JP4929224B2 - Mass spectrometry system - Google Patents

Description

  The present application relates to a mass spectrometry system and a mass spectrometry method.

  The mass analysis method includes a method in which a sample is ionized and analyzed as it is (MS analysis method), and a tandem mass in which a specific sample ion (precursor ion) is selected and dissociated to generate a mass analysis of dissociated ions. There is an analysis method.

In tandem mass spectrometry, ions having a specific mass-to-charge ratio (precursor ion) are selected from dissociated ions, the precursor ions are dissociated, and mass analysis of the generated dissociated ions is repeated. There is a function (function to perform dissociation / mass spectrometry in multiple stages) (note that the n-th stage measurement is hereinafter referred to as MS ⁿ ).

In JP-A-2007-121134 (Patent Document 1), as a method for performing tandem mass spectrometry, an analysis of MS ⁿ measurement results is performed using a plurality of MS ^{n + 1} measurement results. It describes that the analysis should be made more efficient.

JP 2007-121134 A

  In the current mass spectrometry system, when the valence of ions to be detected is increased, the peak interval (1 / valence) of the isotope peak is reduced. As a result, the peaks are buried and it is difficult to separate the peaks, making it difficult to perform valence determination. If the valence of multiply charged ions cannot be determined, it is difficult to measure a mixed sample composed of a plurality of components or an unknown sample. Furthermore, even if it is a known sample, the valence cannot be determined by automatic data analysis, and the experimenter determines the valence based on the information of the measurement sample, which places a heavy burden on the experimenter. .

  Accordingly, an object of the present invention is to provide a mass spectrometry system that can easily determine the valence of multiply charged ions. In addition, such a mass analysis system is capable of accurately detecting multivalent ions and improving the accuracy of duplicate analysis and post-processing.

  A feature of the present invention that solves the above-described problem is that a plurality of ion species having different valences of components having the same mass are determined in a mass spectrometry spectrum of a substance to be measured, and the obtained masses are different and have the same mass The least common multiple is obtained within a certain tolerance range for a plurality of ion species having a valence, and the valence and mass of each ion species are estimated.

  A mass spectrometry system includes a sample separation unit that separates a sample material to be subjected to mass spectrometry from a sample containing a plurality of substances, a mass spectrometer that acquires a mass spectrometry spectrum of the sample material, a sample separation unit, and a mass spectrometer. Control means for controlling.

In the present invention, the mass and valence of each ion species are estimated by obtaining the least common multiple of the M / Z values of a plurality of ion species having the same mass and different valences. Therefore, in a tandem mass spectrometer, a sample material is ionized and mass spectrometry (MS ¹ ) is performed to obtain a mass spectrometry spectrum, and an ion species having at least one M / Z value is obtained from the ionized sample material. Select, dissociate the selected ionic species, perform mass analysis (MS ² ) of the dissociated ionic species to obtain a mass spectrometry spectrum, and have the same mass and different valence from the MS ¹ spectrum Can determine the least common multiple of M / Z values of a plurality of ion species within a predetermined tolerance range, and estimate the mass of the sample compound and the valence of each ion species. .

In addition, another feature of the present invention is that MS ¹ measurement is performed at the time of mass spectrometry measurement of a sample, and one ion species is selected from the spectrum obtained by MS ¹ measurement. MS ² was measured, to identify the peaks that match by comparing the MS ² measurement data and MS ¹ measurement data have the same mass in the spectrum obtained by the measurement of the MS ^1, and different valences of the plurality In this method, the ion species having the same mass and different valences are identified. Furthermore, there is a valence estimation method for estimating the valence of the selected ion species of MS ¹ based on the specified ion species.

  According to the above, the mass spectrometry system capable of estimating the valence of the measurement target required by the user for the multivalent ion peak that is difficult to determine by the valence determination method using the interval between the isotope peaks. Can be provided.

  Hereinafter, the invention of the present application will be described in further detail.

First, a specific example of tandem mass spectrometry is shown. In tandem mass spectrometry, the function of performing dissociation and mass spectrometry in multiple stages has the following steps. (1) A mass spectrometry distribution of a predetermined substance in a sample is measured as mass spectrum data (MS ¹ ). (2) A precursor ion having a certain m / z value is selected from MS ¹ . (3) Dissociate the selected precursor ions. (4) The mass spectrometry data (MS ² ) of dissociated ions dissociated from the precursor ions is measured. (5) Select ions having a certain m / z value from the measured MS ² mass spectrum data. (6) The selected precursor ion is further dissociated, and mass spectrometry data (MS ³ ) of the obtained dissociated ion is measured. (7) Continue to MS ⁿ for these operations.

  By performing dissociation / mass spectrometry in multiple stages as described above, information on the molecular structure of ions before dissociation can be obtained for each dissociation stage, and the structure of ions can be estimated. As the information on the ion structure becomes more detailed, the estimation accuracy of the precursor ion structure improves.

  Various methods for dissociating ions have been reported. Typical examples are shown below. Currently, CID (collision excitation dissociation) is widely used as an ion dissociation method. CID is a technique for fragmenting ions by causing an inert gas such as He gas to collide with sample ions in a mass spectrometer. The degree of fragmentation in the case of proteins depends on the binding energy between amino acid residues. Therefore, the degree of fragmentation and the tendency of dissociation vary depending on the amino acid residue sequence. CID also dissociates modifying groups attached to amino acid residues, such as post-translational modifications. In CID, since only a part of dissociation according to the sequence occurs, the amount of information obtained is small. Therefore, for the analysis of peptides and proteins with large mass, peptides consisting of more than ten amino acid residues (molecular weight: about 3000, valence: up to 5 valences) that have been fragmented by digesting the protein before measurement are used. It is mainstream to perform the measurement.

  As other ion dissociation methods, there are dissociation techniques such as ECD (electron capture dissociation) and ETD (electron transfer dissociation) which cause fragmentation of ions by exchange of electrons, and these have been put into practical use in recent years. Unlike CID, ECD does not depend on the amino acid sequence, and dissociation occurs evenly in the main chain between amino acid residues excluding the N-terminal side of proline. Therefore, analysis is possible even for ions having a large mass (molecular weight: several thousand to 10,000) compared to CID. However, since the detection range of m / z is limited (up to 2000 or the like), in order to measure ions having a molecular weight of about 10,000, it is necessary to ionize more multivalently and detect within the detection range. In this case, it is necessary to analyze ions having a valence of at least 6 or more. For this reason, in ETD and ECD, the measurement of more multivalent ions is indispensable as compared with the measurement using the mainstream CID. Further, in ECD, dissociation occurs only in the main chain of the amino acid, so that the post-translational modification group can be analyzed while leaving the amino acid residue.

By analyzing data acquired by mass spectrometry, it is possible to estimate the structure of the measurement object. Here, information necessary for data analysis is the valence and mass of the precursor ion. In the mass spectrometry spectrum, the horizontal axis represents m / z (mass / valence), and the vertical axis represents ion intensity. In the vicinity of the ion peak of each component, an isotope peak containing an isotope of an element constituting an amino acid such as ¹³ C, ¹⁸ O, or ¹⁵ N is detected. Since the mass difference between the ion peak of each component and its isotope peak is 1, the isotope peak interval is detected by 1 / valence. Therefore, the valence can be determined from the interval between the isotope peaks. For example, when the measurement object is a peptide such as a protein digest, the obtained mass spectrometry spectrum is subjected to data analysis using database search or denovo sequencing analysis to identify the original sequence.

  Since the interval between isotope peaks is detected by 1 / valence, the valence of ions can be determined from this interval. Even with ECD and ETD, it is possible to determine the valence of ions from the peak interval, but as the valence of ions increases, the isotope peak interval becomes narrower, making it difficult to separate peaks and determine the correct valence. Become. When the peak separation accuracy is low, the valence determination accuracy is low, and the components cannot be discriminated (discriminated), so that it is difficult to measure a mixed sample composed of a plurality of components. In addition, even a single component sample cannot be determined as to what valent ion species appears, and therefore, only a known sample whose mass is known in advance can be measured. In addition, since the valence cannot be automatically determined in the data analysis after the analysis is completed, the experimenter must manually determine and specify the valence from the information (for example, mass) of the measurement sample. A lot of burden is placed on the experimenter.

  Examples of the present invention are shown below.

In Example 1, when mass spectrometry was performed on an unknown sample containing only a single component, a plurality of ion species having the same mass and different valences were used from the MS ¹ spectrum, and An example of performing valence determination will be described. FIG. 1 shows a multivalent ion determination flow using only a single component MS ¹ spectrum.
(1) First, an MS ¹ spectrum is measured using a peptide or the like as a measurement target.
(2) Next, noise is removed from the MS ¹ spectrum, for example, by removing a signal having a certain intensity or less.
(3) Next, peak determination is performed on the remaining signal in the MS ¹ spectrum to create an ion species list. When the object to be measured is an ion with a high valence (for example, about 10 valences), one broad peak is detected (see the lower diagram in FIG. 2, an example of a 9-valent ion), and individual isotope peaks are separated. The valence cannot be determined from the isotope peak. Therefore, as shown in FIG. 3, each representative value of the broad peak composed of the isotope peak group is set as the value of the ion species list. As a representative value of the broad peak of the isotope peak group, a value that is the maximum intensity or a minimum m / z value after removing noise (M / Z = 580 in the case of FIG. 9) is used. Here, m is the ion mass, and z is the charge valence of the ion. The minimum m / z value after removing the noise is a value at which the content of the isotope peak is minimum in the detected isotope peak group. The above process is repeated for all the peaks to obtain an ion species list (X _{1 to} X ₅ ).
(4) Next, from the obtained ion species list (X _{1 to} X ₅ ), the valence is determined for the ion species having the smallest m / z, that is, the ion species (X ₁ ) having the largest valence. Assume. Here, the first assumed valence is an upper limit valence to be considered during the analysis, and can be specified by the user. In the case of an unknown sample, if the upper limit is set large (for example, 50 values), almost all possibilities can be examined.
(5) Next, the mass M of the ion species is determined from the ion species having the assumed valence and the maximum valence (ion species having the minimum m / z). This time, since a sample consisting of only a single component is measured, other ionic species also have the same mass. For other ion species, the valence z is derived from the mass M and the m / z value of each ion species.
(6) Here, since the created ion species list is a representative value of the isotope peak group, z is not necessarily an integer. For this reason, the valence z obtained for each ion species is rounded to the integer Z. Next, from the obtained Z, the mass of each ion species is recalculated, and the difference Δm from the mass of the ion species assuming the valence is evaluated. If Δm of all ion species is within the tolerance, it is assumed that the assumption is correct, and the assumed valence is determined.
(7) On the other hand, if Δm is greater than or equal to the tolerance, the initially assumed valence is changed (−1) and assumed again, and the same process is repeated.
(8) For each ion species, when Δm is less than or equal to the tolerance, the valence of each ion species is determined. In addition, the mass of the component is determined from the obtained valence.

  Here, the tolerance is specified by the user, or the mass difference between the monoisotopic mass and the isotope peak having the maximum intensity in the theoretical isotope distribution obtained from the mass M. When the measurement target is a protein or peptide, the average amino acid (averagine) mass (112.59) and constituent element ratio (C: 4.9384 H: 7.7583 N: 1.35777 O: 1.5994 S: 0 0.0417) it is possible to determine the theoretical isotope distribution of the components. From the theoretical isotope distribution, the isotopic peak and the monoisotopic mass with the maximum intensity are known. In this example, the mass difference between the monoisotopic mass obtained from the theoretical isotope distribution and the isotopic peak having the maximum intensity was used.

  Since it is highly possible that the obtained mass is an isotope peak having the maximum intensity, it is possible to estimate the monoisotopic mass. However, when the monoisotopic mass is estimated by this processing, it is desirable to average or integrate a plurality of spectra in order to reduce spectral signal variations.

In Example 2, an example in which a valence is determined using an MS ¹ spectrum and an MS ^{n + 1} (n ≧ 1) spectrum when a sample in which a plurality of components are mixed is measured will be described. FIG. 4 shows a flow for determining the valence using the MS ¹ spectrum and the MS ² spectrum. The MS ² spectrum is obtained by selecting one of the ions detected in the MS ¹ spectrum, dissociating with ECD or ETD, and then performing mass spectrometry.

First, noise removal is performed on each of the MS ¹ spectrum and the MS ² spectrum in the same manner as in Example 1 to create an ion species list of the isotope peak group. Since a plurality of components are detected in the MS ¹ spectrum, it is necessary to separate only the components having the same mass. In addition, it is difficult to distinguish ions having the same mass but different valences from only the spectra of MS ¹ and MS ² .

As shown in FIG. 5, in the MS ² spectrum measured by dissociation by ECD or ETD, an ion species in which only the valence of the precursor ion is decreased is detected together with the dissociated ion from which the precursor ion is dissociated. These ionic species are hereinafter referred to as valence-reducing ionic species. By collating the MS ¹ spectrum and the MS ² spectrum, only the component having the same mass is separated from the spectrum, and the valence-reduced ion species is determined. At this time, the determined valence is displayed on the MS ¹ and MS ² spectra according to the determination result, and the same component is displayed in the same color. Thereby, the experimenter can obtain information on how many kinds of components are currently mixed. When a large number of components are detected at the same time using this information as an index, it is desirable to change the separation conditions in the pretreatment stage such as liquid chromatography.

The user can specify the tolerance when collating each ion species. As a result of the collation, the matched ion species are stored in the same component list obtained from the MS ¹ spectrum and the MS ² spectrum, and the valence determination shown in the first embodiment is performed.

Even in the case of a sample in which a plurality of components are mixed as in this example, MS ² analysis is performed on the peak to be analyzed, and comparison with the MS ¹ spectrum enables separation of target sample component information and value. A number determination can be made. Therefore, by performing the valence determination according to the present embodiment during the measurement, it is possible to measure a sample in which a plurality of components are mixed. Further, MS ² mass spectrometry can be performed on different components by discriminating the same component, and more components can be analyzed in one measurement from avoiding duplicate analysis on the same component. Alternatively, by performing MS ² analysis on ions having the same component but different valences, the amount of information per component increases, and the reliability of the measurement result is improved. These priorities can be specified by the user before measurement. In addition, when dissociation is performed by ECD or ETD, valence-reduced ion species are generated in the MS ² spectrum. Since this valence-reduced ion species is not fragmented from the precursor ion, it not only contains no information about the structure of the precursor ion, but may also lead to incorrect structural information due to the presence of the valence-reduced ion species. Will increase. For this reason, it is desirable to exclude information on valence-reduced ion species before analysis. If the sample is a known substance, it is easy for an experimenter to exclude valence-reduced ion species information from the MS ² spectrum. The burden is due to the difficulty of number determination. In this embodiment, by comparing the MS ¹ spectrum and the MS ² spectrum, the valence-reduced ion species can be determined during the measurement, and these pieces of information can be excluded before the analysis. As a result, it is possible to improve the accuracy of data analysis after the analysis and reduce the burden on the experimenter.

  In Example 3, a mass spectrometry system using the valence determination method of Examples 1 and 2 will be described. FIG. 6 is a flowchart of the mass spectrometry system of the present embodiment.

  A configuration example of the mass spectrometry system is shown in FIG. The mass spectrometry system of the present embodiment includes a pretreatment system for fragmenting and separating a sample, an ionization unit for ionizing the sample, a mass analysis unit for dispersing the ionized sample according to mass and valence, and dispersed sample ions. Ion detection unit that detects the data, data analysis unit that processes the result of mass analysis of the sample, display unit that displays the mass analysis result of the data analysis, and control that controls these according to the conditions input to the user input unit Part.

  The sample to be analyzed is pretreated in a pretreatment system such as liquid chromatography. For example, when a sample to be analyzed is a protein, it is converted into a peptide fragmented by a digestive enzyme in a pretreatment system, and then separated by gas chromatography (GC) or liquid chromatography (LC).

  Next, the sample is ionized by the ionization unit, the sample ions are transported and incident on the mass analysis unit, and are separated according to the mass-to-charge ratio m / z of the ions by the mass analysis unit. The separated ions are detected by the ion detection unit, the detection result is arranged and processed by the data processing unit, and the mass analysis data as the analysis result is displayed on the display unit. The entire series of mass analysis processes (sample pretreatment, sample ionization, transport and incidence of sample ion beam to the mass spectrometer, mass separation process, ion detection, and data processing) are controlled by the controller. The

By performing valence determination of multivalent ions in real time during mass spectrometry measurement, analysis can be performed according to the valence determination result. Hereinafter, using FIG. 6, the MS ¹ analysis is performed once and then the MS ² analysis is performed twice, and the valence determination of the multiply charged ions shown in Examples 1 and 2 is performed in real time during the measurement. An example of implementing and controlling the next analysis content will be described.
(1) First, MS ¹ analysis is performed to obtain an MS ¹ spectrum. Among the obtained spectra, the ion species having the maximum intensity is selected as the precursor ion. At this stage, the valence of the selected ionic species is unknown.
(2) MS ² analysis is performed on the selected precursor ions. Noise removal is performed on the obtained MS ² spectrum.
(3) All detected peaks from which noise has been removed are checked against the immediately preceding MS ¹ spectrum. If there is a coincidence peak as a result of the collation, the valence is determined according to the first embodiment.
(4) Since there is no coincidence peak as a result of collation, the valence determination cannot be performed. Therefore, the ion species having the second highest intensity (if the ion species having the nth highest intensity is being analyzed, the n + 1th MS ² analysis is performed on the ion species with high intensity), and if there is a coincident peak, the valence is determined according to Example 1.

Such a valence determination can be performed in real time during measurement. By making it real time, the next MS ² analysis content can be selected based on the result of the valence determination. Among a plurality of ion species included in the MS ¹ spectrum, the same component can be analyzed or avoided although the valence is different. In the case of avoidance, by reducing the number of times of analysis for the same component, the number of times of analyzing another component increases, and more components can be analyzed by one measurement. Also, when analyzing ion species of the same component with different valences, the amount of information for one component is increased by performing MS ² analysis on ions with the same components but with different valences. , Improve the reliability of the results. The user can specify which is prioritized before or during measurement. The above processing is repeated until the analysis end time is reached.

It should be noted that the total processing time from the step of comparing MS ¹ and MS ² to the step of determining the next analysis content is preferably within 10 milliseconds so as not to adversely affect the overall measurement throughput. For shortening the processing time, means such as efficient peak determination and reduction of the data amount are effective.

FIG. 5 is a schematic diagram of a flow of valence determination processing using a plurality of ion species having the same mass and different valences according to the first embodiment of the present invention. It is an isotope peak distribution of divalent and nonvalent ions. It is the schematic of the ion species list | wrist produced from the isotope peak group in the 1st Example of this invention. According to a second embodiment of the present invention, it is a schematic diagram of the flow of the charge number determination processing using the MS ¹ spectrum and MS ² spectra. FIG. 4 is a schematic diagram of an MS ² spectrum to be matched with an MS ¹ spectrum according to a second embodiment of the present invention. It is the schematic of the flow of the mass spectrometry system using the valence determination method of Example 1 and Example 2 in the 3rd Example of this invention. It is the schematic of the whole mass spectrometry system which measures mass spectrometry data by the 3rd example of the present invention.

Claims (9)

The sample material is ionized and mass spectrometry (MS ¹ ) is performed to obtain a mass spectrometry spectrum.
Select an ion species having at least one M / Z value from the ionized sample material, dissociate the selected ion species, and perform mass analysis (MS ² ) of the dissociated ion species to obtain a mass spectrometry spectrum A tandem mass spectrometry method that repeats selection of ion species from dissociated ion species, dissociation, and measurement of M / Z value,
(A) identifying a plurality of ion species of the same compound having the same mass and different valence numbers;
(B) Among these ionic species, the valence is assumed for the ionic species having the maximum valence,
(C) Calculate the mass M from the assumed valence and the M / Z value of the ionic species with the maximum valence,
(D) The valence of each ionic species is obtained by rounding off the quotient of the M / Z value of each ionic species other than the ionic species that maximizes the calculated mass M and valence.
(E) Calculate the mass of each ionic species other than the ionic species having the maximum valence from the obtained valence and M / Z value,
(F) Obtain the difference between these masses and masses M, respectively.
(G) It is determined whether all the obtained differences are within a predetermined tolerance range,
(H) The valence assumed in step (b) is changed until all the mass differences of the ion species obtained in (f) in step (g) are within the tolerance range (b) to (g The mass spectrometric method is characterized by repeatedly performing the step (1). The mass spectrometric method according to claim 1 ,
A plurality of ionic species of the same compound having the same mass and different valence numbers are collated with respect to the MS ¹ spectrum and the MS ^{n + 1} (n ≧ 1) spectrum to determine a plurality of peaks matching between the spectra; Identify each matching peak as multiple ion species of components with the same mass and different valence, and determine or specify the ion species for the next MS ² mass analysis from the identified ion species A mass spectrometric method characterized by determining an ion species to be subjected to the next MS ² mass spectrometry from those other than the ion species thus obtained. The mass spectrometric method according to claim 1 ,
The method of identifying a plurality of ion species of the same compound having the same mass and different valence numbers is performed at a time between MS ¹ mass analysis and MS ² mass analysis. The mass spectrometric method according to claim 3 ,
A time period between the MS ¹ mass analysis and the MS ² mass analysis is 10 milliseconds or less. 2. The mass spectrometric method according to claim 1, wherein said tolerance is a monoisotopic peak and an isotope having a maximum intensity from a theoretical isotope distribution obtained from a mass estimated for components having the same mass. A mass spectrometric method characterized by identifying a body peak and determining a mass difference between the identified monoisotopic peak and the isotope peak. In the mass spectrometry method according to claim 1, characterized in that the MS ² to eliminate the valence reduction ions from the ion type information of the spectrum, analyzing the MS ² spectra based on the ionic species remaining in the ion species information Mass spectrometry method . 2. The mass spectrometric method according to claim 1 , wherein a monoisotopic mass of each ion species is estimated using a theoretical isotope distribution from the valence of each ion species of the assumed MS ^1. Mass spectrometry method . The sample material is ionized and mass spectrometry (MS ¹ ) is performed to obtain a mass spectrometry spectrum.
Select an ion species having at least one M / Z value from the ionized sample material, dissociate the selected ion species, and perform mass analysis (MS ² ) of the dissociated ion species to obtain a mass spectrometry spectrum In a mass spectrometry method for ionizing a sample material again and performing mass spectrometry (MS ¹ ) to obtain a mass spectrometry spectrum,
At the time between the MS ² mass analysis and the second MS ¹ mass analysis, the MS ¹ spectrum and the MS ² spectrum are matched to determine multiple peaks that match between the spectra, and each matching peak has the same mass. And identified as a plurality of ionic species of components having different valences,
(A) identifying a plurality of ion species of the same compound having the same mass and different valence numbers;
(B) Among these ionic species, the valence is assumed for the ionic species having the maximum valence,
(C) Calculate the mass M from the assumed valence and the M / Z value of the ionic species with the maximum valence,
(D) Obtaining the valence of each ion species by rounding off the quotient of the m / z value of each ion species other than the ion species having the maximum mass M and valence calculated,
(E) Calculate the mass of each ionic species other than the ionic species having the maximum valence from the obtained valence and M / Z value,
(F) Obtain the difference between these masses and masses M, respectively.
(G) It is determined whether all the obtained differences are within a predetermined tolerance range,
(H) The valence assumed in step (b) is changed until all the mass differences of the ion species obtained in (f) in step (g) are within the tolerance range (b) to (g The mass spectrometric method is characterized by repeatedly performing the step (1). The mass spectrometric method according to claim 8 ,
In the above-mentioned MS ¹ mass spectrometry again, the measurer consists of an ion species having the same valence as the ion species analyzed by MS ² before or during the measurement of MS ² mass spectrometry, or other components. A mass spectrometric method characterized in that it can be measured by specifying an ion species.

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