JP4821400B2 - Structural analysis system - Google Patents

Description

  The present invention relates to a system for analyzing the structure of various samples using mass spectrometry. Specifically, for example, mass analysis is performed on a sample containing a peptide mixture, and the amino acid sequence of each peptide is determined based on the obtained data. The present invention relates to a structural analysis system suitable for this.

In recent years, protein structures and functions have been rapidly analyzed as post-genomic research. As one of such protein structure and function analysis methods, protein expression analysis and primary structure analysis using mass spectrometers have been widely performed. Quadrupole ion traps (Quadrupole Ion Trap) : QIT), collision induced decomposition (Collision Induced Dissociation: CID), etc., so-called MS / MS analysis (or MS ⁿ analysis), which captures and cleaves ions corresponding to a specific peak, demonstrates its power. In general, MS / MS analysis, first, the specific mass to charge ratio from the analyte ions having (m / z) were selected as the precursor ion cleaves the precursor ions by CID. Thereafter, mass analysis of ions (product ions) generated by cleavage can obtain information on the mass and chemical structure of the target ions.

When the amino acid sequence of a protein is identified using MS / MS (or MS ⁿ ) analysis as described above, the protein is first digested with an appropriate enzyme to form a mixture of peptide fragments, and then the peptide mixture. Is subjected to mass spectrometry (MS analysis). At this time, since stable isotopes having different masses exist in elements such as carbon (C) and nitrogen (N) constituting each peptide, even if the peptides are composed of the same amino acid sequence, the isotope composition a plurality of peaks of different mass to charge ratios appearing on the mass spectrum by difference. The plurality of peaks include a peak of an ion composed of only an isotope having a maximum natural abundance ratio (referred to as a main ion or a monoisotopic ion), and a peak of an ion including other isotopes (isotope ions) These form a peak group consisting of a plurality of peaks arranged at intervals of 1 [Da]. In the present invention, this is called an isotope peak group. An example of an isotope peak group on the mass spectrum is shown in FIG.

  Subsequently, a set of isotope peaks derived from a single peptide is selected as a precursor ion from the mass spectrometry data (MS mass spectrum data) of the peptide mixture as described above, and the precursor ion is cleaved. The ions (product ions) thus obtained are subjected to mass spectrometry (MS / MS analysis).

  The amino acid sequence of the test peptide can be identified by subjecting the thus obtained mass spectrum pattern by MS / MS analysis or the mass spectrum pattern by MS analysis to a database search. Moreover, the amino acid sequence of each peptide can also be estimated by mathematically analyzing these mass spectrum patterns using computer software called a De Novo sequence.

  Conventionally, when identifying amino acid sequences by the above-mentioned methods, if isotopic peak groups derived from different peptides are close to each other on the mass spectrum by MS analysis, it corresponds to the peak derived from the desired peptide As a result, ions corresponding to peaks derived from other peptides close to the peak were selected as precursor ions, and accurate identification could not be performed. On the other hand, with the recent development of high-mass resolution ion trap technology, ions corresponding to a single peak can be selected as a precursor ion from a group of isotope peaks appearing at intervals of 1 [Da]. Since it became possible, even when a part of isotopic peak groups derived from different peptides overlap each other, ions corresponding to the peaks derived from each peptide are individually selected from among them, and MS / MS By performing the analysis, it has become possible to accurately separate and identify each peptide (see, for example, Non-Patent Document 1).

  However, when there is a large overlap of isotope peak groups derived from different peptides, even if a single peak is selected from the isotope peak group and the corresponding ions are subjected to MS / MS analysis as described above, Since the single peak itself was originally obtained from a mixture of ions derived from multiple types of peptides, interpretation of the MS / MS mass spectrum was complicated, and accurate amino acid sequence identification was possible. There was a problem that I could not.

In order to solve these problems, the inventor of the present application described in Non-Patent Document 2 the isotope peak groups on the mass spectrum obtained by MS / MS analysis according to the number of peaks constituting each isotope peak group. A method has been proposed in which a class list is created, a peak list in which mass-to- charge ratios of peaks are collected for each class, and amino acid sequences are identified based on the peak list. According to this method, it is possible to expect improvement in identification accuracy of individual peptides as compared with the conventional technique.

However, even this method has the following problems. That is, in the plurality of peaks composing a set of isotopic peak group one, the larger the mass to charge ratio of number number number MS / MS peaks of isotopes contained in the selected precursor ions, high mass the intensity of the peak corresponding to the low mass to charge ratio ions as compared with the peak corresponding to the charge ratio ions in a relatively small tendency. For this reason, the intensity of the peak having a relatively low mass-to- charge ratio is too small, and it may be buried in measurement noise, making it difficult to clearly detect the peak. In such a case, the number of peaks constituting the isotope peak group cannot be accurately grasped, and there is a risk of incorrect classification.

As an example, FIG. 8 shows a part of the results of MS / MS analysis by selecting a peak group starting from the mass-to- charge ratio 1046 obtained by MS analysis or a part thereof as a precursor ion, specifically, the mass-to- charge ratio. An enlarged view of the mass spectrum near 932 is shown. In this FIG. 8 (a) ~ (d) are peaks of mass-to-charge ratio from 1046 to 1048 (all), one peak of mass to charge ratio 1046 only, one peak of mass to charge ratio 1047 only, mass-1048 A spectrum obtained by performing MS / MS analysis by selecting ions corresponding to only one peak of each as a precursor ion is shown. At this time, the number of peaks appearing in the MS / MS mass spectrum is actually 1 in (b), 2 in (c), and 3 in (d), depending on the number of isotopes contained in the precursor ion. It should be a book. However, in (d), an isotope peak group of mass spectrum by MS / MS analysis performed by selecting an ion corresponding to a peak with a mass to charge ratio of 1048 as a precursor ion has the smallest mass to charge ratio (m / z = 932) The intensity of the peak is almost the same level as the measurement noise, and it is substantially difficult to detect this as a peak. As a result, even if this result is seen, for example, it is difficult to clearly distinguish whether the number of peaks is 2 or 3.

High-Pep Institute Okinawa Lab Establishment Commemorative Conference Handout, February 19, 2005, Shimadzu Corporation Sugawara, Iwamoto, “Peak Classification in MS / MS Using High Precursor Resolution Ion Trap”, 53rd ASMS Conference , WP345 (2005)

  That is, in the conventional method as described above, even if each isotope peak group appearing on the MS / MS mass spectrum is classified according to the number of peaks constituting the isotope peak group, Since it is difficult to accurately grasp itself, it is difficult to maintain the accuracy of classification, and as a result, the accuracy of identification of amino acid sequences may be reduced.

  The present invention has been made to solve these problems. For example, the isotope peak groups derived from a plurality of peptides are overlapped, and a certain isotope in the isotope peak group is present. Even when the intensity of the peak that appears when MS / MS analysis is performed by selecting a body peak group or a part thereof as a precursor ion, the amino acid sequence of each peptide can be identified with high accuracy. It is to provide a structural analysis system that can.

The structural analysis system according to the present invention, which has been made to solve the above problems, is a structural analysis system for identifying the amino acid sequence or base sequence of a sample using mass spectrometry,
A structural analysis system for identifying the amino acid sequence or base sequence of a sample using mass spectrometry,
a) MS analysis of the sample is performed to obtain an MS mass spectrum, and a part of the isotope peaks appearing in the MS mass spectrum is selected, and ions corresponding to the peaks are used as precursor ions. Mass spectrometry means for performing MS / MS analysis to obtain an MS / MS mass spectrum;
b) The isotope peak group appearing in the MS / MS mass spectrum obtained by the mass spectrometric means is included in the precursor ion that is based on the intensity ratio of a plurality of peaks included in each isotope peak group. By estimating the number of isotopes other than the main isotope, it is classified into a plurality of classes corresponding to the number one-to-one , and monoisotopic peaks within the isotope peak group are determined for each class , A peak list creating means for creating a peak list that lists at least the mass-to-charge ratio of the monoisotopic peak ;
c) sequence identification means for identifying the amino acid sequence or base sequence of the sample based on the peak list created by the peak list creation means;
It is characterized by having.

  In the present invention, the “sample” can be various samples such as a biological sample derived from a living body or an artificially synthesized sample. As a typical example, the sample is a peptide mixture, It can be set as the structure analysis system for identifying the amino acid sequence of a peptide mixture.

In the present invention, “MS / MS analysis” means that a predetermined precursor ion is cleaved, and the obtained ion (product ion) is separated and detected based on a mass-to- charge ratio. It includes not only MS / MS analysis but also so-called MS ⁿ analysis in which the target molecule is cleaved multiple times.

  In addition, as the “partial peak” in the mass spectrometry means, a plurality of peaks may be selected. However, in order to facilitate data analysis, a single peak is selected from the group of isotope peaks. It is desirable to select to perform MS / MS analysis. Furthermore, it is preferable to select the single peak having the maximum ionic strength from the group of isotope peaks obtained by MS analysis.

The “mass analysis means” is not particularly limited in its form or method, but a typical example is a mass spectrometer equipped with a three-dimensional quadrupole ion trap, in which precursor ions are cleaved inside the ion trap. Can be performed. In particular, in order to perform MS / MS analysis by selecting a single peak, this is a mass spectrometer capable of performing MS / MS analysis (or MS ⁿ analysis) by selecting a precursor ion with high resolution. Although necessary, an IT-TOF type configuration combining a three-dimensional quadrupole ion trap and a time-of-flight mass separation unit meets these conditions. In addition, the ion source of the mass spectrometer is preferably configured to ionize a sample by a matrix assisted laser desorption / ionization (MALDI) method. Alternatively, known surface-assisted laser desorption / ionization (SALDI) such as desorption / ionization on (porous) silicon (DIOS) or other known laser ionization methods May be used.

  The “sequence identification means” is for identifying the sequence of a sample from which these peaks are derived, for example, the amino acid sequence of a peptide, based on the peak list created by the peak list creation means. Examples of methods for identifying an amino acid sequence from a peak list include a method of searching a known peptide with a matching spectral pattern by querying the peak list with a database, and a mathematical analysis of each peak list to obtain the amino acid sequence. Various well-known methods such as a method for estimating a sequence can be used.

In the MS / MS analysis using only a part of the isotope peak group by the mass spectrometry means as a precursor ion, the number of peaks constituting the isotope peak group of the product ion generated by cleavage of the precursor ion is the number of the precursor ion. It is limited by the maximum number of isotopes other than the main isotope contained in. However, in some cases, the intensity of some of the peaks constituting the isotope peak group is very small, and it may not be clearly recognized as a peak. Therefore, in the structural analysis system according to the present invention, the peak list creating means includes a plurality of isotope peak groups that constitute each isotope peak group for each of a plurality of isotope peak groups appearing on the MS / MS mass spectrum by the mass analyzing means. Classification is performed by estimating the number of isotopes other than the main isotope contained in the precursor ion that is the basis of the MS / MS analysis based on the intensity ratio of the peaks.

Specifically, for example, it is assumed that a predetermined number (1, 2,...) Of isotopes is included in the elemental composition of the precursor ion and the MS / MS analysis is performed under the assumption. The elemental composition corresponding to the peak included in the isotope peak group obtained in step 1 is estimated, and the intensity ratio of each peak is calculated. That is, this is a theoretical intensity ratio under the assumption of the number of isotopes other than the main isotope . Then, the peak intensity ratio in the isotope peak group appearing on the measured MS / MS mass spectrum is compared with the above theoretical intensity ratio. Assign to the class of the number of isotopes other than the main isotope . In this way, each isotope peak group is classified into each class, and a peak list using the mass-to- charge ratio of the peak as information for each class is created.

Since the peak intensity ratio is used to classify the isotope peak group, for example, the intensity of the peak with the smallest mass-to- charge ratio among the plurality of peaks constituting a certain isotope peak group is very small and clearly Even if it is difficult to identify the presence of a peak, it is known that the intensity of the peak corresponding to the mass-to- charge ratio is originally small based on the peak intensity ratio, so this isotope peak group is accurately classified. It becomes possible.

According to the structural analysis system of the present invention, for example, in the MS mass spectrum obtained by MS analysis of a peptide mixture, even when isotope peaks derived from different peptides overlap , other than the main isotope contained in the selected precursor ion Based on the number of isotopes, isotope peaks derived from individual peptides appearing on the MS / MS mass spectrum can be accurately classified. As a result, an accurate peak list can be provided for identification of amino acid sequences and base sequences, so that amino acid sequences and base sequences can be identified with higher accuracy than before.

  Hereinafter, a system for identifying the amino acid sequence of a peptide mixture will be described as an example of the structural analysis system according to the present invention.

FIG. 1 is a schematic configuration diagram of a structural analysis system according to the present embodiment. The structural analysis system of the present embodiment is roughly composed of a mass analysis unit 10 and a control / processing unit 20. The mass analysis unit 10 selects an ion having a predetermined mass-to- charge ratio as a precursor ion and ionizes the sample containing the peptide mixture by MALDI, and cleaves the precursor ion to generate a product ion. A three-dimensional quadrupole ion trap unit 12, a time-of-flight mass separation unit 13 that separates ions in the time direction based on a mass-to- charge ratio , and an ion detector 14 that sequentially detects the separated ions. I have.

  The control / processing unit 20 creates a mass spectrum (MS mass spectrum, MS / MS mass spectrum) by processing the detection signal obtained by the ion detector 14 and the control unit 21 that controls each unit of the mass analysis unit 10. MS data processing unit 22, peak list creation unit 23 that creates a peak list by processing MS and MS / MS mass spectrum data, and sequence identification processing unit 24 that performs amino acid sequence identification processing based on the peak list And an identification database 25 used for identification. The sequence identification processing unit 24 includes, for example, a search engine such as MASCOT (manufactured by Matrix Science), and can perform identification processing using a public genome database or protein database as an identification database 25. Alternatively, a process for mathematically identifying an amino acid sequence may be performed using software such as a De Novo sequence. The function of the control / processing unit 20 can be realized by executing predetermined software installed in a personal computer.

  The overall procedure for identifying the amino acid sequence of the peptide mixture using the structural analysis system of this example will be described below based on the flowchart of FIG.

First, under the control of the control unit 21, MS analysis is performed by the mass analysis unit 10 on the sample containing the peptide mixture (step S <b> 1). As a result, the MS data processing unit 22 creates an MS mass spectrum with the horizontal axis representing the mass-to- charge ratio and the vertical axis representing the intensity. In this MS mass spectrum, a plurality of isotope peak groups derived from different peptides appear. Now, as shown in FIG. 4, in the obtained MS mass spectrum, an isotope peak group derived from peptide A having a mass-to- charge ratio of 4000 [Da] and derived from peptide B having a mass-to- charge ratio of 4001 [Da] It is assumed that the isotope peak groups overlap.

Next, for example, an ion corresponding to a peak having the highest intensity is selected as a precursor ion from the isotope peak group (step S2). For example, in the example of FIG. 4, a peak (4003 [Da]) having a mass-to- charge ratio of 4003 [Da] in which the fourth peak of peptide A and the third peak of peptide B overlap is selected as the precursor ion.

  Then, under the control of the control unit 21, the mass analysis unit 10 performs MS / MS analysis of the ions selected as the precursor ions (step S3). That is, the precursor ions are selected by the ion trap unit 12 and then cleaved, and the product ions generated by the cleavage of the precursor ions are separated by the mass separation unit 13 and detected by the ion detector 14. Then, the MS data processing unit 22 creates an MS / MS mass spectrum.

The peak (4003 [Da]) on the MS mass spectrum selected as the precursor ion in step S2 has a mass to charge ratio of 3 [Da] larger than the peak of the main ion of peptide A (4000 [Da]). some reason, the isotopic ion of peptide a contained in the peak, so that the isotope other than the main isotope is contained up to three. Similarly, since the peak has a mass-to- charge ratio of 2 [Da] larger than the peak of the main ion of peptide B (4001 [Da]), the isotope ion of peptide B contained in the peak is the main isotope. 2 isotopes other than are included at the maximum. Therefore, since the product ions derived from peptide A generated by cleavage of the peak contain isotopes other than 0 to 3 main isotopes, the product ions derived from peptide A are MS / MS masses. It should form an isotope peak group (quadruple peak) consisting of four peaks on the spectrum. Similarly, since the product ions derived from peptide B each contain isotopes other than 0 to 2 main isotopes, there are 3 product ions derived from peptide B on the MS / MS mass spectrum. This should form an isotope peak group (triple peak) consisting of the peaks.

Next, in the MS / MS mass spectrum, a peak group including a peak having an intensity equal to or higher than a predetermined value and appearing every 1 [Da] is regarded as an isotope peak group (step S4). If the peak previously selected as the precursor ion is an overlap of peaks derived from different peptides, the maximum number of isotopes other than the main isotope contained in the precursor ion may be different between the peptides. The product ions generated from the precursor ions form isotope peak groups having different numbers of peaks for each peptide on the MS / MS mass spectrum. Therefore, in principle, if isotopic peak groups on the MS / MS mass spectrum are classified based on the difference in the number of peaks, only isotopic peak groups of product ions derived from the same peptide are included in each class. Will be included.

However, in reality, there is a possibility that the peak intensity is quite small as described above. In such a case, the existence of the peak cannot be determined deterministically. I can't. Therefore, here we estimate the number of isotopes other than the main isotope contained in the precursor ion after reliably identifying low-intensity peaks by using the intensity ratio of multiple peaks contained in the isotope peak group. Then, classification is performed based on the classification (step S5). A specific method will be described later.

Once classified, in each class of MS / MS mass spectrum, monoisotopic peaks (ion peaks composed of isotopes having the highest natural abundance ratio) within the isotope peak group according to a predetermined algorithm. A peak list describing the mass-to- charge ratio and the like is created (step S6).

  As described above, the peak list La for the peptide A and the peak list Lb for the peptide B are obtained, and the peak lists La and Lb are delivered to the sequence identification processing unit 24. The sequence identification processing unit 24 identifies the amino acid sequences of peptide A and peptide B by searching the database for each of the peak lists La and Lb or by performing mathematical analysis processing of the MS / MS mass spectrum pattern by De Novo sequence or the like. (Step S7).

Subsequently, the process of step S5 will be described in more detail with reference to the flowchart of FIG. First, the element composition of the selected precursor ion is estimated (step S11). Then, the variable n is set to 1 as an initial value (step S12), and it is assumed that the precursor ion includes n isotopes other than the main isotope , that is, one is initially included (step S13). Under the assumption, the composition of the peak obtained by the MS / MS analysis is estimated, and the intensity ratio of each peak of the isotope peak group appearing in the MS / MS mass spectrum is theoretically calculated (step S14).

  Assuming an isotope peak group consisting of four peaks P1, P2, P3, and P4 as shown in FIG. 5, the peak intensity ratio is the ratio U1: U2 of the intensities U1 to U4 of the peaks P1 to P4: U3: U4. For comparison as will be described later, the maximum intensity (U4 in this case) may be set to 1, and the others may be normalized. That is, the intensity ratio is U1 / U4: U2 / U4: U3 / U4: 1. Then, a correlation value Tn indicating a correlation between the obtained intensity ratio by theoretical calculation and each peak intensity ratio of the isotope peak group on the MS / MS mass spectrum by actual measurement is calculated (step S15). The higher the similarity of the peak intensity ratio, the higher the correlation value.

Next, it is determined whether or not the variable n matches a certain default value N (step S16). The predetermined value N is a value that limits the maximum number of isotopes other than the main isotope contained in the precursor ion. For example, the precursor ion includes isotopes other than one, two, or three main isotopes. If this is assumed, N = 3 is set. If the variable n has not reached N, a value obtained by adding 1 to n is set as a new n (step S17), and the process returns to step S13. On the other hand, if the variable n has reached N in step S16, the correlation value T1 to TN calculated so far is extracted as the maximum value Tmax (step S18). Then, it is determined whether or not the maximum value Tmax is greater than or equal to a certain threshold value (step S19).

If the maximum value Tmax does not exceed the threshold value, it cannot be concluded that the peak intensity ratio obtained by actual measurement is similar to the peak intensity ratio obtained by theoretical calculation even if the maximum value Tmax is used. Therefore, if NO in step S19, the isotope peak group is classified into a precursor-derived peak list class that does not contain isotopes other than the main isotope (step S21). On the other hand, in the case of YES in step S19, the isotope peak group is classified into a class of precursor-derived peak lists including the number of isotopes other than the main isotope giving the maximum value Tmax (step S20). . That is, for example, when the precursor ion includes two isotopes other than the main isotope (n = 2), the correlation value T2 with respect to the peak intensity ratio calculated is Tmax, and Tmax is equal to or greater than the threshold value. In this case, this isotope peak group in the MS / MS mass spectrum is classified into a class derived from a precursor ion including two isotopes.

A specific example of the processing described with reference to FIG. Here, the case of analyzing angiotensin II (Angiotensin II: amino acid sequence = [DRVYIHPF], mass to charge ratio = 1046.5 [Da]) is exemplified here. Here, an ion corresponding to a peak having a mass-to- charge ratio of 1047.20 [Da] is selected as a precursor ion.

In order to calculate the isotope peak intensity ratio, it is necessary to estimate the element composition constituting the precursor ion. Senko et al. (Michel W. Senko et.al, “Determination of Monoisotopic, Mathis and Ion Populations, etc.) proposed to estimate the constituent elements from the mass-to- charge ratio of the observed peaks. For Large Biomolecules from Resolved Isotopic Distributions ”, Journal of American Soc. Mass Spectom., 6, pp.229 -233, (1995)) The Averagine model can be used. Averagine model intended to determine the average composition of amino acids than the statistical analysis of the protein _database, the _{C 4.9384 H 7.7583 N 1.3577 O 1.4773} S 0.0417 a unit amino acid sequence. This unit is 111.0543 [Da] when calculated by the elemental mass of monoisotopic. For example, the elemental composition used to calculate the isotope peak group at 1000 [Da] is 9.0046 (= 1000 / 111.0543) units after the number of constituent elements of the Averagine model is rounded to an integer (C ₄₄ H ₇₀ N ₁₂ O ₁₃ , mass-to- charge ratio = 974.5185 [Da]), and determined by appropriately increasing / decreasing the number of H to approach a given mass value (C ₄₄ H ₉₅ N ₁₂ O ₁₃ , mass-to- charge ratio = 999. 7142 [Da]).

When the above Averagine model is used, the elemental composition estimated from the mass-to- charge ratio of the precursor ion (1047.20 [Da]) is C ₅₀ H ₇₁ N ₁₃ O ₁₂ . Assuming that the peak due to the precursor ion is the second isotope peak in the MS mass spectrum, the precursor ion includes one isotope other than the main isotope (that is, n = 1). Therefore, under this assumption, the elemental composition of product ions obtained by cleaving the precursor ions is ¹² C ₄₉ ¹³ C ₁ H ₇₁ N ₁₃ O ₁₂ , C ₅₀ ¹ H ₇₀ ² H ₁ N ₁₃ O ₁₂ , ^{_{C 50 H 71 14 N 12 15}} N 1 O 12, or _believed to ^{_{C 50 H 71 N 13 16 O}} 11 17 O 1, is either. At this time, when the strength ratio of these molecules is obtained by a well-known binomial theorem, the strength of C ₅₀ H ₇₁ N ₁₃ O ₁₂ is 0.5840 (= (0.9893) ⁵⁰ ), and ¹² C ₄₉ ¹³ C ₁ The strength of H ₇₁ N ₁₃ O ₁₂ is 0.3158 (= 50 (0.9893) ⁴⁹ × 0.0107), and if the strength of C ₅₀ H ₇₁ N ₁₃ O ₁₂ is 1, ¹² C ₄₉ ¹³ C ₁ H The strength of ₇₁ N ₁₃ O ₁₂ is 0.5408.

Similarly, when calculating the intensity ratio of other molecules,
C ₅₀ ¹ H ₇₀ ² H ₁ N ₁₃ O ₁₂ : 0.01050 (= 71 (0.99985) ⁷⁰ × 0.00015 / (0.99985) ⁷¹ )
^{_{C 50 H 71 14 N 12 15}} N 1 O 12: 0.0480 (= 13 (0.99632) 12 × 0.00368 / (0.99632) 13)
C ₅₀ H ₇₁ N ₁₃ ¹⁶ O ₁₁ ¹⁷ O ₁ : 0.0046 (= 12 (0.99757) ¹¹ × 0.000383 / (0.99757) ¹² )
It becomes.

When the Averagine model is used, the elemental composition estimated from the mass-to- charge ratio 931.9 [Da] of the isotope peak group on the MS / MS mass spectrum obtained by actual measurement is C ₄₆ H ₆₆ N ₁₃ O _9. . Assuming that a precursor ion having an elemental composition of ¹² C ₄₉ ¹³ C ₁ H ₇₁ N ₁₃ O ₁₂ is cleaved and a product ion having an elemental composition of C ₄₆ H ₆₆ N ₁₃ O ₉ is generated, the product ion is The probability of not including isotopes other than the main isotope is the probability of selecting 46 out of 49 non- isotopes other than the main isotope when selecting 46 out of 50 carbons (C). because there, 49/50 × 48/49 × ... × 4/5, a rewritten when ₄₉ C _46/50 C _46. ^Therefore, 12 the peak intensity of ^{_{C 49 13 C 1 H 71 N}} 13 O 12 from the monoisotopic ion becomes _{(49 C 46/50 C 46} ) × 0.5408. Similarly, when the peak intensity of monoisotopic ions derived from other molecules is calculated, the monoisotopic peak intensity of this product ion can be calculated by the following equation.
_{(49 C 46/50 C 46} ) × 0.5408 + (70 C 66/71 C 66) × 0.0105 + (12 C 12/13 C 12) × 0.048 + (11 C 9/12 C 9) × 0 .0046 = (4/50) × 0.5408 + (5/71) × 0.0105 + (1/13) × 0.048 + (3/12) × 0.0046 = 0.0488
In the same way, the peak intensity of a product ion containing one isotope other than the main isotope can be calculated by the following formula.
{1- (4/50)} × 0.5408 + {1- (5/71)} × 0.0105 + {1- (1/13)} × 0.048 + {1- (3/12)} × 0 .0046 = 0.5551

Accordingly, assuming that MS / MS analysis was performed using an ion containing one isotope other than the main isotope (that is, n = 1) as a precursor ion, the MS / MS mass spectrum on the mass-to- charge ratio of 931.9 [Da] The intensity ratio of the isotope peak group is 0.0488: 0.5551, that is, 0.0879: 1. This is the theoretical peak intensity ratio when it is assumed that the precursor ion contains an isotope other than one main isotope .

Similarly, assuming that MS / MS analysis was performed using an ion containing two isotopes other than the main isotope (that is, n = 2) as a precursor ion, an MS / MS mass spectrum at a mass-to- charge ratio of 931.9 [Da] The intensity ratio of the upper isotope peak group is 0.0410: 0.1602: 1. This is the theoretical peak intensity ratio when it is assumed that the precursor ion contains isotopes other than the two main isotopes .

Now, when the isotope peak group shown in FIGS. 8C and 8D is redrawn with the maximum peak intensity being 1, the result is as shown in FIGS. 6A and 6B. In FIG. 6A, the peak intensity ratio of two peaks appearing at an interval of mass-to- charge ratio 1 [Da] is theoretical when the precursor ion is assumed to contain one isotope other than the main isotope. It is in good agreement with the peak intensity ratio. On the other hand, the peak intensity ratio of the three peaks appearing at an interval of mass-to- charge ratio 1 [Da] in FIG. 6B is theoretical when it is assumed that the precursor ion contains two isotopes other than the main isotope. The peak intensity ratio is in good agreement. Therefore, even if the peak intensity is small and the level is almost buried in measurement noise, each isotope peak group can be accurately classified to create a peak list.

  By using the classification method based on the peak intensity ratio as described above, for example, when an isotope peak group consisting of two peaks is extracted, a product using monoisotopic ions as precursor ions Whether there are two peaks based on ions, or whether there are two peaks based on a product ion whose precursor ion is an ion containing one isotope, that is, whether it is a pseudo isotope peak group. It is also possible to determine.

Further, by applying the above method, the reliability of identification can be improved as follows. That is, ions corresponding to the second (= M + 1 [Da]) and the third (= M + 2 [Da]) single peaks in the isotope peak group starting from the peak whose mass to charge ratio is M [Da]. MS / MS analysis is performed using each as a precursor ion. From the MS / MS analysis in which the ion corresponding to the third peak is executed as a precursor ion, the peak list assuming that the precursor ion created using the above classification includes two isotopes other than the main isotope. Thus, a peptide having a mass to charge ratio of M [Da] can be identified. In the MS / MS analysis in which the ion corresponding to the second peak is used as the precursor ion, the mass to charge ratio is M [Da] from the peak list assuming that the precursor ion contains one isotope other than the main isotope . A peptide can be identified. If a peptide having the same M [Da] can be identified by MS / MS analysis using the ion corresponding to the third or second peak as a precursor ion, it can be considered that the identification reliability of this peptide is high. In this way, identification of the same peptide can be attempted using multiple MS / MS analyses.

  Moreover, the said Example is only an example of this invention, and it is clear that even if it changes suitably, amends, and is added within the meaning of this invention, it is included in the claim of this application.

1 is a schematic configuration diagram of a structural analysis system that is an embodiment of the present invention. The flowchart which shows the whole procedure at the time of identifying the amino acid sequence of a peptide mixture using the structural analysis system of a present Example. The flowchart at the time of classifying the isotope peak group on the MS / MS mass spectrum in the structural analysis system of a present Example. The figure which shows the state which isotope peaks derived from a different peptide overlap on MS mass spectrum. The figure for demonstrating the intensity ratio of the peak which comprises an isotope peak group. The figure which redrawn the isotope peak group shown in FIG.8 (c) and (d) as the maximum peak intensity | strength to 1. FIG. The figure which shows an example of the isotope peak group on MS mass spectrum. The figure which shows a part of result of having selected a part of isotope peak group obtained by MS analysis as a precursor ion, and having performed MS / MS analysis.

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 ... Mass analysis part 11 ... Ionization part 12 ... Ion trap part 13 ... Mass separation part 14 ... Ion detector 20 ... Control / processing part 21 ... Control part 22 ... MS data processing part 23 ... Peak list creation part 24 ... Sequence identification Processing unit 25 ... Identification database (DB)

Claims (5)

A structural analysis system for identifying the amino acid sequence or base sequence of a sample using mass spectrometry,
a) MS analysis of the sample is performed to obtain an MS mass spectrum, and a part of the isotope peaks appearing in the MS mass spectrum is selected, and ions corresponding to the peaks are used as precursor ions. Mass spectrometry means for performing MS / MS analysis to obtain an MS / MS mass spectrum;
b) The isotope peak group appearing in the MS / MS mass spectrum obtained by the mass spectrometric means is included in the precursor ion that is based on the intensity ratio of a plurality of peaks included in each isotope peak group. By estimating the number of isotopes other than the main isotope, it is classified into a plurality of classes corresponding to the number one-to-one , and monoisotopic peaks within the isotope peak group are determined for each class , A peak list creating means for creating a peak list that lists at least the mass-to-charge ratio of the monoisotopic peak ;
c) sequence identification means for identifying the amino acid sequence or base sequence of the sample based on the peak list created by the peak list creation means;
A structural analysis system comprising:
  The structural analysis system according to claim 1, wherein the sample is a peptide mixture and identifies an amino acid sequence of the peptide mixture.   3. The structural analysis according to claim 2, wherein the mass spectrometric means performs MS / MS analysis by selecting a single peak from a group of isotope peaks obtained by MS analysis of a peptide mixture. system.   4. The structural analysis system according to claim 3, wherein a peak having the maximum ionic strength is selected from the group of isotope peaks obtained by MS analysis of the peptide mixture as the single peak. The mass spectrometer is a mass spectrometer equipped with a three-dimensional quadrupole ion trap, and cleaves precursor ions inside the ion trap. The structural analysis system described in Crab.

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