Sugar chain mass spectrometry data analysis device and method, medium

Abstract

A sugar chain mass spectrometry data analysis device, method, and medium. It is determined whether a sialic acid-containing sugar chain is derivatized as intended. The sugar chain mass spectrometry data analysis device includes a sugar chain information acquisition unit that acquires, based on a sugar chain composition, a plurality of sugar chains having a core structure composed of a plurality of monosaccharide residues of a plurality of types and mass information corresponding thereto; a mass variation factor setting unit that sets a mass variation factor that causes a mass variation of the sugar chain; and a sugar chain theoretical mass calculation unit that calculates, based on the mass variation factor, a sugar chain theoretical mass-to-charge ratio of each sugar chain after the mass variation has occurred. The mass variation factor setting unit sets an ion species or salt that can be generated on a carboxyl group of a sialic acid of a sialic acid-containing sugar chain included in an analysis target and a sialic acid linking method that can cause a mass variation corresponding to a derivatization process. The sugar chain theoretical mass calculation unit estimates a mass variation amount according to a combination of the ion species or salt and the sialic acid linking method.

Description

TECHNICAL FIELD

[0001] The present application relates to a sugar chain mass spectrometry data analysis device and a sugar chain mass spectrometry data analysis program. BACKGROUND

[0002] The biosynthesis process of biological molecules such as proteins and peptides in living organisms is precisely controlled. Therefore, it is considered that biological molecules modified with sugar chains generated in the process of biosynthesis play an important role in life activities, and in recent years, a large number of specific reports on the sugar chain molecule level have been made particularly in relation to physiological functions or diseases. As a result, it is expected that elucidation of the structure of sugar chains that modify biological molecules related to various processes in life phenomena will be helpful for elucidation of life phenomena, development of new drugs, and diagnosis.

[0003] Sialic acid, which is one of sugars, is considered to be an important substance in quality management of proteins, information transmission in nerves, or recognition between cells, but recently the following view is being elucidated: the difference in the attachment mode, i.e., the linkage mode, of sialic acid residues to the end of a sugar chain is important for such biological activities. Therefore, it is important to grasp the linkage mode of sialic acid contained in the sugar chain that modifies biological molecules for understanding the function of sugar chains in life activities.

[0004] For example, in the case of humans, as the linkage mode of sialic acid, α2,3 linkage and α2,6 linkage are mainly known. It is known that the linkage mode changes with the cancerization of cells, and it is proposed to use the linkage mode as a biomarker or for quality management of biological medicines and the like. However, since sugar chain isomers containing only sialic acid different in linkage mode are not different in mass, it is difficult to discriminate the linkage mode by mass spectrometry, which is widely used for sugar chain analysis. In addition, since sialic acid is unstable and easily dissociates from a sugar chain at the time of mass spectrometry or its pretreatment, the detection sensitivity and quantitativeness are low.

[0005] Therefore, in order to stabilize the structure of sialic acid and enable discrimination of the difference in linkage mode from the results of mass spectrometry, a method of derivatizing a sugar chain specific to the linkage mode of sialic acid has been developed. For example, in Patent Literature 1, Non-Patent Literature 1, a method is disclosed in which a lactone is generated as a derivative of a sugar chain having α2,3-sialic acid and an amide is generated as a derivative of a sugar chain having α2,6-sialic acid by reacting an analysis target sample containing a sugar chain with a dehydration condensing agent containing an amine such as isopropylamine and a carbodiimide. In addition, several other derivatization methods are also described in the above-mentioned literature.

[0006] The sample containing the sialic acid-containing sugar chain obtained by processing using the derivatization method specific to the sialic acid linkage mode described above is analyzed using a matrix-assisted laser desorption ionization (MALDI)-time-of-flight type mass spectrometry analyzer (TOF MS) or the like, and the data collected thereby are analyzed by a computer, whereby the composition and structure of the sialic acid-containing sugar chain can be determined. For example, a computer program for analyzing the structure of a sugar chain from data obtained by mass spectrometry of a sugar chain is described in Patent Literature 2.

[0007] As for the sialic acid-containing sugar chain obtained by processing using the derivatization method specific to the sialic acid linkage mode, peaks (hereinafter referred to as "ion peaks") are detected on the mass spectrum in the vicinity of the theoretical mass-to-charge ratio of the sugar chain after an increase or decrease in mass corresponding to each sialic acid linkage mode due to derivatization. For example, if it is a derivatization method that can distinguish between α2,3 and α2,6 binding types, the mass of the sugar chain increases by an amount corresponding to the mass that is different for each of the two linkage modes. Thus, in a case where N sialic acids are contained in one sugar chain structure, N+1 (= N+1 C N ) ion peaks of different mass-to-charge ratios can be detected.

[0008] In the computer program described in Patent Literature 2, the structure of the sialic acid-containing sugar chain obtained by processing using the derivatization method specific to the sialic acid linkage mode is determined by comparing the mass-to-charge ratios of the ion peaks in the mass spectrometry data obtained for the sample containing the sialic acid-containing sugar chain with a plurality of theoretical mass-to-charge ratios of sugar chains that are set in advance corresponding to the derivatization method.

[0009] Prior Art Documents

[0010] Patent Literature

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

[0012] Patent Literature 2: International Publication No. WO2020 / 079878

[0013] Non-Patent Literature

[0014] Non-Patent Literature 1: N. de Haan et al., Glycomics studies using sialic acid derivatization and mass spectrometry, Nature Reviews Chemistry, 2020, 4(5), 229-242 SUMMARY

[0015] Problem to be solved by the invention

[0016] In the above-described computer program, a plurality of sugar chain theoretical mass-to-charge ratios are set on the premise that all of the sialic acids contained in the sialic acid-containing sugar chains are derivatized in a prescribed linkage, and the sugar chain theoretical mass-to-charge ratios are compared with the mass-to-charge ratios of the ion peaks in the mass spectrometric data. In addition, depending on the conditions under which the derivatization method specific to the sialic acid linkage is performed, a part of the sialic acid-containing sugar chains contained in the sample sometimes remains in a state in which the sialic acids in the prescribed linkage are not derivatized and remain as they are, or in a state in which the derivatization of the sialic acids in the prescribed linkage is incomplete. Such sialic acid-containing sugar chains exist as impurities in the sample. The impurities present in the sample can interfere with the analysis of the sugar chain structure.

[0017] The present invention was made to solve the above-described problem, and aims to enable the determination of whether or not the sialic acid-containing sugar chains obtained by performing the derivatization treatment specific to the sialic acid linkage are derivatized as intended.

[0018] Solution to the problem

[0019] The sugar chain mass spectrometric data analysis device according to the first aspect of the present invention for analyzing mass spectrometric data obtained by performing mass spectrometry on a sample containing a sialic acid-linked sugar chain or a molecule modified by the sugar chain, which is obtained by performing a derivatization treatment specific to the sialic acid linkage, includes:

[0020] a sugar chain composition setting section for setting the composition of the sugar chain to be analyzed;

[0021] a sugar chain information acquisition section for acquiring a plurality of sugar chains having a core structure composed of a plurality of monosaccharide residues of a plurality of types and mass information corresponding thereto, based on the composition of the sugar chain set by the sugar chain composition setting section;

[0022] a mass variation factor setting section for setting a mass variation factor that causes a mass variation of the sugar chain; and

[0023] a sugar chain theoretical mass calculation section for calculating a sugar chain theoretical mass-to-charge ratio after the mass variation for each of the sugar chains acquired by the sugar chain information acquisition section, based on the mass variation factor set by the mass variation factor setting section,

[0024] wherein the mass variation factor setting section sets, as the mass variation factor, an ion species or a salt that can be generated on a carboxyl group of a sialic acid possessed by the sialic acid-linked sugar chain contained in the sample to be analyzed, and a sialic acid linkage that can be subjected to a mass variation corresponding to the derivatization treatment,

[0025] The sugar chain theoretical mass calculation section has a mass change calculation section that calculates a mass change amount estimated from the combination of the ion species and the sialic acid linkage mode or the combination of the salt and the sialic acid linkage mode.

[0026] In addition, a second aspect of the present application, which has been achieved in order to solve the above-described problems, is a sugar chain mass spectrometry data analysis program for analyzing mass spectrometry data obtained by performing mass spectrometry on a sample containing a sugar chain to which sialic acid is attached or a molecule modified by the sugar chain, which has been subjected to a derivatization treatment specific to a sialic acid linkage mode, the sugar chain mass spectrometry data analysis program being for causing a computer to function as:

[0027] a sugar chain composition setting section for setting a composition of a sugar chain that is an analysis target;

[0028] a sugar chain information acquisition section that acquires a plurality of sugar chains having a core structure composed of a plurality of monosaccharide residues of a plurality of types and mass information corresponding thereto, based on the composition of the sugar chain set by the sugar chain composition setting section;

[0029] a mass change factor setting section for setting a mass change factor that causes a mass change of the sugar chain; and

[0030] a sugar chain theoretical mass calculation section that calculates a sugar chain theoretical mass-to-charge ratio after the mass change has occurred, for each sugar chain acquired by the sugar chain information acquisition section, based on the mass change factor set by the mass change factor setting section,

[0031] wherein the mass change factor setting section sets, as the mass change factor, an ion species or a salt that can be generated on a carboxyl group of sialic acid possessed by the sugar chain to which sialic acid is attached contained in the analysis target, and a sialic acid linkage mode that can undergo a mass change corresponding to the derivatization treatment,

[0032] the sugar chain theoretical mass calculation section has a mass change calculation section that calculates a mass change amount estimated from the combination of the ion species and the sialic acid linkage mode or the combination of the salt and the sialic acid linkage mode.

[0033] Effects of the Invention

[0034] According to the present application, in the case where the sugar chain as an analysis target is subjected to a derivatization treatment specific to the mode of attachment of sialic acid in order to perform structural analysis of a sugar chain having a sialic acid attached thereto, in addition to being able to provide the user with a plurality of sugar chains (sugar chain derivatives) having different mass-to-charge ratios corresponding to differences in the mode of attachment of sialic acid, which are generated by derivatization of sialic acid, it is also possible to provide the user with information on the mass-to-charge ratios of impurities expected to be generated due to incomplete derivatization of sialic acid or non-occurrence of derivatization. Thus, the user can judge whether or not the processing conditions such as temperature, pH, and the like in the derivatization treatment are appropriate by collating the information on the mass-to-charge ratios provided with the mass spectrometric analysis data (mass spectrum data). BRIEF DESCRIPTION OF DRAWINGS

[0035] Figure 1 is a schematic structural block diagram of a sugar chain mass spectrometric analysis data analysis system as one embodiment of the present application.

[0036] Figure 2 is a flowchart of the analysis processing procedure of the sugar chain mass spectrometric analysis data analysis system of the present embodiment.

[0037] Figure 3 is a diagram showing an example of a label for the user to set the kind of sialic acid attachment mode-specific derivatization in the sugar chain mass spectrometric analysis data analysis system of the present embodiment.

[0038] Figure 4 is a diagram showing an example of a label for the user to set the composition of the sugar chain as an analysis target in the sugar chain mass spectrometric analysis data analysis system of the present embodiment.

[0039] Figure 5 is a diagram showing an example of a label for the user to set the ionization conditions in the sugar chain mass spectrometric analysis data analysis system of the present embodiment.

[0040] Figure 6 is a diagram showing an example of a label for the user to set the salt formation conditions in the sugar chain mass spectrometric analysis data analysis system of the present embodiment.

[0041] Figure 7 is a diagram showing an example of the mass-to-charge ratio value of the ion peak set as an analysis target.

[0042] Figure 8 is a diagram showing an example of a mass spectrum obtained for a sample containing an analysis target. DETAILED DESCRIPTION

[0043] Hereinafter, a sugar chain structure analysis system as one embodiment of the present application will be described with reference to the accompanying drawings.

[0044] Figure 1is a schematic configuration block diagram of the sugar chain structure analysis system of the present embodiment. As shown in Figure 1 the system is provided with a mass spectrometry analysis section 1 that performs measurement on a sample, a data analysis section 2 that performs analysis processing, and an input section 3 and a display section 4 as a user interface. The data analysis section 2 includes a data storage section 21, a peak detection section 22, and a sugar chain structure analysis section 23. The sugar chain structure analysis section 23 includes a sugar chain composition setting section 24, a sugar chain information acquisition section 25, a mass variation factor setting section 26, a sugar chain theoretical mass calculation section 27, a data collation section 28, and a display processing section 29 as functional blocks. Further, the mass variation factor setting section 26 includes a reducing terminal labeling condition setting section 261, a derivatization condition setting section 262, an ionization condition setting section 263, and a salt formation condition setting section 264 as lower-level functional blocks, and the sugar chain theoretical mass calculation section 27 includes a mass variation calculation section 271 as a lower-level functional block.

[0045] The mass spectrometry analysis section 1 is not particularly limited in its mode, but it is generally desirable that the mass accuracy and mass resolution be high, and therefore a time-of-flight type mass spectrometry analysis device (TOFMS) or a Fourier transform ion cyclotron resonance mass spectrometry analysis device (FT-ICRMS) or the like is useful. In addition, as the ionization method of the mass spectrometry analysis device, in addition to being able to use a matrix-assisted laser desorption ionization (MALDI) method, an electrospray ionization (ESI) method, a probe electrospray ionization (PESI) method, or the like can be used, but based on the point that a monovalent ion is mainly produced, the MALDI method is preferable.

[0046] In addition, as described later, in the sugar chain structure analysis including the number and the linking mode of the sialic acid that modifies the sugar chain, in most cases, a general mass spectrometry analysis by a cleavage operation without ions is sufficient, but in order to perform structure analysis on a sugar chain that is bound to a peptide or the like, a MSn analysis with n being 2 or more is generally performed to acquire MSn spectrum data. In this case, as the mass spectrometry analysis section 1, a mass spectrometry analysis device having a function of causing ions to cleave by collision-induced dissociation (CID) or the like, such as an ion trap, a collision cell, or the like, is used. n n In this case, as the mass spectrometry analysis section 1, a mass spectrometry analysis device having a function of causing ions to cleave by collision-induced dissociation (CID) or the like, such as an ion trap, a collision cell, or the like, is used.

[0047] In the present system, the entity of the data analysis section 2 is a general personal computer or a higher-performance workstation, and by causing a dedicated data processing program installed on such a computer to operate in the computer, the functions as described above are realized. Figure 1 ​The functions of each of the illustrated functional blocks. This data processing program corresponds to the sugar chain structure analyzing program to which the present application pertains. In this case, the input section 3 is a keyboard, a pointing device (mouse or the like) attached to a computer, and the display section 4 is a monitor also attached to the computer. The above-described data processing program can be provided to the user incorporated in a non-transitory recording medium such as a CD-ROM, a DVD-ROM, a memory card, a USB memory (dongle), or the like. Alternatively, it can also be provided to the user in the form of data transmission via a communication line such as the Internet.

[0048] In performing sugar chain structure analysis by the system of the present embodiment, a sample containing a sugar chain (sugar chain sample) is subjected to pretreatment based on sialic acid linkage mode-specific derivatization, and mass spectrometric analysis is performed on the pretreated sample by the mass spectrometric analysis section 1 to obtain mass spectrometric data over a prescribed mass-to-charge ratio range. Here, as an example, the sugar chain sample is subjected to sialic acid linkage mode-specific derivatization in the presence of a dehydration condensing agent containing an amine and a carbodiimide as described in Patent Document 1, Non-Patent Document. In this case, in the case where the sialic acid contained in the sugar chain is of the a2,3 binding type, a lactone is formed as a derivative, and in the case where the sialic acid contained in the sugar chain is of the a2,6 binding type, an amide is formed as a derivative. Even if the composition of the original sugar chain is the same, the mass of the derivative is different. The mass spectrometric data obtained by the mass spectrometric analysis section 1 with respect to the pretreated sample based on derivatization as described above is input to the data analysis section 2 and saved to the data storage section 21.

[0049] In the data analysis section 2, the peak detection section 22 detects peaks with respect to the collected mass spectrometric data in accordance with a prescribed algorithm, obtains the mass-to-charge ratio values and signal intensities of each peak, and creates a peak list. Then, the created peak list is temporarily saved in the data storage section 21. This peak list becomes the data that is the analysis target of the sugar chain structure analysis section 23.

[0050] In performing sugar chain structure analysis, if the user performs a prescribed operation from the input section 3, the sugar chain structure analysis section 23 displays a main window on the screen of the display section 4 after accepting the operation. The main window is composed of a plurality of tabs that can be switched. Figure 3 is a diagram showing the main window 50 in a state where the "Sialic Acid Modification" setting tab 52, which is one of the tabs, is open. In addition to this "Sialic Acid Modification" setting tab 52, a "Data" setting tab 53, a "Residue" setting tab 54, a "Labelling" setting tab 55, an "Ion Species" setting tab 56, a "Salt Formation" setting tab 57, a result display (in this case, a peak list) 58, and a "Sialic Acid Modification" setting window 59 are prepared. Figure 3The tab 58 is a "Results" tab, and the tabs can be switched simply by clicking any tab in the tab switching area 51.

[0051] The tabs will be described below.

[0052] The "Results" tab 58 is a tab for displaying the analysis results of the sugar chain structure, and the other tabs are tabs for setting conditions for analysis or the like before analysis is performed or for instructing analysis to be performed.

[0053] The "Data" setting tab 53 is a tab for the user to select a peak list to be analyzed and to input values such as a mass tolerance of measurement data. By this input, a tolerance when the mass-to-charge ratio of an ion peak actually measured is compared with a theoretical mass-to-charge ratio of a sugar chain, which will be described later, is determined.

[0054] The "Residue" setting tab 54 is a tab for the user to set the composition of a sugar chain to be analyzed, and is displayed by the sugar chain composition setting section 24. Figure 4 is a view showing a setting target area 541 displayed in the main window 50 by opening the "Residue" setting tab 54. With respect to the "Residue" setting tab 54, in the setting target area 541, for example, as the types of sugars, the names of monosaccharides (Hexose, N-Acetylhexosamine, Deoxyhexose, N-Acetyl neuraminic acid, N-Glycolyl neuraminic acid, KDN (2-keto-3-deoxy-D-glycero-D-galacto-nononic acid), Pentose) and their abbreviations, and the masses thereof, and the names of molecules that increase the mass of a sugar chain by modification (Phosphate, Sulfate, Acetate) and their abbreviations, and the masses thereof are displayed, and the number thereof can be set. Further, in a case where the number is set to 0, it means that the sugar or molecule is not contained. Thus, by inputting only the number of the types of sugars contained in a sugar chain to be analyzed and the like, the user can easily set the composition of a sugar chain.

[0055] In addition, in a case where a sugar not displayed in the setting target area 541 is contained in a sugar chain to be analyzed, the user inputs the name, the mass of the residue, and the number thereof. Thus, even in a case where a sugar that is not so common is contained, the composition of a sugar chain can be set by an easy operation.

[0056] Further, in Figure 4In the figure, as a sugar chain component, it is shown to be configured to contain 5 hexoses, 4 N-acetylglucosamines, and 2 N-acetylneuraminic acids.

[0057] The “Marking” setting label 55 is a label used by the user to select the type of modification mainly performed by labeling, such as fluorescent marking. Regarding the “Marking” setting label 55, in its setting target area (not shown), for example, 2-aminobenzamide or pyridylamine can be selected as the marking substance.

[0058] The “Sialic Acid Modification” setting label 52 is a label used by the user to select the type of sialic acid linkage-specific derivatization, and is displayed by the derivatization condition setting unit 262. Figure 3 The main window 50 is shown with the "Sialic Acid Modification" setting tab 52 open. Regarding the "Sialic Acid Modification" setting tab 52, radio buttons can be used to select from five predefined sialic acid linkage specific derivatization methods and undefined sialic acid linkage specific derivatization methods in the setting object area 521. Figure 3 One of the "Other modifications" in the text. For specific derivatization of five given sialic acid linkages, the mass change based on the changes in the known chemical formulas corresponding to the two linkages, namely α2,3 binding and α2,6 binding, was defined. Figure 3 The "Mass Change" option allows users to easily select derivatizations, such as combinations of isopropylamidation and methylamidation, intended for high-frequency use in publications. Furthermore, in... Figure 3 In the example, as a specific derivatization of sialic acid linkage, a state in which a combination of isopropylamidation and methylamidation is selected is shown.

[0059] Furthermore, when using undefined derivatizing reagents other than those already defined, after selecting "Other Modifications," the user inputs the mass change based on the changes in the chemical formulas corresponding to α2,3 binding and α2,6 binding, respectively. This allows for easy analysis even when using new or less common derivatizing reagents not previously considered. Additionally, when the user... Figure 3 When the "Default" button 522 is clicked on the "Sialic Acid Modification" setting label 52 shown in the figure, the system returns to the default state where the information in the setting object area 521 is registered to a pre-specified file.

[0060] Furthermore, when the user clicks the "Save Settings" button 523 on the "Sialic Acid Modification" setting label 52, the information set in the setting target area 521 at that time is saved to a pre-specified setting information saving file. On the other hand, when the user clicks the "Load Settings" button 524, the information saved in the setting information saving file is automatically set in the setting target area 521. Thus, for example, it is possible to perform parsing under the same conditions as the parsing implemented in the past with a simple operation.

[0061] The "Ion Type" setting label 56 is a label used by the user to select the polarity, valence, and ion type of the ion when the sugar chain is ionized, and is displayed by the ionization condition setting unit 263. Figure 5 The image shows the setting object area 561 displayed in the main window 50 when the "Ion Type" setting tab 56 is open. Regarding the "Ion Type" setting tab 56, in the setting object area 561, as the ion type, positive ions can be selected as protons (H+). + ) addition, sodium ions (Na + ) addition, potassium ions (K + ) addition, negative ions can selectively combine with protons (H) - ) Desorption, chloride ions (Cl - ) addition, phosphate ions (H2PO4) - ) addition, hydrogen sulfate ions (HSO4) - ) addition, hexafluorophosphate ion (PF6) - One of the additive methods. In Figure 5 The text shows the selection of sodium ions (Na+). + The state of addition. In addition, in the setting object area 561, the user can select the name of ion type other than those mentioned above by inputting the name of the ion type and its mass.

[0062] The “Salt Formation” setting label 57 is a label used by the user to set the substitution of a portion of the sugar chain structure without a change in valence, and is displayed by the salt formation condition setting unit 264. Figure 6 The setting area 571 displayed in the main window 50 is shown when the "Salt Formation" setting tab 57 is open. In this setting area 571, it is possible to select whether to replace protons with sodium, potassium, or lithium. Figure 6 The diagram shows the state in which a proton is replaced by a sodium atom.

[0063] Next, the process of parsing glycan mass spectrometry analysis data using this system will be explained.

[0064] The user sets the analysis conditions in the tabs of the main window 50 as described above (step 1).

[0065] When the setting operation of the analysis conditions is completed, and the user clicks the "Analyze" button 525 on the "Sialic acid modification" setting tab 52 (or another setting tab), the operation is accepted, and the sugar chain structure analysis section 23 actually starts the analysis process under the set analysis conditions.

[0066] That is, first, the sugar chain information acquisition section 25 calculates the theoretical mass of the sugar chain based on the kinds and numbers of sugars or the like indicating the sugar chain composition set in the setting target area 541 of the "Residue" setting tab 54 (step 2). In addition, the sugar chain information acquisition section 25 estimates the combination of sialic acid linkage modes from the kinds and numbers of sugars or the like indicating the sugar chain composition, and calculates a plurality of sugar chain structures. For example, for an N-type sugar chain (hereinafter referred to as A2 glycan) having two branches containing two sialic acids and all sialic acid linkage modes, if a sugar chain composed of five hexoses, four N-acetylhexosamines, and two N-acetylneuraminic acids is set as the object sugar chain of sialic acid linkage mode-specific derivatization, the theoretical mass of the sugar chain is calculated by adding the theoretical mass of each of the hexoses, the N-acetylhexosamines, and the N-acetylneuraminic acids by the number of each.

[0067] Next, the sugar chain theoretical mass calculation section 27 calculates the theoretical mass-to-charge ratio when the reducing end marker, derivatization, ionization, or salt formation or the like set as the mass variation factor is performed for each sugar chain having a different structure (step 3).

[0068] For example, as the labeling condition of the reducing end of the sugar chain, labeling based on 2-aminobenzamide (AB labeling, +120.0687 Da) is selected. In addition, as other mass variation factors, the contents shown in the setting target areas of Figure 3 , Figure 5 , and Figure 6 are set. That is, as the sialic acid linkage mode-specific derivatization reagent, a derivatization reagent that methylamidates α2,3-bound sialic acid and isopropylamidates α2,6-bound sialic acid is selected, the polarity of ionization is set to positive, and sodium ion (Na + ) addition is selected as the ion species. Furthermore, in the case where the polarity of ionization is selected to be negative, regardless of the selected ion species, an anion is generated due to the loss of a proton from the carboxyl group of the sialic acid remaining due to incomplete derivatization or non-derivatization.

[0069] In addition, if a sodium salt is selected as the salt formation condition, a part of the carboxyl group of the sialic acid remaining due to incomplete derivatization or non-derivatization can form a sodium salt. The substitution of a proton to sodium is assumed to be selected.

[0070] The mass change calculation section 271 assumes four states for each of the carboxyl groups of the two sialic acids contained in the A2 glycan, namely, an unreacted state, a state substituted to a sodium salt, a state subjected to methyl amidation in the case of an a2,3-bound sialic acid, and a state subjected to isopropyl amidation in the case of an a2,6-bound sialic acid, and calculates the mass-to-charge ratio of each state, based on the mass change factor set as described above. Specifically, there are one kind of unreacted sugar chain (i.e., the sugar chain as the starting material), three kinds of the sugar chain as the target object subjected to complete derivatization, and six kinds of the sugar chain (incomplete reactant) generated due to incomplete derivatization, for a total of ten kinds (= 1 + 3 + 6) of the sugar chain. 2+4-1 C 4-1 ) of the theoretical mass-to-charge ratio of the sugar chain is calculated. A list of the theoretical mass-to-charge ratios calculated in the case of the monovalent ion is shown in Table 1. Figure 7

[0071] Next, the data comparison section 28 compares the mass-to-charge ratio values (measured values) of the ion peaks listed in the given peak list with the theoretical mass-to-charge ratios (theoretical values) calculated in Step 3, one by one. Here, the deviation of the measured value from the theoretical value of the mass-to-charge ratio value is calculated for each ion peak (Step 4), and in the case where the deviation converges to a pre-set allowable error, the ion peak is determined as the sugar chain species corresponding to the theoretical value (Step 5).

[0072] Next, the display processing section 29 creates a screen for displaying the comparison result obtained by the data comparison section 28 and the mass spectrum obtained by adding the theoretical value of the determined sugar chain species to the vicinity of the corresponding ion peak, and displays the screen on the result display tab 58 when the user opens the tab (Step 6).

[0073] Figure 8 A schematic view of the mass spectrum obtained using a matrix-assisted laser desorption ionization mass spectrometry device (MALDI-MS) for the Na adduct ion of the AB-labeled A2 glycan generated by performing sialic acid linkage pattern-specific derivatization is shown in Table 2. As shown in Table 2, the theoretical value m / z is displayed in the vicinity of each ion peak. Among them, the ion peaks of m / z 2392, 2420, and 2448 are consistent with the m / z of the monovalent ion of the target object, and the ion peaks detected in the vicinity of these ion peaks are consistent with the m / z of the monovalent ion of the unreacted object and the incomplete reactant. In addition, the ion peaks of m / z 2394, 2422, and 2450 are consistent with the m / z of the monovalent ion of the sodium salt of the target object, and the ion peaks detected in the vicinity of these ion peaks are consistent with the m / z of the monovalent ion of the sodium salt of the unreacted object and the incomplete reactant. Figure 8 Figure 8 ​​In the present embodiment, in order to easily confirm that ion peaks of unreacted substances and incompletely reacted substances are generated in addition to ion peaks of the target substance, the ion peaks of the target substance are indicated by solid lines and the ion peaks of the unreacted substances and the incompletely reacted substances are indicated by dotted lines.

[0074] As described above, according to the sugar chain mass spectrometry data analysis system of the present embodiment, it is possible to easily confirm that unreacted substances and incompletely reacted substances are generated by the derivatization treatment performed in order to grasp the sialic acid linkage mode. Furthermore, in a case where the intensity of the ion peak corresponding to the unreacted substances and the incompletely reacted substances is a size that cannot be ignored with respect to the intensity of the ion peak of the target substance, it is possible to use this as a judgment material for reconsidering the conditions of the derivatization treatment.

[0075] [Various Modes]

[0076] As understood by those skilled in the art, the above-described embodiments are specific examples of the following modes.

[0077] (First Mode) One mode of the present application is a sugar chain mass spectrometry data analysis device for analyzing mass spectrometry data obtained by performing mass spectrometry on a sample containing a sugar chain to which a sialic acid is attached or a molecule modified by the sugar chain, which has been subjected to derivatization treatment specific to a sialic acid linkage mode, the sugar chain mass spectrometry data analysis device comprising:

[0078] a sugar chain composition setting section for setting a composition of a sugar chain that is an analysis target;

[0079] a sugar chain information acquisition section for acquiring, based on the composition of the sugar chain set by the sugar chain composition setting section, a plurality of sugar chains having a core structure composed of a plurality of monosaccharide residues of a plurality of types and mass information corresponding thereto;

[0080] a mass variation factor setting section for setting a mass variation factor that causes a mass variation of the sugar chain; and

[0081] a sugar chain theoretical mass calculation section for calculating, based on the mass variation factor set by the mass variation factor setting section, a sugar chain theoretical mass-to-charge ratio after the mass variation has occurred for each sugar chain acquired by the sugar chain information acquisition section,

[0082] wherein the mass variation factor setting section sets, as the mass variation factor, an ion species or a salt that can be generated on a carboxyl group of a sialic acid possessed by the sugar chain to which the sialic acid is attached contained in the analysis target, and a sialic acid linkage mode that can correspond to the derivatization treatment and cause a mass variation,

[0083] The sugar chain theoretical mass calculation section has a mass change calculation section that calculates a mass change amount estimated from the combination of the ion species and the sialic acid linkage mode or the combination of the salt and the sialic acid linkage mode.

[0084] (Third item) The second aspect of the present application is a sugar chain mass spectrometry data analysis program for analyzing mass spectrometry data obtained by performing mass spectrometry on a sample containing a sialic acid-linked sugar chain or a molecule modified by the sugar chain, which has been subjected to a derivatization treatment specific to a sialic acid linkage mode, the sugar chain mass spectrometry data analysis program causing a computer to function as:

[0085] a sugar chain composition setting section for setting a composition of a sugar chain that is an analysis target;

[0086] a sugar chain information acquisition section that acquires a plurality of sugar chains having a core structure composed of a plurality of monosaccharide residues of a plurality of types and mass information corresponding thereto, based on the composition of the sugar chain set by the sugar chain composition setting section;

[0087] a mass change factor setting section for setting a mass change factor that causes a mass change of a sugar chain; and

[0088] a sugar chain theoretical mass calculation section that calculates a sugar chain theoretical mass-to-charge ratio after the mass change has occurred, for each sugar chain acquired by the sugar chain information acquisition section, based on the mass change factor set by the mass change factor setting section,

[0089] wherein the mass change factor setting section sets, as the mass change factor, an ion species or a salt that can be generated on a carboxyl group of a sialic acid possessed by a sialic acid-linked sugar chain contained in the analysis target, and a sialic acid linkage mode that can undergo a mass change corresponding to the derivatization treatment,

[0090] the sugar chain theoretical mass calculation section has a mass change calculation section that calculates a mass change amount estimated from the combination of the ion species and the sialic acid linkage mode or the combination of the salt and the sialic acid linkage mode.

[0091] According to the present application, in the case where the sugar chain as an analysis target is subjected to a derivatization treatment specific to the mode of attachment of sialic acid in order to perform structural analysis of the sugar chain having a sialic acid attached thereto, in addition to being able to provide the user with a plurality of sugar chains (sugar chain derivatives) having different mass-to-charge ratios corresponding to differences in the mode of attachment of sialic acid, which are generated by derivatization of sialic acid, it is also possible to provide the user with information on the mass-to-charge ratios of impurities expected to be generated due to incomplete derivatization of sialic acid or non-occurrence of derivatization. Thus, the user can judge whether or not the processing conditions such as temperature and pH in the derivatization treatment are appropriate by comparing the information on the mass-to-charge ratios provided with the mass spectrometric analysis data (mass spectrum data).

[0092] (Second) In the sugar chain mass spectrometric analysis data analysis device of the first, further has:

[0093] a data comparison section that performs comparison of whether or not the ion of the sugar chain theoretical mass-to-charge ratio calculated by the sugar chain theoretical mass calculation section is included in the mass spectrometric data; and

[0094] a comparison result display section that displays the comparison result obtained by the data comparison section.

[0095] (Fourth) In the sugar chain mass spectrometric analysis data analysis program of the third, further causes the computer to function as:

[0096] a data comparison section that performs comparison of whether or not the ion of the sugar chain theoretical mass-to-charge ratio calculated by the sugar chain theoretical mass calculation section is included in the mass spectrometric data; and

[0097] a comparison result display section that displays the comparison result obtained by the data comparison section.

[0098] According to the sugar chain mass spectrometric analysis data analysis device of the second and the sugar chain mass spectrometric analysis data analysis program of the fourth, it is possible to easily confirm whether or not the sialic acid-attached sugar chain obtained by performing the derivatization treatment specific to the mode of attachment of sialic acid or the molecule modified by the sugar chain contains the sialic acid-attached sugar chain incompletely derivatized or the sialic acid-attached sugar chain not derivatized.

[0099] Explanation of Reference Signs

[0100] 1: mass spectrometry section; 2: data analysis section; 21: data storage section; 22: peak detection section; 23: sugar chain structure analysis section; 24: sugar chain composition setting section; 25: sugar chain information acquisition section; 26: mass variation factor setting section; 261: reducing end labeling condition setting section; 262: derivatization condition setting section; 263: ionization condition setting section; 264: salt formation condition setting section; 27: sugar chain theoretical mass calculation section; 271: mass variation calculation section; 28: data comparison section; 29: display processing section; 3: input section; 4: display section.

Claims

1. A sugar chain mass spectrometry data analysis device for analyzing mass spectrometry data obtained by performing mass spectrometry on a sample containing a sialic acid-linked sugar chain or a molecule modified by the sugar chain, which is obtained by performing a derivatization treatment specific to a sialic acid linkage mode, the sugar chain mass spectrometry data analysis device comprising: a sugar chain composition setting section for setting a composition of a sugar chain that is an analysis target; a sugar chain information acquisition section for acquiring, based on the composition of the sugar chain set by the sugar chain composition setting section, a plurality of sugar chains having a core structure composed of a plurality of monosaccharide residues of a plurality of types and mass information corresponding thereto; a mass change factor setting section for setting a mass change factor that causes a mass change of the sugar chain; and a sugar chain theoretical mass calculation section for calculating, based on the mass change factor set by the mass change factor setting section, a sugar chain theoretical mass-to-charge ratio after the mass change has occurred for each of the sugar chains acquired by the sugar chain information acquisition section, for a sugar chain that is completely derivatized by the derivatization treatment, a sugar chain that is not completely derivatized by the derivatization treatment, and a sugar chain that is not reacted to the derivatization treatment, respectively, wherein the mass change factor setting section sets, as the mass change factor, an ion species or a salt that can be generated on a carboxyl group of a sialic acid possessed by the sialic acid-linked sugar chain contained in the analysis target, and a sialic acid linkage mode that can cause a mass change corresponding to the derivatization treatment, the sugar chain theoretical mass calculation section has a mass change calculation section that calculates a mass change amount of the sugar chain that is completely derivatized by the derivatization treatment based on the sialic acid linkage mode, and calculates a mass change amount of the sugar chain that is not completely derivatized by the derivatization treatment based on a combination of the ion species and the sialic acid linkage mode or a combination of the salt and the sialic acid linkage mode. Further comprising: a data comparison section that performs a comparison of whether or not ions of the sugar chain theoretical mass-to-charge ratio calculated by the sugar chain theoretical mass calculation section are included in the mass spectrometry data; and a comparison result display section that displays a comparison result obtained by the data comparison section.

3. A medium recording a sugar chain mass spectrometry data analysis program, which is a non-transitory computer-readable medium, the sugar chain mass spectrometry data analysis program being for analyzing mass spectrometry data obtained by performing mass spectrometry on a sample containing a sialic acid-linked sugar chain or a molecule modified by the sugar chain, which is obtained by performing a derivatization treatment specific to a sialic acid linkage mode, the sugar chain mass spectrometry data analysis program being for causing a computer to execute the following steps: a sugar chain composition setting step of setting a composition of a sugar chain that is an analysis target; a sugar chain information acquisition step of acquiring, based on the composition of the sugar chain set by the sugar chain composition setting step, a plurality of sugar chains having a core structure composed of a plurality of monosaccharide residues of a plurality of types and mass information corresponding thereto; a mass change factor setting step of setting a mass change factor that causes a mass change of the sugar chain; and a sugar chain theoretical mass calculation step of calculating, based on the mass change factor set by the mass change factor setting step, a sugar chain theoretical mass-to-charge ratio after the mass change has occurred for each of the sugar chains acquired by the sugar chain information acquisition step, for a sugar chain that is completely derivatized by the derivatization treatment, a sugar chain that is not completely derivatized by the derivatization treatment, and a sugar chain that is not reacted to the derivatization treatment, respectively. ​ ​ ​ ​ ​ 2. The sugar chain mass spectrometric analysis data analysis device according to claim 1, characterized by, ​ ​ ​ ​ ​ ​ ​ ​ the sugar chain theoretical mass calculation step calculates, for each of the sugar chains obtained by the sugar chain information acquisition step, a sugar chain theoretical mass-to-charge ratio after the mass change, with respect to a sugar chain that is completely derivatized by the derivatization treatment, a sugar chain that is not completely derivatized by the derivatization treatment, and a sugar chain that is not reacted to the derivatization treatment, respectively, based on the mass change factor set by the mass change factor setting step, wherein, in the mass change factor setting step, an ion species or a salt that can be generated on a carboxyl group of a sialic acid possessed by a sialic acid-attached sugar chain contained in the analysis target, and a sialic acid attachment mode that can be changed in mass corresponding to the derivatization treatment, are set as the mass change factor, the sugar chain theoretical mass calculation step includes a mass change calculation step in which a mass change amount of a sugar chain that is completely derivatized by the derivatization treatment is calculated based on the sialic acid attachment mode, and a mass change amount of a sugar chain that is not completely derivatized by the derivatization treatment is calculated based on a combination of the ion species and the sialic acid attachment mode or a combination of the salt and the sialic acid attachment mode.

4. The medium having the sugar chain mass spectrometric analysis data analysis program recorded, according to Claim 3, wherein, the computer is further caused to execute the following steps: a data comparison step of comparing whether or not ions of the sugar chain theoretical mass-to-charge ratio calculated by the sugar chain theoretical mass calculation step are included in the mass spectrometry data; and a comparison result display step of displaying a comparison result obtained by the data comparison step.

5. A sugar chain mass spectrometry data analysis method for analyzing mass spectrometry data obtained by performing mass spectrometry on a sample containing a sialic acid-attached sugar chain or a molecule modified by the sugar chain obtained by performing a derivatization treatment specific to a sialic acid attachment mode, the sugar chain mass spectrometry data analysis method including the following steps: a sugar chain composition setting step of setting a composition of a sugar chain as an analysis target; a sugar chain information acquisition step of acquiring, based on the composition of the sugar chain set by the sugar chain composition setting step, a plurality of sugar chains having a core structure composed of a plurality of monosaccharide residues of a plurality of types and mass information corresponding thereto; a mass change factor setting step of setting a mass change factor of a mass change of a sugar chain; a sugar chain theoretical mass calculation step of calculating, for each of the sugar chains obtained by the sugar chain information acquisition step, a sugar chain theoretical mass-to-charge ratio after the mass change, with respect to a sugar chain that is completely derivatized by the derivatization treatment, a sugar chain that is not completely derivatized by the derivatization treatment, and a sugar chain that is not reacted to the derivatization treatment, respectively, based on the mass change factor set by the mass change factor setting step, wherein, in the mass change factor setting step, an ion species or a salt that can be generated on a carboxyl group of a sialic acid possessed by a sialic acid-attached sugar chain contained in the analysis target, and a sialic acid attachment mode that can be changed in mass corresponding to the derivatization treatment, are set as the mass change factor, ​ The sugar chain theoretical mass calculation step includes a mass variation calculation step in which a mass variation amount of a sugar chain that is completely derivatized by the derivatization treatment is calculated based on the sialic acid linkage pattern, and a mass variation amount of a sugar chain that is not completely derivatized by the derivatization treatment is calculated based on the combination of the ion species and the sialic acid linkage pattern or the combination of the salt and the sialic acid linkage pattern.

6. The method according to claim 5, wherein Further comprising the steps of: a data comparison step of comparing whether or not the ion of the sugar chain theoretical mass-to-charge ratio calculated by the sugar chain theoretical mass calculation step is included in the mass spectrometry data; and a comparison result display step of displaying the comparison result obtained by the data comparison step.

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