Methods and apparatus for resolving sialic acid-containing glycan chains.

Abstract

This invention relates to a method for analyzing glycans containing sialic acid, comprising: a comparison condition setting step (S1) for setting glycan comparison conditions; a peak detection step (S2) for detecting representative peaks for each isotope peak group based on mass spectrometry data; a composition estimation step (S3) for estimating the glycan composition and determining glycan composition candidates for each detected representative peak according to the glycan comparison conditions; a peak group detection step (S4) for detecting isomer peak groups containing multiple peaks that are estimated to contain sialic acid and glycan compositions other than sialic acid, based on the detected representative peaks; a glycan composition filtering step (S5) for filtering glycan composition candidates by applying defined limiting conditions related to identity to each peak contained in the isomer peak group; and a display step (S6-S7) for generating and displaying a list of glycan composition candidates in a manner that allows visual identification of the filtered glycan composition candidates and other glycan composition candidates.

Description

Technical Field

[0001] This invention relates to a method and apparatus for analyzing glycans using quality analysis. More specifically, it relates to an analytical method and apparatus capable of structurally analyzing glycans containing sialic acid, including the binding mode of sialic acid. Furthermore, the term "glycan" here refers not only to standalone glycans but also to glycans modified with biomolecules such as proteins, peptides, lipids, and nucleic acids; that is, it also includes modified forms of glycans. Background Technology

[0002] In life sciences, new drug development, and medicine, the analysis of glycans is a significant field. Among these, determining the binding mode of sialic acid in glycans containing sialic acid is a crucial aspect of glycan structure analysis. Against this backdrop, to efficiently analyze the structures of sialic acid-containing glycans, including differences in binding modes, using quality analysis, methods for specifically modifying the binding mode of sialic acid have been developed. For example, Patent Document 1 and Non-Patent Document 1 disclose a method for specifically modifying the binding mode of sialic acid, named SALSA (Sialic Acid Linkage-Specific Alkylamidation).

[0003] The SALSA method utilizes the differences in the reactions of carboxylic acids that constitute α2,3-sialic acid and α2,6-sialic acid with amines to form amides, thereby enabling the isopropylamidation and methylamidation of various sialic acids. Through this derivative oxidation, a 28-Da mass difference is generated between α2,3-sialic acid and α2,6-sialic acid, allowing for the identification of α2,3-sialic acid and α2,6-sialic acid based on the mass analysis results.

[0004] Patent Document 1 discloses a method for analyzing glycans based on mass spectrometry data obtained through mass analysis using SALSA as a pretreatment method. In this method, for the three sialic acid-binding isomer ion peaks of glycans containing sialic acid detected at 28-Da intervals in the mass spectrometer, the composition is compared one by one based on the type and number of monosaccharides, thereby estimating the glycan composition. Then, from the glycan composition candidates obtained through this estimation, the peaks containing two or more sialic acids and showing the largest mass are extracted as candidates with higher suitability. These candidates with higher suitability and other candidates whose suitability as sialic acid-containing glycan isomers are visually distinguished and displayed in a summary table (e.g., refer to Patent Document 1). Figure 6 (Figure 8, etc.)

[0005] Existing technical documents

[0006] Patent documents

[0007] Patent Document 1: International Publication No. 2017 / 145496

[0008] Non-patent literature

[0009] Non-Patent Literature 1: Takashi Nishikaze et al., 6, "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

[0010] The technical problem that the invention aims to solve

[0011] The analytical method described in Patent Document 1 is a structural analysis method targeting glycans containing a specific type of sialic acid, such as N-acetylneuraminic acid (Neu5Ac). However, there are many known molecular types of sialic acid. Besides N-acetylneuraminic acid, N-hydroxyacetylneuraminic acid (Neu5Gc) or deaminoneuraminic acid (Kdn) are also known as major sialic acids, and the distribution of each molecular type is species- or tissue-specific. In principle, glycans containing sialic acid other than Neu5Ac can be analyzed in the same way as glycans containing Neu5Ac. However, typically, the presence of other molecular types is quite small compared to the most abundant molecular type. Therefore, even with specific modifications to the sialic acid binding mode, peaks corresponding to the desired binding mode combination are often not detected on mass spectrometry. For this reason, it is difficult to select compositional candidates for such sialic acid-containing glycans.

[0012] This invention was made to solve such a technical problem, and its main objective is to provide a method and apparatus for analyzing sialic acid-containing sugar chains that can improve the efficiency of the operation of analyzing sugar chains containing various types of sialic acid and also improve the accuracy of the analysis.

[0013] Solution to the above technical problems

[0014] The present invention, which addresses the aforementioned technical problems, provides a method for analyzing sialic acid-containing glycan chains. This method utilizes mass spectrometry data obtained from mass analysis of a sample containing sialic acid-containing glycan chains with specifically modified sialic acid binding mechanisms, or molecules modified by such glycans. The method comprises:

[0015] The process of setting comparison conditions involves setting the sugar chain comparison conditions.

[0016] The peak detection process involves detecting representative peaks for each isotope peak group based on the mass spectrometry data.

[0017] The composition estimation process involves estimating the sugar composition of each representative peak detected by the peak detection process according to the sugar chain comparison conditions and determining the sugar chain composition candidates.

[0018] The peak cluster detection process involves detecting isomer peak clusters that contain the same number of peaks presumed to be sialic acid and the same number of peaks composed of sugar chains other than sialic acid, based on the representative peaks detected by the peak detection process.

[0019] In the glycan composition filtering process, for each peak contained in the isomer peak group, the candidate glycan compositions are screened by applying specified limiting conditions related to the number of sialic acids contained, the number of sialic acid binding modes, the type of sialic acid, and the identity of glycan compositions other than sialic acid.

[0020] The display process generates and displays a visually identifiable overview of the glycan composition candidates, including those filtered by the glycan composition filtering process and other glycan composition candidates.

[0021] The present invention, which addresses the aforementioned technical problems, provides a solution for an apparatus for analyzing sialic acid-containing glycan chains. This apparatus uses mass spectrometry data obtained from mass analysis of a sample containing a sialic acid-containing glycan chain with a specifically modified sialic acid binding mode, or a molecule modified by such a glycan chain, and comprises:

[0022] The alignment condition setting unit accepts the user's specification of glycan alignment conditions and sets the glycan alignment conditions accordingly.

[0023] The peak detection unit detects representative peaks for each isotope peak group based on the mass spectrometry data.

[0024] The composition estimation unit estimates the glycan composition and obtains candidate glycan compositions for each representative peak detected by the peak detection unit according to the glycan comparison conditions.

[0025] The peak group detection unit detects isomer peak groups containing multiple peaks that are presumed to be sialic acid and have the same composition of sugar chains other than sialic acid, based on the representative peaks detected by the peak detection unit.

[0026] The glycan composition filtering section filters the glycan composition candidates by applying specified limiting conditions related to the number of sialic acids contained in each peak of the isomer peak group, the number of sialic acid binding modes, the type of sialic acid, and the identity of glycan compositions other than sialic acid.

[0027] The display processing unit generates and displays a list of glycan composition candidates, including those filtered by the glycan composition filtering unit and other glycan composition candidates, in a visually identifiable manner.

[0028] Invention Effects

[0029] According to the above-described scheme of the present invention, even in sialic acid-containing glycans where the glycan composition other than sialic acid has the same number of sialic acid components but the sialic acid binding mode differs from the type of sialic acid, and where no ion peak corresponding to a portion of the proposed binding mode combination is detected for a certain type of sialic acid, it is still possible to accurately select candidate glycan compositions containing that specific type of sialic acid and present them to the user. This improves the efficiency of experimental work such as MS / MS analysis for verifying the appropriateness of the proposed candidate composition of sialic acid-containing glycans, as well as the analysis of the data collected therefrom, enabling faster and more accurate structural analysis of sialic acid-containing glycans. Attached Figure Description

[0030] Figure 1 This is a block diagram of an embodiment of a glycan decomposition system including the glycan decomposition apparatus containing sialic acid of the present invention.

[0031] Figure 2 This is a flowchart illustrating the parsing process in the glycan parsing system of this embodiment.

[0032] Figure 3 This is an example of mass spectrometry, and an explanatory diagram of glycan composition analysis based on peaks detected in the mass spectrometer.

[0033] Figure 4 It shows the... Figure 3 The figure shown is a graph of the peak groups detected by mass spectrometry and the classification results of the peaks in each group.

[0034] Figure 5 It shows the... Figure 4 The figure shows an example of a list of candidate sugar chain compositions obtained from the six peaks.

[0035] Figure 6 It shows the... Figure 4 The figure shows an example of a list of candidate sugar chain compositions obtained from the six peaks. Detailed Implementation

[0036] In the above-described embodiments of the present invention, the molecules modified by sugar chains containing sialic acid are, for example, biomolecules such as proteins, peptides, lipids, and nucleic acids modified by sugar chains containing sialic acid.

[0037] Furthermore, as a prerequisite for the above-mentioned scheme of the present invention, namely the specific modification of the sialic acid binding mode, the typical method is the SALSA method disclosed in the above-mentioned Patent Document 1 and Non-Patent Document 1, but it is not limited to this. Any method that identifies the specific chemical modification (derivation) of the sialic acid binding mode by the quality difference for at least two different sialic acid binding modes, such as α2,3-binding, α2,6-binding, and further α2,8-binding, is acceptable.

[0038] Furthermore, in the above-described embodiments of the present invention, there is no particular limitation on the type of mass analysis apparatus used for mass analysis of samples containing sugar chains such as sialic acid. For example, ion trap type mass analysis apparatus, linear ion trap type mass analysis apparatus, TOF / TOF type mass analysis apparatus, quadrupole time-of-flight (Q-TOF type) mass analysis apparatus, quadrupole ion trap type mass analysis apparatus, Fourier transform ion cyclotron resonance mass analysis apparatus, etc., can be used. In order to estimate the sugar chain composition based on the mass value, a device with high mass accuracy is desirable.

[0039] Hereinafter, with reference to the accompanying drawings, an embodiment of a glycan decomposition system including a decomposition apparatus for implementing the decomposition method for glycans containing sialic acid of the present invention will be described.

[0040] Figure 1 This is a schematic block diagram of the glycan chain analysis system.

[0041] like Figure 1 As shown, the system includes a quality analysis unit 1 that performs quality analysis on the sample, an analysis control unit 2 that controls the quality analysis unit 1, a data parsing unit 3 that performs parsing processing on the data obtained through the quality analysis, an input unit 4 that serves as a user interface, and a display unit 5.

[0042] The data analysis unit 3 includes a data storage unit 30, a peak detection unit 31, a glycan alignment condition setting unit 32, an isomer peak group detection unit 33, a glycan composition estimation unit 34, a glycan composition filtering unit 35, a glycan composition candidate list generation unit 36, a display processing unit 37, and a precursor ion selection and acceptance unit 38 as functional modules. The glycan alignment condition setting unit 32 includes a glycan alignment condition storage unit 320 as a lower-level functional module.

[0043] While there are no particular limitations on the method of mass analysis unit 1, when performing MS / MS analysis as described later, a mass analysis apparatus capable of dissociating ions using collision-induced dissociation (CID) or similar methods, such as ion traps or collision cells, can be used. Furthermore, in order to estimate the composition based on the mass-to-charge ratio as described later, high accuracy and resolution of the m / z value are desirable. Therefore, time-of-flight mass separators and Fourier transform ion cyclotron resonance mass separators are more suitable as mass separators.

[0044] In addition, the quality analysis unit 1 may not be a separate quality analysis device, but may be a liquid chromatograph quality analysis device (LC-MS), or it may be a configuration in which multiple samples are prepared by separating or partitioning the eluent obtained by separating components by liquid chromatograph, and the quality analysis device is used to perform quality analysis on each of the multiple samples.

[0045] In this system, the entity of the data parsing unit 3 is a personal computer or a higher-performance workstation. By enabling a dedicated data processing program installed on such a computer to operate, the following is achieved: Figure 1 The functions of each module are shown. In this case, the input unit 4 is a keyboard or pointing device (mouse, etc.) attached to the computer, and the display unit 5 is a monitor also attached to the computer.

[0046] The following is through Figures 2-6 The process of parsing sialic acid-containing glycans in this glycan parsing system is explained with reference to experimental examples. Figure 2 This is a flowchart illustrating the process of glycan parsing and processing implemented with the data parsing unit 3 as the center.

[0047] When resolving glycans containing sialic acid using this glycan analysis system, the sample containing glycans or molecules modified by glycans containing sialic acid (glycopeptides, glycolipids, etc.) undergoes a pretreatment based on sialic acid-binding specific chemical modification. As a sialic acid-binding specific modification method, the SALSA method described in Non-Patent Document 1, etc., can be used, but it is not limited to this method. As mentioned above, in the SALSA method, even if the glycan composition is otherwise identical, the mass of the modified product will differ by 28 Da between the case where the sialic acid contained in the glycan is α2,3-binding and the case where it is α2,6-binding.

[0048] Next, for the pretreatment sample that has undergone specific chemical modification based on sialic acid binding, mass analysis is performed by the mass analysis unit 1. Mass spectrometry data within the specified m / z range obtained through mass analysis is sent from the mass analysis unit 1 to the data parsing unit 3 and stored in the data storage unit 30.

[0049] If the user specifies the data of the object to be parsed through the input unit 4 and instructs the parsing to be performed, then in the data parsing unit 3, according to Figure 2 The process shown will begin parsing and processing.

[0050] The glycan alignment condition setting unit 32 displays the prescribed glycan alignment condition setting screen on the display unit 5, accepting user input for glycan alignment conditions (step S1). However, it is also possible to automatically set default glycan alignment conditions without relying on user input. In addition to glycan alignment conditions, peak detection conditions used to detect peaks from mass spectrometry data can also be accepted by the user. The input or default glycan alignment conditions or peak detection conditions are saved in the glycan alignment condition saving unit 320.

[0051] Specifically, the glycan alignment conditions can be set to include, for example, the specific modification method for sialic acid binding used, the mass tolerance for estimating glycan composition, the assumed ion types, and the types and number of sugar residues (including sialic acid) used for alignment.

[0052] On the other hand, peak detection conditions can be set to include, for example, conditions such as signal strength or SN ratio, which serve as a threshold for identifying a peak.

[0053] If substantive analysis begins, the peak detection unit 31 reads the mass spectrometry data of the analysis target stored in the data storage unit 30, and detects a single isotope ion peak as the representative peak of each isotope peak group according to the peak detection conditions described above. Typically, in biologically derived molecules such as glycans, the peak with the smallest m / z value among multiple isotope ion peaks appearing at 1 Da intervals can be detected as a single isotope ion peak. Then, the peak detection unit 31 calculates the m / z value of each detected ion peak and generates a peak list (step S2). Alternatively, the average (centroid) m / z value of multiple isotope ion peaks can be calculated instead of the m / z value of a single isotope ion peak. That is, for each isotope ion peak group originating from the same glycan, the m / z value representing that group can be calculated.

[0054] Next, the glycan composition estimation unit 34 estimates the glycan composition for each peak listed in the peak list generated in step S2 according to the glycan comparison conditions stored in the glycan comparison condition storage unit 320, and determines glycan composition candidates (step S3). Specifically, under the limitation of the types and number of sugar residues specified as glycan comparison conditions, glycan compositions that match the m / z values ​​of the ion peaks are compared one by one within a specified quality accuracy range. Then, the matched glycan compositions are taken as glycan combination candidates corresponding to the ion peak. When glycan composition candidates are obtained, it is not limited to selecting only one candidate for a single ion peak; sometimes multiple candidates may be obtained.

[0055] Next, the isomer peak group detection unit 33 detects isomer peak groups from the peaks generated in step S2 that contain the same number of peaks presumed to be sialic acid and the same composition of sugar chains other than sialic acid (step S4).

[0056] Since the SALSA method is used here for sialic acid binding mode specific modification, corresponding to the SALSA method, adjacent ion peaks with an m / z difference of 28 Da between α2,6-sialic acid and α2,3-sialic acid are detected from the peak list. Furthermore, multiple detected peaks are determined to be a group of peaks of the same sialic acid binding isomer, that is, a group composed of isomers containing sialic acid that share the same number of sialic acids, the same type of sialic acid, and the same sugar chains other than sialic acid. The permissible error for the peak spacing used for this detection can be, for example, set to m / z 0.1. When determined to be a group of isomer peaks, the spacing between adjacent peaks varies depending on the sialic acid binding mode specific modification method used. Naturally, the number of ion peaks contained in a group of isomer peaks varies depending on the number of sialic acids in the sialic acid-containing sugar chain, the binding mode of the sialic acid, etc.

[0057] Furthermore, the isomer peak group detection unit 33 determines whether the ion peaks contained in multiple different isomer peak groups of the same sialic acid are arranged at intervals corresponding to the m / z value of the difference in the mass of sugar residues between different types of sialic acids as specified as the sugar chain comparison condition. Multiple isomer peak groups containing ion peaks that meet this m / z value interval are identified as peak groups of different sialic acid binding isomers, that is, peak groups containing the same number of sialic acids as the sugar chain composition other than sialic acids, but differing only in the type of sialic acid. Then, the ion peaks contained in all peak groups of different sialic acid binding isomers with the same number of sialic acids as the sugar chain composition other than sialic acids are identified as isomer ion peak groups.

[0058] Here, a specific example based on experiments is shown. In the experimental example conducted by the inventors of this invention, a mixture of glycans obtained by cleaving and concentrating the N-type glycans of fetuin, a blood glycoprotein of fetal bovine blood, with PNGase, one of the deglycanases, was used as a sample. Sialic acid in the sample was specifically modified for sialic acid binding using the SALSA method, and then the reduced ends of the glycans were labeled with anthranilic acid (AA tagging) to prepare the sample to be analyzed. Furthermore, the labeling modification is a pretreatment used to promote ionization in negative ion mode. Then, the sample thus obtained was analyzed using a matrix-assisted laser desorption / ionization trap time-of-flight mass spectrometry (MALDI-IT-TOFMS) in negative ion mode to obtain mass spectrometry data.

[0059] Furthermore, in the above experimental example, the sugar chain alignment conditions were determined as follows.

[0060] Sialic acid binding mode specific modification method: SALSA method

[0061] Labeling method: AA labeling

[0062] Permissible mass tolerance for estimating sugar composition: m / z 0.2

[0063] Permissible error for mass interval in isomer peak group detection: m / z 0.1

[0064] Types of ions: proton desorption ions

[0065] The types and numbers of sugar residues in the comparison targets are set as follows: Hexose 3–15, HexNAc 2–14, fucose (dHex) 0–2, Neu5Ac (sialic acid) 0–5, Neu5Gc (sialic acid) 0–5.

[0066] A portion of the mass spectra obtained through the above embodiments is shown in Figure 3 .exist Figure 3 Within the m / z range shown, through processing in step S2 of the mass spectrometry, m / z 3038.1, m / z 3066.2, m / z 3082.2, m / z 3094.2, m / z 3110.2, and m / z 3122.2 were detected as single isotopic ion peaks. Furthermore, based on the peak list containing these peaks, through processing in step S4, i.e., detecting peak groups arranged at intervals of 28 Da between α2,6-sialic acid and α2,3-sialic acid, two groups of isotopic sialic acid binding isomer peaks were detected, containing four monovalent ion peaks (m / z 3038.1, m / z 3066.2, m / z 3094.2, and m / z 3122.2) and two monovalent ion peaks (m / z 3082.2 and m / z 3110.2).

[0067] It was confirmed that peaks contained in a subset of the two groups of isomeric sialic acid binding isomers were arranged at m / z intervals of 16, which corresponds to the mass difference of sugar residues in the two sialic acids specified as glycan alignment criteria: N-acetylneuraminic acid (Neu5Ac) and N-hydroxyacetylneuraminic acid (Neu5Gc). Therefore, the two groups of isomeric sialic acid binding isomers were detected as isomeric peak groups of different sialic acid binding isomers, and all peaks contained in these isomeric peak groups of different sialic acid binding isomers were detected as isomeric peak groups with the same number of sialic acids and the same composition of glycans other than sialic acids.

[0068] Furthermore, as described above, the isomer peak group detection unit 33 classifies multiple peaks arranged according to the mass difference of sugar residues of different types of sialic acid into peaks with the same peak sequence number #n. In the above experimental example, such as Figure 4 As shown, the peak with the smallest mass value among all peaks in the two different sialic acid binding isomer peak groups "cluster 1" and "cluster 2" (cluster 1 and cluster 2), i.e., m / z 3038.1, is classified as peak sequence number #1 and belongs to peak group 1 (cluster 1) of the same sialic acid binding isomer. The peak with a mass value 28 Da greater than this peak and the second smallest mass value in the peak group, m / z 3066.2, is classified as peak sequence number #2. The peak with a mass value of m / z 3082.18, detected with a mass difference of 16 Da relative to this m / z 3066.2, is classified as the same peak sequence number #2 and belongs to peak group 2 (cluster 2) of the same sialic acid binding isomer. Similarly, the peaks at m / z 3094.2 and m / z 3110.2 are classified as peak sequence number #3, and the peak at m / z 3122.2 is classified as peak sequence number #4.

[0069] In addition, in this example, although a heterosial sialic acid binding isomer peak group was found among the multiple peaks contained in the two groups of homosial sialic acid binding isomer peak groups, it is also possible to detect a single glycan peak containing heterosial sialic acid with a residue mass difference of heterosial sialic acid in a portion of the peaks contained in a group of homosial sialic acid binding isomer peak groups, thereby forming a heterosial sialic acid binding isomer peak group.

[0070] return Figure 2 The flowchart continues to explain.

[0071] Next, for each glycan composition candidate obtained in step S3, the glycan composition filtering unit 35 applies the following limiting conditions related to the number of sialic acid, the number of sialic acid binding modes, the type of sialic acid, and the identity of glycan compositions other than sialic acid, thereby screening glycan composition candidates respectively (step S5).

[0072] <Qualification>

[0073] (1) In this example, four peak sequence numbers are obtained by the isomer peak group detection unit 33. Therefore, it is appropriate to regard the sugar chain composition candidate containing sialic acid as a sugar chain containing sialic acid, where each peak contains the same number of sialic acid molecules and the composition other than sialic acid is the same. Generally, when N peak sequence numbers are obtained, the sugar chain corresponding to the original peak contains N-1 or more sialic acid molecules.

[0074] (2) Furthermore, based on the relative masses of each peak, the peak classified as peak sequence number #1 should contain at least three α2,3-binding sialic acids; the peak classified as peak sequence number #2 should contain at least two α2,3-binding sialic acids and at least one α2,6-binding sialic acid; the peak classified as peak sequence number #3 should contain at least one α2,3-binding sialic acid and at least two α2,6-binding sialic acids; and the peak classified as peak sequence number #4 should contain at least three α2,6-binding sialic acids. In addition, peaks classified as isomeric peaks of the same sialic acid binding isomer group 2 should have a sugar chain composition with the same peak sequence number in isomeric peaks of the same sialic acid binding isomer group 1, where one sialic acid is obtained by replacing Neu5Ac with Neu5Gc.

[0075] In addition, for Figure 3 The aforementioned limiting conditions are also recorded for each peak.

[0076] Through the screening performed by the glycan composition filtering unit 35, the number of glycan composition candidates is typically greatly reduced. Corresponding to each peak in the isomer peak group detected in step S4, the glycan composition candidate list generation unit 36 ​​collects the glycan composition candidates estimated in step S3 and generates a glycan composition candidate list associated with each peak in the isomer peak group. Furthermore, the glycan composition candidate list generation unit 36 ​​displays the glycan composition candidates screened in step S5 (remaining in the filtering) and the unscreened glycan composition candidates in the glycan composition candidate list in a visually identifiable manner (step S6).

[0077] Then, the display processing unit 37 displays the generated list of candidate sugar chains on the screen of the display unit 5 (step S7).

[0078] Figure 5 and Figure 6 This is an example of a list of glycan composition candidates obtained from the six peaks in the above experimental example. In this example, the glycan composition candidates "compositions" selected as appropriate by the glycan composition filter 35 are shown in bold, while other glycan composition candidates are shown in thin, light-colored text. The method of display is not limited to this; for example, two glycan composition candidates can be shown using characters of different colors, or the background color can be changed, in ways that are less likely to cause visual errors. Furthermore, depending on user specifications, only appropriate glycan composition candidates can be selectively displayed, or only inappropriate glycan composition candidates can be selectively displayed.

[0079] Figure 5 and Figure 6The "Priority Methods" column on the right side of the document distinguishes between plausible glycan composition candidates deemed plausible by the method described in Patent Document 1 and implausible glycan composition candidates deemed implausible. However, this "Priority Methods" column is only for comparison between the method of this embodiment and methods in the prior art, and does not include the glycan composition candidate list displayed in the apparatus of this embodiment.

[0080] exist Figure 5 and Figure 6 In this embodiment, by comparing the distinction between bold / fine text in the "Composition" column with the distinction between "plausible / implausible" in existing methods, it can be found that some sugar composition candidates that are considered plausible in existing methods are considered implausible in the method of this embodiment.

[0081] Specifically, the appropriateness estimates of the glycan composition candidates for peaks m / z 3082.18 and m / z 3110.21 belonging to "cluster 2" differ between the prior art and the method of this embodiment. Figure 5 and Figure 6 In this study, it can be confirmed that the sugar composition candidates that are considered appropriate in the prior art and are shown in the dashed line in the existing method column are screened as inappropriate in the method of this embodiment.

[0082] Even according to existing methods, it is highly valuable in that appropriate screening of the proposed glycan composition candidates within glycans belonging to "cluster 1" can be performed. On the other hand, in the method of this embodiment, since uniform limiting conditions are used among glycan groups containing different types of sialic acid, appropriate glycan compositions can be screened even when only a portion of the binding isomer peaks of glycans containing any type of sialic acid are detected. Therefore, it has the advantage of being able to appropriately screen candidates for a wider range of glycans compared to existing methods.

[0083] When experimentally determining the glycan composition or structure for a peak with multiple glycan composition candidates, MS / MS analysis targeting the ion corresponding to that peak is required. In this case, the user, for example, indicates any peak as a precursor ion from the displayed mass spectrum or glycan composition candidate list. The precursor ion selection receiver 38 then receives this operation and selects the indicated ion peak as the precursor ion for MS / MS analysis.

[0084] The selected information is sent to the analysis control unit 2, which controls the mass analysis unit 1 to perform MS / MS analysis targeting the selected precursor ion. Specifically, product ion scanning measurement using the CID plasma dissociation method is performed. Thus, the mass analysis unit 1 performs MS / MS analysis on a sample containing glycans specifically modified by sialic acid binding, acquiring MS / MS mass spectrometry data. In the MS / MS mass spectrometry, since multiple product ion peaks originating from the target sialic acid-containing glycans are observed, the user can verify which of the multiple glycan composition candidates is appropriate, or verify whether a single glycan composition candidate is appropriate, based on the m / z value of this peak.

[0085] As described above, the glycan analysis system according to this embodiment can efficiently analyze the structure of glycans containing sialic acid, including the sialic acid binding mode.

[0086] In addition, as long as the substantive processing content in each step is not affected, appropriate adjustments can be made. Figure 2 The flowchart shown illustrates the sequence of steps. For example, the processing order of steps S3 and S4 can be interchanged; for instance, the setting of the glycan comparison conditions in step S1 can be implemented at any time before the glycan composition estimation in step S3.

[0087] Furthermore, in the isomer peak group detection unit 33, regardless of whether multiple sialic acids are specified as sugar residues for comparison in the sugar chain comparison condition setting unit 32, isomer peak groups of different sialic acid binding modes can be detected for all combinations of selectable sialic acids. In the sugar chain comparison condition setting unit 32, combinations of sialic acids with mass differences that match the detected peaks are added to the sugar residues of the comparison target, and sugar chain composition estimation is performed.

[0088] Furthermore, the above embodiments are merely one example of the present invention, and appropriate modifications, alterations, additions, etc., made within the scope of the spirit of the present invention should also be included within the scope of the claims of the present invention.

[0089] [Various options]

[0090] Those skilled in the art will understand that the above exemplary embodiments are specific examples of the following schemes.

[0091] (Item 1) A technical solution of the method for analyzing sialic acid-containing glycans of the present invention is a method for analyzing sialic acid-containing glycans based on mass spectrometry data obtained from mass analysis of a sample containing a sialic acid-containing glycan with a specifically modified sialic acid binding mode or a molecule modified by such glycan. The method comprises:

[0092] The process of setting comparison conditions involves setting the sugar chain comparison conditions.

[0093] The peak detection process involves detecting representative peaks for each isotope peak group based on the mass spectrometry data.

[0094] The composition estimation process involves estimating the sugar composition and determining candidate sugar compositions for each representative peak detected by the peak detection process, based on the sugar chain comparison conditions.

[0095] The peak cluster detection process involves detecting isomer peak clusters that contain the same number of peaks presumed to be sialic acid and the same number of peaks composed of sugar chains other than sialic acid, based on the representative peaks detected by the peak detection process.

[0096] In the glycan composition filtering process, for each peak contained in the isomer peak group, the candidate glycan compositions are screened by applying specified limiting conditions related to the number of sialic acids contained, the number of sialic acid binding modes, the type of sialic acid, and the identity of glycan compositions other than sialic acid.

[0097] The display process generates and displays a list of glycan composition candidates, including those filtered by the glycan composition filtering process and other glycan composition candidates, in a visually identifiable manner.

[0098] (Item 3) Furthermore, one technical solution of the analytical apparatus for glycans containing sialic acid according to the present invention is an analytical apparatus for analyzing glycans containing sialic acid based on mass spectrometry data obtained from mass analysis of a sample containing glycans containing sialic acid with a specifically modified sialic acid binding mode or molecules modified by such glycans, comprising:

[0099] The alignment condition setting unit accepts the user's specification of glycan alignment conditions and sets the glycan alignment conditions accordingly.

[0100] The peak detection unit detects representative peaks for each isotope peak group based on the mass spectrometry data.

[0101] The composition estimation unit estimates the glycan composition and obtains candidate glycan compositions for each representative peak detected by the peak detection unit according to the glycan comparison conditions.

[0102] The peak group detection unit detects isomer peak groups containing multiple peaks that are presumed to be sialic acid and have the same composition of sugar chains other than sialic acid, based on the representative peaks detected by the peak detection unit.

[0103] The glycan composition filtering section filters the glycan composition candidates by applying specified limiting conditions related to the number of sialic acids contained in each peak of the isomer peak group, the number of sialic acid binding modes, the type of sialic acid, and the identity of glycan compositions other than sialic acid.

[0104] The display processing unit generates and displays a list of glycan composition candidates, including those filtered by the glycan composition filtering unit and other glycan composition candidates, in a visually identifiable manner.

[0105] In the method for analyzing sialic acid-containing sugar chains described in item 1, the steps are not limited to being performed in the order described above, and their execution order can be appropriately replaced. For example, the isomer peak group detection step can be performed before the composition estimation step. In this case, it is sufficient to estimate the composition only for the peaks detected by the peak detection unit that are included in the peak group, without causing any obstacles to subsequent processing.

[0106] According to the method for analyzing sialic acid-containing glycans described in item 1 and the apparatus for analyzing sialic acid-containing glycans described in item 3, even in glycans containing sialic acid where the composition of glycans other than sialic acid is the same as the number of sialic acid present, but the binding mode of sialic acid differs from the type of sialic acid, and where no ion peak corresponding to a certain type of sialic acid combination corresponding to the expected binding mode is detected, it is still possible to accurately screen out candidate glycan compositions containing that specific type of sialic acid and present them to the user. This improves the efficiency of experimental work such as MS / MS analysis for verifying the suitability of the proposed candidate composition of sialic acid-containing glycans, and the analysis of the data collected therefrom, enabling faster and more accurate structural analysis of sialic acid-containing glycans.

[0107] (Item 2) In the glycan analysis method containing sialic acid described in Item 1, it can be set such that, in the peak group detection step, for sialic acid of an unspecified type, isomer peak group detection can also be attempted, and based on the isomer peak group information obtained as a result, the unspecified type of sialic acid is added to the comparison object in the glycan comparison conditions, and the isomer peak group detection process is performed again.

[0108] According to the method for analyzing sialic acid-containing glycans described in item 2, even if the sample contains sialic acid-containing glycans of a type not previously conceived by the user, the structure of the sialic acid-containing glycan can be analyzed.

[0109] Explanation of reference numerals in the attached figures

[0110] 1. Quality Analysis Department

[0111] 2. Analysis and Control Department

[0112] 3 Data Analysis Department

[0113] 30 Data Storage Department

[0114] 31 Peak Detection Department

[0115] 32 Glycan Alignment Condition Setting Section

[0116] 320 sugar chain alignment condition preservation section

[0117] 33 Isomer Peak Group Detection Department

[0118] 34. Estimated composition of sugar chains

[0119] The filtration section consists of 35 sugar chains.

[0120] List of 36 Glycan Composition Candidates (Generation Department)

[0121] 37 Display Processing Unit

[0122] 38 Precursor Ion Selective Acceptor

[0123] 4 Input Section

[0124] 5. Display section.

Claims

1. A method for resolving sialic acid-containing glycans, comprising a method for resolving sialic acid-containing glycans based on mass spectrometry data obtained from mass analysis of a sample containing a sialic acid-containing glycan with a specifically modified sialic acid binding mode, or a molecule modified by such a glycan, characterized in that... have: The process of setting comparison conditions involves setting the sugar chain comparison conditions. The peak detection process involves detecting representative peaks for each isotope peak group based on the mass spectrometry data. The composition estimation process involves estimating the sugar composition and determining candidate sugar compositions for each representative peak detected by the peak detection process, based on the sugar chain comparison conditions. The peak cluster detection process involves detecting isomer peak clusters that contain the same number of peaks presumed to be sialic acid and the same number of peaks composed of sugar chains other than sialic acid, based on the representative peaks detected by the peak detection process. In the glycan composition filtering process, for each peak contained in the isomer peak group, the candidate glycan compositions are screened by applying specified limiting conditions related to the number of sialic acids contained, the number of sialic acid binding modes, the type of sialic acid, and the identity of glycan compositions other than sialic acid. In the display step, a visually identifiable overview of the glycan composition candidates, including those filtered by the glycan composition filtering step and other glycan composition candidates, is generated and displayed. The isomer peak groups detected in the peak group detection process include peak groups of the same sialic acid-binding isomers and peak groups of different sialic acid-binding isomers.

2. The method for decomposing sialic acid-containing sugar chains as described in claim 1, characterized in that, In the peak group detection process, for sialic acid of unspecified type, isomer peak group detection is also attempted. Based on the isomer peak group information obtained as a result, the unspecified type of sialic acid is added to the comparison object in the sugar chain comparison conditions, and the isomer peak group detection process is performed again.

3. A device for resolving sialic acid-containing glycans, comprising a device for resolving sialic acid-containing glycans based on mass spectrometry data obtained from mass analysis of a sample containing a sialic acid-containing glycan with a specifically modified sialic acid binding mode, or a molecule modified by such glycan, characterized in that, have: The alignment condition setting unit accepts the user's specification of glycan alignment conditions and sets the glycan alignment conditions accordingly. The peak detection unit detects representative peaks for each isotope peak group based on the mass spectrometry data. The composition estimation unit estimates the glycan composition and obtains candidate glycan compositions for each representative peak detected by the peak detection unit according to the glycan comparison conditions. The peak group detection unit detects isomer peak groups containing multiple peaks that are presumed to be sialic acid and have the same composition of sugar chains other than sialic acid, based on the representative peaks detected by the peak detection unit. The glycan composition filtering section filters the glycan composition candidates by applying specified limiting conditions related to the number of sialic acids contained in each peak of the isomer peak group, the number of sialic acid binding modes, the type of sialic acid, and the identity of glycan compositions other than sialic acid. The display processing unit generates and displays a visually identifiable overview of the glycan composition candidates, including those filtered by the glycan composition filtering unit and other glycan composition candidates. The peak groups detected by the peak group detection unit include peak groups of isomers of the same sialic acid binding and peak groups of isomers of different sialic acid binding.

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