JP2009168695A - Three-dimensional structure prediction method, three-dimensional structure prediction program, and mass spectrometer - Google Patents

Abstract

【Task】
By using a mass spectrometer, a predetermined binding site important for formation of a three-dimensional structure of a protein or peptide is predicted, and acquisition of three-dimensional structure information is realized.
[Solution]
As a pretreatment method of mass spectrometry, a cleaving agent that cleaves a predetermined bond important for the formation of a three-dimensional structure is added to a protein or peptide sample to prepare it as a measurement sample, and a cleaving agent is added. The measurement sample processed in the two ways is measured with a mass spectrometer, and the obtained measurement data is compared and analyzed to obtain a predetermined binding site. Predict.
[Selection] Figure 1

Description

  The present invention relates to a three-dimensional structure prediction method, a three-dimensional structure prediction program, and a mass spectrometer, and more particularly to a technique suitable for a three-dimensional structure analysis of proteins or peptides and a post-translational modification site.

  In general pretreatment methods for mass spectrometry, protein three-dimensionality is added by adding a reducing agent that reduces disulfide bonds between cysteine residues in proteins and peptides, or a denaturing agent that cleaves hydrogen bonds. A bond that is important for forming a structure is cleaved and processed so that peptide fragmentation of a protein by a proteolytic enzyme or the like proceeds efficiently. Therefore, the obtained mass spectrometry data does not include protein three-dimensional structure information. On the other hand, in Non-Patent Document 1, mass spectrometry is performed without breaking the disulfide bond of the target sample, and a database search is performed on the MS / MS spectrum database constructed based on the amino acid sequence of the target sample to predict the disulfide bond. To do.

Jiaxi Wang, Bin Ma, Weiwu Chen “Disulfide bonded Dipeptide Analysis with PEAKS and Q-TOF Mass Spectrometry” ASMS 2007, June 3-7, P.6-P.7

  Proteins can exhibit their functions in living bodies by taking a unique three-dimensional structure. Therefore, in order to elucidate what function the protein performs in the living body, it is important to analyze the three-dimensional structure.

  The formation of the three-dimensional structure of a protein is greatly influenced by disulfide bonds and hydrogen bonds between cysteine residues that are present at distant positions on the amino acid sequence. For functional expression, post-translational modifications such as sugar chains and phosphate groups are important.

  In current mass spectrometers, the process of deciding important bonds for protein and peptide structure formation at the pre-processing stage is performed, so information about such binding sites is lost and the three-dimensional structure cannot be estimated. There is a problem. Furthermore, although the above-mentioned Non-Patent Document 1 describes mass spectrometry related to disulfide bonds, it is not considered when the amino acid sequence of the target sample whose structure is predicted is unknown when the MS / MS spectrum database is constructed. In addition, there is a problem that it does not correspond to the prediction of a bond or a post-translational modification site important for forming a protein three-dimensional structure other than a disulfide bond.

  One object of the present invention is to mass-analyze a sample while maintaining an important bond for formation of a three-dimensional structure, predict a binding site and post-translational modification site from the obtained data, and realize acquisition of three-dimensional structure information. That is.

  One feature of the present invention is that a first data file obtained by mass spectrometry using a first method of adding a cleaving agent that cleaves a bond important for conformation formation or a bond at a post-translational modification site, and In the method of comparative analysis of the second data file obtained by mass spectrometry using the second method in which the above-mentioned cleavage agent is not added. This realizes a method for predicting a binding site important for the formation of a three-dimensional structure of a protein or peptide and a post-translational modification site by a mass spectrometer.

  Another feature of the present invention is that (1) in the three-dimensional structure analysis method, pretreatment is performed by the first method and the second method, and each sample is measured by a mass spectrometer, so that proteins and peptides can be analyzed. Three-dimensional structure information is obtained.

  Another feature of the present invention is that (2) a three-dimensional structure analysis program searches a database of mass spectrometry data of a sample preprocessed by the first method to identify an amino acid sequence. From the identified amino acid sequence, the mass when an amino acid residue involved in a bond important for three-dimensional structure formation forms a bond, or the peptide containing an amino acid residue that is post-translationally modified is post-translationally modified Find the mass when. Comparing this mass with the mass spectrometry data of the sample pretreated by the second method, and searching for a matching mass, predicts a site that forms an important bond in the three-dimensional structure or a site that is post-translationally modified To do.

  According to one aspect of the present invention, it is possible to predict a binding site or post-translational modification site important for the formation of a three-dimensional structure of a protein or peptide from mass spectrometry data.

  In addition, according to another aspect of the present invention, it is possible to easily analyze the structure of a protein without complicated processing and examination of conditions, which contributes to the elucidation of the structure and function of a protein that has been difficult until now. it can.

  An example of an embodiment of the present invention will be described with reference to FIGS.

  FIG. 1 is a diagram showing a flow of processing according to the embodiment of the present invention.

  Sample (protein or peptide) 101 is pretreated 102 by two methods. One is a first method of cleaving a predetermined bond by adding a cleaving agent. The other is a second method in which a predetermined bond is not cleaved without adding a cleaving agent. Pretreatment is performed in these two ways, and the peptide is fragmented. The sample processed in the two ways is subjected to mass spectrometry 103, and a first data file and a second data file are obtained as respective measurement data. By comparing and analyzing the first data file and the second data file 104, information on the predicted predetermined binding site 105 is obtained. Hereinafter, each stage will be described in detail.

1. Pre-processing FIG. 2 is a schematic diagram illustrating an example of pre-processing according to the embodiment of the present invention.

  The left flow on the left side of FIG. 2 represents the first method and is the same as the normal pretreatment method of the chromatograph mass spectrometer. A sample 201 that is a protein or peptide is subjected to a cleaving agent addition process 202 for adding a cleaving agent. After cleaving a predetermined bond, in the case of a protein sample, an enzyme digestion process 203 is performed to obtain a peptide fragment, and a measurement sample 204 And

  The right flow on the right side of FIG. 2 represents the second method, which is a preprocessing method that does not break the predetermined bond. In the case of a protein sample, the sample 201 which is protein or peptide is subjected to an enzyme digestion process 203 to obtain a measurement sample 204.

2. Mass Spectrometry FIG. 3 is a schematic diagram illustrating a configuration example of a chromatographic mass spectrometer according to an embodiment of the present invention.

  The pretreated sample 301 is separated according to the retention time by using the separation column 303 of the liquid chromatograph section 302 and by interaction such as adsorption / distribution. Then, after ionization by the ion source 305 of the mass spectrometer main body 304, the mass is separated by the mass separator 306, and the mass number and its signal intensity are detected by the detector 307. This detection data is taken into a data processing device 308 having a computer, and includes information on retention time of each peptide fragment, mass-to-charge ratio m / z (m is ion mass, z is the number of ions), and signal intensity. Data is obtained. The mass separation apparatus 306 may be an apparatus that performs mass separation such as a magnetic field type, a quadrupole type, and a time-of-flight type in addition to the ion trap method shown in the figure.

3. Data Analysis FIG. 4 is an explanatory diagram of the comparative analysis of measurement data in the embodiment of the present invention.

  The first data file 401, which is the measurement data of the sample preprocessed by the first method, is subjected to database search 402 using existing database search software, and peptide peptide amino acid sequence identification 403 is performed. And extraction 404 of the peptide containing the amino acid residue which concerns on a predetermined bond is performed from the amino acid sequence of the identified peptide. A mass-to-charge ratio is calculated when it is assumed that the extracted peptide forms a predetermined bond, and a first mass-to-charge ratio list is created 405.

  On the other hand, a second mass-to-charge ratio list is created 407 from the second data file 406, which is measurement data of the sample preprocessed by the second method. A mass-to-charge ratio comparison process 408 for comparing the mass-to-charge ratios of the second mass-to-charge ratio list and the first mass-to-charge ratio list created 405 in the first mass-to-charge ratio list 405 Do. Then, a matching mass-to-charge ratio extraction 409 is performed. Thereby, the prediction 410 of the amino acid residue which forms a predetermined bond is performed. In this way, it is possible to predict the three-dimensional structure of a protein or peptide by identifying amino acid residues that form a predetermined bond. Each step of reference numerals 404, 405, 408, 409, and 410 in FIG. 4 will be described in more detail with reference to FIGS.

FIG. 5 is a diagram showing details of the comparative analysis shown in FIG. From the identification result 502 of the peptide amino acid sequence by the database search, which is a result obtained by identifying the amino acid sequence of the peptide by searching the database of the first data file 501, the peptide P _i containing the amino acid residue involved in the predetermined binding And the peptide P _j , the mass M (P _{i, j} ) 503 is calculated. For this mass M (P _{i, j} ) 503, the mass-to-charge ratio when the charge z = 2, 3, 4, 5 is calculated, and the calculated value M (P _{i, j} ) / z ( z = 2, 3, 4, 5). This calculated mass-to-charge ratio M (P _{i, j} ) / z (z = 2, 3, 4, 5) 504 and the mass-to-charge ratio m / z _n 505 obtained from the second data file A comparison process 506 to be compared is executed. In the comparison processing 506, when there is M (P _{i, j} ) / z that matches within ± 1.0 with respect to m / z _n (YES in the figure), this is related to a predetermined combination of P _i and P _j. A predicted result 507 that a predetermined bond is formed between amino acid residues is obtained.

FIG. 6 is a diagram showing details of the comparative analysis shown in FIG. 5 by symbols. Reference numeral 601 indicates a mass M (P _{i, j} ) when it is assumed that the peptides P _i and P _j including amino acid residues involved in the predetermined bond form a predetermined bond, and the mass is obtained by the brute force combination of the peptides. . Reference numeral 602 represents a calculated value M (P _{i, j} ) / z of the mass-to-charge ratio when the charge z = 2, 3, 4, 5, and reference numeral 603 represents the mass pair obtained from the second data file. a charge ratio m / z _n. The calculated value M (P _{i, j} ) / z 602 of the mass-to-charge ratio when the charge z = 2, 3, 4, 5 and the mass-to-charge ratio m / z _n 603 obtained from the second data file are as follows: Will be compared.

  Embodiment 1 of the present invention will be described below with reference to the drawings.

  As Example 1, a case of identifying a disulfide (SS) binding site that plays an important role in the formation of a three-dimensional structure of bovine serum albumin (BSA) will be described. In FIG. 7, an amino acid sequence 701 is an amino acid sequence of bovine serum albumin (BSA) and includes 35 cysteine residues. Reference numeral 702 indicating the disulfide bond site of bovine serum albumin indicates that 17 S—S bonds are formed by 34 cysteine residues.

  First, preprocessing in the first embodiment will be described. In the first method in the pretreatment, dithiothreitol as a reducing agent is added as a cleaving agent for cleaving the SS bond. The cysteine residue that has been reduced to a thiol group is alkylated with iodoacetamide. Then, BSA is peptide fragmented using a proteolytic enzyme such as trypsin. This is prepared as a measurement sample. In the second method, peptide fragmentation is performed with a proteolytic enzyme without adding a cleaving agent, and this is prepared as a measurement sample.

  The two types of samples were subjected to mass analysis using a nanoLC / IT-TOF (nano liquid chromatograph / ion trap-time-of-flight type) mass spectrometer (NanoFrontierL (Hitachi High-Tech)). Obtain a second data file.

  Next, data analysis will be described. The first data file is searched with database search software Mascot. As a result, the amino acid sequences of 23 peptide fragments containing 32 cysteine residues are identified. As confirmation, a Mascot search of the second data file revealed that no peptide fragment containing a cysteine residue was identified and that the SS bond was not cleaved.

For the 23 peptide fragments obtained as a result of the database search, the mass M (P _{i, j} ) when SS bonds are formed in a brute force combination is calculated. From this M (P _{i, j} ), the mass-to-charge ratio M (P _{i, j} ) / z when z = 2, 3, 4, 5 is calculated.

M (P _{i, j} ) / z calculated from the first data file is compared with m / z _n having a signal intensity of 500 or more in the second data file, and the difference is ± 1. Extract those that are within 0. Then, it is predicted that an SS bond is formed between the extracted cysteines of peptides P _i and P _j having a value of M (P _{i, j} ) / z.

FIG. 8 shows an example of data for predicting the disulfide bond site of BSA. As an example 801 of the mass-to-charge ratio M (P _{i, j} ) / z calculated from the first data file of bovine serum albumin, the peptide of mass 1033.5 identified from the first data file of bovine serum albumin Assuming that P _i and peptide P _{j of} mass 1493.6 form a disulfide bond, its mass is calculated to be 2357.0, and this mass-to-charge ratio M (P _{i, j} ) / z is z = 5 472.4.

This is because the second data file for bovine serum albumin has m / z _n 473.3345 as shown in Example 802 of the mass-to-charge ratio m / z _n obtained from the second data file for bovine serum albumin. Peptides P _i and P _j are predicted to form SS bonds.

As shown in Chart 803 showing that the 114th cysteine residue and the 125th cysteine residue of bovine serum albumin form a disulfide bond, the 125th cysteine residue of peptide P _i and the peptide P _j The 114th cysteine residue actually forms an S—S bond, indicating that the present invention is effective.

  As described above, according to the present invention, the three-dimensional structure of a protein or peptide can be easily predicted, and not only the mass or primary structure of a protein or peptide but also a secondary structure or tertiary structure can be analyzed by a mass spectrometer. The effect is obtained.

An example of the disclosure of this specification is listed below:
1. A first step in which a cleaving agent is added to a sample having a protein or peptide to cleave and process a predetermined bond; and a second step in which the cleaved agent is not added to the sample and the predetermined bond remains. And a step of predicting a predetermined binding site by comparing mass spectrometry data relating to the measurement sample obtained in the first step and the measurement sample obtained in the second step. A three-dimensional structure prediction method for predicting a three-dimensional structure by having the structure.

  2. Above 1. Wherein the predetermined bond is an important bond for a protein or peptide to form a three-dimensional structure, or a bond at a post-translational modification site, and the cleavage agent is a substance that dissociates the predetermined bond. 3D structure prediction method.

  3. 2. The important bonds for forming a three-dimensional structure of the protein or peptide are disulfide bonds, hydrogen bonds, ionic bonds, and hydrophobic interactions. The bonds at the post-translational modification site are phosphorylation sites and sugars. The method for predicting a three-dimensional structure, wherein the substance that is a chain modification site and the like and dissociates the predetermined bond is a substance that exhibits a reducing property with respect to a disulfide bond, a denaturant that cleaves a hydrogen bond, or the like.

  4). An amino acid residue related to the predetermined binding identified from the mass spectrometry data of the measurement sample obtained by using the first step of cleaving a predetermined bond and processing the sample having a protein or peptide by adding a cleavage agent A procedure for calculating a predicted mass observed when a disulfide bond is formed between amino acid residues involved in the predetermined bond of two or more peptides based on the mass of the peptide containing the predicted mass and the sample The computer is subjected to a procedure for comparing the mass spectrometric data of the measurement sample obtained by using the second step of processing without adding the cleaving agent and leaving a predetermined bond and searching for a matching mass. 3D structure prediction program

  5). 4. above. The three-dimensional structure prediction program according to claim 1, wherein the program predicts a three-dimensional structure by predicting the predetermined binding site.

  6). In a mass spectrometer having a mass analysis unit for separating a sample into components, ionizing the separated components to analyze mass, and a data processing unit for processing analysis data from the mass analysis unit, a protein or peptide A process in which a sample obtained by mass spectrometry of a measurement sample obtained by using a first step of cutting and processing a predetermined bond by adding a cutting agent to a sample having a first data file; and the cutting agent for the sample And a process of using a second data file as a result of mass spectrometry of a measurement sample obtained using the second step of processing without adding a predetermined bond and leaving a predetermined bond. .

  The present invention is not limited to the above-described embodiments, and various modifications can be made within the scope of the technical idea.

It is the figure which showed the flow of the process of embodiment of this invention. It is the schematic of the pre-processing which is an example of embodiment of this invention. It is the schematic which shows the structural example of the chromatograph mass spectrometer of embodiment of this invention. It is explanatory drawing of the comparative analysis of the measurement data in embodiment of this invention. It is a figure showing the detail of the comparative analysis shown in FIG. It is the figure which described the detail of the comparative analysis shown in FIG. 5 with the symbol. It is the amino acid sequence and disulfide bond site of bovine serum albumin. It is an example of BSA disulfide bond site prediction.

Explanation of symbols

101 samples (protein or peptide)
102 Pretreatment 103 Mass spectrometry 104 Comparative analysis processing 105 Predicted predetermined binding site 201 Sample 202 Cutting agent addition treatment 203 Enzyme digestion treatment 204 Measurement sample 301 Measurement sample 302 after pretreatment Liquid chromatograph section 303 Separation column 304 Mass spectrometry Main body 305 Ion source 306 Mass separation device 307 Detector 308 Data processing device 401 First data file 402 which is measurement data of the sample preprocessed by the first method 402 Database search 403 Identification of peptide amino acid sequence 404 Predetermined binding Extraction of peptides containing amino acid residues involved 405 Creation of first mass-to-charge ratio list 406 Second data file 407 that is measurement data of the sample pretreated by the second method Create 408 Mass-to-charge comparison process 409 Extraction of mass to charge ratio 410 Prediction of amino acid residues forming a predetermined bond 501 First data file 502 Identification result of peptide amino acid sequence by database search 503 Mass M (P _{i, j} )
504 Calculated mass-to-charge ratio M (P _{i, j} ) / z (z = 2, 3, 4, 5)
505 Mass to charge ratio obtained from the second data file m / z _n
506 Comparison process 507 Prediction result 601 Mass M (P _{i, j} ) when it is assumed that peptides P _i and P _j containing amino acid residues involved in a predetermined bond form a predetermined bond
602 Calculated value of mass-to-charge ratio when charge z = 2, 3, 4, 5 M (P _{i, j} ) / z
603 Mass to charge ratio obtained from the second data file m / z _n
701 Amino acid sequence 702 Bovine serum albumin disulfide bond site 801 Example of mass to charge ratio M (P _{i, j} ) / z calculated from the first data file of bovine serum albumin 802 Second data file of bovine serum albumin Example of Mass to Charge Ratio m / z _n Obtained from 803 Chart showing that 114th cysteine residue and 125th cysteine residue of bovine serum albumin form a disulfide bond

Claims (6)

A first step in which a cleaving agent is added to a sample having a protein or peptide to cleave and process a predetermined bond; and a second step in which the cleaved agent is not added to the sample and the predetermined bond remains. A pretreatment step comprising:
Predicting a predetermined binding site by comparing mass spectrometry data relating to the measurement sample obtained in the first step and the measurement sample obtained in the second step;
A three-dimensional structure prediction method for predicting a three-dimensional structure by having In claim 1,
The predetermined bond is an important bond for a protein or peptide to form a three-dimensional structure, or a bond at a post-translational modification site, and the cleavage agent is a substance that dissociates the predetermined bond. Structure prediction method. In claim 2,
The important bonds for the protein or peptide to form a three-dimensional structure are disulfide bonds, hydrogen bonds, ionic bonds, and hydrophobic interactions, and the bonds at the post-translational modification site are phosphorylation sites and sugar chain modifications. The method for predicting a three-dimensional structure, wherein the substance that is a site or the like and dissociates the predetermined bond is a substance that exhibits reducibility with respect to a disulfide bond or a denaturant that cleaves a hydrogen bond. An amino acid residue related to the predetermined binding identified from the mass spectrometry data of the measurement sample obtained by using the first step of cleaving a predetermined bond and processing the sample having a protein or peptide by adding a cleavage agent A procedure for calculating a predicted mass observed when a disulfide bond is formed between amino acid residues involved in the predetermined bond of two or more peptides, based on the mass of the peptide containing;
Compare the predicted mass with the mass analysis data of the measurement sample obtained using the second step of processing the sample without adding the cleaving agent and leaving a predetermined bond, and search for a matching mass. A three-dimensional structure prediction program for causing a computer to execute a procedure. In claim 4,
The program is a three-dimensional structure prediction program that predicts a three-dimensional structure by predicting the site of the predetermined bond. In a mass spectrometer having a mass analysis unit for separating a sample into components, ionizing the separated component to analyze mass, and a data processing unit for processing analysis data from the mass analysis unit,
A process of adding a cleaving agent to a sample having a protein or peptide and cleaving a predetermined bond to process the first measurement step obtained by mass spectrometry of a measurement sample obtained using the first step;
Performing a process of using a second sample data file as a result of mass spectrometry of a measurement sample obtained using the second step of processing the sample without adding the cleaving agent and leaving a predetermined bond. Characteristic mass spectrometer.

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