JP5655758B2 - Mass spectrometer - Google Patents

Description

The present invention identifies each substance by performing mass spectrometry on a sample obtained by separating and fractionating various substances by a liquid chromatograph (LC), a gas chromatograph (GC), a capillary electrophoresis apparatus (CE) or the like. More particularly, the present invention relates to a mass spectrometer capable of performing MS ⁿ analysis (n is an integer of 2 or more).

In fields such as life science research, medical care, and drug development, it has become increasingly important to comprehensively identify various substances such as proteins, peptides, nucleic acids, and sugar chains in biological samples. Such a comprehensive analysis method especially for proteins and peptides is called Shotgun Proteomics. For such analysis, an analysis method combining a chromatography such as LC, GC, or CE and a tandem mass spectrometer (MS ^n- type mass spectrometer) is very effective.

As a comprehensive identification technique for various substances in a biological sample using a tandem mass spectrometer, the procedure of one technique that has been conventionally known is as follows (see Non-Patent Document 1).
[Step 1] Various substances contained in the sample to be analyzed are separated by LC, CE, etc., and a large number of samples are prepared by fractionating and fractionating the eluate (in this case, by fractionation and fractionation). Each sample obtained will be referred to as a “fractional sample”). In addition, when fractionating and fractionating a sample, fractionation is performed at a predetermined time interval or fractionation so that a certain amount of sample solution is collected, so that all substances will not leak. It is made to be contained in any fraction sample (for example, refer patent document 1).

[Step 2] An MS ¹ spectrum is obtained for each fraction sample, and a peak that is considered to be derived from the substance group to be identified is selected on the MS ¹ spectrum.
[Step 3] The peak selected in Step 2 above is set as a precursor ion, MS ² analysis is performed on the fractionated sample, and a database search or a de novo sequence search is performed based on the result, and it is included in the fractionated sample. To identify
[Step 4] If a specific substance is not identified with sufficient accuracy, another peak found on the MS ¹ spectrum is set as a precursor ion and MS ² analysis is performed, or higher order MS ⁿ analysis of n (that is, n is 3 or more) is executed, and substances contained in the fractionated sample are identified by database search or de novo sequence search based on the result.
[Step 5] The processes of Steps 2 to 4 are performed on each of the plurality of fractionated samples, and various substances contained in the original sample are comprehensively identified.

In a separation method using a column such as LC or GC, various substances in a sample are separated according to the time required for passing through the column (retention time, hereinafter referred to as “RT”). In this case, the profile representing the total ion intensity detected by the MS ¹ analysis along the retention time is generally called TIC (total ion chromatogram), focusing on a specific mass-to-charge ratio m / z, A profile representing the ionic strength of the mass-to-charge ratio along the retention time is called MC (mass chromatogram).

In the analytical method for exhaustive identification as described above, in order to identify a substance with high accuracy, the number of species contained in the same fraction sample should be small (preferably only one). desirable. For this purpose, the time for collecting one fraction sample may be shortened, but in this case, the number of fractions increases. When MS ⁿ analysis and identification processing are performed on all fraction samples to comprehensively identify the substances contained in the original sample, the time and effort required for analysis and processing increases as the number of fractions increases. It will be something. In particular, when analyzing different fractionated samples, it is necessary to change the sample mechanically or to transport the sample to be analyzed to the analysis position. As the number of fraction samples increases, the analysis efficiency decreases significantly.

In general, since not all fraction samples contain significant substances, in fact, it is not always necessary to analyze all fraction samples even with comprehensive identification. Therefore, in order to comprehensively and efficiently identify the substances contained in the original sample, it is necessary to preferentially select a fraction sample that has a high possibility of containing the substance to be identified and perform MS ⁿ analysis. is important. As one method for that purpose, it is conceivable that TIC corresponding to the total amount of substances contained in each fraction sample is monitored, and the fraction sample corresponding to the retention time of the peak appearing in TIC is preferentially analyzed by MS ^n. . However, when MC and TIC in the mass-to-charge ratio of the substance to be identified are compared, the peak generation positions (that is, retention times) of the two do not always match.

FIG. 6 is a diagram illustrating an example of measurement results of TIC for a certain sample and MC of a substance that is m / z 1956 included in the sample. In this example, a large peak occurs at about RT = 6100 in MC, whereas a peak does not appear at the same RT in TIC, and the occurrence positions of both peaks do not coincide. In such a case, even if a fraction sample corresponding to the retention time of the peak appearing on the TIC is selected as an object of MS ² analysis, the fraction sample contains a substance that is an identification target at a sufficient concentration. It may not be. On the other hand, in order to select a fraction sample containing a substance to be identified at a sufficient concentration, a fraction sample corresponding to the retention time of a peak appearing in MC for the substance may be selected. However, in the state where the substance contained in the fraction sample is unknown, it is impossible to determine which mass-to-charge ratio MC should be used. Therefore, selecting a fraction sample using MC is not possible. Can not.

In order to solve the above problem, a method described in Non-Patent Document 2 has been known. That is, first, a linked peak list in which peak lists (or MS ¹ spectra) at each RT are arranged is created. FIG. 5 is a three-dimensional graph of this linked peak list, where retention time (RT) and mass-to-charge ratio are two axes on the paper surface, and the signal intensity of each peak is another axis of color and its shading. Indicated by The holding time range sandwiched between two horizontal dotted lines in FIG. 5 is the time range of TIC and MC shown in FIG. 6, and the vertical dotted line in FIG. 5 is the MC of m / z1956. Is the peak data corresponding to.

Next, peak detection is performed on the three-dimensional graph on a two-dimensional plane. Hereinafter, this peak detection is referred to as “2D peak detection”, and the peak detected thereby is referred to as “2D peak”. As shown in FIG. 7, 2D peaks are observed at several mass-to-charge ratios in RT corresponding to a certain fraction (RT indicated by a horizontal dotted line in FIG. 7). Since it can be determined that the fractionated sample thus obtained contains at least one of the substances to be identified, this fractionated sample is selected as an object of MS ² analysis. Furthermore, the 2D peak found at this time (the peak indicated by the circled circle in FIG. 7) is a precursor ion candidate for MS ² analysis for identifying the substance contained in the fraction, It becomes an object to perform MS ⁿ analysis in which the number of n is sequentially increased (the number of cleavage stages is increased) until the substance contained in the fraction is identified.

In the fraction sample selection process based on the 2D peak detection described above, a 2D peak having a signal intensity exceeding a predetermined threshold is extracted as a precursor ion candidate for MS ² analysis. Therefore, the number of 2D peaks to be extracted varies depending on the threshold value. As a result, the number of selected fraction samples and the number of precursor ion candidates to be listed also increase or decrease depending on the threshold value. When the threshold value is small, a 2D peak having a relatively small signal intensity is also selected, so that it is possible to reduce the list-up leakage of precursor ion candidates. On the other hand, since the number of fraction samples to be subjected to MS ⁿ analysis increases, the labor of analysis and processing increases, leading to a decrease in analysis efficiency. On the other hand, if the threshold value is increased, the number of fraction samples selected for MS ⁿ analysis can be reduced, but the number of precursor ion candidates is also reduced, so substance identification in the selected fraction sample is possible. The possibility of failure will increase.

JP 2010-14559 A

Edward M Marcotte, “How do shotgun proteomics algorithms identify proteins?”, Nature Biotechnology (Nature Biotechnology) 25, 2007, p.755-757 Jurugen Cox and one other, "Max Quant Enable High Identification Rates, Individualized Peaby-Range Mass Accurate and Proteome-Wide Protein・ Quantification (MaxQuant enable high peptide identification rates, individualized ppb-range mass accuracies and proteome-wide protein quantification), Nature Biotechnology 26, 2008, p.1367-1372

The present invention has been made in order to solve the above-mentioned problems, and the object of the present invention is, for example, by 2D peak detection as described above on a two-dimensional plane having two axes of retention time and mass-to-charge ratio. when extracting the precursor ion candidates fractionated sample and MS ⁿ analysis of interest of MS ⁿ analysis, while achieving savings in labor and time for analysis by suppressing as much as possible the number of fractions sample to be analyzed, It is an object of the present invention to provide a mass spectrometer capable of eliminating precursor ion candidate leaks, reducing identification failures, and improving identification accuracy.

In order to solve the above problems, the present invention provides an MS ⁿ analysis for a plurality of fraction samples prepared by separating and fractionating various substances contained in a sample according to separation parameters. n is an integer greater than or equal to 2), respectively,
a) Based on the MS ¹ spectral data obtained by performing MS ¹ analysis on all fractionated samples, a two-dimensional plane with the separation parameters and the mass-to-charge ratio as two axes is formed and orthogonal to the plane. A virtual three-dimensional graph having the signal intensity on the axis to be generated is created, and the first threshold value which is a relative value based on the signal intensity of the peak having the maximum signal intensity in the three-dimensional graph and the first threshold defines a small second threshold value than first threshold, Oite signal strength to the three-dimensional graph extracts the peak is not less than the first threshold value, corresponding to the position on the two-dimensional plane peak top of the peak appears A fraction sample selection means for selecting the fraction sample as a fraction sample to be analyzed by MS ⁿ ,
the separation parameter b) Oite signal strength to the three-dimensional graph corresponding to fraction sample selected by the previous SL fractionation sample selection means comprising at least before Symbol second threshold peak with a peak top present A precursor ion candidate selection means for extracting and selecting a mass-to-charge ratio corresponding to the peak top of the peak as a precursor ion candidate;
c) For the fraction sample selected by the fraction sample selection means, one of the precursor ion candidates selected corresponding to the fraction sample by the precursor ion candidate selection means is set as a precursor ion. MS ⁿ analysis execution means for performing MS ⁿ analysis;
and identification means for performing identification of a substance contained in the fraction sample based on the MS ⁿ analysis execution result by d) said MS ⁿ analysis executing means,
It is characterized by having.

  When the means for separating various substances contained in the sample uses a column such as LC or GC, the separation parameter is time (retention time). Further, when the means for separating various substances contained in the sample is CE, the separation parameter is mobility. That is, when the means for separating various substances contained in the sample is LC, for example, a plurality of fractionated samples are collected (sorted) at different holding times (strictly in a predetermined holding time range). It is.

In the mass spectrometer according to the present invention, each of the fraction sample selection means and the precursor ion candidate selection means is a virtual three-dimensional graph with the separation parameter, the mass-to-charge ratio, and the signal intensity as three axes. The peak is detected by determining whether or not the signal intensity is greater than or equal to the threshold value. However, the first threshold for detecting a peak used for selecting a fraction sample to be analyzed for MS ⁿ from a large number of fraction samples is used for selecting a precursor ion candidate for MS ⁿ analysis. Larger than the second threshold for detecting the peak to be detected. That is, since the former detects peaks with a stricter standard than the latter, the fraction sample to be subjected to MS ⁿ analysis is limited to a fraction sample containing some substance at a relatively high concentration. On the other hand, since the standard for detecting a peak that is a precursor ion candidate is relatively loose, the number of precursor ion candidates listed for one fraction sample tends to be relatively large.

That is, in general, even when the number of fraction samples to be subjected to MS ⁿ analysis is small, there is a high possibility that a plurality of candidate precursor ions that are useful for identification of substances in each fraction sample are listed. . In general, even if the identification of contained substances is not successful using the result of MS ² analysis using the peak having the highest signal intensity among the peaks in the MS ¹ spectrum as a precursor ion, the same MS ¹ spectrum is used. Among them, the contained substance may be successfully identified using the result of MS ² analysis using a peak whose signal intensity is not maximum as a precursor ion. Therefore, by listing a plurality of precursor ion candidates for a certain fraction sample, it is possible to increase the probability that the substance contained in the fraction sample can be identified.

Preferably, in the mass spectrometer according to the present invention, when the substance contained in the fraction sample cannot be identified by the identification unit, the MS ⁿ analysis execution unit is configured to select the precursor ion candidate selection unit for the fraction sample. run the MS ⁿ analysis by another one was set to precursor ions of the precursor ion candidate selected by said identification means, the fractionation sample based on the MS ⁿ analysis execution result for the different precursor ion It may be configured to try to identify contained substances.

As described above, a plurality of precursor ion candidates are usually given to one fraction sample, and even when substance identification cannot be performed from the MS ⁿ analysis result in which a certain one precursor ion is set, another precursor ion is selected. There is a possibility that substance identification can be performed from the set MS ⁿ analysis result. Therefore, according to the preferable configuration, it is possible to further increase the probability that the substance contained in the fractionated sample can be identified.

In the mass spectrometer according to the present invention, after the substance identification for all the fraction samples selected by the fraction sample selection means is completed, the fraction sample selection means sets the first threshold value to be higher than the second threshold value. A peak is extracted after decreasing in a large range, and a fraction sample corresponding to a position on the two-dimensional plane where a peak top of a newly extracted peak appears is selected as a fraction sample for MS ⁿ analysis, The selected fraction sample may be configured to execute processing by the precursor ion candidate selection means, the MS ⁿ analysis execution means, and the identification means.

In such a configuration, every time the first threshold value is decreased in a range larger than the second threshold value, the fraction sample selection means, the precursor ion candidate selection means, the MS ⁿ analysis execution means, and the processing by the identification means Is repeated, and the repetition may be terminated when the number of substances to be identified is saturated or when the degree of increase in the number of newly identified substances is greatly reduced as compared to before. Of course, regardless of the number of substances to be identified, the above repetition may be terminated based on the number of repetitions or the processing time.

In the above configuration, since the first threshold value for detecting the peak used for selecting the fraction sample to be analyzed for MS ⁿ is selected from a large number of fraction samples, the fraction sample to be selected is gradually reduced. The total number (total number) of will increase. That is, the application range of the MS ⁿ analysis and the identification process is expanded from a fraction sample having a high possibility of containing a substance to be identified to a fraction sample having a low possibility. For this reason, even when a sufficient number of substance identifications have not been made with only the fraction sample obtained with respect to the initial setting of the first threshold value, with a relatively small number of repetitions, that is, the MS ⁿ analysis target. It becomes possible to identify most of the substances contained in the original sample without increasing the number of fraction samples.

In the mass spectrometer according to the present invention, n may be fixed to 2 and only MS ² analysis may be executed as MS ⁿ analysis. However, depending on the target substance, database search is performed only by one dissociation operation. It may not be a small fragment that can be identified. Therefore, when the substance cannot be identified from the MS ² analysis result, the MS ³ analysis is performed using, for example, the peak of the signal intensity as the precursor ion in the MS ² spectrum obtained by the MS ² analysis, and the substance is obtained from the MS ³ analysis result. The substance may be identified from the MS ⁿ analysis result by performing MS ⁿ analysis in which n is 3 or more.

According to the mass spectrometer of the present invention, by performing MS ⁿ analysis on a relatively small number of fraction samples among a large number of fraction samples obtained by fractionating and fractionating the original sample. Thus, it becomes possible to accurately identify a large number of substances contained in the original sample. Accordingly, it is possible to reduce identification errors while performing efficient analysis while saving labor and time required for analysis.

The schematic block diagram of LC / MALDI-MS by one Example of this invention. Conceptual view of a process for selecting the MS ² analyte fraction samples in LC / MALDI-MS of the present embodiment. In LC / MALDI-MS of a present Example, the flowchart which shows the process and control operation | movement at the time of identifying comprehensively the substance contained in the fraction sample prepared in each well on a sample plate. Shows the experimental result of the relationship between the number of peptides identified as the number of selected well is the subject of the MS ² analysis. The figure which made the connection peak list into the three-dimensional graph. The figure which shows the example of a measurement result with TIC with respect to a certain sample, and MC of the substance which is m / z1956 contained in this sample. The figure which shows the relationship between the 2D peak detected and a fraction.

  Hereinafter, an LC / MALDI-MS according to an embodiment of the present invention will be described with reference to the accompanying drawings. FIG. 1 is a schematic configuration diagram of the LC / MALDI-MS of the present embodiment.

The LC / MALDI-MS of the present embodiment fractionates and fractionates the LC part 1 that separates various substances in the liquid sample according to the holding time, and the sample containing the substance separated in the LC part 1, respectively. A preparative fractionation unit 2 for preparing different fractional samples, a sample exchange unit 3 for selecting one of a plurality of fractional samples for mass spectrometry, and mass spectrometry (MS ¹ analysis, MS section 4 that performs MS ⁿ analysis in which n is 2 or more), data processing section 5 that processes data obtained by MS section 4, and LC section 1 and fractionation fractionation section 2 An analysis control unit 6 that controls the operation and controls the operation of the sample exchange unit 3 and the MS unit 4 based on the processing result by the data processing unit 5, an input unit 7 for setting analysis conditions, and the analysis result The display part 8 to display is provided.

The MS unit 4 includes a MALDI ion source, an ion trap that holds ions and has a function of separating ions according to a mass-to-charge ratio and a function of dissociating ions by collision-induced dissociation (CID), and various types of ions emitted from the ion trap This includes a time-of-flight mass spectrometer that separates and detects ions according to the mass-to-charge ratio, etc., and allows not only MS ¹ analysis but also MS ⁿ analysis that repeats ion selection and ion dissociation. However, when only MS ¹ analysis and MS ² analysis need be performed (when MS ⁿ analysis where n is 3 or more is not required), a simpler one such as a triple quadrupole mass spectrometer can be used. An MS ^n- type mass spectrometer having the configuration can be used.

The data processing unit 5 includes functional blocks such as a spectrum data collection unit 51, a 2D peak detection unit 52, an MS ⁿ analysis control information generation unit 53, and an identification processing unit 54. The data processing unit 5 and the analysis control unit 6 use, for example, a personal computer as a hardware resource, and execute the dedicated control / processing software installed in the personal computer, thereby realizing the above functional blocks. Can be configured.

  The LC unit 1 separates various substances contained in the injected sample in the time direction. Therefore, the substance contained in the eluate coming out of the LC unit 1 changes with time. The preparative fractionation unit 2 fractionates the eluate at predetermined time intervals, and sequentially drops the fractionated eluate into different wells formed on the sample plate for MALDI. To prepare. That is, the fraction samples in each well contain substances that sequentially have different retention times, more precisely, retention times that fall within a certain retention time range. Of course, since there is a range of time for a certain substance to elute in the eluate, one substance may be contained in a plurality of fraction samples.

A sample plate on which a large number of fractionated samples have been prepared in the preparative fractionation unit 2 is conveyed to the sample exchange unit 3, and the sample exchange unit 3 is controlled by the MALDI ion source of the MS unit 4 under the control of the analysis control unit 6. The sample plate is moved so that the fraction sample to be analyzed comes to a position where ionization is performed (that is, a laser light irradiation position for MALDI). Thereby, MS ¹ analysis and MS ⁿ analysis are performed on an arbitrary fraction sample among a large number of fraction samples.

As described above, processing and control operations when exhaustively identifying substances contained in the fraction sample prepared in each well on the sample plate will be described with reference to FIGS. FIG. 3 is a flowchart showing an operation procedure of this comprehensive identification process and control, and FIG. 2 is a conceptual diagram of a process when selecting a fraction sample to be analyzed by MS ² .

First, MS ¹ analysis is performed by the MS unit 4 for each of the fraction samples prepared in each well on the sample plate, and the spectrum data collection unit 51 acquires MS ¹ spectrum data (step S1). ). The spectrum data collection unit 51 performs peak detection processing on each MS ¹ spectrum, collects peak information including the mass-to-charge ratio of the peak and the signal intensity, and creates a peak list. Then, the peak lists obtained for each fractionated sample are linked, and a virtual three-dimensional graph as shown in FIG. 5 is created based on the linked peak list (step S2). In this three-dimensional graph, the retention time (RT) and the mass-to-charge ratio (m / z) are two axes orthogonal to each other, and the signal intensity is taken in a direction orthogonal to the two axes. In FIG. 5, the signal intensity axis is indicated by the color and its shading as described above.

The 2D peak detector 52 performs characteristic 2D peak detection on the virtual three-dimensional graph. First, the repetition variable N is set to ₁ , and threshold values θ ₁ , θ ₂ ,..., Which are criteria for determining the signal intensity are set as appropriate (step S3). These threshold values are indicated as% values when the signal intensity of the peak having the maximum signal intensity in the virtual three-dimensional graph is set to 100%, and the magnitude relationship is θ ₁ >θ2>...>Θm>. (See FIG. 2C). These θ ₁ , θ 2,... Are first threshold values, and ω is a second threshold value. The 2D peak detector 52 uses θ _N as the first threshold, and detects a 2D peak having a signal intensity equal to or higher than the first threshold θ _N in the virtual three-dimensional graph (step S4). When S4 is executed immediately after step S3, since N = 1, a 2D peak having a signal intensity equal to or greater than the first threshold value θ ₁ is detected. As shown in FIGS. 2 (a) and 2 (b), θ ₁ is sufficiently large (close to 100%), and therefore, there are usually few peaks having a signal intensity of θ ₁ or more among all peaks.

Next, the MS ⁿ analysis control information generation unit 53 determines a fraction sample corresponding to the retention time of the peak top of the 2D peak newly detected in step S4 as a fraction sample to be analyzed by MS ² (step S5). ). Since a plurality of 2D peaks detected in step S4 may correspond to the same fraction sample, the number of fraction samples to be analyzed for MS ² becomes the number of detected 2D peaks or less. . Since the first threshold value θ ₁ is initially set high, the number of 2D peaks detected in step S4 at this time is small, and the number of fraction samples selected in step S5 is relatively small. On the other hand, since the signal intensity of the detected 2D peak is high, it is presumed that the fraction sample selected here is highly likely to contain some substance to be identified.

Next, the 2D peak detection unit 52, for one of the plurality of fraction samples selected in step S5, on a two-dimensional plane including two axes of retention time and mass-to-charge ratio in the three-dimensional graph, A 2D peak having a peak top in the fractionated sample and having a signal intensity equal to or higher than the second threshold value ω is detected. Then, the MS ⁿ analysis control information generation unit 53 lists the 2D peak detected here as a precursor ion candidate for MS ² analysis of the one fractionated sample (step S6). As shown in FIG. 2A, since the second threshold value ω is much smaller (close to 0%) than the first threshold value θ ₁ , the 2D peak is more easily detected in step S6 than in step S4. Therefore, normally, in addition to the 2D peak detected in step S4, one or more 2D peaks not detected in step S4 are extracted for one fraction sample. That is, a plurality of precursor ion candidates, which is a relatively large number, are listed for one fraction sample. Thereby, possibility that the precursor ion derived from the substance contained in the fraction sample may be avoided from leaking candidates is increased.

The fraction sample and the precursor ion candidate list obtained in step S6 are given to the analysis control unit 6, and the analysis control unit 6 selects one of the precursor ion candidates listed for the fraction sample as a precursor ion. The sample exchange unit 3 and the MS unit 4 are controlled so as to execute the MS ² analysis set in (1). Spectral data collecting unit 51 collects the MS ² spectral data obtained by the MS ² analysis (step S7). The identification processing unit 54 identifies a substance contained in the fractionated sample by a predetermined algorithm such as MS / MS ion search or de novo sequencing using the MS ² analysis result, that is, MS ² spectrum data (step S8).

If the substance identification is successful (for example, when MS / MS ion search is used, the compound is specified with a sufficiently high reliability score or expected value), the process proceeds from step S9 to S10. move on. On the other hand, if the identification fails, the process proceeds from step S9 to S14, and the MS ⁿ analysis control information generation unit 53 determines whether there is another precursor ion candidate listed for the same fraction sample. . If there are other precursor ion candidates, only the precursor ion is changed for the same fraction sample (step S15), the process returns to step S7, and MS ² analysis in which the precursor ion is set is executed. Therefore, if the substance cannot be identified from the MS ² analysis result, the same fractionation is repeated until the substance is identified by repeating steps S7 → S8 → S9 → S14 → S15 → S7. MS ² analysis for dissociating different precursor ions from the sample is sequentially performed.

If the substance identification cannot be performed even if the MS ² analysis is performed on all the precursor ion candidates listed for a certain fraction sample, the process proceeds from step S14 to S16, and the MS ⁿ analysis control information generation unit No. 53 is determined in step S5, and it is determined whether there is another fraction sample that has not been subjected to MS ² analysis and identification processing yet. If there is another fraction sample, the process returns to step S6, and MS ² analysis and identification processing are executed for the other one unprocessed fraction sample in the same manner as described above.

  If identification of a substance contained in a certain fraction sample is successful, the process proceeds from step S9 to S10, and it is determined whether or not the number of substances identified so far is sufficient. If the number of types of substances contained in the original sample is known, or if it is determined that the substance is OK if a predetermined number or more of substances are identified in advance, the “sufficient” number is set to the predetermined number. As long as it is determined as described above, when the number of types of contained substances is unknown, it is not possible to determine pass / fail by the value of the number. Therefore, in that case, the cumulative value of the number of substances to be identified is obtained sequentially, and when the change in the cumulative value becomes smaller or no longer changes, or the number of newly identified substances is obtained in order, and the growth is increased. It may be determined that the number of substances to be identified has been saturated (that is, most of the substances have been identified), for example, when it has stopped.

When the identification number of the substance is determined to be insufficient in step S10, or when it is determined that there is no more fraction samples MS ² analysis in step S16, increments the variable N (step S11). Then, it is determined whether or not the variable N has reached a predetermined upper limit value (step S12). If the variable N has not reached the upper limit value, the process returns to step S4. The reason why the upper limit value of the variable N is determined in step S12 is to avoid an abnormally long analysis time even if the number of identified substances is insufficient.

When the process returns from step S12 to S4, the variable N is increased by 1 from the previous time. Therefore, when the process of step S4 is the second time, 2D peak detection using θ ₂ as the first threshold is performed. FIG. 2 (b), the as shown in (c), because theta ₂ is less than theta _1, as a new 2D peak was not detected when the first threshold value was theta ₁ above is detected become. Accordingly, a new fraction sample is also selected in step S5 according to the retention time of the peak top of the new 2D peak. However, when the peak top of the newly detected 2D peak is already present in the fraction sample that has been subjected to MS ² analysis, this fraction sample is excluded from the selection target. When a new fraction sample that has not yet been subjected to MS ² analysis is selected, the processing of steps S6 to S8 is performed on the fraction sample, and the substances contained in the new fraction sample are identified. .

Until it is determined that the number of substances identified in step S10 is sufficient, or until the variable N reaches the upper limit value in step S12, the process of steps S11 → S12 → S4 →. Accordingly, as shown in FIG. 2 (c), the first threshold value for detecting the 2D peak for selecting the fraction sample is gradually lowered, and the range of the fraction sample to be analyzed by MS ² is expanded. . However, as the range of the fraction sample to be analyzed by MS ² is expanded, the possibility that a substance is identified in a new fraction sample becomes lower. That is, many substances are identified while the variable N is relatively small. If it is determined that the number of substances identified in step S10 is sufficient, or if it is determined in step S12 that the variable N has reached the upper limit value, the identification processing unit 54 displays all the identification results. The data is output from the unit 8, and the process is terminated.

In the above description, it is assumed that substance identification is performed from the MS ² analysis result. However, for example, when a post-translationally modified protein is targeted, an MS ⁿ analysis result in which n is 3 or more is used. Otherwise, material identification may not be performed accurately. Therefore, when the substance cannot be identified from the MS ² analysis result, the MS ³ analysis is performed using, for example, the peak of the signal intensity as the precursor ion in the MS ² spectrum obtained by the MS ² analysis, and the substance is obtained from the MS ³ analysis result. For example, identification may be performed, and an MS ⁿ analysis in which n is 3 or more may be performed, and identification of a substance may be attempted from the MS ⁿ analysis result.

Next, an experimental example in which a sample containing a plurality of types of peptides is analyzed by the LC / MALDI-MS described above will be described. FIG. 4 shows the relationship between the cumulative number of selected wells (fractional samples) and the number of identified peptides when the above-described method according to the present invention and the conventional method are used. Here, the conventional method is a method in which the 2D peak detected at step S4 in step S6 in FIG. 3 is not performed, and the 2D peak detected at step S4 is used as a precursor ion candidate as it is. The parameters in this experimental example are θ ₁ = 15, θ ₂ = 10, θ ₃ = 9, θ ₅ = 8,..., Θ ₈ = 4, θ ₉ = ω = 3. Furthermore, the dotted line in FIG. 4 represents the number of peptides expected to be identified when a well to be analyzed for MS ² is selected without performing 2D peak detection.

As can be seen from FIG. 4, in the method of the present invention, a larger number of peptides are identified with a smaller number of well selection (in this example, about 150 or less) compared to the conventional method. For example, in order to identify 15 out of 18 peptides, in the method of the present invention, about 60 wells may be analyzed by MS ² , whereas in the conventional method, about 100 wells need to be analyzed by MS ^2. is there. Thus, according to the method of the present invention, the time until the same number of peptides is identified can be reduced.

  It should be noted that the above embodiment is an embodiment of the present invention, and it is a matter of course that modifications, corrections, and additions may be appropriately made within the scope of the present invention, and included in the scope of the claims of the present application.

1 ... LC 2 ... partial share image portion 3 ... sample exchange unit 4 ... MS unit 5 ... data processing unit 51 ... spectral data acquisition unit 52 ... 2D peak detector 53 ... MS ⁿ analysis control information generating unit 54 ... identification process Unit 6 ... Analysis control unit 7 ... Input unit 8 ... Display unit

Claims (4)

MS ⁿ analysis (n is an integer of 2 or more) can be performed on multiple fraction samples prepared by separating and fractionating various substances contained in the sample according to the separation parameters. A mass spectrometer,
a) Based on the MS ¹ spectral data obtained by performing MS ¹ analysis on all fractionated samples, a two-dimensional plane with the separation parameters and the mass-to-charge ratio as two axes is formed and orthogonal to the plane. A virtual three-dimensional graph having the signal intensity on the axis to be generated is created, and the first threshold value which is a relative value based on the signal intensity of the peak having the maximum signal intensity in the three-dimensional graph and the first threshold defines a small second threshold value than first threshold, Oite signal strength to the three-dimensional graph extracts the peak is not less than the first threshold value, corresponding to the position on the two-dimensional plane peak top of the peak appears A fraction sample selection means for selecting the fraction sample as a fraction sample to be analyzed by MS ⁿ ,
the separation parameter b) Oite signal strength to the three-dimensional graph corresponding to fraction sample selected by the previous SL fractionation sample selection means comprising at least before Symbol second threshold peak with a peak top present A precursor ion candidate selection means for extracting and selecting a mass-to-charge ratio corresponding to the peak top of the peak as a precursor ion candidate;
c) For the fraction sample selected by the fraction sample selection means, one of the precursor ion candidates selected corresponding to the fraction sample by the precursor ion candidate selection means is set as a precursor ion. MS ⁿ analysis execution means for performing MS ⁿ analysis;
and identification means for performing identification of a substance contained in the fraction sample based on the MS ⁿ analysis execution result by d) said MS ⁿ analysis executing means,
A mass spectrometer comprising: The mass spectrometer according to claim 1,
When the substance contained in the fraction sample cannot be identified by the identification means, the MS ⁿ analysis execution means determines another fraction of the precursor ion candidates selected by the precursor ion candidate selection means for the fraction sample. MS ⁿ analysis is performed in which one precursor ion is set, and the identification means attempts to identify a substance contained in the fraction sample based on the MS ⁿ analysis execution result for the different precursor ions. Mass spectrometer. The mass spectrometer according to claim 1 or 2,
After the substance identification for all the fraction samples selected by the fraction sample selection means is completed, the fraction sample selection means decreases the first threshold value within a range larger than the second threshold value, and then peaks. A fraction sample corresponding to the position on the two-dimensional plane where the peak top of the newly extracted peak appears is selected as a fraction sample for MS ⁿ analysis, and the selected fraction sample is A mass spectrometer that performs processing by a precursor ion candidate selection unit, the MS ⁿ analysis execution unit, and the identification unit. The mass spectrometer according to claim 3,
Each time the first threshold value is decreased in a range larger than the second threshold value, the process by the fraction sample selection means, the precursor ion candidate selection means, the MS ⁿ analysis execution means, and the identification means is repeated and identified. A mass spectrometer characterized in that the repetition is terminated at a stage where the number of substances to be saturated is saturated or the degree of increase in the number of newly identified substances is greatly reduced as compared to before.

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