JP2005257483A - Differential method of analyzing proteome - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To directly analyze the measured result of the LC/MS analysis of a mixture material, obtained by proteome experiment or the like or a protein sample which is low in purity or the like. <P>SOLUTION: The differential method of analyzing proteome includes a process (1) Z-SCORE (Zscore) calculated from the peak of the mass analysis data of liquid chromatograph purified protein or the average value is set as a criterion to select an effective peak and a process (2) of intensifying the effective peak value selected in the process (1) to a range divided by the constant range of a molecular weight and the constant range of a holding time, to make it display the same as an image or as a numerical value. <P>COPYRIGHT: (C)2005,JPO&NCIPI

Description

In the differential analysis of the proteome, the present invention selects only effective peak values by using a statistical method and detects qualitative or quantitative changes in the measurement results detected by mass spectrometry of liquid chromatographed proteins. The present invention relates to a method, an apparatus, and a program that can be used.
Specifically, effective peaks are selected from all peaks in the mass spectrometry data of liquid chromatograph purified protein using the Z score as a criterion, and the peak values selected earlier are aggregated by dividing them by molecular weight and retention time. The present invention relates to a method for identifying the peptide.

A complete set of proteins produced by genomic genes, the generic term for proteins that function in vivo is called the proteome.
Proteome research is a comprehensive study of the relationship between genomic information and the protein produced by it, in light of life activity. Specifically, it analyzes the function of the discovered gene, analyzes the regulatory mechanism of the produced protein, Research on the interaction between proteins and elucidation of the relationship between diseases and pathological conditions and the function of proteins are the issues.
Among these, differential analysis that finds proteins whose expression changes between two or more samples makes it easy to identify the causative gene from thousands of proteins, and targets that select appropriate molecules as targets for drug discovery It is attracting attention as a useful search technique in the field of validation.
Examples of differential analysis include isotope labeling and subtractive methods, such as two-dimensional electrophoresis and mass spectrometry (hereinafter abbreviated as MS), mass spectrometry (hereinafter referred to as LC / MS) of liquid chromatographed proteins. It is analyzed using measurement results such as (omitted). In particular, LC / MS analysis is an excellent technique for further purifying highly purified protein materials by liquid chromatography and identifying the molecular weight of the materials.

Washbum P. M. et al., Anal. Chem. 2002, 74, 1650-1657 Chelius D. et al., J. of Proteome Research 2002, 1, 317-323

LC / MS analysis is useful for the analysis of protein samples with extremely high levels of separation and purification. However, when a mixture obtained in a proteome experiment or the like is analyzed as data, many peptides appear as peaks, and base noise beyond that appears. As a result, the identification of individual peptides by visualization has been very difficult with the standard software provided on the instrument.
In addition to the problems described above, the individual peaks in the LC / MS measurement results are very sharp, so that the differential analysis of the proteome by two-dimensional electrophoresis or the like is a simple subtraction and difficult to process.
In addition, in order to increase the degree of separation and purification of protein samples, it is necessary to spend a lot of time and money. Therefore, it is desirable to easily analyze a crudely purified sample and obtain a highly accurate analysis result. .

The object of the present invention is to directly analyze the measurement results of LC / MS analysis of a mixture material obtained by proteome experiments or the like, or a protein sample of low purity, etc. (1) Data noise processing To select effective peaks, (2) make up for the difficulty of differential processing due to peak sensitivity, (3) perform numerical processing without relying on visual judgment, and enable objective judgment. .
In Japan, it is difficult to analyze directly because of a lot of noise. By making it possible to directly analyze the behavior of the differential from the mixture data obtained by proteomic experiments or the measurement results of low-purity protein samples, As a result of analyzing the behavior of differential processing, many peptides appearing as a result appear as peaks, and more base noise appears, so the software provided as standard in the instrument allows individual peptides to be visualized by simple visualization. Identification was very difficult.

As a result of intensive studies, the present inventors tried to directly compare LC / MS measurement results, and by using statistical methods, the noise was greatly reduced from a huge number of peaks, and only effective peaks were selected. Qualitative or quantitative changes within the range divided by molecular weight and retention time can be detected.
Furthermore, by direct comparison of effective peaks, the peptides in the mixture were limited, and the target peptide could be identified by differential analysis.
That is, the gist of the present invention is as follows.
[1] Proteome differential analysis method including the following steps;
(1) The effective peak is selected based on the Z score (Zscore) calculated from the peak of the mass spectrometry data of the liquid chromatograph purified protein or the average value as a criterion,
(2) The effective peak values selected in (1) are aggregated and displayed as an image or numerical value in a range divided by a certain range of molecular weight and a certain range of retention time.
[2] The differential analysis method for proteome according to claim 1, wherein a natural logarithm is calculated by excluding a peak having a negative score of the Z score or the average value, and a peak having a positive result of processing is selected as an effective peak.
[3] A proteome differential analysis method including the following procedures;
(1) The Z score (Zscore) of each peak intensity is calculated from all the peaks detected by mass spectrometry of the liquid chromatograph purified protein, the natural logarithm is calculated except for the negative peak of the Z score, and the processing result is Select positive peaks as effective peaks,
(2) Using the grouping method including the following steps (i) to (iv) for the effective peaks selected in (1), the peak values are grouped.
(i) Select one peak from the list of all effective peaks to make one group.
(ii) There is a molecular weight (m / z) peak with a constant error range next to the minimum retention time peak belonging to the group formed in (i), or a constant error range equal to the peak next to the maximum retention time. If there are peaks of molecular weight (m / z), add those peaks to the group formed in (i),
(iii) Repeat the operation of (ii), and if there is no corresponding peak, return to (i).
(iv) Terminate if there are no peaks that do not belong to the group,
(3) A peak group having a retention time of 10 minutes to 300 minutes selected in (2) is selected,
A valid peak value,
(4) Compare two different peak data in the effective peak values selected in (3), and extract common peak data.
(4) The measurement results of the peak value of the sample subjected to differential analysis processed in (1) to (4) are divided and displayed with the molecular weight and retention time as axes, and the common part is extracted.
(6) A differential analysis is performed using the intensity of the effective peak obtained by the above procedure.
[4] The method according to claim 3, wherein the molecular weight within a certain error range is ± 0.5 Da to ± 0.1 Da.
[5] The method according to any one of claims 1 to 3, wherein the molecular weight is 50 to 2000.
[6] The method according to any one of claims 1 to 3, wherein the holding time is 30 minutes to 300 minutes.
[7] The method according to any one of claims 1 to 3, wherein the differential analysis method is an isotope labeling method or a subtractive method.
[8] A computer-readable program that causes a computer to execute the method according to any one of claims 1 to 7.
[9] A proteome differential analyzer for performing steps including the following procedures;
(1) a step of selecting an effective peak using a Z score or an average value calculated from a peak of mass spectrometry data of liquid chromatograph purified protein as a criterion,
(2) The step of aggregating the effective peak values selected in (1) in a range divided by a certain range of molecular weight and a certain range of retention time and displaying the image or numerical value.
[10] The differential analysis device for proteome according to claim 7, wherein a natural logarithm is calculated except for a peak having a negative value of Z score, and a peak having a positive result of processing is selected as an effective peak.
[11] A proteome differential analyzer for carrying out steps including the following procedures;
(1) The Z score (Zscore) of each peak intensity is calculated from all the peaks detected by mass spectrometry of the liquid chromatograph purified protein, the natural logarithm is calculated except for the negative peak of the Z score, and the processing result is Selecting positive peaks as effective peaks,
(2) Grouping peak values using the grouping method including the following steps (i) to (iv) for the effective peaks selected in (1):
(i) Select one peak from the list of all effective peaks and make it a group.
(ii) There is a molecular weight (m / z) peak with a constant error range next to the minimum retention time peak belonging to the group formed in (i), or a constant error range equal to the peak next to the maximum retention time. If there are peaks of molecular weight (m / z), add those peaks to the group formed in (i),
(iii) repeating the operation of (ii) and returning to (i) if there is no corresponding peak,
(iv) The step that ends if there is no peak that does not belong to the group,
(3) A peak group having a retention time of 10 minutes to 300 minutes selected in (2) is selected,
A step for a valid peak value,
(4) comparing two different peak data in the effective peak values selected in (3), and extracting common peak data;
A step of extracting and displaying a common part by dividing and displaying the measurement result of the peak value of the sample subjected to the differential analysis processed in (1) to (4) with the molecular weight and the retention time as axes.
(5) A step of performing a differential analysis using the effective peak intensity obtained by the above procedure.
[12] The apparatus according to claim 11, wherein the molecular weight within a certain error range is ± 0.5 Da to ± 0.1 Da.
[13] The apparatus according to any one of claims 9 to 11, having a molecular weight of 50 to 2000.
[14] The apparatus according to any one of [9] to [11], wherein the holding time is 30 minutes to 300 minutes.
[15] The apparatus according to any one of claims 9 to 11, wherein the differential analysis method is an isotope labeling method or a subtractive method.
[16]

  According to the present invention, it became possible to select effective peaks directly from the LC / MS measurement result data. As a result, it was possible to obtain a gentle intensity increase / decrease surface that was easy to visualize. In addition, the difficulty of differential processing due to peak sharpness can be compensated, and a clear difference due to the differential can be detected. According to the present invention, the rough behavior of the differential processing can be detected quickly, and it has become a useful technique for marker detection in the case of proteomic processing with HTS.

Below, the definition of the phrase as described in the claim in this invention is demonstrated.
"Liquid chromatograph purified protein mass spectrometry (LC / MS)" means measuring protein mass using a device such as a protein mass spectrometer coupled to a liquid chromatograph (LC). That means. Mass spectrometry (hereinafter referred to as MS) is to analyze a mass of ions by introducing a sample to be analyzed by ionization, creating a difference for each mass by an electric force or a magnetic force. The MS, including the mass spectrometer, which performs the above analysis, includes ion trap MS, Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR / MS) MS, ion scan MS, Q-TOF MS, etc. Can be mentioned. These may be analyzed by only one method, or may be analyzed by connecting a plurality of MSs (hereinafter, MS / MS) and a liquid chromatograph (hereinafter, LC / MS / MS analysis). .
The “Z score” generally refers to a Z score that is one of statistical analysis indices. One of the indicators of the strength of the co-occurrence relationship (mutual connection) between two words. The larger the value, the stronger the connection. The formula for calculating the Z score can be expressed as follows, for example.

「一定誤差範囲の分子量」とは、質量分析器の検出感度を考慮した検出ピークの許容範囲をいう。分析機によって検出感度はが異なるり、例えば、イオントラップ型質量分析器の場合は±0.5Da、四重極飛行時間ハイブリッド型質量分析器の場合は±0.1Da等が挙げあられる。
「保持時間」とは、分子量（m/z)のピークが検出されるために所有する時間をいう。１０分以下のものはノイズピークと考えられるので、本発明では１０分以上ピーク検出に所有されたピーク値について解析に用いる。好ましくは、３０〜３００分の保持時間が挙げられる。

“Molecular weight within a certain error range” refers to an allowable range of detection peaks in consideration of detection sensitivity of a mass analyzer. The detection sensitivity varies depending on the analyzer, for example, ± 0.5 Da for an ion trap mass analyzer, ± 0.1 Da for a quadrupole time-of-flight hybrid mass analyzer, and the like.
“Retention time” refers to the time possessed in order to detect the peak of molecular weight (m / z). Since a peak of 10 minutes or less is considered as a noise peak, in the present invention, a peak value owned by peak detection for 10 minutes or more is used for analysis. Preferably, the holding time of 30 to 300 minutes is mentioned.

A first aspect of the present invention relates to a proteome differential analysis method.
The analysis method of the present invention makes it possible to compensate for the difficulty of differential processing by selecting effective peaks by performing noise processing from the LC / MS measurement results by statistical methods and performing visualization. In addition, it is possible to detect a clear difference due to the differential. .
The data output by the general-purpose software used in the analysis method of the present invention may be a text file, for example, data such as Mascot, Sonar MS / MS, LC / MS used in proteome analysis and the like. . Hereinafter, the method of the present invention will be described with reference to FIGS.

FIG. 1 is a flowchart for explaining the method of claim 1 of the present invention. The method according to claim 1 is effective in significantly reducing noise by using a Z-score or average value judgment result from a huge number of peaks in direct comparison data of LC / MS measurement results. Only peaks were selected, and qualitative changes in a range divided by a certain range of molecular weight and retention time could be detected.
101, the Z score or average value of each peak intensity is calculated from all the peaks detected by mass spectrometry of the liquid chromatograph purified protein, and the natural logarithm is calculated except for the negative Z score peak at 102, 103 In step 104, the peak data is statistically determined. In step 104, a peak whose processing result is a positive value is selected as an effective peak.
In 105, the effective peak values selected in 104 in the range divided by the arbitrarily determined molecular weight and retention time of the peaks selected by the methods 101 to 104 are collected.
At 106, the groups aggregated at 105 are compared.
The effective peak group collected in 106 is described by text data, image data, etc., and is displayed so as to be visually easy. Examples of the display method include a table format and a graph, and a graph display is preferable. In addition, by changing the color tone for each peak aggregation and displaying it, it is possible to easily make a visual judgment and detect a clear difference due to a differential. In addition, by selecting the grid, it was possible to enlarge it, and it became possible to investigate more detailed differences. A display example is shown in FIG.

Further, the analysis of the effective peak group aggregated at 106 was examined without regard to visual judgment, and aggregation by numerical processing was examined. The present inventors have found that the accuracy of the molecular weight measurement of the mass spectrometer is very high, so the error is small. However, the retention time of liquid chromatography has been found to vary slightly depending on the situation. For example, an ion trap type or a quadrupole time-of-flight hybrid type has a molecular weight in a certain range according to the accuracy of the mass analyzer, and a certain constant retention. By consolidating the peaks that fall within the time range into one group, the group could be simplified.
By comparing the groups, it is possible to quickly determine the difference between the two spectra and to apply the differential analysis in a proteomic experiment.
In addition, noise is greatly reduced from the huge peaks of LC / MS measurement results, and only effective peaks are selected to detect qualitative changes in a range divided by a certain range of molecular weight and retention time. This suggests that direct comparison of LC / MS measurement results is possible.
Examples of the “differential analysis” method used in the present invention include an isotope labeling method and a subtractive method. The isotope labeling method compares the peak areas of the two using the difference in the detected molecular weight (m / z) due to the presence of isotopes in the labeled peptide in the same sample. It is a technique. For example, in the case of ¹⁸ O labeling, if the molecular weight (m / z) is detected as monovalent, the distance between the two is 4 Da, and if it is detected as bivalent, it is compared using the difference of 2 Da. .
The subtractive method is a method in which peak areas are compared between different samples by using a pair of peaks whose retention time and molecular weight (m / z) are close to each other.

Next, the method according to claim 3 of the present invention will be described with reference to FIGS. In the method according to claim 3, the effective peak value selected by removing noise is further compared with the peak by the retention time, and the target peak is detected, thereby limiting the peptides in the mixture, It was suggested that the differential peptide can be used to identify or quantify the target peptide.
First, assuming that an effective peak (301a) obtained by removing and selecting noise peaks by the method 101 to 104 in FIG. 1 appears for several seconds, a common part of a plurality of experimental results is acquired and the peak is obtained. Grouped values.
In 301 of FIG. 3, one is selected from the peak data of 301a,
At 302, adjacent peaks in a molecular weight (m / z) range with a constant retention time are grouped as a group. The fixed holding time may be 10 minutes or more, and the range is preferably 30 minutes to 300 minutes.
The target peaks were grouped as one large peak group if the molecular weights of the peaks detected over a plurality of locations with a retention time of several minutes were within a certain error range and were adjacent to each other. The fixed molecular weight range is 10 to 2000, preferably 50 to 2000.
In 303, the peak values that do not appear in a certain period of time are excluded from the peak group grouped in 302, and the peak values that are close to each other in the other peak values are grouped as a group (301b).
If the operation of 302-303 is repeated and there is no applicable peak, it will return to 301, and 301-303 will be repeated until grouping of all the peak groups is completed, and let the grouped peak group be 305. If there is no peak that does not belong to the group, the process ends.
In 401 of FIG. 4, the retention time of the peak data grouped in 305 of FIG. 3 is analyzed.
At 402, a sample having a retention time of 10 minutes or more is selected and set as an effective peak value (403). The holding time should just be 10 minutes or more, Preferably the range of 10 minutes-300 minutes is mentioned, More preferably, the range of 30 minutes-300 minutes is mentioned.
Repeat steps 401-403 until all peaks have been analyzed.
FIG. 5 compares adjacent peak values in the molecular weight (m / z) range having a constant retention time for the effective peak value obtained in 403 in FIG. 4 (501), and further groups to extract a common peak, (502) An effective peak is set (503). The effective peak group obtained in 503 is described by text data, image data, etc., and is displayed so as to be visually easy.
The differential analysis processed by the above method is performed.
Examples of the display method include a table format and a graph, and a graph display is preferable. Further, by displaying the color tone changed for each grouping of peaks, it is possible to easily make a visual judgment and detect a clear difference due to the differential. A display example is shown in FIG.

Display Example FIG. 6 is a result of examining whether or not a target peptide can be identified by directly comparing LC / MS measurement results of a mixture of several kinds of peptides by the procedures of 301 to 503.
These results suggest that, in LC / MS differential analysis, it is possible to detect the target candidate peak pair by comparing the peaks on the premise that the molecular weight error is small but the retention time is significantly different. It was.

The second aspect of the present invention is an apparatus for executing the analysis method of the present invention.
An apparatus for executing the analysis method of the present invention is shown in FIG. Reference numeral 701 denotes an apparatus for inputting data output by general-purpose software used in the analysis method of the present invention. The apparatus may be an electronic computer, and may be a server, such as a personal computer (hereinafter referred to as a PC), and the capacity of the computer is not limited. The operation system for operating the program for executing the analysis method of the present invention may also be general-purpose software (for example, Microsoft Windows (registered trademark) series).
In the apparatus 702, z-SCORE is determined for the data input in 701, and in 702, an effective peak is extracted based on the determination result in 701. Based on the determinations 301 to 305 in FIG. 3 by the apparatus 703, the effective peak values are grouped and output to 704.
In 705, the data output in 704 is selected according to the determination criteria 402 to 403 in FIG.
The apparatus 706 performs the data analysis of 501 to 502 in FIG. 5, and outputs the execution results executed by the apparatuses 701 to 206 of the apparatus 707.
The output result can be displayed on the apparatus 707 and can be printed on a recording medium such as paper, and can be printed on a magnetic medium such as FD, MO, CD-RW, and DVD-RW, or on a recording medium such as a magnetic optical disk and an optical disk. It can also be output.
The devices 701 to 707 may be all integrated devices, separated devices, partially integrated devices, or a combination thereof.
A third aspect of the present invention is a program that can be read by a computer for the analysis method of the present invention.
A program for executing the analysis methods 101 to 502 in the flowcharts of FIGS. 1 to 5, which may be a module according to the algorithm procedure shown in FIGS. A combination of written modules may be used. These are recorded on a recording medium such as a magnetic disk.
Examples of the analysis method of the present invention will be given below. However, the present invention is not limited to the present embodiment.

1) Recomparison of results with the HGH + β casein trypsin digestion system
Effective peaks were grouped before a direct comparison of the HGH + β casein trypsin digestion system. This assumes that the target peak is detected in multiple locations with a retention time of several minutes, and if the molecular weight indicated by the detected peak is within a certain error range and is adjacent, these peaks will be Combined as one large peak. Based on this conversion data, both were compared. Then, it was investigated how much tolerance could be given to subtract both peaks.
FIG. 8 shows a result of tryptic digestion of HGH, FIG. 9 shows a result of trypsin digestion of HGH + β casein, and FIG. 10 shows a result of superimposing FIGS. Looking at FIG. 10, pattern similarity was observed at several points, and it was thought that it was possible to match by setting the tolerance of retention time around 1 minute and giving that of molecular weight as 1 Da.

Comparison of several types of peptides in a mixed system The HGH + β casein trypsin digestion system is too complex and inappropriate for the verification of this program. We measured LCQ and tried comparative analysis of the results.
FIG. 11 shows the results of plotting the MS (LCQ) results obtained by mixing seven types of peptides, Bradykinin, Dynarphin, Substance P, Renin Substrate, Angiotensin, Leu-enkepharin, and Fibrinopeptide, with noise treatment. Here, most of the peaks appearing after the retention time of up to 10 minutes and after 43 minutes are noise that appears even in the blank. FIG. 12 shows that the intended seven peptides are assigned from the numerical data after the noise processing.
Fibrinopeptide appeared just around 44 minutes and overlapped with the noise inherent in this measurement system, so it was judged that detection of Fibrinopeptide was difficult. In addition, the noise could be largely eliminated by assuming that the peptide of interest appeared over at least 3 scans but not longer than 10 minutes (FIG. 13).

Quantitative comparison in ¹⁸ O mixed system The target peak pair was selected based on the peptide information identified from the Mascot search results for the pre-processed 18O mixed LCQ peak data, and the quantitative performance was examined. .
FIG. 14 is a plot of the ¹⁸ O LCQ measurement results after noise processing. FIG. 15 shows the 16O and 18O isotope pairs extracted from the Mascot analysis of the sample.

  According to the present invention, it became possible to select effective peaks directly from the LC / MS measurement result data. As a result, it was possible to obtain a gentle intensity increase / decrease surface that was easy to visualize. In addition, the difficulty of differential processing due to peak sharpness can be compensated, and a clear difference due to the differential can be detected. According to the present invention, the rough behavior of the differential processing can be detected quickly, and it has become a useful technique for marker detection in the case of proteomic processing with HTS.

FIG. 1 is a flowchart showing the procedure of the analysis method of the present invention. Figure 2 shows a hard copy of the screen displaying the analysis results of general-purpose software FIG. 3 is a flowchart showing the grouping procedure of the analysis method of claim 3 of the present invention. FIG. 4 is a flowchart showing a procedure for selecting effective peak values in the analysis method of claim 3 of the present invention. FIG. 5 is a flowchart showing the effective peak value selection procedure of the analysis method of claim 3 of the present invention. FIG. 6 is a view showing the result of analysis by the method of the present invention. FIG. 7 is a diagram showing the configuration of an apparatus for executing the analysis method of the present invention. FIG. 8 shows the analysis and display of the system in which tryptic digestion of HGH in Example 1 was performed. FIG. 9 is a diagram showing an analysis and display of a system in which trypsin digests HGH + β casein in Example 1. FIG. 10 is a superposition of FIGS. 8 and 9. FIG. 11 shows LC / MS results of the seven peptide mixtures of Example 2. FIG. 12 shows the result of displaying the attribution of the seven peptides of Example 2. FIG. 13 is an LC / MS plot of the seven-peptide mixture after noise treatment in Example 2. FIG. 14 is a plot of the 18O measurement results of Example 3. FIG. 15 is a plot of the attribution of 16O and 18O peptide pairs as Mascot results.

Claims (15)

Proteome differential analysis method including the following steps;
(1) Select an effective peak using a Z score (Zscore) calculated from all peaks of mass spectrometry data of liquid chromatograph purified protein or an average value as a criterion,
(2) The effective peak values selected in (1) are aggregated and displayed as an image or numerical value in a range divided by a certain range of molecular weight and a certain range of retention time.   The differential analysis method for a proteome according to claim 1, wherein a natural logarithm is calculated except for a peak having a negative value as a determination criterion for a Z score, and a peak having a positive value as a processing result is selected as an effective peak. Proteome differential analysis method including the following steps;
(1) The Z score (Zscore) of each peak intensity is calculated from all the peaks detected by mass spectrometry of the liquid chromatograph purified protein, the natural logarithm is calculated except for the negative peak of the Z score, and the processing result is Select positive peaks as effective peaks,
(2) For the effective peaks selected in (1), the peak values are grouped by the grouping method including the following steps (i) to (iv).
(i) Select one peak from the list of all effective peaks to make one group.
(ii) There is a molecular weight (m / z) peak with a constant error range next to the minimum retention time peak belonging to the group formed in (i), or a constant error range equal to the peak next to the maximum retention time. If there are peaks of molecular weight (m / z), add those peaks to the group formed in (i),
(iii) Repeat the operation of (ii), and if there is no corresponding peak, return to (i).
(iv) Terminate if there are no peaks that do not belong to the group,
(3) A peak group having a retention time of 10 minutes to 300 minutes selected in (2) is selected,
A valid peak value,
(4) Compare two different peak data in the effective peak values selected in (3), and extract common peak data.
(5) The peak value measurement results of the sample subjected to differential analysis processed in (1) to (4) are divided and displayed with the molecular weight and retention time as axes, and the common part is extracted.
(6) A differential analysis is performed using the intensity of the effective peak obtained by the above procedure.   The method according to claim 3, wherein the molecular weight within a certain error range is ± 0.5 Da to ± 0.1 Da. The method according to any one of claims 1 to 3, wherein the molecular weight is 50 to 2000. The method according to any one of claims 1 to 3, wherein the holding time is 30 minutes to 300 minutes. The method according to claim 1, wherein the differential analysis method is an isotope labeling method or a subtractive method.   A computer-readable program for causing a computer to execute the method according to claim 1. Proteome differential analyzer for performing steps including the following procedures;
(1) a step of selecting an effective peak using a Z score calculated from a peak of mass spectrometry data of a liquid chromatograph purified protein as a criterion;
(2) The step of aggregating the effective peak values selected in (1) in a range divided by a certain range of molecular weight and a certain range of retention time and displaying the image or numerical value.   10. The differential analysis apparatus for proteome according to claim 9, wherein a natural logarithm is calculated by excluding a peak having a negative value as a determination criterion of a Z score, and a peak having a positive result of processing is selected as an effective peak. Proteome differential analyzer for performing steps including the following procedures;
(1) The Z score (Zscore) of each peak intensity is calculated from all the peaks detected by mass spectrometry of the liquid chromatograph purified protein, the natural logarithm is calculated except for the negative peak of the Z score, and the processing result is Selecting positive peaks as effective peaks,
(2) Grouping the peak values by the grouping method including the following steps (i) to (iv) for the effective peaks selected in (1):
(i) selecting one peak from the list of all effective peaks to form one group;
(ii) There is a molecular weight (m / z) peak with a constant error range next to the minimum retention time peak belonging to the group formed in (i), or a constant error range equal to the peak next to the maximum retention time. Adding any peaks of molecular weight (m / z) to the group formed in (i), if any.
(iii) repeating the operation of (ii) and returning to (i) if there is no corresponding peak,
(iv) a step that ends if there is no peak that does not belong to the group;
(3) A peak group having a retention time of 10 minutes to 300 minutes selected in (2) is selected,
A step for a valid peak value,
(4) comparing two different peak data in the effective peak values selected in (3), and extracting common peak data;
A step of extracting and displaying a common part by dividing and displaying the measurement result of the peak value of the sample subjected to the differential analysis processed in (1) to (4) with the molecular weight and the retention time as axes.
(5) A step of performing a differential analysis using the effective peak intensity obtained by the above procedure. The apparatus according to claim 11, wherein the molecular weight within a certain error range is ± 0.5 Da to ± 0.1 Da. The apparatus according to any one of claims 9 to 11, having a molecular weight of 50 to 2,000. The apparatus according to any one of claims 9 to 11, wherein the holding time is 30 minutes to 300 minutes. The apparatus according to any one of claims 9 to 11, wherein the differential analysis method is an isotope labeling method or a subtractive method.

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