JP2006284509A - Mass spectrometric system - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a mass spectrometric system capable of identifying precisely an amino acid sequence of each peptide, even when isotope peak groups derived from the plurality of peptides are overlapped. <P>SOLUTION: This system is provided with a mass analytical part for selecting one part of peaks from the isotope peak groups obtained by MS measurement of a peptide mixture to carry out the MS/MS measurement, and a control/processing part for controlling the mass analytical part and for analyzing a data. The isotope peak groups obtained by the MS/MS measurement (S3) of the peptide mixture are classified in response to the number of the peaks constituting each of the isotope peak groups (S5), and a peak list is prepared in every of respective classes (S6). A product ion derived from the individual peptide is discriminated even when the isotope peak groups derived from the different peptides are overlapped, by using te peak list classified by this manner, and the amino acid sequence is thereby identified precisely. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a mass spectrometry system for mass-analyzing a sample containing a peptide mixture and identifying the amino acid sequence of each peptide based on the obtained data.

In recent years, protein structures and functions have been rapidly analyzed as post-genomic research. As one of such protein structure and function analysis methods, protein expression analysis and primary structure analysis using mass spectrometers have been widely performed. Quadrupole ion traps (Quadrupole Ion Trap) A so-called MS / MS analysis (or MS ⁿ analysis) that captures and cleaves a specific peak by means of QIT) or collision induced decomposition (CID) is effective. In general, in MS / MS analysis, first, an ion having a specific mass-to-charge ratio (m / z) is selected as a precursor ion from an analysis object, and the precursor ion is cleaved by CID. Thereafter, mass analysis of ions (product ions) generated by cleavage can obtain information on the mass and chemical structure of the target ions.

When the amino acid sequence of a protein is identified by MS / MS (or MS ⁿ ) as described above, the protein is first digested with an appropriate enzyme to form a mixture of peptide fragments, and then the peptide mixture is subjected to mass spectrometry. Provide. At this time, since stable isotopes having different masses exist in the elements constituting each peptide, a plurality of peaks having different mass-to-charge ratios depending on the isotopic composition of peptides having the same amino acid sequence. Occurs (FIG. 5). The plurality of peaks are composed of peaks of ions (main ions) composed only of isotopes having the maximum natural abundance ratio, and peaks of ions containing other isotopes (isotope ions), which are spaced by 1 Da. A peak group consisting of a plurality of peaks arranged in the form of is formed (in the present invention, this is called an isotope peak group).

  Subsequently, a set of isotope peaks derived from a single peptide is selected as a precursor ion from the mass spectrometry data (MS data) of the peptide mixture as described above, and the precursor ion is cleaved. Mass analysis (MS / MS analysis) of the generated ions (product ions) is performed.

  The amino acid sequence of the test peptide can be identified by subjecting the product ion spectrum pattern obtained as described above or the precursor ion spectrum pattern to a database search. Moreover, the amino acid sequence of each peptide can also be estimated (DeNovo sequence) by mathematically analyzing these spectral patterns using computer software.

  Conventionally, when amino acid sequences are identified by the above-described method, if isotopic peak groups derived from different peptides are close to each other on the MS data, the peaks together with the peak derived from the desired peptide are displayed. A peak derived from another adjacent peptide was selected as a precursor ion, and accurate identification could not be performed. However, with the development of high-resolution ion trap technology in recent years, it has become possible to select a single peak from a group of isotope peaks appearing at 1 Da intervals and use it as a precursor ion. Even when some of the body peak groups overlap, separation / identification of each peptide can be performed by selecting individual peaks from each peptide and performing MS / MS. (Non-Patent Document 1).

High-Pep Institute Okinawa Lab Establishment Commemorative Conference Handout, February 19, 2005, Shimadzu Corporation

  However, when there is a large overlap of isotopic peak groups derived from different peptides, even if a single peak is selected from the isotopic peak group as described above and MS / MS analysis is performed, Is in a state where ions derived from a plurality of types of peptides coexist, it complicates interpretation of MS / MS data and cannot identify an exact amino acid sequence.

  That is, the problem to be solved by the present invention is that mass spectrometry that can identify or estimate the amino acid sequence of each peptide with high accuracy even when isotope peaks derived from a plurality of peptides overlap. Is to provide a system.

A mass spectrometry system according to the present invention made to solve the above problems is a mass spectrometry system for identifying an amino acid sequence of a peptide mixture in a test sample,
a) mass spectrometry means for performing MS / MS measurement by selecting some of the isotope peaks obtained by MS measurement of the peptide mixture;
b) A peak list in which isotope peak groups obtained by MS / MS measurement by the mass spectrometry means are classified according to the number of peaks constituting each isotope peak group, and a peak list is created for each class. Creating means;
c) sequence identification means for identifying or estimating an amino acid sequence based on each peak list created by the peak list creation means;
It is characterized by having.

  Here, it is desirable that the mass spectrometric means comprises a mass spectrometer equipped with a quadrupole ion trap.

In the present invention, “MS / MS measurement” means that a predetermined precursor ion is cleaved, and the obtained ion (product ion) is separated and detected based on a mass-to-charge ratio. It shall include not only the MS / MS analysis to be performed but also so-called MS ⁿ analysis in which the target molecule is cleaved multiple times.

  In addition, as the “partial peaks”, a plurality of peaks may be selected. However, in order to facilitate data analysis, a single peak is selected and MS / MS measurement is performed. It is desirable to make it. Furthermore, it is desirable to select the single peak having the maximum ionic strength from the group of isotope peaks obtained by MS measurement.

  According to the mass spectrometry system of the present invention having the above-described configuration, the amino acid sequences of individual peptides can be obtained with high accuracy even in the case where isotope peaks derived from different peptides overlap in the MS data of the peptide mixture. Can be identified or estimated.

As a mass spectrometry means according to the mass spectrometry system of the present invention, a mass spectrometer capable of performing MS / MS analysis (or MS ⁿ analysis) by selecting a precursor ion with high resolution is used. As such a mass spectrometer, for example, a time-of-flight mass spectrometer equipped with a quadrupole ion trap can be suitably used. In addition, it is desirable that the mass spectrometer performs ionization of a sample by a matrix assisted laser desorption / ionization (MALDI) method. However, the configuration of the mass spectrometric means in the mass spectrometric system of the present invention is not limited to this, and the precursor ions may be selected and cleaved by other means, the sample may be ionized, and the mass may be separated.

  In the mass spectrometry system of the present invention, the peak list creating means classifies a plurality of isotope peak groups appearing on the MS / MS data by the mass analyzing means based on the number of peaks constituting each peak group. A peak list is created for each class. In the MS / MS measurement using only a part of the isotope peak group by the mass spectrometry means as a precursor ion, the number of peaks constituting the isotope peak group of the product ion generated by cleavage of the precursor ion is the number of the precursor ion. It is limited by the maximum number of isotopes contained in (details will be described later). Therefore, when a peak in which peaks derived from different peptides overlap is selected as a precursor ion and the maximum number of isotopes in the precursor ion is different for each peptide, each isotope peak group on the MS / MS data Isotope peaks derived from different peptides can be distinguished from each other on the basis of the number of peaks constituting.

  The sequence identification means identifies the amino acid sequence of the peptide from which those peaks are derived based on the peak list created by the peak list creation means. As a method for identifying an amino acid sequence from the peak list, for example, by querying the peak list with a database, a known peptide having a matching spectral pattern can be searched, or each peak list can be analyzed mathematically. A method for estimating the amino acid sequence can be used.

  FIG. 1 shows a schematic configuration of a mass spectrometry system according to the present embodiment. The mass spectrometry system of the present embodiment is roughly composed of a mass analysis unit 10 and a control / processing unit 20. The mass spectrometric unit 10 selects an ion having a predetermined mass-to-charge ratio as a precursor ion and an ionization unit 11 that ionizes a sample containing a peptide mixture by the MALDI method, and generates product ions by cleaving the precursor ion. A quadrupole ion trap unit 12 and a time-of-flight mass separation unit 13 that separates and detects ions based on a mass-to-charge ratio are provided.

  The control / processing unit 20 processes a signal from a control unit 21 that controls each unit of the mass analysis unit 10 and an ion detector 14 provided in the mass separation unit 13 to obtain a mass spectrum (MS data, MS / MS data). ) And a data processing unit 22 for performing a predetermined analysis such as selection of a precursor ion, creation of a peak list described later, identification of an amino acid sequence, and the like. The control / processing unit 20 is connected to the Internet, and can search public genome databases, protein databases 30 and the like using a search engine such as MASCOT (Matrix Science). The function of the control / processing unit 20 can be realized by installing predetermined software on a personal computer.

  Hereinafter, the procedure for identifying the amino acid sequence of the peptide mixture using the mass spectrometry system of the present example will be described based on the flowchart of FIG.

1. MS measurement First, MS measurement is performed using a test sample containing a peptide mixture (S1). At this time, as shown in FIG. 3, it is assumed that an isotope peak group derived from peptide A having the following sequence and an isotope peak group derived from peptide B overlap on the obtained MS data. .
Peptide A (4000.00 Da); IADHHYALVPPVGGLTPGTATEYEVLLDGTGVWPPPSR
Peptide B (4001.00 Da); SGTLTYEAAVHQLSEAGVGQSTAVGIGGDDPVNGTNFIDVLK

2. Selection of Precursor Ion The peak (4003 Da) having the highest intensity is selected as the precursor ion from the isotope peak group in FIG. 3 (S2). This peak is obtained by overlapping the fourth peak of peptide A and the third peak of peptide B.

3. MS / MS Measurement The precursor ions are selected and cleaved by the ion trap unit 12, and the product ions generated by the cleavage of the precursor ions are separated and detected by the mass separation unit 13 (S3). Here, since the peak (4003 Da) on the MS data selected as the precursor ion has m / z 3 larger than the peak (4000 Da) of the main ion of peptide A, peptide A contained in the peak This isotope ion contains at most three isotopes other than the main isotope (hereinafter simply referred to as isotopes). Similarly, since the peak has a larger m / z 2 than the peak of the main ion of peptide B (4001 Da), the isotope ion of peptide B contained in the peak contains at most two isotopes. Will be. Therefore, 0 to 3 isotopes are included in the product ions derived from peptide A generated by the cleavage of the peak. Therefore, the product ions derived from peptide A are 4 in the MS / MS data. An isotope peak group consisting of peaks (quadruple peaks) is formed. Similarly, since the product ion derived from peptide B contains 0 to 2 isotopes, the product ion derived from peptide B has an isotope peak group consisting of 3 peaks (triple peak). ).

4). Extraction of Isotope Peak Group Next, in the MS / MS data, a peak group including a peak having an intensity equal to or higher than a predetermined value and a peak appearing every 1 Da is extracted (S4).

5. Classification according to the number of isotope peaks The extracted peak group is classified based on the number of peaks constituting the isotope peak group (S5). Here, the isotope peak group of product ions (b10, b20, b30, b37) derived from peptide A and peptide B is a class A in which 4 peaks are collected and a peak is 3 in number. Are classified into class B (FIG. 4).

  In a normal analysis, even if a peak selected as a precursor ion is an overlap of peaks derived from different peptides, the peak corresponding to each peptide is known. Therefore, the number of isotope peaks that appear on the MS / MS data cannot be predicted in advance. However, as long as the maximum number of isotopes contained in the precursor ions is different among the peptides, the product ions generated from the precursor ions are isotopic peak groups having different numbers of peaks for each peptide on the MS / MS data. Form. Therefore, if the isotope peak groups on the MS / MS data are classified based on the difference in the number of peaks, each class includes only the isotope peak groups of product ions derived from the same peptide. .

6). Creating a peak list for each class In the MS / MS data of each class, determine the monoisotopic peak (the peak of the ion composed of the isotope with the highest natural abundance ratio) within the isotope peak group according to a predetermined algorithm, A peak list describing the mass-to-charge ratio and the like is created (S6).

7). Amino acid sequence identification for each peak list As described above, a peak list A for peptide A and a peak list B for peptide B are obtained, so a database search for each peak list or a DeNovo sequence by MS / MS data mathematical analysis, etc. To identify or estimate the amino acid sequences of peptide A and peptide B (S7).

  As described above, according to the mass spectrometry system of the present invention, even if isotope peak groups derived from different peptides are overlapped, a part of the peaks is selected as a precursor ion from the peak group, and By classifying isotope peak groups on MS / MS data based on the number of peaks, isotopic peak groups derived from different peptides can be easily distinguished, so only peaks derived from individual peptides are described. The amino acid sequence can be identified using the peak list. Therefore, compared to the conventional case where identification is performed using a peak list in which peaks derived from a plurality of peptides are mixed, false positives can be reduced, and a highly accurate identification result can be obtained.

BRIEF DESCRIPTION OF THE DRAWINGS The schematic block diagram which shows one Example of the mass spectrometry system of this invention. The flowchart which shows the procedure of the amino acid sequence identification using the mass spectrometry system of the Example. The figure which shows MS data in the mass spectrometry system of the Example. The figure which shows the classification result of MS / MS data in the mass spectrometry system of the Example. The figure which shows an example of MS data of a peptide mixture.

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 ... Mass analysis part 11 ... Ionization part 12 ... Ion trap part 13 ... Mass separation part 14 ... Ion detector 20 ... Control / processing part 21 ... Control part 22 ... Data processing part 30 ... Database

SEQ ID NO: 1: amino acid sequence of peptide A SEQ ID NO: 2: amino acid sequence of peptide B

Claims (4)

A mass spectrometry system for identifying the amino acid sequence of a peptide mixture in a test sample, comprising:
a) mass spectrometry means for performing MS / MS measurement by selecting some of the isotope peaks obtained by MS measurement of the peptide mixture;
b) A peak list in which isotope peak groups obtained by MS / MS measurement by the mass spectrometry means are classified according to the number of peaks constituting each isotope peak group, and a peak list is created for each class. Creating means;
c) sequence identification means for identifying or estimating an amino acid sequence based on each peak list created by the peak list creation means;
A mass spectrometric system comprising:   The mass spectrometry according to claim 1, wherein the mass spectrometric means performs MS / MS measurement by selecting a single peak from a group of isotope peaks obtained by MS measurement of a peptide mixture. system.   The mass spectrometry system according to claim 2, wherein a peak having the maximum ionic strength is selected from the group of isotope peaks obtained by MS measurement of a peptide mixture as the single peak. The mass spectrometric system according to any one of claims 1 to 3, wherein the mass spectrometric means comprises a mass spectroscope equipped with a quadrupole ion trap.

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