JP2008039608A - Mass spectrometry system - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a mass spectrometry system which avoids duplicate analysis of a peptide kind originated in the same protein. <P>SOLUTION: In the first-time analysis, a protein candidate stored already in a protein database among proteins included in a sample is estimated in real time from MS<SP>2</SP>mass spectrum. In the second-time analysis, when selecting parent ion, a peptide included in the protein candidate is removed from a selection target for the parent ion in peaks of MS mass spectrum. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a mass spectrometry system and a mass spectrometry method, and more particularly to a mass spectrometry system and an analysis method for analyzing biopolymers such as proteins and polypeptides.

  In a general mass spectrometry method, a target sample is ionized and the ions are sent to a mass spectrometer. In the mass spectrometer, the ion intensity is measured for each mass-to-charge ratio (m / z) comprising the ratio of mass (m) to valence (z) with respect to the transmitted ions. As a result, a mass spectrum in which the horizontal axis represents the mass-to-charge ratio (m / z) and the vertical axis represents the ionic strength can be obtained.

Mass spectrometry includes MS analysis that ionizes a sample and measures it as it is, and tandem mass spectrometry that selects and dissociates a specific ion after measurement after ionizing and measuring the sample. In tandem mass spectrometry, ions that are the targets of dissociation are called parent ions. Tandem mass spectrometry has a function of performing dissociation and mass spectrometry in multiple stages, such as selecting a parent ion further from a mass spectrum obtained from dissociated ions and dissociating it for mass analysis. Hereinafter, the n-th stage mass spectrometry is referred to as MS ⁿ mass spectrometry. In addition, mass spectrum obtained by ionizing and measuring a sample is selected from MS mass spectrum, and mass spectrum obtained by measuring dissociated parent ions selected from MS mass spectrum is selected from MS ² mass spectrum and MS ² mass spectrum. A mass spectrum obtained by measuring the dissociated parent ion is called an MS ³ mass spectrum.

  Most mass spectrometers that can perform tandem mass spectrometry have a function of automatically selecting a parent ion from an MS mass spectrum to perform ion dissociation.

  For a sample containing a lot of various components such as a biological sample, a system combining a chromatograph and a mass spectrometer is frequently used. In a chromatograph, components in a sample are temporally separated due to differences in the degree of adsorption of substances on a column, etc., so that ion species that are difficult to separate with a mass spectrometer can be separated.

  In the mass spectrometry system, analysis is repeatedly performed in one measurement. At this time, peptide species derived from the same protein are analyzed by dissociating them in duplicate. Duplicate analysis of the same components wastes samples and time. Therefore, Patent Document 1 and Patent Document 2 describe methods for avoiding duplicate analysis of the same component.

  (1) In the method of Patent Document 1, information (ion mass, ion intensity, mass spectrum quality, etc.) of ions that have been dissociated up to N times in tandem mass spectrometry is registered in real time as an internal database. Using the information in this internal database, parent ion selection is limited in the N + 1 first measurement.

(2) In the method of Patent Document 2, the selection of parent ions is limited using retention time in tandem mass spectrometry. Specifically, the amino acid sequence is predicted from the MS ² mass spectrum data, and the retention time predicted from the amino acid sequence is compared with the actual retention time. As a result of the comparison, if they match, the amino acid sequence is regarded as highly reliable data, and the measurement is terminated. If they do not match, dissociation of other parent ions or MS ³ mass spectrometry is performed to further acquire information.

JP 2005-91344 A JP-A-2005-241251

  In the method described in Patent Document 1, only the components having the same mass can limit the selection of the parent ion. There are several peptides that are produced by enzymatic degradation of proteins. If a protein can be sufficiently predicted from the analysis of a certain peptide, it can be said that it is efficient to analyze a peptide other than the peptide derived from the protein. You can only restrict selection.

  In addition, in the method described in Patent Document 2, as in Patent Document 1, it is only a component of a certain mass that can restrict the selection of parent ions, and it is possible to restrict the selection of peptides derived from the same protein. Can not.

  An object of the present invention is to provide a mass spectrometric system that can avoid dissociating and analyzing peptide species derived from the same protein.

According to the present invention, in the first analysis, protein candidates already stored in the protein database among proteins contained in the sample are estimated in real time using the MS ² mass spectrum. In the second analysis, when the parent ion is selected, a peptide other than the peptide contained in the protein candidate is selected as the parent ion among the peaks of the MS mass spectrum.

  According to the present invention, it is possible to avoid overlapping and dissociating and analyzing peptide species derived from the same protein.

  Examples of the present invention are shown below. An outline of a mass spectrometer according to the present invention will be described with reference to FIG. The mass spectrometer of this example includes a sample separation unit 12, an ionization unit 13, a mass analysis unit 14, an ion detection unit 15, a control unit 16, a user input unit 17, a display unit 18, a data processing unit 19, and a protein database 20. Have

As described above, in the mass spectrometry method, a sample is ionized, mass analyzed to obtain an MS mass spectrum, and one peptide is selected as a parent ion from the MS mass spectrum, There are tandem mass spectrometry methods that dissociate and mass analyze to obtain MS ² mass spectra. Tandem mass spectrometry is effective in estimating the structure of a parent ion because molecular structure information of the parent ion can be obtained for each dissociation stage. Hereinafter, tandem mass spectrometry will be described.

  The sample separation unit 12 includes a liquid chromatograph (LC), a gas chromatograph (GC), and the like. The ionization unit 13 performs ionization using an ionization method such as electrospray ionization (ESI), atmospheric pressure chemical ionization (APCI), electron ionization (EI), and chemical ionization (CI). Among these ionization methods, an ionization method is selected according to the sample to be analyzed and the purpose of the analysis.

  The mass spectrometer 14 is configured by a quadrupole mass spectrometer capable of performing tandem mass spectrometry, an ion trap mass spectrometer, or the like. The dissociation method of the parent ion includes a collision dissociation method and an electron capture dissociation method. The collision dissociation method is a method in which a buffer gas such as helium collides with ions to dissociate, and the electron capture / dissociation method dissociates by irradiating low energy electrons and capturing a large amount of low energy electrons in the parent ion. Is the method. The ion detector 15 measures the ion intensity for each mass-to-charge ratio (m / z).

The control unit 16 controls a series of analysis processes of tandem mass spectrometry. The user input unit 17 is a user interface for controlling analysis processing and devices. The display unit 18 displays an analysis result such as a mass spectrum. The data processing unit 19 generates an MS mass spectrum, selects a parent ion, generates an MS ² mass spectrum, and the like. The protein database 20 stores known data about proteins.

  With reference to FIG. 2, an outline of a general process for measuring a protein by tandem mass spectrometry will be described. First, in step S101, a solution containing a sample is introduced into the sample separation unit 12. In step S102, the sample separation unit 12 separates the sample. As described above, sample separation methods include liquid chromatograph (LC) and gas chromatograph (GC). FIG. 3A shows the result of sample separation. As described above, the peak of the intensity of the components separated in time appears from the difference in the degree of adsorption to the column and the like. Thus, the temporal separation result of the sample is referred to as a chromatogram 301.

In step S103, the ionization unit 13 ionizes the sample thus separated in time. In step S104, the mass analyzer 14 performs mass analysis. This is called MS mass spectrometry. In step S105, the result of MS mass spectrometry is obtained. This is called an MS mass spectrum. In step S106, one peptide is selected as the parent ion from the MS mass spectrum and dissociated. Usually, the peak having the highest intensity among the peaks of the MS mass spectrum is selected as the parent ion. In step S107, the mass analyzer 14 performs mass analysis on the dissociated ions. This is referred to as MS ² mass spectrometry. In step S108, the result of MS ² mass spectrometry is obtained. This is referred to as the MS ² mass spectrum.

In the MS mass spectrum and the MS ² mass spectrum, the horizontal axis represents a mass-to-charge ratio (m / z), and the vertical axis represents an ion intensity peak (thin bar graph). The MS ² mass spectrum includes information for estimating the molecular structure of the protein of the sample, and the protein can be estimated based on this information.

From ionization in step S103, MS mass analysis in step S104, acquisition of MS mass spectrum in step S105, selection and dissociation of parent ion in step S106, MS ² mass analysis in step S107, acquisition of MS ² mass spectrum in step S108 This series of processing is called analysis. In step S109, it is determined whether or not reanalysis is performed. When reanalysis is performed, the process returns to step S103. When the reanalysis is not performed, the process proceeds to step S110. In step S110, post-processing is performed on the MS ² mass spectrum. In post-processing, noise removal, peak determination, isotope peak removal, and the like are performed, and a protein database 20 in which information on known proteins is accumulated is searched for protein identification.

FIG. 3A shows an example of the chromatogram 301 obtained by the sample separation unit 12 as described above. The chromatogram includes component intensity peaks separated in time. FIG. 3B shows the relationship between the chromatograph 301 and the analysis cycle. The analysis 302 is a series of processes from the ionization in step S103 in FIG. 2 to the acquisition of the MS ² mass spectrum in step S108. As shown in the figure, when the chromatogram is obtained, it can be seen that the analysis 302 is repeatedly performed for each component. The analysis time 303 required for one analysis 302 varies depending on analysis conditions, apparatus conditions, and the like. In order to perform many analyzes 302 by one measurement, it is desirable that the analysis time 303 is short.

By repeatedly performing the analysis 302 on the chromatograph 301 obtained by temporal separation in this way, MS ² mass spectra of various ions can be acquired. In this way, it is common to accumulate protein structural information contained in a sample.

In the tandem mass spectrometry method, the accuracy of the protein identification result and the number of proteins to be identified depend on the MS ² mass spectrum, and how to select a parent ion from the MS mass spectrum is important.

Therefore, in the present invention, when the MS ² mass spectrum is acquired in the first analysis, the amino acid is decoded and the protein database is searched in real time. When the same amino acid sequence exists in the protein database 20, it is considered that the protein from which the amino acid sequence is derived has already been experimented. Therefore, in the second analysis, all peptides derived from the protein are excluded from the parent ion selection targets. That is, a peptide other than the peptide derived from the protein is selected as a parent ion. In this way, it is possible to avoid duplicate measurement of peptides derived from proteins already stored in the protein database 20.

A first example of the tandem mass spectrometry method according to the present invention will be described with reference to FIGS. 4, 5, and 6. Steps S201 to S208 may be the same as the conventional tandem mass spectrometry method described with reference to FIG. That is, in step S201, a solution containing a sample is introduced into the sample separation unit 12. In step S202, the sample separation unit 12 separates the sample. In step S203, the ionization unit 13 ionizes the sample. In step S204, the mass spectrometer 14 performs MS mass spectrometry. In step S205, an MS mass spectrum is obtained. In step S206, one peptide is selected as a parent ion from the MS mass spectrum and dissociated. In step S207, the mass spectrometer 14 performs MS ² mass spectrometry on the dissociated parent ion. In step S208, an MS ² mass spectrum is obtained.

In step S209, the data processing unit 19 estimates protein candidates using the MS ² mass spectrum. That is, the amino acid sequence is decoded from the MS ² mass spectrum and searched from the protein database 20. As a search result, proteins that are estimated to contain the amino acid sequence are listed. In this way, a list of protein candidates is obtained. The protein candidate is obtained by searching the protein database 20. It is considered that sufficient data on such proteins has been obtained. Therefore, it is not necessary to analyze such protein candidates in duplicate. Therefore, in the second analysis, peptides included in the protein candidates are excluded from parent ion selection targets.

  An example of a protein candidate estimation method will be described later with reference to FIG. The data processing unit 19 calculates the estimation accuracy of each protein candidate.

  In step S210, the data processing unit 19 determines whether there is a protein candidate whose estimation accuracy is greater than the threshold value X. If there is a protein candidate whose estimation accuracy is greater than the threshold value X, the process proceeds to step S211, and if there is no protein candidate whose estimation accuracy is greater than the threshold value X, the first analysis is terminated and the next second analysis is performed. Move.

  In step S211, the data processing unit 19 acquires peptide information of protein candidates whose estimation accuracy is greater than the threshold value X. That is, the mass of a peptide derived from a protein candidate whose estimation accuracy is larger than the threshold value X is obtained. Details of the peptide information acquisition process will be described later with reference to FIG.

  In step S212, the data processing unit 19 registers peptide information. That is, the peptide mass (m) is registered in the peptide list 600. The peptide list 600 is a list of peptide names 601, their amino acid sequences 602, and their masses 603, as shown in FIG. 6 (a).

Thus, the protein candidate is estimated using the MS ² mass spectrum, and when the acquisition and registration processing of the peptide information related to the estimated protein candidate is completed, the first analysis is completed, and the next analysis is started.

  The second analysis of the first example of the mass spectrometry method according to the present invention will be described with reference to FIG. In step S301, the ionization unit 13 ionizes the sample. In step S302, the mass spectrometer 14 performs MS mass spectrometry. In step S303, an MS mass spectrum is obtained.

  In step S304, the data processing unit 19 excludes the data included in the peptide list 600 from the MS mass spectrum peaks from the selection target of the parent ion. FIG. 6B shows an MS mass spectrum. In this example, the mass of the data marked with “x” is included in the peptide list. Therefore, the data marked with x is excluded from the parent ion selection targets.

In step S305, one peptide is selected as a parent ion from the MS mass spectrum and dissociated. As described above, the same data as the data in the peptide list 600 is excluded from the peak of the MS mass spectrum. In step S306, the mass spectrometer 14 performs MS ² mass spectrometry on the dissociated parent ion. In step S307, an MS ² mass spectrum is obtained. In step S308, the MS ² mass spectrum is post-processed to identify the protein.

  Thus, the second analysis is completed. Comparing the first analysis in FIG. 4 and the second analysis in FIG. 5, the second analysis in FIG. 5 is different in that the process of step S304 is added.

  The main part of the mass spectrometry method of the present invention will be described with reference to FIG. FIG. 6A shows an example of the peptide list 600. The peptide list 600 includes data of a peptide name 601, a peptide sequence 602, and a peptide sequence mass 603, and is created for each protein candidate. Therefore, a protein name is assigned to each peptide list 600. In the MS mass spectrum obtained in step S303, the horizontal axis represents the mass-to-charge ratio (m / z), and the vertical axis represents the ion intensity peak (thin bar graph). According to the present invention, in the second and subsequent analyses, the peptide spectrum 600 is referred to in step S306 before the parent ion selection process in step S305, and the same mass spectrum as the mass (m) registered there. Exclude peaks. Data registered in the peptide list 600 is obtained from the protein database 20. Therefore, it is considered that sufficient information has already been obtained regarding proteins derived from such peptides. Therefore, duplicate analysis can be avoided by removing the peptides registered in the peptide list 600 from the selection target of the parent ion.

  In the example of FIG. 6, the mass of the peak marked with “x” in the MS mass spectrum is included in the peptide list. Therefore, the peak marked with x is removed from the parent ion selection target.

  The horizontal axis of the MS mass spectrum peak is the mass-to-charge ratio (m / z). On the other hand, the peptide mass is registered in the peptide list. Therefore, in order to compare the two, it is necessary to convert the mass-to-charge ratio (m / z) of the peak of the MS mass spectrum into the mass (m). Therefore, the valence (z) may be obtained from the peak interval between the isotope peaks appearing on the MS mass spectrum and multiplied by the mass-to-charge ratio (m / z).

  Further, the mass of the MS mass spectrum peak and the mass of the peptide list do not need to be completely coincident with each other. For example, when the mass m of the peak of the MS mass spectrum is obtained, a mass range (m ± Δm) around that is set. When the mass of the peptide list is included in the mass range (m ± Δm), it may be determined that they match.

Details of the protein candidate estimation process in step S209 of FIG. 4 will be described with reference to FIG. In step S71, the data processing unit 19 decodes the amino acid sequence from the MS ² mass spectrum. That is, the amino acid sequence of the peptide selected as the parent ion is decoded. The MS ² mass spectrum to be subjected to amino acid decoding includes not only the MS ² mass spectrum acquired in this analysis but also the MS ² mass spectrum acquired in the previous analysis. For example, if the current analysis is the second analysis, the MS ² mass spectrum acquired from the first time to the second time is used.

For each analysis, the amino acid sequence may be deciphered from the MS ² mass spectrum obtained at that time, but for each analysis, the amino acid sequence of all MS ² mass spectra obtained from the previous time to this time is analyzed. Decryption may be performed.

A de novo sequencing method may be used for decoding the amino acid sequence. In this method, the amino acid sequence is decoded by reading the peak interval present in the MS ² mass spectrum.

In step S72, the data processing unit 19 searches the protein database 20 using the decrypted amino acid sequence as a key. That is, a protein containing the amino acid sequence obtained by decoding from the MS ² mass spectrum is searched from the protein database 20 and used as a protein candidate.

  The data processing unit 19 creates a protein candidate list 700. The protein candidate list 700 includes a protein name 701, its estimated accuracy 702, and an amino acid sequence 703 constituting the protein.

In this example, the estimation accuracy is the content of the amino acid sequence decoded from the MS ² mass spectrum for amino acids contained in the protein obtained by searching from the protein database 20. For example, the estimation accuracy of “protein 1” is 30%. This is 30% of the amino acid sequence included in “Protein 1” decoded from the MS ² mass spectrum, and the remaining 70% is an amino acid sequence other than the amino acid sequence decoded from the MS ² mass spectrum. It means that.

  With reference to FIG. 8, the acquisition process of the peptide information regarding the protein candidate of step S211 will be described. In the peptide information acquisition process, the protein candidate is decomposed into peptides. However, protein candidates already decomposed in the past analysis and registered in the peptide list are excluded because they are duplicates. Proteins are cleaved and digested by digestive enzymes. This fragmented product is a peptide. Since the amino acid cleaved by the digestive enzyme is determined, it is understood how the protein is peptided by the digestive enzyme.

  FIG. 8 shows an example using trypsin as a digestive enzyme. When trypsin is used as a digestive enzyme, the amino acid sequence of the protein is cleaved between lysine (K) and arginine (R). As illustrated, the protein candidate 801 is cleaved by trypsin, and a plurality of peptides 802 are generated. Since the mass of amino acids contained in these peptides 802 is known, the mass of each peptide can be calculated 803. In step S212, the protein name, amino acid name, amino acid sequence, and mass are registered in the peptide list 600 for each protein candidate.

FIG. 9 shows an example of a user interface (UI) of the mass spectrometry system according to the present invention, that is, an input screen of the display unit 18. The input screen 900 of this example includes a peak exclusion execution condition 901 and a protein search condition 904. The peak exclusion execution condition 901 includes an estimation accuracy threshold value (%) 902 and a mass matching accuracy (amu) 903. The protein search condition 904 includes MS ² analytical mass accuracy (amu) 905, digestive enzyme 906, protein database 907, and taxonomy 908.

The peak exclusion execution condition 901 is a field for setting parameters relating to the entire analysis. The estimation accuracy threshold value (%) 902 is a field for setting a threshold value X used in the estimation accuracy comparison process in step S210 of FIG. The mass coincidence accuracy (amu) 903 is used in the process of removing the data in the peptide list from the peaks of the MS mass spectrum in step S304 in FIG. That is, this is a field for designating the mass range (Δm) when comparing the MS ² mass spectrum peak and the peptide mass of the peptide list.

The protein search condition 904 is a field for setting parameters and the like regarding processing for estimating protein candidates from the MS ² mass spectrum in step S209 of FIG. The MS ² analysis mass accuracy (amu) 905 is a field for designating a mass error range in which the peak interval of the MS ² mass spectrum and the amino acid mass are considered to coincide in the decoding process of the amino acid sequence in step S71 of FIG. The digestive enzyme field 906, the protein database field 907, and the classification (Taxnomy) field 908 are fields for designating conditions for searching the protein database 20 in the database search processing in step S72 of FIG. In this example, three conditions are used as search conditions for the protein database 20, but other conditions such as the type of apparatus used and protein modification conditions can also be used. However, the digestive enzyme 906 is an indispensable designation item because it is also used when acquiring a peptide sequence from a protein amino acid sequence in the peptide information acquisition process of step S211.

FIG. 10 shows an example of a user interface (UI) of the mass spectrometry system according to the present invention, that is, an output screen of the display unit 18. The output screen 1000 of this example includes a protein list 1001, a chromatogram display unit 1005, an MS mass spectrum display unit 1006, and an MS ² mass spectrum display unit 1007. In the protein list 1001, proteins 1002 are displayed in order, and finally 1003 is displayed. The protein 1002 is the protein 701 in the protein candidate list 700 shown in FIG.

When one of the proteins 1002 is clicked, among the amino acid sequences included in the protein candidate, the amino acid sequence 1004 decoded from the MS ² mass spectrum is displayed. When one of the protein candidates 1002 is double-clicked, as shown in FIG. 11, the entire amino acid sequence 1100 of the protein is displayed in another window. For example, 30% of the amino acid sequence included in “Protein 2” is an amino acid sequence decoded from the MS ² mass spectrum, and 70% is an amino acid sequence other than the amino acid sequence decoded from the MS ² mass spectrum. Clicking on “Protein 2” displays a 30% amino acid sequence, and double-clicking on “Protein 2” displays all amino acid sequences in a separate window.

In the other field 1003, an amino acid sequence that is not derived from any protein among amino acid sequences decoded from the MS ² mass spectrum is displayed.

When one of the amino acid sequences 1004 is selected, it is switched to bold display, and the MS ² mass spectrum used when decoding this amino acid sequence is displayed on the MS ² mass spectrum display unit 1007 to select the parent ion. The MS mass spectrum used sometimes is displayed on the MS mass spectrum display unit 1006, and the chromatogram which is the result of the sample separation is displayed on the chromatogram display unit.

On the MS mass spectrum screen 1006, the peak selected as the parent ion is displayed in a color different from the other peaks, and the value of the mass is displayed. In the MS ² mass spectrum screen 1007, the amino acid sequence read with respect to the peak interval is displayed.

Next, a second example of the mass spectrometry method according to the present invention will be described. In the first example shown in FIGS. 4 and 5, in step S209, the amino acid sequence was decoded from the MS ² mass spectrum using the de novo sequencing method, and the protein candidate was estimated. In this example, an MS ² mass spectrum for testing is prepared in advance in the protein database 20 from known proteins. And MS ² mass spectrum for this assay, by comparing the MS ² mass spectrum obtained in the analysis, to estimate the protein candidates. For example, MS / MS Ion Search in MASCOT is used.

Next, a third example of the mass spectrometry method according to the present invention will be described. In the first example shown in FIGS. 4 and 5, two-stage mass analysis of MS mass spectrometry and MS ² mass spectrometry was performed. In this example, when the estimation accuracy is low in the protein candidate estimation process in step S209, three or more stages of mass analysis are performed. As described above, the details of the protein candidate estimation process in step S209 are shown in FIG. In the decoding process of the amino acid sequence in step S71 in FIG. 7, when the amino acid sequence cannot be sufficiently decoded from the MS ² mass spectrum, MS ³ mass spectrometry is performed. That is, one parent ion is selected from the MS ² mass spectrum, dissociated, and MS ³ mass spectrometry is performed.

  According to this example, in the decoding process of the amino acid sequence in step S71 of FIG. 7, a threshold value N for the number of decoding amino acid sequences is set. The threshold value N may be a fixed value, but is preferably set by the user on the input screen 900 shown in FIG.

In the protein candidate estimation process in step S209, the number of amino acids obtained by decoding the MS ² mass spectrum is compared with a threshold value N. If the threshold value N is not satisfied, MS ³ mass spectrometry is performed. As described with reference to FIG. 7, in step S71, the data processing unit 19 decodes the amino acid sequence from the MS ² mass spectrum and the MS ³ mass spectrum. If the number of decoding is greater than or equal to the threshold value N, the process proceeds to step S210. When the number of decoding is less than the threshold value N, MS ⁴ mass spectrometry is performed. The amino acid sequence is decoded from the MS ² mass spectrum, the MS ³ mass spectrum, and the MS ⁴ mass spectrum. Thus, MS ⁿ mass spectrometry is repeated until the number of decoding is equal to or greater than the threshold value N. Thus, the MS ² mass spectrometry, it is possible to cope for ions such as not sufficiently decomposed.

  Next, a fourth example of the mass spectrometry method according to the present invention will be described. In Example 1 shown in FIGS. 4 and 5, in step S211, peptide information is obtained for a protein candidate with high estimation accuracy, and the mass (m) of the peptide is registered in the peptide list 600 in step S212. . However, in this example, when a known protein is included in the sample, it is registered in the peptide list. For other proteins, peptide information is acquired and registered by the method shown in FIG. In the case where the same sample is repeatedly analyzed, the past peptide list can be continuously used to avoid duplicate analysis and to perform efficient sample measurement.

  A fifth example of the mass spectrometry method according to the present invention will be described with reference to FIGS. In the first example, in step S305, one peak was selected from the peaks of the MS mass spectrum and used as the parent ion. In one analysis, only the information on the amino acid sequence of one peptide appearing in the MS mass spectrum is obtained. Therefore, information on peaks other than the peak selected as the parent ion among the peaks of the MS mass spectrum is not effectively used. Therefore, in this example, the information of the MS mass spectrum is used efficiently.

  The first analysis of the fifth example of the mass spectrometry method according to the present invention will be described with reference to FIG. Steps S401 to S409 may be the same as steps S201 to S209 of the first example shown in FIG.

  According to this example, in step S410, peptide information is acquired for all estimated protein candidates. In the first example shown in FIG. 4, peptide information is obtained for protein candidates whose estimation accuracy is greater than the threshold value X. However, in this example, peptide information is obtained for all protein candidates.

  In step S411, the peptide information is divided into two peptide lists. The estimation accuracy of the protein candidate is compared with a preset threshold value Y. In step S <b> 412, the mass of the protein candidate peptide whose estimation accuracy is greater than the threshold Y is registered in the first peptide list. In step S413, the mass of the peptide candidate protein whose estimation accuracy is equal to or less than the threshold Y is registered in the second peptide list. Thus, the first analysis is completed, and then the second analysis is performed.

  A second analysis of the fifth example of the mass spectrometry method according to the present invention will be described with reference to FIG. In step S501, the ionization unit 13 ionizes the sample. In step S502, the mass spectrometer 14 performs MS mass spectrometry. In step S503, an MS mass spectrum is obtained.

  In step S504, the data processing unit 19 checks whether or not a peak corresponding to the mass of the peptide in the second peptide list exists in the MS mass spectrum. That is, the peptide content of the second peptide list in the MS mass spectrum is calculated. In step S505, a protein candidate whose content exceeds the threshold value Z is extracted, and the mass of the peptide is transferred from the second peptide list to the first peptide list. In this example, even if the peptides are in the second peptide list, if the content ratio with respect to the MS mass spectrum exceeds the threshold Z in the second analysis, the protein candidate is stored in the protein database 20. It is thought that. Therefore, it is not necessary to analyze such protein candidates in duplicate. Thus, the peptides transferred to the first peptide list are excluded from the parent ion selection targets.

  In step S506, the data processing unit 19 excludes data included in the first peptide list from the MS mass spectrum peaks from the parent ion selection targets. In step S507, one peptide is selected as a parent ion from the MS mass spectrum and dissociated. As described above, the same data as the data of the first peptide list is excluded from the peak of the MS mass spectrum.

  The processing from step S508 to step S510 may be the same as the processing from step S306 to step S308.

  In this way, in this example, it is possible to determine whether there are other peptides derived from protein candidates by effectively using the information of the MS mass spectrum obtained in the second analysis.

  A sixth example of the mass spectrometry method according to the present invention will be described with reference to FIG. In the fifth example, in the second analysis, peptides having a high content in the MS mass spectrum are transferred to the first peptide list. In this example, in the second analysis, the peptides in the second peptide list are used to set the priority of selection of parent ions.

  In this example, the first analysis is the same as in the fifth example. That is, steps S401 to S413 in FIG. 12 are the first analysis.

  The second analysis of the sixth example of the mass spectrometry method according to the present invention will be described with reference to FIG. In step S601, the ionization unit 13 ionizes the sample. In step S602, the mass spectrometer 14 performs MS mass spectrometry. In step S603, an MS mass spectrum is obtained.

  In step S604, the data processing unit 19 excludes the data in the first peptide list from the peaks of the MS mass spectrum from the selection targets of the parent ions. In step S605, the data processing unit 19 sets priorities for selecting parent ions for the remaining peaks of the MS mass spectrum. The priority is compared to the mass of peptides in the second peptide list for all remaining peaks in the MS mass spectrum. Give bonus to matching peaks. For each protein candidate, such comparison and bonus are given, and the cumulative value of the bonus for each peak is obtained. The order in which the cumulative value is large is set as a priority order for selection as a parent ion. Here, the bonus given when the mass of the peak coincides with the mass of the peptide may be a fixed value, but it preferably varies depending on conditions such as the estimation accuracy of the protein candidate. For example, a high bonus is set for protein candidates with high estimation accuracy, and a low bonus is set for protein candidates with low estimation accuracy. In this way, by setting a bonus based on the estimation accuracy of protein candidates and giving priority, it is possible to select a peptide derived from a protein that is likely to be contained in a sample as a parent ion it can.

  In step S606, the data processing unit 19 selects a parent ion and dissociates it. The selection target of the parent ion is the remaining peaks excluding the data in the first peptide list among the peaks of the MS mass spectrum. From these peaks, the peak with the highest priority is selected as the parent ion.

  Steps S607 to S609 may be the same as the processes of steps S508 to S510 in FIG.

  The example of the present invention has been described above, but the present invention is not limited to the above-described example, and various modifications can be easily made by those skilled in the art within the scope of the invention described in the claims. Will be understood.

It is a figure which shows the structure of the mass spectrometry system of this invention. It is a figure which shows the outline of a process of the conventional tandem mass spectrometry method. It is a figure which shows the chromatogram obtained by a sample separation part, and the repetition of an analysis. It is a figure which shows the outline of the 1st analysis process of the 1st example of the tandem mass spectrometry method of this invention. It is a figure which shows the outline of the 2nd analysis process of the 1st example of the tandem mass spectrometry method of this invention. It is a figure for demonstrating the process excluded from the selection object of a parent ion from the peak of MS mass spectrum in the analysis process of the 2nd time of the 1st example of the tandem mass spectrometry method of this invention. It is a figure for demonstrating the estimation process of a protein candidate in the 1st analysis process of the 1st example of the tandem mass spectrometry method of this invention. It is a figure for demonstrating the acquisition process of peptide information in the 1st analysis process of the 1st example of the tandem mass spectrometry method of this invention. It is a figure which shows the example of the input screen of the mass spectrometry system of this invention. It is a figure which shows the example of the output screen of the mass spectrometry system of this invention. It is a figure which shows the example which displays the amino acid sequence of an analysis result in the output screen of the mass spectrometry system of this invention. It is a figure for demonstrating the 1st analysis process of the 5th example of the tandem mass spectrometry method of this invention. It is a figure for demonstrating the 2nd analysis process of the 5th example of the tandem mass spectrometry method of this invention. It is a figure for demonstrating the 2nd analysis process of the 6th example of the tandem mass spectrometry method of this invention.

Explanation of symbols

DESCRIPTION OF SYMBOLS 12 ... Sample separation part, 13 ... Ionization part, 14 ... Mass spectrometry part, 15 ... Ion detection part, 16 ... Control part, 17 ... User input part, 18 ... Display part, 19 ... Data processing part, 20 ... Protein database

Claims (20)

Sample separation unit that separates the sample, ionization unit that ionizes the separated sample, mass analysis unit that performs mass analysis of the ionized sample, and ion detection that measures ion intensity for each mass-to-charge ratio (m / z) And a protein database storing data on proteins,
Ionization treatment for ionizing a sample, MS mass spectrometry treatment for obtaining an MS mass spectrum by mass analysis of the ionized sample, parent ion selection and dissociation treatment for selecting a parent ion from a peak of the MS mass spectrum, and when executing dissociated MS ² mass analysis process to obtain the MS ² mass spectrum parent ion by mass spectrometry, by repeating the analysis including,
In the first analysis, using the MS ² mass spectrum, protein candidates stored in the protein database among the proteins contained in the sample are estimated in real time,
In the second analysis, when selecting a parent ion, a peptide other than the peptide contained in the protein candidate is selected as a parent ion among the peaks of the MS mass spectrum. The mass spectrometry system according to claim 1, wherein in the first analysis, when the protein candidate is estimated, an amino acid sequence is decoded from a peak of the MS ² mass spectrum, and the decoded amino acid sequence is used as a search key for the protein database. A mass spectrometry system characterized by searching.   The mass spectrometry system according to claim 1, wherein the estimation accuracy of the protein candidate is calculated in the first analysis, and when the parent ion is selected in the second analysis, the estimation among the peaks of the MS mass spectrum. A mass spectrometric system, wherein a peptide other than a peptide included in a protein candidate with accuracy greater than a predetermined threshold is selected as a parent ion.   4. The mass spectrometric system according to claim 3, wherein the estimation accuracy is represented by a ratio including an amino acid sequence decoded from an MS mass spectrum with respect to all amino acid sequences of protein candidates searched from the protein database. Mass spectrometry system.   The mass spectrometry system according to claim 3, wherein in the first analysis, information on peptides included in protein candidates whose estimation accuracy is greater than a predetermined threshold is obtained to create a peptide list, and in the second analysis, A mass spectrometric system characterized in that, when performing ion selection, a peptide other than the peptides included in the peptide list is selected as a parent ion out of MS mass spectrum peaks.   6. The mass spectrometry system according to claim 5, wherein, in the first analysis, when obtaining the peptide information, the protein candidate is decomposed by an enzyme, and the mass of the obtained peptide is calculated from the mass of each amino acid. Mass spectrometry system.   6. The mass spectrometry system according to claim 5, wherein, in the first analysis, when the peptide information is acquired, if a known protein is included in the sample, the peptide of the known protein is registered in the peptide list. A mass spectrometry system characterized by: 2. The mass spectrometry system according to claim 1, wherein when the protein candidate is estimated in the first analysis, an MS ² mass spectrum for a test for a known protein is obtained in advance and registered in the protein database. and MS ² mass spectrum of use, the mass spectrometry system by comparing the MS ² mass spectrum obtained by the first analysis, characterized in that to estimate the protein candidates. The mass spectrometry system according to claim 1, wherein in the first analysis, when estimating a protein candidate from the MS ² mass spectrum, if the estimation accuracy is low, a parent ion is selected from the peak of the MS ² mass spectrum. mass spectrometry system according to claim parent ion selection and dissociation process, and to perform the MS ³ mass spectrometry process, to obtain the MS ³ mass spectrum with mass spectrometry parent ions dissociated dissociates Te. The mass spectrometry system of claim 1,
In the first analysis, the estimation accuracy of the protein candidate is calculated, information on the peptide included in the protein candidate is obtained, and information on the peptide candidate protein whose estimation accuracy is greater than a predetermined threshold among the protein candidates. Register in the first peptide list, register information on the protein candidates whose estimated accuracy is less than the predetermined threshold among the protein candidates in the second peptide list, and select the parent ion in the second analysis A mass spectrometry system, wherein a peptide other than the peptides in the first peptide list is selected as a parent ion from the MS mass spectrum peaks.   The mass spectrometry system according to claim 10, wherein in the second analysis, the content of the peptide of the second peptide list with respect to the peak of the MS mass spectrum is calculated, and the peptide whose content exceeds a predetermined threshold is calculated. A mass spectrometry system, wherein the peptide list is transferred from the second peptide list to the first peptide list.   The mass spectrometry system according to claim 10, wherein in the second analysis, when the parent ion is selected, the peptides of the first peptide list are removed from the selection target of the parent ion among the peaks of the MS mass spectrum, Furthermore, a priority order for selecting a parent ion for the remaining peaks of the MS mass spectrum is set.   13. The mass spectrometric system according to claim 12, wherein in the second analysis, when setting the priority order for selecting a parent ion, the remaining peaks of the MS mass spectrum are compared with the peptides in the second peptide list and matched. Bonus is given to each peak, and such comparison and bonus are given for each protein candidate, and the cumulative value of the bonus for every remaining peak in the MS mass spectrum is obtained. A mass spectrometry system having the above-mentioned priority order. Sample separation unit that separates the sample, ionization unit that ionizes the separated sample, mass analysis unit that performs mass analysis of the ionized sample, and ion detection that measures ion intensity for each mass-to-charge ratio (m / z) And a protein database storing data on proteins,
Ionization treatment for ionizing a sample, MS mass spectrometry treatment for obtaining an MS mass spectrum by mass analysis of the ionized sample, parent ion selection and dissociation treatment for selecting a parent ion from a peak of the MS mass spectrum, and when executing dissociated MS ² mass analysis process to obtain the MS ² mass spectrum parent ion by mass spectrometry, by repeating the analysis including,
In the first analysis, protein candidates already stored in the protein database among the proteins contained in the sample from the MS ² mass spectrum are estimated in real time, the estimation accuracy of the protein candidates is calculated, and the protein candidates are calculated. Information on peptides included in the protein candidates, information on peptides of the protein candidates whose estimated accuracy is greater than a predetermined threshold among the protein candidates is registered in the first peptide list, and the estimated accuracy of the protein candidates is predetermined Registering information on peptide candidates that are less than the threshold value in the second peptide list,
In the second analysis, when the parent ion is selected, the peptides in the first peptide list are excluded from the parent ion selection targets among the MS mass spectrum peaks, and further, the remaining peaks in the MS mass spectrum are added. A mass spectrometry system characterized in that a priority order for selecting a parent ion is set.   15. The mass spectrometry system according to claim 14, wherein in the second analysis, the content of the peptide of the second peptide list with respect to the peak of the MS mass spectrum is calculated, and the peptide whose content exceeds a predetermined threshold is calculated. A mass spectrometry system, wherein the peptide list is transferred from the second peptide list to the first peptide list.   15. The mass spectrometry system according to claim 14, wherein in the second analysis, when setting the priority for selecting a parent ion, the remaining peaks of the MS mass spectrum are compared with the peptides in the second peptide list and matched. The mass spectrometry system characterized in that the order in which there are many peaks is the priority.   15. The mass spectrometry system according to claim 14, wherein in the second analysis, when setting the priority for selecting a parent ion, the remaining peaks of the MS mass spectrum are compared with the peptides in the second peptide list and matched. Bonus is given to each peak, and such comparison and bonus are given for each protein candidate, and the cumulative value of the bonus for every remaining peak in the MS mass spectrum is obtained. The mass spectrometry system is characterized in that priority is selected as a parent ion. Ionization treatment for ionizing a sample, MS mass spectrometry treatment for obtaining an MS mass spectrum by mass analysis of the ionized sample, parent ion selection and dissociation treatment for selecting a parent ion from a peak of the MS mass spectrum, and In a tandem mass spectrometry method for identifying a protein contained in a sample by repeating an analysis including MS ² mass spectrometry processing for obtaining an MS ² mass spectrum by mass spectrometry of dissociated parent ions,
In the first analysis, protein candidates already stored in the protein database among the proteins contained in the sample are estimated in real time from the MS ² mass spectrum,
In the second analysis, when a parent ion is selected, a peptide other than the peptide contained in the protein candidate is selected as a parent ion among MS mass spectrum peaks.   The tandem mass spectrometry method according to claim 18, wherein in the first analysis, the estimation accuracy of the protein candidate is calculated, information on the peptide contained in the protein candidate is obtained, and the estimation accuracy of the protein candidates is Register information on peptides of protein candidates that are larger than a predetermined threshold in the first peptide list, and register information on peptides of protein candidates whose estimated accuracy is less than the predetermined threshold among the protein candidates in the second peptide list. In the second analysis, the content of the peptide of the second peptide list with respect to the peak of the MS mass spectrum is calculated, and the peptide whose content exceeds the predetermined threshold is calculated from the second peptide list. When transferring to the peptide list and selecting the parent ion, the peak of the MS mass spectrum Of, tandem mass spectrometry method characterized by selecting peptides other than peptides contained in the first peptide listed as parent ion.   19. The tandem mass spectrometry method according to claim 18, wherein in the second analysis, the peptides in the first peptide list are excluded from the selection targets of the parent ions, and the parent ions are further excluded from the remaining peaks of the MS mass spectrum. A tandem mass spectrometry method characterized by setting a priority order for selecting.

Images