JP2022056935A - Molecule structure analysis system and molecule structure analysis method - Google Patents

Abstract

To perform the structure analysis of as many similar structure molecules as possible under the constraints of sample quantity and time.SOLUTION: Provided is a system for analyzing the structure of similar structure molecules. The system comprises: a mass analysis unit (1); desired mass spectrum peak selection units (240, 25, 22); reference spectrum acquisition units (3, 242, 25, 4) for acquiring a peak reference MSn spectrum from a preliminarily prepared information source; a control unit (20) for repeatedly executing an MSn analysis on a sample, using an ion having m/z in one peak among selected peaks as a precursor ion, as well as terminating repetition of the MSn analysis when a command described later is received; and spectrum assessment units (21, 23) for integrating the data with which MSn analysis is carried out, assessing the similarity of a measured MSn spectrum to the reference MSn spectrum, and outputting a command for analysis termination to the control unit when it is assessed that the similarity is high.SELECTED DRAWING: Figure 1

Description

The present invention relates to a molecular structure analysis system that analyzes the molecular structure of an organic compound by using mass spectrometry. The molecular structure analysis system according to the present invention is particularly suitable for analyzing the molecular structure of sugar chain-modified compounds such as glycoproteins and glycolipids.

Although many proteins and lipids exist in the living body, recent studies have revealed that most of them are expressed and regulated by some modification. In particular, glycoproteins and glycolipids in which sugar chains are bound to proteins and lipids are also called glycoconjugates, and are important for various in vivo phenomena such as cell-cell interaction, signal transduction, development / differentiation, fertilization, and diseases. It is becoming known that it plays a key role.

The structures of sugar chains that modify proteins and lipids are extremely diverse, and the structures have a great influence on the functions of proteins and lipids. Therefore, it is very important to distinguish and analyze differences in sugar chain structure in glycoproteins and glycolipids, and in recent years, mass spectrometry, especially MS ⁿ analysis (n is an integer of 2 or more) is widely used for such analysis. Has been done.

For example, when glycoprotein is the subject of analysis, generally, a free sugar chain mixture obtained by cutting out a sugar chain from a glycoprotein or a glycopeptide mixture obtained by decomposing a glycoprotein using a proteolytic enzyme is used. , Subject to mass analysis. When analyzing a glycopeptide mixture, the glycopeptide is separated or purified in advance for each peptide having the same amino acid sequence by using ion exchange, reverse phase liquid chromatograph (LC), or the like. , It is common to prepare a sample to be analyzed.

As disclosed in Non-Patent Document 1, when the glycopeptide mixture sample prepared as described above is mass-analyzed, a plurality of peaks having mass differences derived from sugars are observed in the mass spectrum. These peaks are peaks derived from glycopeptides in which sugar chains having different structures are bound to one common peptide. Since the main structures of peptides are common, these glycopeptides can be said to be similar structural molecules. In Non-Patent Document 1, in order to confirm the product ion derived from the sugar chain of one glycopeptide, one peak presumed to be derived from sugar is selected in the mass spectrum, and the mass-to-charge ratio of the peak (strictly speaking). Is an oblique letter "m / z", but is referred to as "mass-to-charge ratio" or " ^m / z", which is commonly used in this specification). .. This makes it possible to obtain product ion information useful for structural analysis of glycopeptides.

"Glycopeptide analysis using small MALDI digital ion trap mass spectrometer" MALDIminiTM-1 "", [Online], [Search on July 7, 2020], Shimadzu Corporation, Internet <URL: https: // www .an.shimadzu.co.jp/apl/vaccine/b100.pdf ＞

As can be seen from the example described in Non-Patent Document 1, many glycopeptide-derived peaks appear in the mass spectrum obtained for the glycopeptide mixture sample. In order to perform a comprehensive structural analysis of glycopeptides, it is desirable to perform MS ² analysis on all glycopeptide-derived peaks observed in the mass spectrum and obtain MS ² spectra. However, in general, time constraints and sample volume constraints limit the number of glycopeptide-derived peaks for which MS ² analysis can be performed.

That is, in the mass spectrometer described in Non-Patent Document 1, an ion source using the Matrix Assisted Laser Desorption / Ionization (MALDI) method is used, but in the MALDI method, the laser light is used. The amount of ions generated by irradiation is not very large, and the amount of ions varies widely. Therefore, the mass spectrum is acquired by repeating a large number of operations of irradiating the sample with laser light and performing mass spectrometry, and integrating the data obtained for each mass spectrometry. Since a part of the sample evaporates every time the laser beam is irradiated to the sample, if the number of times of mass spectrometry is increased, the prepared sample may be exhausted. This is the reason why the amount of sample is limited.

The present invention has been made in view of the above problems, and its main purpose is a molecular structure analysis system and a molecular structure analysis method capable of collecting product ion information about as many similar structural molecules as possible from the same sample. Is to provide. Another object of the present invention is to provide a molecular structure analysis system and a molecular structure analysis method capable of collecting product ion information about many similar structural molecules from the same sample within a limited time. be.

One aspect of the molecular structure analysis system according to the present invention, which has been made to solve the above problems, is a molecular structure analysis for analyzing the structures of a plurality of similar structural molecules contained in a sample, which are similar in structure to each other. It ’s a system,
A mass spectrometer capable of MS ⁿ analysis (n is an integer of 2 or more),
In the mass spectrum obtained for the sample, a peak selection unit that selects one or more peaks presumed to be derived from the target similar structural molecule, and a peak selection unit.
A reference spectrum acquisition unit that acquires one or more reference MS ⁿ spectra corresponding to the peaks selected or selected by the peak selection unit from a source prepared in advance, and a reference spectrum acquisition unit.
The sample was repeatedly subjected to MS ⁿ analysis using an ion having a mass-to-charge ratio at one of the peaks selected by the peak selection unit as a precursor ion, and was instructed by the spectrum determination unit described later. A control unit that controls the mass spectrometry unit so as to end the repetition of the MS ⁿ analysis at times.
The data obtained each time MS ⁿ analysis is performed under the control of the control unit is integrated, and the measured MS ⁿ spectrum obtained by the integration and the peak of the MS ⁿ analysis target acquired by the reference spectrum acquisition unit are used. A spectrum determination unit that determines the similarity with the corresponding reference MS ⁿ spectrum and issues a command to end the analysis to the control unit when the similarity is determined to be high.
It is equipped with.

Further, one aspect of the molecular structure analysis method according to the present invention, which has been made to solve the above problems, is a molecule for analyzing the structure of a plurality of similar structural molecules contained in a sample, which are similar in structure to each other. It is a structural analysis method
In the mass spectrum obtained for the sample, a peak selection step for selecting one or more peaks presumed to be derived from the target similar structural molecule, and a peak selection step.
A reference spectrum acquisition step of acquiring one or more reference ^MSn spectra corresponding to the peaks selected or selected in the peak selection step from a source prepared in advance, and a reference spectrum acquisition step.
An analysis execution step of repeatedly executing MS ⁿ analysis (n is an integer of 2 or more) using an ion having a mass-to-charge ratio at one of the peaks selected in the peak selection step as a precursor ion for the sample. When,
The data obtained each time the MS ⁿ analysis is performed in the analysis execution step is integrated, and the measured MS ⁿ spectrum obtained by the integration and the reference corresponding to the peak of the MS ⁿ analysis target acquired in the reference spectrum acquisition step. A spectrum determination step for determining the similarity with the MS ⁿ spectrum and ending the repetition of the MS ⁿ analysis when the similarity is determined to be high.
Is included.

Here, "plurality of similar structural molecules having similar structures to each other" refers to a group of molecules in which the main structure of the molecule is common and the incidental structure bonded to the main structure is different. That means. The above-mentioned peptides and proteins having the same amino acid sequence are the main structures, and the glycopeptides and glycoproteins having a sugar chain incidental structure are examples of the above-mentioned similar structural molecules. In addition, other complex sugar chains such as glycolipids are also examples of similar structural molecules.

Further, in the molecular structure analysis system according to the present invention, the "mass spectrometry unit capable of MS ⁿ analysis" includes, for example, a MALDI method, a Laser Desorption / Ionization (LDI) method, and a surface-assisted laser desorption / ionization (LDI) method. Mass spectrometry using an ion source using an ionization method that irradiates a sample with laser light such as the Surface Assisted Laser Desorption / Ionization (SALDI) method, or an ionization method that irradiates a sample with particle beams such as ion beams and neutral particle beams. It can be a device.

Further, in the present invention, the expressions "peak selected or selected by the peak selection unit" and "peak selected or selected in the peak selection step" refer to peak selection by the peak selection unit. This is because it may be performed prior to the acquisition of the reference MS ⁿ spectrum by the reference spectrum acquisition unit or after the acquisition of the reference MS ⁿ spectrum by the reference spectrum acquisition unit. That is, in the former case, the peak selection by the peak selection unit is performed first, and one or more reference ^MSn spectra corresponding to the peaks selected by the peak selection unit are acquired. On the other hand, in the latter case, the reference MS ⁿ spectrum is acquired by the reference spectrum acquisition unit, and then the peak selection unit corresponding to the acquired reference MS ⁿ spectrum is selected by the peak selection unit. In either case, as a result, the peak selected by the peak selection unit and the reference MS ⁿ spectrum acquired by the reference spectrum acquisition unit correspond to each other.

In the above aspect of the molecular structure analysis system and the analytical structure analysis method according to the present invention, MS ⁿ analysis using a certain ion as a precursor ion is repeatedly executed, and a mass having a predetermined mass-to-charge ratio range is executed for each MS ⁿ analysis. When the spectrum data is obtained, the data is integrated to create an actually measured MS ⁿ spectrum. In this case, the signal intensity of each peak on the measured MS ⁿ spectrum gradually increases each time the MS ⁿ analysis is performed. If the compound molecule corresponding to the peak of interest for MS ⁿ analysis (the peak selected as the precursor ion) is the same as the compound molecule determined as the reference MS ⁿ spectrum for that peak, then to some extent the data When the integration progresses and the spectral pattern of the measured MS ⁿ spectrum becomes clear, it is determined that the similarity between the measured MS ⁿ spectrum and the reference MS ⁿ spectrum is high. In response, the repetition of the MS ⁿ analysis is terminated.

Conventionally, in general, the number of times data is integrated is predetermined, and the measured MS ⁿ spectrum after the predetermined number of times is integrated is used for structural analysis. On the other hand, in the present invention, it is usually determined that the measured MS ⁿ spectrum in which data is integrated less than a predetermined number of times has high similarity to the reference MS ⁿ spectrum, and is therefore necessary for structural analysis. The number of data integrations required to collect product ion information, that is, the number of MS ⁿ analysis executions, is small. Once product ion information for one peak observed on the mass spectrum has been collected, repeated MS ⁿ analysis for another peak observed on the same mass spectrum may be performed. These ^MSn analyzes can be repeated as long as time permits or as long as the amount of sample allows.

Therefore, according to the above aspect of the molecular structure analysis system and the analytical structure analysis method according to the present invention, it is possible to collect product ion information for as many similar structural molecules as possible from the same sample. In addition, product ion information about many similar structural molecules can be collected from the same sample within a limited time. As a result, structural analysis of compounds such as glycolipides and glycolipids can be efficiently performed.

The block block diagram of the molecular structure analysis system which is one Embodiment of this invention. The flowchart which shows the analysis procedure in the molecular structure analysis system of this embodiment. The flowchart which shows the analysis procedure in the molecular structure analysis system of this embodiment. Explanatory drawing of analysis operation in the molecular structure analysis system of this embodiment.

Hereinafter, an embodiment of the molecular structure analysis system according to the present invention will be described with reference to the accompanying drawings. FIG. 1 is a block configuration diagram of a main part of the molecular structure analysis system of the present embodiment.

This molecular structure analysis system controls the operations of the mass spectrometric unit 1 that executes mass spectrometry (MS ¹ analysis and MS ² analysis) on the sample and collects data, and the mass spectrometric unit 1 and the mass spectrometric unit 1. A control / processing unit 2 that analyzes and processes the data collected in the above, a spectrum database (DB) 3 that stores standard mass spectra of various compound molecules in advance, an input unit 4 that is a user interface, and a display unit. 5 and.

The mass spectrometer 1 is a MALDI mass spectrometer such as a MALDI ion trap type mass spectrometer or a MALDI ion trap flight time type mass spectrometer. The mass spectrometer 1 has a chamber 10 that is evacuated by a vacuum pump (not shown), and inside the chamber 10, a sample stage 11, an ion optical system 15 including an extraction electrode, a mirror 13, and ion dissociation capable of MS ⁿ analysis. -The separation unit 16 and the detection unit 17 are arranged. Further, a laser beam irradiation unit 14 is arranged on the outside of the chamber 10. When the mass spectrometer 1 is a MALDI ion trap type mass spectrometer, the ion dissociation / separation unit 16 is an ion trap. When the mass spectrometer 1 is a MALDI ion trap time-of-flight mass spectrometer, the ion dissociation / separation unit 16 is an ion trap and a time-of-flight mass spectrometer.

The control / processing unit 2 has, as functional blocks, an analysis control unit 20, a spectrum integration unit 21, an ion peak detection unit 22, a spectrum similarity calculation unit 23, a display processing unit 24, an instruction reception unit 25, and a structural analysis unit 26. ,including. The display processing unit 24 includes an actually measured MS ¹ spectrum display processing unit 240, an actually measured MS ² spectrum display processing unit 241 and a reference MS ² spectrum display processing unit 242 as lower functional blocks.

In this device, the substance of the control / processing unit 2 is a general-purpose personal computer or a workstation with higher performance, and each of the above functions is performed by operating a dedicated control / processing program installed in such a computer on the computer. The function of the block can be realized. However, the spectrum DB 3 may be included in the control / processing unit 2 or may be an external database not included in the control / processing unit 2. In the latter case, the database server may be accessed through a network line such as the Internet, and only necessary data may be acquired from the database server.

The mass spectrometer 1 is not limited to a mass spectrometer using a MALDI ion source, but a mass spectrometer equipped with an ion source by ionization using laser light other than the MALDI method such as the LDI method and the SALDI method. Can be used. Further, for example, a mass spectrometer equipped with an ion source by ionization using a particle beam such as an ion beam or a neutral particle beam, such as an ion source used in a secondary ion mass spectrometry method, can also be used.

Further, the ion dissociation operation in the ion dissociation / separation unit 16 can typically utilize low-energy collision-induced dissociation (CID), but is not limited to this, and high-energy CID and light can be used. , A method of ion dissociation using a radical species or the like can also be used.

Next, an example of the analysis operation using the molecular structure analysis system of the present embodiment will be described with reference to FIGS. 2 to 4 in addition to FIG. 2 and 3 are flowcharts showing the analysis procedure. FIG. 4 is an explanatory diagram of the analysis operation.

Here, as an example, it is assumed that the analysis target is glycoprotein. A glycopeptide mixture is obtained by degrading a glycoprotein with a proteolytic enzyme. Then, using reverse phase LC, ion exchange, or the like for this glycopeptide mixture, each peptide having the same amino acid sequence is separated or purified to obtain a glycopeptide mixture sample. This glycopeptide mixture sample contains glycopeptides in which sugar chains of various structures are bound to peptides having the same amino acid sequence. A predetermined matrix is added to this sample to prepare a sample S on the sample plate 12.

As shown in FIG. 1, when the sample plate 12 on which the sample S is formed is placed on the sample stage 11 and the user instructs the input unit 4 to execute the analysis, the vacuum pump (not shown) performs vacuum exhaust. The inside of the chamber 10 is brought into a predetermined vacuum state. After that, the analysis control unit 20 controls each unit of the mass spectrometry unit 1 and executes a normal mass spectrometry (MS ¹ analysis) for the sample S (step S1).

That is, the laser light emitted from the laser light irradiation unit 14 in a pulsed manner irradiates the sample S on the sample plate 12 through the mirror 13. Upon irradiation with laser light, the compound in the sample S is ionized, and the generated ions are introduced into the ion dissociation / separation unit 16 via the ion optical system 15. At this time, ion dissociation is not performed, and the ion dissociation / separation unit 16 separates the introduced various ions according to the mass-to-charge ratio. The detection unit 17 sequentially detects the separated ions according to the mass-to-charge ratio and outputs an ionic strength signal according to the amount of ions.

The above-mentioned mass spectrometry for the sample S is performed a plurality of times (usually a considerably large number of times), and the relationship between the mass-to-charge ratio and the ion intensity over a predetermined mass-to-charge ratio range is determined for each mass spectrometry. The indicated mass spectrum data is input to the spectrum integration unit 21. The spectrum integration unit 21 creates a final mass spectrum for the sample S by integrating the mass spectrum data one after another. The ion peak detection unit 22 detects peaks in the mass spectrum according to a predetermined algorithm to obtain a mass-to-charge ratio value of each peak, and further detects an ion peak having a specific mass-to-charge ratio difference among a large number of peaks. (Step S2).

The glycopeptide mixture sample to be analyzed mainly has peptides having the same amino acid sequence as a common main structure, and the structure of the non-reducing end of the sugar chain is different from this main structure, or a modification to the sugar chain. Includes various glycopeptides to which sugar chains with different structures are bound, such as different structures. Therefore, in general, two or more ion peaks having a mass-to-charge ratio difference corresponding to a known difference in the structure of the non-reducing end of the sugar chain and a difference in the modified product with respect to the sugar chain are observed in the mass spectrum. Therefore, by searching for peaks with mass-to-charge ratio differences derived from known sugar chain constituents, it is possible to detect similar structural molecules, that is, ion peaks corresponding to glycopeptides having a common peptide. can.

The actual measurement MS ¹ spectrum display processing unit 240 clearly displays the ion peak group detected in step S2 on the screen of the display unit 5 on the created actual measurement mass spectrum (step S3). FIG. 4A is an example of the actually measured mass spectrum displayed at this time. In FIG. 4A, the peak marked with a circle near the peak top is an ion peak that is a candidate for a similar structural molecule. Further, ◯ and Δ in the figure are mass-to-charge ratio differences derived from known constituents of sugar chains.

The user (operator) confirms this mass spectrum on the display screen, selects one or more peaks as precursor ions, and instructs the input unit 4 (step S4). In the example of FIG. 4A, it is assumed that one peak indicated by an arrow is selected by the user. The instruction receiving unit 25 receives this instruction and instructs the reference MS ² spectrum display processing unit 242 to indicate the mass-to-charge ratio value of the peak instructed. When there are a plurality of specified peaks, the reference MS ² spectrum display processing unit 242 extracts one of them according to predetermined conditions (for example, in descending order of mass-to-charge ratio), and has one specified peak. If it is only, one of them is unconditionally extracted (step S5).

The reference MS ² spectrum display processing unit 242 accesses the spectrum DB3, and based on the indicated mass-to-charge ratio value, one or more compound molecules (in this case, sugar) corresponding to the above-mentioned one extracted peak. Search for the peptide) and obtain the standard MS ² spectrum associated with the compound molecule. Then, the information of the compound molecule is displayed on the screen of the display unit 5 together with the MS ² spectrum (step S6). When there are a plurality of compound molecules as a search result and there are a plurality of displayed MS ² spectra, the user selects one of the plurality of MS ² spectra as a reference MS ² spectrum (step). S7). If there is only one MS ² spectrum as the search result, it becomes the reference MS ² spectrum as it is.

FIG. 4C is an example of this reference MS ² spectrum. In the reference MS ² spectrum, peaks of various product ions generated by dissociation of precursor ions are observed. In FIG. 4C, the precursor ions are shown by dotted lines, but some of the precursor ions are not dissociated depending on the dissociation conditions such as the energy applied to the ions during collision-induced dissociation. Can also be left.

Information on the mass-to-charge ratio of the peak extracted in step S5 is sent to the analysis control unit 20, and each unit of the mass spectrometry unit 1 starts the MS ² analysis using the mass-to-charge ratio as the precursor ion. Is controlled (step S8).

As a result, in the mass spectrometry unit 1, the laser beam is irradiated to the sample S, and the compound-derived ions generated from the sample S are introduced into the ion dissociation / separation unit 16. At this time, in the ion dissociation / separation unit 16, precursor ions are first selected, and other ions are excluded. Ions with selected specific mass-to-charge ratios are dissociated by collision-induced dissociation to produce various product ions. The product ions are separated according to the mass-to-charge ratio, and the detection unit 17 detects the product ions in order and outputs an ionic strength signal according to the amount of ions.

Data representing the MS ² spectrum (product ion spectrum) over a predetermined mass-to-charge ratio range can be obtained in response to a single laser beam irradiation. The amount of ions produced by a single laser beam irradiation is relatively small, and the amount of product ions corresponding to a specific precursor ion is even smaller. Therefore, the intensity of each product ion peak is not very high in the MS ² spectrum. Therefore, the spectrum integration unit 21 integrates the MS ² spectrum data obtained for each MS ² analysis and creates an actually measured MS ² spectrum. Therefore, each time the MS ² analysis is performed, the measured MS ² spectrum is updated (step S9). The measured MS ² spectrum display processing unit 241 displays the measured MS ² spectrum updated by data integration on the screen of the display unit 5 as shown in FIG. 4 (B). The signal strength of each peak on the displayed measured MS ² spectrum gradually increases over time.

Each time the measured MS ² spectrum is updated, the spectrum similarity calculation unit 23 determines the similarity of the spectral pattern between the updated measured MS ² spectrum and one reference MS ² spectrum by an appropriate algorithm known. The calculation is performed according to the dot product method or the like (step S10). Similarity is indicated as a scoring value by using any of the scoring techniques known as library retrieval techniques for peptides and metabolites. Then, the spectrum similarity calculation unit 23 determines whether or not the calculated score value is equal to or higher than a predetermined threshold value (step S11).

When calculating the similarity, the peak pattern shape of the spectrum is important, and the intensity value of each peak is not important. Therefore, for example, the peak with the maximum ionic strength on the measured MS ² spectrum is used for each. The similarity can be calculated after standardizing the peak intensities. Further, the measured MS ² spectrum display processing unit 241 displays the calculated score value of the similarity in the measured MS ² spectrum displayed on the screen of the display unit 5. Of course, the score value of the similarity may be displayed separately from the measured MS ² spectrum.

Even if the peak extracted in step S5, that is, the precursor ion to be analyzed in MS ² is an ion derived from the compound selected as the reference MS ² spectrum, the background noise remains as long as the number of data integrations is small. Due to the influence, the similarity does not increase so much and falls below the threshold value. If No is determined in step S11, the spectrum integration unit 21 determines whether or not the number of times of data integration has reached a predetermined upper limit value (step S12), and if not, returns to step S9. .. Therefore, the processes of steps S9 to S12 are repeated until the similarity of the MS ² spectra exceeds the threshold value or the number of times of data integration reaches the upper limit value.

When the precursor ion to be analyzed in MS ² is an ion derived from a compound selected as the reference MS ² spectrum, when the number of data integration increases and the intensity of each peak in the measured MS ² spectrum becomes higher than a certain level, The similarity becomes considerably large and exceeds the threshold. When the similarity exceeds the threshold value, the spectral similarity calculation unit 23 records the result and notifies the analysis control unit 20 of the end of the analysis. As a result, the process proceeds from step S11 to S13, and the analysis control unit 20 ends the repetition of the MS ² analysis.

On the other hand, for example, when the precursor ion to be analyzed in MS ² is different from the ion derived from the compound selected as the reference MS ² spectrum, the similarity does not increase so much even if the number of times of data integration increases, and the similarity thereof. The number of times data is integrated reaches the upper limit without exceeding the threshold value. That is, if Yes is determined in step S12, the spectrum integration unit 21 records the result and notifies the analysis control unit 20 of the end of the analysis. As a result, the process proceeds from step S12 to S13, and the analysis control unit 20 ends the repetition of the MS ² analysis.

That is, it is determined as Yes in step S11 and the repetition of the MS ² analysis is completed when the product ion information necessary for the structural analysis of one compound molecule in the sample S is obtained. On the other hand, it is determined as Yes in step S12 and the repetition of the MS ² analysis is terminated when the acquisition of the product ion information necessary for the structural analysis of one compound molecule in the sample S fails.

The reference MS ² spectrum display processing unit 242 determines whether or not there are other precursor ions selected in step S4 (step S14), returns to step S5 if any, and returns to step S5 for another precursor ion in step S5. The subsequent processing is executed again. As a result, when the user specifies multiple peaks and precursor ions on the MS ¹ spectrum, the precursor ions are targeted for MS ² analysis one by one and the product ion information required for structural analysis is collected. It will be. That is, product ion information about a plurality of pseudo-structured molecules contained in the sample S is sequentially collected.

Further, as described above, when it is determined to be Yes in step S12, the information necessary for the structural analysis of the compound molecule corresponding to the designated precursor ion has not been acquired. Therefore, it is also possible to have the user select another reference MS ² spectrum for the same precursor ion so that the processing after step S8 is performed again. This calculates the similarity between the measured MS ² spectrum and another reference MS ² spectrum, so if the selected precursor ion is an ion derived from a compound corresponding to this other reference MS ² spectrum, then the precursor ion It is possible to obtain product ion information necessary for structural analysis of the compound molecule corresponding to the above.

However, the same result is obtained by allowing the user to select a plurality of reference MS ² spectra in step S7, calculating the similarity between the plurality of reference MS ² spectra and one actually measured MS ² spectrum, and calculating the similarity thereof. It can also be obtained by ending the MS ² analysis when one of the plurality of similarities exceeds the threshold. According to this method, the number of actual MS ² analyzes is reduced, which is advantageous for both shortening the analysis time and reducing the consumption of the sample.

The threshold value as a reference for determining the similarity in step S11 depends on the score adopted as the similarity. Therefore, it is advisable to determine the threshold value in advance according to the score of similarity. Therefore, for example, the distribution of the score of the degree of similarity between the MS ² spectrum of a specific glycopeptide and the MS ² spectrum targeting other peaks (including a glycopeptide having a similar structure and a molecule having a dissimilar structure) is experimentally conducted in advance. It is advisable to set a threshold value appropriately so that the specificity and sensitivity to similar structural molecules are not low in the score distribution.

When the series of processes as described above is completed, the structural analysis unit 26 uses the reference MS ² spectrum for which the similarity is determined to be equal to or higher than the threshold value or the product ion information obtained from the reference MS 2 spectrum for each precursor ion. The structure of the compound molecule corresponding to the precursor ion can be estimated.

As described above, in the molecular structure analysis system of the present embodiment, it is possible to collect product ion information necessary for structural analysis while reducing the number of times of integration of data obtained by MS ² analysis. Since the number of MS ² analyzes for collecting product ion information required for structural analysis of one compound molecule (glycide in the above example) can be reduced, many samples S can be analyzed within a fixed time unless the sample S is exhausted. Product ion information of the compound molecule of the above can be obtained. In addition, product ion information of a larger number of compound molecules can be obtained while the same amount of sample S is consumed.

In the above description, the user selects the reference MS ² spectrum after the user specifies one or more peaks as precursor ions on the actually measured mass spectrum, but the order can be changed. That is, the user may select the reference MS ² spectrum of the target compound, and then specify the peak estimated to correspond to the target compound on the actually measured mass spectrum.

Further, in the description of the above embodiment, the structural analysis of the glycopeptide has been carried out, but the present invention is not limited to this, and the present invention is applied to the overall structural analysis of similar structural molecules having a common main structure such as glycolipids. be able to.

Further, the above-described embodiments and modifications are merely examples of the present invention, and it is natural that the present invention is included in the claims even if modifications, modifications, additions, etc. are appropriately made within the scope of the present invention.

[Various aspects]
It will be understood by those skilled in the art that the above-mentioned exemplary embodiments are specific examples of the following embodiments.

(Clause 1) One aspect of the molecular structure analysis system according to the present invention is a molecular structure analysis system for analyzing the structures of a plurality of similar structural molecules contained in a sample, which are similar in structure to each other.
A mass spectrometer capable of MS ⁿ analysis (n is an integer of 2 or more),
In the mass spectrum obtained for the sample, a peak selection unit that selects one or more peaks presumed to be derived from the target similar structural molecule, and a peak selection unit.
A reference spectrum acquisition unit that acquires one or more reference MS ⁿ spectra corresponding to the peaks selected or selected by the peak selection unit from a source prepared in advance, and a reference spectrum acquisition unit.
The sample was repeatedly subjected to MS ⁿ analysis using an ion having a mass-to-charge ratio at one of the peaks selected by the peak selection unit as a precursor ion, and was instructed by the spectrum determination unit described later. A control unit that controls the mass spectrometry unit so as to end the repetition of the MS ⁿ analysis at times.
The data obtained each time MS ⁿ analysis is performed under the control of the control unit is integrated, and the measured MS ⁿ spectrum obtained by the integration and the peak of the MS ⁿ analysis target acquired by the reference spectrum acquisition unit are used. A spectrum determination unit that determines the similarity with the corresponding reference MS ⁿ spectrum and issues a command to end the analysis to the control unit when the similarity is determined to be high.
It is equipped with.

(Section 6) Further, one aspect of the molecular structure analysis method according to the present invention is a molecular structure analysis method for analyzing the structures of a plurality of similar structural molecules contained in a sample, which are similar in structure to each other. hand,
In the mass spectrum obtained for the sample, a peak selection step for selecting one or more peaks presumed to be derived from the target similar structural molecule, and a peak selection step.
A reference spectrum acquisition step of acquiring one or more reference ^MSn spectra corresponding to the peaks selected or selected in the peak selection step from a source prepared in advance, and a reference spectrum acquisition step.
An analysis execution step of repeatedly executing MS ⁿ analysis (n is an integer of 2 or more) using an ion having a mass-to-charge ratio at one of the peaks selected in the peak selection step as a precursor ion for the sample. When,
The data obtained each time the MS ⁿ analysis is performed in the analysis execution step is integrated, and the measured MS ⁿ spectrum obtained by the integration and the reference corresponding to the peak of the MS ⁿ analysis target acquired in the reference spectrum acquisition step. A spectrum determination step for determining the similarity with the MS ⁿ spectrum and ending the repetition of the MS ⁿ analysis when the similarity is determined to be high.
Is included.

Here, although n can theoretically take an integer of 2 or more, n is usually 2.

For example, in a mass spectrometer equipped with a MALDI ion source, not only the MS ⁿ spectrum but also a normal mass spectrum is created by integrating the data obtained by a plurality of mass analyzes. In general, the number of times mass spectrometry is executed, that is, the number of times data is integrated is predetermined, and the measured mass spectrum or the measured MS ⁿ spectrum after the predetermined number of times of integration is used for analysis. On the other hand, in the present invention, the similarity between the measured MS ⁿ spectrum and the reference MS ⁿ spectrum is determined even during data integration, and if it is determined that the similarity is high, the MS ⁿ analysis for the precursor ion at that time is repeated. Is discontinued.

According to the molecular structure analysis system described in the first item and the analytical structure analysis method described in the sixth item, product ion information about as many similar structural molecules as possible can be collected from the same sample. In addition, product ion information about many similar structural molecules can be collected from the same sample within a limited time. As a result, structural analysis of compounds such as glycolipides and glycolipids can be efficiently performed.

(Item 2) In the molecular structure analysis system according to item 1, the peak selection unit is
In the mass spectrum obtained for the sample, a mass spectrum display processing unit that clearly displays a peak presumed to be derived from a molecule having a similar structure on the display unit, and a mass spectrum display processing unit.
An instruction receiving unit that allows the user to specify one or more peaks on the displayed mass spectrum and selects the specified peak as a peak derived from a similar structural molecule.
Can be included.

(Section 7) Similarly, in the molecular structure analysis method according to Section 6, the peak selection step is performed.
In the mass spectrum obtained for the sample, a mass spectrum display step of clearly displaying a peak presumed to be derived from a molecule having a similar structure and displaying it on the display unit, and a mass spectrum display step.
An instruction acceptance step that allows the user to specify one or more peaks on the displayed mass spectrum and selects the specified peak as a peak derived from a similar structural molecule for the purpose.
Can be included.

For example, when the sample is a purified glycopeptide mixture, a plurality of peaks having a mass-to-charge ratio difference due to a difference in the structure of sugar chains are usually observed in the mass spectrum. According to the molecular structure analysis system described in the second item and the analytical structure analysis method described in the seventh item, the user confirms this mass spectrum and then selects the peak of interest by referring to, for example, the mass-to-charge ratio difference. However, it is possible to narrow down the peak and collect product ion information. As a result, it is possible to prevent the ^MSn analysis from being performed for peaks that the user is not interested in, and it is possible to suppress wasteful consumption of the sample and shorten the analysis time.

(Clause 3) In the molecular structure analysis system according to the first or second paragraph, the reference spectrum acquisition unit is
The source of the information is a database containing ^MSn spectra of similar structural molecules related to compound molecules that are the purpose of analysis.
Have the user select an MS ⁿ spectrum selected by the peak selection unit or corresponding to the selected peak from the database, or have the user select one or more MS n spectra from a plurality of MS ⁿ spectra extracted from the database. The MS ⁿ spectrum can be selected.

(Item 8) Similarly, in the molecular structure analysis method according to the item 6 or 7, the reference spectrum acquisition step is performed.
The source of the information is a database containing ^MSn spectra of similar structural molecules related to compound molecules that are the purpose of analysis.
Have the user select from the database the MS ⁿ spectra selected or corresponding to the peaks selected in the peak selection step, or have the user select one or more MS n spectra from a plurality of MS ⁿ spectra extracted from the database. The MS ⁿ spectrum can be selected.

The database itself may or may not be included in this system. That is, it is also possible to use an external database that is generally provided.
According to the molecular structure analysis system described in Section 3 and the analytical structure analysis method described in Section 8, the user can determine the compound whose structure is desired to be grasped, and thus whether or not the target compound molecule is contained in the sample. Can be grasped quickly. Further, when a plurality of molecules having similar structures having the same or similar mass-to-charge ratio exist in the database, it is possible to determine which molecule is similar to the product ion information at the user's discretion.

(Item 4) In the molecular structure analysis system according to any one of the items 1 to 3, the reference MS ² spectrum and the actually measured MS ² spectrum are displayed on the screen of the display unit. A processing unit may be further provided.

(Section 9) Similarly, in the molecular structure analysis method according to any one of paragraphs 6 to 8, the reference MS ² spectrum and the measured MS ² spectrum are displayed on the screen of the display unit. The display step may further include.
The molecular structure analysis method according to any one of claims 6 to 8.

According to the molecular structure analysis system described in Section 4 and the analytical structure analysis method described in Section 9, the user screens a reference MS ² spectrum and an actually measured MS ² spectrum that changes over time. Can be compared above. Thereby, for example, when the measured MS ² spectrum is clearly different from the intention, or conversely, when it can be confirmed that they are visually similar, the user himself / herself instructs to terminate the repetition of the MS ⁿ analysis. Can be done.

(Item 5) Further, in the molecular structure analysis system according to any one of the items 1 to 4, the spectrum determination unit calculates a degree of similarity indicating the similarity of the MS ⁿ spectrum numerically, and the similarity is calculated. It may further include a display processing unit that displays the similarity in association with the mass spectrum or the measured ^MS2 spectrum.

(Item 10) Similarly, in the molecular structure analysis method according to any one of the items 6 to 9, in the spectrum determination step, the similarity indicating the similarity of the MS ⁿ spectra is calculated. It may further include a display step of displaying the similarity in association with the mass spectrum or the measured ^MS2 spectrum.

According to the molecular structure analysis system described in the fifth item and the analytical structure analysis method described in the sixth item, the user can numerically confirm the similarity between the reference MS ² spectrum and the actually measured MS ² spectrum. As a result, the user himself / herself can judge the similarity and accurately instruct the termination of the repetition of the MS ⁿ analysis.

1 ... Mass spectrometric unit 10 ... Chamber 11 ... Sample stage 12 ... Sample plate 13 ... Mirror 14 ... Laser light irradiation unit 15 ... Ion optical system 16 ... Ion dissociation / separation unit 17 ... Detection unit 2 ... Control / processing unit 20 ... Analysis Control unit 21 ... Spectral integration unit 22 ... Ion peak detection unit 23 ... Spectral similarity calculation unit 24 ... Display processing unit 240 ... Actual measurement MS ¹ Spectrum display processing unit 241 ... Actual measurement MS ² Spectrum display processing unit 242 ... Reference MS ² Spectrum display Processing unit 25 ... Instruction receiving unit 26 ... Structural analysis unit 3 ... Spectrum DB
4 ... Input unit 5 ... Display unit

Claims (10)

A molecular structure analysis system for analyzing the structures of multiple similar structural molecules contained in a sample that are similar in structure to each other.
A mass spectrometer capable of MS ⁿ analysis (n is an integer of 2 or more),
In the mass spectrum obtained for the sample, a peak selection unit that selects one or more peaks presumed to be derived from the target similar structural molecule, and a peak selection unit.
A reference spectrum acquisition unit that acquires one or more reference MS ⁿ spectra corresponding to the peaks selected or selected by the peak selection unit from a source prepared in advance, and a reference spectrum acquisition unit.
The sample was repeatedly subjected to MS ⁿ analysis using an ion having a mass-to-charge ratio at one of the peaks selected by the peak selection unit as a precursor ion, and was instructed by the spectrum determination unit described later. A control unit that controls the mass spectrometry unit so as to end the repetition of the MS ⁿ analysis at times.
The data obtained each time MS ⁿ analysis is performed under the control of the control unit is integrated, and the measured MS ⁿ spectrum obtained by the integration and the peak of the MS ⁿ analysis target acquired by the reference spectrum acquisition unit are used. A spectrum determination unit that determines the similarity with the corresponding reference MS ⁿ spectrum and issues a command to end the analysis to the control unit when the similarity is determined to be high.
A molecular structure analysis system equipped with. The peak selection unit
In the mass spectrum obtained for the sample, a mass spectrum display processing unit that clearly displays a peak presumed to be derived from a molecule having a similar structure on the display unit, and a mass spectrum display processing unit.
An instruction receiving unit that allows the user to specify one or more peaks on the displayed mass spectrum and selects the specified peak as a peak derived from a similar structural molecule.
The molecular structure analysis system according to claim 1. The reference spectrum acquisition unit is
The source of the information is a database containing ^MSn spectra of similar structural molecules related to compound molecules that are the purpose of analysis.
Have the user select an MS ⁿ spectrum selected by the peak selection unit or corresponding to the selected peak from the database, or have the user select one or more MS n spectra from a plurality of MS ⁿ spectra extracted from the database. The molecular structure analysis system according to claim 1 or 2, wherein the MS ⁿ spectrum is selected. The molecular structure analysis system according to any one of claims 1 to 3, further comprising a display processing unit that displays the reference MS ² spectrum and the actually measured MS ² spectrum on the screen of the display unit. The spectrum determination unit further includes a display processing unit that calculates a similarity indicating the similarity of the MS ⁿ spectrum numerically and displays the similarity in association with the mass spectrum or the measured MS ² spectrum. The molecular structure analysis system according to any one of 4 to 4. It is a molecular structure analysis method for analyzing the structure of a plurality of similar structural molecules contained in a sample, which are similar in structure to each other.
In the mass spectrum obtained for the sample, a peak selection step for selecting one or more peaks presumed to be derived from the target similar structural molecule, and a peak selection step.
A reference spectrum acquisition step of acquiring one or more reference ^MSn spectra corresponding to the peaks selected or selected in the peak selection step from a source prepared in advance, and a reference spectrum acquisition step.
An analysis execution step of repeatedly executing MS ⁿ analysis (n is an integer of 2 or more) using an ion having a mass-to-charge ratio at one of the peaks selected in the peak selection step as a precursor ion for the sample. When,
The data obtained each time the MS ⁿ analysis is performed in the analysis execution step is integrated, and the measured MS ⁿ spectrum obtained by the integration and the reference corresponding to the peak of the MS ⁿ analysis target acquired in the reference spectrum acquisition step. A spectrum determination step for determining the similarity with the MS ⁿ spectrum and ending the repetition of the MS ⁿ analysis when the similarity is determined to be high.
Molecular structure analysis method including. The peak selection step is
In the mass spectrum obtained for the sample, a mass spectrum display step of clearly displaying a peak presumed to be derived from a molecule having a similar structure and displaying it on the display unit, and a mass spectrum display step.
An instruction acceptance step that allows the user to specify one or more peaks on the displayed mass spectrum and selects the specified peak as a peak derived from a similar structural molecule for the purpose.
6. The molecular structure analysis method according to claim 6. The reference spectrum acquisition step is
The source of the information is a database containing ^MSn spectra of similar structural molecules related to compound molecules that are the purpose of analysis.
Have the user select from the database the MS ⁿ spectra selected or corresponding to the peaks selected in the peak selection step, or have the user select one or more MS n spectra from a plurality of MS ⁿ spectra extracted from the database. The molecular structure analysis method according to claim 6 or 7, wherein the MS ⁿ spectrum is selected. The molecular structure analysis method according to any one of claims 6 to 8, further comprising a display step of displaying the reference MS ² spectrum and the actually measured MS ² spectrum on the screen of the display unit. 6. The spectrum determination step further includes a display step of calculating a similarity indicating the similarity of the MS ⁿ spectrum numerically and displaying the similarity in association with the mass spectrum or the measured MS ² spectrum. 9. The molecular structure analysis method according to any one of 9.

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