JP2007278712A5 - - Google Patents

Description

Amino acid sequence analysis system using mass spectrometry

  The present invention relates to an amino acid sequence analysis system for mass-analyzing a test sample containing a peptide mixture and estimating the amino acid sequence of each peptide using mass spectrum data obtained thereby.

In recent years, protein structures and functions have been rapidly analyzed as post-genome research. As one of such protein structure / function analysis methods (proteome analysis), protein expression analysis and primary structure analysis using mass spectrometers have been widely performed. Quadrupole ion traps So-called MS ⁿ analysis (n is an integer of 2 or more), which captures and cleaves a specific peak by, for example, collision induced decomposition (CID), is effective. In general, in MS ² (= MS / MS) analysis, first, an ion having a specific mass ( strictly m / z) is selected from an analysis object as a precursor ion, and the precursor ion is cleaved by CID. Then, information on the mass and chemical structure of the target ion can be obtained by mass analysis of ions (product ions) generated by cleavage.

When the amino acid sequence of a protein is identified by MS ⁿ analysis as described above, first, the protein is digested with an appropriate enzyme to form a mixture of peptide fragments, and then the peptide mixture is subjected to mass spectrometry. At this time, since stable isotopes having different masses exist in the elements constituting each peptide, a plurality of peaks having different masses are generated even if the peptides have the same amino acid sequence due to the difference in the isotopic composition. The plurality of peaks are composed of peaks of ions (main ions) composed only of isotopes having the maximum natural abundance ratio, and peaks of ions containing other isotopes (isotope ions), which are spaced by 1 Da. An isotope peak group consisting of a plurality of peaks arranged in a row is formed.

Subsequently, from a mass spectrum data of the peptide mixture as described above, a set of isotope peaks derived from a single peptide is selected as a precursor ion, and ions obtained by cleaving the precursor ion ( Product ion) mass analysis (MS ² analysis). In addition, when the cleavage operation is not performed into a sufficiently small fragment by one cleavage operation, the cleavage operation may be performed a plurality of times.

  Based on the mass spectrum pattern of the product ion obtained as described above and the mass spectrum pattern of the precursor ion, amino acid sequence identification is performed using a search engine such as MASCOT provided by Matrix Science. By executing the database search, the amino acid sequence of the test peptide can be determined. Alternatively, the amino acid sequence of the test peptide can be estimated by performing mathematical operations based on the mass spectrum pattern by executing various analysis software called a De Novo sequence on a computer.

As one of the amino acid sequence estimation methods using the de novo sequence as described above, a method described in Non-Patent Document 1 is conventionally known. This utilizes a mass spectrum obtained by MS ² analysis and a mass spectrum obtained by MS ³ analysis in which one-step cleavage is performed. Briefly, the partial sequence of the peptide is estimated by using the fact that the ions observed in both MS ² analysis and MS ³ analysis are fragment ions having the same end (C-terminal or N-terminal). and it estimates the amino acid sequence of the entire peptide binds a partial sequence was determined by the MS ³ analysis. However, the ions observed in both the MS ² analysis and the MS ³ analysis are only part of the product ions generated by cleavage of the actual peptide, and the information for analysis is not sufficient by itself. Therefore, a complementary spectrum obtained by reversing the mass spectrum obtained by MS ³ analysis (hereinafter simply referred to as MS ³ mass spectrum) with respect to the position of the precursor ion at the time of MS ³ analysis is used. Thus, attempts have been made to increase the types of fragment ion peaks to be collected.

JP 2004-12355 A Z. Zhang and one other member, “De Novo Peptide Sequencing by Two-Demensional Fragment Correlation Mass Spectrometry '', Analytical Chemistry, Vol. 72, No. 11, June 1 2000, pp. 2337-2350

However, in the conventional method described in the above document, the ion peak on the collected mass spectrum is a mixture of fragment ion peaks having different ends (that is, C-terminal and N-terminal). Indistinguishable from terminal fragment ions. Therefore, even if mass information (peak data) relating to such fragment ions is input to de novo sequence analysis software and calculation is performed, there is a problem that the amino acid sequence cannot always be estimated with high accuracy.

  The present invention has been made to solve these problems, and the object of the present invention is to facilitate the estimation of amino acid sequences of proteins and peptides based on mass spectrum data and to increase the accuracy of the estimation. To provide an analysis system.

The present invention made to solve the above problems is an amino acid sequence analysis system for estimating the amino acid sequence of a test sample using mass spectrometry,
a) A peak appearing in a mass spectrum obtained by MS ^n-1 analysis (n is an integer of 2 or more) of a test sample is selected, and MS ⁿ analysis is performed by using the ion corresponding to the peak as a precursor ion. mass spectrometry ^means for acquiring an ⁿ mass spectrum, selecting a peak appearing in the MS ⁿ mass spectrum, performing MS ^{n + 1} analysis using an ion corresponding to the peak as a precursor ion, and obtaining an MS ^{n + 1} mass spectrum;
b) In addition to extracting peaks commonly appearing in the MS ⁿ mass spectrum and MS ^{n + 1} mass spectrum obtained by the mass analyzing means and collecting mass information of ions corresponding to the peaks, the MS ^{n + 1} mass spectrum The mass spectrum after performing either or both of the shift processing of the mass difference of the precursor ion of the ⁿ analysis and the MS ^{n + 1} analysis and the folding processing based on the mass position of the precursor ion of the MS ^{n + 1} analysis Peak list creation that collects the mass information of ions corresponding to the peaks extracted from both MS and MS ⁿ mass spectra, and creates a peak list that aggregates the collected mass information for each end of the amino acid sequence Means,
c) sequence estimation means for estimating an amino acid sequence based on the peak list created by the peak list creation means and aggregated for each amino acid sequence end;
It is characterized by having.

The “mass analysis means” is not particularly limited in its form or method, but as a typical example, 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 ⁿ analysis by selecting a single peak, it is necessary to be a mass spectrometer capable of performing MS ⁿ analysis by selecting a precursor ion with high resolution. The configuration of the IT-TOF type that combines the heavy ion trap and the time-of-flight mass separation unit meets these conditions. Further, the ion source of the mass spectrometer is preferably configured to ionize a sample by, for example, a matrix assisted laser desorption / ionization (MALDI) method.

In addition, the above “sequence estimation means” can perform amino acid sequence estimation based on a so-called de novo sequence, and by executing predetermined software on a computer, a given peak list (a set of mass information) is displayed. Based on this, the amino acid sequence can be estimated by mathematical calculation processing.

In the amino acid sequence analysis system according to the present invention, n can be an appropriate integer of 2 or more, but typically n = 2, MS ⁿ is MS ² (= MS / MS), MS ^{n + 1} is MS ³ .

In the amino acid sequence analysis system according to the present invention, the peak list creating means (1) extracts peaks that appear in common in the MS ⁿ mass spectrum and the MS ^{n + 1} mass spectrum, and collects mass information of ions corresponding to the peaks. (2) The MS ^{n + 1} mass spectrum is subjected to MS ⁿ analysis and MS ^{n + 1} analysis by shifting the precursor ion mass difference, and then the peaks that appear in both the mass spectrum and the MS ⁿ mass spectrum are extracted. Then, mass information of ions corresponding to the peak is collected, and (3) the MS ^{n + 1} mass spectrum is subjected to a folding process based on the mass position of the precursor ion of the MS ^{n + 1} analysis, and the mass spectrum after the processing is performed. mass information of the corresponding ions to the peak by extracting peaks appearing in common to the MS ⁿ mass spectrum and Collected, further (4) MS n + ¹ to the mass spectrum by extracting a peak appearing in common to the mass spectrum and the MS ⁿ mass spectrum after the treatment after performing the above shift processing and folding processing corresponding to the peak Collect mass information of ions.

Since both the shift process and the folding process have the effect of exchanging the terminal sequence of fragment ions corresponding to the peak appearing on the mass spectrum (that is, the N terminal or C terminal), the above (1) and (4) The peak at the same end as the end of the fragment ion corresponding to the peak appearing in the MS ^{n + 1} mass spectrum is extracted, and the mass information of the ion corresponding to the peak is collected. On the other hand, in the above (2) and (3), a peak at the end different from the end of the fragment ion corresponding to the peak appearing in the original MS ^{n + 1} mass spectrum is extracted, and the mass information of the ion corresponding to the peak is obtained. Collected. Thus, since mass information about fragment ions at each end is collected, it can be divided into ends and aggregated into two peak lists.

In this way, mass information of more fragment ions than before can be collected for each terminal. The de novo sequence estimation means estimates fragments (partial sequences) whose bonds are cleaved by cleavage at each end of the amino acid sequence. Therefore, if mass information of fragment ions is given for each end in advance, the sequence is estimated accordingly. Becomes easier and accuracy is improved. Thereby, according to the amino acid sequence analysis system of the present invention, it is possible to estimate the amino acid sequence of a protein or peptide with higher accuracy than before, and the reliability of the analysis result is improved.

Its Furthermore, in order to increase the number of mass information to be collected, the peak list creating means, in further has at least perform the folding processing to the MS n ^{+ 1} mass spectra relative to the mass position of MS n ^{+ 1} analysis of precursor ions After extracting peaks that appear in both the processed mass spectrum and the MS ⁿ mass spectrum, the peak returned to the original MS ^{n + 1} mass spectrum is obtained by performing a reverse conversion to the folding process and corresponds to the peak. The mass information of ions to be collected can be added to the peak list. That is, the peak extracted by the above (3) and (4) is converted to the reverse of the folding process to obtain the peak returned to the original MS ^{n + 1} mass spectrum, and the mass information of the ion corresponding to the peak To collect. This inverse transformation also has the effect of switching the terminal sequence of fragment ions corresponding to the peak appearing on the mass spectrum, so that mass information can be obtained for each terminal. Thereby, the estimation of the amino acid sequence is further facilitated, and the estimation accuracy can be improved.

In addition, the peak list creation means preferably reduces unnecessary information as much as possible when aggregating the peak list for each terminal as described above. For example, the mass information collected for each terminal is within the allowable range of the measured mass . It is better to perform the process of combining the things that can be regarded as the same in one. Moreover, it is good to delete the thing corresponding to a desalting ion or a dehydration ion.

In addition, as a precursor ion in MS ^{n + 1} analysis, it is common to select a precursor ion having the highest peak intensity on the MS ⁿ mass spectrum, but it is better to select an ion corresponding to another peak as a precursor ion. In some cases, the accuracy of sequence estimation may increase.

Therefore, the amino acid sequence analysis system according to the present invention further includes a reliability determination unit that determines the reliability of the sequence estimation by the sequence estimation unit, and when the reliability determination unit determines that the reliability is low. The mass analyzing means selects another peak among the peaks appearing in the MS ⁿ mass spectrum, performs an MS ^{n + 1} analysis using an ion corresponding to the peak as a precursor ion, and the peak list creating means includes: The peak list may be created based on the new MS ^{n + 1} mass spectrum and the MS ⁿ mass spectrum.

According to this configuration, even if the initially selected precursor ion is not suitable for the estimation of the amino acid sequence, a higher reliability amino acid can be obtained based on the result of selecting another precursor ion and performing MS ^{n + 1} analysis. Sequence estimation can be performed.

  Hereinafter, an embodiment of an amino acid sequence analysis system according to the present invention will be described with reference to the drawings.

FIG. 1 is a schematic configuration diagram of an amino acid sequence analysis system according to this embodiment. The 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 as a precursor ion by ionizing a sample containing a peptide mixture by the MALDI method, and generates a product ion by cleaving the precursor ion. A quadrupole ion trap unit 12, a time-of-flight mass separation unit 13 that separates ions in the time direction based on mass , and an ion detector 14 that sequentially detects the separated ions are provided. The mass spectrometric unit 10 can perform MS ² analysis involving at least one cleavage operation and MS ³ analysis involving two cleavage operations.

The control / processing unit 20 creates a mass spectrum (MS ² mass spectrum, MS ³ 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. An MS data processing unit 22, a peak list creation unit 23 for creating a peak list by performing processing as described later on the MS ² and MS ³ mass spectrum data, and an amino acid sequence estimation process based on the peak list It consists of a sequence estimation processing unit 24 to perform and a database 25 used in the sequence estimation.

  The sequence estimation processing unit 24 is a function achieved by executing analysis software called a so-called de novo sequence on a computer. The functions of the control / processing unit 20 other than the sequence estimation processing unit 24 can also be realized by executing predetermined software installed in the personal computer.

  The procedure for estimating the amino acid sequence of the peptide mixture using the amino acid sequence analysis system will be described by taking as an example the case where a tryptic digest of myoglobin (GLSDGEWQQVLNVWGK) is analyzed. FIG. 2 is a flowchart showing a procedure for estimating the amino acid sequence, and FIG. 3 is a schematic diagram showing a processing procedure of mass spectrum data for creating a peak list.

First, under the control of the control unit 21, the mass analysis unit 10 performs MS ² analysis on a test sample containing a peptide mixture, using ions having a peak at a mass of 1815.8 Da as a precursor ion as a result of MS analysis. (Step S1). As a result, the MS data processing unit 22 creates an MS ² mass spectrum with the horizontal axis representing mass and the vertical axis representing intensity.

Next, an ion having a peak at a mass of 1444.4 Da (y12 ion (C-terminal fragment ion); GEWQQVLNVWGK) on the MS ² mass spectrum is selected as a precursor ion, and MS ³ analysis is performed (step S2).

In the MS ² mass spectrum obtained in step S1 and the MS ³ mass spectrum obtained in step S2, the peaks that appear in common within the measurement mass tolerance Δm / z are extracted, and the masses of ions corresponding to these peaks are extracted. A peak list is collected (step S3). Since the precursor ion of MS ³ analysis is the y ion (C-terminal fragment ion) as described above, the peak appearing in the MS ³ mass spectrum is an ion of the same series (that is, the C-terminal series) as the precursor ion, and step S3 Through the above process, a peak list in which mass information of MS ² fragment ions that are the same series as the precursor ion for MS ³ analysis (C-terminal series in this example) is collected is created. The peak list created here is referred to as a common peak list PC (see FIG. 3A).

Then, the mass of the precursor ion of the MS ² analysis (1815.8Da) the mass difference between the MS ³ analysis of the precursor ion mass (1444.4Da) only (371.4Da), each appearing in MS ³ mass spectrum The peak is shifted to the higher mass side on the mass axis (this shifted mass spectrum will be referred to as the MS ³ shift mass spectrum). Then, in the MS ² mass spectrum and the MS ³ shift mass spectrum, peaks that appear in common within the tolerance Δm / z are extracted, and a peak list in which mass information of ions corresponding to these peaks is collected (step) S4). Since the C-terminal series is changed to the N-terminal series by the shift process, mass information of MS ² fragment ions that are different from the precursor ion for MS ³ analysis (N-terminal series in this example) is collected by the process of step S4. A peak list is created. The peak list created here will be referred to as a common peak list notPC (see FIG. 3B).

Next, the MS ³ mass spectrum is folded left and right based on the mass of the precursor ion (1815.8 Da) of MS ² analysis (this folded mass spectrum is referred to as an MS ³ reverse mass spectrum). However, strictly speaking, it is necessary to consider the proton mass (Mproton: 1.0073 Da), so the mass of each peak on the MS ³ reverse mass spectrum is calculated by the following equation (1).
M3rev = M2pre + Mproton-M3 (1)
M3rev: MS ³ each peak of mass on the reverse mass spectrum M2pre: MS ³ analysis time of precursor ion mass M3: MS ³ mass of each peak on the mass spectrum <br/> Then, MS ² mass spectrum and MS ^{In 3} reverse mass spectra, peaks that appear in common within the tolerance Δm / z are extracted, and a peak list in which mass information of ions corresponding to these peaks is collected is created (step S5). Since the folding process also converts the C-terminal fragment ion into a pair of N-terminal fragment ions, the MS in the sequence different from the precursor ion for MS ³ analysis (N-terminal series in this example) is obtained by the process in step S5. A peak list collecting mass information of ^two fragment ions is created. The peak list created here will be referred to as a bare peak list MS2notPC (see FIG. 3C).

Furthermore, the mass that has been collected in the pair peak list MS2notPC extracts peaks when returned by inverse transformation, respectively (1) to the value of the original MS ³ mass spectrum, the ion mass and their corresponding peak A peak list in which information is collected is created (step S6). By this processing, a peak list is created in which mass information of MS ³ fragment ions of the same series (C-terminal series in this example) as the precursor ions for MS ³ analysis is collected. The peak list created here is called a pair peak list MS3PC (see FIG. 3D).

Also the MS ² mass spectrum obtained in step S1, MS ³ for relative mass spectrum and aliasing processing in the shift processing and S5 in the step S4 and the MS ³ shift / reverse mass scan spectrum was made in succession, the allowable measurement mass Peaks that appear in common within the error Δm / z are extracted, and a peak list in which mass information of ions corresponding to these peaks is collected is created (step S7). By this processing, a peak list is created in which MS ² fragment ions of the same series (in this example, C-terminal series) as the precursor ions for MS ³ analysis are collected. The peak list created here will be referred to as a pair peak list MS2PC (see FIG. 3 (e)).

Furthermore, the masses collected in the above pair peak list MS2PC are extracted by returning the values on the original MS ³ mass spectrum by inverse transformation of equation (1), and the masses of ions corresponding to those peaks are extracted. A peak list in which information is collected is created (step S8). By this processing, a peak list is created in which mass information of MS ³ fragment ions of a series different from the precursor ion for MS ³ analysis (N-terminal series in this example) is collected. The peak list created here is referred to as a pair peak list MS3notPC (see FIG. 3F).

Through the above processing, six types of peak lists are obtained. The steps S3 to S8 do not need to proceed in this order. Since these are either the same peak list of the terminal series as the precursor ion for MS ³ analysis or the peak list of a different terminal series, the mass information collected in each peak list is converted into two peak lists for each terminal series. Aggregate (step S9).

That is, the six types of peak lists are aggregated into two types: a peak list including a common peak PC, a pair peak MS2PC, and a pair peak MS3PC, and a peak list including a common peak notPC, a pair peak MS2notPC, and a pair peak MS3notPC. At this time, in each peak list, the peaks that match within the allowable error Δm / z are combined into one. Moreover, peaks with mass differences corresponding to _CO, NH _{3, H 2} O or the like to pair of peaks are deleted. That is, the common peak list includes a / x series fragment ions and desalted and dehydrated ions, but the pair peaks collect only the b / y series fragment ions. Therefore, priority is given to the pair peak, and a / x series fragment ions and desalted / dehydrated ions are removed.

As described above, the masses of ions corresponding to the necessary peaks among the various peaks appearing on the mass spectrum obtained by MS ² analysis and MS ³ analysis are classified into two types of peak lists, b series and y series. The This peak list is delivered to the sequence estimation processing unit 24, and the sequence estimation processing unit 24 performs amino acid sequence estimation using de novo sequence analysis software based on the two types of peak lists (step S10). Although the estimation method will not be described in detail here, generally, such software also outputs an index value indicating the reliability of the obtained result at the same time. Therefore, it is determined whether or not the reliability is equal to or higher than a predetermined threshold (step S11). If the reliability is less than the threshold, the process returns to step S2, and among the peaks appearing in the MS ² mass spectrum. Another precursor ion is selected, MS ³ analysis is performed, and the processes of steps S3 to S10 are repeated.

If it is determined that the reliability is equal to or higher than the threshold value (YES in step S11), it is determined that the amino acid sequence estimation is sufficiently reliable, the process is terminated, and the result is output to the display unit or the like. (Step S12). Alternatively, MS ³ analysis may be performed on all possible peaks to obtain the reliability of amino acid sequence estimation based on the MS ³ analysis, and the amino acid sequence estimation result obtained with the highest reliability may be output.

Above example, i.e. the MS ³ analysis and using the precursor ion of the MS ² analysis and mass 1444.4Da with precursor ions of mass 1815.8Da, peaks in a conventional manner (the method according to Non-Patent Document 1) list (common When peak PC) was prepared, 13 fragment ions (y4 / y5 / y6 / y7 / y10 / y12 / y5-NH ₃ / y6-NH ₃ / y7-NH ₃ / y8-NH ₃ out of 31) were prepared. _{/ y9-NH 3 / y10-} NH 3 / y12-NH 3) was only contains.

On the other hand, when the above-described method according to the present invention is used, 11 fragment ions (y3 / y4 / y5 / y6 / y7 / y10 / y12 / y9) are obtained in the peak list of the same C-terminal series as the precursor ions. _{-NH 3 / y10-NH 3 /} y12-NH 3 / x7) contains, precursor ions with different N-terminal sequences of 27 of 13 amino fragment ion peak lists (b6 / b8 / b9 / b10 / b11 / _{b12 / b13 / b14 / b7-} H 20 / b9-H 20 / b14-NH 3 / b15-NH 3 / c8) were included. That is, fragment ions (y3 / y8 ions and 13 N-terminal fragment ions) that could not be extracted by the conventional method can be extracted. Further, other than b / y-series fragment ions _{(y5-NH 3 / y6-} NH 3 / y7-NH 3 / y10-NH 3) can be removed. Thus, mass information of a large number of fragment ions can be collected for each terminal series, so that amino acid estimation by a de novo sequence can be easily performed, and the reliability of the estimation can be increased.

  As a modification of the above method, when the peak list is aggregated in step S9, the minimum amino acid mass (glycine 57.02 Da) is compared with a highly reliable peak (for example, a pair peak with dehydration and desalting ions). A method of excluding peaks in the range is also conceivable.

  Further, the above-described embodiment is merely an example of the present invention, and it is obvious that the present invention is encompassed in the scope of the claims of the present application even if appropriate modifications, corrections, additions, etc. are made within the scope of the present invention.

For example, in the above-described embodiment, the peak list is created based on the MS ² mass spectrum and the MS ³ mass spectrum. However, the peak list may be created by using the peaks of the mass spectrum having a large number of cleavage operations. . Further, in the above embodiment, six types of peak lists are created and then aggregated into two by end. For example, four peak lists excluding the peak list obtained in steps S6 and S8 are obtained, and two by end are obtained. May be aggregated into one.

BRIEF DESCRIPTION OF THE DRAWINGS The schematic block diagram of the amino acid sequence analysis system which is one Example of this invention. The flowchart which shows the procedure which estimates the amino acid sequence of a peptide mixture using the system of a present Example. The schematic diagram which shows the process sequence of the mass spectrum data for peak list preparation.

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 preparation part 24 ... Sequence estimation Processing unit 25 ... database

Claims (7)

An amino acid sequence analysis system for estimating an amino acid sequence of a test sample using mass spectrometry,
a) A peak appearing in a mass spectrum obtained by MS ^n-1 analysis (n is an integer of 2 or more) of a test sample is selected, and MS ⁿ analysis is performed by using the ion corresponding to the peak as a precursor ion. mass spectrometry ^means for acquiring an ⁿ mass spectrum, selecting a peak appearing in the MS ⁿ mass spectrum, performing MS ^{n + 1} analysis using an ion corresponding to the peak as a precursor ion, and obtaining an MS ^{n + 1} mass spectrum;
b) In addition to extracting peaks commonly appearing in the MS ⁿ mass spectrum and MS ^{n + 1} mass spectrum obtained by the mass analyzing means and collecting mass information of ions corresponding to the peaks, the MS ^{n + 1} mass spectrum The mass spectrum after performing either or both of the shift processing of the mass difference of the precursor ion of the ⁿ analysis and the MS ^{n + 1} analysis and the folding processing based on the mass position of the precursor ion of the MS ^{n + 1} analysis Peak list creation that collects the mass information of ions corresponding to the peaks extracted from both MS and MS ⁿ mass spectra, and creates a peak list that aggregates the collected mass information for each end of the amino acid sequence Means,
c) sequence estimation means for estimating an amino acid sequence based on the peak list created by the peak list creation means and aggregated for each amino acid sequence end;
An amino acid sequence analysis system comprising: The peak list creating means may further commonly to the MS n ^{+ 1} Mass spectrum of the processed after having at least execute the folding processing on mass spectrum relative to the mass position of MS n ^{+ 1} analysis of precursor ions and MS ⁿ mass spectrum After extracting the appearing peaks, the peak returned to the original MS ^{n + 1} mass spectrum is obtained by performing the reverse conversion to the folding process, and the mass information of the ions corresponding to the peaks is added to the peak list. The amino acid sequence analysis system according to claim 1.   The amino acid sequence analysis system according to claim 1 or 2, wherein n = 2.   The amino acid sequence analysis system according to any one of claims 1 to 3, wherein the sequence estimation means performs amino acid sequence estimation based on a de novo sequence. The apparatus further includes a reliability determination unit that determines the reliability of the sequence estimation by the sequence estimation unit, and when the reliability determination unit determines that the reliability is low, the mass analysis unit includes the MS ⁿ mass Another peak is selected from the peaks appearing in the spectrum, MS ^{n + 1} analysis is performed using the ion corresponding to the peak as a precursor ion, and the peak list creating means includes the new MS ^{n + 1} mass spectrum and the MS ⁿ mass spectrum. The amino acid sequence analysis system according to claim 3, wherein a peak list is created based on: The amino acid sequence analysis system according to claim 1 or 2, wherein the peak list creation means collects mass information collected for each terminal into one that can be regarded as identical within an allowable range of measured mass . 3. The amino acid sequence analysis system according to claim 1, wherein the peak list creating unit deletes mass information collected for each terminal and corresponding to desalted ions or dehydrated ions. 4.