JP2022063454A - Organic compound structure analysis system and organic compound structure analysis assisting program - Google Patents

Abstract

To provide a sugar peptide structure analysis system that heightens comprehensiveness including a sugar peptide the elution time of which drifts.SOLUTION: Provided is a system for analyzing the structure of an organic compound that is modified by a modification molecule. The system comprises: chromatograph units 10, 11 for separating in time the compound included in a target sample by a column and then fractionating an eluent, thus preparing a fractionated sample; a mass analysis unit 12 for executing mass analysis on the fractionated sample and acquiring a mass spectrum; spectrum integration units 21, 22 for integrating mass spectra that correspond to a plurality of fractionated samples included in the range of elution time and obtaining an integrated mass spectrum; a candidate peak detection unit 23 for detecting a peak where the m/z difference equals a prescribed value and thereby finding a candidate peak; a fractionated sample identification unit 24 for identifying a fractionated sample; and a display processing unit 25 for displaying, on a display unit, the m/z information of the candidate peak and information that indicates the fractionated sample identified in correspondence to the peak.SELECTED DRAWING: Figure 1

Description

The present invention relates to a structural analysis system for analyzing the structure of an organic compound using mass analysis, and a computer program for analyzing the structure of an organic compound using a computer. The structural analysis system and the structural analysis support program according to the present invention can be used for structural analysis of an organic compound having a structure in which a modified molecule is modified with various modified molecules having similar structures, and in particular, glycoprotein and sugar. It is suitable for structural analysis of sugar chain-modified organic compounds such as peptides and glycolipids.

Recent studies have revealed that many proteins and lipids exist in the living body, but most of them do not have sufficient functions by themselves and can exert their functions by undergoing some modification. Has been done. 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 known to play 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), has been widely used for such analysis. ing.

When a 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, it is recommended to use a reverse-phase liquid chromatograph or the like to separate various glycopeptides into groups having the same peptide skeleton, and then prepare a sample to be analyzed. It is common.

As disclosed in Non-Patent Document 1, mass spectrometry of a glycopeptide mixture sample prepared as described above reveals a plurality of peaks having mass differences derived from sugar residues in the mass spectrum. .. These peaks are peaks derived from glycopeptides in which sugar chains having different structures are bound to a common peptide. In Non-Patent Document 1, in order to confirm the product ion derived from the sugar chain of one glycopeptide, a peak presumed to be derived from the glycopeptide is selected in the mass spectrum, and the mass-to-charge ratio of the peak (strictly speaking). Is the oblique letter "m / z", but in this specification, MS ² analysis is performed using the precursor ion as the precursor ion (referred to as "mass-to-charge ratio" or "m / z"). This makes it possible to obtain product ion information useful for structural analysis of glycopeptides and sugar chains.

Japanese Unexamined Patent Publication No. 2017-181404

"Glycopeptide analysis using small MALDI digital ion trap mass spectrometer" MALDIminiTM-1 "", [online], [Search on September 4, 2020], Shimadzu Corporation, Internet <URL: https: // www .an.shimadzu.co.jp/apl/vaccine/b100.pdf ＞

When comprehensively analyzing glycopeptide variants (a group of glycopeptides having the same peptide skeleton but different sugar chain structures) for various peptides, the eluate that elutes from the column of the liquid chromatograph is usually discharged every predetermined time. In other words, time fractionation is performed, and a sample for Matrix Assisted Laser Desorption / Ionization (MALDI) is prepared for each of the fractionated eluates to prepare a MALDI mass spectrometer. (For example, refer to Patent Document 1 and the like). As a result, a mass spectrum can be obtained for each fraction unit. Therefore, as described above, the precursor ion is determined after detecting the peak having a mass difference derived from the sugar residue for each mass spectrum, and MS ² analysis is performed. Can be done.

However, when separating glycopeptides by liquid chromatograph, even if the peptide skeleton is the same, a relatively large difference in elution time may occur due to the difference in sugar chains. For example, there is often a large difference in elution time between a glycopeptide containing an acidic sugar and a glycopeptide not containing an acidic sugar. When such glycopeptides are analyzed, glycopeptides having the same peptide skeleton may be fractionated separately. In that case, even if an attempt is made to detect a peak derived from a glycopeptide by the above-mentioned method, the detection omission increases, and the completeness of the structural analysis of the glycopeptide may decrease.

The present invention has been made in view of these problems, and an object thereof is, for example, a structure of a glycopeptide or a sugar chain by a liquid chromatograph mass spectrometry method for a sample which is a mixture of a glycopeptide and a sugar chain. It is to provide a structural analysis system for organic compounds and a computer program for that purpose, which can enhance the comprehensiveness of structural analysis of glycopeptide variants and provide useful structural analysis information to users when performing analysis. ..

One aspect of the organic compound structure analysis system according to the present invention, which has been made to solve the above problems, is an organic compound for analyzing the structure of an organic compound in which a molecule to be modified is modified by a modified molecule, which is contained in a sample. It is a structural analysis system of
A chromatograph unit that separates the compound contained in the target sample in time by a column, fractionates the eluate, and prepares a fractionated sample for each fraction.
A mass spectrometric unit that performs mass spectrometry on each of the fractionated samples and acquires a mass spectrum,
A spectrum integration unit obtained by the mass spectrometer to obtain an integrated mass spectrum by integrating mass spectra corresponding to a plurality of fractionated samples included in a predetermined elution time range, and a spectrum integration unit.
A candidate peak detection unit for obtaining a candidate peak derived from a target organic compound by detecting a peak having a mass-to-charge ratio difference of a predetermined value in the integrated mass spectrum.
A fractionated sample identification unit that identifies a fractionated sample in which the candidate peak is significantly observed, and a fractionated sample specifying unit.
A display processing unit that displays mass-to-charge ratio information of the candidate peak and information indicating a fractionated sample specified by the fractionated sample specifying unit corresponding to the peak on the display unit.
To prepare for.

In addition, the organic compound structure analysis support program according to the present invention, which was made to solve the above problems, separates the compound contained in the sample in time by a column, fractionates the eluent, and fractionates the eluate. Uses mass spectrometric data obtained by a measurement unit including a liquid chromatograph unit that prepares a fractionated sample for each, and a mass spectrometric unit that performs mass spectrometry on the fractionated sample to acquire a mass spectrometric sample. This is a structural analysis support program for organic compounds used to analyze the structure of an organic compound in which the modified molecule is modified by the modified molecule, which is contained in the sample.
A spectrum integration function unit that integrates mass spectra corresponding to multiple fractionated samples included in a predetermined elution time range to obtain an integrated mass spectrum, and
A candidate peak detection function unit for obtaining a candidate peak derived from a target organic compound by detecting a peak having a mass-to-charge ratio difference of a predetermined value in the integrated mass spectrum.
The fractional sample identification function unit that identifies the fractional sample in which the candidate peak is significantly observed, and the fractional sample identification function unit.
A display processing function unit for displaying the mass-to-charge ratio information of the candidate peak and information indicating the fractionated sample specified by the fractionation sample specifying function unit corresponding to the peak on the display unit.
To operate.

The mass spectrometric unit in the present invention sequentially analyzes a plurality of fractionated samples prepared by fractionation in the liquid chromatograph unit, and typically, by irradiating the sample with a laser beam, the sample is contained. A mass spectrometer using an ionization method such as the MALDI method for ionizing components, the Laser Desorption / Ionization (LDI) method, and the Surface Assisted Laser Desorption / Ionization (SALDI) method is used. be able to.

When the target organic compound is a glycopeptide, the modified molecule is a sugar chain and the modified molecule is a peptide in the present invention. When the target organic compound is a glycolipid, the modified molecule is a sugar chain and the modified molecule is a lipid in the present invention.

For example, when the target organic compound is a glycopeptide, in the above-mentioned embodiment of the organic compound structure analysis system and the organic compound structure analysis support program according to the present invention, the same is used when separating or purifying with a liquid chromatograph. Different types of glycopeptides with a peptide skeleton, i.e., glycopeptide variants, are derived from such glycopeptide variants, even if the dissolution times of the glycopeptide variants vary widely and they are fractionated separately. The peak can be searched comprehensively. As a result, MS ⁿ analysis of many glycopeptide variants can be performed to collect structural information on glycopeptides and sugar chains, improving the comprehensiveness and accuracy of structural analysis of glycopeptides and sugar chains. be able to.

That is, according to the above aspect of the organic compound structure analysis system and the organic compound structure analysis support program according to the present invention, the target organic compound and the organic compound in which the modified molecule has various similar structures are included. The structural analysis of the modified molecule can be carried out comprehensively and accurately.

The block block diagram of the glycopeptide structure analysis system which is one Embodiment of this invention. The flowchart which shows the analysis procedure in the glycopeptide structure analysis system of this embodiment. The schematic diagram for demonstrating the relationship between the separation state by the LC part and the prepared sample in the glycopeptide structure analysis system of this embodiment. The explanatory view of the analysis process in the glycopeptide structure analysis system of this embodiment. The figure which shows an example of the display screen in the glycopeptide structure analysis system of this embodiment. The figure which shows an example of the display screen in the glycopeptide structure analysis system of this embodiment.

Hereinafter, the glycopeptide structure analysis system according to the embodiment of 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 glycopeptide structure analysis system of the present embodiment.

This glycopeptide structure analysis system includes a measurement unit 1, a data processing unit 2 that processes data obtained by the measurement unit 1, a control unit 3 that controls the operation of the measurement unit 1, and an operation unit 4 that is a user interface. And a display unit 5.

The measuring unit 1 is a liquid chromatograph unit (hereinafter referred to as “LC unit”) 10 including a column (not shown) for separating substances in the sample, and an eluent elution from the column outlet of the LC unit 10 for a specified time. Fraction / spotting unit 11 for preparing a sample for MALDI by fractionating each (fraction time Δt) and dropping the fractionated eluate together with a matrix onto a sample plate, and for each prepared sample. It includes a MALDI mass spectrometric unit (hereinafter referred to as “MALDI-MS unit”) 12 that performs mass spectrometry (MS ¹ analysis and MS ² analysis), respectively.
As the MALDI-MS unit 12, for example, the MALDI digital ion trap mass spectrometer “MALDI mini ^TM -1” disclosed in Non-Patent Document 1 can be used, but the method is not limited thereto.

The data processing unit 2 has, as functional blocks, a data storage unit 20, an MS ¹ spectrum creation unit 21, an MS ¹ spectrum integration unit 22, a sugar chain mass difference analysis unit 23, a well identification unit 24, a display processing unit 25, and an MS ² precursor. The ion selection processing unit 26 is included. Further, the display processing unit 25 includes an integrated mass spectrum display processing unit 250, a glycopeptide-containing information display processing unit 251 and a glycopeptide candidate display processing unit 252 as lower functional blocks.

In this system, the substance of the data processing unit 2 and the control unit 3 is a general-purpose personal computer or a workstation with higher performance, and by operating a dedicated control / processing program installed in such a computer on the computer. The functions of each of the above functional blocks can be realized.

Next, an example of the analysis operation using the glycopeptide structure analysis system of the present embodiment will be described with reference to FIGS. 2 to 5. FIG. 2 is a flowchart showing the analysis procedure. FIG. 3 is a schematic diagram for explaining the relationship between the separated state by the LC unit and the prepared sample. FIG. 4 is an explanatory diagram of the analysis process. FIG. 5 is a diagram showing an example of a display screen of the display unit 5.

Here, the subject of analysis is glycoprotein, and a glycopeptide / sugar chain mixture is obtained as a sample by decomposing the glycoprotein with a proteolytic enzyme. This glycopeptide / sugar chain mixture sample contains a glycopeptide in which sugar chains having various structures are bound to a peptide having the same amino acid sequence (that is, the same peptide skeleton).

The glycopeptide / sugar chain mixture sample is introduced into the LC unit 10 of the measuring unit 1, and the components (glycopeptide, sugar chain, etc.) in the sample are temporally separated while passing through the column of the LC unit 10. .. Various glycopeptides and peptides from which sugar chains have been eliminated are separated for each peptide using a reverse phase column, but even if the peptides are common, the retention times of the glycopeptides may differ due to differences in sugar chains. be. Therefore, the peptides are roughly separated by the difference in peptides, and some glycopeptides are finely separated by the difference in the sugar chain which is a modification of the same peptide.

The fractionation / spotting unit 11 fractionates the eluate from the column outlet at predetermined time Δt specified by the control unit 3, and all or part of the fractionated eluate is used as one well of the sample plate. Drop into. In addition, a predetermined matrix is added dropwise to each well and dried to dryness to prepare a sample. However, during the period when it is known that the eluate from the column outlet does not contain the substance to be analyzed, the fractionated eluate may be discarded without preparing the sample.

In FIG. 3, (A) is an example of a chromatogram (actually, this chromatogram is not prepared) obtained when the eluate eluted from the column outlet of the LC unit 10 is detected, and (B) is a sample plate. It is a schematic diagram at the time of forming a sample in each well 101 on 100. Here, 6 × 4 = 24 wells 101 are provided on the sample plate 100. In this example, as shown in FIG. 3A, the eluate is fractionated every fractionation time Δt, and a sample is prepared from a part of the fractionated eluate. That is, in FIG. 3A, the eluates fractionated during the t1, t2, t3, t4, and t5 periods are sequentially ordered into wells 101 having well numbers A1, A2, A3, A4, and B1. Samples are prepared by dropping and adding a matrix to each well 101.

For example, when a sample containing a different fractionated sample is prepared in each of the plurality of wells 101 on one sample plate 100, the sample plate is loaded into the MALDI-MS unit 12, and the MALDI-MS unit 12 is each sample. Mass spectrometry (MS ¹ analysis) is performed in order. This MS ¹ analysis provides mass spectral data showing ionic strength over a predetermined mass-to-charge ratio range.

In the data processing unit 2, the data storage unit 20 stores one mass spectrum data group (data indicating a change in ionic strength in a predetermined mass-to-charge ratio range) for each well. In general, this mass spectrum data group is a combination of data obtained by performing MS ¹ analysis multiple times on the same sample.

When the user instructs the operation unit 4 to start the analysis while the mass spectrum data group corresponding to each of a large number of fractionated samples obtained from one sample is stored in the data storage unit 20, the data processing unit 2 will perform the analysis. The following processing is executed.

The MS ¹ spectrum creation unit 21 sequentially reads out the mass spectrum data group from the data storage unit 20, and creates a mass (MS ¹ ) spectrum for each mass spectrum data group, that is, for each well on the sample plate (step). S1). In this mass spectrum, peaks derived from glycopeptides and peptides contained in the sample, sugar chains, and the like appear. For example, in the conventional general analysis method described in Non-Patent Document 1 and the like, a peak corresponding to a mass-to-charge ratio difference derived from a component of a sugar chain is searched for in this mass spectrum, and a peak derived from a glycopeptide is found.

On the other hand, in this system, the MS ¹ spectrum integration unit 22 refers to a plurality of fractionated samples, that is, samples included in one elution time range ΔT for each preset elution time range ΔT (> Δt). The MS ¹ spectrum thus obtained is integrated to create an integrated mass spectrum (step S2). In the example of FIG. 3, since one elution time range ΔT contains five wells, for example, five obtained for samples in wells numbered A1, A2, A3, A4, and B1. The MS ¹ spectra are integrated (see FIGS. 4A and 4B). This integrated mass spectrum can be a total value obtained by adding peak intensities for each mass-to-charge ratio. Further, instead of the total value of the peak intensities, other representative values such as the average value and the maximum value may be used.

The elution time range ΔT may be set so as to include the elution time (retention time) of various glycopeptides having a common peptide as much as possible in consideration of the variation. On the other hand, if the elution time range ΔT is made too large, there is a high possibility that glycopeptides having different peptides are included in the elution time range ΔT, but usually, glycopeptides having different peptides during the treatment described later. Since they can be separated from each other, it does not matter practically.

The sugar chain mass difference analysis unit 23 detects peaks for each integrated mass spectrum according to a predetermined algorithm, and obtains the mass-to-charge ratio value of each peak. Then, a peak having a known mass-to-charge ratio difference of sugar residues (marked with ◯ and Δ in FIG. 4B) is detected among a large number of detected peaks, and the detected peak is a glycopeptide candidate. It is set to the peak (step S3). In the example of FIG. 4B, five glycopeptide candidate peaks are selected in the integrated mass spectrum.

For example, even if the peptide skeleton (modified molecule) is the same, depending on the structure of the added sugar chain (modified molecule), the glycopeptide has a chromatographic peak A and a chromatographic peak B shown in FIG. 3 (A). The elution time may be significantly different. In the example of FIG. 3, the glycopeptide corresponding to chromatographic peak B is contained in the sample in the wells of numbers A1, A2, and A3, but is not included in the sample in the well of number B1. Conversely, the glycopeptide corresponding to chromatographic peak A is not included in the samples in the wells of numbers A1, A2, A3, but is included in the samples in the wells of number B1. Therefore, for example, in the MS ¹ spectrum obtained for the sample in the well of No. B1, even if the search for the peak having the mass-to-charge ratio difference of the known sugar residue is performed, the sugar corresponding to the chromatographic peak A is searched. Peptide-derived peaks may not be detected.

On the other hand, in the integrated mass spectrum in which a plurality of MS ¹ spectra are integrated, a peak derived from a glycopeptide corresponding to chromatographic peak A and a peak derived from a glycopeptide corresponding to chromatographic peak B appear together. Therefore, by searching for a peak having a known mass-to-charge ratio difference of sugar residues with respect to the integrated mass spectrum, it is possible to detect a peak derived from a glycopeptide having a common peptide with high comprehensiveness. ..

Next, the well identification unit 24 confirms the peak of the corresponding mass-to-charge ratio in each MS ¹ spectrum before integration for each of the plurality of glycopeptide candidate peaks detected based on one integrated mass spectrum, and the sugar thereof. A well in which a peptide candidate peak is significantly observed is determined (step S4).

Specifically, when there is a peak in the mass-to-charge ratio of the glycopeptide candidate peak in each MS ¹ spectrum before integration for each glycopeptide candidate peak, and the signal strength of the peak is equal to or higher than a predetermined threshold value. It is determined that the glycopeptide candidate peak is present in the MS ¹ spectrum. For example, the glycopeptide candidate peak detected in the integrated mass spectrum shown in FIG. 4 (B) is compared with the peak detected in the MS ¹ spectrum corresponding to well A1 shown in FIG. 4 (C), and the signal intensity of the peak is increased. It is determined that four peaks P _B , P _C , P _D , and P _E above the threshold are observed in well A1. The threshold value for determining the signal strength of the peak may be a value predetermined by the manufacturer or a value appropriately set by the user. Further, in the above processing, it can be determined that one glycopeptide candidate peak exists in a plurality of MS ¹ spectra, but in that case, only one MS ¹ spectrum having the highest signal intensity of the peak may be selected. ..

Further, the well identification unit 24 determines wells included in one group of glycopeptides having a common peptide based on the well identification result for the glycopeptide candidate peak detected for each integrated mass spectrum, and determines the wells included in one group of glycopeptides in common, and for each group. Is assigned the group identification numbers GP1, GP2, .... As mentioned above, it is possible that some of the groups that are back and forth in time may overlap.

After that, the integrated mass spectrum display processing unit 250 creates an integrated mass spectrum that clearly indicates the glycopeptide candidate peak, while the glycopeptide-containing information display processing unit 251 is a sugar belonging to the same group (the peptide is common). A well arrangement diagram showing the positions of the wells in which the peptide candidate peaks are observed is created, and the contained sample display screen in which they are arranged together is displayed on the display unit 5 (step S5).

Further, the glycopeptide candidate display processing unit 252 describes the mass-to-charge ratio value of the glycopeptide candidate peak together with the number of the well in which the glycopeptide candidate peak is observed and the number for identifying the group of the glycopeptide candidate peaks. Is created, and this table is displayed in the content sample display screen (step S6).

FIG. 5 is an example of the contained sample display screen 200 created and displayed by the processing of steps S5 and S6. In the contained sample display screen 200, an integrated mass spectrum display frame 210, a well arrangement diagram 220, and a glycopeptide candidate peak table 230 are arranged.
The integrated mass spectrum created by the integrated mass spectrum display processing unit 250 is displayed in the integrated mass spectrum display frame 210, and the mass-to-charge ratio value ( _{PA, P B ,} of the glycopeptide candidate peak) is displayed in the integrated mass spectrum. ...) And the mass-to-charge ratio difference (◯, Δ) between adjacent glycopeptide candidate peaks on the mass-to-charge ratio axis are specified. The well arrangement diagram 220 is a schematic plan view of a sample plate in which the positions of the wells are indicated by ◯, and the wells in which glycopeptide candidate peaks belonging to the same group are detected are shown in the same color. Further, in the glycopeptide candidate peak table 230, the retention time (RT), the number for identifying the group of glycopeptide candidate peaks (GP cluster), and the mass-to-charge ratio value of the glycopeptide candidate peak (MS2 precursor) are shown in the order of well numbers. ) And is shown.

The user finds the glycopeptide candidate peak of interest on the contained sample display screen 200, selects the mass-to-charge ratio value of one or more glycopeptide candidate peaks in the MS ² precursor column 231 in the glycopeptide candidate peak table 230, and operates. The selection is instructed in the part 4 (step S7). Specifically, the glycopeptide candidate peak can be selected by clicking the mass-to-charge ratio value with a pointing device such as a mouse. By using the extracted ion chromatogram display function described later, the user can more easily and accurately select the glycopeptide candidate peak.

In response to the above-mentioned selection instruction, the MS ² precursor ion selection processing unit 26 sets the mass-to-charge ratio value of the selected glycopeptide candidate peak as the precursor ion for MS ² analysis (step S8). Further, the MS ² precursor ion selection processing unit 26 sends the information of the precursor ion and the information of the well number corresponding to the row instructed to be selected to the control unit 3. The control unit 3 controls the MALDI-MS unit 12, moves the sample at the specified well number position to the measurement position, and then executes the MS ² analysis targeting the specified precursor ion (step). S9). Thereby, it is possible to obtain an MS ² spectrum in which the partial structure of the target glycopeptide candidate is reflected, that is, the product ion peak corresponding to such the partial structure is observed. When a plurality of glycopeptide candidate peaks are instructed to be selected in step S7, the processes of steps S8 and S9 may be repeated.

In the MALDI method, when a sample is irradiated with laser light, the portion exposed to the laser light and the components in the surrounding sample are volatilized or scattered and disappear. Therefore, the number of times a good analysis can be performed on one sample is limited to some extent, and it is not appropriate to repeat the MS ² analysis targeting a large number of glycopeptide candidates for the same sample. In contrast, in this system, when one glycopeptide candidate peak is observed in multiple wells, the user can easily confirm this in the glycopeptide candidate peak table 230 and select a well to perform MS ² analysis. Glycopeptide candidate peaks can be selected and instructed to be appropriately dispersed. This allows MS ² analysis of each glycopeptide candidate peak to be performed without sample depletion.

In this way, in this system, it is possible to reduce analysis omissions and perform highly comprehensive structural analysis for many types of glycopeptide variants in which peptides are common. In particular, when separation and purification of glycopeptides were performed using a liquid chromatograph, MS ² analysis was surely performed for glycopeptides whose elution time was significantly different from that of other glycopeptides, and the results were obtained. The structure can be analyzed based on.

Further, in the glycopeptide structure analysis system of the present embodiment, the following extracted ion chromatogram display function is added so that the user can easily and accurately select one or a plurality of glycopeptide candidate peaks. be able to. FIG. 6 is a diagram showing another example of the contained sample display screen 200 created and displayed by the processing of steps S5 and S6 in FIG. In this example, as shown in FIG. 6, in addition to the integrated mass spectrum display frame 210, the well arrangement diagram 220, and the glycopeptide candidate peak table 230 already described, the extracted ion chromatogram is displayed in the contained sample display screen 200. The frame 240 is arranged.

As described above, the integrated mass spectrum display processing unit 250 creates an integrated mass spectrum in which the glycopeptide candidate peak is clearly indicated and displays it in the integrated mass spectrum display frame 210, but at the same time, it is clearly indicated in the integrated mass spectrum. An extracted ion chromatogram with a mass-to-charge ratio of one or more glycopeptide candidate peaks is prepared and displayed in the extracted ion chromatogram display frame 240. As shown in FIG. 6, when there are a plurality of glycopeptide candidate peaks (five in this example), a plurality of extracted ion chromatograms corresponding to each peak are overlaid with different display colors. Alternatively, as shown in FIG. 6, the images may be overlaid with a slight shift in the vertical axis (strength axis) direction.

In addition to simply displaying the extracted ion chromatogram corresponding to each glycopeptide candidate peak, the extracted ion chromatogram corresponding to the glycopeptide candidate peak specified by the user can be visually combined with other extracted ion chromatograms. It is also possible to have a function to highlight a specific extracted ion chromatogram so that it can be clearly distinguished. For example, the example of FIG. 6 is a state in which the user indicates one _glycopeptide candidate peak PD in the MS ² precursor column 231 of the glycopeptide candidate peak table 230. Then, in response to this instruction, the integrated mass spectrum display processing unit 250 highlights the extracted ion chromatogram corresponding to the indicated _glycopeptide candidate peak PD. Actually, only the chromatogram may be displayed by increasing the brightness of the display or displaying it with a thick line. This allows the user to confirm the temporal change in the content of the glycopeptide candidate peak that is being selected as the precursor ion, and to compare it with the temporal change in the content of other glycopeptide candidate peaks. can do. Further, the indication of the glycopeptide candidate peak to be highlighted can be given not only in the glycopeptide candidate peak table 230 but also on the integrated mass spectrum.

Furthermore, instead of designating one or more glycopeptide candidate peaks, by designating a particular well on the glycopeptide candidate peak table 230 or on the well arrangement diagram 220, the sugar corresponding to that designated well. It is also possible to highlight the extracted ion chromatogram at the peptide candidate peak. For example, when well A3 is specified on the glycopeptide candidate peak table 230 or on the well arrangement diagram 220, all the extracted ion chromatograms at the corresponding three glycopeptide candidate peaks P _B , _PC , and P _D are included. It will be highlighted. This allows the user to easily grasp the extracted ion chromatogram of the glycopeptide candidate peak that can be observed in a certain well.

When displaying the extracted ion chromatogram on the extracted ion chromatogram display frame 240, it is preferable to also display information indicating the division time division corresponding to each well as shown in FIG. 3A. .. This makes it easier to understand the relationship between the amount of glycopeptide candidates and the wells.
Further, as a matter of course, the extracted ion chromatogram may be displayed on a separate screen such as a pop-up screen instead of the contained sample display screen 200.

In the glycopeptide structure analysis system of the present embodiment, the glycopeptide candidate peak is presented to the user, and MS ² analysis is performed on the glycopeptide candidate peak selected according to the user's instruction. In parallel with displaying the peptide candidate peaks, glycopeptide candidate peaks may be automatically selected in the order according to a predetermined algorithm, and MS ² analysis targeting the selected peaks may be performed. ..

Further, in the system of the above embodiment, the MALDI-MS unit 12 is used as the mass spectrometry unit in the measurement unit 1, but various types of mass spectrometers can be used. For example, the ion source is generally an ion source using an ionization method such as the LDI method or the SALDI method in addition to the MALDI method, but an ion source by various other ionization methods may be used. In addition to the ion trap, the mass separator may be a quadrupole mass filter, a time-of-flight mass separator, or the like. Further, the MALDI-MS unit 12 can perform MS ⁿ analysis with n of 2 or more by itself, but uses a mass spectrometer having a structure that cannot perform MS ⁿ analysis, and the MS ⁿ analysis after the precursor ion is determined is performed. It may be executed by another mass spectrometer not included in the measuring unit 1.

Further, although the system of the above embodiment was intended to perform structural analysis of glycopeptides, the present invention can be used for structural analysis of various organic compounds other than glycopeptides. Typically, it is particularly useful for compounds modified with sugar chains such as glycoproteins and glycolipids, but it is an organic compound in which the modified molecule is modified by the modified molecule, and the structural variation of the modified molecule is used. It is useful for compounds in which there are many similar organic compounds.

Furthermore, 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 organic compound structure analysis system according to the present invention is an organic compound structure analysis system for analyzing the structure of an organic compound in which a molecule to be modified is modified by a modified molecule, which is contained in a sample. There,
A chromatograph unit that separates the compound contained in the target sample in time by a column, fractionates the eluate, and prepares a fractionated sample for each fraction.
A mass spectrometric unit that performs mass spectrometry on each of the fractionated samples and acquires a mass spectrum,
A spectrum integration unit obtained by the mass spectrometer to obtain an integrated mass spectrum by integrating mass spectra corresponding to a plurality of fractionated samples included in a predetermined elution time range, and a spectrum integration unit.
A candidate peak detection unit for obtaining a candidate peak derived from a target organic compound by detecting a peak having a mass-to-charge ratio difference of a predetermined value in the integrated mass spectrum.
A fractionated sample identification unit that identifies a fractionated sample in which the candidate peak is significantly observed, and a fractionated sample specifying unit.
A display processing unit that displays mass-to-charge ratio information of the candidate peak and information indicating a fractionated sample specified by the fractionated sample specifying unit corresponding to the peak on the display unit.
To prepare for.

(Section 7) Further, in one aspect of the organic compound structural analysis support program according to the present invention, the substance contained in the sample is temporally separated by a column, the eluent is fractionated, and the fraction is fractionated. Using the mass spectrometric data obtained by the measurement unit including the liquid chromatograph unit that prepares the image sample and the mass spectrometric unit that performs mass spectrometry on the fractionated sample to acquire the mass spectrometric sample, respectively. It is a structural analysis support program for organic compounds used to analyze the structure of organic compounds in which modified molecules are modified by modified molecules contained in a sample, and is a computer.
A spectrum integration function unit that integrates mass spectra corresponding to multiple fractionated samples included in a predetermined elution time range to obtain an integrated mass spectrum, and
A candidate peak detection function unit for obtaining a candidate peak derived from a target organic compound by detecting a peak having a mass-to-charge ratio difference of a predetermined value in the integrated mass spectrum.
The fractional sample identification function unit that identifies the fractional sample in which the candidate peak is significantly observed, and the fractional sample identification function unit.
A display processing function unit for displaying the mass-to-charge ratio information of the candidate peak and information indicating the fractionated sample specified by the fractionation sample specifying function unit corresponding to the peak on the display unit.
To operate.

According to the organic compound structure analysis system described in the first item and the organic compound structure analysis support program described in the seventh item, for example, when separating or purifying in the chromatograph section, the same peptide skeleton is used. Covers peaks derived from such glycopeptide variants, even when the elution times of the various types of glycopeptides, i.e. glycopeptide variants, vary widely and are fractionated separately. Can be searched for. As a result, MS ⁿ analysis of many glycopeptide variants can be performed to collect structural information on glycopeptides and sugar chains, improving the comprehensiveness and accuracy of structural analysis of glycopeptides and sugar chains. be able to.

(Item 2) The organic compound structure analysis system according to item 1 further includes a selection processing unit that allows the user to select a candidate peak as a precursor ion for MS ² analysis on the display screen by the display processing unit. Can be.

(Item 8) Further, in the organic compound structure analysis support program described in item 7, a computer is further displayed to the user on the screen displayed by the display processing function unit, and a candidate peak is displayed to the user as a precursor ion for MS ² analysis. It can be operated as a selection processing function unit to be selected as.

According to the organic compound structure analysis system described in Section 2 and the organic compound structure analysis support program described in Section 8, the user can use the displayed candidate peak information and the fractionated sample in which it is observed. After confirming the above information, MS ² analysis can be performed by designating one or more candidate peaks derived from the target compound of interest, and product ion information can be obtained. In particular, by making it possible to select the fraction sample to be subjected to the MS ² analysis, it is possible to perform the MS ² analysis with the candidate peak as the precursor ion for the fraction sample selected by the user. As a result, MS ² analysis can be performed without biasing to a specific fractionated sample, so that it is possible to avoid exhaustion of the fractionated sample and to obtain product ion information for many similar compounds (compounds having the same modified molecule but different modified molecules). Can be collected.

(Clause 3) In the structural analysis system for an organic compound according to the first or second paragraph, the display processing unit displays the integrated mass spectrum on the screen of the display unit, and the integrated mass spectrum is displayed. Information about the modified molecule corresponding to the mass-to-charge ratio difference between the plurality of candidate peaks detected in the above can be displayed on the integrated mass spectrum.

(Item 9) Further, in the organic compound structure analysis support program according to the item 7 or 8, the display processing function unit displays the integrated mass spectrum on the screen of the display unit, and the display processing function unit displays the integrated mass spectrum on the screen of the display unit. Information about the modified molecule corresponding to the mass-to-charge ratio difference between the plurality of candidate peaks detected in the integrated mass spectrum can be displayed on the integrated mass spectrum.

According to the organic compound structure analysis system described in Section 3 and the organic compound structure analysis support program described in Section 9, the user can use information on the modified molecule displayed on the integrated mass spectrum, for example, sugar residue. You can find the candidate peaks you are interested in from the basic information. As a result, product ion information can be preferentially collected from the candidate peaks of interest, and structural analysis of the compound or modified molecule can be advanced.

(Clause 4) In the structural analysis system for an organic compound according to any one of paragraphs 1 to 3, the mass spectrometric unit is a sample that is a fractionated sample formed in each well on a plate. Is irradiated with laser light to ionize the components in the sample for mass spectrometry.
The information indicating the fractionated sample is the identification number of the well on the plate.
The display processing unit may display the mass-to-charge ratio information of the candidate peak in association with the identification number of the well in which the candidate peak is detected.

(Section 10) Further, in the structural analysis support program for an organic compound according to any one of paragraphs 7 to 9, the mass spectrometric unit is the fractionated sample formed in each well on the plate. The sample is irradiated with laser light, and the components in the sample are ionized for mass spectrometry.
The information indicating the fractionated sample is the identification number of the well on the plate.
The display processing function unit may display the mass-to-charge ratio information of the candidate peak in association with the identification number of the well in which the candidate peak is detected.

In the organic compound structure analysis system according to the fourth item and the organic compound structure analysis support program according to the tenth item, the mass spectrometric unit is based on an ionization method such as the MALDI method, the LID method, and the SALDI method. It is equipped with an ion source.

According to the organic compound structure analysis system described in Section 4 and the organic compound structure analysis support program described in Section 10, candidate peaks derived from the target organic compound are found in a large number of wells on the sample plate. The user can grasp the relationship between the observed well position and the mass-to-charge ratio value of the candidate peak at a glance.

(Clause 5) In the structural analysis system for an organic compound according to any one of paragraphs 1 to 4, the display processing unit is of one of the candidate peaks when the candidate peak is one. A single extracted ion chromatogram at the mass-to-charge ratio, or if there are a plurality of candidate peaks, a plurality of extracted ion chromatograms at each mass-to-charge ratio of the plurality of peaks are overlaid and displayed on the display unit. Can be.

(Clause 6) In the organic compound structure analysis system according to paragraph 5, the display processing unit receives instructions from the user regarding information displayed on the display unit other than the extracted ion chromatogram, and the instructions are given. The extracted ion chromatogram of a particular candidate peak corresponding to can be highlighted.

(Clause 11) In the structural analysis support program for an organic compound according to any one of paragraphs 7 to 10, the display processing function unit is one of the candidate peaks when it is one. A single extracted ion chromatogram at the mass-to-charge ratio of the peak, and if there are a plurality of candidate peaks, a plurality of extracted ion chromatograms at the mass-to-charge ratio of each of the plurality of peaks are overlaid on the display. It can be displayed.

(Clause 12) In the organic compound structure analysis support program according to paragraph 11, the display processing function unit receives instructions from the user regarding information displayed on the display unit other than the extracted ion chromatogram, and receives instructions from the user. Extracted ion chromatograms of specific candidate peaks corresponding to the indication can be highlighted.

According to the organic compound structural analysis system according to the fifth or sixth paragraph and the organic compound structural analysis support program according to the eleventh or twelfth paragraph, the user can use the content of each compound candidate. After confirming the extracted ion chromatogram showing the temporal change, the candidate peak to be the precursor ion for MS ² analysis can be selected. As a result, the accuracy of selection of candidate peaks can be improved, and unnecessary execution of MS ² analysis can be avoided.

1 ... Measurement unit 10 ... Liquid chromatograph unit 11 ... Fractionation / spotting unit 12 ... MALDI mass spectrometry unit 2 ... Data processing unit 20 ... Data storage unit 21 ... MS ¹ spectrum creation unit 22 ... MS ¹ spectrum integration unit 23 ... Sugar Chain mass spectrometric analysis unit 24 ... Well identification unit 25 ... Display processing unit 250 ... Integrated mass spectrum display processing unit 251 ... Glycopeptide-containing information display processing unit 252 ... Glycopeptide candidate display processing unit 26 ... MS ² Precursor ion selection processing unit 3 ... Control unit 4 ... Operation unit 5 ... Display unit

Claims (12)

It is a structural analysis system for organic compounds that analyzes the structure of organic compounds in which the molecule to be modified is modified by the modified molecule, which is contained in the sample.
A chromatograph unit that separates the compound contained in the target sample in time by a column, fractionates the eluate, and prepares a fractionated sample for each fraction.
A mass spectrometric unit that performs mass spectrometry on each of the fractionated samples and acquires a mass spectrum,
A spectrum integration unit obtained by the mass spectrometer to obtain an integrated mass spectrum by integrating mass spectra corresponding to a plurality of fractionated samples included in a predetermined elution time range, and a spectrum integration unit.
A candidate peak detection unit for obtaining a candidate peak derived from a target organic compound by detecting a peak having a mass-to-charge ratio difference of a predetermined value in the integrated mass spectrum.
A fractionated sample identification unit that identifies a fractionated sample in which the candidate peak is significantly observed, and a fractionated sample specifying unit.
A display processing unit that displays mass-to-charge ratio information of the candidate peak and information indicating a fractionated sample specified by the fractionated sample specifying unit corresponding to the peak on the display unit.
A structural analysis system for organic compounds. A selection processing unit that allows the user to select a candidate peak as a precursor ion for MS ² analysis on the display screen by the display processing unit.
The organic compound structure analysis system according to claim 1. The display processing unit displays the integrated mass spectrum on the screen of the display unit, and provides information on the modified molecule corresponding to the mass-to-charge ratio difference between the plurality of candidate peaks detected in the integrated mass spectrum. The structural analysis system for an organic compound according to claim 1 or 2, which is displayed on the integrated mass spectrum. The mass spectrometric unit irradiates a sample, which is a fractionated sample, formed in each well on a plate with a laser beam, ionizes the components in the sample, and performs mass spectrometry.
The information indicating the fractionated sample is the identification number of the well on the plate.
The organic compound according to any one of claims 1 to 3, wherein the display processing unit displays the mass-to-charge ratio information of the candidate peak in association with the identification number of the well in which the candidate peak is detected. Structural analysis system. The display processing unit displays a single extracted ion chromatogram at the mass-to-charge ratio of the one candidate peak when there is one candidate peak, and when there are a plurality of candidate peaks, the display processing unit of the plurality of peaks. The structural analysis system for an organic compound according to any one of claims 1 to 4, wherein a plurality of extracted ion chromatograms at each mass-to-charge ratio are overlaid and displayed on a display unit. The display processing unit receives an instruction from a user in information displayed on the display unit other than the extracted ion chromatogram, and highlights the extracted ion chromatogram of a specific candidate peak corresponding to the instruction. 5. The organic compound structure analysis system according to 5. After temporally separating the substances contained in the sample with a column, the eluent is fractionated, and mass spectrometry is performed on the liquid chromatograph unit that prepares the fractionated sample for each fraction and the fractionated sample. Using the mass spectrometric data obtained by the mass spectrometric unit to acquire the mass spectrogram and the measurement unit including the mass spectrometric unit, the structure of the organic compound in which the modified molecule is modified by the modified molecule contained in the sample is analyzed. It is a structural analysis support program for organic compounds used to support computer use.
A spectrum integration function unit that integrates mass spectra corresponding to multiple fractionated samples included in a predetermined elution time range to obtain an integrated mass spectrum, and
A candidate peak detection function unit for obtaining a candidate peak derived from a target organic compound by detecting a peak having a mass-to-charge ratio difference of a predetermined value in the integrated mass spectrum.
The fractional sample identification function unit that identifies the fractional sample in which the candidate peak is significantly observed, and the fractional sample identification function unit.
A display processing function unit for displaying the mass-to-charge ratio information of the candidate peak and information indicating the fractionated sample specified by the fractionation sample specifying function unit corresponding to the peak on the display unit.
A program to support structural analysis of organic compounds. The structure of the organic compound according to claim 7, wherein the computer is further operated as a selection processing function unit that allows the user to select a candidate peak as a precursor ion for MS ² analysis on the screen displayed by the display processing function unit. Analysis support program. The display processing function unit displays the integrated mass spectrum on the screen of the display unit, and information on the modified molecule corresponding to the mass-to-charge ratio difference between the plurality of candidate peaks detected in the integrated mass spectrum. The structural analysis support program for an organic compound according to claim 7 or 8, which displays the above on the integrated mass spectrum. The mass spectrometric unit irradiates a sample, which is a fractionated sample, formed in each well on a plate with a laser beam, ionizes the components in the sample, and performs mass spectrometry.
The information indicating the fractionated sample is the identification number of the well on the plate.
The organic compound according to any one of claims 7 to 9, wherein the display processing function unit displays the mass-to-charge ratio information of the candidate peak in association with the identification number of the well in which the candidate peak is detected. Structural analysis support program. The display processing function unit displays a single extracted ion chromatogram at the mass-to-charge ratio of one candidate peak when there is one candidate peak, and the plurality of peaks when there are a plurality of candidate peaks. The structural analysis support program for an organic compound according to any one of claims 7 to 10, wherein a plurality of extracted ion chromatograms at each mass-to-charge ratio are overlaid and displayed on a display unit. The display processing function unit receives an instruction from the user in the information displayed on the display unit other than the extracted ion chromatogram, and highlights the extracted ion chromatogram of a specific candidate peak corresponding to the instruction. Item 11 is a structural analysis support program for organic compounds.

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