JP2008076406A - Method for protein structure analysis, protein structure analyzer, program, and recording medium - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a highly sensitive and quick actual measuring technique for stereostructure information, capable of evaluating a stereostructure predicted result, and capable of providing structure information useful for predicting the structure. <P>SOLUTION: The technique of the present invention fragments an objective protein for predicting the stereostructure to measure a fragmentation spectrum, determines assignment information of a fragmentation ion in an amino acid sequence of the objective protein, based on the measured fragmentation spectrum, specifies a portion contacting with a solvent in the amino acid sequence of the objective protein, according to the measured fragmentation spectrum and the assignment information of the fragmentation ion, determines solvent contacting moiety information in the amino acid sequence of the objective protein, according to the portion, predicts the stereostructure as to the objective protein, calculates reference vibration as to the objective protein, and outputs the predicted stereostructure prediction data, the determined solvent contacting moiety information, and a calculation result by the reference vibration calculation, while correlated each other thereamong. <P>COPYRIGHT: (C)2008,JPO&INPIT

Description

  The present invention relates to a protein structure analysis method, a protein structure analysis apparatus, a program, and a recording medium, and in particular, can supplement protein three-dimensional structure data obtained by a calculation method with data obtained by a biochemical experimental method. The present invention relates to a protein structure analysis method, a protein structure analysis apparatus, a program, and a recording medium.

  Whole genome sequence analysis is proceeding in many biological species including humans, and the sequence information is made into a database (Non-patent Document 1). Here, it is possible to identify and predict gene functions to some extent from genome sequences, but there are a large number of genes whose functions cannot be predicted only by sequence information. A gene is actually a function of a protein translated from its DNA sequence. In the case of a gene whose function is unknown, the function is predicted by first searching for a protein whose function is known with high homology in the amino acid sequence of the protein. However, there are many cases where a protein with a known function does not exist. In that case, it is possible to search for a protein having a high three-dimensional structure similarity or to predict a function by predicting a new (de novo) three-dimensional structure.

  In order for a protein to exert its function, it is essential to have a three-dimensional structure suitable for it. Therefore, it is possible to estimate the function more precisely by comparing the homology of the three-dimensional structure than comparing the homology of the amino acid sequences.

  As a method for predicting a three-dimensional structure from a protein amino acid sequence whose three-dimensional structure is unknown, a homology modeling method is generally used. This is a computational scientific method mainly consisting of the following four steps.

(1) When an arbitrary amino acid sequence (target sequence) is given, a protein having a sequence similar to the target sequence (reference protein) is searched from a three-dimensional structure database such as PDB (Protein Data Bank). Homology search) to give alignment (sequence alignment) between the target sequence and similar sequences.

  In order to perform this database search and alignment, there are computer software such as FASTA, PSI-BLAST, and LIBRA. FASTA is a program that matches 20 alphabetic character sequences that mean 20 kinds of natural amino acids, and has high homology (corresponding to about 30% or more of amino acid matching, equivalent to about 0.01 or less in FASTA e value). It is said that a highly reliable model can be constructed by constructing a three-dimensional structure with respect to a reference protein.

  Also, in PSI-BLAST, matching of character sequences is performed in the same manner. However, instead of information on whether or not characters match, the degree of matching of characters called a profile is replaced for each region on the character sequence of related proteins. It has the property of optimizing the alignment by calculating it as a matrix and then repeatedly calculating it.

  LIBRA is a program based on the 3D-1D method (also called the threading method), and searches for similar sequences using a known three-dimensional structure as a probe. Therefore, the search algorithm is clearly different from FASTA and PSI-BLAST. Therefore, there may be a case where similarities between sequences different from FASTA and PSI-BLAST can be pointed out.

(2) If the alignment calculated by FASTA, PSI-BLAST, LIBRA, etc. is used, the corresponding relationship for each amino acid between the target sequence and the similar sequence is determined. Based on this relationship, the three-dimensional coordinates of the reference protein Create three-dimensional coordinates for each amino acid.

(3) When there is no amino acid corresponding to the target sequence side, the amino acid coordinates at that position on the reference protein side are not used. Conversely, when there is no amino acid corresponding to the reference protein side, The amino acid coordinates on the target sequence are created by searching for appropriate ones from a protein fragment coordinate database prepared in advance.

(4) In the construction of protein coordinates according to the above (2) and (3), structurally inappropriate gaps, collisions and distortions may occur between amino acid residues. Eliminate structural distortion. Depending on the modeling software, in order to smoothly eliminate this structural distortion, the calculation and search processing of (2) to (4) is not performed on all protein atoms at the same time, but in steps. . That is, it is performed first on the α carbon atom forming the protein skeleton, then on the main chain atom containing the α carbon atom, and finally on the entire protein atom containing the side chain atom.

  As described above, if alignment with the target sequence is obtained, the three-dimensional structure can be predicted and constructed.

Patent application WO00 / 39326 Gerardo Jimenez-Sanchez, Nature 409, 853-855 (2001) Zhang, Z .; and Smith, D.A. L. (1993) Protein Sci. , 2, 522-531. Akashi, S .; , And Takao, K .; , (2000) Protein Sci, 9, 2497-2505. , Tomoko Akashi “Abstracts of the 48th General Meeting of Mass Spectrometry” p. 208 (2000) Yamada N .; Suzuki E .; , Hirayama K .; (2002) Rapid Commun. Mass Spectrom. 16, 293-299.

  However, the three-dimensional structure of the protein constructed by In Silico by computational science has not been verified by experiments. Here, methods for experimentally measuring the three-dimensional structure information of proteins include X-ray crystal structure analysis, electron microscope, and NMR, but these methods are technically difficult to obtain the structure. In addition, time-consuming and labor-intensive work is required.

  Here, in recent years, methods for obtaining three-dimensional structure information of proteins using hydrogen-deuterium (H / D) exchange reaction and mass spectrometry (MS) have been reported. For example, a technique using an H / D exchange reaction is disclosed in Patent Document 1 (Methods for quantifying heavy hydrogen levels at specific peptide linkages in protein and peptides) and Non-Patent Document 2.

  In these methods, the protein dissolved in a solution prepared from heavy water is sampled over time, digested with pepsin in an acidic solution, and the resulting peptide is measured by LC / MS. The deuteration rate is calculated from the rate. Thus, it can be seen that the place where the deuteration rate is fast is a part where the solvent contact rate is high or the fluctuation is large, and the part where the deuteration rate is slow is a part where the solvent contact rate is low.

  However, even in this method, there is a problem that a large number of samplings and LC / MS with a long measurement time must be performed. In addition, there is a problem that the three-dimensional structure of the protein is remarkably changed during pepsin digestion and LC / MS measurement, and thus there is a high possibility that H / D scrambling occurs within and between peptides. Regarding the latter problem, in recent years, the capillary skimmer CID method, which is a fragmentation method using FTICR-MS (Fourier transform ion cyclotron mass spectrometer), is used to fragment proteins instead of pepsin, and to perform H / D exchange. In addition, there is a report that protein-protein interaction site information has been obtained (Non-Patent Document 3), but there is still a problem that a large number of samplings are still necessary.

  In addition, Yamada et al. Reported that protein-protein interaction site information was obtained by a method combining the fragmentation method by FTICR-MS, the hydrogen-deuterium exchange method (H / D exchange method), and affinity chromatography. (Non-Patent Document 4). In this method, interaction site information can be obtained with a small amount of sampling.

  As described above, in conventional protein tertiary structure prediction, a three-dimensional structure is constructed using a computer from an amino acid sequence, but it is a virtual structure and has not been verified by experiments. In order to evaluate in comparison with the three-dimensional structure, the structural analysis must be performed using X-ray crystal structure analysis or NMR. For this purpose, preparation of a large amount of protein, crystallization, stable isotope labeling and the like must be performed, which requires much labor and time.

  Further, the method of combining MS and H / D exchange reaction is a method that can obtain protein three-dimensional structure information with high sensitivity but low accuracy, but still requires time and labor for experiments.

  Accordingly, an object of the present invention is to provide a highly sensitive and quick measurement technique for three-dimensional structure information, which can evaluate a three-dimensional structure prediction result and provide structural information useful for structure prediction.

  In order to solve the above problems, the present inventor has developed a method for rapidly and highly sensitively obtaining three-dimensional structure information of a protein with respect to a fragmentation technique by mass spectrometry (MS) and structure information obtained therefrom. We examined in detail. As a result, the conventional method based on the H / D exchange reaction still has problems such as a large number of samplings and the occurrence of H / D scrambling.

  Therefore, for fragmentation by MS, measurement of Hexapore assisted capillary skimmer CID method (hereinafter referred to as “hexapole CID method”) and infrared multiphoton dissociation method (hereinafter referred to as “IRMPD method”). As a result of careful examination of the method and the fragmentation spectrum obtained by the method, it was found that fragmentation reflecting the secondary structure, tertiary structure, fluctuation, etc. of the protein occurred.

  That is, it has been found that a strong portion having a secondary structure such as α-helix or β-strand in the protein structure is less likely to be fragmented, and a highly mobile portion such as a loop is likely to be fragmented.

  Furthermore, the candidate protein used for three-dimensional structure prediction is narrowed down by comparing with the secondary structure of the candidate protein with the known three-dimensional structure ranked using the 3D-1D method from the amino acid sequence of the protein whose three-dimensional structure is desired. I was able to.

  In addition, the predicted structure could be evaluated by referring the protein fragmentation data by MS to the predicted structure by the calculation method.

  The present invention has been completed based on these various findings.

  In order to achieve the above-described object, a protein structure analysis method, a protein structure analysis apparatus, and a program according to the present invention include a fragmentation spectrum measurement step of measuring a fragmentation spectrum by fragmenting a target protein that predicts a three-dimensional structure ( The fragmentation ion attribution information for the amino acid sequence of the target protein is determined based on the fragmentation spectrum measurement means) and the fragmentation spectrum measured by the fragmentation spectrum measurement step (fragmentation spectrum measurement means). By the attribution information determination step (attribute information determination means) and the attribute information of the fragmented ions determined by the attribute information determination step (attribute information determination means), the fragment ions in the amino acid sequence of the target protein Identify the fragmented part and An easy-cleavage region information determination step (easy-cleavage region information determination means) for determining easy-cleavage region information in the amino acid sequence of the target protein according to a place, and a three-dimensional structure prediction step (three-dimensional structure prediction) for predicting a three-dimensional structure of the target protein Means), the three-dimensional structure prediction data predicted by the three-dimensional structure prediction step (three-dimensional structure prediction means), and the easy-cleavage area information determined by the easy-cleavage area information determination step (easy-cleavage area information determination means) And a processing result output step (processing result output means) that outputs the data in correspondence with each other.

  According to this method, apparatus, and program, ions ionized by an electrospray ionization method or the like for a target protein for predicting a three-dimensional structure are fragmented into fragmented ions by a hexapole CID method or the like, and a fragmentation spectrum is measured. Based on the measured fragmentation spectrum, the fragmentation ion attribution information for the target protein amino acid sequence is determined, and the fragmentation ion attribution information is fragmented into fragmentation ions in the target protein amino acid sequence. The target region is identified, the easy-cleavage region information in the amino acid sequence of the target protein is determined, the three-dimensional structure is predicted for the target protein, and the predicted three-dimensional structure prediction data corresponds to the determined easy-cleavage region information. Output (for example, graphic display When displaying a three-dimensional structure of a protein with an unknown three-dimensional structure (including a gene encoding it), it is possible to make a high-precision prediction by taking into account the measured three-dimensional structure information. It becomes like this.

  The protein structure analysis method, protein structure analysis apparatus, and program according to the next invention are the above-described processing result output step (processing result output means) in the protein structure analysis method, protein structure analysis apparatus, and program described above. The three-dimensional structure prediction data is graphically displayed by any one of a wire model, a ribbon model, a pipe model, a ball and stick model, or a space filling model, and the three-dimensional structure prediction corresponding to the easy-to-cut region information is displayed. It is characterized by being displayed in association with a data display portion.

  This more specifically shows an example of the processing result output. According to this method, apparatus, and program, the three-dimensional structure prediction data is graphically displayed by one of a wire model, a ribbon model, a pipe model, a ball and stick model, or a space filling model, and an easy-to-cut area Information is displayed in association with the display portion of the corresponding three-dimensional structure prediction data (for example, a link is set, displayed on the model, and a specific pattern pattern such as shading or diagonal lines corresponding to easy-to-cut area information is displayed on the model. Therefore, it becomes possible to display such information visually and easily, and the user can intuitively determine the reliability of the three-dimensional structure prediction data.

  The protein structure analysis method, protein structure analysis apparatus, and program according to the next invention are the above-described processing result output step (processing result output means) in the protein structure analysis method, protein structure analysis apparatus, and program described above. Is characterized by displaying the corresponding three-dimensional structure prediction data display portion in different colors according to the easy-to-cut region information.

  This more specifically shows an example of the processing result output. According to this method, apparatus, and program, when the graphic display or the table display is performed, the display portion of the corresponding three-dimensional structure prediction data corresponding to the easy-to-cut area information is displayed in different colors. Therefore, the user can intuitively determine the reliability of the three-dimensional structure prediction data.

  The protein structure analysis method, protein structure analysis apparatus, and program according to the next invention are the protein structure analysis method, protein structure analysis apparatus, and program described above, in the three-dimensional structure prediction step (three-dimensional structure prediction means). Is compared with the easy-to-cut region information determining step (the easy-to-cut region information determining unit), and the cutting specified by the easy-to-cut region information is compared. A prediction structure evaluation information determination step (prediction structure evaluation information determination means) for evaluating the prediction structure portion corresponding to the easy region and determining prediction structure evaluation information, and the prediction structure evaluation information determination step (prediction structure evaluation information determination means) Predictive structure evaluation information output for outputting predicted structure evaluation information determined by -Up further comprises a (predicted structure evaluation information output unit) (comprises) can be characterized.

  According to this method, apparatus, and program, the predicted three-dimensional structure prediction data is compared with the determined easy-cleavage region information, and the predicted structure corresponding to the easy-cleavage region specified by the easy-cleavage region information Since the part is evaluated and the predicted structure evaluation information is determined and the determined predicted structure evaluation information is output, the predicted structure predicted by the computer can be evaluated based on the biochemical experiment data. The prediction accuracy can be remarkably improved.

  This also greatly improves the accuracy of function prediction based on the three-dimensional structure when conducting functional analysis of interesting genes and proteins found by genome sequence analysis, expression profiling analysis using DNA chips, proteome analysis, etc. In genome sequence analysis, the function can be efficiently estimated by performing function prediction in consideration of the three-dimensional structure of a gene / protein whose function is unknown. Further, this makes it possible to efficiently design drugs such as inhibitors, improve activity by protein engineering, and design functional modifications.

  The protein structure analysis method, the protein structure analysis apparatus, and the program according to the next invention are the reference vibration for performing the reference vibration calculation for the target protein in the protein structure analysis method, the protein structure analysis apparatus, and the program described above. The processing result output step (processing result output means) further includes (comprises) a calculation step (reference vibration calculation means), and the three-dimensional structure prediction data corresponding to the calculation result of the reference vibration calculation step (reference vibration calculation means) It is characterized by being displayed in association with the display part.

  According to this method, apparatus, and program, the reference vibration calculation is performed for the target protein, and the reference vibration calculation result (for example, information on fluctuation) is displayed in association with the display portion of the corresponding three-dimensional structure prediction data (for example, Since the vector is displayed on the three-dimensional structure model), the fluctuation information can be considered as the easy-to-cut region. That is, the prediction model can be evaluated by obtaining fluctuation information from the reference vibration calculation result and comparing it with the MS actual measurement data.

  The protein structure analysis method, the protein structure analysis device, and the program according to the next invention are the above-described fragmentation spectrum measurement step (fragmentation spectrum measurement) in the protein structure analysis method, protein structure analysis device, and program described above. Means) include a hexapole CID method, a nozzle skimmer CID method, a capillary skimmer CID method, a SORI-CID method, and a collision activated dissociation (CID) method including a multipole store auxiliary capillary skimmer CID method, an IRMPD method, an in-source method The target protein is fragmented into fragment ions by at least one of decomposition (ISD) method, post-source decomposition (PSD) method, surface induced dissociation (SID) method, ECD method, and BIRD method. Measure And wherein the door.

  This shows one example of the fragmentation spectrum measurement more specifically. According to this method, apparatus, and program, a hexapole CID method, a nozzle skimmer CID method, a capillary skimmer CID method, a SORI-CID method, and a multipole store-assisted capillary skimmer CID method are used for a target protein that predicts a three-dimensional structure. At least one of collision activated dissociation (CID) method, IRMPD method, in-source decomposition (ISD) method, post-source decomposition (PSD) method, surface-induced dissociation (SID) method, ECD method, and BIRD method Since the target protein is fragmented into fragmented ions by the method and the fragmentation spectrum is measured, fragmented ions reflecting the three-dimensional structure of the target protein can be efficiently created.

  The protein structure analysis method, the protein structure analysis device, and the program according to the next invention are the above-described fragmentation spectrum measurement step (fragmentation spectrum measurement) in the protein structure analysis method, protein structure analysis device, and program described above. (Means) is characterized in that the target protein is fragmented by an enzymatic reaction and the fragmentation spectrum is measured.

  This shows one example of the fragmentation spectrum measurement more specifically. According to this method, apparatus, and program, since the target protein is fragmented by an enzymatic reaction and the fragmentation spectrum is measured, the fragmentation reflecting the three-dimensional structure of the target protein can be efficiently performed. Become.

  A protein structure analysis method, a protein structure analysis apparatus, and a program according to the next invention include a fragmentation spectrum measurement step (fragmentation spectrum measurement means) that measures a fragmentation spectrum by fragmenting a target protein for which a three-dimensional structure is predicted. , An attribution information determination step (identification information) for determining the attribution information of the fragmentation ion to the amino acid sequence of the target protein based on the fragmentation spectrum measured by the fragmentation spectrum measurement step (fragmentation spectrum measurement means) Determination means), the fragmentation spectrum measured by the fragmentation spectrum measurement step (fragmentation spectrum measurement means), and the fragmentation ions determined by the attribution information determination step (attribute information determination means) According to attribution information A solvent contact residue information determination step (solvent contact residue information determination step) that identifies a position in contact with a solvent in the amino acid sequence of the protein and determines solvent contact residue information in the amino acid sequence of the target protein according to the position Determination means), a three-dimensional structure prediction step (three-dimensional structure prediction means) for predicting a three-dimensional structure of the target protein, three-dimensional structure prediction data predicted by the three-dimensional structure prediction step (three-dimensional structure prediction means), and the solvent contact A processing result output step (processing result output means) for outputting the corresponding information to the solvent contact residue information determined by the residue information determination step (solvent contact residue information determination means). And

  According to this method, apparatus, and program, fragmentation is performed on a target protein for which a three-dimensional structure is predicted, a fragmentation spectrum is measured, and based on the measured fragmentation spectrum, fragmentation ions for the amino acid sequence of the target protein are measured. Determine the attribution information, identify the location in contact with the solvent in the amino acid sequence of the target protein according to the measured fragmentation spectrum and the determined attribution information of the fragmented ion, and the amino acid of the target protein according to the location Solvent contact residue information in the sequence is determined, the three-dimensional structure is predicted for the target protein, and the predicted three-dimensional structure prediction data and the determined solvent contact residue information are output in correspondence with each other (eg, graphic display, table) List of proteins, etc.). When performing three-dimensional structure prediction of containing the gene) to, by adding the actually measured three-dimensional structure information, it is possible to perform a highly accurate prediction.

  The protein structure analysis method, protein structure analysis apparatus, and program according to the next invention are the above-described processing result output step (processing result output means) in the protein structure analysis method, protein structure analysis apparatus, and program described above. Displays the three-dimensional structure prediction data graphically by any one of a wire model, a ribbon model, a pipe model, a ball and stick model, or a space-filling model, and the solvent-contacting residue information corresponds to the corresponding three-dimensional structure It is characterized by being displayed in association with the display portion of the prediction data.

  This more specifically shows an example of the processing result output. According to this method, apparatus, and program, the three-dimensional structure prediction data is graphically displayed by one of a wire model, a ribbon model, a pipe model, a ball and stick model, or a space filling model, and a solvent contact residue is displayed. Display the base information in association with the display portion of the corresponding three-dimensional structure prediction data (for example, set a link, display on the model, and display a specific pattern pattern such as shading or diagonal lines corresponding to the solvent contact area information on the model. Therefore, such information can be displayed visually and in an easily understandable manner, and the user can intuitively determine the reliability of the three-dimensional structure prediction data.

  The protein structure analysis method, protein structure analysis apparatus, and program according to the next invention are the above-described processing result output step (processing result output means) in the protein structure analysis method, protein structure analysis apparatus, and program described above. Is characterized by displaying the corresponding three-dimensional structure prediction data in a color-coded manner in accordance with the solvent contact residue information.

  This more specifically shows an example of the processing result output. According to this method, apparatus, and program, when performing graphic display or table display, the display portion of the corresponding three-dimensional structure prediction data corresponding to the solvent contact residue information is displayed in different colors. It becomes possible to display visually and intelligibly, and the user can intuitively determine the reliability of the three-dimensional structure prediction data.

  The protein structure analysis method, protein structure analysis apparatus, and program according to the next invention are the protein structure analysis method, protein structure analysis apparatus, and program described above, in the three-dimensional structure prediction step (three-dimensional structure prediction means). Is compared with the solvent contact residue information determined in the solvent contact residue information determination step (solvent contact residue information determination means), and the solvent contact residue information is compared. A predicted structure evaluation information determination step (predicted structure evaluation information determination means) that evaluates the predicted structure portion corresponding to the specified solvent-contacting residue and determines predicted structure evaluation information, and the predicted structure evaluation information determination step (predicted structure) Predicted structure evaluation information output unit for outputting the predicted structure evaluation information determined by the evaluation information determining means) -Up further comprises a (predicted structure evaluation information output unit) (comprises) can be characterized.

  According to this method, apparatus, and program, the predicted three-dimensional structure prediction data is compared with the determined solvent contact residue information to correspond to the solvent contact residue specified by the solvent contact residue information. The predicted structure evaluation information is determined and the predicted structure evaluation information is determined and the determined predicted structure evaluation information is output, so that the predicted structure predicted by the computer can be evaluated based on the biochemical experiment data. Thus, the prediction accuracy can be remarkably improved.

  This also greatly improves the accuracy of function prediction based on the three-dimensional structure when conducting functional analysis of interesting genes and proteins found by genome sequence analysis, expression profiling analysis using DNA chips, proteome analysis, etc. In genome sequence analysis, the function can be efficiently estimated by performing function prediction in consideration of the three-dimensional structure of a gene / protein whose function is unknown. Further, this makes it possible to efficiently design drugs such as inhibitors, improve activity by protein engineering, and design functional modifications.

  The protein structure analysis method, the protein structure analysis apparatus, and the program according to the next invention are the reference vibration for performing the reference vibration calculation for the target protein in the protein structure analysis method, the protein structure analysis apparatus, and the program described above. The processing result output step (processing result output means) further includes (comprises) a calculation step (reference vibration calculation means), and the three-dimensional structure prediction data corresponding to the calculation result of the reference vibration calculation step (reference vibration calculation means) It is characterized by being displayed in association with the display part.

  According to this method, apparatus, and program, the reference vibration calculation is performed for the target protein, and the reference vibration calculation result (for example, information on fluctuation) is displayed in association with the display portion of the corresponding three-dimensional structure prediction data (for example, Since the vector display is performed on the three-dimensional structure model, fluctuation information can be considered as the solvent contact area. That is, the prediction model can be evaluated by obtaining fluctuation information from the reference vibration calculation result and comparing it with the MS actual measurement data.

  The protein structure analysis method, the protein structure analysis device, and the program according to the next invention are the above-described fragmentation spectrum measurement step (fragmentation spectrum measurement) in the protein structure analysis method, protein structure analysis device, and program described above. Means) after subjecting the target protein to hydrogen-deuterium exchange reaction, fragmentation and measurement of the fragmentation spectrum, and the solvent contact residue information determination step (solvent contact residue information determination means) The target protein according to the fragmentation spectrum measured by the fragmentation spectrum measurement step (fragmentation spectrum measurement means) and the attribution information of the fragmentation ion determined by the attribution information determination step (attribute information determination means) At each amino acid residue in the above amino acid sequence Determine the deuteration rate, identify the position in contact with the solvent in the amino acid sequence of the target protein according to the deuteration rate, and determine the solvent contact residue information in the amino acid sequence of the target protein according to the position. It is characterized by determining.

  This shows one example of the fragmentation spectrum measurement more specifically. According to this method, apparatus, and program, after performing a hydrogen-deuterium exchange reaction on the target protein, the fragmentation spectrum is measured and the measured fragmentation spectrum and the determined fragmentation ion are determined. In accordance with the attribution information of the target protein, the deuteration rate at each amino acid residue in the amino acid sequence of the target protein is determined, the location in contact with the solvent in the amino acid sequence of the target protein is identified according to the deuteration rate, and the location Accordingly, the solvent contact residue information in the amino acid sequence of the target protein is determined according to the above, so that the solvent contact information can be efficiently obtained.

  The protein structure analysis method, the protein structure analysis device, and the program according to the next invention are the above-described fragmentation spectrum measurement step (fragmentation spectrum measurement) in the protein structure analysis method, protein structure analysis device, and program described above. Means) after chemically modifying the target protein, fragmenting and measuring the fragmentation spectrum, and the solvent contact residue information determination step (solvent contact residue information determination means) includes the fragmentation spectrum measurement step The amino acid sequence of the target protein according to the fragmentation spectrum measured by (fragmentation spectrum measurement means) and the attribution information of the fragmentation ions determined by the attribution information determination step (attribute information determination means) Chemical modification at each amino acid residue Characterized in that, in accordance with the chemical modification portion, a position in contact with the solvent in the amino acid sequence of the target protein is identified, and solvent contact residue information in the amino acid sequence of the target protein is determined in accordance with the position. And

  This shows one example of the fragmentation spectrum measurement more specifically. According to this method, apparatus, and program, after subjecting a target protein to chemical modification, a fragmentation fragmentation spectrum is measured, and according to the measured fragmentation spectrum and the determined fragmentation ion assignment information , Determine the chemical modification part of each amino acid residue in the amino acid sequence of the target protein, identify the position in contact with the solvent in the amino acid sequence of the target protein according to the chemical modification part, and according to the position, the amino acid sequence of the target protein Since the solvent contact residue information in is determined, the solvent contact information can be efficiently obtained.

  The protein structure analysis method, the protein structure analysis device, and the program according to the next invention are the above-described fragmentation spectrum measurement step (fragmentation spectrum measurement) in the protein structure analysis method, protein structure analysis device, and program described above. Means) include a hexapole CID method, a nozzle skimmer CID method, a capillary skimmer CID method, a SORI-CID method, and a collision activated dissociation (CID) method including a multipole store auxiliary capillary skimmer CID method, an IRMPD method, an in-source method The target protein is fragmented into fragment ions by at least one of decomposition (ISD) method, post-source decomposition (PSD) method, surface induced dissociation (SID) method, ECD method, and BIRD method. Measure And wherein the door.

  This shows one example of the fragmentation spectrum measurement more specifically. According to this method, apparatus, and program, a hexapole CID method, a nozzle skimmer CID method, a capillary skimmer CID method, a SORI-CID method, and a multipole store-assisted capillary skimmer CID method are used for a target protein that predicts a three-dimensional structure. At least one of collision activated dissociation (CID) method, IRMPD method, in-source decomposition (ISD) method, post-source decomposition (PSD) method, surface-induced dissociation (SID) method, ECD method, and BIRD method Since the target protein is fragmented into fragmented ions by the method and the fragmentation spectrum is measured, fragmented ions reflecting the three-dimensional structure of the target protein can be efficiently created.

  The protein structure analysis method, the protein structure analysis device, and the program according to the next invention are the above-described fragmentation spectrum measurement step (fragmentation spectrum measurement) in the protein structure analysis method, protein structure analysis device, and program described above. (Means) is characterized in that the target protein is fragmented by an enzymatic reaction and the fragmentation spectrum is measured.

  This shows one example of the fragmentation spectrum measurement more specifically. According to this method, apparatus, and program, it is possible to efficiently obtain solvent contact site information and the like by fragmenting the target protein by enzymatic reaction and combining the H / D exchange method and the chemical modification method. It becomes like this.

  A protein structure analysis method, a protein structure analysis apparatus, and a program according to the next invention include a fragmentation spectrum measurement step (fragment) for fragmenting a complex of a target protein and a compound for predicting a three-dimensional structure and measuring a fragmentation spectrum. Fragmentation spectrum measurement means) and the fragmentation ion attribution information for the target protein and / or the compound are determined based on the fragmentation spectrum measured by the fragmentation spectrum measurement step (fragmentation spectrum measurement means). Determined by the attribute information determination step (attribute information determination means), the fragmentation spectrum measured by the fragmentation spectrum measurement step (fragmentation spectrum measurement means), and the attribute information determination step (attribute information determination means). The above fragmentation ion An interaction interface information determination step (interaction interface information determination means) for determining interaction interface information regarding the target protein and / or the compound according to the information, and a three-dimensional structure for predicting a three-dimensional structure of the target protein and / or the compound Determined by the structure prediction step (stereostructure prediction means), the three-dimensional structure prediction data predicted by the three-dimensional structure prediction step (stereostructure prediction means), and the interaction interface information determination step (interaction interface information determination means). And a processing result output step (processing result output means) that outputs the interaction interface information in association with each other.

  According to this method, apparatus, and program, a complex of a target protein and a compound (for example, a protein, a low molecular compound, or a nucleic acid) that predicts a three-dimensional structure is fragmented, and a fragmentation spectrum is measured and measured. The fragmentation ion attribution information for the target protein and / or compound is determined based on the fragmentation spectrum, and the target protein and / or the fragmentation ion identification information is determined according to the measured fragmentation spectrum and the determined fragmentation ion attribution information. The interaction interface information about the compound is determined, the three-dimensional structure is predicted for the target protein and / or compound, and the predicted three-dimensional structure prediction data and the determined interaction interface information are output in correspondence with each other (eg, graphic 3D structure unknown protein When performing a three-dimensional structure estimation of the (containing the gene encoding the same), by adding the actually measured three-dimensional structure information, it is possible to perform a highly accurate prediction.

  The protein structure analysis method, protein structure analysis apparatus, and program according to the next invention are the above-described processing result output step (processing result output means) in the protein structure analysis method, protein structure analysis apparatus, and program described above. Displays the three-dimensional structure prediction data graphically with respect to the target protein and / or the compound by any one of a wire model, a ribbon model, a pipe model, a ball and stick model, or a space filling model, and the interaction The interface information is displayed in association with the display portion of the corresponding three-dimensional structure prediction data.

  This more specifically shows an example of the processing result output. According to this method, apparatus, and program, the three-dimensional structure prediction data is obtained for a target protein and / or compound by any one of a wire model, a ribbon model, a pipe model, a ball and stick model, or a space filling model. Graphic display (for example, including docking simulation) and display the interaction interface information in association with the display part of the corresponding 3D structure prediction data (for example, set a link, display on the model, interaction interface on the model) This makes it possible to display such information visually and in an easy-to-understand manner, so that users can intuitively understand the reliability of 3D structure prediction data. Can be judged automatically.

  The protein structure analysis method, protein structure analysis apparatus, and program according to the next invention are the above-described processing result output step (processing result output means) in the protein structure analysis method, protein structure analysis apparatus, and program described above. Is characterized in that the display portion of the corresponding three-dimensional structure prediction data of the target protein and / or the compound is displayed in different colors according to the interaction interface information.

  This more specifically shows an example of the processing result output. According to this method, apparatus, and program, when graphic display or table display is performed, the display portion of the above-described three-dimensional structure prediction data corresponding to the target protein and / or compound is displayed in different colors according to the interaction interface information. Therefore, it becomes possible to display such information visually and easily, and the user can intuitively determine the reliability of the three-dimensional structure prediction data.

  The protein structure analysis method, protein structure analysis apparatus, and program according to the next invention are the protein structure analysis method, protein structure analysis apparatus, and program described above, in the three-dimensional structure prediction step (three-dimensional structure prediction means). Is compared with the interaction interface information determined in the interaction interface information determination step (interaction interface information determination means), and the interaction information specified by the interaction interface information is compared. A predicted structure evaluation information determining step (predicted structure evaluation information determining means) for evaluating the predicted structure portion corresponding to the working interface and determining predicted structure evaluation information, and the predicted structure evaluation information determining step (predicted structure evaluation information determining means). Predictive structure evaluation information output for outputting predicted structure evaluation information determined by -Up further comprises a (predicted structure evaluation information output unit) (comprises) can be characterized.

  According to the method, the apparatus, and the program, the predicted three-dimensional structure prediction data is compared with the determined interaction interface information, and the predicted structure corresponding to the interaction interface specified by the interaction interface information is compared. Since the part is evaluated and the predicted structure evaluation information is determined and the determined predicted structure evaluation information is output, the predicted structure predicted by the computer can be evaluated based on the biochemical experiment data. The prediction accuracy can be remarkably improved.

  This also greatly improves the accuracy of function prediction based on the three-dimensional structure when performing functional analysis of interesting genes and proteins found by genome sequence analysis, expression profiling analysis using DNA chips, proteome analysis, etc. In genome sequence analysis, the function can be efficiently estimated by performing function prediction in consideration of the three-dimensional structure of a gene / protein whose function is unknown. Furthermore, from the model structure and active site (interaction site) information, drug design for inhibitors and the like, improvement of activity by protein engineering, and design of functional modifications are possible.

  The protein structure analysis method, protein structure analysis apparatus, and program according to the next invention are the above-described protein structure analysis method, protein structure analysis apparatus, and program. A reference vibration calculation step (reference vibration calculation means) for performing calculation is further included (provided), and the processing result output step (processing result output means) corresponds to the calculation result of the reference vibration calculation step (reference vibration calculation means). It is characterized by being displayed in association with the display portion of the three-dimensional structure prediction data.

  According to this method, apparatus, and program, the reference vibration calculation is performed on the target protein and / or compound, and the reference vibration calculation result (for example, fluctuation information) is associated with the display portion of the corresponding three-dimensional structure prediction data. Since the information is displayed (for example, a vector is displayed on the three-dimensional structure model), information on fluctuation can be considered as the interaction interface region. That is, the prediction model can be evaluated by obtaining fluctuation information from the reference vibration calculation result and comparing it with the MS actual measurement data.

  The protein structure analysis method, the protein structure analysis device, and the program according to the next invention are the above-described fragmentation spectrum measurement step (fragmentation spectrum measurement) in the protein structure analysis method, protein structure analysis device, and program described above. Means), after performing a hydrogen-deuterium exchange reaction on the complex of the target protein and the compound, fragmenting, measuring the fragmentation spectrum, and determining the interaction interface information (determination of interaction interface information) Means) the fragmentation spectrum measured by the fragmentation spectrum measurement step (fragmentation spectrum measurement means), and the attribution of the fragmentation ions determined by the attribution information determination step (attribute information determination means). According to the information, the target protein and / or above Determine the deuteration rate in the compound, identify the interaction interface in the target protein and / or the compound according to the deuteration rate, and interact interface information in the target protein and / or the compound according to the interaction interface It is characterized by determining.

  This shows one example of the fragmentation spectrum measurement more specifically. According to this method, apparatus, and program, after a hydrogen-deuterium exchange reaction is performed on a complex of a target protein and a compound, the fragmentation is measured and the fragmentation spectrum is measured, the measured fragmentation spectrum, and Determining the deuteration rate in the target protein and / or compound according to the determined fragmentation ion assignment information, identifying the interaction interface in the target protein and / or compound according to the deuteration rate, and determining the interaction Since the interaction interface information in the target protein and / or compound is determined according to the interface, the interaction interface information can be efficiently obtained.

  The protein structure analysis method, the protein structure analysis device, and the program according to the next invention are the above-described fragmentation spectrum measurement step (fragmentation spectrum measurement) in the protein structure analysis method, protein structure analysis device, and program described above. Means) after chemically modifying the complex of the target protein and the compound, fragmenting and measuring the fragmentation spectrum, the interaction interface information determination step (interaction interface information determination means), According to the fragmentation spectrum measured by the fragmentation spectrum measurement step (fragmentation spectrum measurement means) and the attribution information of the fragmentation ions determined by the attribution information determination step (attribute information determination means) The target protein and / or the above compound Determining a chemical modification moiety, identifying an interaction interface in the target protein and / or the compound according to the chemical modification moiety, and determining interaction interface information in the target protein and / or the compound according to the interaction interface It is characterized by that.

  This shows one example of the fragmentation spectrum measurement more specifically. According to this method, apparatus, and program, after chemical modification is performed on a complex of a target protein and a compound, a fragmented fragmentation spectrum is measured, and the measured fragmentation spectrum and the determined fragment are determined. In accordance with the attribute information of the cation ion, a chemical modification moiety in the target protein and / or compound is determined, an interaction interface in the target protein and / or compound is identified according to the chemical modification moiety, and the target protein and / or in accordance with the interaction interface Since the interaction interface information in the compound is determined, the interaction interface information can be efficiently obtained.

  The protein structure analysis method, the protein structure analysis device, and the program according to the next invention are the above-described fragmentation spectrum measurement step (fragmentation spectrum measurement) in the protein structure analysis method, protein structure analysis device, and program described above. Means) include a hexapole CID method, a nozzle skimmer CID method, a capillary skimmer CID method, a SORI-CID method, and a collision activated dissociation (CID) method including a multipole store auxiliary capillary skimmer CID method, an IRMPD method, an in-source method Fragmentation of the complex of the target protein and the compound by at least one of decomposition (ISD) method, post-source decomposition (PSD) method, surface induced dissociation (SID) method, ECD method, and BIRD method Fragmented into ions above And measuring the fragmented spectrum.

  This shows one example of the fragmentation spectrum measurement more specifically. According to this method, apparatus, and program, a hexapole CID method, a nozzle skimmer CID method, a capillary skimmer CID method, a SORI-CID method, and a multipole store-assisted capillary skimmer CID method are used for a target protein that predicts a three-dimensional structure. At least one of collision activated dissociation (CID) method, IRMPD method, in-source decomposition (ISD) method, post-source decomposition (PSD) method, surface-induced dissociation (SID) method, ECD method, and BIRD method Since the complex of the target protein and the compound is fragmented into fragment ions by the method and the fragmentation spectrum is measured, fragment ions reflecting the three-dimensional structure of the target protein can be efficiently created.

  The protein structure analysis method, the protein structure analysis device, and the program according to the next invention are the above-described fragmentation spectrum measurement step (fragmentation spectrum measurement) in the protein structure analysis method, protein structure analysis device, and program described above. (Means) is characterized in that the complex of the target protein and the compound is fragmented by an enzymatic reaction and the fragmentation spectrum is measured.

  This shows one example of the fragmentation spectrum measurement more specifically. According to this method, apparatus, and program, fragmentation is performed on a complex of a target protein and a compound by an enzymatic reaction, and the interaction interface information is efficiently obtained by combining the H / D exchange method and the chemical modification method. To be able to get to.

  The present invention also relates to a recording medium, and the recording medium according to the present invention is a recording medium for causing a computer to execute the program described above.

  According to this recording medium, by causing the computer to read and execute the recording medium, the program described above can be realized using the computer, and the same effect as each of the programs can be obtained. it can.

  Hereinafter, embodiments of a protein structure analysis method, a protein structure analysis apparatus, a program, and a recording medium according to the present invention will be described in detail with reference to the drawings. Note that the present invention is not limited to the embodiments.

  In particular, in the following embodiment, an example in which the present invention is applied to interleukin-6 and the like will be described. However, the present invention is not limited to this case, and can be similarly applied to all proteins.

[Outline of the present invention]
Hereinafter, the outline of the present invention will be described, and then the configuration and processing of the present invention will be described in detail. FIG. 1 is a principle configuration diagram showing the basic principle of the present invention.

  The present invention generally has the following basic features. First, a three-dimensional structure prediction target protein (hereinafter referred to as “target protein”) for which a three-dimensional structure is predicted is fragmented by various fragmentation methods or enzyme digestion methods using MS, and a fragmentation spectrum is measured.

  The target protein is measured by MS using at least one of an ionization method such as an electrospray ionization method, a laser spray ionization method, a sonic spray ionization method, an atmospheric pressure chemical ionization method, a MALDI method, and other equivalent ionization methods. After ionization by one method, the generated ions are subjected to hexapole CID method (similar method to nozzle skimmer CID method, capillary skimmer CID method, multipole store auxiliary capillary skimmer CID method), SORI-CID. Collision-induced dissociation (CID) method, IRMPD method, in-source decomposition (ISD) method, post-source decomposition (PSD), surface-induced dissociation (SID) method, ECD (electron supplemental dissociation) method, BIRD (black body) Infrared radiation dissociation method In addition, the target protein may be fragmented into fragment ions by measuring at least one of the corresponding fragmentation methods, and the fragmentation spectrum may be measured, or the target protein may be obtained by enzymatic digestion using pepsin or trypsin. The protein may be fragmented and the fragmentation spectrum may be measured.

  The capillary skimmer CID method is an in-source fragmentation method in which fragmentation is induced by using the voltage between the capillary and the skimmer in the ionization and ion introduction section (S. Akashi, Anal. Chem., 71, 4974-4980). 1999)).

  Further, the hexapole CID method is a method for further improving the efficiency of the capillary skimmer CID method by storing ions in the hexapole for a certain period of time (KA, Sannes-Lowery, J. Am. Soc. Mass Spectrom. , 11, 1-9 (2000)).

  Further, the IRMPD method is a method of inducing fragmentation by irradiating a carbon dioxide laser to ions (see CP Dufresne, J. Am. Soc. Mass Spectrom., 9, 1222-1225 (1998)). ).

  Then, each fragment is assigned in the fragmentation spectrum obtained using the above method. That is, based on the measured fragmentation spectrum, the attribution information of the fragmented ions with respect to the amino acid sequence of the target protein is determined. Here, the amino acid sequence of the target protein may be used when stored in the PDB, and the target protein can be obtained by using an amino acid sequence identification method such as MALDI-TOF-MS or MS / MS. May be identified.

  And the location fragmented by the fragmentation ion in the amino acid sequence of the target protein is specified by the determined fragmentation ion attribution information, and easy-cleavage region information in the amino acid sequence of the target protein is determined according to the location.

  Here, FIG. 6 is a conceptual diagram when determining fragmentable ion attribution information for human interleukin-6 (IL-6) and determining easy-cleavage region information. In FIG. 6, fragmented ion fragments when fragmented by the IRMPD method are indicated by dotted lines, and fragmented ion fragments when fragmented by the hexapole CID method are indicated by solid lines. In addition, easy-cleavage region information obtained based on the cleavage sites of these fragmented ions is indicated by hatching.

  Then, a three-dimensional structure is predicted for the target protein. Here, the three-dimensional structure prediction may use any of the existing three-dimensional structure prediction methods such as a homology modeling method, a molecular simulation method, an ab initio method, a secondary structure prediction method, a 3D-1D method, and a threading method. Hereinafter, a three-dimensional structure prediction method based on sequence homology will be described as an example.

  Here, FASTA (Pearson WR, Methods Enzymol, 266, 227-258, 1996), PSI-BLAST (Schaffer AA, Wolf YI, Ponting CP, Koonin EV, Aravind L and Altschul SF, Bioinformatics, 12, 1000-101, aI. , K., Protein Engineering, 10, 339-351, 1997), RBS-BLAST (Schaffer AA, olf YI, Ponting CP, Koonin EV, Aravind L and Altschul SF, Bioinformatics, 12, 1000-1011, 1999), IMPALA (A. A. Schaffet et al., Bioinformatics, 1000 (101), 1999 (101), 15 (19), 1999). HMMER (R. Durbin, S. Eddy, A. Krogh, and G. Mitchison, Cambridge University Press, 1998, SR Eddy. Bioinformatics, 14, 755-763, 1998), or ClustalW (Th. D., D. G. Higgins, and T. J. Gibson, Nuclei c Acids Res., 22, 4673-4680, 1994) and the like may be used.

  It is preferable to use a database obtained by processing PDB (Protein Data Base) in advance as a search target. The processing database is created by classifying all protein sequences registered in the PDB into classes having a sequence homology (homology) of 95% or more, and extracting the most experimental accuracy of each class as a representative sequence. By using this processing database, the efficiency of the homology search is increased, and the diversity and variety of the three-dimensional structures searched can be ensured even when the number of searches is limited.

  From the output result of the search program, pairwise alignments between the three-dimensional structure prediction target protein and the reference protein sequence are individually created for the number of searches. For each pair-wise alignment, a secondary structure region in the three-dimensional structure of the reference protein is identified and queried on the reference protein sequence. Multiple alignment may be performed based on the three-dimensional structure prediction target protein and a plurality of reference protein sequences.

  Then, the easy-to-cut region information obtained from the MS fragmentation data is referred to the alignment result displaying the secondary structure information. That is, both ends of the fragmented ion obtained by MS are sites that are easily cleaved and are sites that are highly likely to correspond to loop portions with high mobility in terms of the three-dimensional structure. On the other hand, since the part sandwiched between both ends of the fragmented ions obtained by MS is a part where fragmentation hardly occurs, it corresponds to a part having a secondary structure such as α helix or β strand. It is a site that is considered highly likely.

  Then, the candidate proteins are evaluated (ranked) in consideration of information on all fragmented ions.

  Subsequently, based on the three-dimensional structure of the highly evaluated candidate protein, the three-dimensional structure of the protein to be predicted is predicted by the homology modeling method. Here, as a three-dimensional structure prediction program, “FAMS” (Ogata, K. and Umeyama, H., J. Mol. Graphics Mod. 18, 258-272, 2000), Swiss-Model (Peitsch MC., Biochem. Soc.Trans.24,274-279, 1996), CPHmodels (O. Lund, K. Friand, J. Gorodkin, H. Bohr, J. Bohr, J. Hansen, and S. Brunak., Protein Engineering, 10, Protein Engineering). 1241-1248, 1997), “SAM-T98” (J. Park, K. Karplus, C. Barrett, R. Hughhey, D. Haussler, T. Hubbard, and C. C. othia, JMB 284 (4), 1201-1210, 1998), “MODELLER” (A. Sali, L. Potterton, F. Yuan, H. van Vlijmen, and M. Karplus, Proteins, 23, 318-326, 1995). ) Etc. may be adopted. In addition, the three-dimensional structure may be disclosed or referred to in Daniel Fischer et al., “CAFASP2 The Second Critical Assessment of Full Automated Structure Prediction Methods, PROTEINS, 5; 171-183 (2001)”.

  For the candidate protein, the predicted three-dimensional structure prediction data and the determined easy-cleavage region information are output in correspondence with each other (for example, graphic display, list display by table, etc.). For example, the three-dimensional structure prediction data is displayed graphically as a wire model, ribbon model, pipe model, ball-and-stick model, or space filling model, and the three-dimensional structure prediction data corresponding to the easy-to-cut area information is displayed. It may be displayed in association with the part (for example, a link is set, displayed on the model, a specific pattern such as shading or diagonal lines corresponding to the easy-to-cut area information is displayed on the model, etc.), or a graphic display When the table is displayed, the display portion of the corresponding three-dimensional structure prediction data may be displayed in different colors according to the easy-to-cut area information.

  The predicted three-dimensional structure prediction data is compared with the determined easily cleavable region information, and the predicted structure evaluation information corresponding to the easily cleavable region specified by the easily cleavable region information is evaluated to determine the predicted structure evaluation information. The determined predicted structure evaluation information is output. That is, the obtained predicted structure and MS fragmentation data are queried to evaluate the predicted structure.

  Further, for example, for a candidate protein with high homology, it is possible to perform evaluation with higher accuracy by measuring the fragmentation spectrum by MS and comparing it with the fragmentation spectrum of the three-dimensional structure prediction target protein.

  Furthermore, the present invention performs a hydrogen-deuterium exchange reaction on a target protein or complex for which a three-dimensional structure is predicted, fragments into fragmented ions, and measures a fragmentation spectrum.

  Here, FIG. 19 shows that a complex of human interleukin-6 (IL-6) and anti-IL-6 antibody is subjected to hydrogen-deuterium exchange reaction and fragmented by electrospray ionization. It is the conceptual diagram at the time of fragmenting into ion and measuring a fragmentation spectrum.

  Then, based on the measured fragmentation spectrum, the fragmentation ion attribution information for the amino acid sequence of the target protein is determined, and the target protein is determined according to the measured fragmentation spectrum and the determined fragmentation ion attribution information. The deuteration rate at each amino acid residue in the amino acid sequence is determined.

  Here, FIG. 20 is a conceptual diagram when the deuteration rate is obtained from the fragmentation spectrum of human IL-6 subjected to the hydrogen-deuterium exchange reaction. As shown in FIG. 20, the fragmentation spectrum (upper stage) of human IL-6 that had undergone a hydrogen-deuterium exchange reaction and the fragmentation spectrum of human IL-6 that had not undergone a hydrogen-deuterium exchange reaction The deuteration rate (D conversion rate) is determined from the change in the center of gravity value (lower), and the deuteration rate is calculated from the deuteration rate obtained over time.

  Then, in accordance with the deuteration rate at each amino acid residue in the determined amino acid sequence, the location in contact with the solvent in the amino acid sequence of the target protein is specified, and the solvent contact residue in the amino acid sequence of the target protein according to the location Determine information. For the solvent contact residue information, for example, a site where the deuteration rate is slow compared to a control experiment in which hydrogen-deuterium exchange is not performed may be determined as the solvent contact residue information.

  In the case of a complex, interaction interface information in the amino acid sequence of the target protein is determined according to the deuteration rate at each amino acid residue in the determined amino acid sequence. As for the interaction interface information, for example, a site where the deuteration rate is slow may be determined as the interaction interface information as compared with the case where measurement is performed when the complex is not formed alone. Alternatively, a site where the deuteration rate is slow compared to a control experiment in which hydrogen-deuterium exchange is not performed may be determined as interaction interface information.

  Thus, the determined solvent contact residue information and interaction interface information are output together with the prediction data, or used when determining the predicted structure evaluation information. That is, the predicted three-dimensional structure prediction data is output in association with the determined solvent contact residue information and interaction interface information (for example, graphic display, list display by table, etc.), and the predicted three-dimensional structure Comparing the predicted data with the determined solvent contact residue information and interaction interface information to support the interaction interface specified by the solvent contact residue and interaction interface information specified by the solvent contact residue information The predicted structure portion to be evaluated is evaluated to determine the predicted structure evaluation information, and the determined predicted structure evaluation information is output.

  Here, FIG. 12 graphically displays the three-dimensional structure prediction data with a ribbon model for human interleukin-6 (IL-6), and color-codes the corresponding three-dimensional structure prediction data display portion according to the easy-to-cut region information. It is a figure which shows an example of the display screen in the case of displaying. In FIG. 12, the easy-to-cut region information is shown in black, and the interaction interface information is shown in gray.

  FIG. 17 is a diagram illustrating an example of a display screen when a list of processing results is displayed using a table. As shown in this figure, the processing result list display screen includes, for example, a display area MA-1 for displaying amino acid residue numbers of proteins, and a display area MA- for displaying easy-to-cut areas determined according to the present invention. 2. Display area MA-3 for displaying solvent contact residue information determined by the present invention, display area MA-4 for displaying interaction interface information determined by the present invention, three-dimensional structure by structure prediction method A display area MA-5 for displaying structure prediction data (for example, secondary structure), a display area MA-6 for displaying prediction structure evaluation information for the three-dimensional structure prediction data determined by the present invention, and the like are included. It consists of As shown in the figure, these pieces of information may be displayed in a table format for each amino acid residue of the protein. Further, when displaying the interaction interface information, it is desirable to distinguish and display one-to-many interactions (for example, a display method of A, B, C,... May be adopted). .

  In addition, it is considered that information on fluctuation is included as a factor that causes an easy cutting region and a region where H / D replacement is easy. Therefore, according to the present invention, it is also possible to perform a reference vibration calculation as fluctuation information, compare the reference vibration calculation result with MS data, and evaluate the model. Therefore, the present invention performs the reference vibration calculation for the target protein using various known computer programs (for example, the molecular orbit calculation program MOPAC (product name) of Fujitsu Limited (trademark), etc.), and the reference vibration calculation result (For example, fluctuation information) is displayed in association with the display portion of the corresponding three-dimensional structure prediction data (for example, vector display is performed on the three-dimensional structure model), so that the fluctuation information can be considered as the easy-to-cut region. It becomes like this. That is, the prediction model can be evaluated by obtaining fluctuation information from the reference vibration calculation result and comparing it with the MS actual measurement data.

[System configuration]
First, the configuration of this system will be described. FIG. 2 is a block diagram showing an example of the configuration of the system to which the present invention is applied, and conceptually shows only the portion related to the present invention in the configuration. This system is generally configured by connecting a protein structure analysis apparatus 100 and an external system 200 that provides an external database such as sequence information and an external program such as a homology search to be communicable via a network 300. Has been. Further, the protein structure analysis apparatus 100 is configured such that the fragmentation spectrum information of the mass spectrometer 400 can be acquired via the input device 112 or the network 300.

  In FIG. 2, a network 300 has a function of mutually connecting the protein structure analysis apparatus 100 and the external system 200, and is, for example, the Internet.

  In FIG. 2, an external system 200 is connected to the protein structure analysis apparatus 100 via a network 300, and is a web for executing external programs such as homology search and motif search for an external database related to sequence information and the like for users. Has the function of providing a site.

  Here, the external system 200 may be configured as a WEB server, an ASP server, or the like, and the hardware configuration may be configured by an information processing apparatus such as a commercially available workstation or a personal computer and an accessory device thereof. Good. Each function of the external system 200 is realized by a CPU, a disk device, a memory device, an input device, an output device, a communication control device, and the like in the hardware configuration of the external system 200 and a program for controlling them.

  In FIG. 2, the mass spectrometer 400 includes a collision activated dissociation (CID) method including a hexapole CID method, a nozzle skimmer CID method, a capillary skimmer CID method, a SORI-CID method, and a multipole store auxiliary capillary skimmer CID method, The fragmentation spectrum is measured by fragmenting into fragmented ions by methods such as IRMPD, in-source decomposition (ISD), post-source decomposition (PSD), surface-induced dissociation (SID), ECD, or BIRD. To do.

  In FIG. 2, the protein structure analysis apparatus 100 schematically includes a control unit 102 such as a CPU that comprehensively controls the entire protein structure analysis apparatus 100, a communication device such as a router connected to a communication line (not shown). ) Connected to the communication control interface unit 104, the input / output control interface unit 108 connected to the input device 112 and the output device 114, and the storage unit 106 for storing various databases and tables. These units are communicably connected via an arbitrary communication path. Further, the protein structure analysis apparatus 100 is communicably connected to the network 300 via a communication device such as a router and a wired or wireless communication line such as a dedicated line.

  Various databases, files and tables (fragmented spectrum data file 106a to processing result data file 106f) stored in the storage unit 106 are storage means such as a fixed disk device, and various programs and tables used for various processes, Stores files, databases, web page files, etc.

  Among the components of the storage unit 106, the fragmented spectrum data file 106a is a fragmented spectrum data storage unit that stores the fragmented spectrum data acquired from the mass spectrometer 400.

  The easy-to-cut area information file 106b is easy-to-cut area information storage means for storing easy-to-cut area information and the like.

  The solvent contact residue information / interaction interface information file 106c is interaction interface information storage means for storing solvent contact residue information, interaction interface information, and the like.

  The 3D structure prediction data file 106d is a 3D structure prediction data storage unit that stores 3D structure prediction data predicted by the 3D structure prediction process.

  The predicted structure evaluation information file 106e is a predicted structure evaluation information storage unit that stores predicted structure evaluation information and the like.

  The processing result data file 106f is processing result data storage means for storing information about processing results.

  In addition, as other information, a protein information database storing protein amino acid sequence information and structure information may be recorded in the storage unit 106 of the protein structure analysis apparatus 100. This protein information database may be an external database accessed via the Internet, such as a PDB, or may be copied, stored with original sequence information, or unique clustering information. Or an in-house database created by adding annotation information or the like.

  In FIG. 2, the communication control interface unit 104 performs communication control between the protein structure analysis apparatus 100 and the network 300 (or a communication apparatus such as a router). That is, the communication control interface unit 104 has a function of communicating data with other terminals via a communication line.

  In FIG. 2, the input / output control interface unit 108 controls the input device 112 and the output device 114. Here, as the output device 114, in addition to a monitor (including a home TV), a speaker can be used (hereinafter, the output device 114 may be described as a monitor). As the input device 112, a keyboard, a mouse, a microphone, or the like can be used. The monitor also realizes a pointing device function in cooperation with the mouse.

  In FIG. 2, the control unit 102 has a control program such as an OS (Operating System), a program defining various processing procedures, and an internal memory for storing necessary data. Information processing for executing various processes is performed. The control unit 102 is functionally conceptually divided into a fragmentation spectrum data acquisition unit 102a, an attribution information determination unit 102b, an easily cleavable region information determination unit 102c, a solvent contact residue information determination unit 102d, an interaction interface information determination unit 102e, The structure prediction processing unit 102f, the prediction structure evaluation information determination unit 102g, the processing result output unit 102h, the prediction structure evaluation information output unit 102i, the deuteration rate determination unit 102j, and the reference vibration calculation processing unit 102k are configured. Yes.

  Among these, the fragmentation spectrum data acquisition unit 102a fragments the target protein for which a three-dimensional structure is predicted using MS or an enzyme, and acquires fragmentation spectrum data from the mass spectrometer 400 that measures the fragmentation spectrum. This is a generalized spectral data acquisition means.

  Further, the attribution information determination unit 102b is an attribution information determination unit that determines the attribution information of fragmented ions for the amino acid sequence of the target protein based on the fragmentation spectrum data acquired by the fragmentation spectrum data acquisition unit.

  In addition, the easy-cleavage region information determination unit 102c identifies the location fragmented into fragmented ions in the amino acid sequence of the target protein based on the fragmentation ion attribution information determined by the attribution information determination means, This is an easily cleavable region information determining means for determining easily cleavable region information in the amino acid sequence of a protein.

  The solvent contact residue information determination unit 102d is in contact with the solvent in the amino acid sequence of the target protein according to the deuteration rate at each amino acid residue in the amino acid sequence determined by the deuteration rate determination means. Solvent contact residue information determination means for specifying a location and determining solvent contact residue information in the amino acid sequence of the target protein according to the location.

  Further, the interaction interface information determination unit 102e determines the interaction interface information in the amino acid sequence of the target protein according to the deuteration rate at each amino acid residue in the amino acid sequence determined by the deuteration rate determination means. It is an interaction interface information determination means.

  Further, the three-dimensional structure prediction processing unit 102f uses the existing three-dimensional structure prediction method such as a homology modeling method, a molecular simulation method, an ab initio method, a secondary structure prediction method, a 3D-1D method, and a threading method. This is a three-dimensional structure prediction means for predicting a three-dimensional structure of a protein.

  Further, the predicted structure evaluation information determination unit 102g compares the three-dimensional structure prediction data predicted by the three-dimensional structure prediction unit with the easy-to-cut region information determined by the easy-to-cut region information determination unit, and uses the easy-to-cut region information. Predicted structure evaluation information determining means for evaluating the predicted structure portion corresponding to the specified easily cleavable region and determining predicted structure evaluation information, three-dimensional structure prediction data predicted by the three-dimensional structure prediction means, and solvent contact residue information determination Predicted structure evaluation information that compares the solvent contact residue information determined by the means, evaluates the predicted structure portion corresponding to the solvent contact residue specified by the solvent contact residue information, and determines the predicted structure evaluation information Determination means, three-dimensional structure prediction data predicted by three-dimensional structure prediction means, and interaction interface determined by interaction interface information determination means By comparing the distribution, it is predicted structure evaluation information determining means for determining a predicted structure evaluation information evaluated for predicted structural portion corresponding to the interaction interface identified by the interaction interface information.

  The processing result output unit 102h is a processing result output unit that outputs the three-dimensional structure prediction data predicted by the three-dimensional structure prediction unit and the easy-to-cut region information determined by the easy-to-cut region information determination unit in association with each other. Processing result output means for outputting the three-dimensional structure prediction data predicted by the structure prediction means and the solvent contact residue information determined by the solvent contact residue information determination means in correspondence with each other, and the three-dimensional prediction predicted by the three-dimensional structure prediction means It is a processing result output means for outputting the structure prediction data and the interaction interface information determined by the interaction interface information determination means in association with each other.

  The predicted structure evaluation information output unit 102i is a predicted structure evaluation information output unit that outputs the predicted structure evaluation information determined by the predicted structure evaluation information determination unit.

  In addition, the deuteration rate determination unit 102j determines whether the fragmentation spectrum data measured by the fragmentation spectrum data acquisition unit and the fragmentation ion attribution information determined by the attribution information determination unit are included in the amino acid sequence of the target protein. Is a deuteration rate determining means for determining a deuteration rate at each amino acid residue.

  The reference vibration calculation processing unit 102k is a reference vibration calculation unit that performs reference vibration calculation for the target protein. Details of processing performed by each of these units will be described later.

System processing
Next, an example of processing of the system in the present embodiment configured as described above will be described in detail with reference to FIG.

[Main processing]
First, details of the main process will be described with reference to FIG. FIG. 3 is a flowchart showing an example of main processing of the system according to the present embodiment.

  First, a fragmentation spectrum is measured for the target protein by the mass spectrometer 400 (step SA-1). That is, the target protein solution is continuously injected into the mass spectrometer 400 with a syringe pump, the target protein is fragmented into fragmented ions by the hexapole CID method or the like, and the fragmentation spectrum is measured.

  Here, FIG. 4 is a flowchart showing an example of measuring a fragmented spectrum by the hexapole CID method in the present embodiment.

  First, the capillary outlet voltage is increased (step SB-1).

  In order to improve the fragmentation efficiency, the ion trap time in the hexapole may be increased (step SB-2).

  Then, MS ionization and detection parameters (for example, capillary voltage, trap time in hexapole, etc.) are adjusted so that the protein is fragmented (step SB-3).

  Then, the fragmentation spectrum is captured and stored by the MS (step SB-4).

  FIG. 5 is a flowchart showing an example of measuring a fragmented spectrum by the IRMPD method in the present embodiment.

  First, an IR laser (10.9 μm) is irradiated to ions existing in the MS (for example, in a hexapole or ion trap) (step SC-1).

  In order to improve the fragmentation efficiency, the ion trap time in the hexapole may be increased (step SC-2).

  Then, MS ionization and detection parameters (for example, capillary voltage, trap time in hexapole, IR laser energy, IR laser irradiation time, etc.) are adjusted so that the protein is fragmented (step SC-3).

  Then, the fragmentation spectrum is captured and stored by the MS (step SC-4).

  The fragmentation spectrum may be obtained by other nozzle skimmer CID method, capillary skimmer CID method, multipole store auxiliary capillary skimmer CID method, ECD method, BIRD method, or SORI-CID method.

  Returning to FIG. 3 again, the protein structure analysis apparatus 100 acquires the fragmentation spectrum data from the mass spectrometer 400 via the input device 112 or the network 300 by the processing of the fragmentation spectrum data acquisition unit 102a, and fragments. It stores in the spectrum data file 106a (step SA-2).

  Next, the protein structure analysis apparatus 100 determines the attribution information of the fragmented ions with respect to the amino acid sequence of the target protein based on the measured fragmentation spectrum by the processing of the attribution information determination unit 102b. The portion fragmented into fragment ions in the amino acid sequence of the target protein is identified from the fragmentation ion attribution information determined by the processing of the determination unit 102c, and the easy-cleavage region information in the amino acid sequence of the target protein according to the portion Is determined (step SA-3).

  Next, the protein structure analysis apparatus 100 stores the easy-cleavage region information in the easy-cleavage region information file 106b by the processing of the easy-cleavage region information determination unit 102c (step SA-4).

  Further, the fragmentation spectrum of the target protein or complex thereof is measured by the hydrogen-deuterium exchange method (step SA-5).

  Here, FIG. 7 is a flowchart showing an example of measuring a fragmented spectrum by the hydrogen-deuterium exchange method in the present embodiment.

  First, the target protein is dissolved or deuterated in a buffer solution (step SD-1).

  Then, while sampling over time, the buffer is acidified to lower the temperature and the H / D exchange reaction rate is lowered (step SD-2).

  Then, the target protein is digested with pepsin under acidic conditions (step SD-3).

  And a peptide digest is extracted (step SD-4) and MALDI-TOF-MS or LC / MS / MS is performed (step SD-5).

  FIG. 8 is a flowchart showing an example in the case where a protein is fragmented as it is with MS by the hydrogen-deuterium exchange method in this embodiment and a fragmentation spectrum is measured.

  First, the target protein is dissolved or substituted with deuterium in a buffer solution (step SE-1).

  Then, the fragmentation spectrum is measured by ESI-MS (step SE-2).

  Then, the fragmentation spectrum is captured and stored while sampling over time (step SE-3).

  FIG. 9 is a flowchart showing an example of fragmenting a protein complex and measuring a fragmentation spectrum by the hydrogen-deuterium exchange method in the present embodiment.

  First, the protein complex is dissolved in a buffer or substituted with deuterium (step SF-1).

  Then, while sampling over time, the buffer solution is acidified to lower the temperature, and the H / D exchange reaction rate is lowered (step SF-2).

  Then, the target protein is digested with pepsin under acidic conditions (step SF-3).

  And a peptide digest is extracted (step SF-4) and MALDI-TOF-MS or LC / MS / MS is performed (step SF-5).

  FIG. 10 is a flowchart showing an example of fragmenting the complex of proteins A and B and measuring the fragmentation spectrum by the hydrogen-deuterium exchange method in the present embodiment.

  First, proteins A and B are separately dissolved or substituted with deuterium in a buffer solution and allowed to stand for a predetermined time (t1) (step SG-1).

  Then, proteins A and B are mixed to form a complex and left for a certain time (t2) (step SG-2).

  And it substitutes or dilutes with a light water buffer solution (step SG-3).

  Then, it is allowed to stand for a certain time (T = t1 + t2), and an H / D reverse exchange reaction (off change) is performed (step SG-4).

  Then, the fragmentation spectrum is measured by ESI-MS (step SG-5).

  Returning to FIG. 3 again, the protein structure analysis apparatus 100 acquires the fragmentation spectrum data from the mass spectrometer 400 via the input device 112 or the network 300 by the processing of the fragmentation spectrum data acquisition unit 102a, and fragments. It stores in the spectrum data file 106a (step SA-6).

  Next, the protein structure analysis apparatus 100 determines the attribution information of the fragmented ions for the amino acid sequence of the target protein based on the measured fragmentation spectrum by the processing of the attribution information determination unit 102b, and the deuteration rate By the processing of the determination unit 102j, the deuteration rate at each amino acid residue in the amino acid sequence of the target protein is determined according to the measured fragmentation spectrum and the determined fragmentation ion attribution information.

  Here, the deuteration rate is obtained as follows. First, when the attribution information determination unit 102b determines the attribution information of the peptide, the deuteration rate determination unit 102j obtains the deuteration rate for each amino acid residue by dividing the increase in mass by the number of exchangeable protons. Then, the deuteration rate determining unit 102j determines the deuteration rate of the amino acid residue of each peptide sequence based on the deuteration rate.

  Then, the solvent contact residue information determination unit 102d identifies a position in contact with the solvent in the amino acid sequence of the target protein according to the deuteration rate at each amino acid residue in the determined amino acid sequence, and according to the position Solvent contact residue information in the amino acid sequence of the target protein is determined (step SA-7) and stored in the solvent contact residue information / interaction interface information file 106c (step SA-8). For the solvent contact residue information, for example, a site where the deuteration rate is slow compared to a control experiment in which hydrogen-deuterium exchange is not performed may be determined as the solvent contact residue information.

  Further, the interaction interface information determination unit 102e determines interaction interface information in the amino acid sequence of the target protein according to the deuteration rate at each amino acid residue in the determined amino acid sequence (step SA-7), and the solvent The contact residue information / interaction interface information file 106c is stored (step SA-8). As for the interaction interface information, for example, a site where the deuteration rate is slow may be determined as the interaction interface information as compared with the case where measurement is performed when the complex is not formed alone. Alternatively, a site where the deuteration rate is slow compared to a control experiment in which hydrogen-deuterium exchange is not performed may be determined as interaction interface information.

  Further, the protein structure analyzing apparatus 100 predicts the three-dimensional structure of the target protein by the processing of the three-dimensional structure prediction processing unit 102f (step SA-9), and stores the predicted three-dimensional structure prediction data in the three-dimensional structure prediction data file 106d. (Step SA-10).

  The protein structure analysis apparatus 100 outputs the easily cleavable region information, the solvent contact residue information, and the interaction interface information determined in this way together with the prediction data or when determining the prediction structure evaluation information. Use.

  That is, the processing result output unit 102h outputs the predicted three-dimensional structure prediction data in association with the determined easy-cleavage region information, solvent contact residue information, and interaction interface information (step SA-11).

  Further, the predicted structure evaluation information determination unit 102g compares the predicted three-dimensional structure prediction data with the determined cleavable region information, solvent contact residue information, and interaction interface information (step SA-12), Predictive structure evaluation is performed by evaluating the predicted structure portion corresponding to the interaction interface specified by the easy contact region specified by the easy access region information, the solvent contact residue specified by the solvent contact residue information or the interaction interface information. Information is determined (step SA-13).

  Further, the predicted structure evaluation information output unit 102i outputs the determined predicted structure evaluation information (step SA-14). This completes the main process.

[Example 1]
Next, details of the first embodiment of the present invention will be described with reference to FIG. FIG. 13 is a flowchart showing an example of the first embodiment of the system according to the present invention.

  First, the fragmentation spectrum of the target protein (or complex) is measured by the mass spectrometer 400 (step SJ-1). Moreover, you may measure a fragmentation spectrum, performing hydrogen-deuterium exchange reaction about object protein (or complex). Then, the protein structure analysis apparatus 100 acquires fragmentation spectrum data from the mass spectrometry apparatus 400 via the input device 112 or the network 300 by the processing of the fragmentation spectrum data acquisition unit 102a, and stores the fragmentation spectrum data file 106a. Store.

  Next, the protein structure analysis apparatus 100 determines fragmentation ion attribution information for the amino acid sequence of the target protein based on the measured fragmentation spectrum by the processing of the attribution information determination unit 102b (step SJ-2). In addition, the protein structure analysis apparatus 100 identifies the location fragmented into fragment ions in the amino acid sequence of the target protein based on the fragmentation ion attribution information determined by the processing of the easy-cleavage region information determination unit 102c. The easy-cleavage region information in the amino acid sequence of the target protein is determined according to the location.

  In addition, when the hydrogen-deuterium exchange reaction is performed, the protein structure analysis apparatus 100 uses the process of the deuteration rate determination unit 102j to measure the measured fragmentation spectrum and the determined fragmented ions. According to the attribution information, the deuteration rate at each amino acid residue in the amino acid sequence of the target protein is determined. Then, the solvent contact residue information determination unit 102d identifies a position in contact with the solvent in the amino acid sequence of the target protein according to the deuteration rate at each amino acid residue in the determined amino acid sequence, and according to the position Solvent contact residue information in the amino acid sequence of the target protein is determined and stored in the solvent contact residue information / interaction interface information file 106c. In addition, the interaction interface information determination unit 102e determines interaction interface information in the amino acid sequence of the target protein according to the deuteration rate at each amino acid residue in the determined amino acid sequence, and determines solvent contact residue information / mutual The data is stored in the working interface information file 106c.

  In addition, the protein structure analysis apparatus 100 performs homology search on the target protein using a protein database such as PDB by the processing of the three-dimensional structure prediction processing unit 102f, and extracts a protein with a known structure that becomes a template three-dimensional structure. (Step SJ-3).

  Then, the three-dimensional structure prediction processing unit 102f performs alignment between the target protein and the template structure known protein by a method such as the 3D-1D method (step SJ-4), and uses the three-dimensional structure model data of the target protein. Certain 3D structure prediction data is created and stored in the 3D structure prediction data file 106d (step SJ-5).

  Then, the processing result output unit 102h outputs the predicted three-dimensional structure prediction data in association with the determined easy-cleavage region information, solvent contact residue information, and interaction interface information (step SJ-6).

  Further, the predicted structure evaluation information determination unit 102g compares the predicted three-dimensional structure prediction data with the determined easy-cleavage region information, solvent contact residue information, and interaction interface information, and specifies the easy-cleavage region information. The predicted structure evaluation information is determined by evaluating the predicted structure portion corresponding to the interaction interface specified by the solvent contact residue or the interaction interface information specified by the easily cleavable region or the solvent contact residue information. Also, the predicted structure evaluation information output unit 102i outputs the determined predicted structure evaluation information (step SJ-7). This completes the process of the first embodiment.

[Example 2]
Next, details of the second embodiment of the present invention will be described with reference to FIG. FIG. 14 is a flowchart showing an example of Embodiment 2 of the present system according to the present invention.

  First, the fragmentation spectrum of the target protein (or complex) is measured by the mass spectrometer 400 (step SK-1). Moreover, you may measure a fragmentation spectrum, performing hydrogen-deuterium exchange reaction about object protein (or complex). Then, the protein structure analysis apparatus 100 acquires fragmentation spectrum data from the mass spectrometry apparatus 400 via the input device 112 or the network 300 by the processing of the fragmentation spectrum data acquisition unit 102a, and stores the fragmentation spectrum data file 106a. Store.

  Next, the protein structure analysis apparatus 100 determines fragmentation ion attribution information for the amino acid sequence of the target protein based on the measured fragmentation spectrum by the process of the attribution information determination unit 102b (step SK-2). In addition, the protein structure analysis apparatus 100 identifies the location fragmented into fragment ions in the amino acid sequence of the target protein based on the fragmentation ion attribution information determined by the processing of the easy-cleavage region information determination unit 102c. The easy-cleavage region information in the amino acid sequence of the target protein is determined according to the location.

  In addition, when the hydrogen-deuterium exchange reaction is performed, the protein structure analysis apparatus 100 uses the process of the deuteration rate determination unit 102j to measure the measured fragmentation spectrum and the determined fragmented ions. According to the attribution information, the deuteration rate at each amino acid residue in the amino acid sequence of the target protein is determined. Then, the solvent contact residue information determination unit 102d identifies a position in contact with the solvent in the amino acid sequence of the target protein according to the deuteration rate at each amino acid residue in the determined amino acid sequence, and according to the position Solvent contact residue information in the amino acid sequence of the target protein is determined and stored in the solvent contact residue information / interaction interface information file 106c. In addition, the interaction interface information determination unit 102e determines interaction interface information in the amino acid sequence of the target protein according to the deuteration rate at each amino acid residue in the determined amino acid sequence, and determines solvent contact residue information / mutual The data is stored in the working interface information file 106c.

  In addition, the protein structure analysis apparatus 100 performs homology search on the target protein using a protein database such as PDB by the processing of the three-dimensional structure prediction processing unit 102f, and extracts a protein with a known structure that becomes a template three-dimensional structure. (Step SK-3).

  If the template protein cannot be extracted, the three-dimensional structure prediction processing unit 102f creates three-dimensional structure prediction data that is data of the three-dimensional structure model of the target protein by a technique such as a molecular simulation method. It stores in the structure prediction data file 106d (step SK-4).

  Then, the processing result output unit 102h outputs the predicted three-dimensional structure prediction data and the determined easy-cleavage region information, solvent contact residue information, and interaction interface information in association with each other (step SK-5).

  Further, the predicted structure evaluation information determination unit 102g compares the predicted three-dimensional structure prediction data with the determined easy-cleavage region information, solvent contact residue information, and interaction interface information, and specifies the easy-cleavage region information. The predicted structure evaluation information is determined by evaluating the predicted structure portion corresponding to the interaction interface specified by the solvent contact residue or the interaction interface information specified by the easily cleavable region or the solvent contact residue information. Also, the predicted structure evaluation information output unit 102i outputs the determined predicted structure evaluation information (step SK-6). This completes the process of the second embodiment.

[Example 3]
Next, details of the third embodiment of the present invention will be described with reference to FIG. FIG. 15 is a flowchart showing an example of the third embodiment of the system according to the present invention.

  First, the fragmentation spectrum of the target protein (or complex) is measured by the mass spectrometer 400 (step SL-1). Moreover, you may measure a fragmentation spectrum, performing hydrogen-deuterium exchange reaction about object protein (or complex). Then, the protein structure analysis apparatus 100 acquires fragmentation spectrum data from the mass spectrometry apparatus 400 via the input device 112 or the network 300 by the processing of the fragmentation spectrum data acquisition unit 102a, and stores the fragmentation spectrum data file 106a. Store.

  Next, the protein structure analysis apparatus 100 determines the attribution information of the fragmented ions with respect to the amino acid sequence of the target protein based on the measured fragmentation spectrum by the processing of the attribution information determination unit 102b (step SL-2). In addition, the protein structure analysis apparatus 100 identifies the location fragmented into fragment ions in the amino acid sequence of the target protein based on the fragmentation ion attribution information determined by the processing of the easy-cleavage region information determination unit 102c. The easy-cleavage region information in the amino acid sequence of the target protein is determined according to the location.

  In addition, when the hydrogen-deuterium exchange reaction is performed, the protein structure analysis apparatus 100 uses the process of the deuteration rate determination unit 102j to measure the measured fragmentation spectrum and the determined fragmented ions. According to the attribution information, the deuteration rate at each amino acid residue in the amino acid sequence of the target protein is determined. Then, the solvent contact residue information determination unit 102d identifies a position in contact with the solvent in the amino acid sequence of the target protein according to the deuteration rate at each amino acid residue in the determined amino acid sequence, and according to the position Solvent contact residue information in the amino acid sequence of the target protein is determined and stored in the solvent contact residue information / interaction interface information file 106c. In addition, the interaction interface information determination unit 102e determines interaction interface information in the amino acid sequence of the target protein according to the deuteration rate at each amino acid residue in the determined amino acid sequence, and determines solvent contact residue information / mutual The data is stored in the working interface information file 106c.

  In addition, the protein structure analysis apparatus 100 performs a homology search on the target protein using a protein database such as PDB by the processing of the three-dimensional structure prediction processing unit 102f, and a candidate protein of a protein with a known structure that becomes a template three-dimensional structure Is extracted (step SL-3).

  Then, the three-dimensional structure prediction processing unit 102f performs alignment between the target protein and the candidate protein by a method such as the 3D-1D method (step SL-4).

  Then, the processing result output unit 102h outputs the amino acid sequence and structure data of the candidate protein in association with the determined cleavable region information, solvent contact residue information, and interaction interface information (step SL-5). .

  The predicted structure evaluation information determination unit 102g compares the structure data of the candidate protein with the determined cleavable region information, solvent contact residue information, and interaction interface information, and is identified by the cleavable region information. The predicted structure evaluation information is determined by evaluating the predicted structure portion corresponding to the interaction interface specified by the solvent contact residue or the interaction interface information specified by the easy-cleavage region or the solvent contact residue information. The predicted structure evaluation information output unit 102i outputs the determined predicted structure evaluation information. Thereby, the coincidence between the structure data of each candidate protein and the experimental data of the target protein can be ranked, and the candidate protein with the highest evaluation can be narrowed down (step SL-6).

  Next, the three-dimensional structure prediction processing unit 102f creates three-dimensional structure prediction data, which is the data of the three-dimensional structure model of the target protein, using the candidate protein with the highest evaluation as a template protein, and stores it in the three-dimensional structure prediction data file 106d ( Step SL-7).

  Then, the model structure is evaluated by the same method as in Step SJ-6 and Step SJ-7 of Example 1 (Step SL-8). This completes the process of the third embodiment.

[Example 4]
Next, details of the fourth embodiment of the present invention will be described with reference to FIG. FIG. 16 is a flowchart showing an example of Embodiment 4 of the system according to the present invention.

  First, the protein structure analysis apparatus 100 performs a homology search on a target protein using a protein database such as PDB by the processing of the three-dimensional structure prediction processing unit 102f, and extracts a protein with a known structure that becomes a template three-dimensional structure. (Step SM-1).

  If the template protein cannot be extracted, the three-dimensional structure prediction processing unit 102f creates three-dimensional structure prediction data that is data of the three-dimensional structure model of the target protein by a technique such as a molecular simulation method. It stores in the structure prediction data file 106d (step SM-2 to step SM-3).

  Then, the fragmentation spectrum of the target protein (or complex) and the template protein is measured by the mass spectrometer 400 (step SM-1 and step SM-4). Further, the fragmentation spectrum may be measured while performing a hydrogen-deuterium exchange reaction on the target protein (or complex) and the template protein. Then, the protein structure analysis apparatus 100 acquires fragmentation spectrum data from the mass spectrometry apparatus 400 via the input device 112 or the network 300 by the processing of the fragmentation spectrum data acquisition unit 102a, and stores the fragmentation spectrum data file 106a. Store.

  Next, the protein structure analysis apparatus 100 determines the attribution information of the fragmented ions for the amino acid sequences of the target protein and the template protein based on the measured fragmentation spectrum by the processing of the attribution information determination unit 102b (step SM). -2) In addition, the protein structure analysis apparatus 100 uses the fragmentation ion assignment information determined by the processing of the easy-cleavage region information determination unit 102c to fragment the fragmented ions in the amino acid sequences of the target protein and the template protein. The identified site is identified, and easy-cleavage region information in the amino acid sequence of the target protein is determined according to the location.

  In addition, when the hydrogen-deuterium exchange reaction is performed, the protein structure analysis apparatus 100 uses the process of the deuteration rate determination unit 102j to measure the measured fragmentation spectrum and the determined fragmented ions. According to the attribution information, the deuteration rate at each amino acid residue in the amino acid sequences of the target protein and the template protein is determined. Then, the solvent contact residue information determination unit 102d identifies a portion in contact with the solvent in the amino acid sequences of the target protein and the template protein according to the deuteration rate at each amino acid residue in the determined amino acid sequence. The solvent contact residue information in the amino acid sequence of the target protein is determined according to the location, and stored in the solvent contact residue information / interaction interface information file 106c. Further, the interaction interface information determination unit 102e determines interaction interface information in the amino acid sequences of the target protein and the template protein according to the deuteration rate at each amino acid residue in the determined amino acid sequence, and the solvent contact residue The base information / interaction interface information file 106c is stored.

  Then, the processing result output unit 102h outputs the predicted three-dimensional structure prediction data of the target protein and the template protein in association with the determined cleavable region information, solvent contact residue information, and interaction interface information ( Step SM-5).

  Further, the predicted structure evaluation information determination unit 102g compares the predicted three-dimensional structure prediction data with the determined easy-cleavage region information, solvent contact residue information, and interaction interface information, and specifies the easy-cleavage region information. The predicted structure evaluation information is determined by evaluating the predicted structure portion corresponding to the interaction interface specified by the solvent contact residue or the interaction interface information specified by the easily cleavable region or the solvent contact residue information. Also, the predicted structure evaluation information output unit 102i outputs the determined predicted structure evaluation information (step SM-6). This completes the process of the fourth embodiment.

  In Example 4, the case where the template protein cannot be extracted in the homology search in step SM-2 has been described as an example. However, if the template protein can be extracted in the homology search, the template protein is extracted. Experimental values are collected in step SM-4.

[Example 5]
Next, details of the fifth embodiment of the present invention will be described with reference to FIGS.

(1) Preparation of Recombinant Human Interleukin-6 (IL-6) IL-6 was expressed and purified using Escherichia coli according to literature methods (D. Eima, Biotechnol. Bioeng., 62, 301-). 10 (1999)).

(2) FTICR-MS measurement of IL-6 All measurements were performed by the gas assisted dynamic trapping (GADT) method. The GADT method is a method of introducing argon gas into the cell as a trap gas in a pulse to temporarily increase the trapping voltage and lower the kinetic energy of the ions when trapping the ions in the measurement cell. . As a result, ions can be efficiently trapped in the cell, and the sensitivity is improved.

Measuring instruments and analysis conditions are as follows.
Mass spectrometer: 7T self-shielded superconducting magnet Fourier transform type mass spectrometer Apex II (manufactured by Bruker Daltonics);
Flow rate: 60 μl / h;
• Dry gas: 20 psi, 150 ° C;
Nebulizing gas: 30 psi;
Argon gas pressure: 7.8 Toor;
-Hexapole accumulation time: 1 second;
Capillary outlet voltage: 106.5 kV (MS spectrum), 150 kV (IRMPD), 220 kV (Hexapol CID); and Number of data acquisition points: 512K.

(3) Fragmentation of IL-6 by IRMPD method and hexapole CID method In the IRMPD method, ions confined in the ion cyclotron cell were irradiated with a carbon dioxide laser for 300 milliseconds. The hexapole CID method was able to induce fragmentation by increasing the capillary outlet voltage and setting the accumulation time in the hexapole to 1-3 seconds using the same pulse sequence as in the normal measurement. All FTICR-MS spectra were analyzed using Xmass, software that came with the instrument. For the assignment of the fragmented ions, software Frag-Pro (manufactured by msmssoft) was used.

(4) Homology search by 3D-1D method and PSI-BLAST The amino acid sequence of human interleukin-6 (IL6) was obtained from PIR (protein amino acid sequence database). The number of amino acid residues including the presequence was 212 residues. As a result of performing a homology search by 3D-1D method using LIBRA, the top 30 PDB file lists are 1alu, 1lki, 1au1_A, 1nf1_A, 1qkm_A, 1jnk, 1pme, 1dg9_A, 1b5l, 1bgc, 1yrg_A, 2eups_A, 2eups , 1bg0, 1ial_A, 2cnd, 1cjs_A, 1wer, 7odc_A, 1eqf_A, 1f6f_A, 1dxy, 1edt, 1gse_A, 1qr2_A, 1llp, 1qi7_A, 2bid_A, 2ljr_A, 2ljr_A1

  In addition, as a result of the homology search under the condition that the e value is 0.01 or less using PSI-BLAST, 1IL6, 1ALU, 1GNC, 1BGE_B, 1BGC, 1RHG_C, 1I1R_B were obtained as a PDB file list. In this case, the top six cases are GCSF (Granulocyte Colony Stimulating Factor), which is a protein that has already been pointed out to be similar to IL6 itself or three-dimensional structure, and therefore any reference protein can be a candidate for three-dimensional structure prediction. I was able to confirm it.

(5) Inquiry of secondary structure information and MS fragmentation data for alignment Therefore, for each of the 30 types of alignment obtained by LIBRA homology search, secondary structure information and MS fragmentation data of the reference protein are obtained. A program to inquire at the same time was created and the results were displayed for each alignment. FIG. 18 is a diagram illustrating an example of a processing result in the fifth embodiment (B is combined after A in the drawing). As shown in this figure, the processing result display screen includes, for example, a display area MB-1 for displaying the amino acid sequence of the target protein, a display area MB-2 for displaying the amino acid sequence of the template protein, and a three-dimensional structure. It includes a display area MB-3 for displaying secondary tertiary structure prediction data, which is an example of prediction data, and a display area MB-4 for displaying fragments of fragmented ions of experimental data. . In FIG. 18, the secondary structure in MB-3 indicates that “1” is an α helix, “2” is a β strand, and “4” is a loop region.

(6) Evaluation of candidate protein A program for evaluating (ranking) candidate proteins is created for each alignment from secondary structure information and information on all fragmentation data. It is confirmed that IL6 itself (1alu) is the most effective candidate protein for Gd, and that the next effective candidate protein is GCSF (1bgc), a protein that has already been pointed out to be similar in three-dimensional structure to IL6 did it.

  Here, FIG. 11 is a flowchart showing an example of analyzing a fragmented spectrum in the present embodiment.

  First, the attribution information determination unit 102b refers to the fragmentation spectrum stored in the fragmentation spectrum data file 106a, and the m / z of each ion in the fragmentation spectrum and valence information (if the resolution is low, The number may not be known) (step SH-1).

  Then, the attribution information determination unit 102b obtains the attribution of each fragmented ion to the amino acid sequence, and searches, extracts, and displays candidate peptide fragments based on the m / z (+ valence) of each ion (step SH). -2).

  Then, the easy-to-cut area information determination unit 102c extracts a part that can be easily cut and stores it in the easy-to-cut area information file 106b as easy-to-cut area information (step SH-3).

  Then, as shown in FIG. 12 described above, the processing result output unit 102h controls and displays the three-dimensional structure display software so as to change the color of the cut site based on the easy-to-cut region information (step SH-4). ). This completes the processing of the fifth embodiment.

[Example 6]
Next, details of the sixth embodiment of the present invention will be described with reference to FIG.

(1) Measurement and assignment of fragmentation spectrum of ubiquitin by FTICR-MS Use a sample obtained by dissolving bovine-derived ubiquitin (Sigma Code No. U6253) in 1% acetic acid-50% methanol aqueous solution to 5 pmol / μL. did. All measurements were performed by the GADT method.

Measuring instruments and analysis conditions are as follows.
Mass spectrometer: 7T self-shielded superconducting magnet Fourier transform mass spectrometer equipped with a nanoelectrospray ion source Apex II (Bruker Daltonics);
-Flow rate: 200 nl / min;
Dry gas: 5 psi, 200 ° C .;
-Hexapole accumulation time: 0.6 seconds;
-Capillary voltage: 1800V;
Capillary outlet voltage: 90V; and Number of data acquisition points: 512K.

  In this example, ubiquitin fragmentation could be induced using the noise skimmer CID method with a high capillary exit voltage. Analysis of FTICR-MS spectrum and assignment of fragmented ions were performed in the same manner as in Example 5. The amino acid sequence was obtained from PIR.

(2) Query of 3D structure and MS fragmentation data The 3D structure of ubiquitin has already been clarified. Obtain a ubiquitin structure file (PDB file; 1UBI.pdb) from PDB (Protein 3D Structure Data Bank), and use 3D structure display software “ViewerLite (product name)” (manufactured by Accelrys (company name)). As shown in FIG. 26, the three-dimensional structure of ubiquitin was displayed as a ribbon model. FIG. 26 is a diagram showing an example of the three-dimensional structure of bovine-derived ubiquitin in Example 6 of the present system according to the present invention. In FIG. 26, α-helix is displayed in red, β-strand is displayed in light blue, and a fragmentation site obtained by MS is displayed in yellow. As a result, it was found that the α-helix ends and β-strands are likely to be fragmented, and the α-helix and β-strands having a secondary structure tend not to be generated. This completes the description of the sixth embodiment.

[Example 7]
Next, details of the seventh embodiment of the present invention will be described with reference to FIG.

(1) Measurement and assignment of fragmentation spectrum of alcohol dehydrogenase by FTICR-MS 1% acetic acid-50% methanol aqueous solution of yeast-derived alcohol dehydrogenase (Code No. made by Sigma) to 5 pmol / μL. Dissolved samples were used. All measurements were performed by the GADT method.

Measuring instruments and analysis conditions are as follows.
Mass spectrometer: 7T self-shielded superconducting magnet Fourier transform mass spectrometer equipped with a nanoelectrospray ion source Apex II (Bruker Daltonics);
-Flow rate: 200 nl / min;
Dry gas: 5 psi, 200 ° C .;
-Hexapole accumulation time: 1 second;
-Capillary voltage: 1750V;
-Capillary outlet voltage: 170V; and-Data acquisition point number: 512K.

  In this example, the fragmentation of alcohol dehydrogenase could be induced using the hexapole CID method by increasing the voltage at the capillary outlet. Analysis of FTICR-MS spectrum and assignment of fragmented ions were performed in the same manner as in Example 5. The amino acid sequence was obtained from PIR.

(2) Modeling of alcohol dehydrogenase and inquiry of MS fragmentation data A three-dimensional structure model of alcohol dehydrogenase was created using fully automated protein modeling software FAMS. Three-dimensional structure display software “ViewerLite (product name)” (manufactured by Accelrys (company name)) was used to display the three-dimensional structure of alcohol dehydrogenase as a ribbon model as shown in FIG. FIG. 27 is a diagram showing an example of the three-dimensional structure of a yeast-derived alcohol dehydrogenase in Example 7 of the present system in the present invention. In FIG. 27, the α helix is displayed in red, the β strand is displayed in light blue, and the fragmentation site obtained by MS is displayed in yellow. As a result, it can be easily seen visually that the model structure has many fragmentation sites at the ends of the α helix and β strands and tends to be less inside the α helix and β strands. Matched well. This completes the description of the seventh embodiment.

[Example 8: Three-dimensional structure display of protein of interest based on experimental results]
Next, details of the eighth embodiment of the present invention will be described with reference to FIGS.
FIG. 21 is a flowchart showing an example of the eighth embodiment of the present system according to the present invention.

  In this example, as shown in FIG. 21, the results of expression analysis such as mRNA expression profiling by a DNA microarray, the identification result by MS obtained by proteome analysis (for example, Mascot, SEQUEST, etc.), comparative genome analysis Results, protein interaction analysis (eg, yeast two-hybrid method or TAP method (Anuj Kumer, Nature, 415, 123-124 (2002))), metabolic pathways such as KEGG, etc. By specifying an identification number such as an accession number of an interesting gene on a map or signal transmission map (for example, clicking on the screen), a three-dimensional structure database (for example, PDB) or a model structure database (for example, FAMSBase) From) Body structure data is retrieved / extracted and displayed using a three-dimensional structure display program (for example, RasMol).

  Below, Anabaena sp. The case where the correlation between the increase / decrease in the transcription amount and the three-dimensional structure of the genome of the PCC7120 strain is described as an example. This embodiment is a display program for clarifying the correlation between increase / decrease in transcription amount and three-dimensional structure by linking the amount of mRNA transcription by DNA microarray and the three-dimensional structure database. It is.

  FIG. 22 is a diagram showing an example of a display screen displaying the difference in expression level by DNA microarray for each color. Small squares on this screen indicate genes one by one, and “red”, “blue”, and “green” indicate “increase”, “decrease”, and “invariance” of the transfer amount, respectively. "And" light "indicate" present "or" no "of sequence homology with a three-dimensional structure database (for example, PDB).

  Here, when the user clicks on one of the cells corresponding to the interesting gene, as shown in FIG. 23, the SCOP (Structural Classification of Proteins) classification number and the ID (identification number) such as the PDB of the template are displayed. In addition, a click button (the underlined portion in FIG. 23) indicating the PDB data with high homology (the top five in the example of this figure) is displayed.

  When the user clicks the click button, an alignment display screen as shown in FIG. 24 or a three-dimensional structure display screen by graphic software such as RasMol as shown in FIG. 25 can be displayed.

  According to this example, identification of notable genes and proteins displayed on the display by transcriptome analysis using DNA microarray, proteome analysis (protein identification results by MS, etc.), genome analysis (bioinformatics), etc. When a number (accession number or the like) is clicked, the 3D structure can be extracted from the 3D structure and structure prediction database and displayed in 3D. This completes the process according to the eighth embodiment.

[Other embodiments]
Although the embodiments of the present invention have been described so far, the present invention can be applied to various different embodiments in addition to the above-described embodiments within the scope of the technical idea described in the claims. May be implemented.

  For example, the case where the protein structure analysis apparatus 100 performs processing in a stand-alone form has been described as an example. However, the protein structure analysis apparatus 100 performs processing in response to a request from a client terminal configured in a separate housing from the protein structure analysis apparatus 100, You may comprise so that the process result may be returned to the said client terminal.

  In the above-described embodiments, the case where the solvent contact residue, interaction site interface information, and the like are obtained using the hydrogen-deuterium exchange method has been described as an example. However, the present invention is limited to such a case. For example, it may be obtained by a chemical modification method or the like. For example, a stable isotope 15N can be selectively introduced into a glutamine residue of a protein using transglutaminase (N. Shimba, N. Yamada, K. Yokoyama, E. Suzuki, Anal. Biochem., 301, 123-127 (2002)). By combining this method with the above-described embodiment, the difference in the degree of solvent contact for each glutamine residue can be determined from the labeling rate, so that the solvent contact residue, interaction site interface information, etc. can be determined from hydrogen-deuterium. It can be obtained in the same manner as when the exchange method is used.

  In the above-described examples, the case where fragmentation is performed by MS has been described as an example. However, the present invention is not limited to such a case, and may be obtained using, for example, an enzyme digestion method. Good. That is, the protein may be fragmented by an enzymatic reaction using digestive enzymes such as pepsin and trypsin.

  The three-dimensional structure prediction processing unit 102f may perform prediction using a prediction program provided in the protein structure analysis apparatus 100. Alternatively, the three-dimensional structure prediction processing unit 102f creates three-dimensional structure prediction data using a prediction program provided in the external system 200. The structure prediction data may be acquired via the network 300.

  In addition, among the processes described in the embodiment, all or part of the processes described as being performed automatically can be performed manually, or all of the processes described as being performed manually are performed. Alternatively, a part can be automatically performed by a known method.

  In addition, the processing procedures, control procedures, specific names, information including parameters such as various registration data and search conditions, screen examples, and database configurations shown in the above documents and drawings, unless otherwise specified. It can be changed arbitrarily.

  Moreover, regarding the protein structure analysis apparatus 100, each illustrated component is functionally conceptual and does not necessarily need to be physically configured as illustrated.

  For example, the processing functions of each unit or each device of the protein structure analysis apparatus 100, particularly the processing functions performed by the control unit 102, are all or any part thereof are assigned to a CPU (Central Processing Unit) and the CPU. It can be realized by a program that is interpreted and executed, or can be realized as hardware by wired logic. The program is recorded on a recording medium to be described later, and is mechanically read by the protein structure analysis apparatus 100 as necessary.

  That is, in the storage unit 106 such as a ROM or an HD, computer programs for giving instructions to the CPU and performing various processes in cooperation with an OS (Operating System) are recorded. This computer program is executed by being loaded into a RAM or the like, and constitutes the control unit 102 in cooperation with the CPU. The computer program may be recorded in an application program server connected to the protein structure analysis apparatus 100 via an arbitrary network 300, and may be downloaded in whole or in part as necessary. It is.

  The program according to the present invention can also be stored in a computer-readable recording medium. Here, the “recording medium” is an arbitrary “portable physical medium” such as a flexible disk, a magneto-optical disk, a ROM, an EPROM, an EEPROM, a CD-ROM, an MO, and a DVD, and is incorporated in various computer systems. Program in a short time, such as a communication line or carrier wave when transmitting a program via any “fixed physical medium” such as ROM, RAM, HD, or a network such as LAN, WAN, or the Internet The “communication medium” that holds

  The “program” is a data processing method described in an arbitrary language or description method, and may be in any format such as source code or binary code. The “program” is not necessarily limited to a single configuration, but is distributed in the form of a plurality of modules and libraries, or in cooperation with a separate program represented by an OS (Operating System). Including those that achieve the function. Note that a well-known configuration and procedure can be used for a specific configuration for reading a recording medium, a reading procedure, an installation procedure after reading, and the like in each device described in the embodiment.

  Various databases and the like (fragmented spectrum data file 106a to processing result data file 106f) stored in the storage unit 106 are memory means such as RAM and ROM, fixed disk devices such as hard disks, storage means such as flexible disks and optical disks. It stores various programs, tables, files, databases, web page files, etc. used for various processes and website provision.

  The protein structure analyzing apparatus 100 connects a peripheral device such as a printer, a monitor or an image scanner to an information processing apparatus such as an information processing terminal such as a known personal computer or workstation, and the method of the present invention is connected to the information processing apparatus. You may implement | achieve by mounting the software (a program, data, etc. are included) which implement | achieve.

  Further, the specific form of dispersion / integration of the protein structure analysis apparatus 100 is not limited to that shown in the figure, and all or part of the protein structure analysis apparatus 100 may be functionally or physically dispersed / arbitrary in arbitrary units according to various loads. Can be integrated and configured. For example, each database may be independently configured as an independent database device, and a part of the processing may be realized by using CGI (Common Gateway Interface). For example, the protein structure analysis apparatus 100 may be configured integrally with the mass spectrometry apparatus 400.

  The network 300 has a function of mutually connecting the protein structure analysis apparatus 100 and the external system 200. For example, the Internet, an intranet, a LAN (including both wired and wireless), a VAN, a personal computer, and the like. Communication networks, public telephone networks (including both analog / digital), leased line networks (including both analog / digital), CATV networks, IMT2000 system, GSM system, PDC / PDC-P system, etc. It may include any one of a mobile circuit switching network / mobile packet switching network, a wireless paging network, a local wireless network such as Bluetooth, a PHS network, a satellite communication network such as CS, BS, or ISDB. That is, this system can transmit and receive various data via any network regardless of wired or wireless.

  As described above in detail, according to the present invention, ions ionized by an electrospray ionization method or the like for a target protein for predicting a three-dimensional structure are fragmented into fragmented ions by a hexapole CID method or the like, and a fragmentation spectrum is measured. Then, based on the measured fragmentation spectrum, the assignment information of the fragment ion to the amino acid sequence of the target protein is determined, and the fragment information is fragmented in the amino acid sequence of the target protein by the determined assignment information of the fragment ion. And the cleavable region information in the amino acid sequence of the target protein is determined according to the portion, the three-dimensional structure is predicted for the target protein, the predicted three-dimensional structure prediction data and the determined cleavable region information (For example, graphic display, Therefore, when predicting the three-dimensional structure of proteins with unknown three-dimensional structures (including the genes that encode them), it is possible to make a highly accurate prediction by taking into account the measured three-dimensional structure information. A protein structure analysis method, a protein structure analysis apparatus, a program, and a recording medium can be provided.

  Further, according to the present invention, the three-dimensional structure prediction data is graphically displayed by any one of a wire model, a ribbon model, a pipe model, a ball and stick model, or a space filling model, and the easy-to-cut area information is supported. Since it is displayed in association with the display part of the 3D structure prediction data (for example, setting a link, displaying on the model, displaying a specific pattern pattern such as shading or diagonal lines corresponding to the easy-to-cut area information on the model, etc.) A protein structure analysis method, a protein structure analysis apparatus, a program, and a program that can display such information visually and in an easily understandable manner, and allow the user to intuitively determine the reliability of the three-dimensional structure prediction data. A recording medium can be provided.

  Further, according to the present invention, when the graphic display or the table display is performed, the display portion of the corresponding three-dimensional structure prediction data is displayed in different colors according to the easy-to-cut area information. It is possible to provide a protein structure analysis method, a protein structure analysis apparatus, a program, and a recording medium that can be displayed, and allow the user to intuitively determine the reliability of the three-dimensional structure prediction data. .

  Further, according to the present invention, the predicted three-dimensional structure prediction data is compared with the determined easy-cleavage area information to evaluate the predicted structure portion corresponding to the easy-cut area specified by the easy-cleavage area information. The predicted structure evaluation information is determined and the determined predicted structure evaluation information is output, so that the predicted structure predicted by the computer can be evaluated based on the biochemical experiment data, and the prediction accuracy is remarkably improved. A protein structure analysis method, a protein structure analysis apparatus, a program, and a recording medium that can be improved can be provided.

  This also greatly improves the accuracy of function prediction based on the three-dimensional structure when performing functional analysis of interesting genes and proteins found by genome sequence analysis, expression profiling analysis using DNA chips, proteome analysis, etc. In genome sequence analysis, the function can be efficiently estimated by performing function prediction in consideration of the three-dimensional structure of a gene / protein whose function is unknown. Further, this makes it possible to efficiently design drugs such as inhibitors, improve activity by protein engineering, and design functional modifications.

  In addition, according to the present invention, the reference vibration calculation is performed on the target protein, and the reference vibration calculation result (for example, fluctuation information) is displayed in association with the display portion of the corresponding three-dimensional structure prediction data (for example, the three-dimensional structure model). Since the vector display is performed above), the fluctuation information can be considered as the easy-to-cut region. That is, it is possible to provide a protein structure analysis method, a protein structure analysis apparatus, a program, and a recording medium capable of obtaining fluctuation information from the reference vibration calculation result and evaluating the prediction model by comparing with the MS actual measurement data. it can.

  In addition, according to the present invention, the collision activity including the hexapole CID method, the nozzle skimmer CID method, the capillary skimmer CID method, the SORI-CID method, and the multipole store auxiliary capillary skimmer CID method for the target protein for predicting the three-dimensional structure. The above-mentioned object by at least one of chemical dissociation (CID) method, IRMPD method, in-source decomposition (ISD) method, post-source decomposition (PSD) method, surface-induced dissociation (SID) method, ECD method, and BIRD method Since the protein is fragmented into fragment ions and the fragmentation spectrum is measured, a protein structure analysis method, a protein structure analysis apparatus, a program, and a protein structure analysis method capable of efficiently creating fragment ions reflecting the three-dimensional structure of the target protein Can provide recording media .

  In addition, according to the present invention, since the target protein is fragmented by an enzymatic reaction and the fragmentation spectrum is measured, the protein structure analysis method and protein that can efficiently perform fragmentation reflecting the three-dimensional structure of the target protein A structural analysis device, a program, and a recording medium can be provided.

  Further, according to the present invention, the target protein for which the three-dimensional structure is predicted is fragmented, the fragmentation spectrum is measured, and the attribution information of the fragmented ions for the amino acid sequence of the target protein is determined based on the measured fragmentation spectrum. Then, in accordance with the measured fragmentation spectrum and the determined fragmentation ion attribution information, the portion in contact with the solvent in the amino acid sequence of the target protein is specified, and the solvent contact in the amino acid sequence of the target protein according to the location Residue information is determined, the three-dimensional structure of the target protein is predicted, and the predicted three-dimensional structure prediction data and the determined solvent contact residue information are output in correspondence with each other (for example, graphic display, list display by table, etc.) ) Because of the protein whose structure is unknown (including the gene that encodes it) When performing the body structure prediction, by adding the actually measured three-dimensional structure information, how protein structure analysis that can be performed with high accuracy prediction, protein structure analysis apparatus, a program, and can provide a recording medium.

  Further, according to the present invention, the predicted three-dimensional structure prediction data and the determined solvent contact residue information are compared, and the predicted structure portion corresponding to the solvent contact residue specified by the solvent contact residue information Since the predicted structure evaluation information is determined and the determined predicted structure evaluation information is output, the predicted structure predicted by the computer can be evaluated based on the biochemical experiment data. It is possible to provide a protein structure analysis method, a protein structure analysis apparatus, a program, and a recording medium that can significantly improve accuracy.

  According to the present invention, after subjecting the target protein to hydrogen-deuterium exchange reaction, fragmentation and fragmentation spectrum are measured, and according to the measured fragmentation spectrum and the determined fragmentation ion attribution information. , Determine the deuteration rate at each amino acid residue in the amino acid sequence of the target protein, identify the location in contact with the solvent in the amino acid sequence of the target protein according to the deuteration rate, and Since the solvent contact residue information in the amino acid sequence is determined, it is possible to provide a protein structure analysis method, a protein structure analysis apparatus, a program, and a recording medium capable of efficiently obtaining solvent contact information.

  Further, according to the present invention, after subjecting the target protein to chemical modification, the fragmentation and fragmentation spectrum is measured, and according to the measured fragmentation spectrum and the determined fragmentation ion attribution information, Determine the chemically modified part at each amino acid residue in the amino acid sequence, identify the part in contact with the solvent in the amino acid sequence of the target protein according to the chemical modified part, Since the base information is determined, it is possible to provide a protein structure analysis method, a protein structure analysis apparatus, a program, and a recording medium that can efficiently obtain solvent contact information.

  In addition, according to the present invention, a complex of a target protein and a compound (for example, a protein, a low molecular compound, a nucleic acid, etc.) that predicts a three-dimensional structure is fragmented, a fragmentation spectrum is measured, and the measured fragmentation spectrum is measured. To determine the fragmentation ion attribution information for the protein and / or compound of interest, and to interact with the protein and / or compound according to the measured fragmentation spectrum and the determined fragmentation ion attribution information Interface information is determined, the three-dimensional structure is predicted for the target protein and / or compound, and the predicted three-dimensional structure prediction data and the determined interaction interface information are output in correspondence with each other (for example, by graphic display or by table) Proteins with unknown three-dimensional structure (registration that codes this) Provided protein structure analysis method, protein structure analysis apparatus, program, and recording medium capable of performing high-precision prediction by taking into account the actually measured three-dimensional structure information. can do.

  Further, according to the present invention, the three-dimensional structure prediction data is graphically displayed on the target protein and / or compound by any one of a wire model, a ribbon model, a pipe model, a ball and stick model, or a space filling model (for example, , Including docking simulation) and displaying the interaction interface information in association with the display portion of the corresponding three-dimensional structure prediction data (for example, setting a link, displaying on the model, corresponding to the interaction interface information on the model) This makes it possible to display such information visually and easily, and the user intuitively determines the reliability of the three-dimensional structure prediction data. Protein structure analysis method, protein structure analysis apparatus, program, Beauty, it is possible to provide a recording medium.

  Further, according to the present invention, when the graphic display or the table display is performed, the display portion of the three-dimensional structure prediction data corresponding to the target protein and / or compound is displayed in different colors according to the interaction interface information. Protein structure analysis method, protein structure analysis apparatus, program, and record that can display information visually and in an easy-to-understand manner, and allow users to intuitively determine the reliability of three-dimensional structure prediction data A medium can be provided.

  Further, according to the present invention, the predicted three-dimensional structure prediction data is compared with the determined interaction interface information, and the predicted structure portion corresponding to the interaction interface specified by the interaction interface information is evaluated. The predicted structure evaluation information is determined and the determined predicted structure evaluation information is output, so that the predicted structure predicted by the computer can be evaluated based on the biochemical experiment data, and the prediction accuracy is remarkably improved. A protein structure analysis method, a protein structure analysis apparatus, a program, and a recording medium that can be improved can be provided.

  Further, according to the present invention, the complex of the target protein and the compound was subjected to a hydrogen-deuterium exchange reaction, and then fragmented to measure the fragmentation spectrum, and the measured fragmentation spectrum was determined. Determining the deuteration rate of the target protein and / or compound according to the fragmentation ion assignment information, identifying the interaction interface in the target protein and / or compound according to the deuteration rate, and determining the target protein according to the interaction interface Since interaction interface information in a compound and / or compound is determined, a protein structure analysis method, a protein structure analysis apparatus, a program, and a recording medium capable of efficiently obtaining interaction interface information can be provided.

  Further, according to the present invention, after the chemical modification of the complex of the target protein and the compound, the fragmentation fragmentation spectrum is measured, and the measured fragmentation spectrum and the assignment of the determined fragmentation ion are determined. According to the information, a chemical modification moiety in the target protein and / or compound is determined, an interaction interface in the target protein and / or compound is identified according to the chemical modification moiety, and an interaction in the target protein and / or compound is determined according to the interaction interface Since the interface information is determined, it is possible to provide a protein structure analysis method, a protein structure analysis apparatus, a program, and a recording medium that can efficiently obtain interaction interface information.

  Furthermore, according to the present invention, since the complex of the target protein and the compound is fragmented by an enzyme reaction and the fragmentation spectrum is measured, the protein capable of efficiently performing the fragmentation reflecting the three-dimensional structure of the target protein A structure analysis method, a protein structure analysis apparatus, a program, and a recording medium can be provided.

  As described above, the protein structure analysis method, the protein structure analysis apparatus, the program, and the recording medium according to the present invention supplement the protein three-dimensional structure data obtained by the calculation method with the data obtained by the biochemical experimental method. can do.

  As a result, the protein structure analysis method, protein structure analysis apparatus, program, and recording medium according to the present invention are extremely useful in the bioinformatics field for performing analysis of protein mass analysis and three-dimensional structure.

  The present invention can be widely implemented in many industrial fields, particularly in fields such as pharmaceuticals, foods, cosmetics, medicine, and gene expression analysis, and is extremely useful.

It is a principle block diagram which shows the basic principle of this invention. It is a block diagram which shows an example of a structure of this system with which this invention is applied. It is a flowchart which shows an example of the main process of this system in this embodiment. It is a flowchart which shows an example in the case of measuring a fragmentation spectrum by the hexapole CID method in this embodiment. It is a flowchart which shows an example in the case of measuring a fragmentation spectrum by the IRMPD method in this embodiment. It is a conceptual diagram at the time of obtaining | requiring the fragmentation ion attribution information about human interleukin-6 (IL-6), and determining easy-cleavage area | region information. It is a flowchart which shows an example in the case of measuring a fragmentation spectrum by the hydrogen-deuterium exchange method in this embodiment. It is a flowchart which shows an example in the case of fragmenting protein by MS as it is by the hydrogen-deuterium exchange method in this embodiment, and measuring a fragmentation spectrum. It is a flowchart which shows an example in the case of fragmenting a protein complex by the hydrogen-deuterium exchange method in this embodiment, and measuring a fragmentation spectrum. It is a flowchart which shows an example in the case of fragmenting the complex of protein A and B by the hydrogen-deuterium exchange method in this embodiment, and measuring a fragmentation spectrum. It is a flowchart which shows an example in the case of analyzing a fragmentation spectrum in a present Example. Display screen for human interleukin-6 (IL-6), in which three-dimensional structure prediction data is displayed graphically by a ribbon model, and the corresponding three-dimensional structure prediction data is displayed in different colors according to easy-to-cut region information It is a figure which shows an example. It is a flowchart which shows an example of Example 1 of this system in this invention. It is a flowchart which shows an example of Example 2 of this system in this invention. It is a flowchart which shows an example of Example 3 of this system in this invention. It is a flowchart which shows an example of Example 4 of this system in this invention. It is a figure which shows an example of the display screen in the case of performing list display by a table about a processing result. It is a figure which shows an example of the process result in Example 5. FIG. A complex (complex) of human interleukin-6 (IL-6) and anti-IL-6 antibody is subjected to hydrogen-deuterium exchange reaction, fragmented into fragmented ions by electrospray ionization, and fragmented It is a conceptual diagram at the time of measuring a spectrum. It is a conceptual diagram at the time of calculating | requiring a deuteration rate from the fragmentation spectrum of human IL-6 which performed the hydrogen-deuterium exchange reaction. It is a flowchart which shows an example of Example 8 of this system in this invention. It is a figure which shows an example of the display screen which displayed the difference in the expression level by the DNA microarray of Example 8 of this system in this invention according to the color. It is a screen which displays the gene information which the user of Example 8 of this system in this invention selected. It is a display screen of alignment of Example 8 of this system in the present invention. It is a display screen of the three-dimensional structure by the graphic software of Example 8 of this system in the present invention. It is a figure which shows an example of the three-dimensional structure of the bovine origin ubiquitin in Example 6 of this system in this invention. It is a figure which shows an example of the three-dimensional structure of the yeast origin alcohol dehydrogenase in Example 7 of this system in this invention.

Explanation of symbols

100 Protein structure analyzer
102 Control unit
102a Fragmentation spectrum data acquisition unit
102b Attribution information determination unit
102c Easy-to-cut area information determination unit
102d Solvent contact residue information determination unit
102e Interaction interface information determination unit
102f Three-dimensional structure prediction processing unit
102g Predictive structure evaluation information determination unit
102h Processing result output part
102i Predictive structure evaluation information output unit
102j Deuteration rate determination unit
102k reference vibration calculation processing unit
104 Communication control interface unit
106 Storage unit
106a Fragmentation spectrum data file
106b Easy cutting area information file
106c Solvent contact residue information / interaction interface information file
106d 3D structure prediction data file
106e Predictive structure evaluation information file
106f Processing result data file
108 Input / output control interface
112 Input device
114 Output device 200 External system 300 Network 400 MS

Claims (6)

Fragmentation spectrum measurement step of measuring a fragmentation spectrum by fragmenting a target protein that predicts a three-dimensional structure;
Based on the fragmentation spectrum measured by the fragmentation spectrum measurement step, an attribution information determination step for determining the attribution information of the fragmented ions for the amino acid sequence of the target protein,
According to the fragmentation spectrum measured by the fragmentation spectrum measurement step and the attribution information of the fragmentation ion determined by the attribution information determination step, the amino acid sequence of the target protein is in contact with a solvent. A solvent contact residue information determination step for identifying a location and determining solvent contact residue information in the amino acid sequence of the target protein according to the location;
A three-dimensional structure prediction step for predicting a three-dimensional structure of the target protein;
A reference vibration calculation step for performing a reference vibration calculation for the target protein;
The three-dimensional structure prediction data predicted in the three-dimensional structure prediction step, the solvent contact residue information determined in the solvent contact residue information determination step, and the calculation result in the reference vibration calculation step are output in association with each other. Processing result output step,
A protein structure analysis method comprising: Fragmentation spectrum measurement step of fragmenting a complex of a target protein and a compound that predicts a three-dimensional structure and measuring a fragmentation spectrum;
An attribution information determination step for determining attribution information of fragmented ions for the target protein and / or the compound based on the fragmentation spectrum measured by the fragmentation spectrum measurement step;
According to the fragmentation spectrum measured by the fragmentation spectrum measurement step and the attribution information of the fragmentation ion determined by the attribution information determination step, interaction interface information regarding the target protein and / or the compound is obtained. An interaction interface information determination step to determine;
A three-dimensional structure prediction step of predicting a three-dimensional structure of the target protein and / or the compound;
A reference vibration calculation step for performing a reference vibration calculation for the target protein and / or the compound;
Processing for outputting the three-dimensional structure prediction data predicted in the three-dimensional structure prediction step, the interaction interface information determined in the interaction interface information determination step, and the calculation result in the reference vibration calculation step in association with each other A result output step;
A protein structure analysis method comprising: Fragmentation spectrum data acquisition means for obtaining a fragmentation spectrum data from a mass spectrometer that has fragmented a target protein for predicting a three-dimensional structure and has measured the fragmentation spectrum;
Based on the fragmentation spectrum data acquired by the fragmentation spectrum data acquisition means, attribution information determination means for determining attribution information of fragmented ions for the amino acid sequence of the target protein;
According to the fragmentation spectrum measured by the fragmentation spectrum measurement means and the attribution information of the fragmentation ions determined by the attribution information determination means, the amino acid sequence of the target protein is in contact with a solvent. A solvent contact residue information determining means for identifying a location and determining solvent contact residue information in the amino acid sequence of the target protein according to the location;
A three-dimensional structure prediction means for predicting a three-dimensional structure of the target protein;
A reference vibration calculation means for performing a reference vibration calculation for the target protein;
The three-dimensional structure prediction data predicted by the three-dimensional structure prediction means, the solvent contact residue information determined by the solvent contact residue information determination means, and the calculation result by the reference vibration calculation means are output in association with each other. Processing result output means,
A protein structure analyzing apparatus comprising: Fragmentation spectrum data acquisition means for fragmenting a complex of a target protein and a compound that predicts a three-dimensional structure and acquiring fragmentation spectrum data from a mass spectrometer that measures the fragmentation spectrum;
Attribution information determination means for determining the attribution information of fragmented ions for the target protein and / or the compound based on the fragmentation spectrum data acquired by the fragmentation spectrum data acquisition means,
In accordance with the fragmentation spectrum measured by the fragmentation spectrum measurement means and the attribution information of the fragmentation ions determined by the attribution information determination means, interaction interface information regarding the target protein and / or the compound is obtained. An interaction interface information determining means for determining;
A three-dimensional structure prediction means for predicting a three-dimensional structure of the target protein and / or the compound;
A reference vibration calculation means for performing a reference vibration calculation for the target protein and / or the compound;
Processing for outputting the three-dimensional structure prediction data predicted by the three-dimensional structure prediction means, the interaction interface information determined by the interaction interface information determination means, and the calculation result by the reference vibration calculation means in association with each other A result output means;
A protein structure analyzing apparatus comprising: Fragmentation spectrum data acquisition step of obtaining a fragmentation spectrum data from a mass spectrometer that has fragmented a target protein that predicts a three-dimensional structure and has measured the fragmentation spectrum;
Based on the fragmentation spectrum data acquired by the fragmentation spectrum data acquisition step, an attribution information determination step for determining the attribution information of fragmentation ions for the amino acid sequence of the target protein;
According to the fragmentation spectrum measured by the fragmentation spectrum measurement step and the attribution information of the fragmentation ion determined by the attribution information determination step, the amino acid sequence of the target protein is in contact with a solvent. A solvent contact residue information determination step for identifying a location and determining solvent contact residue information in the amino acid sequence of the target protein according to the location;
A three-dimensional structure prediction step for predicting a three-dimensional structure of the target protein;
A reference vibration calculation step for performing a reference vibration calculation for the target protein;
The three-dimensional structure prediction data predicted in the three-dimensional structure prediction step, the solvent contact residue information determined in the solvent contact residue information determination step, and the calculation result in the reference vibration calculation step are output in association with each other. Processing result output step,
A computer program for causing a computer to execute a protein structure analysis method including: Fragmentation spectrum data acquisition step of obtaining a fragmentation spectrum data from a mass spectrometer that has fragmented a complex of a target protein and a compound that predicts a three-dimensional structure and measured the fragmentation spectrum;
Based on the fragmentation spectrum data acquired by the fragmentation spectrum data acquisition step, an attribution information determination step for determining attribution information of fragmentation ions for the target protein and / or the compound,
According to the fragmentation spectrum measured by the fragmentation spectrum measurement step and the attribution information of the fragmentation ion determined by the attribution information determination step, interaction interface information regarding the target protein and / or the compound is obtained. An interaction interface information determination step to determine;
A three-dimensional structure prediction step of predicting a three-dimensional structure of the target protein and / or the compound;
A reference vibration calculation step for performing a reference vibration calculation for the target protein and / or the compound;
Processing for outputting the three-dimensional structure prediction data predicted in the three-dimensional structure prediction step, the interaction interface information determined in the interaction interface information determination step, and the calculation result in the reference vibration calculation step in association with each other A result output step;
A computer program for causing a computer to execute a protein structure analysis method including: