JP2008170346A - Mass analyzing system - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a mass analysis system, capable of analyzing trace amounts of components contained in a sample, without having to remeasure and extend the analysis time for ions per analysis case. <P>SOLUTION: In the tandem-type mass analyzing system, a database is searched, when the candidate of parent ions for MS<SP>2</SP>mass analysis is selected from among the peak ions of an MS<SP>1</SP>mass spectrum and the components that are not stored in the database or the component that are stored in the database but not identified in the arrangement of molecules with a predetermined number N or larger is selected as parent ions, and MS<SP>2</SP>mass analysis is performed, until a predetermined number N or larger of the rows of molecules are identified, with respect to the selected parent ions. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a tandem mass spectrometry system suitable for mass spectrometry of biopolymers such as peptides and sugar chains, chemical molecules, dioxins, ions containing explosives, and the like, and particularly suitable for detecting trace components. The present invention relates to a tandem mass spectrometry system.

In general mass spectrometry, a sample to be measured is ionized and various generated ions are sent to a mass spectrometer. In the mass spectrometer, the ion intensity is measured for each mass-to-charge ratio m / z, which is the ratio of the mass number m of ions to the valence number z, and a mass spectrum is generated. In the mass spectrum, the horizontal axis represents the mass-to-charge ratio m / z, and the vertical axis represents the ion intensity peak (ion peak). Thus, mass analysis of an ionized sample as it is is called MS ¹ mass spectrometry.

In tandem mass spectrometry, an ion peak having a specific mass-to-charge ratio m / z value is selected from the ion peaks detected by MS ¹ mass spectrometry, and the ion collides with a gas molecule. Dissociate and decompose by, for example. Thus, mass analysis is performed on the generated dissociated ion species, and a mass spectrum is similarly generated. The ionic species selected in this way is called a parent ion or a precursor ion. This mass spectrometry is called MS ² mass spectrometry.

In the tandem mass spectrometry, the selection and dissociation of the parent ion and the mass analysis of the dissociated ions are repeatedly performed to generate a mass spectrum. Hereinafter, the mass analysis of the n-th stage is referred to as MS ^{n + 1} mass spectrometry, the mass spectrum obtained by the MS ^{n + 1} mass spectrometry will be referred to as the MS ^{n + 1} mass spectra.

Tandem mass spectrometry can be used to analyze the amino acid sequence of a peptide contained in a protein. Known methods for reading amino acid sequences from MS ² mass spectra include database search methods and de novo methods. In many cases, an unknown protein resulting from a disease is not usually present in a database. Therefore, when analyzing peptides derived from such proteins, the de novo method is used. The de novo method is a technique for identifying an amino acid sequence based on a mass spectrum peak and a mass-to-charge ratio m / z, and is known by those skilled in the art, and detailed description thereof is omitted here. Computer software that realizes the de novo method is commercially available.

When analyzing by the de novo method using a normal personal computer, it takes several seconds per case. Therefore, the process of identifying the amino acid sequence from the MS ² mass spectrum using the de novo method is usually performed after all the analyzes are completed. Therefore, it is called post-processing. That is, it is difficult to execute the analysis by the de novo method in real time. For example, even in the method described in Patent Document 1, post-processing is performed after completion of all analysis.

Special table 2005-510732

As described above, in the analysis by the de novo method, the analysis time of ions per case is constant. Therefore, there is a limit to the number of ions that can be selected as parent ions in MS ² mass spectrometry. Usually, when selecting parent ions, they are selected in order of higher ion intensity than the MS ¹ mass spectrum. Therefore, ions having a small peak intensity in the MS ¹ mass spectrum, that is, trace ions cannot be selected. That is, only the data of components contained in a large amount can be obtained as post-processing data.

  The conventional method is suitable for analyzing a component contained in a large amount in a sample, but cannot be used for analyzing a component contained in a trace amount in a sample. The component contained in a large amount in the sample is usually a known peptide in many cases, but the trace component is often an unknown peptide.

  In the conventional method, when analyzing an unknown peptide contained in a trace amount in a sample, it is necessary to perform remeasurement or to extend the analysis time of ions per case.

  An object of the present invention is to provide a mass spectrometry system capable of analyzing a trace component contained in a sample without performing remeasurement and without extending the analysis time of ions per case. is there.

According to the tandem mass spectrometry system of the present invention, when a candidate for a parent ion for MS ² mass analysis is selected from ions in a peak of the MS ¹ mass spectrum, the database is searched and components not stored in the database are selected. Alternatively, a component that is stored in the database but for which a sequence of molecules of a predetermined number N or more has not been identified is selected as a parent ion, and a molecule sequence of a predetermined number N or more is selected for each of the selected parent ions. MS ² mass spectrometry is performed until identified.

  According to the present invention, it is possible to realize a mass spectrometer capable of performing qualitative analysis of a peptide derived from a small amount of unknown protein desired by a user without waste of measurement time.

  A first example of the tandem mass spectrometry system of the present invention will be described with reference to FIG. The tandem mass spectrometry system of the present invention is capable of mass spectrometry of biopolymers such as peptides and sugar chains, drug molecules, dioxins, ions containing explosives, etc. Will be described.

  The tandem mass spectrometry system of this example has an internal database 10 that stores data relating to the peptides derived from the identified proteins, a pretreatment system 11 that decomposes and fractionates the sample protein into polypeptide sizes, and ionizes the peptides The ionization unit 12, the mass analysis unit 13 that separates ions according to the mass-to-charge ratio m / z, the ion detection unit 14 that detects the separated ions and generates a mass spectrum, and organizes and processes data such as the mass spectrum The data processing unit 15 includes a display unit 16 that displays mass analysis data that is an analysis result, a control unit 17 that controls processing of these components, and an input unit 18 for a user to input data and commands.

A protein as a sample is first introduced into the pretreatment system 11. The pretreatment system 11 has a liquid chromatograph (LC). In the pretreatment system 11, proteins are decomposed into polypeptide sizes by digestive enzymes, and separated and fractionated in time according to the difference in the adsorption power of liquid chromatograph (LC). A gas chromatograph may be used instead of a liquid chromatograph (LC).
In MS ¹ mass spectrometry, the peptide is ionized by the ionization unit 12 and sent to the mass analysis unit 13. The ionization unit 12 may be ESI (Electro Spray Ionization) or MALDI (Matrix Assisted Laser Desorption Ionization). The ions are separated by the mass analyzer 13 according to the mass-to-charge ratio m / z. Here, m is the ion mass, and z is the charge valence of the ion. An MS ¹ mass spectrum is generated by the ion detector 14.

In MS ² mass spectrometry, the parent ion selected from the MS ¹ mass spectrum is dissociated. In this example, as a method for dissociating the parent ion, a collision dissociation method is used in which the parent ion collides with a buffer gas such as helium to dissociate. In this example, a collision cell 13A filled with a neutral gas is provided.

  The mass analyzer 13 captures parent ions in a region having a specific mass-to-charge ratio (m / z) or a specific mass-to-charge ratio (m / z), and puts them together in the collision cell 13A. In order to capture a specific parent ion, for example, a resonance voltage having a predetermined frequency is superimposed on the trap voltage so that ions to be excluded are in a resonance state.

  The parent ions collide with the neutral gas in the collision cell 13A and dissociate. In order for the parent ion to collide with the neutral gas, a voltage having a frequency that resonates with the parent ion is applied. Alternatively, the mass analyzer 13 may be filled with a neutral gas, and the parent ion may collide with the neutral gas in the mass analyzer 13 to be dissociated. In that case, the collision cell 13A becomes unnecessary.

  As a method of dissociating the parent ion, an electron capture dissociation method in which the parent ion is irradiated with low-energy electrons and a large amount of low-energy electrons are captured, the parent ion is irradiated with an ion beam, and electrons are emitted. Alternatively, an electron transfer dissociation method for dissociating the metal by moving it may be used.

The parent ions dissociated by the collision cell 13A are separated by the mass analyzer 13 according to the mass-to-charge ratio m / z. The separated ions are detected by the ion detector 14, and an MS ² mass spectrum is generated. The MS ² mass spectrum is processed by the data processing unit 15, and the mass analysis data as the analysis result is displayed on the display unit 16. Processing by the data processing unit 15 is referred to as post-processing.

  The control unit 17 controls this series of mass analysis processes, that is, ionization of the sample, transport and incidence of the ionized sample to the mass analysis unit 13, dissociation and separation of the parent ion, detection of the parent ion, data processing, and the like. To do.

A fast de novo method may be used to identify amino acid sequences from MS ² mass spectra. The de novo method is well known and will not be described in detail here. Commercially available high-speed de novo software may be used.

The concept of the tandem mass spectrometry method according to the present invention will be described with reference to FIG. 2. Here, the concept will be described in comparison with a conventional tandem mass spectrometry method. In the tandem mass spectrometry method, first, a sample protein is decomposed into peptides, and MS ¹ mass spectrometry is performed to obtain an MS ¹ mass spectrum 200. As shown in the figure, the MS ¹ mass spectrum 200 is a graph in which the horizontal axis represents the mass-to-charge ratio m / z and the vertical axis represents the ion intensity, and appears as a peak for each mass-to-charge ratio m / z.

In the illustrated example, the MS ¹ mass spectrum 200 includes a peak 201 representing peptide A, a peak 202 representing peptide B, and a peak 203 representing peptide C. The height of the peaks 201, 202, 203 of the MS ¹ mass spectrum 200 represents the relative ratio of the content of each peptide A, B, C. In this example, the content of peptide B is the highest, then the content of peptide A is the highest, and the content of peptide C is the lowest.

On the other hand, the internal database 10 stores mass analysis results for the sample. An MS ² mass spectrum 211 when peptide A is selected as the parent ion, an MS ² mass spectrum 212 when peptide B is selected as the parent ion, and an MS ² mass spectrum 213 when peptide C is selected as the parent ion are already obtained. It has been. Further, in the internal database 10, one amino acid sequence is identified from the MS ² mass spectrum 211, five amino acid sequences are identified from the MS ² mass spectrum 212, and an amino acid sequence is identified from the MS ² mass spectrum 213. Is stored. Information indicating that is not identified.

  The greater the number of amino acid sequences identified, the more accurately peptides and proteins can be identified.

  Therefore, in this example, if at least five amino acids can be identified, it is determined that peptides and proteins can be identified with considerable accuracy. There are about 20 kinds of amino acids. Therefore, if five amino acids can be identified, it is considered possible to identify 20 to the fifth power (about 100,000) of peptides or proteins. In the example of FIG. 2, since five amino acids have been identified from peptide B, it is determined that the data regarding peptide B is obtained with sufficient accuracy. Here, the condition is that five amino acids have been identified, but this is merely an example. For example, six amino acids may be identified, and four amino acids are identified. It is good also as a condition.

In the conventional technique, a predetermined number of parent ions are selected from the peaks of the MS ¹ mass spectrum 200 in descending order of the peaks. In recent years, all peaks may be selected as parent ions. In the conventional technique, a parent ion is selected regardless of what data is stored in the internal database 10. In the example of FIG. 2, the peak 202 has the highest intensity. Therefore, peptide B is first selected as the parent ion and MS ² mass spectrometry is performed. Once the MS ² mass spectrum is obtained, it identifies the amino acid. However, the internal database 10 already stores five amino acid sequences derived from peptide B. Therefore, duplicate data may be obtained for peptide B.

Therefore, according to the present invention, a peptide in which five or more amino acid sequences are identified among peptides stored in the internal database 10 is not selected as a parent ion. Therefore, a peptide that is not stored in the internal database 10 or that is stored in the internal database 10 but has less than 5 identified amino acids is selected as a parent ion. Thus, peptides with insufficient data are sequentially selected as parent ions, MS ² mass spectrometry is performed, and an MS ² mass spectrum is obtained. The obtained MS ² mass spectrum is stored in the internal database 10. If there is already data for the same peptide in the internal database 10, it is integrated. Thus, MS ² mass spectrometry is repeated until 5 or more amino acids can be identified. If this is done for all data-deficient peptides, at least five amino acids are finally identified for all peptides stored in the internal database 10. Thus, according to the present invention, since the amino acid sequence can be identified even for a trace amount peptide contained in a protein as a sample, a trace amount peptide contained in an unknown protein can be identified.

In the example of FIG. 2, peptides A and C are parent ions. Which of peptides A and C is selected as the parent ion first and MS ² mass spectrometry is performed is not particularly limited. Among peptides A and C, MS ² mass spectrometry may be performed in order of increasing peak height. Further, as an ion dissociation method for MS ² analysis, ECD or ETD may be selected in addition to CID.

A first example of the tandem mass spectrometry method according to the present invention will be described with reference to FIG. In step S11, the data processing unit 15 obtains the MS ¹ mass spectrum obtained by the MS ¹ mass spectrometry. In step S12, the data processing unit 15 performs peak determination from the MS ¹ mass spectrum. That is, the intensity and mass-to-charge ratio m / z of all peaks included in the MS ¹ mass spectrum are detected. Let Np be the total number of peaks thus obtained. In step S13, isotope peak determination is performed. In the isotope peak determination, since the mass numbers are the same but the valences are different, those having different mass-to-charge ratios m / z are detected. That is, the number of isotopes is subtracted from the total number Np of peaks. The total number Npi of peaks thus obtained does not include the number of isotopes. Note that Npi ≦ Np.

In step S14, each of the total number Npi peaks obtained from the MS ¹ mass spectrum is compared with the data stored in the internal database 10. The data to be compared here are the mass number m and valence z of all peptides expected to be generated when the pre-specified protein in the sample is enzymatically degraded, and the retention time (retention time) of the liquid chromatograph (LC). ), Intensity values of all peaks in the MS ¹ mass spectrum, etc. These pieces of information are referred to herein as peak information.

In step S141, the mass number m and valence z of each peak are calculated, and in step S142, the retention time (holding time) of each peak is calculated. In step S143, the calculated data is compared with the data stored in the internal database 10 to determine whether there is a match. If there is no match, the process proceeds to step S146. If there is a match, the process proceeds to step S144. In step S144, information on the peptide with the matching data is read from the internal database 10, and it is determined whether or not five or more amino acid sequences have been identified for the peptide. If five or more amino acid sequences have been identified, the process proceeds to step S145, and the peptide is excluded from the parent ion target for MS ² mass spectrometry. If five or more amino acid sequences have not been identified, the process proceeds to step S146. In this case, as an ion dissociation method, ECD or ETD may be selected in addition to CID.

In step S146, parent ion candidates for MS ² mass spectrometry are selected. Here, a candidate for a parent ion for MS ² mass spectrometry is a peptide that is not stored in the internal database 10 or that is stored in the internal database 10 but five or more amino acid sequences have been identified. There are no peptides.

In step S15, a parent ion for MS ² mass spectrometry is selected. A parent ion is selected from the parent ion candidates prepared in step S146. In step S16, MS ² mass spectrometry is executed. In this case, as an ion dissociation method, ECD or ETD may be selected in addition to CID. In step S17, the peak intensity of the MS ² mass spectrum is stored in the internal database 10. If the MS ² mass spectrum peak information for the same parent ion is already stored in the internal database 10, it is added to it. In step S18, the peak information of the MS ² mass spectrum is analyzed to identify the amino acid sequence. The amino acid sequence is identified by the de novo method. In step S19, it is determined whether or not the total number of identified amino acid sequences is 5 or more.

If five or more amino acid sequences are not identified, the process proceeds to step S20. In step S20, MS ² mass spectrometry is performed again on the same parent ion. In this case, the measurement is performed under conditions different from the previous MS ² mass spectrometry. For example, the number of integrations may be increased, but the dissociation time may be extended. Thus, more amino acid sequences can be identified by extending the dissociation time. An example of extending the dissociation time will be described later with reference to FIG. Here, when the intensity of the peak of the MS ¹ mass spectrum decreases to 20% of the maximum value, MS ² mass spectrometry is performed by changing to another parent ion. This will be described later with reference to FIG.

Hereinafter, likewise, the peak information of MS ² mass spectrum, integrating the peak information of MS ² mass spectrum for the same parent ions stored in the internal database 10. By repeating this, five or more amino acid sequences are identified in step S19.

If five or more amino acid sequences have been identified, the process returns to step S15 to select the next parent ion. Steps S16 to S19 are repeated for this parent ion. Thus, according to this example, at least 5 amino acid sequences are obtained in the internal database 10 for each of all the parent ions for MS ² mass spectrometry obtained in step S146. Thus, at least 5 amino acid sequences are obtained for each of the total Npi peaks.

A modified example of the first example of the tandem mass spectrometry method according to the present invention will be described with reference to FIG. Here, only a different part from the process of FIG. 3 is demonstrated. In this example, in step S19, it is determined whether or not the total number of identified amino acid sequences is 5 or more and the integrated value of the peak intensity of the MS ² mass spectrum is greater than a predetermined value.

  This will be described with reference to FIGS. 5A and 5B. Here, in the tandem mass spectrometry method of the present invention, the execution of the processing shown in FIG. 3 in real time will be described.

FIG. 5A shows a time chart of the tandem mass spectrometry method of FIG. 3, in which the vertical axis is a voltage applied by the mass analyzer 13 and the horizontal axis is a time graph. First, MS ¹ mass spectrometry is performed from time t1 to time t2, and processing from step S11 to step S14 is performed at a preparation time ΔTp from time t2 to time t3. MS ² mass spectrometry is performed on parent ion A from time t3 to time t4. Using the de novo method at the preparation time ΔTp from time T4 to time t5. Amino acid sequence is identified from MS ² mass spectrum. In this example, it is assumed that at least five parent amino acid sequences can be identified. From time t5 to t6, MS ² mass spectrometry is performed on the next parent ion B.

FIG. 5B shows a time chart of the tandem mass spectrometry method of FIG. 3, in which the vertical axis is a voltage applied by the mass analyzer 13 and the horizontal axis is a time graph. First, MS ¹ mass spectrometry is performed from time t1 to time t2, and processing from step S11 to step S14 is performed at a preparation time ΔTp from time t2 to time t3. MS ² mass spectrometry is performed on parent ion A from time t3 to time t4. The amino sequence is identified from the MS ² mass spectrum at the preparation time ΔTp from time t4 to t5. In this example, three parent ions could be identified. That is, at least 5 parent ions could not be identified. Therefore, MS ² mass spectrometry is repeated for the same parent ion A from time t5 to t6. In this case, a longer dissociation time is set as compared with the previous MS ² mass spectrometry.

The preparation time ΔTp is, for example, about 30 msec. The series of processing shown in FIG. 3 is executed by the data processing unit 15. In order to execute the processing in the data processing unit 15 in the actual measurement time, it is necessary to complete the series of processing shown in FIG. 3 within the preparation time ΔTp as described above. In order to realize such high-speed processing, the data processing unit 15 may be provided with a cache memory or a hard disk, and a parallel computer may be used if necessary. According to this example, the amino acid sequence is identified from the MS ² mass spectrum by the high-speed de novo method within the actual measurement time, and it is determined whether or not the number of identified amino acids is 5 or more. In this way, tandem mass spectrometry can be performed even for a small amount of peptide. Furthermore, a method of separating the high-speed de novo process and the operation sequence may be adopted.

FIG. 6 shows an example of data stored in the internal database 10. The internal database 10 contains the mass number m of the identified peptide, the retention time τ of the liquid chromatograph (LC), the peak intensity of the MS ¹ mass spectrum for each fixed period, the m, z and intensity of the MS ² mass spectrum peak , The amino acid sequence and its number, and the intensity sum of MS ² dissociated ions for the peptide of interest are stored. These data are automatically stored in the internal database 10 after measurement. The storage process of these data in the internal database 10 is preferably performed within the actual measurement time, but when the amount of processing is large, for example, when derivation of protein-derived peptides occurs, the actual measurement time It is not necessary to carry out within.

FIG. 7 is a diagram showing the time change of the peak intensity of the MS ¹ mass spectrum, where the vertical axis represents the peak intensity of the MS ¹ mass spectrum and the horizontal axis represents the time. As described above, when it is not possible to identify five or more amino acid sequences from the MS ² mass spectrum in step S19 in FIG. 3, in step S20 in FIG. Repeat MS ² mass spectrometry. However, the peak intensity of the MS ¹ mass spectrum decreases with time beyond the maximum value. Therefore, when the intensity of the peak of the MS ¹ mass spectrum decreases to 20% of the maximum value, MS ² mass spectrometry is performed by changing to another parent ion.

A second example of the tandem mass spectrometry method of the present invention will be described with reference to FIGS. The tandem mass spectrometry method of this example is different from the first example shown in FIG. 3 in the operation of step S20, and other than that, it may be the same as the first example of FIG. In step S20, MS ² mass spectrometry is performed on the same parent ion. This MS ² mass analysis is performed under different conditions from the previous MS ² mass analysis. In this example, the dissociation voltage is increased compared to the previous MS ² mass spectrometry. For example, it is increased by 50% only for 1 second.

FIG. 9 is a time chart of the tandem mass spectrometry method of FIG. 8, in which the vertical axis is a voltage applied by the mass analyzer 13 and the horizontal axis is a time graph. The time chart of this example is different from the example shown in FIG. 5 in the second MS ² mass analysis, and the MS ¹ mass analysis and the first MS ² mass analysis are the same as the example in FIG. . In the first MS ² mass analysis, at least 5 parent ions could not be identified. Therefore, MS ² mass spectrometry is repeated for the same parent ion A from time t5 to t6. In this case, a higher dissociation voltage is set compared to the previous MS ² mass spectrometry. For example, the dissociation voltage is increased by 50% for 1 second from the time point t5 to t6. By increasing the dissociation voltage in this way, more amino acid sequences can be identified.

Next, a third example of the tandem mass spectrometry method of the present invention will be described. In the first example and the second example described above, the amino acid sequence was identified using the high-speed de novo method in step S18. However, in this example, instead of identifying the amino acid sequence, it is determined whether or not the same data as the MS ² mass spectrum is stored in the internal database 10. Whether or not they are the same may be determined, for example, by comparing the degree of coincidence with a predetermined threshold. When the degree of coincidence is equal to or greater than a predetermined threshold, it is determined that they are the same, and when they are less than the predetermined threshold, it is determined that they are not the same.

If the same data as the MS ² mass spectrum is stored in the internal database 10, the process returns to step S15, and MS ² mass analysis is performed on the next parent ion. If the same data as the MS ² mass spectrum is not stored in the internal database 10, the process proceeds to step S20, and MS ² mass analysis is performed on the same parent ion. In this case, as in the above-described example, any of the number of integrations, the dissociation time, or the dissociation voltage may be increased.

A fourth example of the tandem mass spectrometry method of the present invention will be described with reference to FIG. From the MS ¹ mass spectrum 1001 peak, select the parent ion for MS ² mass analysis. In this case, the mass m or the mass-to-charge ratio m / z of the parent ion has a predetermined width on the horizontal axis, that is, a mass range (mass window). There is no problem if only one type of peptide is included in this mass range. However, it becomes a problem when two or more types of peptides are included. In this case, MS ² mass spectrum 1002 obtained by the MS ² mass spectrometry will include the peak for two or more peptides. In such a case, it is difficult to identify the amino acid sequence from the MS ² mass spectrum.

Therefore, in this example, when the amino acid sequence of two or more peptides can be read from the MS ² mass spectrum 1002 by the de novo method, the mass different from the previous one is selected in the selection of the parent ion for the next MS ² mass analysis. Select the range (mass window). Thereby, even when two or more kinds of peptides are included in one mass range (mass window), the amino acid sequence can be identified with high accuracy.

Next, a fifth example of the tandem mass spectrometry method of the present invention will be described. In this example, MS ¹ mass spectrometry and MS ² mass spectrometry are performed on biological samples such as blood, urine, sputum, sugar chains, and modified peptides, and the obtained data is stored in the internal database 10. According to this example, it is possible to automatically determine a peptide derived from a protein that may cause a lesion or a sugar chain, and to perform a detailed structural analysis.

A second example of the tandem mass spectrometer of the present invention will be described with reference to FIG. The tandem mass spectrometer of this example is different from the first example of FIG. 1 in that an ion trap mass analyzer 20 having an ion trap is used instead of the mass analyzer 13 and the collision cell 13A. Different. The other configuration may be the same as that of the first example of FIG. The ion trap of the mass spectrometer 20 has a function of a collision cell. Therefore, in this example, it is not necessary to provide a collision cell separately. Since the ion trap can perform n-stage (n ≧ 3) tandem analysis, that is, MS ⁿ mass spectrometry, the system for automatically determining the next parent ion as in the present invention is very effective. It is.

  A third example of the tandem mass spectrometer of the present invention will be described with reference to FIG. Compared with the first example of FIG. 1, the tandem type mass spectrometer of this example has an ion trap 22 and a time-of-flight (TOF) mass analyzer 21 instead of the mass analyzer 13 and the collision cell 13A. Is different. The other configuration may be the same as that of the first example of FIG. The ion trap 22 has functions of ion accumulation and parent ion selection, and further has a function of a collision cell. Ions from the ion trap 22 are subjected to mass analysis with high resolution in a time-of-flight (TOF) mass analysis unit 21TOF unit.

When the MS ² mass spectrometry, select and dissociate parent ions at the ion trap 22, by time-of-flight (TOF) mass analyzer 21, MS ² mass spectrometry. In the case of MS ¹ mass spectrometry, ions from the ionization unit 12 pass through an ion trap 22 and are subjected to MS ¹ mass analysis by a time-of-flight (TOF) mass analysis unit 21. Therefore, according to this example, the necessity of tandem analysis can be automatically determined, so that analysis can be performed with very high efficiency.

  A fourth example of the tandem mass spectrometer of the present invention will be described with reference to FIG. The tandem mass spectrometer of this example is different from the third example of FIG. 12 in that a linear trap 23 is used instead of the ion trap 22. The other configuration may be the same as that of the third example of FIG. The linear trap includes pole-shaped quadrupole electrodes, and a neutral gas is filled between the quadrupole electrodes. The linear trap has functions of ion accumulation and parent ion selection, and further has a function of a collision cell. When a linear trap is used, the trap rate of ions is greatly improved (about 8 times). Therefore, according to this example, since the next analysis content is determined based on the high sensitivity data, the determination can be performed with very high accuracy.

A fifth example of the tandem mass spectrometer of the present invention will be described with reference to FIG. The tandem mass spectrometer of this example is different from the third example of FIG. 12 in that a quadrupole (Q pole) 24 and a collision cell 25 are used instead of the ion trap 22. The other configuration may be the same as that of the third example of FIG. In the time-of-flight (TOF) mass spectrometer 21 of this example, basically, only MS ² mass spectrometry can be performed. However, even if the number of peaks in the MS ² mass spectrum obtained by the first MS ² mass analysis is insufficient, it can be replaced with another parent ion (especially by replacing it with a peak having the same mass number but a different valence). MS ² mass spectrometry can be performed repeatedly. In addition, since the necessity can be determined in the actual measurement time, the mass analysis unit of the present embodiment enables further dissociation and analysis, which has been impossible in the past.

  As described above, the tandem mass spectrometer according to the present invention includes a linear trap type (LIT), a linear trap (LIT) —time of flight type (TOF), a quadrupole (Q pole) —time of flight type (TOF), Time of flight (TOF) —Time of flight (TOF) or Orbital-Trap type.

  In the tandem mass spectrometry method of the present invention, a mass correction method for analysis data will be described. In protein shotgun analysis and the like, an external database storing data such as genes and proteins is searched based on mass spectrometry results, and the chemical structure of biopolymers is finally identified. In this case, the higher the mass accuracy of the analyzed ions, the more accurately and efficiently the biopolymer can be identified. For this reason, a time-of-flight (TOF) mass spectrometer or a Fourier transform ion cyclotron resonance (FTICR) mass spectrometer with relatively high mass accuracy is used for such analysis.

  However, for example, the mass accuracy of a time-of-flight (TOF) mass spectrometer may be affected by the room temperature of the place where it is installed. If the mass accuracy fluctuates unexpectedly for some reason, the biopolymer cannot be accurately identified even if an external database is searched.

  Therefore, it is often performed to analyze an internal standard material whose m / z of detection ions is known in advance immediately before the analysis, and to calibrate the m / z of the mass spectrometer based on the analysis result. However, in a liquid chromatograph (LC) type mass spectrometry system that continuously analyzes for many hours, mass accuracy may fluctuate unexpectedly. Therefore, when a known ion whose mass-to-charge ratio m / z is known in advance is detected among the ions detected by mass spectrometry, the mass-to-charge ratio m / z of other detected ions is determined based on that information. Correction may be performed.

When a plurality of known ions are detected, the corrected detection ion mass-to-charge ratio m / z becomes very accurate. The problem with this method is that the analysis data is corrected by a kind of manual operation, so that complexity is required. However, if the internal database 10 stores information such as ion mass m, mass-to-charge ratio m / z, and liquid chromatograph (LC) retention time τ that can be detected in advance, MS ¹ Known ions detected by mass spectrometry can be identified.

  By identifying multiple known ions, temporal fluctuations in the mass-to-charge ratio m / z can be estimated by information processing technology, and the mass-to-charge ratio m / z of the analyzed ions is automatically corrected. can do. This means that even when the mass accuracy of the mass spectrometer fluctuates unexpectedly, data with high mass accuracy can be easily acquired. Further, when using a mass spectrometer having such an information processing technique, it is not always necessary to analyze a known substance before starting analysis, and the burden on the user can be reduced. As described above, it is practically effective to use the information in the internal database 10 not only for control of real-time tandem mass spectrometry but also for calibration and correction of mass-to-charge ratio m / z of analysis data.

  Although the example of the present invention has been described above, 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. It will be understood.

It is a figure which shows the 1st example of the tandem type | mold mass spectrometry system of this invention. It is a figure explaining the concept of the tandem type | mold mass spectrometry method by this invention. It is a figure explaining the 1st example of the tandem type | mold mass spectrometry method by this invention. It is a figure explaining the modification of the 1st example of the tandem type | mold mass spectrometry method by this invention. In a first example of a tandem mass spectrometry method according to the present invention, showing a time chart in the case of performing the MS ² mass spectrometry. In a first example of a tandem mass spectrometry method according to the present invention, showing another example of a time chart when performing MS ² mass spectrometry. It is a figure which shows the example of the data stored in the internal database of the tandem type | mold mass spectrometry system of this invention. MS ¹ is a graph showing a temporal change of the peak intensity of the mass spectrum. It is a figure which shows the 2nd example of the tandem type | mold mass spectrometry method of this invention. It is a figure which shows the time chart of the 2nd example of the tandem-type mass spectrometry method of this invention. It is a figure which shows the 4th example of the tandem mass spectrometry method of this invention. It is a figure which shows the 2nd example of the tandem mass spectrometer of this invention. It is a figure which shows the 3rd example of the tandem-type mass spectrometer of this invention. It is a figure which shows the 4th example of the tandem-type mass spectrometer of this invention. It is a figure which shows the 5th example of the tandem-type mass spectrometer of this invention.

Explanation of symbols

10: Internal database, 11: Pre-processing system, 12: Ionization unit, 13: Mass spectrometry unit, 14: Ion detection unit, 15: Data processing unit, 16: Display unit, 17: Control unit, 18: User input unit, 19: Overall mass spectrometry system, 20: Ion trap mass spectrometer, 21: Time of flight mass analyzer, 22: Ion trap, 23: Linear trap, 24: Quadrupole (Q pole), 25: Collision cell,

Claims (23)

A database storing mass analysis results for the substance to be measured, a chromatograph for preprocessing the substance, an ionization part for ionizing components contained in the substance, an ion dissociation part for dissociating the ionized components, and ions Obtained by the mass analyzer, the ion detector for detecting the ions separated at the mass-to-charge ratio m / z and generating a mass spectrum, and the ion detector. A data processing unit for identifying a molecular sequence included in the component from the obtained mass spectrum, and separating the ions from the ionization unit for each mass-to-charge ratio m / z by the mass analysis unit, and MS ¹ mass spectrometry to obtain the MS ¹ mass spectrum by the detecting unit, select a parent ion peak of the MS ¹ mass spectrum, the parent ion is dissociated by the ion dissociation unit The該解away ions separated for each mass-to-charge ratio m / z by the mass analyzer performs, and MS ² mass spectrometry to obtain an MS ² mass spectrum by the ion detector, it repeated a given stage of the mass analysis Tandem Type mass spectrometry system,
When a candidate for a parent ion for MS ² mass analysis is selected from the ions of a peak of MS ¹ mass spectrum, the database is searched, and components not stored in the database or stored in the database Is selected as a parent ion, and a sequence of MS ² masses until a predetermined number N or more of molecular sequences are identified for each of the selected parent ions. A tandem mass spectrometry system characterized in that analysis is performed. In claim 1 tandem mass spectrometry system as claimed, in the MS ² mass spectrometry, when for one of the parent ion candidate, MS ² performs mass spectrometry, more than the predetermined number N of molecular arrangement is not identified The MS ² mass analysis is repeated again for the same parent ion, and when a molecular sequence of a predetermined number N or more is identified, the MS ² mass analysis is performed for the next parent ion. Tandem mass spectrometry system. In claim 2 tandem mass spectrometry system as claimed, the same parent ions, when repeating the MS ² mass spectrometry again, MS ² mass by changing to a different value from the previous at least one of dissociation time and dissociation voltage A tandem mass spectrometry system characterized by repeated analysis. 3. In the tandem mass spectrometry system according to claim 2, when the MS ² mass analysis is repeated again for the same parent ion, the peak value of the MS ² mass spectrum is reduced from the peak maximum value to a predetermined ratio. A tandem mass spectrometry system characterized in that MS ² mass analysis for the parent ion is stopped and MS ² mass analysis of the next parent ion is performed. 2. The tandem mass spectrometry system according to claim 1, wherein MS ² mass analysis is performed until a predetermined number N or more of molecular sequences are identified and an integrated value of peaks of the MS ² mass spectrum is larger than a predetermined value. A tandem mass spectrometry system. 2. The tandem mass spectrometry system according to claim 1, wherein the database includes an ion molecular sequence read from an MS ¹ mass spectrum peak, an array number, a mass, a valence, a chromatographic retention time, an ionic strength, an MS ² mass. A tandem mass spectrometry system having a mass, a valence, an ion intensity, and an integrated value of ion intensity of each ion in a spectrum. The tandem mass spectrometry system according to claim 1, wherein when the database is searched, the molecular sequence, mass, valence, and retention time read from the peak ion of the MS ¹ mass spectrum are collated with the information in the database. Characteristic tandem mass spectrometry system.   2. The tandem mass spectrometer according to claim 1, wherein the tandem mass spectrometer is a linear trap type (LIT), a linear trap (LIT) -time of flight type (TOF), or a quadrupole (Q pole) -time of flight type. (TOF), time-of-flight (TOF) -time-of-flight type (TOF), orbital-trap type, and CID (Collision Induced Dissociation), ECD (Electron) as ion dissociation means A tandem mass spectrometry system characterized by having any means of capture dissociation (ETD) and ETD (electron transfer dissociation).   2. The tandem mass spectrometry system according to claim 1, wherein the ionization unit is ESI (Electro Spray Ionization) or MALDI (Matrix Assisted Laser Desorption Ionization). 3.   The tandem mass spectrometry system according to claim 1, wherein the substance to be measured is a biological sample, a drug molecule, dioxin, or an explosive. The tandem mass spectrometry system according to claim 1, wherein the substance to be measured is a protein,
When a candidate for a parent ion for MS ² mass spectrometry is selected from the MS ¹ mass spectrum peak, the database is searched, or a peptide not stored in the database or stored in the database but predetermined A peptide whose amino acid sequence of several N or more is not identified is selected as a parent ion, and MS ² mass spectrometry is performed until a predetermined number N or more of amino acid sequences are identified for each of the selected parent ions. A tandem mass spectrometry system characterized by being performed.   12. The tandem mass spectrometric system according to claim 11, wherein the amino acid sequence is collated with a de novo method of reading an amino acid sequence from a difference in mass spectrum or amino acid sequence data of one or more proteins accumulated in the database. A tandem mass spectrometric system characterized in that it is identified by either a search method based on or a combination of both.   The tandem mass spectrometry system according to claim 1, wherein both or one of the spectrum and the dissociation voltage at each time during the measurement are stored in a memory or a hard disk device as a log. 2. The tandem mass spectrometry system according to claim 1, wherein the parent ion candidate selection process from the end of MS ¹ mass analysis to the start of MS ² mass analysis is 10 msec or less, preferably 1 msec or less. A tandem mass spectrometry system that is executed by The tandem mass spectrometry system according to claim 1, wherein when a parent ion candidate for MS ² mass analysis is selected from an MS ¹ mass spectrum peak, a peak ion in a predetermined mass range is selected as a parent ion. A tandem mass spectrometry system. The peptides contained in protein ionized, separated the ions for each mass-to-charge ratio m / z, the MS ¹ mass spectrometry to obtain the MS ¹ mass spectrum by detecting ions said separated, the MS ¹ mass spectrum selecting a parent ion peak, to dissociate parent ions, separating the該解away ions per mass-to-charge ratio m / z, performed, and MS ² mass spectrometry to obtain an MS ² mass spectrum, from the mass spectrum In the tandem mass spectrometry method for identifying the amino acid sequence contained in the peptide,
When a parent ion candidate for MS ² mass spectrometry is selected from the ions of the MS ¹ mass spectrum peak, a database storing mass analysis results for proteins is searched, and peptides not stored in the above database, or A peptide that is stored in the database but whose amino acid sequence of a predetermined number N or more has not been identified is selected as a parent ion, and a predetermined number N or more of amino acid sequences are identified for each of the selected parent ions A tandem mass spectrometry method, characterized in that MS ² mass spectrometry is performed until it is performed. The tandem mass spectrometry method according to claim 16, wherein in the MS ² mass spectrometry, when an amino acid sequence of a predetermined number N or more is identified for one of the parent ion candidates, the next parent ion is determined. On the other hand, MS ² mass spectrometry is performed, and when an amino acid sequence of a predetermined number N or more is not identified, MS ² mass spectrometry is repeated again for the same parent ion. . The tandem mass spectrometry method according to claim 16, wherein MS ² mass spectrometry is performed on one of the parent ion candidates, and when an amino acid sequence of a predetermined number N or more is not identified, the same parent ion is analyzed. Then, when the MS ² mass spectrometry is repeated again and a predetermined number N or more of amino acid sequences are identified, the MS ² mass analysis is performed on the next parent ion. . A database that stores data on peptides derived from proteins identified by mass spectrometry, a chromatograph that preprocesses proteins, an ionization unit that ionizes peptides contained in proteins, and an ion dissociation unit that dissociates ionized peptides A mass analyzer that separates ions for each mass-to-charge ratio m / z, an ion detector that detects ions separated for each mass-to-charge ratio m / z and generates a mass spectrum, and the ion detector A data processing unit for identifying the amino acid sequence contained in the peptide from the mass spectrum obtained by the above, and separating the ions from the ionization unit for each mass-to-charge ratio m / z by the mass analysis unit, Select the parent ion from the MS1 mass spectrum obtained from the MS1 mass spectrum obtained by the ion detector and the MS1 mass spectrum. The parent ion is dissociated by the ion dissociation unit, the該解away ions separated for each mass-to-charge ratio m / z by the mass analyzer, and MS ² mass spectrometry to obtain an MS ² mass spectrum by the ion detector, In a tandem mass spectrometry system that repeats the mass spectrometry by a predetermined stage,
When a parent ion candidate for MS ² mass spectrometry is selected from the ions of the MS ¹ mass spectrum peak, a database storing mass analysis results for proteins is searched, and peptides not stored in the above database, or A peptide that is stored in the database but whose amino acid sequence of a predetermined number N or more has not been identified is selected as a parent ion, and a predetermined number N or more of amino acid sequences are identified for each of the selected parent ions A tandem mass spectrometry system, characterized in that MS ² mass spectrometry is performed until   20. The tandem mass spectrometry system according to claim 19, wherein the predetermined number N is an integer of 4 to 6. The tandem mass spectrometry system according to claim 16, wherein MS ² mass spectrometry is performed until a predetermined number N or more of amino acid sequences are identified for each of the selected parent ions. Tandem mass spectrometry system characterized by ion dissociation using ETD. 18. In the tandem mass spectrometry system according to claim 17, when an amino acid sequence of a predetermined number N or more is not identified, ECD or ETD is used instead of CID in MS ² mass spectrometry performed on the same parent ion. A tandem mass spectrometry system characterized in that ion dissociation is used. 20. The tandem mass spectrometry system according to claim 19, wherein ion dissociation is performed using ECD or ETD instead of CID in MS ² mass spectrometry performed until a predetermined number N or more of amino acid sequences are identified. Tandem mass spectrometry system.

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