Sialic acid-containing sugar chain analysis method and sialic acid-containing sugar chain analysis device

Abstract

To improve the efficiency and increase the accuracy of analysis of a sialic acid-containing sugar chain.SOLUTION: Provided is a method for analyzing a sialic acid-containing sugar chain on the basis of the mass spectrum data of a sample that contains a sialic acid-containing sugar chain having been specifically modified to a sialic acid binding form. This analysis method includes: a step (S2) for detecting a representative peak from the mass spectrum data, for each isotope peak cluster; a step (S3) for detecting an isomer peak cluster that includes a plurality of ion peaks where sialic acid counts and sugar chain compositions other than sialic acid are estimated to be identical; a step (S4) for estimating a sugar chain composition with regard to the detected representative peak; a step (S5) for creating and displaying, on a display unit, a mass spectrum in which an annotation indicating the correspondence between peaks included in one cluster and the peaks observed in the mass spectrum is added; and a step (S6) for creating and displaying, on the display unit, a list of composition candidates in which the estimated sugar chain composition candidates are correlated to the isomer peak cluster.SELECTED DRAWING: Figure 2

Description

[Technical Field]

[0001] The present invention relates to a method and apparatus for analyzing glycans using mass spectrometry, and more specifically to an analytical method and apparatus that are capable of analyzing sialic acid-containing glycans, including the binding mode of sialic acid. Note that the term "glycan" as used herein includes not only glycans that exist independently, but also glycans that modify biomolecules such as proteins, peptides, lipids, and nucleic acids, i.e., modified glycans. [Background technology]

[0002] Glycan analysis is a major theme in fields such as life science, drug discovery, and medicine. In particular, understanding the linkage mode of sialic acid in sialic acid-containing glycans is an important task in glycan structural analysis. Against this background, methods for chemical modification specific to the linkage mode of sialic acid have been developed to efficiently perform structural analysis of sialic acid-containing glycans, including differences in linkage modes, using mass spectrometry. For example, Patent Document 1 and Non-Patent Document 1 disclose a sialic acid linkage-specific modification method known as the SALSA (Sialic Acid Linkage-Specific Alkylamidation) method.

[0003] The SALSA method utilizes the difference in the reactivity of the carboxylic acids that make up α2,3-sialic acid and α2,6-sialic acid when they react with amines to form amides. This derivatization results in a mass difference of 28 Da between α2,3-sialic acid and α2,6-sialic acid, making it possible to distinguish between α2,3-sialic acid and α2,6-sialic acid based on the results of mass spectrometry.

[0004] Patent Document 1 discloses a method for analyzing glycans using mass spectral data obtained by mass spectrometry using the SALSA method as a pretreatment method. In this analytical method, glycan compositions are estimated by performing a brute force search on the three sialic acid linkage isomer ion peaks derived from sialic acid-containing glycans detected at 28 Da intervals in the mass spectrum, using the type and number of monosaccharides as search conditions. From the glycan composition candidates obtained by this estimation, compositions containing two or more sialic acids and two or more α2,6-linkages relative to the peak showing the largest mass-to-charge ratio are extracted as highly plausible composition candidates, and these highly plausible composition candidates can be visually distinguished from other composition candidates whose plausibility as sialic acid-containing glycan isomers is questionable and displayed in a list format (see, for example, Figures 6 and 8 of Patent Document 1). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] International Publication No. 2017 / 145496 [Non-patent literature]

[0006] [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 Summary of the Invention [Problem to be solved by the invention]

[0007] When a sample that has undergone linkage-specific modification such as SALSA is subjected to mass spectrometry, the number of peaks observed in the mass spectrum naturally increases compared to when a sample that has not undergone such pretreatment is subjected to mass spectrometry. Therefore, when a sample contains multiple different types of sialic acid-containing glycans that differ in glycan composition, or when a sample contains sialic acid-containing glycans that have the same number of sialic acids and the same glycan structure other than sialic acid but different types of sialic acid, the number of peaks observed in the mass spectrum increases considerably, and isomer ion peaks derived from different types of sialic acid-containing glycans may be observed mixed together.

[0008] In such cases, the number of composition candidates listed in the list of sialic acid-containing glycan composition candidates is extremely large. Because the list of composition candidates lists the estimated glycan composition candidates in order of the m / z values ​​of the original peaks, as described above, when the number of peaks in the mass spectrum is large and the mass spectrum becomes complex, it becomes difficult to understand the correspondence between the peaks in the mass spectrum and each glycan composition candidate in the composition candidate list. This causes problems for users (analysts), such as difficulty in identifying, for example, the glycan composition candidates corresponding to multiple peaks spaced at 28 Da intervals in the mass spectrum that are presumed to belong to a single isomer peak cluster, or difficulty in identifying the glycan composition candidates of other peaks presumed to belong to the same isomer peak cluster as a peak in the composition candidate list where multiple glycan composition candidates exist. These problems reduce the efficiency of user analysis, making analysis time-consuming and prone to operational errors such as misreading and oversight.

[0009] The present invention has been made to solve these problems, and its main object is to provide a method and apparatus for analyzing sialic acid-containing glycans that can improve the efficiency of the work of analyzing sialic acid-containing glycans and reduce the likelihood of errors, thereby improving the accuracy of the analysis. [Means for solving the problem]

[0010] One aspect of the method for analyzing sialic acid-containing sugar chains according to the present invention, which has been made to solve the above-mentioned problems, is a method for analyzing sialic acid-containing sugar chains based on mass spectrum data obtained by mass spectrometry of a sample containing sialic acid-containing sugar chains that have been modified specifically in a sialic acid binding mode or molecules modified with said sugar chains, comprising: a peak detection step of detecting a representative peak for each isotope peak cluster from the mass spectrum data; a peak cluster detection step of detecting isomeric peak clusters including a plurality of ion peaks that are presumed to have the same number of sialic acids and the same sugar chain composition other than sialic acid from the representative peaks detected in the peak detection step; a composition estimation step of estimating the glycan composition of the representative peak detected in the peak detection step in accordance with predetermined glycan search conditions; a first display processing step of creating and displaying on a display unit an annotated mass spectrum, in which an annotation indicating the correspondence between the peaks included in one isomer peak cluster and the peaks observed in the mass spectrum is added for each isomer peak cluster detected in the peak cluster detection step, or a peak list summarizing the mass-to-charge ratio values ​​of the peaks included in one isomer peak cluster for each isomer peak cluster detected in the peak cluster detection step; a second display processing step of creating a composition candidate list in which the glycan composition candidates obtained in the composition estimation step are associated with at least one isomeric peak cluster detected in the peak cluster detection step, and displaying the list on the display unit simultaneously with or switchably to the annotated mass spectrum or the peak list; It has.

[0011] One aspect of the sialic acid-containing sugar chain analysis device according to the present invention, which has been made to solve the above-mentioned problems, is an analysis device that analyzes the sialic acid-containing sugar chain based on mass spectrum data obtained by mass spectrometry of a sample containing a sialic acid-containing sugar chain that has been modified specifically in a sialic acid linkage mode or a molecule modified by the sugar chain, and a peak detection unit that detects a representative peak for each isotope peak cluster from the mass spectrum data; a peak cluster detection unit that detects isomer peak clusters including a plurality of ion peaks that are presumed to have the same number of sialic acids and the same sugar chain composition other than sialic acid from the representative peaks detected by the peak detection unit; a composition estimation unit that estimates the glycan composition of the representative peak detected by the peak detection unit in accordance with predetermined glycan search conditions; a display processing unit that creates an annotated mass spectrum in which annotations indicating the correspondence between peaks contained in one isomer peak cluster and peaks observed in a mass spectrum are added for each isomer peak cluster detected by the peak cluster detection unit, or a peak list that summarizes the mass-to-charge ratio values ​​of peaks contained in one isomer peak cluster for each isomer peak cluster detected by the peak cluster detection unit, and also creates a composition candidate list in which the glycan composition candidates obtained by the composition estimation unit correspond to at least one isomer peak cluster detected by the peak cluster detection unit, and displays the annotated mass spectrum or the peak list and the composition candidate list on a display unit simultaneously or switchably; Equipped with. [Effects of the Invention]

[0012] In the above-described aspects of the sialic acid-containing glycan analysis method and sialic acid-containing glycan analysis device according to the present invention, the user can intuitively grasp the multiple peaks contained in each isomer peak cluster from the annotated mass spectrum or peak list displayed on the display unit. Then, the user can easily recognize the glycan composition candidates corresponding to each of the multiple peaks contained in one isomer peak cluster from the composition candidate list, and can determine the precursor ion for MS / MS analysis required to verify, for example, which of the multiple candidates is the most appropriate.

[0013] In this way, the present invention can improve the efficiency of the analysis of sialic acid-containing glycans, including the binding mode of sialic acid, and also reduce the likelihood of errors during such analysis, thereby improving the accuracy of the analysis. [Brief explanation of the drawings]

[0014] [Figure 1] FIG. 1 is a block diagram of one embodiment of a glycan analysis system including a sialic acid-containing glycan analysis device according to the present invention. [Figure 2] 1 is a flowchart showing the procedure of analysis processing in the sugar chain analysis system of this embodiment. [Figure 3] FIG. 2 shows an example of an annotated mass spectrum displayed by the glycan analysis system of this embodiment. [Figure 4] FIG. 4 is a diagram showing an example of a peak list corresponding to the annotated mass spectrum shown in FIG. 3. [Figure 5] FIG. 5 is a diagram showing an example of a list of sugar chain composition candidates for some peaks in the peak list shown in FIG. 4. DETAILED DESCRIPTION OF THE INVENTION

[0015] In the present invention, a molecule modified with a sialic acid-containing sugar chain is, for example, a biomolecule such as a protein, peptide, lipid, or nucleic acid modified with a sialic acid-containing sugar chain.

[0016] Furthermore, the sialic acid linkage-specific modification that is the premise of the present invention is typically the SALSA method disclosed in the above-mentioned Patent Document 1 and Non-Patent Document 1, but is not limited thereto. For example, any method may be used as long as it performs sialic acid linkage-specific chemical modification (derivatization) that allows discrimination of at least two or more different sialic acid linkages, such as α2,3-linked, α2,6-linked, and α2,8-linked, based on mass differences.

[0017] Furthermore, the type of mass spectrometer used to perform mass analysis on samples containing sialic acid-containing glycans and the like is not particularly limited, but examples that can be used include ion trap mass spectrometers, linear ion trap mass spectrometers, TOF / TOF mass spectrometers, quadrupole time-of-flight (Q-TOF) mass spectrometers, quadrupole ion trap mass spectrometers, and Fourier transform ion cyclotron resonance mass spectrometers.

[0018] Hereinafter, one embodiment of a glycan analysis system including an analysis device for carrying out the sialic acid-containing glycan analysis method according to the present invention will be described with reference to the accompanying drawings. FIG. 1 is a schematic block diagram of this glycan analysis system.

[0019] As shown in Figure 1, this system includes a mass analysis unit 1 that performs mass analysis on a sample, an analysis control unit 2 that controls the mass analysis unit 1, a data analysis unit 3 that performs analysis processing on data obtained by mass analysis, and an input unit 4 and a display unit 5 that serve as user interfaces.

[0020] The data analysis unit 3 includes, as functional blocks, a data storage unit 30, a peak detection unit 31, a glycan search condition setting unit 32, an isomer peak cluster detection unit 33, a glycan composition estimation unit 34, a glycan composition filtering unit 35, an annotated mass spectrum creation unit 36, a glycan composition candidate list creation unit 37, a display processing unit 38, and a precursor ion selection reception unit 39. The glycan search condition setting unit 32 includes, as a subordinate functional block, a glycan search condition storage unit 320.

[0021] The mass spectrometric section 1 can be of any type, but when performing MS / MS analysis as described below, a mass spectrometric device such as an ion trap or collision cell that has the function of dissociating ions by collision-induced dissociation (CID) or the like is used.

[0022] Furthermore, the mass analysis unit 1 may be a liquid chromatograph mass analyzer (LC-MS) instead of a mass analyzer alone, or may be configured to prepare multiple samples by separating and fractionating the eluate whose components have been separated by liquid chromatography, and then subjecting each of the multiple samples to mass analysis using a mass analyzer.

[0023] In this system, the data analysis unit 3 is actually a personal computer or a more powerful workstation, and a dedicated data processing program installed on such a computer is run on the computer to realize the functions of each functional block shown in Figure 1. In this case, the input unit 4 is a keyboard and pointing device (such as a mouse) attached to the computer, and the display unit 5 is a monitor also attached to the computer.

[0024] The procedure for analyzing sialic acid-containing glycans in the glycan analysis system of this embodiment will be explained below with reference to Figs. 2 to 5, along with experimental examples. Fig. 2 is a flowchart showing the procedure for glycan analysis processing carried out mainly by the data analysis unit 3. Figs. 3 to 5 are examples of graphs and tables that can be displayed on the display unit 5 during the analysis processing.

[0025] When analyzing sialic acid-containing glycans using the glycan analysis system of this embodiment, a sample containing a sialic acid-containing glycan or a molecule (glycopeptide, glycolipid, etc.) modified with a sialic acid-containing glycan is pretreated by sialic acid linkage-specific chemical modification. The treated sample is then subjected to mass analysis in the mass spectrometry unit 1. The sialic acid linkage-specific modification method can be, for example, the SALSA method described in Non-Patent Document 1. As described above, in the SALSA method, the masses of the modified products differ by 28 Da between cases where the sialic acid contained in the glycan is α2,3-linked and cases where the sialic acid is α2,6-linked, even if the glycan composition is otherwise identical. Mass spectral data over a predetermined m / z range obtained by the mass spectrometry are sent from the mass spectrometry unit 1 to the data analysis unit 3 and stored in the data storage unit 30.

[0026] When analysis processing based on mass spectrum data is initiated, first, the glycan search condition setting unit 32 displays a predetermined glycan search condition setting screen on the screen of the display unit 5 and prompts the user to input glycan search conditions (step S1). However, default glycan search conditions may be set automatically, regardless of user input. Furthermore, in addition to the glycan search conditions, the user may also be prompted to input peak detection conditions for detecting peaks from the mass spectrum data. The glycan search conditions and peak detection conditions that have been input or that have been set by default are saved in the glycan search condition saving unit 320.

[0027] The glycan search conditions may include, for example, the sialic acid linkage-specific modification method to be used, the mass tolerance for estimating the glycan composition, the expected ion species, the type and number of sugar residues (including sialic acid) to be searched for, etc. The peak detection conditions may include, for example, the signal intensity or SN ratio, which is the threshold for recognizing a peak.

[0028] When the actual analysis begins, the peak detection unit 31 reads out the mass spectrum data of the analysis target stored in the data storage unit 30 and detects a monoisotopic ion peak as a representative peak for each isotope peak cluster according to the peak detection conditions. Generally, for biological molecules such as glycans, the peak with the smallest m / z value among multiple isotope ion peaks that appear at intervals of 1 Da can be detected as a monoisotopic ion peak. The m / z values ​​of each detected ion peak are then determined, and a peak list is created (step S2). Note that instead of the m / z value of the monoisotopic ion peak, the average (center of gravity) m / z value of multiple isotope ion peaks may be determined. That is, for each group of isotope ion peaks derived from the same glycan, a representative m / z value for that group may be determined.

[0029] In an experiment conducted by the present inventors, the N-glycans that modify fetuin, a glycoprotein in fetal bovine blood, were Deglycosylation The sample was a glycan mixture that had been cleaved using the enzyme PNGase and then concentrated. The sialic acids contained in this sample were modified in a sialic acid linkage-specific manner using the SALSA method, and the reducing ends of the glycans were further labeled with anthranilic acid to prepare the sample for analysis. The labeling was a pretreatment step to promote ionization in negative ion mode. The sample obtained after the pretreatment was then subjected to mass analysis in negative ion mode using a matrix-assisted laser desorption / ionization ion trap time-of-flight mass spectrometer (MALDI-IT-TOFMS) to obtain mass spectral data.

[0030] In the above experimental example, the sugar chain search conditions were determined as follows: Sialic acid linkage specific modification method: SALSA method Mass tolerance for estimating glycan composition: m / z 0.2 Ion species: Deprotonated ions The types and numbers of sugar residues to be searched for: 3-15 hexose, 2-14 HexNAc, 0-2 fucose (dHex), 0-5 Neu5Ac (sialic acid), 0-5 Neu5Gc (sialic acid)

[0031] The mass spectrum shown in FIG. 3 is drawn by extracting only the monoisotopic ion peak detected in step S2 from the mass spectrum data acquired by mass analysis in the above experimental example.

[0032] Next, the isomer peak cluster detection unit 33 detects isomer peak clusters including multiple peaks that are presumed to have the same number of sialic acids and the same sugar chain composition other than sialic acid from the peaks listed in the peak list created in step S2 (step S3).

[0033] Specifically, since the SALSA method is used for pretreatment of sialic acid linkage-specific modification, the isomer peak cluster detection unit 33 detects pairs of adjacent ion peaks between α2,6-sialic acid and α2,3-sialic acid at intervals of 28 Da in m / z difference from the peak list, and determines that each pair is an isomer peak cluster composed of linkage isomers of sialic acid-containing glycans that have the same number of sialic acids and the same glycan composition other than sialic acid. The allowable error for the peak spacing used in this detection can be, for example, m / z 0.1. Naturally, the number of ion peaks contained in one isomer peak cluster pair varies depending on the number of sialic acids contained in the sialic acid-containing glycan, the linkage mode of the sialic acid, etc.

[0034] In the mass spectrum shown in Figure 3, three isomer peak clusters were detected: a first isomer peak cluster (SIALIC#1) containing three peaks with m / z values ​​of m / z 2734.0, m / z 2762.1, and m / z 2790.1, all of which are derived from singly charged ions; a second isomer peak cluster (SIALIC#2) containing four peaks with m / z values ​​of m / z 3038.1, m / z 3066.2, m / z 3094.2, and m / z 3122.2; and a third isomer peak cluster (SIALIC#3) containing two peaks with m / z values ​​of m / z 3082.2 and m / z 3110.2.

[0035] Among the isomer peak clusters, some of the peaks contained in the second isomer peak cluster SIALIC#2 and some of the peaks contained in the third isomer peak cluster SIALIC#3 are spaced at intervals of 16 Da, which corresponds to the mass difference between N-acetylneuraminic acid (Neu5Ac) and N-glycolylneuraminic acid (Neu5Gc). This suggests that the third isomer peak cluster SIALIC#3 may have a glycan composition in which N-acetylneuraminic acid present in ions corresponding to peaks contained in the second isomer peak cluster SIALIC#2 is replaced with N-glycolylneuraminic acid.

[0036] Next, the glycan composition estimation unit 34 estimates the glycan composition for each peak listed in the peak list created in step S2 according to the glycan search conditions stored in the glycan search condition storage unit 320, and obtains glycan composition candidates (step S4). Specifically, under the specified type and number of sugar residues, an exhaustive search is performed for glycan compositions that match the m / z value of the ion peak within a specified mass accuracy range. When glycan composition candidates are obtained, the candidates are not necessarily narrowed down to one, and multiple candidates may be obtained.

[0037] Next, the annotated mass spectrum creation unit 36 ​​creates a mass spectrum with annotations that associate each peak included in the isomer peak cluster detected in step S3 with a peak observed in the mass spectrum. The display processing unit 38 displays the annotated mass spectrum on the screen of the display unit 5 (step S5).

[0038] In Figure 3, an annotation display 100 is added to a mass spectrum showing only the monoisotopic ion peak described above. As shown in Figure 3, the annotation display 100 includes a cluster indication mark 101 that combines horizontal bars covering the m / z range from the smallest m / z value to the largest m / z value among the multiple peaks contained in one isomer peak cluster with vertical bars corresponding to the m / z value positions of each peak. Therefore, from the number of vertical bars in this cluster indication mark 101, the user can intuitively grasp the number of constituent peaks that are useful for estimating the sialic acid content in the isomer peak cluster.

[0039] Furthermore, the vertical position at which each of the multiple cluster indication marks 101 is located in the mass spectrum corresponds to the sum of the signal intensities of the peaks contained in the isomer peak cluster corresponding to that mark 101, and the larger the sum of the signal intensities, the higher the vertical position of the cluster indication mark 101 is located. That is, in the example of Figure 3, the cluster indication mark 101 corresponding to the second isomer peak cluster SIALIC#2, which has the largest sum of the signal intensities of the peaks contained therein, is displayed at the highest vertical position. On the other hand, the cluster indication mark 101 corresponding to the third isomer peak cluster SIALIC#3, which has the smallest sum of the signal intensities of the peaks contained therein, is displayed at the lowest vertical position. The m / z ranges of the cluster indication mark 101 corresponding to the second isomer peak cluster SIALIC#2 and the cluster indication mark 101 corresponding to the third isomer peak cluster SIALIC#3 overlap, but as described above, overlapping of the display of the cluster indication marks 101 can be avoided by shifting the position at which the cluster indication marks 101 are displayed according to a ranking based on an intensity index such as the sum of the signal intensities of the peaks contained in the isomer peak clusters.

[0040] Furthermore, when multiple types of sialic acids are set in the glycan screening conditions, isomer peak clusters that have the same number of sialic acids and glycan composition other than sialic acids, but differ only in the type of sialic acid, are determined to be related isomer peak clusters. For example, as described above, when Neu5Ac and Neu5Gc are set as the types of sialic acids, the mass difference between their sugar residues is 16 Da, so peaks spaced 16 Da apart are presumed to be hetero-sialic acid linkage isomer peaks that have the same number of sialic acids and glycan composition other than sialic acids, but differ only in the type of sialic acid, Neu5Ac and Neu5Gc. 3, an arrow 102 is drawn pointing from the vertical bar of the cluster indication mark 101 corresponding to the second isomer peak cluster SIALIC#2, which has the smaller m / z value, to the vertical bar of the cluster indication mark 101 corresponding to the third isomer peak cluster SIALIC#3, which has the larger m / z value, and an annotation 103 is added indicating that the mass difference Δm is 16 Da, which is the mass difference between Neu5Ac and Neu5Gc. This allows the user to visually and easily grasp the relationship between the peaks included in the second isomer peak cluster SIALIC#2 and the peaks included in the third isomer peak cluster SIALIC#3.

[0041] However, the detection of heterosialic acid binding isomer peaks as described above does not need to be performed in step S5, and may be performed at any time after the isomer peak cluster is detected in step S3 and before the processing of step S5 is completed.

[0042] The annotated mass spectrum creation unit 36 ​​may create a peak list instead of or in addition to the annotated mass spectrum described above, and display this on the screen of the display unit 5. This peak list lists the m / z values ​​of the peaks contained in each isomer peak cluster detected in step S3, and also specifies the relationship between corresponding peaks between the above-described related isomer peak clusters that have the same number of sialic acids and the same glycan composition other than sialic acid, but differ only in the type of sialic acid.

[0043] Figure 4 is a peak list corresponding to the annotated mass spectrum shown in Figure 3. In this peak list 200, "Peak Index" is a number corresponding to the number of sialic acids bonded in a specific manner. When there are multiple isomer peak clusters associated as having different types of sialic acid substitutions, the peaks included in the isomer peak cluster containing the peak with the smallest m / z value among all peaks included in the associated multiple isomer peak clusters are assigned Peak Indexes of 1, 2, ... in ascending order of m / z value. Furthermore, the same Peak Index is assigned to peaks associated as having different types of sialic acid substitutions, i.e., peaks that are presumed to have the same number of sialic acids and glycan composition other than sialic acid, except for the type of sialic acid.

[0044] In the example of Figure 4, among the peaks contained in the mutually related second and third isomer peak clusters SIALIC#2 and SIALIC#3, the peak with the smallest m / z value is the peak in the second isomer peak cluster SIALIC#2 with m / z 3038.1, and this peak is assigned Peak Index = 1. Furthermore, the peak with the second smallest m / z value, m / z 3066.2, in the same second isomer peak cluster SIALIC#2 is assigned Peak Index = 2. Note that the peak with Peak Index = 2 in the second isomer peak cluster SIALIC#2 is referred to as 2-2.

[0045] The peak with the smallest m / z value, m / z 3082.2, in the third isomer peak cluster SIALIC#3, which is presumed to be an ion formed by substitution of N-glycolylneuraminic acid for N-acetylneuraminic acid, has a Peak Index of 2, which is the same as the Peak Index of the peak in the second isomer peak cluster SIALIC#2, the source of the sialic acid substitution, that corresponds to this peak. Furthermore, the Relation column in the peak list 200 contains information indicating the relationship between peaks in mutually related isomer peak clusters. That is, the Relation column contains the number indicating the isomer peak cluster, the Peak Index, and the mass difference between the peaks, as information indicating the correspondence between different sialic acids between the peaks.

[0046] In either the annotated mass spectrum shown in Figure 3 or the peak list shown in Figure 4, users can grasp at a glance the number of peaks contained in each isomer peak cluster and the m / z value of each peak. Furthermore, users can easily grasp the relationships between multiple isomer peak clusters that have the same number of sialic acids and the same glycan composition other than sialic acid, but differ only in the type of sialic acid, and in particular the relationships between the peaks contained in those multiple isomer peak clusters.

[0047] The user then selects one or more isomer peak clusters of interest in the displayed annotated mass spectrum or peak list using the input unit 4 (step S6). For example, in the annotated mass spectrum, the user can select an isomer peak cluster corresponding to an arbitrary cluster indication mark 101 by pointing to that mark 101 with a pointing device. In the peak list, the same selection can be made by pointing to one of the SIALIC# columns with a pointing device.

[0048] In response to the above selection operation, the glycan composition candidate list creation unit 37, for example, collects the glycan composition candidates estimated in step S4 corresponding to each peak included in the selected isomer peak cluster and displays a glycan composition candidate list associated with each peak in the isomer peak cluster. Alternatively, the glycan composition candidate list creation unit 37 may display a list of glycan composition candidates for all isomer peak clusters detected in step S3. In this case, the glycan composition candidates estimated corresponding to each peak included in the selected peak cluster may be displayed in an emphasized manner so as to be distinguishable from the other candidates. The display processing unit 38 displays this glycan composition candidate list in the same window or a separate window simultaneously with the annotated mass spectrum or peak list currently displayed on the display unit 5. Alternatively, the glycan composition candidate list may be displayed instead of the annotated mass spectrum or peak list, i.e., by switching the display (step S7).

[0049] Figure 5 shows a glycan composition candidate list 300 that is displayed when the second and third isomer peak clusters SIALIC#2 and SIALIC#3 are selected in Figure 3 or Figure 4. In reality, the upper table and the lower table in Figure 5 are connected horizontally at the aa position. In other words, this is a horizontally long table. Since the second isomer peak cluster SIALIC#2 and the third isomer peak cluster SIALIC#3 are associated with each other, it is possible to automatically create and display a glycan composition candidate list corresponding to both isomer peak clusters, not only when both the second and third isomer peak clusters SIALIC#2 and SIALIC#3 are selected, but also when only one of them is selected.

[0050] As shown in Figure 5, in this glycan composition candidate list 300, all peaks contained in the two isomer peak clusters SIALIC#2 and SIALIC#3 are classified by Peak Index, and the glycan composition candidates estimated for each peak are comprehensively listed. Furthermore, the glycan composition candidates are classified according to whether the glycan composition other than sialic acid is the same.

[0051] Specifically, in the example of Figure 5, for all peaks contained in the two isomer peak clusters SIALIC#2 and SIALIC#3, sialic acid-containing glycans with a sialic acid number of 3 and a glycan composition other than sialic acid of Hex6HexNAc5, or sialic acid-containing glycans with a sialic acid number of 3 and a glycan composition other than sialic acid of Hex5HexNAC5dHex1, are predicted as glycan composition candidates. Furthermore, for only some peaks, sialic acid-containing glycans with different glycan compositions (Hex5HexNAC7 and Hex6HexNAc3dHex1 in this example) are also predicted as glycan composition candidates.

[0052] For example, for the peak (m / z 3082.2) with Peak Index 2 in the third isomer peak cluster SIALIC#3, if the glycan composition other than sialic acid is assumed to be Hex6HexNAc5, two types of glycan composition candidates are predicted as combinations of sialic acid composition and linkage: NeuAc(α2,3-)2NeuGc(α2,6-)1 and NeuAc(α2,6-)1NeuAc(α2,3-)1NeuGc(α2,3-)1. Whether this ion peak is derived from a mixture of these two types of glycans or from one of the glycans can be determined by performing MS / MS analysis using the ion peak as a precursor ion, as described below.

[0053] In addition, when a user selects an isomer peak cluster in an annotated mass spectrum or peak list, the corresponding characters may be highlighted so that the selected isomer peak cluster and its corresponding or associated peaks stand out in the annotated mass spectrum or peak list.

[0054] Regardless of whether multiple glycan composition candidates have been estimated for a certain peak, when the user specifies an arbitrary peak as a precursor ion in either the annotated mass spectrum, the peak list, or the glycan composition candidate list, the precursor ion selection receiving unit 39 selects the specified ion peak as the precursor ion for MS / MS analysis (step S8).

[0055] This selection information is sent to the analysis controller 2, which controls the mass spectrometer 1 to perform MS / MS analysis targeting the selected precursor ion, specifically, product ion scan measurement using an ion dissociation technique such as CID. As a result, the mass spectrometer 1 performs MS / MS analysis on the sample containing glycans that have been specifically modified with sialic acid linkages, and acquires MS / MS spectral data (step S9).

[0056] In the MS / MS spectrum, multiple product ion peaks derived from the target sialic acid-containing glycan are observed, and based on the m / z values ​​of the peaks, the user can verify which of the multiple glycan composition candidates is valid, or whether one glycan composition candidate is valid (step S10).

[0057] As described above, the sugar chain analysis system of this embodiment can efficiently perform structural analysis of sialic acid-containing sugar chains, including the sialic acid binding mode.

[0058] In the above embodiment, all of the glycan composition candidates estimated corresponding to each peak in step S4 are listed in the glycan composition candidate list 300 as shown in Fig. 5, but it is also possible to narrow down the glycan composition candidates according to specific constraints or assumptions, and display the narrowed down glycan composition candidates and the remaining glycan composition candidates in a format that allows easy visual identification. In this case, the glycan composition filtering unit 35 determines the glycan composition candidates according to preset narrowing conditions, and the glycan composition candidate list creation unit 37 changes the display of the glycan composition candidates based on the narrowing down results.

[0059] The narrowing down conditions in the glycan composition filtering unit 35 can be set as appropriate, and can be, for example, the conditions described in Patent Document 1. In particular, when there are a large number of glycan composition candidates, the burden on the user to check can be reduced by distinguishing between highly valid and less valid candidates and then displaying them.

[0060] Furthermore, if ion peaks derived from glycans that should be observed are not detected in step S2 due to reasons such as low peak signal intensity overall, the isomer peak clusters may not be detected properly, hindering glycan composition estimation. In such cases, the user may be able to increase the number of monoisotopic ion peaks detected in step S2 by, for example, changing the peak detection conditions, which may allow for proper glycan composition estimation.

[0061] It should be noted that the above embodiment is merely an example of the present invention, and it goes without saying that any appropriate modifications, alterations, additions, etc. made within the spirit of the present invention will also fall within the scope of the claims of the present application.

[0062] [Various aspects] It will be appreciated by those skilled in the art that the exemplary embodiments described above are examples of the following aspects.

[0063] (Item 1) One aspect of the method for analyzing sialic acid-containing sugar chains according to the present invention is a method for analyzing sialic acid-containing sugar chains based on mass spectrum data obtained by mass spectrometry of a sample containing sialic acid-containing sugar chains that have been modified specifically in a sialic acid linkage mode or molecules modified with said sugar chains, comprising: a peak detection step of detecting a representative peak for each isotope peak cluster from the mass spectrum data; a peak cluster detection step of detecting isomeric peak clusters including a plurality of ion peaks that are presumed to have the same number of sialic acids and the same sugar chain composition other than sialic acid from the representative peaks detected in the peak detection step; a composition estimation step of estimating the glycan composition of the representative peak detected in the peak detection step in accordance with predetermined glycan search conditions; a first display processing step of creating and displaying on a display unit an annotated mass spectrum, in which an annotation indicating the correspondence between the peaks included in one isomer peak cluster and the peaks observed in the mass spectrum is added for each isomer peak cluster detected in the peak cluster detection step, or a peak list summarizing the mass-to-charge ratio values ​​of the peaks included in one isomer peak cluster for each isomer peak cluster detected in the peak cluster detection step; a second display processing step of creating a composition candidate list in which the glycan composition candidates obtained in the composition estimation step are associated with at least one isomeric peak cluster detected in the peak cluster detection step, and displaying the list on the display unit simultaneously with or switchably to the annotated mass spectrum or the peak list; It has.

[0064] (Item 5) Furthermore, one aspect of the sialic acid-containing sugar chain analyzing device according to the present invention is an analyzing device that analyzes the sialic acid-containing sugar chain based on mass spectrum data obtained by mass spectrometry of a sample containing a sialic acid-containing sugar chain that has been modified specifically to a sialic acid linkage or a molecule modified by the sugar chain, comprising: a peak detection unit that detects a representative peak for each isotope peak cluster from the mass spectrum data; a peak cluster detection unit that detects isomer peak clusters including a plurality of ion peaks that are presumed to have the same number of sialic acids and the same sugar chain composition other than sialic acid from the representative peaks detected by the peak detection unit; a composition estimation unit that estimates the glycan composition of the representative peak detected by the peak detection unit in accordance with predetermined glycan search conditions; a display processing unit that creates an annotated mass spectrum in which annotations indicating the correspondence between peaks contained in one isomer peak cluster and peaks observed in a mass spectrum are added for each isomer peak cluster detected by the peak cluster detection unit, or a peak list that summarizes the mass-to-charge ratio values ​​of peaks contained in one isomer peak cluster for each isomer peak cluster detected by the peak cluster detection unit, and also creates a composition candidate list in which the glycan composition candidates obtained by the composition estimation unit correspond to at least one isomer peak cluster detected by the peak cluster detection unit, and displays the annotated mass spectrum or the peak list and the composition candidate list on a display unit simultaneously or switchably; Equipped with.

[0065] According to the sialic acid-containing glycan analysis method described in Item 1 and the sialic acid-containing glycan analysis device described in Item 5, the user can intuitively grasp the multiple peaks contained in each isomer peak cluster from the annotated mass spectrum or peak list displayed on the display unit. Then, the user can easily recognize the glycan composition candidates corresponding to each of the multiple peaks contained in one or more isomer peak clusters from the composition candidate list, and can determine the precursor ion for MS / MS analysis required to verify, for example, an uncertain glycan composition. This improves the efficiency of the work of analyzing the structure of sialic acid-containing glycans. It also reduces the likelihood of errors during such analysis, improving the accuracy of the analysis.

[0066] (Items 2 and 6) In the sialic acid-containing glycan analysis method described in Item 1 and the sialic acid-containing glycan analysis apparatus described in Item 5, the annotated mass spectrum and the peak list may include information indicating the correspondence of peaks with the same sialic acid binding mode among multiple isomeric peak clusters that are identical in number of sialic acids and composition other than sialic acid, but differ only in type of sialic acid.

[0067] Typical types of sialic acid include N-acetylneuraminic acid, N-glycolylneuraminic acid, and deaminoneuraminic acid. According to the analytical method described in item 2 and the analytical device described in item 6, the user can easily understand the correspondence of peaks between multiple isomer peak clusters that differ only in the type of sialic acid, thereby easily confirming the presence or absence of phenomena that occur in vivo, such as the substitution of N-acetylneuraminic acid with N-glycolylneuraminic acid.

[0068] (Item 3) The method for analyzing sialic acid-containing sugar chains according to item 1 or 2 further comprises: a precursor ion selection step of accepting an operation by a user to select, as a precursor ion, any peak included in any isomer peak cluster in any of the annotated mass spectrum, the peak list, and the composition candidate list displayed on the display unit; an MS / MS analysis execution step of executing MS / MS analysis on the sample, targeting the precursor ion selected in the precursor ion selection step; It may have the following structure:

[0069] (Item 7) The sialic acid-containing sugar chain analysis device according to item 5 or 6, a precursor ion selection receiving unit that receives a user's selection operation of any peak included in any isomer peak cluster in any of the annotated mass spectrum, the peak list, or the composition candidate list displayed on the display unit; an MS / MS analysis execution unit that executes MS / MS analysis on the sample, targeting the precursor ions accepted by the precursor ion selection acceptance unit; The device may further comprise:

[0070] In the precursor ion selection step, the peak to be used as the precursor ion can be easily selected by clicking on the screen of the display unit using a pointing device.

[0071] Generally, when multiple glycan composition candidates are predicted for a given peak, verifying which glycan composition candidate is correct requires performing MS / MS analysis on that peak and confirming the mass-to-charge ratio of the resulting product ions. According to the analysis method described in Section 3 and the analysis device described in Section 7, a user can identify the peak that requires MS / MS analysis, for example, in a list of composition candidates, and then, with simple operations, perform MS / MS analysis with the ion corresponding to that peak set as the precursor ion. This allows for more efficient structural analysis of sialic acid-containing glycans.

[0072] (Item 4) The method for analyzing sialic acid-containing sugar chains according to any one of Items 1 to 3, a peak detection condition resetting step of accepting an operation by a user to change the peak detection conditions after displaying the annotated mass spectrum, the peak list, or the glycan composition candidate list, Reanalysis can be performed by executing the processes of the peak detection step and subsequent steps under the changed peak detection conditions.

[0073] According to the analysis method described in paragraph 4, when an isomer peak cluster cannot be detected due to factors such as low signal intensity of the peak observed in the mass spectrum or a poor S / N ratio, the user can appropriately change the peak detection conditions and perform the analysis again, thereby improving the efficiency of glycan analysis even when the sample condition is poor. [Explanation of symbols]

[0074] 1...Mass spectrometry section 2...Analysis control unit 3...Data analysis section 30...Data storage section 31...Peak detector 32...Glycan search condition setting section 320...Glycan search condition storage section 33...Isomeric peak cluster detection section 34...Glycan composition estimation section 35...Glycan composition filtering section 36...Annotated mass spectrum creation section 37...Glycan composition candidate list creation section 38...Display processing unit 39...Precursor ion selection acceptance unit 4...Input section 5...Display section

Claims

1. An analytical method for analyzing a sialic acid-containing sugar chain based on mass spectrum data obtained by mass spectrometry of a sample containing a sialic acid-containing sugar chain that has been modified specifically to a sialic acid binding mode or a molecule modified by said sugar chain, comprising: a peak detection step of detecting a representative peak for each isotope peak cluster from the mass spectrum data; a peak cluster detection step of detecting isomeric peak clusters including a plurality of ion peaks that are presumed to have the same number of sialic acids and the same sugar chain composition other than sialic acid from the representative peaks detected in the peak detection step; a composition estimation step of estimating the glycan composition of the representative peak detected in the peak detection step in accordance with predetermined glycan search conditions; a first display processing step of creating and displaying on a display unit an annotated mass spectrum, in which an annotation indicating the correspondence between the peaks included in one isomer peak cluster and the peaks observed in the mass spectrum is added for each isomer peak cluster detected in the peak cluster detection step, or a peak list summarizing the mass-to-charge ratio values ​​of the peaks included in one isomer peak cluster for each isomer peak cluster detected in the peak cluster detection step; a second display processing step of creating a composition candidate list in which the glycan composition candidates obtained in the composition estimation step are associated with at least one isomeric peak cluster detected in the peak cluster detection step, and displaying the composition candidate list on the display unit simultaneously with or switchably to the annotated mass spectrum or the peak list; wherein the annotated mass spectrum and the peak list contain information indicating correspondence between peaks with the same sialic acid bonding mode among a plurality of isomeric peak clusters that are identical in number of sialic acids and composition other than the sialic acids but differ only in type of sialic acid.

2. An analytical method for analyzing a sialic acid-containing sugar chain based on mass spectrum data obtained by mass spectrometry of a sample containing a sialic acid-containing sugar chain that has been modified specifically to a sialic acid binding mode or a molecule modified by said sugar chain, comprising: a peak detection step of detecting a representative peak for each isotope peak cluster from the mass spectrum data; a peak cluster detection step of detecting isomeric peak clusters including a plurality of ion peaks that are presumed to have the same number of sialic acids and the same sugar chain composition other than sialic acid from the representative peaks detected in the peak detection step; a composition estimation step of estimating the glycan composition of the representative peak detected in the peak detection step in accordance with predetermined glycan search conditions; a first display processing step of creating and displaying on a display unit an annotated mass spectrum, in which an annotation indicating the correspondence between the peaks included in one isomer peak cluster and the peaks observed in the mass spectrum is added for each isomer peak cluster detected in the peak cluster detection step, or a peak list summarizing the mass-to-charge ratio values ​​of the peaks included in one isomer peak cluster for each isomer peak cluster detected in the peak cluster detection step; a second display processing step of creating a composition candidate list in which the glycan composition candidates obtained in the composition estimation step are associated with at least one isomeric peak cluster detected in the peak cluster detection step, and displaying the composition candidate list on the display unit simultaneously with or switchably to the annotated mass spectrum or the peak list; a precursor ion selection step of accepting an operation by a user to select, as a precursor ion, any peak included in any isomer peak cluster in any of the annotated mass spectrum, the peak list, and the composition candidate list displayed on the display unit; an MS / MS analysis performing step of performing MS / MS analysis on the sample, targeting the precursor ion selected in the precursor ion selecting step; A method for analyzing sialic acid-containing sugar chains comprising:

3. An analytical method for analyzing a sialic acid-containing sugar chain based on mass spectrum data obtained by mass spectrometry of a sample containing a sialic acid-containing sugar chain that has been modified specifically to a sialic acid binding mode or a molecule modified by said sugar chain, comprising: a peak detection step of detecting a representative peak for each isotope peak cluster from the mass spectrum data; a peak cluster detection step of detecting isomeric peak clusters including a plurality of ion peaks that are presumed to have the same number of sialic acids and the same sugar chain composition other than sialic acid from the representative peaks detected in the peak detection step; a composition estimation step of estimating the glycan composition of the representative peak detected in the peak detection step in accordance with predetermined glycan search conditions; a first display processing step of creating and displaying on a display unit an annotated mass spectrum, in which an annotation indicating the correspondence between the peaks included in one isomer peak cluster and the peaks observed in the mass spectrum is added for each isomer peak cluster detected in the peak cluster detection step, or a peak list summarizing the mass-to-charge ratio values ​​of the peaks included in one isomer peak cluster for each isomer peak cluster detected in the peak cluster detection step; a second display processing step of creating a composition candidate list in which the glycan composition candidates obtained in the composition estimation step are associated with at least one isomeric peak cluster detected in the peak cluster detection step, and displaying the composition candidate list on the display unit simultaneously with or switchably to the annotated mass spectrum or the peak list; a peak detection condition resetting step of accepting an operation by a user to change peak detection conditions after displaying the annotated mass spectrum, the peak list, or the glycan composition candidate list; and performing reanalysis by carrying out processing in each of the peak detection step, the peak cluster detection step, the composition estimation step, the first display processing step, and the second display processing step under the peak detection conditions changed in the peak detection condition resetting step.

4. An analytical device for analyzing a sialic acid-containing sugar chain based on mass spectrum data obtained by mass spectrometry of a sample containing a sialic acid-containing sugar chain that has been modified specifically to a sialic acid binding mode or a molecule modified by said sugar chain, comprising: a peak detection unit that detects a representative peak for each isotope peak cluster from the mass spectrum data; a peak cluster detection unit that detects isomer peak clusters including a plurality of ion peaks that are presumed to have the same number of sialic acids and the same sugar chain composition other than sialic acid from the representative peaks detected by the peak detection unit; a composition estimation unit that estimates the glycan composition of the representative peak detected by the peak detection unit in accordance with predetermined glycan search conditions; a display processing unit that creates an annotated mass spectrum in which annotations indicating the correspondence between peaks contained in one isomer peak cluster and peaks observed in a mass spectrum are added for each isomer peak cluster detected by the peak cluster detection unit, or a peak list that summarizes the mass-to-charge ratio values ​​of peaks contained in one isomer peak cluster for each isomer peak cluster detected by the peak cluster detection unit, and also creates a composition candidate list in which the glycan composition candidates obtained by the composition estimation unit correspond to at least one isomer peak cluster detected by the peak cluster detection unit, and displays the annotated mass spectrum or the peak list and the composition candidate list on a display unit simultaneously or switchably; wherein the annotated mass spectrum and the peak list contain information indicating the correspondence between peaks with the same sialic acid bonding mode among a plurality of isomeric peak clusters that are identical in number of sialic acids and composition other than the sialic acids, but differ only in type of sialic acid.

5. An analytical device for analyzing a sialic acid-containing sugar chain based on mass spectrum data obtained by mass spectrometry of a sample containing a sialic acid-containing sugar chain that has been modified specifically to a sialic acid binding mode or a molecule modified by said sugar chain, comprising: a peak detection unit that detects a representative peak for each isotope peak cluster from the mass spectrum data; a peak cluster detection unit that detects isomer peak clusters including a plurality of ion peaks that are presumed to have the same number of sialic acids and the same sugar chain composition other than sialic acid from the representative peaks detected by the peak detection unit; a composition estimation unit that estimates the glycan composition of the representative peak detected by the peak detection unit in accordance with predetermined glycan search conditions; a display processing unit that creates an annotated mass spectrum in which annotations indicating the correspondence between peaks contained in one isomer peak cluster and peaks observed in a mass spectrum are added for each isomer peak cluster detected by the peak cluster detection unit, or a peak list that summarizes the mass-to-charge ratio values ​​of peaks contained in one isomer peak cluster for each isomer peak cluster detected by the peak cluster detection unit, and also creates a composition candidate list in which the glycan composition candidates obtained by the composition estimation unit correspond to at least one isomer peak cluster detected by the peak cluster detection unit, and displays the annotated mass spectrum or the peak list and the composition candidate list on a display unit simultaneously or switchably; a precursor ion selection receiving unit that receives a user's selection operation of any peak included in any isomer peak cluster in any of the annotated mass spectrum, the peak list, or the composition candidate list displayed on the display unit; an MS / MS analysis execution unit that executes MS / MS analysis on the sample, targeting the precursor ions accepted by the precursor ion selection acceptance unit; A sialic acid-containing sugar chain analysis device comprising:

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