JP4929149B2 - Mass spectrometry spectrum analysis method - Google Patents

Description

  The present invention relates to a mass spectrometry data analysis method and a mass spectrometry system.

In mass spectrometry, components are ionized and analyzed as they are (MS analysis method), and ions of a specific component (precursor ions) are selected by mass, and dissociated ions are generated and mass analysis is performed for dissociated ions. There is an analysis method. In tandem mass spectrometry, an ion having a specific mass-to-charge ratio (precursor ion) is selected from the dissociated ions, the precursor ion is further dissociated, and mass analysis of the generated dissociated ions is performed. As described above, there is a function of performing dissociation / mass spectrometry in multiple stages (n-th stage measurement: hereinafter MS ⁿ ). The precursor ion is also called a parent ion. By performing MSn, the structure of the component can be analyzed from the fragment information of the precursor ion, and what is the component from the protein database search using the fragment information or the de novo sequencing method that decodes the structure from the mass between the fragments Can be identified.

  A system combining a chromatography and a mass spectrometer is used to measure a sample in which many components are mixed. In this system, the substance to be measured is separated by time from the difference in the degree of adsorption of the substance on the column by chromatography, and separated by mass by a mass spectrometer. In the case of a compound having a sugar chain isomer or a combination of two amino acids having the same mass as one amino acid, it is difficult to separate them by mass. Due to the nature, most are separated in time by chromatography. In the case of using chromatography, even if the same sample is measured under the same analytical conditions, an ion detection time error of several minutes usually appears. In addition, in a crude sample in which a large number of components are mixed like a biological sample, not only a uniform shift in detection time but also a change in the detection order between components of the same mass may occur.

  The measured mass spectrometry data is analyzed after the analysis is completed. Various methods are used to extract the same component from a plurality of mass spectrometry data.

  WO05 / 050188 (Patent Document 1) discloses a method for extracting the same component using an MSn spectrum as a result of analyzing tandem mass spectrometry data as a method for extracting the same component in mass spectrometry. Tandem mass spectrometry is performed on each of a plurality of samples. A database search is performed on the tandem mass spectrometry data obtained by each measurement. As a result of the database search, the same components are extracted for each sample. In this method, since the same component is extracted from the result of the database search, the accuracy of matching determination is high. However, since only the components that have been subjected to tandem mass spectrometry are targeted for extraction, it is not possible to determine whether or not the components have not been subjected to tandem mass spectrometry. For crude samples containing tens of thousands to hundreds of thousands of components such as biological samples, tandem mass spectrometry is performed on all components detected in one measurement according to the performance of the current mass spectrometer. It is difficult. Furthermore, since the intensity and retention time of the components vary between measurements, it may happen that a component that was tandem mass analyzed in one measurement is not tandem mass analyzed in the next measurement. In such a case, since there is structural information on only one side, the accuracy of association is greatly reduced. In addition, when the component is contained only in a trace amount, the structure may not be specified even if tandem mass spectrometry is performed, or incorrect identification may be performed. In such a case, correct matching cannot be determined.

  Further, extraction of the same component is usually performed for the purpose of quantitative analysis of each component, and the intensity of the MS spectrum is used for quantification of the component. For this reason, in order to improve quantitative accuracy, it is necessary to acquire more MS spectra for each component. On the other hand, the number of MS spectra acquired by performing tandem mass spectrometry decreases, resulting in a decrease in quantitative accuracy.

  For this reason, a method of extracting the same component from only the MS spectrum without performing tandem mass spectrometry has been studied.

WO05 / 050188

  In chromatographic mass spectrometry of biological samples, even if the measurement is performed under the same equipment and the same chromatographic separation conditions, the separation due to mixing of multiple components and the intensity and chromatographic It is difficult to associate the components due to variations in retention time or variations in the sample itself that occur during pre-processing.

  An object of the present invention is to provide a mass spectrometry data analysis method capable of extracting the same component with high accuracy from a plurality of mass spectrometry data measured on a plurality of samples under similar conditions.

One feature of the present invention is a mass spectrometry data analysis method for analyzing a plurality of chromatographic mass spectrometry data obtained by measuring a plurality of samples made of a mixture of a plurality of components in a mass spectrometry data analysis method.
Using information on a specific component, and information on another component that has a mass within a certain range with the specific component and is detected under similar conditions in a certain time range Thus, a correspondence relationship between different chromatographic mass spectrometry data is obtained, and the same component among a plurality of chromatographic mass spectrometry data is obtained from the proportion or situation of the coincidence.

  In the present invention, it is possible to provide a mass spectrometry data analysis method capable of extracting the same component with high accuracy from a plurality of mass spectrometry data measured on a plurality of samples.

  The present invention relates to a method for associating components between a plurality of measurement data with respect to mass spectrometry data separated by liquid or gas chromatography in the case where many components such as a biological sample are mixed. It is about. Examples of the present invention are shown below.

  The mass spectrometry data analysis method of the present invention will be described with reference to FIG. An object of the present invention is to analyze a plurality of chromatographic mass spectrometry data. As the assumed mass analysis data to be analyzed, for example, a plurality of mass analysis data are conceivable as a sample group to be compared with a reference sample group at the time of biomarker search. If each component between the reference sample group and the sample group to be compared can be associated with high accuracy, the quantitative ratio of each component is obtained in the subsequent quantitative analysis, and the biomarker with a variation in the quantitative ratio is obtained. Can be extracted as a candidate.

  In the present invention, at least two or more mass analysis data (A, B, C...) Are read, and after performing MS spectrum processing, mass chromatogram processing, and impurity / noise removal for each, the same component Perform the determination. MS spectrum processing includes noise removal by a certain intensity threshold, peak determination, isotope peak determination, and monoisotopic peak extraction. Next, mass chromatogram processing is performed to evaluate the time axis change of each mass peak intensity.

  Here, the mass chromatogram area of each component is evaluated. Next, noise and impurity data are removed from the shape of the mass chromatogram. For example, components that are instantaneously detected only in the 1MS spectrum and components that are detected for a long time from the start to the end of measurement are removed as noise and impurities. Next, the same component is determined based on the component list obtained from each measurement data.

The determination of the same component will be described with reference to FIG. Based on each component information derived in FIG. 1 (2 to 5), each measurement data component is divided into n on the time axis. The number of divisions here can be freely set by the user. For example, if the 60-minute measurement data is divided into 10 parts, the components detected at each time in each measurement are measured every 6 minutes such as 0 to 6 minutes, 6 to 12 minutes,. Classify. Next, components having the same possibility are extracted. Within the n-th region, components whose mass-to-charge ratio (m / z) and retention time (RT) match within a certain tolerance are extracted. The margin here can be set by the user according to the analysis conditions. Here, m / z tolerance: 1.0 Da, RT tolerance: 2.0 minutes. The components extracted here may be the same component, but in the case of a crude sample such as a biological sample, there is a possibility that an erroneous coincidence determination is made.
Therefore, component information around the extracted components (in the two-dimensional map of m / z and RT shown in FIG. 3) is compared. In FIG. 3, each point plots a component detected at a particular mass at each time. Here, it is assumed that the component α of the sample A is determined. Regarding the component α, it is assumed that in the sample B, β and γ having the same mass are detected in the same time zone. In this case, it is difficult to determine which of β and γ of sample B is the same component. Therefore, the matching component is determined using the component information around α, β, and γ. In FIG. 3, four types of components can be confirmed within a certain range of α (m / z and RT). Since these four types of components are also detected in the sample B, the determination is made based on these four types of components. In the sample A, the components 1 and 3 are detected at a time (RT) earlier than α, and the components 2 and 4 are detected at a later time (RT). On the other hand, in β of sample B, components 1 and 3 are detected at an earlier time than β and components 2 and 4 are detected at a later time, whereas in γ, all components 1 to 4 are detected more than γ. Detected early. For this reason, β has a higher coincidence rate and is extracted as the same component. If there are components with the same number of matches, the match rate of the isotope peak distribution of surrounding match components is determined. Since the detected component has an isotope peak distribution, this information is used. However, here, the component for determining the isotope peak distribution is only a component having an intensity equal to or greater than a certain intensity threshold. This is because, as shown in FIG. 4, the distribution pattern changes depending on the intensity. In the case of a peak with high intensity, almost the same isotope distribution pattern can be obtained, but when the intensity is low, the detection error of the mass spectrometer becomes large and the isotope distribution pattern varies from measurement to measurement. It has been. For this reason, the determination of the isotope peak distribution is performed only for components having a certain intensity or more.

  Next, in FIG. 2, component information around the extracted components is compared, and if there is a matching component, measurement data to be used as a reference is determined. Here, assuming that the component A has the maximum intensity, the measurement data B and C are corrected with the correction data A as a reference. Next, based on the correction result of n, correction is performed on the 1 to n-1 regions. Here, all components are uniformly corrected in the same manner as the correction result of n. For example, there is a component α having the maximum intensity in the region n, the component α of the measurement data B is +0.5 minutes, and the component α of the C is −0.2 minutes based on the retention time of the component α of the measurement data A. Suppose that there was a shift. In this case, the component α of B shifts the holding time to the minus side by 0.5 minutes in order to match the component α of A, and the component α of C shifts the holding time to the plus side by 0.2 minutes. Similarly, for all components in the region 1 to n−1, the holding time is corrected by −0.5 minutes for B and +0.2 minutes for C.

  This process is performed in one direction (start to end or end to start) for all regions. Here, the coincidence determination is desirably corrected in the direction from the analysis end time to the start time. FIG. 5 shows the relationship between the measurement time and the hold time. From FIG. 5, it can be seen that the shift in the retention time becomes smaller in the latter half of the analysis. This is because, at the start of the analysis, variations in each measurement such as the state of adsorption of the sample to the column and the outflow of impurities appear, whereas the solvent concentration is always set to the same condition at the end of the analysis.

  By carrying out the above processing over the entire area, the same component determination is completed, and the results are output in FIGS. The output result is shown in FIG. The output result includes the number of components determined to be the same, RT (reference data retention time), m / z, the mass chromatogram area of each measurement data component, and the number of coincidence determination components. These lists are linked to the raw data, and the peak information of the raw data can be displayed by clicking the component number. Thereby, it is possible to perform the quantitative analysis from the list obtained in the present invention, and after extracting the biomarker candidates, it is possible to evaluate the reliability of the component association from the number of matching components and the raw data.

  Next, a second embodiment of the present invention will be described. A mass spectrometry system of this example is shown in FIG. Internal database 27 for storing data on the peptide derived from the identified protein, pretreatment system 19 for separating the peptide as a sample, ionization unit 20 for ionizing the peptide, mass for separating ions according to the mass-to-charge ratio m / z An analysis unit 21, an ion detection unit 22 for detecting separated ions and generating a mass spectrum, a data processing unit 23 for organizing and processing data such as a mass spectrum, a display unit 24 for displaying mass analysis data as an analysis result, A control unit 25 that controls processing of these components and an input unit 26 for a user to input data and commands are provided.

The pretreatment system 19 has liquid chromatography (LC) and separates samples in time.
Here, a gas chromatograph may be used instead of the liquid chromatograph (LC).

In MS ¹ mass spectrometry, the peptide is ionized by the ionization unit 20 and sent to the mass analysis unit 21. Ions are separated by the mass analyzer 21 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 22.

In MS ² mass spectrometry, precursor ions selected from the MS ¹ mass spectrum are dissociated. In this example, as a precursor ion dissociation method, a collision induced dissociation method is used in which the precursor ions collide with a buffer gas such as helium to dissociate. In this example, a collision cell 21A (collision cell) filled with a neutral gas is provided.

  The mass analyzer 21 captures precursor ions in a region having a specific mass-to-charge ratio (m / z) or a specific mass-to-charge ratio (m / z), and collectively collects them into the collision cell 21A. In order to capture specific precursor ions, for example, a resonance voltage having a predetermined frequency is superimposed and applied to the trap voltage so that ions to be excluded are in a resonance state.

  The precursor ions collide with the neutral gas in the collision cell 21A and dissociate. In order to cause the precursor ions to collide with the neutral gas, a voltage having a frequency that resonates with the precursor ions is applied. The mass analyzer 21 may be filled with a neutral gas, and the precursor ions may collide with the neutral gas in the mass analyzer to be dissociated. In that case, the collision cell 21A becomes unnecessary.

  As a precursor ion dissociation method, the precursor ion is irradiated with low-energy electrons and a large amount of low-energy electrons are captured to dissociate, and the precursor ion is irradiated with an anion beam. You may use the electron transfer dissociation method etc. which dissociate by moving an electron.

Precursor ions dissociated by the collision cell are separated by the mass analyzer according to the mass-to-charge ratio m / z. The separated ions are detected by an ion detector, and an MS ² mass spectrum is generated. The MS ² mass spectrum is processed by the data processing unit 23, and mass analysis data as the analysis result is displayed on the display unit 24. Processing by the data processing unit is called post-processing.

  The control unit 25 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 21, dissociation and separation of the precursor ions, detection of the precursor ions, data processing, and the like. To do.

With reference to FIG. 8, the analysis process in the mass spectrometry system by this invention is demonstrated. In step S29, measurement is started. In step S30, MS ¹ mass spectrometry is performed. From step S31 to step 42, real-time data analysis is performed on the MS ¹ (n ≧ 1) mass spectrum obtained by MS ¹ mass spectrometry. In step S32, peak determination is performed. That is, the peak of the MS ¹ (n ≧ 1) mass spectrum is detected. In step S33, isotope / valence determination is performed. That is, the isotope peak is removed from the peak of the MS ¹ (n ≧ 1) mass spectrum. Let Npi be the number of peaks (monoisotopic peaks) thus obtained that do not contain isotopes. In step S34, one peak is sequentially selected from the Npi peaks, and the intensity thereof is compared with a threshold value. If the intensity of this peak is greater than or equal to the threshold, the process proceeds to step S35. If the intensity of this peak is less than the threshold, the process proceeds to step S41, and the same determination is performed for the next peak. In step S35, the retention time (RT), mass-to-charge ratio (m / z), valence (z), and isotope pattern of the liquid chromatograph are collated with the information stored in the internal database for this peak ion. To do.

In step S36, it is determined whether or not the peak ion information matches the internal database information. If they match, the process proceeds to step S38, and if they do not match, the process proceeds to step S37. In step S38, the matched ions are stored in the memory as a match list. Next, in step S39, the retention time information in the internal database is corrected from the current retention time of ions in the match list. Here, in order to perform correction, it is desirable that there is at least three pieces of ion information. In one or two cases, an erroneous determination may be made with respect to noise, impurities, etc., and it is desirable to perform correction by collating as much information as possible. The minimum number of coincident ions used for correction here can be specified by the user. FIG. 9 shows data stored in the internal database. Here, the corrected retention time is stored, and for the ions detected in the subsequent retention time, matching is performed using the correction value. Further, the holding time of the internal database after that time is rewritten as needed by the coincidence ions detected in each time zone of analysis. On the other hand, ions that do not match the internal database are added to the precursor ion candidate list for MS ² mass spectrometry in step S37.

In step S40, it is determined whether or not collation with the internal database has been completed for all peaks. If completed, the process proceeds to step S42, and if not completed, the process proceeds to step S41. In step S41, the process proceeds to the next peak. In step S42, a precursor ion for MS ² mass spectrometry is determined. MS ² mass spectrometry is sequentially performed on the precursor ions thus determined (step S43).

  In step S44, it is determined whether or not the analysis is completed. If not, the process returns to step S30. If the measurement is finished, the measurement is finished in step S45. In order to realize real-time data analysis from step S42 to step S42, it is necessary to execute the processing from step S32 to step S42 within 10 milliseconds, preferably within 1 millisecond.

  As described above, according to the present invention, not only the component to be associated, but also component information detected in a similar time zone is used for the determination, so that the accuracy of association can be improved. Further, in the present invention, since the holding time is not corrected based on data including a plurality of component information such as MS spectrum and total ion chromatogram, the correspondence is performed for each component, and therefore, detection is performed in the same time zone. It is also possible to associate trace components that tend to be affected by components with high strength. Further, in the present invention, since the MS spectrum is used for the determination, it is possible to associate all the components that have not been subjected to the MS / MS analysis without impairing the quantitative analysis accuracy.

  Although the examples of the present invention have been described above, the present invention is not limited to the above-described examples, and it is understood by those skilled in the art that various modifications can be made within the scope of the invention described in the claims. Easy to understand.

It is a figure which shows the flow of the mass spectrometry data analysis system of this invention. It is a figure which shows the flow of the same component determination of this invention. It is a conceptual diagram of the component matching using the surrounding ion information of this invention. It is a figure which shows the relationship between intensity | strength and distribution of an isotope peak. It is a figure which shows the relationship of the shift | offset | difference in holding time and each holding time. It is a figure which shows the output content of the mass spectrometry data analysis result of this invention. It is a figure which shows the structure of the mass spectrometry system which is the 2nd Example of this invention. It is a figure which shows the flow of the mass spectrometry system which is the 2nd Example of this invention. It is the figure which showed the stored data of the internal database used with the mass spectrometry system which is the 2nd Example of this invention.

Explanation of symbols

1 Start of data analysis 2 Data reading 3 MS spectrum processing 4 Mass chromatogram processing 5 Impurity and noise removal 6 Same component determination 7 Result output 8 End of data analysis 9 Start of same component determination 10 Component information reading 11 Retention time Each component information is divided into n. 12 Components having the same possibility of (m / z, RT) within the nth region are extracted. 13 Component information comparison around the extracted components. Holding time correction 15 RT correction of region n = 1 to n−1 16 n = 1 determination 17 n = n−1
18 End of identical component determination 19 Preprocessing system 20 Ionization unit 21 Mass analysis unit 22 Ion detection unit 23 Data processing unit 24 Display unit 25 Control unit 26 User input unit 27 Internal database 28 Mass analysis system 29 Start of measurement 30 MS ¹ analysis 31 Real-time data analysis 32 Peak determination 33 Isotope / valence determination 34 Precursor ion intensity determination 35 Internal DB verification 36 Match determination 37 Addition to candidate ions 38 Store in match list 39 Match DB internal retention time information correction 40 Verification End determination 41 To next peak 42 Determination of MS ² precursor ion 43 MS ² analysis 44 Analysis end determination 45 End of measurement

Claims (6)

Mass spectrometry data of a first sample containing a desired component from mass spectrometry data measured by a mass spectrometer that separates a sample composed of a mixture of a plurality of components by chromatography and performs mass analysis of the separated mixture And mass spectrometry data analysis method for observing the desired component using the mass spectrometry data of the second sample containing the desired component,
And data of said desired component of said first mass spectrometric data, has a mass within a predetermined range with respect to the mass of the desired ingredients, and, with respect to chromatographic retention time of the desired component To identify the comparison component data having a retention time within a predetermined range,
Each data of the second mass spectrometry data is compared with the data of the desired component of the first mass spectrometry data and the data of the comparison component of the first mass spectrometry data, and the second mass spectrometry data Based on the ratio or situation in which each data and the comparison component data of the first mass spectrometry data match within a predetermined tolerance, which data among the data of the second mass spectrometry data is the desired component A method for analyzing mass spectrometry data, characterized in that the data is specified as follows. The method for analyzing mass spectrometry data according to claim 1 ,
The comparison component data of the second mass spectrometry data is at least one of the mass, valence, peak intensity, isotope peak detection distribution, and chromatography retention time of the comparison component of the first mass spectrometry data. A method for analyzing mass spectrometry data, characterized by using A method for analyzing mass spectrometry data according to claim 1 or 2 ,
The mass analysis data analysis method, wherein the predetermined range of mass and the predetermined range of retention time are designated for each analysis of mass analysis data. A method for analyzing mass spectrometry data according to any one of claims 1 to 3 ,
A method of analyzing mass spectrometry data, comprising outputting a list comparing the mass, valence, intensity, retention time, and isotopic distribution of the first and second mass spectrometry data for the desired component data . A method for analyzing mass spectrometry data according to any one of claims 1 to 4 ,
The comparison component of the mass spectrometry data of the first sample and the comparison component of the mass analysis data of the second sample are compared, and the retention time of the comparison component of one mass analysis data having a strong peak intensity of the comparison component is determined. A method of analyzing mass spectrometry data, wherein the retention time of the comparison component of the other mass spectrometry data is corrected as a reference. A method for analyzing mass spectrometry data according to claim 5 ,
A method of analyzing mass spectrometry data, wherein the retention time of all components detected in the other mass spectrometry data is corrected based on the correction of the retention time of the comparison component.

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