JP2015121500A - Mass spectroscopy method and mass spectroscope apparatus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To improve an accuracy of peptide identification based on a mass spectrum obtained by MALDI-ISD analysis without performing MSanalysis.SOLUTION: A mass spectroscopy method includes: analyzing a sample obtained by adding a reference material to peptide as analysis object and preparing the mixture thereof using a matrix, and a reference sample obtained by only preparing the same reference material using the matrix, by MALDI-ISD analysis respectively; performing mass calibration to each ISD mass spectrum using ion peak positions of the reference material and the matrix and known mass of the materials (S1-S6); and acquiring a difference mass spectrum by subtracting the ISD mass spectrum of the reference sample from the ISD mass spectrum of the sample (S7). Since ion peaks derived from the reference material and the matrix are removed, the fragment ion peak of an analysis object compound can be easily observed. The mass spectroscopy method further includes identifying the peptide by serving the peak information for database retrieval(S8-S9), thereby resulting in improved identification accuracy.

Description

  The present invention relates to a mass spectrometry method using a mass spectrometer equipped with an ion source by a matrix-assisted laser desorption / ionization (MALDI) method, and a mass spectrometer for carrying out the method. More specifically, the present invention relates to a mass spectrometry method and a mass spectrometer suitable for protein and peptide identification and structural analysis.

  In recent years, mass spectrometric techniques involving ion dissociation operations have been actively used for identification and structural analysis of macromolecular compounds derived from living bodies such as proteins, peptides, sugar chains, and nucleic acids. As a technique for dissociating ions, collision-induced dissociation (CID = Collision Induced Dissociation) using an ion trap or a collision cell is often used. However, depending on the type of ion source, in-source decomposition (ISD = In-Source Decay) may be used. In-source decomposition is cleavage occurring in the ionization chamber or the like simultaneously with ionization or immediately after ionization, and well-known is the cleavage of molecular ions during ionization by electron ionization (EI).

Ionization by the MALDI method is generally said to be soft ionization, and it is difficult to cause cleavage at the time of ionization. For example, by increasing the energy at the time of ionization, for example, by increasing the power of laser light applied to the sample, the generated ions It is known that the cleavage of is promoted. In-source decomposition in a MALDI ion source, particularly for proteins and peptides, cleaves the N-Cα bond of the peptide main chain by hydrogen radicals generated from the matrix by laser light irradiation, and c-series ions and z-series It is known that ions are mainly produced. Using such a phenomenon, an analysis of estimating an amino acid sequence of a peptide by analyzing a mass spectrum obtained by in-source decomposition in a MALDI time-of-flight mass spectrometer (MALDI-TOFMS) has been performed.
In the following description, a mass spectrometry method using in-source decomposition in a MALDI ion source is referred to as MALDI-ISD analysis. A mass spectrum obtained by MALDI-ISD analysis is referred to as an ISD mass spectrum.

As described above, MALDI-ISD analysis is an analytical technique useful for identification and structural analysis of proteins and peptides with large molecular weights, but generally in the low mass-to-charge ratio range where many ions generated by in-source decomposition appear. Many ions derived from foreign substances also appear and are often difficult to analyze as they are. Therefore, in the apparatus described in Patent Document 1, one of the ions that appears in the MALDI-ISD analysis is selected as a precursor ion, and MS ² analysis is performed on the ion. In the MS ² spectrum thus obtained, peaks derived from contaminants such as matrix are removed, while an ion peak sequence reflecting the amino acid sequence appears in the low mass to charge ratio range. The amino acid sequence of the peptide can be estimated.

However, since the intensity of ions appearing by MALDI-ISD analysis is generally low, in order to perform MS ² analysis using this as a precursor ion, the concentration of the sample is increased so that a certain level of ion intensity can be obtained. It is necessary to keep. Further, even if the influence of ions derived from impurities is excluded, the intensity of the fragment ion peak appearing in the low mass-to-charge ratio range in the MS ² spectrum is quite low, so it is not always easy to obtain amino acid sequence information from this.

In addition, ion species appearing in MALDI-ISD analysis are mainly c-series and z-series, whereas fragment ion species appearing in MS ² analysis are mainly b-series and y-series ions. Is different. Therefore, in order to estimate the amino acid sequence, it is not possible to adopt a method in which both mass spectra are integrated and a predetermined data analysis method is applied, and the results of independent data analysis for each mass spectrum are integrated. It is necessary and the analysis work is complicated.

Further, since it is necessary to perform MS ² analysis after performing MALDI-ISD analysis, the measurement work takes time. In addition, in order to perform MS ² analysis, a reflectron TOFMS (a device capable of at least time-of-flight mass spectrometry in the reflectron mode) that is more expensive than a linear TOFMS is required. The analysis as described above cannot be performed.

  In addition, such circumstances include not only a mass spectrometer using a MALDI ion source but also a surface-assisted laser desorption / ionization (SALDI) method in which ionization is similarly performed using laser light. The same applies to mass spectrometers using other laser desorption ionization methods. In the SALDI method, a sample is prepared on a plate coated with inorganic nanoparticles such as silver (Ag), gold (Au), copper (Cu), and platinum (Pt), which absorbs the energy of the laser beam, and the laser is applied to this. This is an ionization method in which components in a sample are ionized by irradiating light. Although a matrix is not used in the SALDI method, a peak derived from a contaminant due to the surface of the SALDI plate is often detected in a low mass-to-charge ratio range, which makes it difficult to analyze an ISD mass spectrum.

US Pat. No. 7,396,686

“Development of Next Generation Mass Spectrometry System and Contribution to Drug Discovery and Diagnosis Public Software Mass ++ (Ver.2)”, Advanced Research and Development Support Program, [November 19, 2013 Search], Internet <URL: http: / /www.first-ms3d.jp/achievement/software> Daiki Asakawa and three others, “In-Source Decay Duaring Matrix-Assisted Laser Desorption / Ionization Combined With The Collegiate Process in an FT In-Source Decay during Matrix-Assisted Laser Desorption / Ionization Combined with the Collisional Process in an FTICR Mass Spectrometer ”, Analytical Chemistry, Vol.85 (16), 2013, pp.7809? 7817 “What is suitable as a detector for MALDI-TOFMS?”, Shimadzu Corporation, [December 13, 2013 search], Internet <URL: http://www.first-ms3d.jp/files /MALDI-MS_TechRep/MALDI_TechRepV3.0_05.pdf>

The present invention has been made in view of the above problems, and its object is to identify ions appearing in MALDI-ISD analysis, SALDI-ISD analysis, and the like when identifying peptides or proteins and conducting structural analysis. Provided is a mass spectrometry method capable of estimating an amino acid sequence of a peptide with high accuracy using an ISD mass spectrum without performing MS ² analysis using ions, and a mass spectrometer for performing the method That is.

The mass spectrometric method according to the present invention, which has been made to solve the above problems, is a mass spectrometric method for analyzing a polymer compound using a mass spectroscope equipped with an ion source based on laser desorption ionization. And
a) a sample preparation step of preparing a sample by adding a predetermined standard substance to a sample containing a compound to be analyzed;
b) a reference sample preparation step for preparing a reference sample with the standard substance;
c) a mass spectrum acquisition step of acquiring mass spectra by performing mass spectrometry using in-source decomposition on the sample and the reference sample, respectively,
d) Based on the position of the ion peak derived from the standard substance appearing in the mass spectrum relative to the sample and the reference sample and the known mass-to-charge ratio of the standard substance, the mass-to-charge ratio deviation in each mass spectrum is corrected. A mass calibration step;
e) a subtraction processing step of subtracting the mass spectrum for the reference sample, which is also corrected for the mass-to-charge ratio deviation in the mass calibration step, from the mass spectrum for the sample in which the mass-charge ratio deviation is corrected in the mass calibration step;
f) an analysis processing step for performing analysis of the analysis target compound based on the difference mass spectrum obtained in the subtraction processing step;
It is characterized by having.

A mass spectrometer according to the present invention made to solve the above problems is a mass spectrometer used in the mass spectrometry method according to the present invention, and includes a mass provided with an ion source by laser desorption ionization. In the analyzer
a) Mass spectrometry using in-source decomposition was performed on a sample prepared by adding a predetermined standard substance to a sample containing the compound to be analyzed and a reference sample prepared from the standard substance. A measurement execution unit for acquiring a mass spectrum;
b) Based on the position of the ion peak derived from the standard substance appearing in the mass spectrum relative to the sample and the reference sample and the known mass-to-charge ratio of the standard substance, the mass-to-charge ratio deviation in each mass spectrum is corrected. A mass calibration unit;
c) a subtraction processing unit that subtracts a mass spectrum for a reference sample, which is also corrected for a mass-to-charge ratio deviation by the mass calibration unit, from a mass spectrum for the sample in which the mass-to-charge ratio deviation is corrected by the mass calibration unit;
d) an analysis processing unit that performs analysis of the analysis target compound based on the difference mass spectrum obtained by the subtraction processing unit;
It is characterized by having.

  In the mass spectrometry method and the mass spectrometer according to the present invention, as the laser desorption ionization method, for example, a matrix-assisted laser desorption ionization (MALDI) method or a surface-assisted laser desorption ionization method can be used. When the MALDI method is used, an appropriate matrix is used when preparing the sample and the reference sample.

  In the mass spectrometry method and mass spectrometer according to the present invention, the analysis target compound is typically a protein or a peptide. In that case, in the analysis processing step in the mass spectrometry method according to the present invention, identification or structural analysis of protein or peptide is performed by a technique such as database search such as de novo sequencing or MS / MS ion search.

  In the mass spectrometry method according to the present invention, when a mass spectrometer equipped with a MALDI ion source is used, the sample and the reference sample include the same standard substance and matrix, while the sample includes the analysis target compound and the reference sample includes the analysis target. Contains no compounds. Therefore, the mass spectrum obtained by MALDI-ISD analysis of the sample includes the fragment ion peak generated by in-source decomposition of the target compound, the ion peak derived from the standard substance, and the ion peak derived from the matrix. Appears. On the other hand, in the mass spectrum obtained by MALDI-ISD analysis of the reference sample, an ion peak derived from a standard substance and an ion peak derived from a matrix appear. By performing mass calibration of each mass spectrum in the mass calibration step, the mass-to-charge ratio of the same kind of ion peak in both mass spectra becomes substantially equal. Therefore, in the subtraction process step, subtracting the mass spectrum that has been mass calibrated for the reference sample from the mass spectrum that has been calibrated for the sample, the ion peak derived from the standard substance or the ion peak derived from the matrix common to both mass spectra, that is, Removes peaks derived from contaminants not related to the compound to be analyzed.

  In particular, matrix-derived ion peaks often appear in the low mass-to-charge ratio range with a high signal intensity and easily overlap with the mass-to-charge ratio range of the fragment ion peak of the compound to be analyzed, but these unwanted peaks are eliminated. Thus, it becomes possible to clearly observe the fragment ion peak of the analysis target compound having a relatively low signal intensity. As a result, accurate mass information of various fragments generated by cleavage of the analysis target compound can be used for analysis such as database search, and the analysis target compound can be identified and its structure estimated with high accuracy. be able to.

  In addition, as described above, in a mass spectrometer equipped with a SALDI ion source, a peak derived from a contaminant due to the SALDI plate surface may be detected in a low mass-to-charge ratio range. Then, an undesired peak due to the surface of the sample plate can also be removed, whereby the fragment ion peak of the analysis target compound can be clearly observed.

  In the mass spectrometry method according to the present invention, it is desired to observe the peak of the fragment ion of the compound to be analyzed. If the standard substance is a compound that is easily cleaved by in-source decomposition, the position of the ion peak derived from the standard substance is used. In addition to hindering the mass calibration, the fragment ion peak derived from the standard substance may overlap the fragment ion peak of the compound to be analyzed, and the fragment ion peak derived from the standard substance may not be sufficiently removed. Therefore, it is preferable to use, as a standard substance, a peptide chain in which no fragment ions appear by a laser desorption ionization method such as MALDI method. Specifically, using the fact that cleavage by laser desorption ionization tends not to occur on the N-terminal side of a proline residue, a synthetic peptide containing a polymer structure with a series of proline residues is used as a standard substance. Good.

  Further, in a laser desorption ion source such as MALDI, ion generation efficiency often varies with each laser beam irradiation. For this purpose, the final measurement result is usually obtained by repeating the irradiation of the pulsed laser beam many times and integrating the ion intensity obtained for each laser beam irradiation. However, even if such integration processing is performed, when MALDI-ISD analysis (or SALDI-ISD analysis) on the sample and MALDI-ISD analysis (or SALDI-ISD analysis) on the reference sample are performed with the same laser light power In the mass spectrum, the peak intensity for the same type of ions in the mass spectrum is not necessarily the same, and even if the subtraction process is performed, the ion peak derived from the matrix and the ion peak derived from the standard substance are not completely removed. May end up.

  Therefore, in the mass spectrometry method according to the present invention, the ionization efficiency at the time of execution of MALDI-ISD analysis (or SALDI-ISD analysis) for the reference sample is more than the ionization efficiency at the time of execution of MALDI-ISD analysis (or SALDI-ISD analysis) of the sample. In order to increase the laser light power, the MALDI-ISD analysis (or SALDI-ISD analysis) on the reference sample may be performed at a higher laser light power than the MALDI-ISD analysis (or SALDI-ISD analysis) on the sample. Good. Of course, if the laser beam power is increased too much, the sample may be damaged only and the ion generation efficiency may be lowered. Therefore, it is desirable to examine the appropriate increase amount of the laser beam power in advance. Thereby, for example, a mass spectrum (ISD mass spectrum) in which an ion peak derived from a matrix or an ion peak derived from a standard substance is sufficiently removed can be obtained with certainty.

  Further, for example, for identification of peptides by database search or the like, it is necessary to provide mass information with high accuracy for database search. In addition, it is necessary to detect ions in a wide mass-to-charge ratio range including a low mass-to-charge ratio range. Therefore, as a mass spectrometer used in the mass spectrometry method according to the present invention, a laser desorption ionization time-of-flight mass spectrometer equipped with a saturation resistance detector, for example, a MALDI time-of-flight mass spectrometer (MALDI-TOFMS) Is preferably used.

  MALDI quadrupole ion trap time-of-flight mass spectrometer (MALDI-QIT-TOFMS) using ion trap and MALDI quadrupole time-of-flight mass spectrometer (MALDI-Q-TOFMS) equipped with quadrupole mass filter However, MALDI-ISD analysis of high-mass molecules such as proteins is difficult due to the limitations of the mass-to-charge ratio range in ion traps and quadrupole mass filters. On the other hand, in MALDI-TOFMS, since the measurable mass-to-charge ratio range is very wide, a good ISD mass spectrum suitable for analysis can be acquired.

  In the mass spectrometry method according to the present invention, mass calibration is performed using an ion peak derived from a standard substance. When the MALDI method is used, an ion peak derived from a matrix can also be used for mass calibration. That is, as a preferred embodiment of the mass spectrometry method according to the present invention, when a mass spectrometer equipped with a MALDI ion source is used, in the mass calibration step, an ion peak derived from a standard substance appearing in a mass spectrum for a sample. Further, based on the position of the ion peak derived from the matrix and the known mass-to-charge ratio of the standard substance and the matrix, the deviation of the mass-to-charge ratio in each mass spectrum may be corrected. Thereby, mass calibration with higher accuracy can be performed.

  According to the mass spectrometric method and the mass spectroscope according to the present invention, peaks derived from contaminants not related to the analysis target compound are removed particularly in the low mass to charge ratio range of the ISD mass spectrum. The fragment ion peak derived from the target compound to be analyzed can be clearly observed. Thus, accurate mass information of various fragments of the analysis target compound can be used for processing for identification and structural analysis, so that the accuracy of such analysis can be improved. Further, even if the signal intensity of fragment ions derived from the analysis target compound is relatively low, information on these ion peaks can be obtained with high accuracy, so there is no need to increase the concentration of the sample.

Further, since it is not necessary to perform MS ² analysis again after execution of MALDI-ISD analysis or SALDI-ISD analysis as in the past, the time required for measurement can be shortened, and the analysis throughput is improved. In addition, since an ion cleavage operation using CID or the like is not required, sufficient analysis can be performed with a relatively inexpensive mass spectrometer such as MALDI-linear TOFMS that does not include an ion trap or a collision cell. Furthermore, since it is not necessary to integrate the analysis result based on the ISD mass spectrum and the analysis result based on the MS ² spectrum, the analysis process itself is easy.

The schematic block diagram of one Example of MALDI-TOFMS for enforcing the mass spectrometry method concerning this invention. The flowchart which shows an example of the procedure of the peptide identification using MALDI-TOFMS of a present Example. The schematic of the mass spectrum for demonstrating the peptide identification shown in FIG. The figure which shows the ISD mass spectrum by actual measurement of a standard substance (peptide P14R) and an analysis object compound (ACTH18-39). The ISD mass spectrum (a) of ACTH18-39 before execution of background subtraction processing and the ISD mass spectrum (b) of ACTH18-39 after execution of background subtraction processing. The figure which shows the MS / MS ion search execution result based on the ISD mass spectrum before background subtraction process execution. The figure which shows the MS / MS ion search execution result based on the ISD mass spectrum after background subtraction process execution.

Hereinafter, an embodiment of a mass spectrometry method according to the present invention and a MALDI-TOFMS for carrying out the mass spectrometry method will be described with reference to the accompanying drawings.
FIG. 1 is a schematic configuration diagram of a MALDI-TOFMS according to the present embodiment. First, the configuration and basic operation of this MALDI-TOFMS will be described.

  The MALDI ion source 1 includes a stage 11 on which the sample plate 10 is set, an extraction electrode 13, an acceleration electrode 14, a laser irradiation unit 15, and a reflecting mirror 16. On the sample plate 10, a sample 12a containing an analysis target compound and a predetermined internal standard substance and prepared using a predetermined matrix, and a reference sample containing the same internal standard substance and prepared using the same matrix 12b is held. Here, the compound to be analyzed is a peptide or a protein.

Under the control of the control unit 3, the laser light emitted in a pulse form from the laser irradiation unit 15 is focused on a minute region on the sample 12 a located at a predetermined measurement position via the reflecting mirror 16. The analysis target compound and the internal standard substance are vaporized together with the matrix from the sample 12a by the energy of the laser light, and the analysis target compound and the internal standard substance are ionized at that time. When performing MALDI-ISD analysis, the power of the laser beam is increased as compared with the normal analysis (that is, analysis without in-source decomposition), and the ions are cleaved at the same time or immediately after the analysis target compound is ionized. To promote.
In FIG. 1, the sample 12a is positioned at the measurement position where the laser beam strikes. However, the stage 11 can be moved in two directions of the X axis and the Y axis in FIG. By moving the sample 12b to the measurement position, the reference sample 12b can be irradiated with laser light to measure the reference sample 12b.

  Various ions generated in the vicinity of the surface of the sample 12a (or the reference sample 12b) by laser light irradiation are space between the two by a voltage applied to the extraction electrode 13 and the sample plate 10 from a voltage source (not shown). It is extracted by the action of the electric field formed in Then, the extracted ions start to fly with acceleration energy applied by an acceleration electric field formed in a space between the extraction electrode 13 and the acceleration electrode 14 by a voltage applied to the extraction electrode 13 and the acceleration electrode 14, respectively.

  The TOF unit 2 includes a flight space 21 formed inside the flight tube 20 and a detector 23 that detects ions and outputs a detection signal corresponding to the amount of ions. The detector 23 uses an electron multiplier having saturation resistance. In the mass spectrometry method and apparatus according to the present invention, it is desirable that ions on the low mass-to-charge ratio side can be detected with sufficient sensitivity in order to obtain a good ISD mass spectrum. Therefore, it is preferable to use an electron multiplier having saturation resistance as the detector. As an electron multiplier having saturation resistance, for example, a magnetic ion detector such as Magne TOF manufactured by SGE, or a particle having a kinetic energy exceeding a certain threshold described in Non-Patent Document 3 collides. An improved secondary electron multiplier comprising a combination of a first dynode that emits electrons and a second dynode that emits and multiplies secondary electrons when the emitted electrons collide with each other Etc. can be used.

  As described above, various ions accelerated by the MALDI ion source 1 and sent to the TOF unit 2 fly in a substantially linear shape in the flight space 21 and reach the detector 23. At the time of introduction into the TOF unit 2, each ion has a flight speed corresponding to its mass-to-charge ratio, and ions having a smaller mass-to-charge ratio reach the detector 23 earlier. In this example, the TOF unit 2 is a linear type, but may be a reflectron type, a multi-turn type, or the like.

  The signal output from the detector 23 is converted into digital data by an analog / digital converter (not shown) and input to the data processing unit 4. The data processing unit 4 includes functional blocks such as a spectrum creation unit 41, a mass calibration unit 42, a subtraction processing unit 43, and a peptide identification unit 44. The control unit 3 has a function of controlling each unit in order to execute mass spectrometry on the samples 12a and 12b, and a function of a user interface through the input unit 5 and the display unit 6 provided. It should be noted that at least a part of the control unit 3 and the data processing unit 4 have their functions achieved by using a personal computer as a hardware resource and executing dedicated control / processing software installed in the computer. It can be configured.

  Next, a peptide identification method which is one of mass spectrometry methods using MALDI-TOFMS of the present example will be described with reference to FIGS. FIG. 2 is a flowchart showing an example of this peptide identification procedure, and FIG. 3 is a schematic diagram for explaining data processing in this peptide identification.

  First, a user mixes a solution of a peptide (or protein) that is an analysis target compound with a matrix solution to which an internal standard substance has been added, and drops it into a predetermined well on the sample plate 10 to dry the sample 12a. Is prepared (step S1). In addition, the user prepares the reference sample 12b by dropping the matrix solution to which only the same internal standard substance is added into another well on the same sample plate 10 to dry it (step S2). As an internal standard substance, it is preferable to use a synthetic peptide containing a polymer structure of a proline residue for the reasons described later, and P14R is used here. As the matrix, DHB (2,5-dihydroxybenzoic acid) is used.

  Thereafter, the user sets the sample plate 10 on which the sample 12 a and the reference sample 12 b are formed on the stage 11, and performs a predetermined operation with the input unit 5. In response to this operation, the control unit 3 drives the laser irradiation unit 15 and the like, and executes MALDI-ISD analysis on the sample 12 a on the sample plate 10. That is, the pulsed laser beam emitted from the laser irradiation unit 15 is irradiated onto the sample 12a, and various ions generated from the sample 12a are accelerated and then introduced into the flight space 21 for flight. In the data processing unit 4, the spectrum creation unit 41 receives data obtained by digitizing the detection signal obtained by the detector 23, creates a flight time spectrum indicating the relationship between the flight time and the signal intensity, and obtains it in advance. By converting the flight time into the mass-to-charge ratio based on the calibration information, a mass spectrum as shown in FIG. 3A is created (step S3).

  At this time, in-source decomposition in the MALDI ion source 1 is promoted by making the power of the laser beam for ionization slightly higher than that of normal mass spectrometry. Therefore, the peptide as the analysis target compound is cleaved in the MALDI ion source 1 and a large number of fragment ions are generated. On the other hand, P14R used as an internal standard substance is a peptide containing a polymer structure of a proline residue, and it is known that fragment ions due to in-source decomposition hardly occur on the N-terminal side of the proline residue. Therefore, almost no fragment ions derived from the internal standard substance are generated, and the ISD mass spectrum obtained in step S3 mainly includes the fragment ion peak derived from the analysis target compound, the molecular ion peak derived from the internal standard substance, and the molecule derived from the matrix. An ion peak appears.

  The mass calibration unit 42 extracts the molecular ion peak (m / z 154) of DHB as a matrix and the molecular ion peak (m / z 1534) of P14R as an internal standard, which appear in the obtained ISD mass spectrum. To do. Then, a mass-to-charge ratio deviation is obtained from the positions of these peaks (that is, the actually measured mass-to-charge ratio) and the known mass-to-charge ratio of the matrix and the internal standard substance, and mass calibration is performed to correct this deviation. Thereby, as shown in FIG. 3B, an ISD mass spectrum having a more accurate mass-to-charge ratio axis is obtained for the sample 12a including the analysis target compound (step S4). If the internal standard material is cleaved by in-source decomposition, the molecular ion peak of the internal standard material cannot be accurately captured, which hinders accurate mass calibration. On the other hand, in this peptide identification method, a compound in which in-source decomposition does not easily occur, that is, a compound in which molecular ions are easily observed as they are, is used as an internal standard substance, so that mass calibration can be performed with high accuracy. In addition, by using the molecular ion peak information of the matrix for mass calibration, accurate mass calibration over a wide mass-to-charge ratio range is possible even if only one type of internal standard substance is added.

  Next, when the stage 11 is moved and the reference sample 12b is set at the measurement position, the control unit 3 drives the laser irradiation unit 15 and the like again, and performs MALDI-ISD analysis on the sample 12b on the sample plate 10. Run. Thereby, the spectrum creation part 41 acquires the ISD mass spectrum with respect to the reference sample 12b (step S5). In the MALDI-ISD analysis for the reference sample 12b, the laser light power for ionization is set slightly higher than in the MALDI-ISD analysis for the sample 12a in step S3. As a result, the ion generation efficiency is improved as compared with the MALDI-ISD analysis performed on the sample 12a, and the obtained ion intensity is generally increased.

  As in step S4, the mass calibration unit 42 extracts the molecular ion peak of the matrix and the molecular ion peak of the internal standard substance that appear in the ISD mass spectrum obtained by the MALDI-ISD analysis. A deviation of the mass-to-charge ratio is obtained from the known mass-to-charge ratio of the matrix and the internal standard substance, and mass calibration is performed to correct this deviation. Thereby, an ISD mass spectrum having a more accurate mass-to-charge ratio axis is also obtained for the reference sample 12b that does not include the analysis target compound (step S6).

  When two ISD mass spectra that have been mass calibrated are obtained, the subtraction processing unit 43 subtracts the ISD mass spectrum for the reference sample 12b from the ISD mass spectrum for the sample 12a for each mass-to-charge ratio, that is, background subtraction processing. Is executed (step S7). Such a so-called subtracting function of mass spectrum can use what is installed in various existing software. As an example, there is software for mass spectrometry called “Mass ++” described in Non-Patent Document 1. In this case, after the two mass-calibrated ISD mass spectra are read by “Mass ++”, the subtraction function installed in “Mass ++” is used. Thereby, the ISD mass spectrum for the reference sample 12b is subtracted from the ISD mass spectrum for the sample 12a, and a differential ISD mass spectrum as shown in FIG. 3C can be easily obtained.

  As described above, since the laser light power when the MALDI-ISD analysis is performed on the reference sample 12b is set slightly higher than the laser light power when the MALDI-ISD analysis is performed on the sample 12a, the ISD mass spectrum for the reference sample 12b is set. The signal strength of the upper peak is higher than the signal strength of the peak having the same mass-to-charge ratio on the ISD mass spectrum for the sample 12a. That is, the signal intensity of the ion peak derived from the internal standard substance or matrix that is commonly included in the sample 12a and the reference sample 12b is higher on the ISD mass spectrum than the reference sample 12b. Therefore, the differential ISD mass spectrum from which the ion peaks derived from the internal standard substance and the matrix are almost completely removed can be obtained by the background subtraction process described above.

  Subsequently, the peptide identification unit 44 performs a peak picking process on the differential ISD mass spectrum, collects peak information (mass-to-charge ratio and signal intensity), and creates a peak list (step S8). Then, using the data listed in the peak list, for example, a database search such as MS / MS ion search is performed to estimate the amino acid sequence of the peptide that is the analysis target compound (step S9). For this database search, MS / MS included in existing methods such as Mascot, a database search engine provided by Matrix Science, and X! Tandem, a free database search engine. An ion search can be used. Further, instead of database search, other amino acid sequence estimation methods such as de novo sequencing may be used. The accuracy of the peak information listed in the peak list is high, that is, there is little or no information on ion peaks derived from substances that are not derived from the analysis target compound (so-called false ion peak information). The amino acid sequence of the peptide can be estimated with high accuracy. If the identification result is obtained in this way, the result is displayed on the screen of the display unit 6 through the control unit 3.

In the above embodiment, the peak is subtracted by using the subtraction function installed in “Mass ++” or the like. Instead, the peak itself having the same mass-to-charge ratio in the two mass spectra is used instead. You may make it perform the process to delete.
For example, in Non-Patent Document 2, mass spectrometry is performed on a standard substance and a sample, respectively, using a Fourier transform ion cyclotron resonance mass spectrometer (FT-ICRMS) capable of mass spectrometry with very high accuracy. When the ion species of the mass-to-charge ratio appearing in the mass spectrum acquired for the standard substance appears within the preset mass tolerance in the mass spectrum acquired for the sample, Processing to delete the peak is performed. By such processing, peaks derived from contaminant components such as a matrix can be removed from the mass spectrum of the sample.

  In mass spectrometry using MALDI-TOFMS as described above, mass accuracy as high as that of FT-ICRMS cannot be obtained. However, an error in mass-to-charge ratio of at least two mass spectra is caused by characteristic mass calibration using a standard substance. Since it becomes very small, it is possible to use the peak elimination processing based on only the mass-to-charge ratio value (not using the signal intensity value) as described above, and such processing is substantially equivalent to the processing by the subtraction function. is there.

  Next, an actual measurement example carried out to demonstrate the above-described peptide identification method will be described. In this measurement, the compound to be analyzed was ACTH18-39, which is an adrenocorticotropic hormone, P14R manufactured by SIGMA-ARDRICH was used as an internal standard substance, and DHB was used as a matrix. The instrument used for the measurement is AXIMA-Performance manufactured by Shimadzu Corporation.

  FIG. 4A shows the amino acid sequence of P14R, which is an internal standard substance, and the actually measured ISD mass spectrum. FIG. 4B shows the amino acid sequence of ACTH18-39, which is the analysis target compound, and the actually measured ISD mass. It is a spectrum. In FIG. 4B, a large number of c-series fragment ions mainly generated by in-source decomposition are observed. On the other hand, in FIG. 4A, it can be seen that c-series fragment ions are not observed. This indicates that P14R is a compound that hardly undergoes in-source decomposition. P14R is a kind of peptide and is very close in nature to the compound to be analyzed. Therefore, it is considered that ions derived from P14R exhibit similar behavior to ions derived from the analysis target compound in the process of MALDI-ISD analysis, except for the presence or absence of in-source decomposition, and have similar mass charges under the same measurement conditions. It is estimated that a ratio deviation occurs.

  Therefore, it can be said that P14R is suitable as an internal standard substance for mass calibration in MALDI-ISD analysis using peptides and proteins as analysis target compounds. In addition, it can be presumed that other peptides having similar properties and having a polymer structure in which proline residues are linked are also useful as internal standard substances.

  When a synthetic peptide including a polymer structure in which proline residues are linked as described above is used as a standard substance, it is desirable to appropriately adjust the content of proline residues according to the compound to be analyzed. This is because in order to acquire a precise mass, it is necessary to perform mass calibration by sandwiching the measurement mass-charge range between several standard materials. Since the composition formula of the synthesized proline polymer is fixed, precise mass calculation is possible, and adjustment of the number of proline residues corresponding to the measured mass-charge range is easy. Moreover, it is desirable that a base is added to the N-terminal of the polymer of proline residues. This is because it is known that no MALDI-ISD fragment appears on the N-terminal side of the proline residue, so it can be assumed that the MALDI-ISD fragment does not appear from the proline polymer itself having a base on the N-terminal side. is there. In addition, when a polymer containing a proline residue that does not contain a base is used, a salt adduct may also appear with high strength. Therefore, it is desirable to use a polymer containing a proline residue that contains a base.

  FIG. 5A is an ISD mass spectrum (m / z = 1 to 2400) for a sample containing the compound to be analyzed before the background subtraction process in step S7 is performed. FIG. 5B is a differential ISD mass spectrum (m / z = 1−2400) after performing the background subtraction process. The vertical axis of these mass spectra indicates the absolute value of signal intensity. As described above, the analysis target compound is ACTH18-39, the internal standard substance is P14R, and the matrix is DHB.

As shown in FIG. 5 (a), in the ISD mass spectrum for the sample before the background subtraction process, an ion peak (m / z 137) derived from DHB appears with a large signal intensity, whereas ACTH18-39 The signal intensity of the derived fragment ion peak is relatively low. In such a state, it is difficult to accurately distinguish the ion peak derived from impurities such as DHB and the fragment ion peak derived from ACTH18-39.
On the other hand, as shown in FIG. 5B, in the differential ISD mass spectrum after performing the background subtraction process, the ion peak derived from DHB is almost invisible, and the fragment ion derived from ACTH18-39 which is the analysis target A peak appears with a relatively high signal intensity. In such a state, it is possible to appropriately extract these fragment ion peaks and collect peak information.

  FIG. 6 is a diagram showing a result of an MS / MS ion search using a peak list created based on the ISD mass spectrum before the background subtraction process shown in FIG. FIG. 7 is a diagram showing a result of an MS / MS ion search using a peak list created based on the differential ISD mass spectrum after the background subtraction process shown in FIG. Each of these uses the MS / MS ion search mounted on the mascot described above.

  6 and 7, (a) is a mascot score histogram showing the accuracy of amino acid sequence estimation, (b) is a peak assignment on the mass spectrum, and (c) is an assigned product ion. Ion species are shown. The mascot score is 84 in the case of FIG. 6 and 99 in the case of FIG. 7, and it was confirmed that the score is remarkably improved by performing the background subtraction process. This is because the number of c-series ions assigned was almost the same, but when background subtraction was performed, the a-series ions that appeared weakly could be assigned more finely. It is presumed that the matrix-derived peak that does not originate from the sequence has been removed.

  From the above results, it was confirmed that the peptide identification method using MALDI-TOFMS of this example was effective in improving the identification accuracy of the peptide as the analysis target compound.

In addition, since the said Example is only an example of this invention, even if it corrects, changes, an addition etc. suitably in the range of the meaning of this invention in points other than the above-mentioned description, it is included in the claim of this application. Is clear.
For example, in the above embodiment, the present invention is applied to a mass spectrometer using a MALDI ion source. As already described, a laser desorption ionization method other than the MALDI method, typically an ion source using the SALDI method. Of course, the present invention is applied to a mass spectrometer using the same, and the same effect can be obtained.

DESCRIPTION OF SYMBOLS 1 ... MALDI ion source 10 ... Sample plate 11 ... Stage 12a ... Sample 12b ... Reference sample 13 ... Extraction electrode 14 ... Accelerating electrode 15 ... Laser irradiation part 16 ... Reflector 2 ... TOF part 20 ... Flight tube 21 ... Flight space 23 ... Detector 3 ... Control unit 4 ... Data processing unit 41 ... Spectrum creation unit 42 ... Mass calibration unit 43 ... Subtraction processing unit 44 ... Peptide identification unit 5 ... Input unit 6 ... Display unit

Claims (4)

A mass spectrometry method for analyzing a polymer compound using a mass spectrometer equipped with an ion source by a laser desorption ionization method,
a) a sample preparation step of preparing a sample by adding a predetermined standard substance to a sample containing a compound to be analyzed;
b) a reference sample preparation step for preparing a reference sample with the standard substance;
c) a mass spectrum acquisition step of acquiring mass spectra by performing mass spectrometry using in-source decomposition on the sample and the reference sample, respectively,
d) Based on the position of the ion peak derived from the standard substance appearing in the mass spectrum relative to the sample and the reference sample and the known mass-to-charge ratio of the standard substance, the mass-to-charge ratio deviation in each mass spectrum is corrected. A mass calibration step;
e) a subtraction processing step of subtracting the mass spectrum for the reference sample, which is also corrected for the mass-to-charge ratio deviation in the mass calibration step, from the mass spectrum for the sample in which the mass-charge ratio deviation is corrected in the mass calibration step;
f) an analysis processing step for performing analysis of the analysis target compound based on the difference mass spectrum obtained in the subtraction processing step;
A mass spectrometric method characterized by comprising: The mass spectrometric method according to claim 1,
A mass spectrometry method using a protein or peptide containing a polymer structure of proline as the standard substance. The mass spectrometric method according to claim 1 or 2,
A mass spectrometry method using a laser desorption ionization time-of-flight mass spectrometer equipped with a saturation tolerance detector as the mass spectrometer. In the mass spectrometer which comprises the ion source by the laser desorption ionization method used for the mass spectrometry method in any one of Claims 1-3,
a) Mass spectrometry using in-source decomposition was performed on a sample prepared by adding a predetermined standard substance to a sample containing the compound to be analyzed and a reference sample prepared from the standard substance. A measurement execution unit for acquiring a mass spectrum;
b) Based on the position of the ion peak derived from the standard substance appearing in the mass spectrum relative to the sample and the reference sample and the known mass-to-charge ratio of the standard substance, the mass-to-charge ratio deviation in each mass spectrum is corrected. A mass calibration unit;
c) a subtraction processing unit that subtracts a mass spectrum for a reference sample, which is also corrected for a mass-to-charge ratio deviation by the mass calibration unit, from a mass spectrum for the sample in which the mass-to-charge ratio deviation is corrected by the mass calibration unit;
d) an analysis processing unit that performs analysis of the analysis target compound based on the difference mass spectrum obtained by the subtraction processing unit;
A mass spectrometer comprising:

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