JP2006170710A - Method of extracting peak and program for executing the same method - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a method of extracting peaks in an MS/MS spectrum for efficiently and precisely performing proteome analysis using the MS/MS spectrum and to provide a program for executing the same method. <P>SOLUTION: The method of extracting peaks in a spectrum comprising a plurality of peaks includes: (1) a step for acquiring the spectrum; (2) a step for detecting at least two peaks as one peak group from among the plurality of peaks when at least two peaks are contiguous each having a height value equal to or higher than a prescribed proportion of the maximum value of peak heights; and (3) a step for calculating one typical peak by weighted averaging from each of the respective peak groups. <P>COPYRIGHT: (C)2006,JPO&NCIPI

Description

  The present invention relates to analysis of a mass spectrometry spectrum used for proteome analysis, and more particularly to a method for extracting a peak in an MS / MS spectrum used for proteome analysis and a program for executing the method.

  Currently, human genome analysis has been completed and we are entering the post-genomic era. Here, the “genome” is, for example, the total DNA contained in one cell including all human genes and intergenic regions. In this post-genomic era, it has become increasingly important to isolate and identify proteins that are important as biomolecules in vivo and to further quantify them. In particular, it is necessary to elucidate the functions of many proteins in research and development of disease diagnosis and treatment techniques.

  Conventionally, two-dimensional electrophoresis has been used for proteome analysis of proteins expressed by cells. Here, “proteome analysis” refers to analysis that reveals the relationship between genetic information and various proteins that interact in a complex manner in cells (for example, see Non-Patent Document 1). In other words, proteome analysis refers to a method of comprehensively analyzing all proteins constituting a cell.

  In the above-described two-dimensional electrophoresis, an expressed protein is gel-developed, and the types can be comprehensively identified by cutting out spots corresponding to the target protein. Therefore, two-dimensional electrophoresis is a useful qualitative analysis means in proteome analysis. However, it is pointed out that two-dimensional electrophoresis is not suitable for quantitative analysis of proteins because the amount of protein to be developed is small and errors are likely to occur in the recovery rate at the time of analysis. .

  On the other hand, mass spectrometry is used as another important protein analysis technique. This method is a method for analyzing an accurate mass of a protein or peptide using a mass spectrometer. This mass spectrometer generally includes a device for ionizing proteins and peptides, a mass separation unit for separating the ions according to the masses of the ionized proteins and peptides, and a mass spectrometer for analyzing the mass. . Today, it can be said that the identification of proteins has been greatly facilitated by the mass spectrometer and the database of proteins and the search system connecting them. Therefore, it is possible to comprehensively identify a specific protein mixture (for example, a protein complex that forms a certain complex).

  Currently, the identification of protein mixtures typically uses the MS / MS method. This MS / MS method uses a plurality of mass spectrometry separation units, and one of the ion species produced in the ionization chamber of the first mass separation unit (MS1) for the protein in the sample is subjected to mass analysis of precursor ions. The spectrum is selected as a spectrum (hereinafter sometimes referred to as “MS spectrum”), and the precursor ion is collided with an inert gas such as argon and fragmented in the second mass separation unit (MS2). This is a method for analyzing a protein by detecting a spectrum of product ions. In the MS / SM method, the spectrum of this product ion (hereinafter sometimes referred to as “MS / MS spectrum”) is measured and NCBInr (HYPERLINK "http://www.ncbi.nlm.nih.gov/" http://www.ncbi.nlm.nih.gov/) etc. are identified based on the result of collation with a database to identify a protein. And even if a plurality of proteins coexist in a sample, there is an advantage that a protein can be specified from each precursor ion.

FIG. 1 shows a schematic of the MS spectrum and MS / MS spectrum obtained with a sample of the protein mixture. As shown in FIG. 1, the MS spectrum and the MS / MS spectrum show the spectrum results obtained by measuring the sample digested in the gel with respect to the band cut out from the two-dimensional electrophoresis result with a mass spectrometer. The MS spectrum of the ions is obtained by the aforementioned MS1, and then the MS / MS spectrum of the product ions is obtained by MS2 for each MS spectrum. Subsequently, the protein can be identified by comparing the obtained MS / MS spectrum with a database. In the example shown in FIG. 1, it can be identified as a mixture of protein A and protein B.
Karn, P. Science 270, pp 369-370, 1995

However, the actually measured MS / MS spectrum is a very complex spectrum particularly when a plurality of proteins are mixed in the sample to be measured. Therefore, a problem has been pointed out that the proteome analysis itself using the MS / MS spectrum is not efficient.
Therefore, in view of the above circumstances, the present invention provides a method for extracting a peak in an MS / MS spectrum and a program for executing the method in order to efficiently and accurately perform a proteome analysis using an MS / MS spectrum. For the purpose.

  As a result of intensive studies to solve the above problems, the present inventors can realize efficient proteomic analysis by performing peak extraction based on a specific relational expression in an MS / MS spectrum having a plurality of peaks. As a result, the present invention has been completed.

That is, in the first aspect, the present invention is a method for extracting a peak in a spectrum having a plurality of peaks, and (1) a step of acquiring the spectrum;
(2) When at least two peaks having a height greater than or equal to a predetermined ratio of the maximum peak height are consecutive from the plurality of peaks, the at least two peaks are grouped into one peak group. And (3) a centroid step of calculating one representative peak from each peak group using a weighted average. By this method, unnecessary peaks such as noise can be excluded from the analysis target, and the peak to be analyzed can be narrowed down, thereby realizing efficient analysis.

（ここで、ｍは生体分子の質量であり、ｚは生体分子の電荷であり、Ｉ_ijおよびｍ／ｚ_ijは、それぞれ、前記一つの代表的ピークの強度と、ｍ/ｚの値であり、kは、ピーク群に含まれるｍ／ｚを特定する値であり、iからjの値が含まれ、ｙ_kは、ｍ／ｚ_kに対する強度であり、h₀はピーク強度の最大値を示し、aは前記所定の割合を示す。）
かかる式を用いることで、ＭＳ／ＭＳスペクトルの解析が的確かつ迅速に行うことができる。 According to a preferred aspect of the extraction method of the present invention, the spectrum includes an MS spectrum and / or an MS / MS spectrum expressed in intensity with respect to m / z related to a biomolecule, and the step (3 The weighted average in) is executed according to the following formula.

(Where m is the mass of the biomolecule, z is the charge of the biomolecule, and I _ij and m / z _ij are the intensity of the one representative peak and the value of m / z, respectively. , K is a value specifying m / z included in the peak group, i to j are included, y _k is the intensity with respect to m / z _k , and h ₀ is the maximum peak intensity. A represents the predetermined ratio.)
By using such a formula, the analysis of the MS / MS spectrum can be performed accurately and rapidly.

  According to a preferred aspect of the extraction method of the present invention, (4) using the m / z value of each peak included in the peak group and the m / z values of peaks adjacent to the peak group before and after. And calculating a ratio of the maximum value to the minimum value of the interval between the m / z values of adjacent peaks, and when the ratio exceeds a predetermined value, further includes a step of removing the peak group. By determining whether or not the detection of the peak group is correct, a more accurate peak extraction method is realized.

  According to a preferred aspect of the extraction method of the present invention, (5) a plurality of representative peaks in which the m / z value of the representative peak calculated for each peak group is within a specific value are defined as one peak. The specific value is determined based on the molecular weight of the biomolecule, and the intensity of the one peak after the cluster processing is a representative peak to be clustered. The sum of the intensities is calculated, and the m / z value of the one peak after the cluster processing is calculated by weighted averaging the m / z of the representative peak to be clustered with the intensity as a weight. . In this way, more efficient analysis can be performed by arranging the peaks to be analyzed.

  According to a preferred aspect of the extraction method of the present invention, when one MS / MS spectrum has one data set relating to the spectrum, and a plurality of MS / MS spectra are acquired in the step (1), the step Prior to (2), (a) the difference in m / z of the MS spectrum of the precursor ion of each MS / MS spectrum is within a certain range, and the difference in the acquisition timing of the data set of each MS / MS spectrum is The method further includes the step of performing a merging process to combine a plurality of data sets within a certain range. As described above, by adjusting the data set of the MS / MS spectrum before the peak group is detected, the peak group can be efficiently detected.

  According to the preferable aspect of the extraction method which concerns on this invention, the said process (2)-(4) is performed after the said process (a). By performing the steps (2) to (4) after the merge process, the efficiency of peak extraction can be improved.

（ここで、ｍは生体分子の質量であり、ｚは生体分子の電荷であり、Ｉ_ijおよびｍ／ｚ_ijは、それぞれ、前記一つの代表的ピークの強度と、ｍ/ｚの値であり、kは、ピーク群に含まれるｍ／ｚを特定する値であり、iからjの値が含まれ、ｙ_kは、ｍ／ｚ_kに対する強度であり、h₀はピーク強度の最大値を示し、aは前記所定の割合を示す。）
前記［８］に記載のプログラム、
［１０］（４）前記ピーク群に含まれる各ピークのｍ／ｚの値と、前記ピーク群の前後に隣接するピークのｍ／ｚの値とを用いて、隣接するピークのｍ／ｚの値の間隔の最小値に対する最大値の割合を算出し、該割合が所定の値を超える場合、そのピーク群を除去する工程をさらに、コンピュータに実行させる、前記［８］または［９］に記載のプログラム、
［１１］（５）各ピーク群ごとに算出された代表的ピークのｍ／ｚの値が特定の値以内にある複数の代表的ピークを一つのピークとするように、クラスタ処理する工程をさらに、コンピュータに実行させる、前記［８］ないし［１０］のうち何れか一に記載のプログラム、
［１２］前記特定の値は、生体分子の分子量に基づいて決定され、クラスタ処理後の前記一つのピークの強度は、クラスタ処理の対象となる代表的ピークの強度の総和として算出し、クラスタ処理後の前記一つのピークのｍ／ｚの値は、クラスタ処理の対象となる代表的ピークのｍ／ｚを、前記強度を重みとして加重平均して算出される、前記［１１］に記載のプログラム、
［１３］一つのＭＳ／ＭＳスペクトルがスペクトルに関する一つのデータセットを有し、前記工程（１）にて複数のＭＳ／ＭＳスペクトルを取得する場合、前記工程（２）に先立ち、（ａ）各ＭＳ／ＭＳスペクトルの前駆イオンのＭＳスペクトルのｍ／ｚの差が一定の範囲内にあり、かつ、各ＭＳ／ＭＳスペクトルのデータセットの取得タイミングの差が一定の範囲内にある複数のデータセットを結合するマージ処理をする工程をさらに、コンピュータに実行させる、前記［８］ないし［１２］のうち何れか一に記載のプログラム、
「１４」前記工程（ａ）の後に、前記工程（２）〜（４）をコンピュータに実行させる、前記［８］ないし［１３］のうち何れか一に記載のプログラム、等を提供する。 In the second aspect, the present invention provides [8] a program for executing a method for extracting a peak in a spectrum having a plurality of peaks, (1) a step of acquiring the spectrum, and (2) the plurality of the plurality of peaks. Detecting at least two peaks as one peak group when at least two peaks having a height equal to or greater than a predetermined ratio of the maximum peak height are consecutive from (3) A program for causing a computer to execute an extraction method including a centroid step of calculating one representative peak by weighted average from each peak group,
[9] The spectrum includes an MS spectrum and / or an MS / MS spectrum expressed in intensity with respect to m / z related to a biomolecule, and the weighted average in the step (3) is according to the following formula: Executed,

(Where m is the mass of the biomolecule, z is the charge of the biomolecule, and I _ij and m / z _ij are the intensity of the one representative peak and the value of m / z, respectively. , K is a value specifying m / z included in the peak group, i to j are included, y _k is the intensity with respect to m / z _k , and h ₀ is the maximum peak intensity. A represents the predetermined ratio.)
The program according to [8],
[10] (4) Using the m / z value of each peak included in the peak group and the m / z values of adjacent peaks before and after the peak group, The ratio according to [8] or [9], wherein a ratio of a maximum value to a minimum value of a value interval is calculated, and if the ratio exceeds a predetermined value, a step of removing the peak group is further executed by a computer. Programs,
[11] (5) A step of performing cluster processing so that a plurality of representative peaks whose m / z values of the representative peaks calculated for each peak group are within a specific value are defined as one peak. A program according to any one of [8] to [10], which is executed by a computer,
[12] The specific value is determined based on the molecular weight of the biomolecule, and the intensity of the one peak after cluster processing is calculated as the sum of the intensities of representative peaks to be subjected to cluster processing. The program according to [11], wherein the m / z value of the later one peak is calculated by weighted averaging the m / z of a representative peak to be clustered using the intensity as a weight. ,
[13] When one MS / MS spectrum has one data set relating to the spectrum and a plurality of MS / MS spectra are acquired in the step (1), prior to the step (2), (a) A plurality of data sets in which the difference in m / z of the MS spectrum of the precursor ion of the MS / MS spectrum is within a certain range, and the difference in the acquisition timing of each MS / MS spectrum data set is within the certain range The program according to any one of [8] to [12], further causing a computer to execute a merge process of combining
[14] After the step (a), the program according to any one of the above [8] to [13] is provided, which causes the computer to execute the steps (2) to (4).

  The program according to the present invention causes each step of the extraction method according to the present invention to be executed on a computer. The program according to the present invention can be installed or downloaded in a computer through various recording media such as a CD-ROM, a magnetic disk, and a semiconductor memory, or by downloading via a communication network.

  According to the peak extraction method and the program for executing the method according to the present invention, the analysis of the mass spectrometry spectrum can be performed more efficiently. In particular, the peak extraction method according to the present invention is excellent as peak extraction in a Qq-TOFMS / MS apparatus.

  Embodiments of the present invention will be described with reference to the drawings. The following embodiment is an example for explaining the present invention, and is not intended to limit the present invention only to this embodiment. The present invention can be implemented in various forms without departing from the gist thereof.

  FIG. 2 shows an example of a mass spectrometry spectrum of a biomolecule, for example, a protein, as a spectrum having a plurality of peaks targeted by the present invention. The spectrum shown in FIG. 2 is an MS / MS spectrum and has many peaks including noise, so that it is expected that subsequent analysis is not easy.

  FIG. 3 shows a hardware configuration diagram of the apparatus 10 for executing the peak extraction method according to the present invention. As shown in FIG. 3, a device 10 used in the present invention includes a CPU 12, an input device 14 such as a mouse and a keyboard, a display device 16 including a CRT, a RAM (Random Access Memory) 18, a ROM (Read Only Memory) 20, a portable recording medium driver 22 that accesses a portable recording medium 23 such as a CD-ROM or DVD-ROM, a hard disk device 24, and a communication control interface (I / O) that controls data exchange with the outside. F) 26. The apparatus 10 used in the present invention is connected to the mass spectrometer 30 via the input / output control I / F, and is analyzed by the data obtained by the mass spectrometer 30 and the apparatus 10 used in the present invention. Can communicate with data. As shown in FIG. 3, a personal computer or the like can be used as the apparatus 10 according to the present embodiment.

  The peak extraction method according to the present invention will be described by exemplifying an MS / MS spectrum obtained by the mass spectrometer 30, but the present invention is not limited to the MS / MS spectrum alone.

  Examples of the mass spectrometer 30 used in the present invention include an apparatus for measuring a tandem MS / MS method in which at least two mass separation units are combined. In the tandem MS / MS method, a collision chamber is provided between the first mass spectrometer and the second mass spectrometer, and the precursor ions generated by decomposition in the first mass spectrometer collide with an inert gas. To generate product ions.

  Specifically, two mass separation units will be described as an example. In a preferred embodiment of the present invention, the mass separation unit of the first mass spectrometer is a Q filter type, and the mass separation unit of the second mass spectrometer is used. A Qq-TOFMS / MS apparatus having a TOF type is used. The Q filter type mass spectrometer uses four columnar electrodes arranged in parallel and uses a quadrupole electric field obtained by applying a DC current and an AC current between them to mass-separate ions. It is a mass spectrometer. A mass analysis spectrum is obtained by scanning while varying the voltage applied to the quadrupole. By fixing the voltage, only ions having a specific m / z value can be passed, that is, so-called mass filtering can be performed. On the other hand, the TOF type mass spectrometer is a method for mass-separating ions using the fact that the time required for ions to pass through a vacuum analysis tube of a certain length varies depending on the difference in ion m / z. Mass spectrometer.

  In this way, the MS / MS spectrum data obtained by the mass spectrometer 30 used in the present invention is sent to the apparatus 10 used in the present invention, and desired operation and analysis are performed.

  The aforementioned portable recording medium 23 stores a program for executing the peak extraction method used in the present invention. Therefore, the portable recording medium drive 22 reads out the program from the portable recording medium 23, stores it in the hard disk device 24, and starts it so that the personal computer uses the device in the present invention. 10 can be operated. Alternatively, the program may be downloaded via an external network such as the Internet.

  FIG. 4 is a functional block diagram of the main part of the apparatus 10 according to the present invention. The apparatus 10 includes an input / output unit 51 that acquires an MS spectrum and / or an MS / MS spectrum measured by the mass spectrometer 30, and a data storage unit 52 that stores the MS spectrum and / or the MS / MS spectrum. . Furthermore, the apparatus 10 used in the present invention includes a peak group detection unit 54, a centroid processing unit 55, a peak group removal unit 56, and a cluster processing unit 57 for executing the peak extraction method according to the present invention. In a preferred aspect of the present invention, the apparatus 10 further includes a merge processing unit 53.

  First, in the apparatus 10 used in the present invention, the MS spectrum and / or MS / MS spectrum measured by the mass spectrometer 30 is acquired by the input / output unit 51. The acquired various spectrum data can be stored in a data storage unit as necessary, and can be used in the peak extraction method according to the present invention described later.

  Hereinafter, the peak extraction method according to the present invention will be described using MS / MS spectrum data. Typically, as illustrated in FIG. 2, the MS / MS spectrum has a plurality of peaks. Therefore, by performing the extraction method according to the present invention, the MS / MS spectrum is efficiently extracted, which is useful for the accuracy and speed of proteome analysis.

  FIG. 5 is a flowchart showing an outline of the peak extraction method according to the present invention. As shown in FIG. 5, in step S10, an MS spectrum and / or an MS / MS spectrum measured by the mass spectrometer 30 is acquired. In step S11, the peak group detection unit 54 performs peak group detection on the acquired spectrum based on a certain rule. Next, the centroid processing unit 55 performs centroid processing described later on each detected peak group, and calculates a representative peak from each peak group (see step S12 in FIG. 5). Thereafter, in order to evaluate the validity of the above-described peak group detection, in step S13, the peak group removal unit 56 includes a step of removing unnecessary peak groups from comparison with peaks before and after the detected peak group. Execute. In order to further sort out the peak group from the peak group remaining after the removal, the cluster processing unit 57 collects each peak group based on the m / z value calculated for each peak group. Cluster processing is performed (see step S14), and peaks to be analyzed are efficiently extracted. In a preferred embodiment of the present invention, although not shown in FIG. 5, it is preferable to perform a merge process described later prior to step S11. Below, each process S10-S14 and a merge process process are explained in full detail.

(Peak Group Detection Step in Peak Group Detection Unit 54)
The peak group detection unit 54 is a step of detecting a peak group by combining a plurality of peaks to be analyzed from a certain relational expression from MS / MS spectrum data having a plurality of peaks. FIG. 6 is a diagram for explaining peak group detection in the peak extraction method according to the present invention, and is an enlarged schematic view of the area A in FIG. The MS / MS spectrum shown in FIG. 6 is an MS / MS spectrum acquired by the input / output unit 51, and the spectrum is a spectrum displayed with m / z on the x-axis and intensity on the y-axis.

First, the peak having the maximum peak height (h ₀ ) in one MS / MS spectrum, and the peak having the maximum intensity in the example of FIG. 6 is obtained. Secondly, attention is paid to a peak having the height, that is, a height (intensity) having a value equal to or higher than a predetermined ratio of the intensity. Here, the reason for using the maximum peak as a reference is that the influence of noise or the like is the least in the peak, and it is estimated that the peak itself is reliable. Further, the predetermined ratio is a ratio that can be appropriately selected in view of the accuracy of the subsequent analysis. Third, when at least two peaks having intensities of a predetermined ratio or more are consecutive, the at least two peaks are detected as one peak group. In the example of FIG. 6, the predetermined ratio is set to 0.5, and the peaks surrounded by P ₀ are detected as one peak group.

（ここで、ｍは生体分子の質量であり、ｚは生体分子の電荷であり、Ｉ_ijおよびｍ／ｚ_ijは、それぞれ、前記一つの代表的ピークの強度と、ｍ／ｚの値であり、kは、ピーク群に含まれるｍ／ｚを特定する値であり、iからjの値が含まれ、ｙ_kは、ｍ／ｚ_kに対する強度であり、aは前記所定の割合を示し、図６および図７では０．５として表示されている。） (Centroid processing in the centroid processing unit 55)
The centroid process shown in step S12 is a process of calculating what is detected as the aforementioned peak group as one representative peak. In step S12, a representative peak of the peak group is calculated from the x-axis and y-axis values included in the peak group by a centroid process called weighted average and used for subsequent spectrum analysis. The “centroid process” used in the present invention is a process for calculating a representative peak of a peak group based on the following formula.

(Where m is the mass of the biomolecule, z is the charge of the biomolecule, and I _ij and m / z _ij are the intensity of the one representative peak and the value of m / z, respectively. , K is a value for specifying m / z included in the peak group, i to j are included, y _k is an intensity with respect to m / z _k , a indicates the predetermined ratio, 6 and 7 are displayed as 0.5.)

  Equation (1) is a method of calculating the intensity that is the y-axis, and equation (2) is a method of calculating m / z of the x-axis. In this way, the representative peak can be calculated for each peak group. The y-axis intensity is the sum of peak intensities included in the peak group, and the x-axis m / z is a weighted average value of m / z included in the peak group. Moreover, the centroid process used in the present invention may be a process for calculating a representative peak from each detected peak group as described above, and the above-described calculation method is an example, and equations (1) and ( It is not limited to the calculation method of 2).

FIG. 7 shows an example of representative peak calculation from a peak group calculated by centroid processing in the peak extraction method according to the present invention. From the illustration of FIG. 7, the contents of the centroid processing for one peak group detected in step S11 can be easily understood. Specifically, the maximum intensity of the peak group P ₀ is 22, and when the value of a is 0.5, the peak intensity in the range of m / z _{1 to} m / z ₇ is 50% of the maximum intensity. It is continuous as 11 or more. Then, the peak intensity in the range of _{m / z 1 ~m / z 7} , and displays a coordinate S ₁ to S _7. When a new v-axis is created on the vertical axis based on the intensity 11 that is 50% of the maximum intensity, and the peak intensity in the range of m / z _{1 to} m / z ₇ is recalculated, v1 = 1,. , V7 = 6 (see FIG. 7). Thereafter, the total sum I (P ₀ ) of the intensity displayed on the v-axis is calculated, and m / z (P ₀ ) is calculated by performing a weighted average on the m / z axis. In this manner, the representative peak of the detected peak group can be calculated by the centroid processing used in the present invention.

(Peak removal processing in the peak group removal unit 56)
As described above, step 13 is a step of calculating a representative peak of the detected peak group, evaluating the validity of the detection of the peak group, and removing unnecessary peak groups. In the present invention, peak removal processing is performed using the m / z value of each peak included in the detected peak group and the m / z values of the peaks adjacent to the front and back of the peak group.

（ここで、ｌは、各ピーク群に含まれるｍ／ｚの値と、前記各ピーク群の前後に隣接するｍ／ｚの値を表す。）
式（３）に基づき、前記ｄの値の最小値に対する最大値の割合εが、所定の値を超える場合、かかるピーク群Ｐは除去する。ここで、εの値が大きい場合とは、ピーク群に包含される各ピーク間隔が不規則であり、ピーク自体の信頼性が低いと予想されるため、かかるピーク群を除去する。εの値としては、その後の解析の精度に鑑みるに、適宜選択可能な割合である。かかるピーク群除去により、より精度の高いピーク抽出が可能となる。なお、本発明で用いるピーク群除去処理は、前述のように、ピーク群の検出の妥当性を評価し、不必要なピーク群を除去する処理をいい、前述の算出法は例示であり、式（３）の算出法に限定されるものではない。 FIG. 8 is a diagram for explaining a peak removal step in the peak extraction method according to the present invention. In FIG. 8, m / z of each peak included in the peak group indicated by P is m / z _{1 to} m / z ₄ and is not included in the peak group and is adjacent to the peak group before and after. M / z is represented by m / z ₀ and m / z ₅ . In the present invention, as an example of peak removal, the interval between adjacent m / z values in m / z _{0 to} m / z ₅ is represented by d, and the peak group is removed by the following equation (3). Judge whether or not.

(Here, l represents the value of m / z included in each peak group and the value of m / z adjacent before and after each peak group.)
Based on Expression (3), when the ratio ε of the maximum value to the minimum value of d exceeds a predetermined value, the peak group P is removed. Here, when the value of ε is large, each peak interval included in the peak group is irregular and it is expected that the reliability of the peak itself is low. Therefore, the peak group is removed. The value of ε is a ratio that can be appropriately selected in view of the accuracy of subsequent analysis. Such peak group removal enables more accurate peak extraction. Note that, as described above, the peak group removal process used in the present invention refers to a process for evaluating the validity of detection of peak groups and removing unnecessary peak groups. It is not limited to the calculation method of (3).

(Cluster processing in the cluster processing unit 57)
For each peak group remaining by the above-described peak group removal processing, cluster processing is performed to collect peak groups having a certain relationship. Here, each peak group is, for example, a peak group detected in the A region and the B region shown in FIG. 2 and left by the subsequent peak group removal process. Further, the certain relationship is performed based on the m / z value of the representative peak of each peak group. In the present invention, for example, the difference in the m / z value of the representative peak is 2 Da (Da: biological body). In the case of Dalton, which represents the mass of a molecule), cluster processing is performed so that a representative peak of each peak group becomes one peak. This is because, if the representative peaks are adjacent, it is considered that the precursor ions of the MS / MS spectrum are the same or very close to each other, so that it is possible to extract peaks with high accuracy by performing cluster processing.

  In the case of cluster processing in the present invention, the peak intensity obtained by clustering representative peaks and the value of m / z are the sum of representative peak intensities subjected to cluster processing and the intensity as a weight. Calculated as m / z obtained by weighted averaging. The peaks extracted in this way can be displayed as a peak list using the sum of the representative peak intensities and the m / z value obtained by weighted averaging using the intensities as weights. As described above, the cluster process used in the present invention may be a process that collects representative peaks, and is limited to the properties of precursor ions as long as the parameters suggest that the representative peaks are adjacent. Is not to be done.

(Merge processing by the merge processing unit 53)
In a preferred embodiment of the peak extraction method according to the present invention, the merge processing described below is performed before the peak group detection in step S11. Merge processing means that one MS / MS spectrum has one data set related to the spectrum, and a plurality of MS / MS spectra are acquired.
Before the above-described peak group detection, the data sets of the plurality of MS / MS spectra are combined under certain conditions. The “data set” used here is the MS / MS spectrum shown in FIG. 2 displayed with the m / z axis and intensity as coordinates, and one data set related to one MS / MS spectrum is: The MS / MS spectrum shown in FIG. 2 refers to a series of data displayed with the coordinates. Therefore, if the MS / MS spectrum is different, the data set of the MS / MS spectrum is naturally different.

  The above-mentioned certain conditions used in the present invention are focused on the precursor ion of each MS / MS spectrum, the difference in the m / z value of the precursor ion is within a certain range, and each MS / MS spectrum This is a process of combining data sets when the difference in the acquisition timing of the data sets is within a certain range. Here, the difference in the m / z values of the precursor ions is within a certain range, for example, the difference is within a certain range based on the dalton, and acquisition of a data set of each MS / MS spectrum The difference in timing within a certain range means that, for example, the time when the MS / MS spectrum is acquired is within a certain range with the parent peak as a precursor ion in common. By performing this merging process, it is possible to extract peaks more efficiently by combining spectra having common precursor ions of MS / MS spectra. The merge process used in the present invention may be a process that combines MS / MS spectra based on the precursor ions before the MS / MS spectrum is generated. The difference in m / z value of the aforementioned precursor ions and the MS It is not limited to the process performed by the condition based on the difference in the acquisition timing of the / MS spectrum data set.

  After the merge process in step S11, peak extraction with higher accuracy is realized by performing the above-described steps S11 to S14.

  EXAMPLES Hereinafter, the present invention will be specifically described by way of examples, but these do not limit the scope of the present invention in any way.

1. Preparation of cell lysate
RPMI-1640 medium (Gibco BRL, Grand Island, NY) containing ¹³ C ₆ -leucine (Cambridge Isotope Laboratories, Andover, Mass.) Is based on the SILAC protocol (SE Ong, B. Blagoev, I. Kratchmarova , DB Kristensen, H. Steen, A. Pandey, M. Mann, Mol Cell Proteomics 1 (2002) 376.). Mouse neuroblastoma neuro2a cells were cultured in the above medium for ¹³ C ₆ -leucine labeling. Phosphate buffer containing protease inhibitor cocktail (Roche Diagnostics, Basel, Switzerland) was added to two dishes (15 cm in diameter, 80% confluent), and the protein was extracted using ultrasound. .

2. Preparation of peptide mixture for LCMSMS Proteins from cells were dried and resuspended in 50 mM Tris-HCl buffer (pH 9.0) containing 8 M urea. These mixtures were then reduced, alkylated, digested with Lys-C (Wako, Osaka, Japan), diluted 4-fold with 50 mM aqueous ammonium bicarbonate and then trypsin (Promega Corp., Madison, Wyoming, USA). ). The digestion solution was acidified with TFA, desalted and concentrated by C18-Stage Tips (J. Rappsilber, Y. Ishihama, M. Mann, Anal Chem 75 (2003) 663.).

3. Nano LC-MS / MS analysis
Utilizing QSTAR Pulsar I (ABI / MDS-Sciex, Toronto, Canada) with Shimadzu LC10A gradient pump, HTC-PAL autosampler (CTC Analytics AG, Thuringia, Switzerland) and Valco C2 valve with 150 μm port All samples were analyzed by nano LC-MS / MS. To prepare an analytical column needle with "stone-arch" frit (Y. Ishihama, J. Rappsilber, JS Andersen, M. Mann, J Chromatogr A 979 (2002) 233.) ReproSil C18 filler (3 μm, Dr. Maisch, Ammerbuch, Germany) was filled into a self-pulled needle (100 μm ID, 6 μm opening, 150 mm length) with a cell (Nikkyo Technos, Tokyo, Japan) . A column needle attached to a Velco metal connector with magnet is attached to a Teflon-coated column holder (Nikkyo Technos, Tokyo, Japan), and the Proxeon xyz nanospray interface (Odense, Denmark) has an adjustable spray position. equipped. The injection volume was 3 μL and the flow rate after the three-way splitter was 250 nL / min. As the mobile phase, (A) 0.5% acetic acid and (B) 0.5% acetic acid and 80% acetonitrile were used. The gradient conditions are 5% to 10% for the first 5 minutes, 10% to 30% for the next 5 minutes, 10% to 30% for the next 60 minutes, 30% to 100% for the next 5 minutes, and 100% for the last 10 minutes. A linear gradient was adopted. A spray voltage of 2400 V was applied through a metal connector. In MS scan (1 second), a maximum of 3 peaks were selected in order of intensity, after which MSMS scan was performed every 0.6 seconds for each parent ion. The Information Dependent Acquisition (IDA) function was set for 3 minutes to exclude the parent peak ions scanned earlier. The scan range was m / z 350 ··· 1400.

  FIG. 9 shows one MS spectrum (FIG. 9 (A)) obtained by an example according to the present invention and one MS / MS spectrum (FIG. 9 (B)) obtained from the MS spectrum. On the other hand, FIG. 10 shows the result of analyzing the data of one MS / MS spectrum obtained from the example according to the present invention by the peak extraction method according to the present invention. FIG. 10 (A) shows the intensity with respect to the m / z value of the peak of the raw data obtained from the example according to the present invention. FIG. 10 (B) shows typical peaks calculated from the results shown in FIG. 10 (A) according to the present invention according to the peak group detection and the above formulas (1) and (2). In the centroid processing of the present invention according to the formulas (1) and (2), a was calculated as 0.5 (50%). FIG. 10C shows the result of performing peak group removal from the result obtained in FIG. 10B according to the above equation (3). The value of ε in the above formula (3) was 2.9.

  Finally, from the results obtained in FIG. 10C, peak extraction was performed by cluster processing in the present invention. FIG. 10 (D) shows the peak group remaining after removal of the peak group. From the result obtained in FIG. 10 (C), the difference in the m / z value of the representative peak of each peak group is within 2 Da. In the case, the result after cluster processing in which each peak group is one peak group is shown. Note that the values shown in FIG. 10D are the peak intensity obtained by clustering representative peaks and the m / z values are the sum of the representative peak intensities subjected to cluster processing and the intensity, respectively. Was calculated as m / z obtained by weighted average using as a weight.

  From the above results, according to the peak extraction method according to the present invention and a program for causing a computer to execute the method, a more efficient execution of the analysis of the mass spectrometry spectrum is realized by extracting the useful peaks in the mass spectrometry spectrum. Can be made.

FIG. 1 shows an MS spectrum obtained from a sample of a protein mixture and a schematic diagram of the MS spectrum. FIG. 2 shows an example of a mass spectrometry spectrum of a biomolecule, for example, a protein, as a spectrum having a plurality of peaks targeted by the present invention. FIG. 3 shows a hardware configuration diagram of the apparatus 10 for executing the peak extraction method according to the present invention. FIG. 4 is a functional block diagram of the main part of the apparatus 10 according to the present invention. FIG. 5 is a flowchart showing an outline of the peak extraction method according to the present invention. FIG. 6 is a diagram for explaining peak group detection in the peak extraction method according to the present invention, and is an enlarged schematic view of the area A in FIG. FIG. 7 shows an example of representative peak calculation from a peak group calculated by centroid processing in the peak extraction method according to the present invention. FIG. 8 is a diagram for explaining a peak removal step in the peak extraction method according to the present invention. FIG. 9 shows (A) one MS spectrum obtained by an example according to the present invention, and (B) one MS / MS spectrum obtained from the MS spectrum. Note that the MS / MS spectrum shown in FIG. 9B is obtained by performing MS / MS treatment on a peak having a m / z value of 688.9 and a retention time of 82.562 minutes. It is the MS / MS spectrum obtained. FIG. 10 shows the result of analyzing the MS / MS spectrum data obtained from the example according to the present invention by the peak extraction method according to the present invention. FIG. 10A shows original MS / MS spectrum data. FIG. 10 (B) shows the result of the value of the representative peak detected in the peak group in the peak extraction method according to the present invention for the original MS / MS spectrum data. FIG. 10C shows the result of performing peak group removal from the result obtained in FIG. 10B according to the above equation (3). FIG. 10 (D) shows the peak group remaining after removal of the peak group. From the result obtained in FIG. 10 (C), the difference in the m / z value of the representative peak of each peak group is within 2 Da. In the case, the result after cluster processing in which each peak group is one peak group is shown.

Explanation of symbols

10: Device for executing the peak extraction method according to the present invention, 12: CPU, 14: input device, 16: display device, 18: RAM, 20: ROM, 22: portable recording medium driver, 23: portable recording medium 24: disk device, 26: communication control interface, 30: mass spectrometer

Claims (14)

A method for extracting a peak in a spectrum having a plurality of peaks, comprising:
(1) obtaining the spectrum;
(2) When at least two peaks having a height greater than or equal to a predetermined ratio of the maximum peak height are consecutive from the plurality of peaks, the at least two peaks are grouped into one peak group. Detecting as
(3) A centroid process for calculating one representative peak by weighted average from each peak group,
Extraction method including. The spectrum includes an MS spectrum and / or MS / MS spectrum expressed in intensity relative to m / z associated with a biomolecule;
The weighted average in the step (3) is executed according to the following formula:

(Where m is the mass of the biomolecule, z is the charge of the biomolecule, and I _ij and m / z _ij are the intensity of the one representative peak and the value of m / z, respectively. , K is a value specifying m / z included in the peak group, i to j are included, y _k is the intensity with respect to m / z _k , and h ₀ is the maximum peak intensity. A represents the predetermined ratio.)
The extraction method according to claim 1.   (4) Using the m / z value of each peak included in the peak group and the m / z value of adjacent peaks before and after the peak group, the interval between the m / z values of adjacent peaks The extraction method according to claim 1, further comprising a step of calculating a ratio of the maximum value with respect to the minimum value and removing the peak group when the ratio exceeds a predetermined value. (5) The method further includes a step of performing cluster processing so that a plurality of representative peaks whose m / z values of the representative peaks calculated for each peak group are within a specific value are defined as one peak. Item 4. The extraction method according to any one of Items 1 to 3.   The specific value is determined based on the molecular weight of the biomolecule, and the intensity of the one peak after cluster processing is calculated as the sum of the intensity of representative peaks to be subjected to cluster processing, 5. The extraction method according to claim 4, wherein the m / z value of one peak is calculated by performing a weighted average of m / z of a representative peak to be clustered using the intensity as a weight. When one MS / MS spectrum has one data set related to the spectrum and a plurality of MS / MS spectra are acquired in the step (1), prior to the step (2),
(A) The difference in m / z of the MS spectrum of the precursor ion of each MS / MS spectrum is within a certain range, and the difference in the acquisition timing of the data set of each MS / MS spectrum is within a certain range. The extraction method according to claim 1, further comprising a merge process of combining a plurality of data sets.   The extraction method according to any one of claims 1 to 6, wherein the steps (2) to (4) are performed after the step (a). A program for executing a peak extraction method in a spectrum having a plurality of peaks,
(1) obtaining the spectrum;
(2) When at least two peaks having a height greater than or equal to a predetermined ratio of the maximum peak height are consecutive from the plurality of peaks, the at least two peaks are grouped into one peak group. Detecting as
(3) A centroid process for calculating one representative peak by weighted average from each peak group,
A program for causing a computer to execute an extraction method including: The spectrum includes an MS spectrum and / or MS / MS spectrum expressed in intensity relative to m / z associated with a biomolecule;
The weighted average in the step (3) is executed according to the following formula:

(Where m is the mass of the biomolecule, z is the charge of the biomolecule, and I _ij and m / z _ij are the intensity of the one representative peak and the value of m / z, respectively. , K is a value specifying m / z included in the peak group, i to j are included, y _k is the intensity with respect to m / z _k , and h ₀ is the maximum peak intensity. A represents the predetermined ratio.)
The program according to claim 8.   (4) Using the m / z value of each peak included in the peak group and the m / z values of the adjacent peaks before and after each peak group, the m / z value of the adjacent peak The program according to claim 8 or 9, further comprising the step of calculating a ratio of the maximum value to the minimum value of the interval and removing the peak group when the ratio exceeds a predetermined value. (5) A cluster processing step is further performed in the computer so that a plurality of representative peaks whose m / z values of the representative peaks calculated for each peak group are within a specific value are defined as one peak. The program according to any one of claims 8 to 10, which is executed.   The specific value is determined based on the molecular weight of the biomolecule, and the intensity of the one peak after cluster processing is calculated as the sum of the intensity of representative peaks to be subjected to cluster processing, The program according to claim 11, wherein the value of m / z of one peak is calculated by performing weighted averaging of m / z of a representative peak to be clustered using the intensity as a weight. When one MS / MS spectrum has one data set related to the spectrum and a plurality of MS / MS spectra are acquired in the step (1), prior to the step (2),
(A) The difference in m / z of the MS spectrum of the precursor ion of each MS / MS spectrum is within a certain range, and the difference in the acquisition timing of the data set of each MS / MS spectrum is within a certain range. The program according to any one of claims 8 to 12, further causing a computer to execute a merge process of combining a plurality of data sets.   The program according to any one of claims 8 to 13, which causes a computer to execute the steps (2) to (4) after the step (a).

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