JP6318992B2 - Mass spectrometer - Google Patents

Description

  The present invention relates to a mass spectrometer, and more particularly to a mass spectrometer for identifying proteins, peptides, and the like in a test sample based on data obtained by mass spectrometry.

  In the matrix-assisted laser desorption / ionization (MALDI) method, which is one of the ionization methods in mass spectrometry, a sample (a mixture of a compound to be analyzed and a matrix for ionization assistance) prepared on a sample plate is used. By irradiating the laser beam in a pulsed manner, ions derived from the compound contained in the sample are generated. Ions generated by MALDI-TOFMS, which combines such a MALDI method and time-of-flight mass spectrometer (TOFMS), or ions generated from a MALDI ion source are temporarily held in an ion trap, and ions are dissociated by collision-induced dissociation (CID). MALDI-IT-TOFMS, which is later analyzed by TOFMS, is currently widely used for analyzing biological samples such as proteins, peptides, sugar chains, and lipids.

  In the MALDI method, the amount of ions generated from a sample by one irradiation of laser light is not necessarily large, and the variation in the amount of ions generated every time laser light irradiation is relatively large. Therefore, in the mass spectrometer using the MALDI ion source as described above, the mass analysis for the ions generated by the laser light irradiation is usually repeated a plurality of times, and a predetermined time-of-flight range obtained for each mass analysis (that is, Is integrated with the profile data over the mass-to-charge ratio range), and a mass spectrum for the target sample is obtained from the integrated data (see, for example, Patent Document 1). Therefore, prior to measurement on the sample, the analyst needs to input and set the data integration count as one of the analysis conditions.

  In general, if the number of data integrations is too small, the SN ratio of the mass spectrum is insufficient, and for example, even if an attempt is made to identify a peptide in a sample based on the mass spectrum, the identification accuracy may be lowered. In particular, when the concentration of the target peptide in the sample is low, it is difficult to identify the peptide unless the number of data integration is increased. Even at the same concentration, depending on the amino acid sequence of the peptide, identification may be difficult unless the number of data integration is increased. On the other hand, increasing the number of data integrations improves the quality of the mass spectrum, such as the SN ratio, but increases the measurement time for one sample. When the sample is derived from a living body such as a biological tissue section, if the measurement time is long, the sample may be deteriorated or denatured during the measurement, which may result in an inaccurate result.

  For this reason, it is desirable that the measurement can be performed with an appropriate number of data integrations according to the type and concentration of the target peptide without increasing the number of data integrations more than necessary. However, since the type (amino acid sequence) and concentration of the target peptide are usually unknown, it is not possible to determine the optimal number of data integrations for the sample to be measured prior to measurement. Based on this, an appropriate number of data integrations had to be set.

JP 2010-205460 A US Pat. No. 7,230,235

“MASCOT MS / MS Ions Search”, MATRIX SCIENCE, [August 25, 2014 search], Internet <URL: http://www.matrixscience.com/cgi/search_form.pl?FORMVER= 2 & SEARCH = MIS> Robertson Craig and 1 other, "Tandem: matching proteins with tandem mass spectra (TANDEM), Bioinformatics, Vol.20, No.9, 2004, pp.1466-1467 “Software: QualScore”, Seattle Proteome Center, [Search August 25, 2014], Internet <URL: http://tools.proteomecenter.org/wiki/ index.php? title = Software: QualScore> Hua Xu, and one other, “A Dynamic Noise Level Algorithm for Spectral Screening of Peptide MS (A Dynamic Noise Level Algorithm for Spectral Screening of Peptide MS) / MS Spectra) ”, BMC Bioinformatics, 11: 436, 2010 Ma ZQ and 9 others, "ScanRanker: Quality assessment of tandem mass spectra via sequence tagging", Journal of・ Proteome Research, Vol.10, No.7, 2011, pp.2896-904 Marshall Bern and three others, "Automatic Quality Assessment of Peptide Tandem Mass Spectra", Bioinformatics, Vol.20 ( suppl 1), 2004, pp. 49-54

  The present invention has been made in view of the above problems, and the object of the present invention is to identify the compound according to the type and concentration of a compound such as a peptide to be analyzed contained in a sample. An object of the present invention is to provide a mass spectrometer capable of setting the number of data integrations with little excess or deficiency by determining an appropriate number of data integrations before or during measurement of the sample.

The present invention, which has been made to solve the above problems, performs mass analysis on ions derived from a compound contained in a test sample a plurality of times, and integrates data obtained by each mass analysis. In a mass spectrometer for identifying the compound based on the obtained data,
a) Standard for controlling each part of the apparatus so that the standard compound of known type and concentration in the test sample is subjected to mass analysis a predetermined number of times, and the obtained data is integrated to obtain integrated data for evaluation. A compound measurement control unit;
b) By performing a database search based on the evaluation integrated data obtained under the control of the standard compound measurement control unit, the standard compound is estimated and an index value indicating the accuracy of the estimation is calculated. A standard compound estimator;
c) Based on the success or failure of the compound estimation by the standard compound estimation unit and the index value, an integration number determination unit for determining the appropriate number of data integrations;
It is characterized by having.

  The ionization method and the mass separation method for separating ions according to the mass-to-charge ratio in the mass spectrometer according to the present invention are not particularly limited. For example, as the ionization method, laser desorption without using a matrix in addition to the MALDI method described above. Ionization method (LDI), surface-assisted laser desorption ionization method (SALDI), secondary ion mass spectrometry (SIMS), desorption electrospray ionization method (DESI), electrospray support / laser desorption ionization method (ELDI), etc. May be used. The mass separation method may be a quadrupole mass spectrometer in addition to the time-of-flight mass spectrometer described above.

In addition, the mass spectrometer according to the present invention dissociates ions in one or more stages, such as an ion trap time-of-flight mass spectrometer or a tandem quadrupole mass spectrometer, and masses product ions generated thereby. You may analyze it. In that case, the data obtained by mass spectrometry is MS / MS spectral data or MS ⁿ spectral data (n is an integer of 2 or more)).

In the analysis using the mass spectrometer according to the present invention, for example, a standard compound whose concentration and type are known is added as an internal standard substance to a test sample containing a target compound to be identified. Since the concentration of the standard compound is preferably approximately the same as the concentration of the target compound, when the analyst can predict the concentration of the target compound, the standard compound having the predicted concentration may be used. Then, before executing the measurement for the target compound, the mass analysis for the standard compound is performed a predetermined number of times under the control of the standard compound measurement control unit, and the data obtained by each mass analysis is integrated. The data here is profile data indicating raw signal intensity over a predetermined mass-to-charge ratio range or mass spectrum obtained by waveform processing of the profile data (including the above-mentioned MS / MS spectrum and MS ⁿ spectrum). It is data.

When the integrated data for evaluation is obtained, the standard compound estimation unit estimates a standard compound by executing a database search based on the data. When the target compound or standard compound is a protein or peptide, the database is a database in which amino acid sequences of many proteins and peptides are recorded. For database search, MS / MS ion search, which is one method for estimating the amino acid sequence of a peptide using the mass-to-charge ratio of a product ion obtained from an MS ⁿ spectrum, can be used. As software that uses this method, mascot (MASCOT) and X! TANDEM provided by UK Matrix Science are well known (see Non-Patent Documents 1 and 2).

In MS / MS ion search, all the proteins registered in the protein database are digested with the enzyme used in the pretreatment for actual measurement (actually, one of the search conditions). After calculating the amino acid sequence of the obtained peptide, calculate the mass-to-charge ratio of the theoretical product ion obtained from the amino acid sequence, and agree with the mass-to-charge ratio of the product ion on the MS ⁿ spectrum obtained by actual measurement. Check the degree. In MASCOT, this degree of coincidence is a score, and in X! TANDEM, this degree of coincidence is a value called a hyper score. Furthermore, in X! TANDEM, an expected value (e-value) is calculated based on the hyperscore distribution as an index value that statistically indicates the reliability of the degree of coincidence, and the peptide is identified using this expected value. It can be determined whether or not. Therefore, these scores, hyperscores, expected values, and the like can be used as index values indicating the accuracy of compound estimation.

  Since the standard compound is known, if the compound estimated by the standard compound estimation unit is surely a standard compound, the compound estimation by the database search is successful, otherwise it is a failure. Using the result and the index value obtained with the compound estimation, the integration number determination unit determines the appropriate number of data integrations. For example, if the compound estimation is successful and the index value is equal to or greater than a predetermined threshold, it is determined that the number of data integrations at that time is appropriate, and the number of data integrations during measurement of the target compound is determined. On the other hand, if the compound estimation is unsuccessful or is successful, but the index value is less than a predetermined threshold, it is determined that the SN ratio of the mass spectrum data is insufficient, and the number of data integrations is predetermined. Increase by number.

Even if the number of data integrations is increased by a predetermined number in this way, the SN ratio of the mass spectrum data is not always sufficient for compound estimation in that state.
Therefore, as a preferred embodiment of the mass spectrometer according to the present invention,
When the number of times of data integration is determined based on the success or failure of compound estimation by the standard compound estimation unit and the index value, the number of times of data integration is determined to be insufficient. The compound measurement control unit, the standard compound estimation unit, and the integration number determination unit re-execute the estimation of the compound based on the data that is the result of mass spectrometry at the increased data integration number, and use the result to increase It may be configured to determine the appropriate number of data integrations.

  In this configuration, in order to avoid too many data integration times, the data integration number when the standard compound is first measured, that is, the initial value of the data integration number is kept small, and the data integration number is insufficient. If it is determined, it is preferable to gradually increase the number of data integration. Thereby, it can suppress that the frequency | count of data integration becomes too many more than necessary.

In the mass spectrometer according to the present invention,
d) After the data integration number is determined by the integration number determination unit, the measurement of the target compound in the test sample is performed for the data integration number, and each unit of the apparatus is integrated so that the obtained data is integrated. A target compound measurement control unit for controlling
e) a target compound estimation unit that estimates the target compound by performing a database search based on integrated data obtained under the control of the target compound measurement control unit;
It is good to set it as the structure further provided.
The target compound estimation unit may estimate the target compound in the same manner as the standard compound estimation unit.

  According to this configuration, measurement of the target compound contained in the same test sample can be carried out following the determination of the condition for the number of data integration using the standard compound. As a result, the target compound can be measured under almost the same conditions as in the measurement of the standard compound, for example, the ambient environment, the apparatus state, the sample preparation state, etc., other than the data integration count.

  However, even if the concentrations of the standard compound and the target compound are the same, the optimal number of data integrations is not necessarily the same if the ionization efficiency differs due to the difference in the type of compound (difference in amino acid sequence in the peptide). Don't be.

Therefore, in the mass spectrometer according to the present invention, preferably,
f) a spectral quality evaluation unit for calculating a spectral quality evaluation value based on the mass spectrum obtained for the standard compound;
g) Until the spectrum quality evaluation value calculated based on the mass spectrum obtained for the target compound reaches the value calculated by the spectrum quality evaluation unit, from the data integration number determined by the integration number determination unit And the number of times adjustment unit that further increases the number of times,
And the target compound estimation unit estimates the target compound by performing a database search based on integration data obtained under the data integration count after being adjusted by the integration count adjustment unit, and Good.

  In general database search software such as MASCOT described above, it is described above by evaluating whether product ions such as b series / y series predicted from the amino acid sequence of peptides appear in the mass spectrum. A value indicating the reliability of the identification result, a score, an expected value, and the like are obtained. That is, this evaluation value is a value calculated for the amino acid sequence of a certain peptide, and does not evaluate the quality of the mass spectrum. On the other hand, in the field of proteomics, a method for evaluating the quality of mass spectrum itself is also known.

For example, Patent Document 2 and Non-Patent Document 6 describe a method for evaluating the quality of a spectrum using the relationship between the signal intensity of product ions on the mass spectrum and their rank. These documents describe that this spectral quality evaluation method is effective for preventing the identification of incorrect proteins by using the mass spectrum after measurement for filtering. Non-patent documents 3 to 5 also describe other methods for similarly evaluating the quality of the spectrum. In order to evaluate the spectrum quality, non-patent document 3 uses an MS / MS spectrum feature amount, non-patent document 4 uses an algorithm that dynamically calculates an appropriate SN ratio, Each distribution of signal strength is used.
In the above structure, spectral quality evaluation unit and accumulated number adjustment unit, for example, using any of these techniques, it is possible to determine a spectral quality evaluation value for the mass spectrum of the standard compound.

  For example, when the concentration of the target compound is lower than the concentration of the standard compound, or when the target compound is difficult to ionize compared to the standard compound, the spectrum calculated based on the mass spectrum obtained for the target compound The quality evaluation value is lower than the value calculated by the spectrum quality evaluation unit. In that case, even if the mass spectrum for the target compound at that time is used, the possibility that the target compound is correctly identified is low. Therefore, the integration number adjusting unit increases the quality of the mass spectrum by increasing the number of times further than the number of data integrations determined by the integration number determination unit. This increases the possibility that the target compound can be identified with the minimum number of data integrations according to the type and concentration of the target compound. As a result, as described above, even when the concentration of the target compound is lower than the concentration of the standard compound or when the target compound is less ionized than the standard compound, the target compound cannot be identified. Can be avoided.

  The mass spectrometer according to the present invention can reduce compound identification failures due to insufficient number of data integration. In addition, since data integration can be reduced more than necessary, the measurement time can be shortened, and it is effective in reducing the deterioration and denaturation of the sample due to the time required. Furthermore, since it is not necessary for the analyst himself to set the number of data integrations based on past experience or the like, the burden on the analyst is reduced.

The block diagram of the principal part of the mass spectrometer which is 1st Example of this invention. The flowchart of an example of the characteristic mass spectrometry operation implemented in the mass spectrometer of 1st Example. The block diagram of the principal part of the mass spectrometer which is 2nd Example of this invention. The flowchart of an example of the characteristic mass spectrometry operation implemented in the mass spectrometer of 2nd Example.

  First, a mass spectrometer that is a first embodiment of the present invention will be described with reference to the accompanying drawings. FIG. 1 is a configuration diagram of a main part of the mass spectrometer of the first embodiment.

In the mass spectrometer of the present embodiment, the mass analyzer 1 that performs measurement on a sample and collects data includes a MALDI ion source 11, an ion trap 12, and a time-of-flight mass spectrometer (TOFMS) 13. .
The MALDI ion source 11 irradiates a sample prepared on the sample plate with a laser beam in a pulsed manner, thereby ionizing a compound contained in the sample.
The ion trap 12 is, for example, a three-dimensional quadrupole type ion trap. After temporarily holding the ions by the action of an electric field, the ions having a specific mass-to-charge ratio are selected, and the selected ions are further subjected to collision-induced dissociation. To produce product ions.
The TOFMS 13 separates and detects ions ejected from the ion trap 12 substantially simultaneously at a predetermined timing according to the mass-to-charge ratio. In this way, in the mass spectrometer 1, the mass spectrum (MS ⁿ spectrum) for the product ion generated by cleaving ions derived from a specific compound in the sample, that is, profile data that is the source of the product ion spectrum Can be obtained.

  The control / processing unit 2 controls the operation of the mass analysis unit 1 and processes data obtained by the mass analysis unit 1. The data integration processing unit 21, mass spectrum creation unit 22, database (DB) A search unit 23, a protein sequence database (DB) 24, an identification result evaluation unit 25, an integration number adjustment unit 26, and an analysis control unit 27 are included. The control / processing unit 2 is connected to an input unit 3 operated by an analyst and a display unit 4 for displaying analysis results and the like.

Here, the database search unit 23 can use, for example, database search software such as MASCOT or X! TANDEM described above. As the protein sequence database 24, a publicly available database such as UniProt (Swiss-Prot) can be used.
It should be noted that at least some of the functions of the control / processing unit 2 can be realized by operating dedicated control / processing software installed in a personal computer on the computer.

FIG. 2 is a flowchart of an example of a characteristic mass analysis operation performed in the mass spectrometer of the first embodiment.
The analyst estimates the concentration (content) of the target peptide to be identified, and adds a known standard peptide of the estimated concentration to the target peptide to prepare a test sample for MALDI. Therefore, the test sample formed on the sample plate contains the target peptide and the standard peptide. Here, this standard peptide is used as an internal standard substance.

  Before the measurement is executed, measurement parameters including the number of data integrations and the laser beam power are initialized (step S1). These measurement parameters may be set by the analyst himself / herself from the input unit 3, or in the case where there is no such input, a predetermined default value may be automatically set.

  When measurement is started, first, the analysis control unit 27 controls each unit of the mass analysis unit 1 so as to perform MS / MS analysis on a standard peptide in a test sample. Specifically, the MALDI ion source 11 generates ions by irradiating a test sample with laser light, and the ions are temporarily held in the ion trap 12. Since the mass of the standard peptide is known, a precursor ion is selected so that molecular ions derived from the standard peptide are selectively left, and the product ions are generated by dissociating the ions with CID. Then, the product ions are ejected from the ion trap 12 and introduced into the TOFMS 13, and separated and detected for each flight time depending on the mass-to-charge ratio. In the mass spectrometer 1, such measurement is repeated as many times as the initial value of the number of data integration. For example, when the initial value of the number of data integration is “5”, the MS / MS analysis for the standard peptide is repeated five times in the mass analyzer 1.

  The data integration processing unit 21 obtains integration profile data by adding all the profile data (raw data before waveform processing) obtained by the initial number of data integration times as described above. The mass spectrum creation unit 22 creates a mass spectrum (MS / MS spectrum) for the standard peptide by performing, for example, noise removal, smoothing, centroid processing, etc. on the integrated profile (step S2).

  The database search unit 23 performs peak detection on the created mass spectrum to create a peak list, and performs a database search with reference to the protein sequence database 24 based on the peak list to execute peptide identification processing. By performing a database search, the amino acid sequence of the peptide is estimated, and a score and an expected value indicating its reliability are calculated (step S3).

  Next, the identification result evaluation unit 25 determines whether or not the identification is successful by determining whether or not the peptide estimated in step S3 is a known standard peptide. Furthermore, when it is determined that the identification is successful, it is determined whether or not the score and the expected value calculated for the peptide are each equal to or greater than a predetermined threshold (step S4). If the standard peptide has not been correctly identified, the identification has failed. If the identification has failed, the process proceeds from step S4 to S5. Even if the identification is successful, if either the score or the expected value does not reach the predetermined threshold, the quality of the mass spectrum used for the database search, specifically, the SN ratio may be too low. is there. Therefore, also in this case, the process proceeds from step S4 to S5. Then, the integration number adjustment unit 26 increases the data integration number from the value at that time by a predetermined number, for example, “1” (step S5), and returns to step S2. This predetermined number may be an appropriate value other than “1”.

  When returning from step S5 to S2, the analysis control unit 27 that has received notification from the integration number adjusting unit 26 that the number of data integrations has been increased by a predetermined number applies to the standard peptide in the test sample by the increased number of times. The mass spectrometer 1 is controlled to additionally perform MS / MS analysis. Then, the data integration processing unit 21 adds the predetermined number of newly acquired profile data to the previous integration profile data, thereby acquiring integration profile data corresponding to the increased number of data integrations. Then, a new mass spectrum is obtained by performing waveform processing on the integrated profile data. At this time, since the number of data integration increases, the SN ratio of the obtained mass spectrum should be improved. Thereby, when the process of step S3 is continued, the peptide identification accuracy is increased, and the score and the expected value are also improved. As a result, the probability of being determined as Yes in the determination process in step S4 increases.

  In step S4, when it is determined that the identification is successful and the score and the expected value are both greater than or equal to a predetermined threshold value, the integration number adjusting unit 26 measures the data integration number set at that time when measuring the target peptide. The parameter is determined as a parameter, and the analysis control unit 27 controls the mass analysis unit 1 so as to execute MS / MS analysis on the target compound in the test sample according to the measurement parameter. Therefore, for example, when the number of data integration is “10”, if it is determined as Yes in step S4, the MS / MS analysis for the target compound in the test sample is repeated 10 times. The profile data obtained in each of the 10 MS / MS analyzes is integrated. Then, the mass spectrum creation unit 22 performs waveform processing on the integrated profile data to obtain a mass spectrum for the target peptide (step S6).

  The database search unit 23 performs peak detection on the created mass spectrum of the target peptide to create a peak list, and performs a database search with reference to the protein sequence database 24 based on the peak list. Thereby, the amino acid sequence for the target peptide is estimated, and the candidate peptide is output as the identification result (step S7).

  As described above, in the mass spectrometer of the first embodiment, if the concentration of the target peptide is about the same as that of the standard peptide and the ionization efficiency is also about the same as that of the standard peptide, the target peptide is identified. It is possible to automatically determine the number of data integrations that are almost not excessive or insufficient. As a result, it is possible to avoid an unnecessarily large number of measurements while avoiding the target peptide from failing in identification.

  Next, a mass spectrometer that is a second embodiment of the present invention will be described with reference to the accompanying drawings. FIG. 3 is a configuration diagram of a main part of the mass spectrometer of the second embodiment, and FIG. 4 is a flowchart of an example of a characteristic mass analysis operation performed in the mass spectrometer of the second embodiment.

In FIG. 3, the same components as those of the first embodiment shown in FIG. In the mass spectrometer of the second embodiment, a spectrum quality evaluation unit 28 for evaluating the quality of the mass spectrum created by the mass spectrum creation unit 22 is added to the mass spectrometer of the first embodiment. The evaluation result by the quality evaluation unit 28 is input to the integration number adjusting unit 26.
With reference to FIG. 4, the characteristic mass analysis operation performed in the mass spectrometer of the second embodiment will be described. Steps S11 to S15 in the flowchart shown in FIG. 4 are exactly the same as steps S1 to S5 in the flowchart shown in FIG. That is, the number of times of data integration is determined based on the identification process using the result of measuring the standard peptide contained in the test sample by the processes of steps S11 to S15. In the first embodiment, MS / MS analysis is performed on the target peptide using the determined data integration count as a measurement parameter. In the second embodiment, the data integration count is further adjusted.

That is, when it is determined Yes in step S14, the spectrum quality evaluation unit 28 calculates the spectrum quality evaluation value as a reference value from the MS / MS spectrum for the standard peptide obtained at that time (step S16). The method for calculating the spectrum quality evaluation value is not particularly limited as long as it can reflect the S / N ratio of the mass spectrum, but if any of the methods described in Patent Document 2, Non-Patent Documents 3 to 6, etc. is used. Good. In any case, since these methods evaluate the quality of the mass spectrum itself, it is not directly related to the score or expected value calculated during database search, and even if the score or expected value is high, the spectral quality It is possible that the evaluation value is low.
The spectral quality evaluation value calculated in this way is a spectral quality evaluation value when a standard peptide at a predetermined concentration is accurately identified and the score and expected value at that time are equal to or higher than a predetermined threshold.

  After calculation of the spectral quality evaluation value, in step S17 similar to step S6 in FIG. 2, the MS / MS analysis for the target compound in the test sample is repeated for the number of data integration finally determined in step S15. The data integration processing unit 21 calculates integration profile data, and the mass spectrum creation unit 22 obtains an MS / MS spectrum for the target peptide from the integration profile data.

  Next, the spectrum quality evaluation unit 28 calculates a spectrum quality evaluation value from the MS / MS spectrum for the target peptide by the same method as in step S16 (step S18). Then, the calculated spectral quality evaluation value for the target peptide is compared with the spectral quality evaluation value for the standard peptide, that is, the reference value, and it is determined whether or not the reference value has been reached (step S19). If the spectral quality evaluation value for the target peptide has reached the reference value, it is determined that the data integration count at that time is sufficient, and the process proceeds to step S21. Step S21 is the same processing as step S7 in FIG. 2, and the database search unit 23 performs peak detection on the MS / MS spectrum of the target peptide to create a peak list, and a protein sequence database 24 based on this peak list. Perform database search referring to. Thereby, the amino acid sequence for the target peptide is estimated, and the candidate peptide is output as the identification result.

  If it is determined in step S19 that the spectral quality evaluation value for the target peptide has not reached the reference value, the target peptide may not be correctly identified even if a database search using the MS / MS spectrum at that time is performed. High nature. Therefore, the process proceeds from step S19 to S20, and the integration number adjusting unit 26 increases the data integration number by a predetermined number, for example, “1” from the value at that time, and returns to step S17. That is, the number of data integration is once more increased than the number of times of data integration once determined based on the measurement result of the standard peptide, and the MS / MS analysis for the target peptide is additionally performed. Thus, the profile data obtained by the additionally performed MS / MS analysis is further added to the integrated profile data. Therefore, after returning from step S20 to S17, the spectrum quality evaluation value calculated in step S18 should be higher than the spectrum quality evaluation value at the previous time point.

Then, by repeating steps S17 to S20, the number of data integration is increased until the spectral quality evaluation value of the MS / MS spectrum for the target peptide reaches the reference value, and the spectral quality evaluation value of the MS / MS spectrum for the target peptide is increased. When the reference value is reached, peptide identification using the MS / MS spectrum at that time is attempted.
If the concentration of the target peptide in the test sample is lower than the concentration of the standard peptide, or if the target peptide is difficult to ionize (low ionization efficiency) even if the concentration is the same, it is based on the standard peptide. In some cases, the quality of the MS / MS spectrum, such as the signal-to-noise ratio, is not sufficient with the determined number of data integrations. Even in that case, according to the configuration of the second embodiment, the number of data integration can be further increased, and peptide identification by database search can be performed in a state where the quality of the MS / MS spectrum for the target peptide is sufficiently high. it can. As a result, an appropriate number of data integrations can be set according to the concentration and type (amino acid sequence) of the target peptide, and the excess or deficiency of the number of data integrations can be further reduced.

  In the first and second embodiments, the standard peptide is added to the test sample to be used as the internal standard. However, the standard peptide is placed at a different position on the sample plate separately from the test sample containing the target peptide. A test sample may be measured after preparing a standard sample containing the sample and measuring the standard sample.

  In the above-described examples, the case where the compound is a peptide has been described. However, the target compound is not limited to a peptide, and other biologically derived compounds such as sugars and lipids and other compounds may be used as a database. Any compound that can be identified by searching may be used.

In the present invention, the ion source is not limited to the MALDI ion source, and the mass separation unit is not limited to the TOFMS.
Furthermore, the above-described embodiment is an example of the present invention, and not only the above-described various modifications, but also modifications, corrections, and additions as appropriate within the scope of the present invention are included in the scope of the claims of the present application. It is natural.

DESCRIPTION OF SYMBOLS 1 ... Mass spectrometer 11 ... MALDI ion source 12 ... Ion trap 13 ... Time-of-flight mass spectrometer (TOFMS)
2 ... Control / Processing Unit 21 ... Data Integration Processing Unit 22 ... Mass Spectrum Creation Unit 23 ... Database (DB) Search Unit 24 ... Protein Sequence Database (DB)
25 ... Identification result evaluation unit 26 ... Integration number adjustment unit 27 ... Analysis control unit 28 ... Spectral quality evaluation unit 3 ... Input unit 4 ... Display unit

Claims (4)

Mass spectrometry that identifies a compound based on data obtained by performing mass spectrometry on ions derived from a compound contained in a test sample a plurality of times and integrating the data obtained by each mass analysis. In the device
a) Standard for controlling each part of the apparatus so that the standard compound of known type and concentration in the test sample is subjected to mass analysis a predetermined number of times, and the obtained data is integrated to obtain integrated data for evaluation. A compound measurement control unit;
b) By performing a database search based on the evaluation integrated data obtained under the control of the standard compound measurement control unit, the standard compound is estimated and an index value indicating the accuracy of the estimation is calculated. A standard compound estimator;
c) Based on the success or failure of the compound estimation by the standard compound estimation unit and the index value, an integration number determination unit for determining the appropriate number of data integrations;
A mass spectrometer comprising: The mass spectrometer according to claim 1,
When the number of times of data integration is determined based on the success or failure of compound estimation by the standard compound estimation unit and the index value, the number of times of data integration is determined to be insufficient. The compound measurement control unit, the standard compound estimation unit, and the integration number determination unit re-execute the estimation of the compound based on the data that is the result of mass spectrometry at the increased data integration number, and use the result to increase And determining the appropriate number of times of data integration. The mass spectrometer according to claim 2,
d) After the data integration number is determined by the integration number determination unit, the measurement of the target compound in the test sample is performed for the data integration number, and each unit of the apparatus is integrated so that the obtained data is integrated. A target compound measurement control unit for controlling
e) a target compound estimation unit that estimates the target compound by performing a database search based on integrated data obtained under the control of the target compound measurement control unit;
A mass spectrometer further comprising: The mass spectrometer according to claim 3 ,
f) a spectral quality evaluation unit for calculating a spectral quality evaluation value based on the mass spectrum obtained for the standard compound;
g) Until the spectrum quality evaluation value calculated based on the mass spectrum obtained for the target compound reaches the value calculated by the spectrum quality evaluation unit, from the data integration number determined by the integration number determination unit And the number of times adjustment unit that further increases the number of times,
The target compound estimation unit estimates the target compound by performing a database search based on the integration data obtained under the data integration count after being adjusted by the integration count adjustment unit. Characteristic mass spectrometer.

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