage, the numerical value changes when the sequence coverage changes. Therefore, as described above, when an operation to uncheck the checkbox is performed and the sequence coverage is changed, the numerical value of the MS / MS score in the identification result table 530 also changes accordingly.

[0044] In the example of Fig. 8, the fragment ion d16 is unchecked, so the sequence coverage displayed in the sequence coverage display area 51 is changed to sequence coverage 510 in which the display of fragment ion d16 and the branch leading to that fragment ion have disappeared (see range A indicated by the dashed dotted line in Fig. 9), as shown in Fig. 9. In the case of sequence coverage display in fill mode, when the fragment ion d16 is unchecked, the display of the horizontal bar corresponding to that fragment ion d16 disappears.

[0045] In this way, the user can confirm each of the fragment ions listed in the fragment result table 540 and essentially delete any fragment ions that are determined to be misidentified from the fragment results, thereby displaying more accurate and reliable sequence coverage. Of course, it goes without saying that a fragment ion that has been unchecked in the fragment result table 540 can be restored by checking it again.

[0046] As described above, in determining whether a fragment ion is appropriate, whether or not other isotope peaks are observed around the isotope peak used to identify the fragment ion (adjacent on the m / z axis in the MS / MS spectrum) is very important information. While it is possible to confirm the presence or absence of an isotope peak in an actually measured MS / MS spectrum using the paired spectrum 520, it would be convenient to be able to confirm its presence or absence without checking the paired spectrum 520. Therefore, the device of this embodiment has a mode for displaying the detection results of adjacent isotopes in the "Isotope Position" column 541 of the fragment result table 540.

[0047] The adjacent isotopes referred to here refer to isotope peaks having indices [n-1] and [n+1] relative to an isotope peak with a certain index [n], where the monoisotopic ion peak observed at the leftmost position in the isotope distribution in the MS / MS spectrum is indexed [0], and the indices of the isotope peaks appearing in order to the right are indexed [1], [2], ....

[0048] To automatically detect and display adjacent isotopes, the user selects "detect adjacent isotopes" as a condition for peak detection in the MS / MS spectrum when setting the analysis parameters in step S2. FIG. 10 shows an MS / MS spectrum peak detection setting area 60, which is part of the peak detection setting screen used when setting the analysis parameters. As shown, the MS / MS spectrum peak detection setting area 60 allows users to specify numerical values ​​for the minimum intensity, mass error, minimum mass, and other peak detection conditions for the MS / MS spectrum. Below these input items, a checkbox 61 is provided that allows users to select the "detect adjacent isotopes" mode. When this checkbox 61 is checked and analysis is performed, the spectrum analysis unit 24 detects not only the most bandgap isotope peak but also adjacent isotope peaks when detecting mass peaks in the MS / MS spectrum. The indexes of the detected isotope peaks are then stored.

[0049] For example, in the example shown in FIG. 5 , the most banded peak has index [3], and its adjacent isotopes are not detected at all. Therefore, even if the "detect adjacent isotopes" mode is selected, the index displayed in the "isotope position" column 541 in the fragment result table 540 is only "3," as shown in FIG. 7 . On the other hand, if the paired spectrum display is as shown in FIG. 11 , the index of the most banded peak is [3], and its adjacent isotope peaks with indexes [2] and [4] are both detected in the measured MS / MS spectrum. Therefore, in this case, if the "detect adjacent isotopes" mode is selected, the indexes displayed in the "isotope position" column 541 in the fragment result table 540 are "2, 3, 4." On the other hand, if the "detect adjacent isotopes" mode is not selected, the index displayed in the "isotope position" column 541 in the fragment result table 540 is only "3."

[0050] By utilizing the "detect adjacent isotopes" mode in this way, it is possible to check in the fragment result table 540 whether adjacent isotopes of the isotope peak in the MS / MS spectrum used to identify the fragment ion are observed, without checking the actual MS / MS spectrum. However, the information about isotope peaks displayed in the fragment result table 540 is only for peaks that satisfy the detection conditions, such as minimum intensity and mass error, set by the user in the MS / MS spectrum peak detection setting area 60 shown in FIG. 10. Therefore, if the peak detection conditions are not set appropriately, peaks may be missed, or conversely, non-peaks may be mistaken for peaks. Therefore, it is recommended to check the pair spectrum display as necessary to determine whether the fragment ions are appropriate.

[0051] The device of the above embodiment is merely an example of the present invention, and it is clear that any modifications, changes, or additions made within the spirit of the present invention will fall within the scope of the claims of the present application.

[0052] For example, as described above, the measurement unit 1 may be a mass spectrometer equipped with a MALDI ion source, and the data analysis unit 2 may analyze the ISD spectrum obtained for the sample by the measurement unit 1.

[0053] Furthermore, although the above embodiment is an apparatus for analyzing oligonucleic acids and their impurities, the present invention is also applicable to an apparatus for analyzing peptides and proteins having amino acid sequences.

[0054] Various Aspects It will be apparent to those skilled in the art that the exemplary embodiments described above are examples of the following aspects.

[0055] (Item 1) A mass spectrometry data analysis method according to one aspect of the present invention is a method for analyzing a compound having a sequence structure in which multiple known types of substances are linked together using mass spectrometry, and includes: a fragment estimation step for estimating fragment ions derived from the target compound that correspond to peaks observed in a fragment spectrum obtained for the target compound; a coverage creation step for creating sequence coverage that associates the fragment ions estimated in the fragment estimation step with the sequence structure of the target compound; an analysis result display step for displaying, on the same display screen, a fragment list that displays a list of the fragment ions estimated in the fragment estimation step and allows acceptance / non-acceptance of designation for each fragment ion, and the sequence coverage; a designation receiving step for receiving, by a user, designation for acceptance / non-acceptance of each fragment ion in the displayed fragment list; and a coverage display change step for changing the sequence coverage displayed on the display unit to a sequence coverage that reflects only the fragment ions designated as accepted, in accordance with the user's designation for acceptance / non-acceptance in the designation receiving step.

[0056] (Item 6) Another aspect of the present invention provides a mass spectrometer comprising: a measurement unit that performs mass analysis involving fragmentation on a target compound having a sequence structure in which multiple known types of substances are linked together, to obtain data constituting a fragment spectrum; a fragment estimation unit that estimates fragment ions derived from the target compound that correspond to peaks observed in the fragment spectrum; a coverage creation unit that creates sequence coverage in which the fragment ions estimated by the fragment estimation unit correspond to the sequence structure of the target compound; a display unit that can display images; an analysis result display processing unit that displays, on the same screen of the display unit, a fragment list that displays the fragment ions estimated by the fragment estimation unit and in which a designation of whether to adopt or not adopt each fragment ion can be accepted; and a designation receiving unit that receives a user's designation of whether to adopt or not adopt each fragment ion in the fragment list displayed on the display unit. When a user designates whether to adopt or not adopt a fragment ion via the designation receiving unit, the coverage creation unit creates sequence coverage that reflects only the fragment ions designated as adopted.

[0057] When the user performs an operation to designate one or more fragment ions as unaccepted on the fragment list, the sequence coverage displayed on the screen of the display unit is changed in response to this operation to include the remaining fragment ions, excluding the fragment ions designated as unaccepted by the user. This allows the user to easily correct the coverage information of the base sequence or amino acid sequence using fragment information when analyzing the structure of a polymer compound such as an oligonucleic acid or a peptide, thereby improving its accuracy.

[0058] (2) In the mass spectrometry data analysis method described in 1, the analysis result display step can display a pair of spectra, in which the actually measured fragment spectrum in a predetermined m / z range including the m / z of one fragment ion selected in the displayed fragment list and the theoretical isotope spectrum theoretically calculated from the chemical formula of the fragment ion are arranged in a comparable manner, on the same display screen as the fragment list and the sequence coverage.

[0059] (Item 7) The mass spectrometer described in Item 6 further comprises a theoretical isotope spectrum calculation unit that calculates a theoretical isotope spectrum from the chemical formula of the fragment ion, and the analysis result display processing unit can display a pair spectrum in which the theoretical isotope spectrum and an actually measured fragment spectrum in a predetermined mass-to-charge ratio range that includes the mass-to-charge ratio of one fragment ion selected in the displayed fragment list are arranged so as to be comparable, on the same display screen as the fragment list and the sequence coverage.

[0060] Since a relatively large number of isotope peaks are observed in fragment ions derived from polymeric compounds such as oligonucleic acids, whether or not a group of isotope peaks similar to the theoretically calculated isotope spectrum pattern is actually observed is useful information for determining whether or not the fragment estimation is appropriate. Therefore, according to the mass spectrometry data analysis method described in paragraph 2 or the mass spectrometer described in paragraph 7, a user can easily and accurately determine whether or not the fragment estimation is appropriate by comparing the theoretical isotope spectrum with the actually measured fragment spectrum.

[0061] (Items 3 and 8) In the mass analysis data analysis method described in Item 2 or the mass spectrometer described in Item 7, the paired spectra may be an actually measured fragment spectrum and a theoretical isotope spectrum arranged in line symmetry with a common m / z axis.

[0062] According to the mass analysis data analysis method described in paragraph 3 or the mass spectrometer described in paragraph 8, the m / z axis of the two spectra to be compared is common, so it is possible to check at a glance whether peaks in both spectra exist at the same or very similar m / z values, which makes it possible to more efficiently examine the validity of fragments and reduces the occurrence of operational errors such as misreading.

[0063] (4) The mass spectrometry data analysis method according to any one of paragraphs 1 to 3 further comprises an adjacent isotope detection step of detecting an isotope peak adjacent to an isotope peak having the maximum signal intensity among a group of isotope peaks for each fragment ion in the fragment spectrum, and in the analysis result display step, if the adjacent isotope peak is detectable, information indicating the position of the adjacent isotope peak among the group of isotope peaks can be displayed within the fragment list or separately from the fragment list.

[0064] (Item 9) The mass spectrometer described in any one of Items 6 to 8 further includes an adjacent isotope detection unit that detects an isotope peak adjacent to an isotope peak having the maximum signal intensity among the isotope peak group for each fragment ion in the fragment spectrum, and the analysis result display processing unit can display information indicating the position of the adjacent isotope peak in the isotope peak group within the fragment list or separately from the fragment list when the adjacent isotope peak is detectable.

[0065] The "information indicating the position of the adjacent isotope peak in the group of isotope peaks" refers to an index indicating the position of the adjacent isotope peak from the monoisotopic ion peak in the group of isotope peaks, for example, when the position of the monoisotopic ion peak is used as a reference (e.g., No. 0, No. 1). The mass analysis data analysis method described in Section 4 or the mass spectrometer described in Section 9 makes it possible to confirm whether or not there is an isotope peak adjacent to the isotope peak of interest in the actually measured fragment spectrum, without referring to the paired spectrum described above.

[0066] (Item 5, Item 10) In the mass spectrometry data analysis method according to any one of Items 1 to 4 or the mass spectrometer according to any one of Items 6 to 9, the compound may be an oligonucleic acid.

[0067] Oligonucleic acids are particularly prone to producing multiply charged ions with a wide range of valences, which tends to result in complex fragment spectra. The mass spectrometry data analysis method described in paragraph 1 or the mass spectrometer described in paragraph 6 is particularly effective for analyzing fragment spectra in which a large number of such multiply charged ions are observed and the patterns are complex, and is therefore particularly effective for analyzing oligonucleic acids.

[0068] 1... Measurement section 10... Liquid chromatograph section (LC section) 11... Tandem mass spectrometry section (MS / MS section) 2... Data analysis section 20... Spectrum data storage section 21... Analysis parameter setting section 22... Chromatography peak detection section 23... Spectrum creation section 24... Spectrum analysis section 25... Theoretical isotope spectrum calculation section 26... Analysis result information creation section 27... Analysis result display processing section 28... Operation reception section 3... Input section 4... Display section 50... Analysis result screen 51... Sequence coverage display area 510... Sequence coverage 52... Spectrum display area 520... Paired spectrum 521... Horizontal axis (m / z axis) 522... Measured MS / MS spectrum 523... Theoretical isotope spectrum 53... Identification result display area 530... Identification result table 54... Fragment result display area 540... Fragment result table 541... "Isotope position" column 542: Checkbox 60: MS / MS spectrum peak detection setting area 61: Checkbox

Claims

A method for analyzing a compound having an array structure in which multiple known types of substances are linked together using mass spectrometry, comprising: a fragment estimation step of estimating fragment ions derived from the target compound corresponding to peaks observed in the fragment spectrum obtained for the target compound; a coverage generation step of generating a sequence coverage in which the fragment ions estimated in the fragment estimation step are associated with the sequence structure of the target compound; an analysis result display step of displaying, on the same display screen, a fragment list in which the fragment ions estimated in the fragment estimation step are displayed as a list and in which designation of whether each fragment ion is adopted or not can be accepted, and the sequence coverage; a designation receiving step of receiving a designation by a user as to whether or not to adopt each fragment ion in the displayed fragment list; a coverage display change step of changing the sequence coverage displayed on the display unit to a sequence coverage that reflects only fragment ions designated as adopted, in accordance with a user's designation of adoption / non-adoption in the designation receiving step; A mass spectrometry data analysis method comprising:

2. The mass spectrometry data analysis method according to claim 1, wherein the analysis result display step displays a pair of spectra, in which an actually measured fragment spectrum in a predetermined mass-to-charge ratio range including the mass-to-charge ratio of one fragment ion selected in the displayed fragment list and a theoretical isotope spectrum theoretically calculated from the chemical formula of the fragment ion, are arranged in a comparable manner, on the same display screen as the fragment list and the sequence coverage.

4. The mass spectrometry data analysis method according to claim 3, wherein the paired spectra are obtained by arranging an actually measured fragment spectrum and a theoretical isotope spectrum in line symmetry with a common mass-to-charge ratio axis. 　 The method further includes an adjacent isotope detection step of detecting an isotope peak adjacent to an isotope peak having the maximum signal intensity among the isotope peak group for each fragment ion in the fragment spectrum, 2. The mass spectrometry data analysis method of claim 1, wherein, in the analysis result display step, if the adjacent isotope peak is detectable, information indicating the position of the adjacent isotope peak in the group of isotope peaks is displayed within the fragment list or separately from the fragment list. 　 The method for analyzing mass spectrometry data according to claim 1 , wherein the compound is an oligonucleic acid. 　 a measurement unit that performs mass analysis involving fragmentation on a target compound having a sequence structure in which multiple known types of substances are linked together to obtain data constituting a fragment spectrum; a fragment estimation unit that estimates fragment ions derived from the target compound corresponding to peaks observed in the fragment spectrum; a coverage generation unit that generates a sequence coverage in which the fragment ions estimated by the fragment estimation unit are associated with the sequence structure of the target compound; a display unit capable of displaying an image; an analysis result display processing unit that displays a fragment list in which the fragment ions estimated by the fragment estimation unit are displayed as a list and can accept designation of adoption / non-adoption for each fragment ion, and the sequence coverage on the same display screen of the display unit; a designation receiving unit that receives a designation by a user as to whether or not to adopt each fragment ion in the fragment list displayed on the display unit; wherein the coverage creation unit creates a sequence coverage that reflects only the fragment ions designated as adopted when a user designates whether or not to adopt a fragment ion via the designation receiving unit. 　 The method further includes a theoretical isotope spectrum calculation unit that calculates a theoretical isotope spectrum from the chemical formula of the fragment ions, 7. The mass spectrometer according to claim 6, wherein the analysis result display processing unit displays a pair spectrum in which the measured fragment spectrum and the theoretical isotope spectrum in a predetermined mass-to-charge ratio range including the mass-to-charge ratio of one fragment ion selected in the displayed fragment list are arranged so as to be comparable, on the same display screen as the fragment list and the sequence coverage.

8. The mass spectrometer according to claim 7, wherein the paired spectra are obtained by arranging an actually measured fragment spectrum and a theoretical isotope spectrum in line symmetry with respect to a common mass-to-charge ratio axis. 　 an adjacent isotope detection unit that detects an isotope peak adjacent to an isotope peak having the maximum signal intensity among the isotope peak group for each fragment ion in the fragment spectrum; The mass spectrometer of claim 6, wherein the analysis result display processing unit displays, when the adjacent isotope peak is detectable, information indicating the position of the adjacent isotope peak in the group of isotope peaks within the fragment list or separately from the fragment list. 　 The mass spectrometer according to claim 6 , wherein the compound is an oligonucleic acid.

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