Sugar chain structure analysis device and sugar chain structure analysis program

Abstract

In the sugar chain structure analysis device of one mode of the present invention, one of the multiple tags used to set the analysis conditions is specifically used for setting the conditions for the specific chemical modification of the sialic acid connection mode, so that the user can select the main chemical modification reagent. In the case of selecting these reagents, the mass change of each connection mode is automatically set. In addition, when using chemical modifications other than predetermined chemical modifications, the user can input the mass change of each connection mode in numerical form. When analyzing, the theoretical m / z of multiple candidate sugar chain structures corresponding to a chemical modification and having different m / z values ​​according to the connection mode and the m / z of the measured ion peak are compared at one time. Thus, when the sugar chain structure analysis including the connection mode of sialic acid is performed by chemical modification using a sialic acid connection mode specific chemical modification reagent, the user's operation effort accompanying the analysis can be reduced and the efficiency of the analysis can be improved.

Description

Technical Field

[0001] The present invention relates to a sugar chain structure analysis device for analyzing sugar chain structure by mass spectrometry, and a sugar chain structure analysis program for analyzing sugar chain structure on a computer, and more specifically, to a sugar chain structure analysis device capable of analyzing sugar chain structure including the connection mode of sialic acid connected to the sugar chain, and a sugar chain structure analysis program. It should be noted that the "sugar chain" referred to here includes not only sugar chains existing alone, but also sugar chains in a state of modifying biological molecules such as proteins, peptides, lipids, and nucleic acids, that is, sugar chain modifications. Background Art

[0002] The biosynthesis process of biomolecules such as proteins and peptides in living organisms is finely regulated. Therefore, it is increasingly believed that biomolecules modified with sugar chains generated through the biosynthesis process play an important role in life activities. In recent years, there have been a large number of detailed reports on the sugar chain molecular level, especially regarding the relationship with physiological functions and diseases. Based on this situation, it is expected that the clarification of the structure of sugar chains that modify biomolecules involved in various processes in life phenomena will contribute to the elucidation of life phenomena, new drug development and diagnosis.

[0003] Sialic acid, one of the sugars, is considered to be an important substance for protein quality control, information transmission in nerves, and recognition between cells. Recently, it has been gradually clarified that the way sialic acid residues are attached to the end of sugar chains, that is, the difference in the attachment mode, is important for the above-mentioned biological behaviors. Therefore, understanding the attachment mode of sialic acid to sugar chains is one of the important operations in sugar chain structure analysis.

[0004] For example, it is known that in the case of humans, the main types of sialic acid linkage are α2,3 linkage and α2,6 linkage. It is known that the linkage mode changes as cells become cancerous, and it is expected that the linkage mode can be used as a biomarker or used for quality management of biopharmaceuticals. However, there is no difference in the mass of sugar chain isomers containing sialic acid that differs only in the linkage mode, so it is difficult to distinguish the linkage mode using a mass spectrometry device widely used for sugar chain analysis. In addition, sialic acid is generally easy to dissociate from the sugar chain and is unstable, so there are problems such as low detection sensitivity and poor quantification. Therefore, it is urgently desired to develop a method that can quickly and accurately analyze the linkage mode of sialic acid.

[0005] Against the background of the above situation, in order to stabilize the structure of sialic acid and to distinguish the difference in the connection mode using a high-throughput mass spectrometry device, a sialic acid connection mode-specific chemical modification method has been developed. For example, Patent Document 1 and Non-Patent Document 1 disclose the following method: a sample containing a sugar chain is reacted with an amine such as isopropylamine and a dehydration condensation agent including carbodiimide, thereby generating a lactone from a sugar chain modification having α2,3-sialic acid and generating an amide from a sugar chain modification having α2,6-sialic acid. In addition, several other chemical modification methods are also described in the above documents.

[0006] When analyzing the composition and structure of the sugar chain after being treated by the above-mentioned sialic acid connection mode specific chemical modification method, it is necessary to perform the following operation: using matrix-assisted laser desorption ionization (MALDI)-time of flight mass spectrometry analysis device (TOFMS) and the like to implement mass spectrometry, and using a computer to analyze the data collected by mass spectrometry. In the past, it was known that there were several computer software for obtaining the sugar chain composition and its structure from the data obtained by mass spectrometry. As representative software, there are "Glyco-peakfinder" (refer to non-patent literature 2), "SimGlycan" (registered trademark) (refer to non-patent literature 3), etc.

[0007] "Glyco-peakfinder" is a software that exhaustively investigates the composition of sugar chains that match the mass within a specified tolerance for various combinations of monosaccharides, based on the mass-to-charge ratio of the peak (hereinafter referred to as "ion peak") on the mass spectrum obtained in the actual measurement. On the other hand, "SimGlycan" is a software that searches the database constructed by the manufacturer of the software to find the composition and structure of the sugar chain corresponding to the mass-to-charge ratio of the peak on the MS / MS spectrum obtained in the actual measurement.

[0008] The above-mentioned existing sugar chain analysis software can also analyze sugar chain derivatives including sugar chain modifications. However, these software are not intended to be used for the analysis of sugar chain modifications obtained by sialic acid connection mode specific chemical modification methods. Therefore, for example, when performing an exhaustive search or using a certain sialic acid connection mode specific chemical modification reagent, it is necessary to search for a variety of different modifications generated due to the difference in the connection mode of sialic acid as a variety of monosaccharide residues with different masses. For N kinds of sialic acids, assuming that there are two connection modes, the user himself needs to investigate and add 2×N sialic acid modifications as monosaccharide residues of the search object. Usually, the software sets an upper limit on the number of monosaccharide residues that the user can independently add as the search object, and it may not be possible to search for the combination of all the required types of sialic acids, which becomes a problem. At this time, in order to increase the search object, it is necessary to change the search conditions to perform multiple searches, which will consume a lot of effort and time. In addition, the additional input setting of the modified body is not only time-consuming, but also has the problem of easily causing wrong settings.

[0009] Prior art literature

[0010] Patent Literature

[0011] Patent Document 1: Japanese Patent Application Publication No. 2016-194500

[0012] Non-patent literature

[0013] Non-patent document 1: Takashi Nishikaze and five others, "Differentiation of Sialyl Linkage Isomers by One-Pot Sialic Acid Derivatization for Mass Spectrometry-Based Glycan Profiling", Analytical Chemistry, 2017, Vol. 89, pp. 2353-2360

[0014] Non-patent document 2: K. Maass and four others, "Glyco-peakfinder - De novo composition analysis of glycoconjugates", Proteomics, 2007, Vol. 7, No. 24, pp. 4435-4444

[0015] Non-patent document 3: A. Apte et al., “Bioinformatics in Glycomics: Glycan Characterization with Mass Spectrometric Data Using SimGlycan”, Functional Glycomic, Methods in Molecular Biology, Humana Press, 2010, pp. 269-281 Summary of the invention

[0016] Problem that the invention aims to solve

[0017] The present invention has been made to solve the above-mentioned problems, and its main object is to provide a sugar chain structure analysis device and a sugar chain structure analysis program for a computer, which can reduce the time required by the user to set complicated search conditions when analyzing sugar chain structures including the linkage form of sialic acid based on mass spectrum data of a sample containing sugar chains that have undergone sialic acid linkage form-specific chemical modification, and can efficiently perform analysis.

[0018] Solutions for solving problems

[0019] The sugar chain structure analysis device of the first embodiment of the present invention, which was made to solve the above-mentioned problems, is a sugar chain structure analysis device that analyzes the structure of a sugar chain linked to sialic acid based on mass spectrometry data obtained by mass spectrometry analysis of a sample, wherein the sample has been pretreated with a sialic acid linking form-specific chemical modification reagent and contains a sugar chain linked to sialic acid or a molecule modified with the sugar chain linked to sialic acid.

[0020] The sugar chain structure analysis device comprises:

[0021] A condition setting section for allowing the user to set the ion species to be analyzed, the polarity of the charge of the ion species, and the mass change factor of the sugar chain mass change caused by modification;

[0022] a sugar chain core structure information acquisition unit that acquires various sugar chains having a core structure composed of a plurality of types and a plurality of monosaccharide residues and mass information corresponding thereto;

[0023] a theoretical mass calculation unit for increasing or decreasing the mass of various sugar chains acquired by the sugar chain core structure information acquisition unit based on the mass change factor set in the condition setting unit, thereby obtaining a sugar chain theoretical mass-to-charge ratio; and

[0024] a sugar chain structure estimating unit that compares the theoretical mass-to-charge ratio of the sugar chain with the mass-to-charge ratio of the ion peak in the provided mass spectrum data to estimate the sugar chain structure,

[0025] The condition setting unit includes a sialic acid attachment mode-specific chemical modification corresponding mass change setting unit, wherein the sialic acid attachment mode-specific chemical modification corresponding mass change setting unit allows the user to set the sialic acid attachment mode-specific chemical modification as one of the mass change factors independently of other modifications.

[0026] The theoretical mass calculation unit obtains a theoretical mass-to-charge ratio of the sugar chain obtained by increasing or decreasing the mass for each sialic acid linkage form in accordance with the chemical modification set in the sialic acid linkage form-specific chemical modification-responsive mass change setting unit.

[0027] In addition, the sugar chain structure analysis program of the first embodiment of the present invention, which is made to solve the above-mentioned problems, is a computer program for realizing the functions of the sugar chain structure analysis device of the above-mentioned first embodiment, and is a sugar chain structure analysis program for analyzing the structure of a sugar chain linked to sialic acid based on mass spectrum data obtained by mass spectrometry analysis of a sample, wherein the sample is pretreated with a sialic acid linkage form-specific chemical modification reagent and contains a sugar chain linked to sialic acid or a molecule modified with the sugar chain linked to sialic acid, and the sugar chain structure analysis program causes a computer to operate as the following functional unit:

[0028] A condition setting function section for allowing the user to set the ion species to be analyzed, the polarity of the charge of the ion species, and the mass change factor of the sugar chain mass change caused by modification;

[0029] A sugar chain core structure information acquisition function unit that acquires various sugar chains having a core structure composed of multiple types and a plurality of monosaccharide residues and mass information corresponding thereto;

[0030] a theoretical mass calculation function section for increasing or decreasing the mass of various sugar chains acquired by the sugar chain core structure information acquisition function section based on the mass change factor set by the condition setting function section, thereby obtaining a sugar chain theoretical mass-to-charge ratio; and

[0031] a sugar chain structure estimation functional unit that compares the theoretical mass-to-charge ratio of the sugar chain with the mass-to-charge ratio of the ion peak in the provided mass spectrum data to estimate the sugar chain structure;

[0032] The condition setting function section allows the user to set the sialic acid attachment mode-specific chemical modification, i.e., sialic acid attachment mode-specific chemical modification, as one of the mass change factors, independently of other modifications.

[0033] The theoretical mass calculation function unit obtains the theoretical mass-to-charge ratio of the sugar chain by increasing or decreasing the mass for each sialic acid linkage form according to the set sialic acid linkage form-specific chemical modification.

[0034] It should be noted that the sugar chain structure analysis program of the first embodiment is a program that enables a general personal computer, a computer with higher performance, or a special computer installed in various systems to work. The above program can be stored in a non-temporary storage medium such as a CD-ROM, a DVD-ROM, a memory card, a USB memory (encryption dongle) and provided to the user. Alternatively, it can also be provided to the user in the form of data transmission via a communication line such as the Internet. Of course, when the user first purchases the system, the sugar chain structure analysis program of the above embodiment can also be pre-integrated into the computer included in the system.

[0035] In the present invention, the molecules modified with sugar chains linked to sialic acid are, for example, biomolecules such as proteins, peptides, lipids, and nucleic acids modified with sugar chains linked to sialic acid. In addition, the pretreatment using a sialic acid linkage-specific chemical modification reagent, which is a prerequisite in the present invention, is, for example, the pretreatment disclosed in Patent Document 1 and Non-Patent Document 1, but is not limited thereto, and for example, specific chemical modification for identifying at least two or more different sialic acid linkages among α2,3 linkage, α2,6 linkage, and α2,8 linkage may be performed.

[0036] In addition, the type and form of the mass spectrometer used for mass spectrometry analysis of samples containing sugar chains linked to sialic acid are not particularly limited, and for example, an ion capture mass spectrometer, a linear ion capture mass spectrometer, a TOF / TOF mass spectrometer, a quadrupole-time of flight (Q-TOF) mass spectrometer, a quadrupole-ion capture mass spectrometer, a Fourier transform ion cyclotron resonance mass spectrometer, etc. can be used.

[0037] In the sugar chain structure analysis device of the first embodiment of the present invention, the condition setting unit displays a window for setting the prescribed conditions on the screen of the display unit, for example, and receives information input or selected in the window. At this time, the user can set the ion species as the object of analysis, the polarity of the charge of the ion species, and the mass change factor as the search conditions for sugar chain structure analysis, and in particular, the user can set the connection mode specific chemical modification of sialic acid as one of the mass change factors independently of other modifications. That is, as a modification to the sugar chain, the modification of the reducing end for fluorescent labeling, etc. is generally performed, but in addition, information about the connection mode specific chemical modification can also be set.

[0038] Specifically, for example, the mass change factor based on the ligation mode-specific chemical modification can be set in an independent window or tab different from the main setting screen. In addition, the mass change factor based on the ligation mode-specific chemical modification can be set in one of the display areas divided into a plurality of display areas arranged in one window.

[0039] At the beginning of the analysis, first, the sugar chain core structure information acquisition unit acquires various sugar chains having a core structure composed of multiple monosaccharide residues and the corresponding mass information. In order to obtain information on sugar chains having a core structure, a method of exhaustively finding theoretically possible sugar chains and a method of finding them by database search using an existing sugar chain structure database are specifically considered.

[0040] That is, one embodiment of the sugar chain structure analysis device of the present invention can be configured as follows: the sugar chain core structure information acquisition unit exhaustively infers the hypothesized sugar chain structure under the condition of limiting the types and numbers of monosaccharide residues, and obtains various sugar chains having a core structure and their corresponding mass information.

[0041] As another implementation, the following configuration may be adopted:

[0042] It also has a sugar chain structure database that stores various sugar chain structures and masses.

[0043] The sugar chain core structure information acquisition unit searches the information stored in the sugar chain structure database based on the sugar chain mass information set by the user, thereby obtaining various sugar chains having a core structure.

[0044] The sugar chains obtained by the sugar chain core structure information acquisition unit are sugar chains in a state of no modification, no substitution, no specific substance addition during ionization, etc. In contrast, the theoretical mass calculation function unit increases or decreases the mass based on the mass change factor set by the condition setting unit, thereby calculating the theoretical mass-to-charge ratio of the sugar chain when it is detected as an ion by mass spectrometry. At this time, in the case where the chemical modification is set in the sialic acid linkage mode-specific chemical modification corresponding mass change setting unit, the theoretical mass-to-charge ratio of the sugar chain is calculated by increasing or decreasing the mass differently for each sialic acid linkage mode in accordance with the chemical modification. For example, if it is a chemical modification that can distinguish between α2,3 linkage type and α2,6 linkage type, the mass of the sugar chain increases differently according to these two linkage modes, and in the case where N sialic acids are contained in one core sugar chain structure, N+1 (= N+2 C N ) different theoretical mass-to-charge ratios of sugar chains.

[0045] The sugar chain structure estimation function compares the above-calculated sugar chain theoretical mass-to-charge ratio with the mass-to-charge ratio of the ion peak in the provided mass spectrum data. Then, for each ion peak, the deviation of the mass-to-charge ratio falls within the specified allowable range as the candidate sugar chain corresponding to the ion peak. As described above, for sugar chains with the same original core structure, the mass-to-charge ratios of two or more modified sugar chain ions with different theoretical mass-to-charge ratios of sugar chains due to differences in sialic acid attachment methods are obtained, so the correspondence between the two or more sugar chain ions and the ion peaks can be obtained at one time.

[0046] In addition, the sugar chain structure analysis device of the present invention may have the following configuration: preferably, it further comprises a display processing unit that displays the information of the sugar chain structure estimated by the sugar chain structure estimating unit on a display unit,

[0047] When the estimated sugar chain structure contains sialic acid and the linkage form can be identified, the display processing unit further displays information indicating the linkage form for each sialic acid.

[0048] Specifically, when the chemical modification of α2,3-linked and α2,6-linked types can be distinguished, text information, symbols, etc. indicating "α2,3-" and "α2,6-" can be displayed in the sugar chain composition. This allows users to understand the difference in sialic acid linkage at a glance.

[0049] It should be noted that the sugar chain structure analysis device of the present invention may be implemented as follows: the sialic acid linkage form-specific chemical modification corresponding mass change setting unit can select a predetermined sialic acid linkage form-specific chemical modification, and can input a mass change value for each sialic acid linkage form for other chemical modifications.

[0050] Thus, when using a predetermined sialic acid attachment mode-specific chemical modification that is frequently used, the user can select the sialic acid attachment mode-specific chemical modification by a simple selection operation. In addition, when using a sialic acid attachment mode-specific chemical modification that is less commonly used or has not been used before, the sugar chain structure corresponding to such chemical modification can be estimated by directly inputting the mass change value of each sialic acid attachment mode.

[0051] Effects of the Invention

[0052] According to the present invention, in the sugar chain structure analysis using sialic acid linkage mode-specific chemical modification, by setting one sialic acid linkage mode-specific chemical modification and one analysis process, the structures of multiple sugar chains (sugar chain modifications) with different mass-to-charge ratios due to the difference in the linkage mode of sialic acid contained in one sugar chain can be searched exactly. Thus, the time required for setting cumbersome search conditions and the like in the sugar chain structure analysis including the linkage mode of sialic acid by the user can be reduced. In addition, the time required for the analysis process can be shortened and the analysis throughput can be improved.

[0053] In addition, according to the present invention, when using the main sialic acid attachment mode specific chemical modification reagent, the user only needs to select the user from a plurality of options prepared in advance, and the user no longer needs to investigate and manually input the amount of mass change, etc. Therefore, the operation time can be reduced, and the operation errors caused by manual input can be suppressed. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 This is a schematic block diagram of a sugar chain structure analysis system as one embodiment of the present invention.

[0055] Figure 2 This is a flowchart of the analysis processing steps of the sugar chain structure analysis system of this embodiment.

[0056] Figure 3 This is a diagram showing an example of a label for the user to set a sialic acid linkage-specific chemical modification in the sugar chain structure analysis system of this example.

[0057] Figure 4 It is a diagram showing a display example of the sugar chain structure estimation result of the sugar chain structure analysis system of this example.

[0058] Figure 5 This is a diagram showing an example of the mass-to-charge ratio value of the ion peak to be analyzed.

[0059] Figure 6 This is a diagram showing an example of search conditions set by the user.

[0060] Figure 7 This is a diagram showing an example of constraint conditions when exhaustively determining the core sugar chain structure. DETAILED DESCRIPTION

[0061] First, a sugar chain structure analysis system as one embodiment of the present invention will be described with reference to the drawings.

[0062] Figure 1 : is a schematic block diagram of the sugar chain structure analysis system of this embodiment. Figure 1 As shown, the system comprises: a mass spectrometer analysis unit 1 for performing measurement on a sample, a data analysis unit 2 for performing analysis processing, and an input unit 3 and a display unit 4 as user interfaces. The data analysis unit 2 comprises a data storage unit 21, a peak detection unit 22, and a sugar chain structure analysis unit 23, and the sugar chain structure analysis unit 23 comprises an analysis condition setting unit 24, a sugar chain core structure mass calculation unit 25, a sugar chain theoretical mass-to-charge ratio calculation unit 26, a sugar chain structure estimation unit 27, and an estimation result display processing unit 28 as functional modules. In addition, the analysis condition setting unit 24 comprises a sialic acid connection mode specific chemical modification condition setting unit 241 as a sub-functional module, and the sugar chain theoretical mass-to-charge ratio calculation unit 26 comprises a sialic acid connection mode specific chemical modification corresponding mass change calculation unit 261 as a sub-functional module.

[0063] The method of the mass spectrometry unit 1 is not particularly limited, and high mass accuracy and high mass resolution are usually desired, so a time-of-flight mass spectrometry device (TOFMS) or a Fourier transform ion cyclotron resonance mass spectrometry device (FT-ICRMS) is useful. In addition, as an ionization method of the mass spectrometry device, in addition to the matrix-assisted laser desorption ionization (MALDI) method, an electrospray ionization (ESI) method, a probe electrospray ionization (PESI) method, etc. can also be used. From the perspective of mainly generating monovalent ions, the MALDI method is preferred.

[0064] In addition, as described later, for the analysis of sugar chain structure including the number and linkage pattern of sialic acid modified sugar chains, conventional mass spectrometry without ion dissociation operation is usually sufficient. For example, when analyzing the structure of peptides with sugar chains, MS analysis is usually performed when n is 2 or more. n Analyze and acquire MS n In this case, as the mass spectrometer 1, a mass spectrometer having a function of dissociating ions by collision induced dissociation (CID) or the like such as ion trapping and a collision cell is used.

[0065] In addition, the mass spectrometry analysis unit 1 may use a liquid chromatography-mass spectrometry device (LC-MS) instead of a mass spectrometry device alone, or may be configured as follows: the eluate obtained by component separation by liquid chromatography is fractionated and graded to prepare multiple samples, and the multiple samples are separately subjected to mass spectrometry analysis using a mass spectrometry device.

[0066] In this system, the entity of the data analysis unit 2 is a general-purpose personal computer or a workstation with higher performance. By making a dedicated data processing program installed in such a computer run in the computer, it is possible to realize Figure 1 The functions of each functional module shown in the figure. The data processing program is equivalent to the sugar chain structure analysis program of the present invention. In this case, the input unit 3 is a keyboard and a pointing device (mouse, etc.) attached to the computer, and the display unit 4 is a monitor attached to the same computer. The above-mentioned data processing program can be stored in a non-temporary storage medium such as a CD-ROM, a DVD-ROM, a memory card, a USB memory (encryption dongle) and provided to the user. Alternatively, it can also be provided to the user in the form of data transmission via a communication line such as the Internet.

[0067] When the sugar chain structure analysis is performed by the system of this embodiment, a pre-treatment using a sialic acid connection mode-specific chemical modification is performed on a sample containing sugar chains (sugar chain sample), and a mass spectrometry analysis is performed on the treated sample by a mass spectrometry analysis unit 1, thereby obtaining mass spectrum data of the entire specified mass-to-charge ratio range. Here, as an example of a sialic acid connection mode-specific chemical modification, as described in patent document 1 and non-patent literature, a reaction of the sugar chain sample is performed in the presence of an amine and a dehydration condensation agent including carbodiimide. In this case, when the sialic acid contained in the sugar chain is an α2,3 connection type, as a modified body, a lactone is formed, and when the sialic acid contained in the sugar chain is an α2,6 connection type, as a modified body, an amide is formed, and even if the composition of the original sugar chain is the same, the mass of the modified body is different from each other. The mass spectrum data obtained by the mass spectrometry analysis unit 1 of the sample treated with the chemical modification described above is input to the data analysis unit 2 and stored in the data storage unit 21.

[0068] In the data analysis unit 2, the peak detection unit 22 detects peaks based on a predetermined algorithm for the collected mass spectrum data, obtains the mass-to-charge ratio and signal intensity of each peak, and creates a peak list. Then, the created peak list is temporarily stored in the data storage unit 21. The peak list becomes the analysis target data of the sugar chain structure analysis unit 23.

[0069] When performing sugar chain structure analysis, the user performs a predetermined operation from the input unit 3, and upon receiving the operation, the analysis condition setting unit 24 displays a main window on the screen of the display unit 4. The main window is composed of a plurality of switchable tabs. Figure 3 1 is a diagram showing the main window 50 in which the "sialic acid modification" setting tab 52 as one of the tabs is opened. In addition to the "sialic acid modification" setting tab 52, a "data" setting tab, a "residue" setting tab, a "label" setting tab, a "ion species" setting tab, a "salt formation" setting tab, and a result display ( Figure 3 In the tab switching area 51, the tabs can be switched easily by clicking any tab.

[0070] The result display tab is a tab for displaying the sugar chain structure analysis results, and the other tabs are tabs for setting analysis conditions and the like before analysis is performed, or for instructing the execution of analysis.

[0071] Briefly describe each tag.

[0072] The "Data" setting tab is a tab for the user to select a peak list for analysis and input values ​​such as the mass tolerance of the measurement data. This input determines the tolerance when comparing the mass-to-charge ratio of the measured ion peak with the theoretical mass-to-charge ratio of the sugar chain described later.

[0073] The "residue" setting tag is a tag for users to set the types of sugars and the conditions for the number of each type when estimating the sugar chain composition. In the "residue" setting tag, for example, as the type of sugar, you can set the selection of monosaccharides containing sialic acid such as hexose, N-acetyl hexosamine, deoxyhexose, N-acetyl neuraminic acid, N-glycolyl neuraminic acid, KDN (2-keto-3-deoxy-D-glycero-D-galacto-nononic acid, 2-keto-3-deoxy-D-glycero-D-galacto-nonone acid), pentose, etc., and the selection of phosphorus or sulfur as other molecules, as well as their number conditions. It should be noted that when the number is set to 0, it means that the sugar or molecule is not contained.

[0074] The "label" setting tab is a tab for the user to select the type of modification mainly by labeling such as fluorescent labeling. In the "label" setting tab, for example, 2-aminobenzamide, aminopyridine, etc. can be selected as the labeling substance.

[0075] The "Ion Species" setting tab is a tab for users to select the polarity, valence, and ion species of the ion when the sugar chain is ionized. In the "Label" setting tab, as the ion species, positive ions can be selected to have protons (H + ), adding sodium ions (Na + ), adding potassium ions (K + ), etc., negative ions can choose protons (H - ) separation, addition of chloride ions (Cl - ), add phosphate ions (H2PO4 - )wait.

[0076] The "Salt Formation" setting tab is a tab for the user to set a replacement of a partial structure of a sugar chain without a change in valence. In the "Salt Formation" setting tab, you can choose to replace protons with sodium, potassium, or lithium, and you can also specify the upper limit of the number of replacements.

[0077] The sialic acid-linked form-specific chemical modification condition setting unit 241 displays Figure 3 The "sialic acid modification" setting label 52 shown is a label for the user to select the type of sialic acid attachment mode specific chemical modification. In the "sialic acid modification" setting label 52, as shown in FIG. Figure 3 As shown, in the setting object area 53, you can use the radio button to select No modification ( Figure 3 ), 5 predetermined sialic acid attachment mode-specific chemical modifications, and undefined sialic acid attachment mode-specific chemical modifications ( Figure 3 The five predetermined sialic acid attachment-specific chemical modifications are respectively determined by the mass change amount ( Figure 3 The chemical formula changes are known and correspond to two connection modes, namely α2,3 connection and α2,6 connection. Thus, users can easily select relatively common chemical modifications such as isopropylamine.

[0078] In addition, when using undefined chemical modification reagents other than the specified chemical modification, the user can select "Other modification" and enter the mass change amount based on the chemical formula change corresponding to α2,3 connection and α2,6 connection in numerical form. This allows analysis with simple operations when using new or uncommon chemical modification reagents not envisioned here. It should be noted that when the user selects Figure 3 When the "Default" button 54 is clicked in the "Sialic Acid Modification" setting tab 52 shown, the information in the setting target area 53 is restored to the default state registered in the pre-specified file.

[0079] In addition, when the user clicks the "Save Settings" button 57 in the "Sialic Acid Modification" setting tab 52, the information set in the setting target area 53 is saved in a pre-specified setting information saving file. On the other hand, when the user clicks the "Download Settings" button 56, the information saved in the setting information saving file is automatically set in the setting target area 53. Thus, for example, analysis can be performed under the same conditions as the analysis performed previously by a simple operation.

[0080] As described above, the user appropriately sets the analysis conditions in each tab of the main window 50 (step S1 ). Figure 5 This is a diagram showing an example of a peak list set in the “Data” setting tab. Figure 6 This is an example of setting each item when isopropylamine is used as a sialic acid-linked specific chemical modification reagent. Figure 7 This is an example of the sugar chain search condition set on the "Residue" setting tab. Figure 7 For example, for hexose, it means that the sugar chain containing at least 3 and at most 15 hexoses is searched exhaustively. Also, for sialic acid, it means that the sugar chain containing at most 5 N-acetylneuraminic acids or no N-acetylneuraminic acid is searched exhaustively.

[0081] When the user clicks the "Analyze" button 55 in the "Sialic Acid Modification" setting tab 52 (or other setting tabs), upon receiving the operation, the sugar chain structure analysis unit 23 actually starts the analysis process under the set analysis conditions.

[0082] That is, first, the sugar chain core structure mass calculation unit 25 obtains all hypothetical sugar chain compositions by exhaustively combining the sugar chain search conditions set in the "residue" setting tag. The sugar chain composition does not take into account the sugar chain structure, such as the number of branches, etc. Then, for each hypothetical sugar chain composition, the theoretical mass of the monosaccharide, etc. is added to calculate the theoretical mass of the sugar chain (step S2). The theoretical mass obtained here is the theoretical mass of the sugar chain in a state without modification, etc. and before ionization.

[0083] In the sugar chain theoretical mass-to-charge ratio calculation unit 26, for each sugar chain with different composition, the theoretical mass-to-charge ratio is calculated when ionization is performed with the ion species set as the above-mentioned search condition, and modification or substitution is performed as the above-mentioned search condition (step S3). Figure 6 When isopropylamine is used as a sialic acid attachment mode-specific chemical modification reagent as shown in the setting example, the mass of α2,3-sialic acid increases by 13.0316 and the mass of α2,6-sialic acid increases by 41.0629. Therefore, in the case of a sugar chain containing n α2,3-sialic acids, the mass-to-charge ratio of the sugar chain ion increases by 13.0316×n. On the other hand, in the case of a sugar chain containing n α2,6-sialic acids, the mass-to-charge ratio of the sugar chain ion increases by 41.0629×n. The same is true for tag modification and substitution. Therefore, multiple theoretical mass-to-charge ratios resulting from differences in modification and substitution are calculated for a sugar chain with the same sugar chain composition. That is, when the sugar chain contains at least one sialic acid, the theoretical mass-to-charge ratio reflects the difference in the attachment mode of the sialic acid.

[0084] Next, the sugar chain structure estimation unit 27 compares the mass-to-charge ratio (measured value) of the ion peaks listed in the provided peak list with the theoretical mass-to-charge ratio (theoretical value) of each sugar chain calculated in step S3 in sequence. That is, for each ion peak, the deviation between the measured value and the theoretical value of the mass-to-charge ratio is calculated (step S4), and the sugar chain whose deviation falls within the pre-set allowable error range is extracted as the candidate sugar chain structure corresponding to the ion peak (step S5). Usually, the number of sugar chains (modified bodies) estimated according to theory is very large, so usually one ion peak will be associated with multiple candidate sugar chain structures. In the case where the sugar chain contains sialic acid, the sugar chain structure corresponding to the sugar chain that shows different theoretical mass-to-charge ratios from each other contains information on the connection mode of sialic acid. That is, it has information indicating whether the sialic acid is α2,3-connected or α2,6-connected.

[0085] For each ion peak, one or more candidate sugar chain structures are extracted, and information on the deviation of the mass-to-charge ratio is associated with each candidate sugar chain structure. Here, the estimated result display processing unit 28 creates a screen of a list of candidate sugar chain structures including information on the connection mode of sialic acid for each ion peak, and displays the list of candidate sugar chain structures on the result display tab when the user opens the tab (step S6).

[0086] Figure 4 This is the result of sugar chain structure estimation for sugar chain ions when sialic acid-linked specific chemical modification is performed using isopropylamine. Figure 5 to Figure 7 Sugar chain structure estimation results under the indicated conditions.

[0087] For example, Figure 4 The first candidate sugar chain composition corresponding to the ion peak of m / z 2471.88 contains "Neu5Ac(a2,3)", and is therefore a sugar chain having one α2,3-sialic acid. In addition, the first candidate sugar chain composition corresponding to the ion peak of m / z 2499.92 contains both "Neu5Ac(a2,3)" and "Neu5Ac(a2,6)", and is therefore a sugar chain having one α2,6-sialic acid and one α2,3-sialic acid. In this way, when sialic acid is included in the sugar chain composition, its connection mode is displayed as information, so that the user can obtain not only the number of sialic acids contained in the sugar chain composition, but also the connection mode information of each sialic acid.

[0088] In summary, in order to understand the sialic acid connection mode, the data analysis unit 2 of the sugar chain structure analysis system of this embodiment can obtain not only the number of sialic acids contained in the sugar chain but also the connection mode information of sialic acids by simply selecting the chemical modification reagent used in the pretreatment as one of the search conditions.

[0089] It should be noted that the sugar chain structure analysis system of the above embodiment exhaustively infers the sugar chain structure with the core structure under the restrictions of the types and numbers of sugars, etc. set in advance, but it is also possible to use a pre-made sugar chain structure database to find the sugar chain structure with the core structure. In this case, various sugar chain structures and the corresponding mass information are pre-stored in the sugar chain structure database, and the user sets the range of sugar chain mass as the search condition instead of setting the conditions for inferring the sugar chain composition. The sugar chain core structure mass calculation unit 25 performs database search based on the set mass as the condition and infers the candidate sugar chain structure that meets the condition.

[0090] The above-described embodiment is merely an example of the present invention, and in addition to the above-described modified examples, modifications, corrections, additions, etc. appropriately made within the spirit and scope of the present invention are naturally also included in the scope of the claims of the present application.

[0091] Description of Reference Numerals

[0092] 1…Mass Spectrometry Department

[0093] 2…Data Analysis Department

[0094] 21…Data storage

[0095] 22…Peak detection unit

[0096] 23…Sugar Chain Structure Analysis Department

[0097] 24…Analysis condition setting section

[0098] 241…Sialic acid attachment mode specific chemical modification condition setting section

[0099] 25… Sugar chain core structure mass calculation department

[0100] 26…Sugar chain theoretical mass-to-charge ratio calculation section

[0101] 261…Calculation of mass change corresponding to sialic acid attachment-specific chemical modification

[0102] 27…Sugar chain structure estimation section

[0103] 28…Estimation result display processing unit

[0104] 3…Input

[0105] 4…Display

[0106] 50…Main window

[0107] 51…Tag switching area

[0108] 52…“Sialic acid modification” setting label

[0109] 53…Set the target area

[0110] 54…“Default” button

[0111] 55…Analysis button

[0112] 56…Download Settings button

[0113] 57…“Save Settings” button

Claims

1. A sugar chain structure analysis device, characterized in that: The invention relates to a sugar chain structure analysis device for analyzing the structure of sugar chains linked to sialic acid based on mass spectrometry data obtained by mass spectrometry analysis of a sample, wherein the sample is pre-treated with a sialic acid linking mode-specific chemical modification reagent that produces a different mass change depending on the sialic acid linking mode and contains sugar chains linked to sialic acid or molecules modified by the sugar chains. The sugar chain structure analysis device comprises: A condition setting section for allowing the user to set the ion species to be analyzed, the polarity of the charge of the ion species, and the mass change factor of the sugar chain mass change caused by modification; a sugar chain core structure information acquisition unit that acquires various sugar chains having a core structure composed of a plurality of monosaccharide residues of a plurality of types and mass information corresponding thereto, wherein the sugar chains are sugar chains in an unmodified and pre-ionized state; a theoretical mass calculation unit, which increases or decreases the mass of various sugar chains acquired by the sugar chain core structure information acquisition unit based on the mass change factor set in the condition setting unit, thereby obtaining a sugar chain theoretical mass-to-charge ratio, wherein different mass change values ​​are added to or subtracted from the mass of the sugar chain based on the mass change factor set in the condition setting unit; and a sugar chain structure estimating unit that compares the sugar chain theoretical mass-to-charge ratio with the mass-to-charge ratio of the ion peak in the provided mass spectrum data to estimate the sugar chain structure, The condition setting unit includes a sialic acid attachment mode-specific chemical modification corresponding mass change setting unit, wherein the sialic acid attachment mode-specific chemical modification corresponding mass change setting unit allows a user to set a sialic acid attachment mode-specific chemical modification as one of the mass change factors that produces different mass changes according to the sialic acid attachment mode independently of other modifications. The theoretical mass calculation unit calculates a theoretical mass-to-charge ratio of the sugar chain obtained by increasing or decreasing the mass of each sialic acid linkage mode in accordance with the chemical modification set in the sialic acid linkage mode-specific chemical modification corresponding mass change setting unit, wherein different mass change values ​​are added or subtracted from the mass of each sialic acid linkage mode in accordance with the chemical modification set in the sialic acid linkage mode-specific chemical modification corresponding mass change setting unit.

2. The sugar chain structure analysis device according to claim 1, characterized in that The sugar chain core structure information acquisition unit exhaustively estimates hypothetical sugar chain structures under conditions that restrict the types and numbers of monosaccharide residues, and obtains various sugar chains having a core structure and mass information corresponding thereto.

3. The sugar chain structure analysis device according to claim 1, characterized in that It also has a sugar chain structure database that stores various sugar chain structures and masses. The sugar chain core structure information acquisition unit searches the information stored in the sugar chain structure database based on the sugar chain mass information set by the user, thereby obtaining various sugar chains having a core structure.

4. The sugar chain structure analysis device according to claim 1, characterized in that further comprising a display processing unit configured to display information on the sugar chain structure estimated by the sugar chain structure estimating unit on a display unit, When the estimated sugar chain structure contains sialic acid and the linkage form can be identified, the display processing unit further displays information indicating the linkage form for each sialic acid.

5. The sugar chain structure analysis device according to claim 1, characterized in that The sialic acid attachment form-specific chemical modification-corresponding mass change setting unit can select a predetermined sialic acid attachment form-specific chemical modification, and can input a mass change value for each sialic acid attachment form for other chemical modifications.

6. A storage medium storing a sugar chain structure analysis program, which is a computer-readable non-transitory storage medium, characterized in that: The sugar chain structure analysis program is used to analyze the structure of a sugar chain linked to sialic acid based on mass spectrometry data obtained by mass spectrometry analysis of a sample using a computer, wherein the sample is pretreated with a sialic acid linkage mode-specific chemical modification reagent that produces a different mass change depending on the sialic acid linkage mode and contains a sugar chain linked to sialic acid or a molecule modified by the sugar chain, and the sugar chain structure analysis program causes the computer to operate as the following functional unit: A condition setting function section for allowing the user to set the ion species to be analyzed, the polarity of the charge of the ion species, and the mass change factor of the sugar chain mass change caused by modification; A sugar chain core structure information acquisition function unit that acquires various sugar chains having a core structure composed of a plurality of monosaccharide residues of a plurality of types and mass information corresponding thereto, wherein the sugar chains are sugar chains in an unmodified and pre-ionized state; a theoretical mass calculation function section, which increases or decreases the mass of various sugar chains acquired by the sugar chain core structure information acquisition function section based on the mass change factor set by the condition setting function section, thereby obtaining a sugar chain theoretical mass-to-charge ratio, wherein different mass change values ​​are added to or subtracted from the mass of the sugar chain based on the mass change factor set in the condition setting function section; and a sugar chain structure estimation functional unit that compares the theoretical mass-to-charge ratio of the sugar chain with the mass-to-charge ratio of the ion peak in the provided mass spectrum data to estimate the sugar chain structure, The condition setting function section allows the user to set a sialic acid attachment mode-specific chemical modification as one of the mass change factors that produces different mass changes depending on the sialic acid attachment mode, independently of other modifications. The theoretical mass calculation function unit calculates the theoretical mass-to-charge ratio of the sugar chain obtained by increasing or decreasing the mass of each sialic acid linkage mode corresponding to the set sialic acid linkage mode-specific chemical modification, wherein different mass change values ​​are added or subtracted from the mass of each sialic acid linkage mode corresponding to the set sialic acid linkage mode-specific chemical modification.

7. The storage medium storing a sugar chain structure analysis program according to claim 6, wherein: The sugar chain core structure information acquisition function unit exhaustively estimates hypothetical sugar chain structures under conditions that restrict the types and numbers of monosaccharide residues, and obtains various sugar chains having a core structure and the corresponding mass information.

8. The storage medium storing a sugar chain structure analysis program according to claim 6, wherein: The sugar chain core structure information acquisition function unit searches for information stored in a sugar chain structure database storing various sugar chain structures and masses based on sugar chain mass information set by a user, thereby obtaining various sugar chains having a core structure.

9. The storage medium storing a sugar chain structure analysis program according to claim 6, wherein: further comprising a display processing function unit for displaying information on the sugar chain structure estimated by the sugar chain structure estimating function unit on a screen of a display unit, When the estimated sugar chain structure contains sialic acid and the linkage form can be identified, the display processing function unit further displays information indicating the linkage form for each sialic acid.

10. The storage medium storing a sugar chain structure analysis program according to claim 6, wherein: The condition setting function unit can select a predetermined sialic acid attachment form-specific chemical modification, and can input a mass change value for each sialic acid attachment form for other chemical modifications.

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