JP6665740B2 - Peptide analysis method, peptide analysis device, and peptide analysis program - Google Patents

Description

  The present invention relates to a peptide analysis method, a peptide analysis device, and a peptide analysis program for analyzing isomerization of amino acids constituting a peptide using a liquid chromatograph mass spectrometer.

  It is known that some amino acids of proteins are changed from the L-form to the D-form by aging, and such phenomena may cause various illnesses such as cataracts and Alzheimer's disease that are often seen in old age. Has been pointed out. For these reasons, structural analysis of proteins and peptides has become very important in various fields such as life science, medicine, and drug development. Mass spectrometry has been extremely effective for such analysis, and various methods for analyzing proteins and peptides using mass spectrometry have been researched and developed.

  When performing structural analysis of a protein in which some amino acids are isomerized using mass spectrometry, mass spectrometry is often performed on a peptide obtained by fragmenting the protein using a chromatograph mass spectrometer. Done. At this time, the target peptide is decomposed by using an enzyme that specifically hydrolyzes a D-amino acid or an L-amino acid, and mass spectrometry is performed while separating the obtained decomposed product by chromatography. Check whether the peptide has been degraded.

  Non-Patent Document 1 reports a method for detecting a peptide in which some amino acids are isomerized (hereinafter, referred to as an isomerized peptide) using a liquid chromatograph-electrospray ionization ion trap mass spectrometer. I have.

In this technique, an MS / MS spectrum (MS ² spectrum) of a sample that may contain an isomerized peptide is obtained using an ion trap mass spectrometer, and this spectrum is stored in an MS / MS spectrum recorded in a database. The target peptide is identified by comparing it with the above information, or by obtaining the sequence information of the spectrum with software for estimating the peptide sequence. Next, a chromatogram (MS chromatogram) of the identified peptide is prepared. If only one peak appears in this MS chromatogram, it is determined that the isomerized peptide has not been detected. It is determined that the isomerized peptide may be present. The reason for such determination can be that the isomerized peptide and the non-isomerized peptide have different retention behaviors on the column, and thus can be separately detected by chromatography.

  When it is determined that there is a possibility that the isomerized peptide exists, the degree of coincidence of the MS / MS spectrum corresponding to each of the plurality of peaks of the MS chromatogram is confirmed. It is subjected to an additional experiment for verifying the presence, and if they do not match, it is determined that the isomerized peptide does not exist. That is, since the product ion of the non-isomerized peptide and the product ion of the isomerized peptide include the same amino acid sequence, when multiple peaks in the MS chromatogram indicate the presence of the isomerized peptide, A product ion having the same amino acid sequence is detected from each MS / MS spectrum corresponding to the peak, and the shapes of the MS / MS spectra match.

Fujii N, Sakaue H, Sasaki H, and Fujii N., J Biol Chem., 287; 39992-40002, 2012 Maeda H, Takata T, Fujii N, Sakaue H, Nirasawa S, Takahashi S, Sasaki H. and Fujii N., Anal.Chem., 87; 561-568, 2015

  As an additional verification experiment method when the MS / MS spectra match, for example, a peptide obtained by introducing an isomeric amino acid into a standard peptide (isomerized standard peptide) is synthesized and appears in the MS chromatogram of the isomerized standard peptide. A method for confirming whether the retention time of a peak and the retention time of a peak of a peptide which is a candidate for an isomerized peptide contained in a sample are coincident with each other (Non-patent Document 1), which specifically acts on D-Asp residues. Methods of hydrolyzing a peptide in a sample using peptidase and confirming the decomposition product using LC / MS (Non-Patent Document 2) have been reported. However, the former method requires a step of synthesizing an isomerized standard peptide, and the latter method requires a step of an enzyme reaction. Both steps are complicated and troublesome. In addition, when an enzyme is used, there is a problem that an additional verification experiment cannot be performed without the enzyme.

  The problem to be solved by the present invention is to provide a peptide analysis method, a peptide analysis device, and a peptide analysis program that can confirm the presence of an isomerized peptide without performing complicated operations.

Means for Solving the Problems The present invention made to solve the above-mentioned problems provides a peptide analysis method for analyzing the isomerization of a partial amino acid of a protein or peptide using a chromatograph mass spectrometer capable of MS / MS measurement. So,
a) a step of temporally separating a sample containing a protein or peptide to be analyzed by chromatography,
b) measuring a plurality of MRM transitions corresponding to a precursor ion generated by ionization of a protein or peptide contained in the separated sample, and a plurality of types of fragment ions formed by cleavage of an amino acid at a place where the precursor ion is different; A measurement execution step of executing MRM measurement as a condition;
c) creating a chromatogram at each value of the plurality of MRM transitions based on the data obtained by the MRM measurement;
d) a peak detection step of detecting a peak for each of the plurality of chromatograms,
e) In the case where a plurality of peaks are detected in each of the plurality of chromatograms, by determining whether or not a plurality of peaks are respectively present at the same retention time in common to the plurality of chromatograms. Judging whether or not some amino acids of the protein or peptide to be analyzed are isomerized.

Further, a peptide analysis device according to the present invention made to solve the above-mentioned problem is an apparatus for realizing the above-described peptide analysis method according to the present invention, and is a chromatographic mass spectrometer capable of performing MS / MS measurement. A peptide analyzer for analyzing the isomerization of some amino acids of a protein or peptide, comprising:
a) A sample containing a protein or peptide to be analyzed is temporally separated by chromatography, and a precursor ion generated by ionization of a protein or peptide contained in the separated sample. An analysis control unit that operates the chromatograph mass spectrometer to perform MRM measurement with a plurality of MRM transitions corresponding to a plurality of types of fragment ions formed by cleavage as measurement conditions;
b) a chromatogram creating unit that creates a chromatogram at each value of the plurality of MRM transitions based on the data obtained by the MRM measurement;
c) a peak detection unit that detects a peak for each of the plurality of chromatograms,
d) in the case where a plurality of peaks are detected in each of the plurality of chromatograms, by determining whether or not a plurality of peaks are respectively present at the same retention time in common to the plurality of chromatograms. An isomerization determination unit that determines whether or not some amino acids of the protein or peptide to be analyzed are isomerized.

Furthermore, a peptide analysis program according to the present invention made to solve the above-mentioned problem is a computer program for realizing the peptide analysis method according to the present invention, and a partial amino acid of a protein or peptide. A program operating on a computer for controlling the operation of a chromatograph mass spectrometer capable of performing MS / MS measurement and processing data obtained by the apparatus to analyze isomerization,
a) A sample containing a protein or peptide to be analyzed is temporally separated by chromatography, and a precursor ion generated by ionization of a protein or peptide contained in the separated sample. An analysis control function unit that operates the chromatograph mass spectrometer to perform MRM measurement with a plurality of MRM transitions according to a plurality of types of fragment ions formed by cleavage as measurement conditions;
b) creating a chromatogram at each value of the plurality of MRM transitions based on the data obtained by the MRM measurement, and, when a plurality of peaks are detected in each of the plurality of chromatograms, the plurality of chromatograms; By determining whether a plurality of peaks are present at the same retention time in common for each gram, it is possible to determine whether or not some amino acids of the protein or peptide being analyzed are isomerized. And a judgment function unit.

  In the present invention, examples of a chromatograph constituting a chromatograph / mass spectrometer capable of MS / MS measurement include a gas chromatograph, a liquid chromatograph, and capillary electrophoresis, and a tandem quadrupole type as a mass spectrometer. There are mass spectrometers (also called triple quadrupole mass spectrometers), quadrupole time-of-flight mass spectrometers (QTOF), ion trap mass spectrometers, matrix-assisted laser desorption ionization mass spectrometers, etc. An apparatus combining them can be used.

  In the present invention, a peptide in which some amino acids are isomerized is a peptide in which one or more of a plurality of amino acids constituting the peptide are changed from L-form to D-form or vice versa. Or a peptide in which an α amino acid has been changed to a β amino acid, a peptide in which a β amino acid has been changed to an α amino acid, and the like. In addition, peptides in which optical isomerization and stereoisomerization have occurred, such as peptides in which some amino acids have been changed from L-form α-amino acids to D-form β-amino acids, are also included in peptides in which some amino acids have been isomerized. The same applies to proteins in which some amino acids are isomerized.

  In the present invention, a sample obtained by subjecting a protein sample to peptide fragmentation with a protein digesting enzyme such as trypsin is separated by chromatography and introduced into a mass spectrometer. However, it is also possible to analyze a protein having a relatively small molecular weight without using a digestive enzyme. Hereinafter, a case where a sample in which a protein is fragmented with a peptide will be described, but the same can be said for a case where a protein is used as a sample as it is.

  The peptide in the sample has a configuration in which a plurality of amino acids are peptide-bonded, and ions are generated by electrospray ionization, which is a general ionization method used in a liquid chromatograph mass spectrometer. Therefore, the MRM transition was performed so that ions containing amino acids that are expected to undergo isomerization become precursor ions, and ions generated by cleavage of amino acids at different locations of the precursor ions became fragment ions. Set. That is, a plurality of MRM transitions are set according to the difference in the cleavage site of the amino acid constituting the fragment ion. Note that the expression "amino acid is cleaved at a different position" includes both "different amino acid is cleaved" and "an amino acid is cleaved at a different position". When MRM (Multiple Reaction Ion Monitoring) measurement is performed using a plurality of MRM transitions set as described above as measurement conditions, ions derived from the original peptide are detected in MRM transitions corresponding to the number of amino acids of the fragment ions. Is done.

  When the amino acid of interest is isomerized, both the chromatogram obtained by MRM measurement using one MRM transition as a measurement condition and the chromatogram obtained by MRM measurement using another MRM transition as a measurement condition are given. A plurality of peaks corresponding to the number of the non-isomerized peptide and the isomerized peptide (these are also referred to as “peptide isomers” or “isomeric peptides”) appear. Each gram appears at the same retention time. On the other hand, the intensity of a plurality of peaks appearing in each chromatograph has a value corresponding to the location of cleavage (that is, each MRM transition) when a fragment ion is generated from a precursor ion. Therefore, a chromatogram in a plurality of MRM transitions is created, a plurality of peaks are observed in each chromatogram, and when these plurality of peaks are common to a plurality of chromatograms and are present at the same retention time, attention must be paid. It can be determined that isomerization has occurred in the amino acid to be produced. At this time, the intensity of each peak observed at the same retention time in a plurality of chromatograms will be different.

  In this case, if the chromatograms obtained by the MRM measurement using a plurality of MRM transitions as the measurement conditions are superimposed and displayed on the same coordinate axis, the peaks of each chromatogram appear at the same retention time, It is preferable because the peaks have different intensities at a glance.

  Further, amino acid isomers in the peptide chain differ in the relative position of the side chain carboxyl group with respect to the peptide backbone. This is because each amino acid contains an asymmetric carbon, and the amino acid isomers are in a diastereomeric relationship. Diastereomers can generally be separated by liquid chromatography using a reversed-phase column such as a C18 column or C8 column. Furthermore, the number of carbon atoms constituting the peptide backbone may differ depending on the amino acid isomer. For example, FIG. 8 shows a peptide backbone (rectangular or V-shaped hatched portion) of Dα-Asp, Lα-Asp, Dβ-Asp, and Lβ-Asp, which are isomers of aspartic acid (Asp), and a side chain carboxyl group. (Shaded portion of the ellipse). As shown in this figure, β-Asp (Dβ-Asp, Lβ-Asp) has one more carbon atom constituting the peptide backbone than α-Asp (Dα-Asp, Lα-Asp). These can also be separated by reversed-phase liquid chromatography.

  FIG. 9 shows the currently proposed mechanism of cleavage via the side chain of Asp upon ionization of the peptide. As can be seen from FIGS. 8 and 9, when the amino acid of interest is Asp, and the precursor ion containing Asp is fragmented by cleavage through the side chain of Asp, the fragment ion is formed. Even if the number of amino acids is the same, the energy values for cleavage of fragment ions are different, and therefore, it is considered that the peak intensity when ionizing between isomers is different.

  As described above, in a plurality of chromatograms, it is possible to estimate the site where the isomerized amino acid is present by comparing the intensity ratio of the peaks present at the same retention time.

  According to the peptide analysis method, the peptide analysis device, and the peptide analysis program of the present invention, it is possible to confirm whether or not isomerization has occurred in a part of amino acids constituting a protein or a peptide without performing a complicated operation. can do,

1 is a schematic configuration diagram of one embodiment of a peptide analysis device that performs a peptide analysis method according to the present invention. 5 is a flowchart illustrating a procedure of a peptide analysis process in the peptide analyzer according to the embodiment. The figure which shows the measured MRM chromatogram of the sample containing four types of isomer peptides. FIG. 4 is an enlarged view of a region A shown in FIG. 3. The figure which shows the SIM chromatogram of the sample containing four types of isomeric peptides. FIG. 4 is a view showing the relative intensity at each peak of the MRM chromatogram shown in FIG. 3. The figure which shows the amino acid sequence which comprises a 2nd fragment ion. The figure which shows the relationship of the peak intensity of the isomer of aspartic acid. Explanatory drawing of the cleavage mechanism via the side chain of aspartic acid.

One example of the peptide analysis method according to the present invention will be described with reference to the accompanying drawings. FIG. 1 is a schematic configuration diagram of a peptide analysis device that performs the peptide analysis method according to the present embodiment.
As shown in FIG. 1, the peptide analyzer includes a liquid chromatograph mass spectrometry system (“LC-MS / MS system”) including a liquid chromatograph unit (LC unit) 10 and a mass spectrometer unit (MS / MS unit) 20. It is).

  The LC section 10 includes a mobile phase container 11 in which the mobile phase is stored, a pump 12 for sucking the mobile phase and feeding it at a constant flow rate, and an injector 13 for injecting a predetermined amount of a sample prepared in advance in the mobile phase. And a column 14 for separating various compounds contained in the sample in a time direction. In the case where the LC section 10 performs a so-called gradient separation in which compounds included in a sample are separated while continuously changing the compositions of a plurality of mobile phases, two or more mobile phase containers 11 and two or more pumps 12 are provided. .

  The MS / MS unit 20 is composed of a tandem quadrupole mass spectrometer, and has a step between an ionization chamber 21 at atmospheric pressure and a high vacuum analysis chamber 24 evacuated by a high-performance vacuum pump (not shown). This is a configuration of a multi-stage differential pumping system including first and second intermediate vacuum chambers 22 and 23 whose degree of vacuum is increased. The ionization chamber 21 is provided with an electrospray ionization probe 25 for spraying the sample solution while applying a charge, and a small-diameter heating capillary 26 passes between the ionization chamber 21 and the first intermediate vacuum chamber 22 at the next stage. Communicating. The first intermediate vacuum chamber 22 and the second intermediate vacuum chamber 23 are separated by a skimmer 28 having a small hole at the top. The first intermediate vacuum chamber 22 and the second intermediate vacuum chamber 23 converge ions, respectively. Further, ion guides 27 and 29 for transporting to the subsequent stage are provided. In the analysis chamber 24, a multipole ion guide 32 is sandwiched by a collision cell 31 installed therein, and a pre-stage quadrupole mass filter 30 that separates ions according to the mass-to-charge ratio, and similarly, the ions according to the mass-to-charge ratio A post-stage quadrupole mass filter 33 and an ion detector 34 are provided. The CID gas supply unit 35 supplies a CID gas such as argon or nitrogen into the collision cell 31. The power supply unit 36 applies a predetermined voltage to the electrospray ionization probe 25, the ion guides 27, 29, 32, the quadrupole mass filters 30, 33, and the like.

  The data processing unit 40 includes, as functional blocks, a data collection unit 41, a data storage unit 42, a graph creation unit 43, a quantitative analysis unit 44, and the like. The control unit 50 provided with the input unit 52 and the display unit 53 controls the operation of each unit such as the pump 12 and the injector 13 of the liquid chromatograph unit 10, the power supply unit 36 and the CID gas supply unit 35 of the mass spectrometer 20, respectively. I do. At least a part of the functions of the control unit 50 and the data processing unit 40 is realized by using a personal computer as a hardware resource and executing dedicated control / processing software pre-installed on the computer on the computer. be able to.

The basic MS / MS measurement operation in the LC-MS / MS system of the present embodiment is as follows.
First, the pump 12 sucks the mobile phase from the mobile phase container 11 and sends it to the column 14 at a constant flow rate. When a certain amount of sample solution is introduced into the mobile phase from the injector 13, the sample is introduced into the column 14 along with the flow of the mobile phase, and various compounds in the sample are separated in the time direction while passing through the column 14. Then, it is eluted from the outlet of the column 14 and introduced into the MS / MS unit 2.

In the MS / MS unit 20 , when the eluate from the column 14 reaches the electrospray ionization probe 25, the eluate is sprayed while being charged at the tip of the probe 25. The charged droplets formed by spraying are fragmented and finely divided by the action of electrostatic force due to the applied charge, and in the process, the solvent evaporates and ions derived from the compound fly out.

  The ions thus generated are sent to the first intermediate vacuum chamber 22 through the heating capillary 26, converged by the ion guide 27, and sent to the second intermediate vacuum chamber 23 through the small hole at the top of the skimmer 28. Then, the ions derived from the compound are converged by the ion guide 29 and sent to the analysis chamber 24, and introduced into the space in the longitudinal direction of the pre-stage quadrupole mass filter 30. It should be noted that the ionization method is not limited to the electrospray ionization method, but may be an atmospheric pressure chemical ionization method, an atmospheric pressure photoionization method, or the like.

When performing MS / MS analysis in the MS / MS unit 20 , a predetermined voltage (high-frequency voltage and DC voltage) is applied to each rod electrode of the first-stage quadrupole mass filter 30 and the second-stage quadrupole mass filter 33 from the power supply unit 36, respectively. Is applied, and the CID gas supply unit 35 supplies the collision cell 31 continuously or intermittently with the CID gas. Thereby, among the various ions sent to the pre-stage quadrupole mass filter 30, only ions having a specific mass-to-charge ratio corresponding to the voltage applied to each rod electrode of the pre-stage quadrupole mass filter 30 are included. After passing through the filter 30, the ions are introduced into the collision cell 31 as precursor ions. In the collision cell 31, the precursor ions collide with the CID gas and are cleaved to generate various product ions. When the generated various product ions are introduced into the subsequent quadrupole mass filter 33, only the product ions having a specific mass-to-charge ratio corresponding to the voltage applied to each rod electrode of the latter quadrupole mass filter 33 Pass through the filter 33 and reach the ion detector 34 where they are detected. As the ion detector 34, a pulse count type detector can be used. In this case, pulse signals of a number corresponding to the number of incident ions are output as detection signals.
The detection signal of the ion detector 34 is input to the data processing unit 40, where a mass spectrum and a mass chromatogram are created, and a peptide analysis process described later is executed.

Like the general LC-MS / MS, the system according to the present embodiment also provides multiple reaction monitoring (MRM) measurement, product ion scan measurement, precursor ion scan measurement, and neutral loss scan measurement as the MS / MS measurement mode. Have been.
In the MRM measurement, MS / MS measurement is performed by passing only ions having a predetermined mass-to-charge ratio in each of the first-stage quadrupole mass filter 30 and the second-stage quadrupole mass filter 33. A specific product ion for the precursor ion is detected. In the MRM measurement, it is possible to perform a measurement in which a plurality of channels are set in which the mass-to-charge ratio (m / z) of the precursor ion and the mass-to-charge ratio (m / z) of the product ion are set as one set, and the mass-to-charge ratio of the precursor ion is obtained. A set of the ratio and the mass-to-charge ratio of the product ions is called “MRM transition”. In the peptide analysis processing described below, MRM measurement is used as MS / MS measurement.

  Hereinafter, the peptide analysis process performed using the LC-MS / MS system will be described. FIG. 2 is a flowchart showing the procedure of this processing.

When analyzing whether or not some amino acids of a certain protein or peptide are isomerized, the measurer first uses a standard peptide to perform MS / MS in MRM mode using the above-described LC-MS / MS system. The measurement conditions for measurement are examined (step S1). Note that the standard peptide is a peptide having a known structure, which is generated by cleavage when the protein or peptide to be analyzed is introduced into the MS / MS unit 20 and ionized, and which is isomerized. It refers to one that includes multiple isomers of the predicted amino acid.

  For example, when aspartic acid (D, Asp) is expected to be isomerized for a certain peptide, a plurality of isomers of aspartic acid (for example, four isomers (Dα-Asp, Dβ-Asp, Lα -Asp, Lβ-Asp)) are included in the standard peptide.

When an MS / MS measurement using a plurality of MRM transitions as a measurement condition is performed in the MRM mode using such a standard peptide, an MRM chromatogram is obtained for each value of the plurality of MRM transitions. A plurality of peaks appearing are extracted, and the time at which each peak appears (retention time) and the intensity ratio of the peaks appearing at the same retention time of the plurality of MRM chromatograms are registered in the data storage unit 42 (step S2).
In the case of using an already determined retention time or peak intensity ratio as a measurement condition in analyzing an isomerized peptide, the above-described processing can be omitted.

  Next, when the start of the analysis process using the above-described LC-MS / MS system is instructed for the actual sample containing the protein or peptide to be analyzed, a plurality of MRM transitions are performed in the MRM mode in the same manner as the standard peptide. Is performed, and the data obtained thereby is collected and stored in the data storage unit 42 (step S3). When the measurement is completed, the data is processed by the graph creation unit 43 to create a chromatogram for each value of the plurality of MRM transitions, and a waveform process is performed on the created chromatogram to extract peaks. (Step S4).

Subsequently, a peak table showing the relationship between the peak retention time and the peak intensity of the chromatogram created for each value of the MRM transition is created (step S5), and the peaks appearing at the same retention time in each of the plurality of chromatograms are determined. Linking is performed (step S6). Then, the intensity ratio of the linked peaks is compared with the peak intensity ratio of the standard peptide (Step S7). If the difference between the two intensity ratios exceeds the allowable range (No in Step S8), the isomerized peptide is It is presumed that it does not exist (step S13), and the result is output (step S12).
On the other hand, if the difference between the intensity ratio of the tied peak and the peak intensity ratio of the standard peptide is within the allowable range (Yes in step S8), the retention time of the tied peak is changed to the peak obtained for the standard peptide. The retention time is compared with the retention time (step S9). If the difference is within the allowable range, it is determined that the actual sample contains the same type of isomer as the standard peptide (four types of isomers in the above-described Asp example). Estimate and output the result (steps S11, S12). Further, When the difference exceeds the allowable range, the same is not a standard peptide, the actual sample is estimated to contain isomers and outputs the result (step S14, S 12).

Next, an example in which a peptide containing aspartic acid is specifically analyzed for isomerization of the aspartic acid will be described.
Here, as the peptide containing aspartic acid, a peptide consisting of 11 amino acids whose amino acid sequence is “TVLDSGISEVR” was used. This sample peptide contains multiple isomers of aspartic acid, which are contained in Crystallin, a type of protein present in the lens, and have been pointed out to change from the D-form to the L-form in patients who have developed cataracts. A mixture of the peptides.
The equipment used for the measurement is as follows.
LC-MS / MS: LCMS-8060 manufactured by Shimadzu Corporation
Column: L-column 2 metal free, 3 μm, 150 x 2.0 mm (Chemicals Evaluation and Research Organization) Catalog No. 731020

(1) For a peptide having an amino acid sequence of “TVLDSGISEVR”, four isomers in which aspartic acid (Asp, symbol D) is Lα-Asp, Lβ-Asp, Dα-Asp, and Dβ-Asp are prepared. In these four isomeric peptides, amino acids other than aspartic acid have the same steric structure. Then, 2 mg of each of the prepared four isomers is weighed and dissolved in 50% methanol to prepare a 5 mg / mL stock solution.

(2) Four kinds of stock solutions were mixed, and this mixed solution was diluted with 10% ACN so that the concentration of each peptide was adjusted to 0.5 μm / mL, and this was used as a sample.
(3) This sample is introduced into the LC unit 10 in an amount of 0.2 μL (weight of each isomer peptide per column: 0.1 ng), and LC-MS / MS measurement is performed.
Table 1 shows the measurement conditions in the LC section 10 at this time, and Table 2 shows the measurement conditions for the MS / MS measurement. As shown in Table 1, here, gradient separation is performed to separate peptides contained in the sample while continuously changing the compositions of the mobile phase A and the mobile phase B.

  Among the measurement conditions shown in Table 2, each value of the MRM transition indicates a set of values of the mass-to-charge ratio (m / z) of the precursor ion and the mass-to-charge ratio (m / z) of the product ion. Note that the voltage value applied to each rod of the first-stage quadrupole mass filter 30 and the voltage value applied to each rod of the second-stage quadrupole mass filter 33 may be used as the MRM transition. In this measurement example, the mass-to-charge ratio of the precursor ion is common to the five types of measurement conditions y5 to y9, and the peptide whose amino acid sequence is “TVLDSGISEVR” is passed through the former quadrupole mass filter 30 as a precursor ion. Equivalent to. Even for the same peptide, the mass-to-charge ratio of the precursor ion may be different depending on the difference in valence. On the other hand, the voltage value applied to each rod of the subsequent quadrupole mass filter 33 after each of the measurement conditions y5 to y9 is a peptide having the amino acid sequence shown in FIG. This corresponds to a condition in which a peptide consisting of 5 to 9 amino acids colored with a gray rectangle in the sequence) passes through the subsequent quadrupole mass filter 33 as product ions.

  FIG. 3 shows an MRM chromatogram as a measurement result of the sample, and FIG. 4 shows an enlarged view of a region surrounded by a rectangular frame in FIG. FIG. 5 shows a mass chromatogram obtained by performing MS / MS measurement in the SIM mode.

  As can be seen from FIG. 3, each of the MRM chromatograms obtained at the respective MRM transition values shown in Table 2 has four peaks. These four peaks are the same as the peaks observed in the mass chromatogram obtained by SIM / MS / MS measurement (see FIG. 5). Since the four peaks observed in each MRM chromatogram exist at the same retention time as the four peaks observed in the other MRM chromatograms, these peaks are included in the sample. It can be determined that these are isomeric peptides. According to the analysis of the inventor, the four peaks observed in the MRM chromatogram shown in FIG. 3 are, in order from the left, a peptide containing Lβ-Asp, a peptide containing Lα-Asp, a peptide containing Dβ-Asp, It has been found to correspond to peptides containing Dα-Asp.

FIG. 6 shows four peaks observed in the MRM chromatogram obtained for each value of the MRM transition, specifically, in order from the left, a peptide containing Dα-Asp, a peptide containing Dβ-Asp, The relative intensities of the peaks corresponding to the peptide containing Lα-Asp and the peptide containing Lβ-Asp are shown. Here, the relative intensity is defined as an area ratio with respect to four peaks observed in a mass chromatogram obtained when performing MS / MS measurement in the SIM mode.
As shown in FIG. 6, the peak intensity ratio corresponding to the five measurement conditions is slightly different for each peak corresponding to each isomer peptide, and cleavage between Asp (D) and serine (symbol S) is particularly difficult. The ratio (y8 / y7) between the peak intensity of the MRM chromatogram obtained under the measurement condition y7 and the peak intensity of the MRM chromatogram obtained under the measurement condition y8 in which cleavage occurs between Asp and leucine (symbol L). It can be seen that the four different isomeric peptides are different. Therefore, by using the peak intensity ratio y8 / y7 as an index, it was verified that it is possible to estimate the presence of an isomeric peptide and the position where isomerization occurs (Asp in this example). .

  FIG. 4 is an enlarged view of the vicinity (part A) of the baseline in FIG. Here, peaks of impurities included in the four kinds of isomer peptides prepared as standard peptides were detected. As for these peaks, peaks corresponding to five measurement conditions are detected. Therefore, it can be inferred that these small peaks are also isomeric peptides. In this case, it was possible to infer that the peak of interest was an isomeric peptide without preparing a standard peptide. Therefore, it was verified that the presence of the isomeric peptide can be estimated even when steps S1 and S2 in the flowchart shown in FIG. 2 are omitted.

  The above embodiment is merely an example of the present invention, and can be appropriately changed or expanded within the scope of the present invention. For example, in the above embodiment, a liquid chromatograph was used, but a gas chromatograph may be used.

DESCRIPTION OF SYMBOLS 10 ... Liquid chromatograph part 14 ... Column 20 ... Mass spectrometry part 30 ... Pre-stage quadrupole mass filter 31 ... Collision cell 32 ... Multipole ion guide
33 ... Post-stage quadrupole mass filter 34 ... Ion detector 35 ... CID gas supply unit 36 ... Power supply unit 40 ... Data processing unit 41 ... Data collection unit 42 ... Data storage unit 43 ... Graph creation unit 44 ... Quantitative analysis unit 50 ... Control unit 52 ... Input unit 53 ... Display unit

Claims (5)

A peptide analysis method for analyzing isomerization of a partial amino acid of a protein or peptide using a chromatograph mass spectrometer capable of MS / MS measurement,
a) a step of temporally separating a sample containing a protein or peptide to be analyzed by chromatography,
b) measuring a plurality of MRM transitions corresponding to a precursor ion generated by ionization of a protein or peptide contained in the separated sample, and a plurality of types of fragment ions formed by cleavage of an amino acid at a place where the precursor ion is different; A measurement execution step of executing MRM measurement as a condition;
c) creating a chromatogram at each value of the plurality of MRM transitions based on the data obtained by the MRM measurement;
d) a peak detection step of detecting a peak for each of the plurality of chromatograms,
A case where a plurality of peaks are detected in each of e) said plurality of chromatograms, in common to said plurality of chromatograms, when in the state where a plurality of peaks are present in the same retention time, respectively, the analysis Judging that some amino acids of the target protein or peptide are isomerized, and when not in the above state, judging that none of the amino acids of the protein or peptide being analyzed is not isomerized. A peptide analysis method, comprising: The peptide analysis method according to claim 1,
A peptide analysis method, comprising: an isomerization site estimation step of estimating a site where an isomerized amino acid exists by comparing the intensity ratio of peaks existing at the same retention time of the plurality of chromatograms. . The peptide analysis method according to claim 1 or 2,
A peptide analysis method, comprising a display step of displaying the plurality of chromatograms on a coordinate axis having a retention time as a first axis and an intensity as a second axis. A peptide analyzer for analyzing the isomerization of a partial amino acid of a protein or peptide, comprising a chromatograph mass spectrometer capable of MS / MS measurement,
a) A sample containing a protein or peptide to be analyzed is temporally separated by chromatography, and a precursor ion generated by ionization of a protein or peptide contained in the separated sample. An analysis control unit that operates the chromatograph mass spectrometer to perform MRM measurement with a plurality of MRM transitions corresponding to a plurality of types of fragment ions formed by cleavage as measurement conditions;
b) a chromatogram creating unit that creates a chromatogram at each value of the plurality of MRM transitions based on the data obtained by the MRM measurement;
c) a peak detection unit that detects a peak for each of the plurality of chromatograms,
d) in a case where a plurality of peaks in each of the plurality of chromatograms is detected, in common to said plurality of chromatograms, when in the state where a plurality of peaks are present in the same retention time, respectively, the analysis It is determined that some amino acids of the target protein or peptide are isomerized, and when the target is not in the above-mentioned state, it is determined that none of the amino acids of the target protein or peptide is isomerized. A peptide analyzer comprising: Operates on a computer to control the operation of a chromatograph mass spectrometer capable of MS / MS measurement and to process data obtained by the device to analyze the isomerization of some amino acids of proteins or peptides Program that
a) A sample containing a protein or peptide to be analyzed is temporally separated by chromatography, and a precursor ion generated by ionization of a protein or peptide contained in the separated sample. An analysis control function unit that operates the chromatograph mass spectrometer to perform MRM measurement with a plurality of MRM transitions according to a plurality of types of fragment ions formed by cleavage as measurement conditions;
b) creating a chromatogram at each value of the plurality of MRM transitions based on the data obtained by the MRM measurement, and, when a plurality of peaks are detected in each of the plurality of chromatograms, the plurality of chromatograms; in common to gram, when in the state where a plurality of peaks are present in the same retention time, respectively, it is determined that a portion of the amino acid of a protein or peptide which is the analysis object is isomerized, when not in the state An isomerization determination function unit that determines that none of the amino acids of the protein or peptide to be analyzed is isomerized .

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