JP2015152556A - Sequencing method and apparatus of amino acid of protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To accurately estimate the sequence of the terminal portion of a protein and also improve the reliability of the sequence estimation of other parts than the terminal parts even in the case of using protein DB based on the cDNA.SOLUTION: Based on the amino acid sequence on the protein DB based on the cDNA, multiple amino acid sequences from each end of which one amino acid residue is removed is created among the amino acid sequences of a predetermined length from the distal end of the terminal portion for each protein and a terminal sequence DB is created in association with each protein. Based on the result of the MSanalysis of the peptide mixture derived from the target protein, the MS/MS ion search using the terminal sequence DB is performed. Since, on the terminal sequence DB, the sequence which matches the amino acid sequence of the terminal portion of the measured protein from which a signal peptide is left out is recorded, the terminal peptide can be also identified and it is possible to identify the whole protein by matching the search results using the Swiss-Prot.

Description

  The present invention relates to a method for determining an amino acid sequence of a protein using mass spectrometry data obtained by mass spectrometry of a test sample containing a peptide mixture derived from a protein to be analyzed, and an apparatus therefor, Specifically, the present invention relates to a method and apparatus for determining the amino acid sequence of the terminal portion such as the N-terminus or C-terminus of a protein.

In recent years, proteome analysis methods for comprehensive analysis of proteins have been widely used, and there are significant technological advances. In the field of proteome analysis, a database search method is well known as a protein identification technique using a mass spectrometer such as MALDI-TOFMS (matrix-assisted laser desorption / ionization time-of-flight mass spectrometer). In general, in the database search method, the mass-to-charge ratio of peaks appearing in the MS ⁿ spectrum obtained by performing MS ⁿ analysis (n is an integer of 2 or more) on a sample containing a peptide mixture derived from a protein to be analyzed ( m / z) Check the peak list that collects information (and possibly intensity information) against the list of mass-to-charge ratios that are theoretically calculated from the protein information registered in the protein database. The amino acid sequence of the peptide is determined based on the degree, and the original protein is identified based on the result (that is, the amino acid sequence of the entire protein is determined).

  MS / MS Ion Search included in Mascot provided by Matrix Science, UK, as a search engine used for database search as described above. Is well known (see Non-Patent Document 1).

  On the other hand, databases used for determining the amino acid sequence of such proteins are provided by various research institutions and universities and are actually used. One such database is a database created on the basis of complementary DNA (hereinafter referred to as “cDNA” in accordance with common practice). This database contains amino acid sequence information of each protein determined based on data obtained by reverse transcription of messenger ribonucleic acid generally called mRNA. In recent years, the progress of genome analysis has been remarkable, and the database for protein identification based on cDNA that reflects the results is quite substantial compared to the database containing amino acid sequence information of known proteins elucidated by actual measurement etc. It has become.

  However, when the amino acid sequence of the terminal portion such as the N-terminal or C-terminal of the protein is identified by the above MS / MS ion search, a database created from cDNA is often used under normal search conditions. Can not be determined. The reason for this will be briefly described with reference to FIGS. 11 and 12 are diagrams for explaining a processing procedure when determining the amino acid sequence of a protein.

  That is, as described above, the cDNA-based database contains amino acid sequence information determined based on the reverse transcription data of mRNA, and this information contains the coding region (5 ′ untranslated region) of the original mature mRNA. Amino acid residues are arranged at both the N-terminal and C-terminal to the end of the protein (translated into a protein sandwiched between a start codon and a stop codon between the 3 ′ untranslated region and the 3 ′ untranslated region). On the other hand, in proteins that are actually present in the living body, in many cases, after translation from mRNA to protein, post-translational modifications such as removal of the signal peptide on the N-terminal side occur, or even on the C-terminal side. Or a single amino acid residue may be cleaved. For this reason, as shown in FIG. 11A, proteins existing in the living body exist in a shorter state than the amino acid sequences in the database based on cDNA.

  In FIG. 11 (a), “1-” and “-A” (here, A is X) indicate that the “amino acid sequence registered in the database” is A It shows that it consists of amino acid residues. In the figure, “M” is a methionine residue, which is an amino acid residue encoded by the start codon of mRNA. Although there are very rare organisms in which the base sequence encoding valine serves as the start codon, in many organisms the start codon encodes a methionine residue, so the amino acid encoded by the start codon is methionine hereinafter. The explanation will be given assuming that there is. “P-” and “-B” (here, B is Y) in the lower diagram of FIG. 11 (a) are “when the N-terminal of the“ amino acid sequence registered in the database ”is 1,” It shows that the N-terminus of the “actual amino acid sequence of the protein” starts with the p-th amino acid residue and ends with the B (<A) -th amino acid residue. The same applies to FIG. 2, FIG. 3, FIG. 7, FIG. 8, and FIG.

When a protein as shown in FIG. 11 (a) is enzymatically digested, the bond is cleaved at a position peculiar to the enzyme to generate a mixture of a plurality of peptides. The peptide mixture is subjected to mass spectrometry (for example, MS ² analysis), and the result is subjected to database search using MS / MS ion search. At this time, the type of enzyme used for enzyme digestion is set as one of the search conditions.

In the execution process of MS / MS ion search, amino acid sequences of various peptide fragments generated by cleaving the amino acid sequences of proteins in the database at known cleavage positions corresponding to the types of enzymes are calculated. And the amino acid sequence is collated with the amino acid sequence estimated from the mass spectrometry result (for example, MS ² spectrum result) (refer FIG.11 (b)). At this time, except for the terminal portion of the amino acid sequence of the original protein, there are calculated amino acid sequences corresponding to the amino acid sequences based on the actual measurement results, and these peptides are identified. However, as described above, in the actual protein, the signal peptide at the N-terminal is dropped or several amino acid residues are dropped at the C-terminal, so the amino acid sequence of the peptide fragment at the terminal is derived from a database based on cDNA. The calculated amino acid sequence generally does not match. As a result, as shown in FIG. 11C, the amino acid sequence of the terminal portion of the protein cannot be determined by the MS / MS ion search.

  Therefore, conventionally, when a database based on cDNA is generally used, “None” is designated in one enzyme selection condition in the search conditions. As described above, when an enzyme is designated as an enzyme selection condition, the amino acid sequence obtained as a result of specific cleavage by the enzyme is collated with a database.

  On the other hand, when “None” is designated as the enzyme selection condition, as shown in FIG. 12B, the amino acid sequences of the respective proteins in the database are all included in the amino acid sequences cleaved at two arbitrary positions. Database matching is performed on the database. The actual terminal amino acid sequence of a protein rarely matches the amino acid sequence of a terminal portion of a protein in a cDNA-based database, but it is often a part of the amino acid sequence of a protein in the cDNA-based database. If it matches. Therefore, as shown in FIG. 12C, by specifying “None” as the enzyme selection condition, it is possible to determine the amino acid sequence of the terminal portion of the protein, thereby identifying the protein. .

However, these protein identification methods have the following problems.
That is, a huge number of proteins are recorded in a database based on cDNA, and the number of amino acid sequences generated by cleaving at any two positions in the long amino acid sequence of the protein is very large. Therefore, in this case, the number of amino acid sequences to be subjected to database collation is quite enormous. In MS / MS ion search, “Expectation Value” (hereinafter referred to as “Expected Value” in this specification) indicating the degree of erroneous identification is output as an index value indicating the reliability of the identification result. However, the value depends on the size of the database that needs to be collated. The larger the database size, the worse the misidentification prediction value. Therefore, as described above, when "None" is specified as the enzyme selection condition, if the number of amino acid sequences to be collated becomes enormous, the erroneous identification prediction value deteriorates, and reliable identification cannot be performed. There is a problem.

  FIG. 13 shows identification results when MS / MS ion search is executed with “None” specified as the enzyme selection condition and when MS / MS ion search is executed with “Trypsin” specified as the enzyme selection condition. It is the figure which compared the misidentification prediction value which is. The fact that the horizontal bar corresponding to “Trypsin” is not drawn in FIG. 13 means that identification could not be performed. This figure shows that if "None" is specified as the enzyme selection condition, the amino acid sequence of the N-terminal peptide can be identified, but the misidentification prediction value other than at the N-terminal specifies "Trypsin" as the enzyme selection condition. It turns out that it is definitely worse (larger) than the case.

"Searching uninterpreted MS / MS data", [online], UK Matrix Science Ltd., [February 4, 2014 search] , Internet <URL: http://www.matrixscience.com/help/mis_help.html>

  The present invention has been made to solve the above-mentioned problems, and its object is to estimate the amino acid sequence of a target protein by database search such as MS / MS ion search using a database based on cDNA. In addition, it is possible to accurately determine the amino acid sequence of the terminal portion, which is often partially removed in the actual protein, and to estimate the amino acid sequence other than the terminal portion with high accuracy. An object is to provide a method and apparatus for determining the amino acid sequence of a protein.

The first aspect of the method for determining the amino acid sequence of a protein according to the present invention, which has been made to solve the above problems, is to perform a database search based on mass spectral information obtained by mass spectrometry of a peptide mixture derived from a target protein. In the method for determining the amino acid sequence of a protein that determines the amino acid sequence of the target protein by performing or provides information for determining the amino acid sequence,
a) A database in which amino acid sequences of known proteins are recorded, and the position S of the N-terminal amino acid residue of the amino acid sequence of each known protein is 1 ≦ S ≦ p (provided that on the mRNA encoding the known protein) The first amino acid residue corresponding to the start codon of the coding region of p, and the p-th amino acid residue of the N-terminal protein present in the living body based on the base sequence information of the coding region on the mRNA encoding the known protein On the basis of amino acid sequence information obtained from a database of known proteins, and an amino acid sequence consisting of amino acid residues of a predetermined length from the N-terminal for each known protein, and the predetermined length The predetermined length obtained by sequentially removing amino acid residues in the amino acid sequence from the N-terminal end in order. Seek terminal near the following amino acid sequences in length, and N-terminal sequence database creation step of creating an N-terminal sequence database by the amino acid sequence,
b) Database search for estimating the amino acid sequence of the terminal portion of the target protein by collating the mass spectrum information derived from the target protein with the amino acid sequence recorded in the N-terminal sequence database or information obtained from the sequence. Steps,
It is characterized by having.

The second aspect of the protein amino acid sequence determination method according to the present invention, which has been made to solve the above problems, is to perform a database search based on mass spectral information obtained by mass spectrometry of a peptide mixture derived from a target protein. In the method for determining the amino acid sequence of a protein that determines the amino acid sequence of the target protein by performing or provides information for determining the amino acid sequence,
a) A database in which amino acid sequences of known proteins are recorded, and the position Y of the C-terminal amino acid residue of each amino acid sequence of each known protein is q ≦ Y ≦ L (provided that on the mRNA encoding the known protein) 1st amino acid residue corresponding to the start codon of the coding region, and q-th amino acid residue C-terminal amino acid residue of the protein in the living body based on the base sequence information of the coding region on the mRNA encoding the known protein Obtained from a database of known proteins consisting of an amino acid sequence that is the Lth amino acid residue corresponding to the codon adjacent to the 5 'upstream of the stop codon of the coding region on the mRNA encoding the known protein) Based on the amino acid sequence information obtained, each known protein has a predetermined length from the C-terminus. An amino acid sequence consisting of amino acid residues is extracted, and amino acid residues in the amino acid sequence of a predetermined length are sequentially removed from the most terminal on the C-terminal side to obtain a near-terminal amino acid sequence having a length shorter than the predetermined length. C-terminal sequence database creation step of creating a C-terminal sequence database from the amino acid sequence;
b) Database search for estimating the amino acid sequence of the terminal portion of the target protein by collating the mass spectrum information derived from the target protein with the amino acid sequence recorded in the C-terminal sequence database or information obtained from the sequence Steps,
It is characterized by having.

Moreover, the 1st aspect of the amino acid sequence determination apparatus of the protein based on this invention made | formed in order to solve the said subject is for embodying the 1st aspect of the amino acid sequence determination method of the protein based on the said invention. Information for determining an amino acid sequence of the target protein or determining the amino acid sequence by performing a database search based on mass spectral information obtained by mass spectrometry of a peptide mixture derived from the target protein. In a protein amino acid sequencing apparatus that provides
a) A database in which amino acid sequences of known proteins are recorded, and the position S of the N-terminal amino acid residue of the amino acid sequence of each known protein is 1 ≦ S ≦ p (provided that on the mRNA encoding the known protein) The first amino acid residue corresponding to the start codon of the coding region of p, and the p-th amino acid residue of the N-terminal protein present in the living body based on the base sequence information of the coding region on the mRNA encoding the known protein The amino acid residue in the amino acid sequence consisting of amino acid residues of a predetermined length from the N-terminus for each known protein, created based on the amino acid sequence information obtained from the database of known proteins consisting of amino acid sequences The amino acids in the vicinity of the end having a length equal to or shorter than the predetermined length in which the groups are sequentially removed from the N-terminal side from the most end. And N-terminal sequence database acid sequence has been recorded,
b) Database search for estimating the amino acid sequence of the terminal portion of the target protein by collating the mass spectrum information derived from the target protein with the amino acid sequence recorded in the N-terminal sequence database or information obtained from the sequence. And
It is characterized by having.

The second aspect of the protein amino acid sequence determination apparatus according to the present invention, which has been made to solve the above-mentioned problems, is to realize the second aspect of the protein amino acid sequence determination method according to the present invention. Information for determining an amino acid sequence of the target protein or determining the amino acid sequence by performing a database search based on mass spectral information obtained by mass spectrometry of a peptide mixture derived from the target protein. In a protein amino acid sequencing apparatus that provides
a) A database in which amino acid sequences of known proteins are recorded, and the position Y of the C-terminal amino acid residue of each amino acid sequence of each known protein is q ≦ Y ≦ L (provided that on the mRNA encoding the known protein) 1st amino acid residue corresponding to the start codon of the coding region, and q-th amino acid residue C-terminal amino acid residue of the protein in the living body based on the base sequence information of the coding region on the mRNA encoding the known protein Obtained from a database of known proteins consisting of an amino acid sequence that is the Lth amino acid residue corresponding to the codon adjacent to the 5 'upstream of the stop codon of the coding region on the mRNA encoding the known protein) For each known protein created based on the amino acid sequence information obtained from the C-terminus A C-terminal sequence database in which amino acid residues in the amino acid sequence consisting of amino acid residues of a fixed length are removed in order from the most terminal end on the C-terminal side, and the amino acid sequences near the terminal of the predetermined length or less are recorded. When,
b) Database search for estimating the amino acid sequence of the terminal portion of the target protein by collating the mass spectrum information derived from the target protein with the amino acid sequence recorded in the C-terminal sequence database or information obtained from the sequence And
It is characterized by having.

  1st aspect of the amino acid sequence determination method of the protein which concerns on this invention, and 1st aspect of the amino acid sequence determination apparatus of the protein which concerns on this invention determine the amino acid sequence of N terminal part, The protein which concerns on this invention The second aspect of the amino acid sequence determination method of the present invention and the second aspect of the protein amino acid sequence determination apparatus according to the present invention determine the amino acid sequence of the C-terminal portion.

  In the method and apparatus for determining the amino acid sequence of a protein according to the present invention, the known protein database is created based on, for example, a protein database created based on genomic data, and more specifically, based on complementary DNA that is one type of genomic data. Or a database created based on a comprehensive analysis of nucleic acid information. These protein databases contain amino acid sequences that do not take into account (do not reflect) the loss of signal peptides that often occur in the body, that is, protein amino acid sequences that are theoretical in nature. For this reason, in the protein database, the amino acid residues at the end of the protein are aligned to the end based on the base sequence of RNA. However, it is not necessary that both amino acid residues at both ends are aligned to the end based on the RNA base sequence, and the amino acid residue at the end portion on the side where the amino acid sequence is to be determined extends to the end based on the RNA base sequence. It is enough if they are ready.

  As the known protein database, a protein database constructed by directly measuring the amino acid sequence of a protein using Edman degradation, mass spectrometry, or the like can be used. In particular, in the case of databases created based on proteins biosynthesized by prokaryotes such as E. coli, post-translational modifications such as cleavage of the N-terminal signal peptide after translation that occur in proteins biosynthesized by eukaryotes occur. It may be longer than the end of a protein biosynthesized by eukaryotes.

  In the first aspect of the protein amino acid sequence determination method according to the present invention, in the N-terminal sequence database creation step, and in the second aspect of the protein amino acid sequence determination method according to the present invention, in the C-terminal sequence database creation step. Based on the amino acid sequence information of the proteins recorded in the above-mentioned known protein database, an amino acid sequence consisting of amino acid residues having a predetermined length is extracted from the most terminal of the N-terminal or C-terminal part of each protein. The “predetermined length” at this time depends on the search method used in the database search step.

Specifically, when using MS / MS ion search for product ion information obtained by MS ⁿ analysis, the amino acid sequence of the terminal peptide including the terminal portion such as the N-terminal and C-terminal can be estimated. That's fine. Therefore, the “predetermined length” may be longer than the length of the signal peptide to be dropped. Actually, not all of the signal peptides have been elucidated. Therefore, a length that allows for a predetermined margin with respect to the expected length of the signal peptide (approximately 35 amino acid residues or less) is determined in advance. Just keep it. Of course, since the presence of a signal peptide having a length of more than 35 amino acid residues has also been confirmed, the “predetermined length” may be set longer.

On the other hand, when peptide mass fingerprinting (PMF) targeting ion information obtained by mass spectrometry using only MS ¹ ions that have not been cleaved by collision-induced dissociation or the like is used, the N-terminal and C-terminal are used. Search is not possible unless the amino acid sequence includes not only the terminal portion but also the amino acid sequence other than the terminal portion. Therefore, the “predetermined length” may be, for example, the full length of the protein, or an amino acid sequence length shorter than the full length by a predetermined number (however, it is a sufficient length for PMF).

  In any case, in the N-terminal sequence database creation step or the C-terminal sequence database creation step, one amino acid sequence of a predetermined length including the most terminal amino acid residue for one kind of protein is sequentially ordered from the most terminal. A plurality of near-terminal amino acid sequences obtained by removing amino acid residues one by one are obtained, and this is associated with, for example, a protein and made into a database. For example, assuming that the amino acid residues from the most terminal to the Nth are removed to the maximum, N types of amino acid sequences near the terminal having different lengths are obtained for each type of protein. This can be regarded as an amino acid sequence obtained by simulating the loss of the signal peptide at the end of the protein that occurs in the living body.

  In the database search step, the mass spectrum information derived from the target protein is collated with the amino acid sequence recorded in the N-terminal sequence database or the C-terminal sequence database or information obtained from the sequence, whereby the N-terminal side in the target protein is Alternatively, the amino acid sequence of the terminal portion on the C-terminal side is estimated. Here, “information obtained from amino acid sequence” means, for example, ion information or product ion information (typically mass) corresponding to a fragment obtained by cleaving the amino acid sequence near the terminal at a position specific to the designated enzyme. Charge ratio value). As described above, since the amino acid sequence near the terminal is an amino acid sequence generated by pseudo-removing the amino acid residue from the most terminal, the signal peptide itself dropped in the actually measured target protein is unknown. However, it is highly likely that the amino acid sequence corresponding to the amino acid sequence of the terminal portion on one terminal side or the amino acid sequence including the terminal portion in the target protein is recorded in the N-terminal sequence database or the C-terminal sequence database. . Thereby, in the database search step, the amino acid sequence of the terminal portion of the target protein, which has been difficult to identify by the conventional method, can be estimated with high accuracy.

  In this way, if the amino acid sequence of at least one terminal portion is estimated, it can be used to estimate the amino acid sequence of the entire protein, or useful information for the analyst himself to estimate the amino acid sequence. Can be presented.

As described above, the database search can be performed using either MS / MS ion search or PMF.
That is, in one aspect of the method for determining an amino acid sequence of a protein according to the present invention,
The mass spectrum information is product ion information based on an MS ⁿ analysis result where ⁿ is an integer of 2 or more,
The database search step uses MS / MS ion search to match the product ion information with the product ion information obtained from each amino acid sequence recorded in the terminal sequence database, thereby determining the amino acid at the terminal portion of the target protein. The sequence can be deduced.

In another aspect of the method for determining an amino acid sequence of a protein according to the present invention,
The predetermined length is the total length of the protein, and the mass spectrum information is ion information based on a mass analysis result using only MS ¹ ions,
The database search step uses peptide mass fingerprinting to match the ion information with the ion information obtained from each amino acid sequence recorded in the terminal sequence database to obtain an amino acid sequence including the terminal portion of the target protein. It can be estimated.

  According to the method and apparatus for determining the amino acid sequence of a protein according to the present invention, it is possible to determine with high accuracy the amino acid sequence of the terminal portion from which a signal peptide that has often been removed in an actual protein is dropped. . In addition, compared to conventional methods such as MS / MS ion search that specifies “None” as the enzyme search condition, the number of amino acid sequences that are subject to database verification can be reduced. The reliability of the estimation can be increased. Thereby, estimation of the amino acid sequence of the target protein, that is, accuracy of identification of the target protein can be improved.

BRIEF DESCRIPTION OF THE DRAWINGS The whole block diagram of the protein identification system which is 1st Example for enforcing the amino acid sequence determination method of the protein based on this invention. The figure which shows an example of the amino acid sequence recorded in the N terminal sequence database in the protein identification system of 1st Example. Explanatory drawing of the process sequence at the time of determining the amino acid sequence of protein in the protein identification system of 1st Example. The figure which shows an example of the search condition setting screen (a) of a mascot, and the search result (b) when determining the amino acid sequence of protein in the protein identification system of 1st Example. In the protein identification system of the first embodiment, the misidentification prediction value obtained when MS / MS ion search was performed for each of 10 types of AGP-derived glycopeptides using the N-terminal sequence database and Swiss-Prot. FIG. The whole block diagram of the protein identification system which is 2nd Example for enforcing the amino acid sequence determination method of the protein based on this invention. The figure which shows an example of the amino acid sequence recorded in the N terminal sequence database for PMF in the protein identification system of 2nd Example. Explanatory drawing of the process sequence at the time of determining the amino acid sequence of protein in the protein identification system of 2nd Example. The figure which shows an example of the search condition setting screen (a) of a mascot, and its search result (b) when determining the amino acid sequence of a protein by the peptide mass fingerprinting (PMF) method using an N-terminal sequence database. The figure which shows an example of the search condition setting screen (a) of a mascot, and its search result (b) when determining the amino acid sequence of protein in the protein identification system of 2nd Example. Explanatory drawing of the conventional process sequence at the time of determining the amino acid sequence of protein by MS / MS ion search. Explanatory drawing of the conventional process sequence at the time of determining the amino acid sequence of protein by MS / MS ion search. The figure which compared the misidentification prediction value in the case where "None" is designated as an enzyme selection condition of MS / MS ion search, and the case where "Trypsin" is designated.

[First embodiment]
Hereinafter, a first embodiment of a protein identification system for carrying out a method for determining amino acids of a protein according to the present invention will be described with reference to the accompanying drawings.

FIG. 1 is an overall configuration diagram of the protein identification system of the first embodiment.
The protein identification system of this example includes a mass spectrometer 1, a data processing unit 2, an input unit 3, and a display unit 4.

The mass spectrometer 1 is not particularly limited in its configuration, but high mass accuracy and mass resolution are required, as well as collision-induced dissociation (CID) and infrared multiphoton absorption dissociation (IRMPD). , Photo-induced dissociation (PID = Photo-Induced Dissociation), electron capture dissociation (ECD), electron transfer dissociation (ETD = Electron Transfer Dissociation), post-source decomposition (PSD = Post-Source Decay), in-source decomposition Since it is necessary to perform MS ⁿ analysis with (ISD = In-Source Decay) or the like, an ion trap time-of-flight mass spectrometer or TOF / TOF mass spectrometer is preferable.

The data processing unit 2 is a characteristic component for carrying out the amino acid determination method for a protein according to the present invention, and includes an MS ⁿ spectrum data collection unit 20, a peak information extraction unit 21, a database search unit 22, and an identification protein database. 23, a functional block such as a known information reading unit 24, a terminal sequence database creation / management unit 25, and a terminal sequence database 26. The data processing unit 2 can be configured mainly by a general-purpose personal computer, for example, and main functions are realized by executing dedicated control / processing software installed in the computer on the computer. Can be. The database search unit 22 may typically use the MS / MS ion search included in the mascot, but the database search engine that can be used is not limited to this.

  As the identification protein database 23 included in the data processing unit 2, any database generally used for protein identification can be used. On the other hand, the reference protein database 5 placed outside the data processing unit 2 in FIG. 1 is a cDNA-based protein database, such as Swiss-Prot (UniProt). Of course, a protein database based on cDNA may be used as the identification protein database 23. Here, the identification protein database 23 included in the data processing unit 2 is also Swiss-Prot. The reference protein database 5 is not a cDNA-based protein database, but a database created based on comprehensive analysis of nucleic acid information using a next-generation sequencer, etc., and further, Edman degradation, mass spectrometry, etc. A protein database constructed by directly measuring the amino acid sequence of a protein may be used.

  In the protein identification system of this embodiment, the terminal sequence database 26 is constructed as follows prior to actual analysis processing. That is, the known information reading unit 24 reads a known amino acid sequence of a protein from one or both of the reference protein database 5 and the identification protein database 23. The amino acid sequence read at this time is an amino acid sequence in which the amino acid residues are aligned up to the end based on the RNA base sequence, that is, the terminal peptide peptide is bound. The terminal sequence database creating / managing unit 25 creates a terminal sequence database 26 in which terminal amino acid sequences of various lengths are recorded, in which the amino acid sequence of the terminal portion of the protein partially exists based on the read amino acid sequence. create.

FIG. 2 is an example of amino acid sequences recorded in the terminal sequence database 26.
In this example, m = 50th from the N-terminal end (position of methionine residue M in FIG. 2) in the amino acid sequence of the full length protein including the signal peptide read from the protein database based on cDNA. Extract the amino acid sequence up to. A sequence list of peptides including all the amino acid sequences from the jth to the mth, where j is all integers from 1 to m in the amino acid sequence in which the m amino acid residues are connected, create.

  That is, as shown in FIG. 2, an amino acid sequence in which the first to 50th amino acid residues are present, an amino acid sequence in which the second to 50th amino acid residues are present,..., 50th to 50th A 50 amino acid sequence including an amino acid sequence in which an amino acid residue is present (that is, only the 50th amino acid residue is present) is obtained as a “near terminal amino acid sequence” in the present invention. It is indexed, and information on proteins that match that, that is, proteins having such amino acid sequences at the end, entry names, accession numbers, etc., is made into a database. The terminal sequence database 26 is constructed by performing the same processing for all the proteins read from the databases 5 and 23. In other words, the terminal sequence database 26 is a database in which an actual protein is often dropped, and the amino acid sequence itself is an amino acid sequence of a terminal portion assuming that an unknown signal peptide is dropped. is there.

The protein to be analyzed is decomposed into peptide fragments by pretreatment using an appropriate digestive enzyme such as trypsin. This peptide mixture is a mixture of a peptide containing a terminal amino acid sequence of a protein and a peptide containing only an internal amino acid sequence. This mixture of peptide fragments is subjected to analysis by the mass spectrometer 1 as a test sample. In the mass spectrometer 1, mass analysis is performed on the test sample, ions that are estimated to be peptide-derived molecular ions are extracted from the mass spectrum obtained thereby, and the ions are set as precursor ions. Perform MS ² analysis. In some cases, MS ⁿ analysis in which n is 3 or more is performed. These are the same as the conventional methods.

MS ⁿ spectrum data collecting unit 20 temporarily stores to collect and MS ⁿ spectra data obtained by the mass spectrometer 1. The peak information extraction unit 21 extracts, for example, a peak appearing on the MS ² spectrum for each precursor ion, that is, product ion information (mass-to-charge ratio m / z and signal intensity) based on the collected MS ⁿ spectrum data. This product ion information is input to the database search unit 22, the type of enzyme used for enzyme digestion (for example, trypsin) is designated as an enzyme selection condition that is one of the search conditions, and the identification protein database 23 and terminal sequences are specified. By executing a database search using the database 26, an amino acid sequence corresponding to product ion information is estimated.

  The process at this time will be described with reference to FIG. As shown in FIG. 3 (a), the amino acid sequence of the protein stored in the protein database based on cDNA is aligned to the end of the methionine residue M side (N terminal side). On the other hand, the signal peptide of the same terminal part is omitted from the amino acid sequence of the actually measured protein. In general, the length of the signal peptide is unknown, but as far as it is currently known, most of the signal peptides have about 35 amino acid residues or less. Therefore, if the actually measured protein is recorded in the protein database based on the original cDNA, the amino acid sequence that matches the amino acid sequence of the terminal portion on the N-terminal side of the actually measured protein is the terminal sequence database 26. (See FIG. 3B).

  Therefore, when the database search unit 22 collates the input product ion information with the product ion information predicted to appear from the amino acid sequence in the terminal sequence database 26, in most cases, one or more including a protein that is a correct answer. Candidates are listed. In other words, in MS / MS ion search, when a terminal sequence database is designated as the database to be used and the enzyme used as the enzyme selection condition is designated and the search is executed, the N-terminus that does not contain (dropped out) the signal peptide. For peptides, a list of matching amino acid sequences in the terminal sequence database 26 is obtained.

  On the other hand, for amino acid sequences other than the terminal portion of the original protein, the amino acid sequence can be estimated using the normal identification protein database 23 instead of the terminal sequence database 26. This is the same as the conventional protein identification method described with reference to FIG. Thus, the terminal portion of the actually measured protein is identified using the terminal sequence database 26, and the portion other than the terminal portion is identified using the normal identification protein database 23. Thus, the amino acid sequence of the full length of the protein can be estimated and the protein can be identified (see FIG. 3 (c)). These database search results and a comprehensive database search result combining them are output to the display unit 4, and an analyst who has confirmed this finally determines the amino acid sequence of the protein to be analyzed, Is identified.

  Next, the effect of the characteristic amino acid determination method in the protein identification system of the present embodiment will be described using actual analysis processing results based on actual measurement results.

  FIG. 4 is a diagram showing an example of a mascot search condition setting screen (a) and a search result display screen (b) when an MS / MS ion search is performed using the terminal sequence database 26. In this example, “NTERM_human” (N-terminal sequence database uniquely constructed) and the existing Swiss-Prot are simultaneously specified as the database. In addition, trypsin used for digestion is designated as an enzyme selection condition (“Enzyme”). When MS / MS ion search was performed under these conditions, a plurality of N-terminal glycopeptides derived from AGP (Acid GlycoProtein) were identified (A1G1_HUMAN_19, A1G2_HUMAN_19).

  FIG. 5 shows a comparison of misidentification prediction values obtained as a result of MS / MS ion search for each of 10 types of AGP-derived glycopeptides using the N-terminal sequence database and Swiss-Prot.

When a normal enzyme (“Tripsin”) is designated as an enzyme selection condition without using the N-terminal sequence database, an overall low misprediction prediction value (that is, a value indicating a prediction that there are few misidentifications) is used. Although it was possible to identify the peptides, GP1 and GP1 ^* located at the N-terminus were not identified. On the other hand, when MS / MS ion search is performed by specifying “None” as the enzyme selection condition without using the N-terminal sequence database, the N-terminal glycopeptide GP1-1 that was not identified when the enzyme was specified was used. , GP1-1 ^* , GP1-2, and GP1-2 ^* are all identified. On the other hand, however, the misidentification prediction value tended to be about 1000 times larger (that is, misidentification increased) than when the enzyme type was designated as the enzyme selection condition.

In contrast to these conventional methods, MS / MS ion search is used for MS ² spectra of glycopeptides derived from AGP, using an N-terminal sequence database as described above and a normal protein database (Swiss-Prot). In the same manner as when “None” was designated as the enzyme selection condition, glycopeptides GP1-1, GP1-1 ^* , GP1-2, and GP1-2 ^* derived from the N-terminus were identified. Furthermore, the erroneous identification prediction value is improved by about 10 to 100 times compared to the case where “None” is designated as the enzyme selection condition. This is considered to be due to the following reasons.

  As described above, the terminal sequence database used in the protein identification system of the first embodiment includes amino acid residues for the amino acid sequences from the terminal end to the m-th based on the base sequence of RNA in one protein. Everything you cut off one by one is listed. Therefore, as shown in FIG. 2, the degree of increase in the number of amino acid sequences per protein is at most m times (in the example of FIG. 2, m = 50). On the other hand, when “None” is designated as the enzyme selection condition, (x + 1) X / 2 amino acid sequence variations appear in a protein having an amino acid sequence with x amino acid residues (x> 1). To do. Here, x is the number of amino acid residues in the entire amino acid sequence, and m ≦ x, and therefore m ≦ x <(x + 1) X / 2, which is more than when “None” is designated as the enzyme selection condition. When the N-terminal sequence database is used, the number of amino acid sequences in the database to be verified is always smaller.

  Usually, the substantial size of the database when “None” is designated as the enzyme selection condition is several hundred to several thousand times the size when a specific enzyme is designated as the enzyme selection condition. On the other hand, the substantial size of the database when the N-terminal sequence database is used can be several tens of times the size when the database is not used. As described above, since there is a large difference in the substantial size of the databases that need to be collated, as a result, the erroneous identification prediction value in the protein identification method used in the protein identification system of the first embodiment is the enzyme selection. Compared to the case where “None” is used as a condition, this is a significant improvement.

As described above, in the identification of a glycopeptide containing the N-terminal amino acid sequence, the amino acid determination method using the protein identification system of the first embodiment is sufficiently reliable as compared with the case where “None” is designated as the enzyme selection condition. It was confirmed that a highly reliable identification result was obtained.
In the terminal sequence database 26 illustrated in FIG. 3, the amino acid residue of the terminal portion on the C-terminal side opposite to the N-terminal for which the amino acid sequence is to be determined up to the end based on the base sequence of RNA. Although it is assumed that they are aligned, this need not be the case, and even if the length is shorter than the C-terminal side end of the target protein to be identified, there is no problem in identification.

[Second Embodiment]
Next, a protein identification system according to a second embodiment that implements the protein amino acid determination method according to the present invention will be described with reference to the accompanying drawings.

When the terminal sequence database used in the protein identification system of the first embodiment is used, the amino acid sequence of the terminal portion of the protein can be identified by MS / MS ion search, but the peptide mass often used with MS / MS ion search is used. Assignment cannot be performed by the fingerprinting (PMF) method. This is because MS ² analysis is not performed in the PMF method, and the peak list of the mass spectrum obtained for the enzyme digest of the protein is collated with the database to identify the protein, so the N-terminal amino acid sequence is not the terminal part. This is because it is not assigned as one protein unless it is included in one amino acid sequence together with other amino acid sequences. If “None” is designated as the enzyme selection condition, that is, if a specific enzyme is not designated, protein identification cannot be performed by the PMF method, and the result “Data not shown” is output.

  FIG. 9 is a diagram showing an example of a mascot search condition setting screen (a) and a search result (b) when an attempt is made to identify a protein by the PMF method using the N-terminal sequence database in the system of the first embodiment. It is.

  That is, in the case of the PMF method, it is necessary to use a dedicated terminal sequence database for the PMF method. The protein identification system of the second embodiment is a system for performing protein identification by the PMF method. FIG. 6 is an overall configuration diagram of the protein identification system of the second embodiment, FIG. 7 is a diagram showing an example of amino acid sequences recorded in the N-terminal sequence database for PMF in the protein identification system of the second embodiment, and FIG. It is explanatory drawing of the process sequence at the time of determining the amino acid sequence of protein in the protein identification system of 2 Example.

In FIG. 6, the same or corresponding components as those in the system of the first embodiment shown in FIG. In the system of the second embodiment, the mass spectrometer 1 is a general time-of-flight mass spectrometer or quadrupole mass spectrometer that does not execute ion dissociation by CID or the like. The mass spectrum data collection unit 27 collects mass spectrum data, and the peak information extraction unit 21 extracts MS ¹ ion information corresponding to the peak appearing in the mass spectrum. The database search unit 22 performs a database search based on this MS ¹ ion information (particularly, mass-to-charge ratio value), not product ion information.

  The known information reading unit 24 reads the amino acid sequence information of each protein from the reference protein database 5 based on, for example, cDNA, and the PMF terminal sequence database creation / management unit 28 includes the full length or C-terminal including a signal peptide for each protein. An amino acid sequence including a terminal amino acid sequence for PMF is generated based on an amino acid sequence of a predetermined length lacking a part of the amino acid sequence on the side, and this is collected to create a terminal sequence database 29 for PMF.

  The difference between the terminal sequence database 26 in the first embodiment and the terminal sequence database 29 for PMF in the second embodiment is apparent when FIG. 2 and FIG. 7 are compared. That is, the terminal sequence database 26 in the first embodiment is a database in which amino acid sequences of only the terminal portion of the protein having a maximum sequence length of m are collected. On the other hand, the PMF terminal sequence database 29 in the second embodiment has a maximum sequence length of the full length X of the original protein or a predetermined length Y lacking a part of the amino acid sequence on the C-terminal side. Is a database in which amino acid sequences including both a terminal portion and a portion other than the terminal are collected, the minimum of which is Xm or Ym (m = 30 in this example).

  Also in the example shown in FIG. 7, the number of amino acid sequences generated for one type of protein is m, and m amino acid sequences having different sequence lengths are similarly generated for each protein, and the end sequence for PMF is generated. Recorded in database 29.

  The database search unit 22 searches for the protein corresponding to the peak information of the given mass spectrum using such a PMF end sequence database 29. As shown in FIG. 8 (a), the N-terminal signal peptide is usually dropped in the actually measured protein, but it matches the terminal portion in the dropped state (however, the other terminal portion does not match). The amino acid sequence is recorded in the terminal sequence database 29 for PMF. Therefore, as shown in FIG. 8 (b), the N-terminal peptide from which the signal peptide has been dropped is also identified. In this example, the other C-terminal peptide is not identified, but most of the peptides other than the C-terminal peptide including the N-terminal peptide have been identified, so that the original protein can be identified with high accuracy.

  FIG. 10 is a diagram showing an example of a mascot search condition setting screen (a) and a search result display screen (b) when the PMF method is performed using the PMF terminal sequence database 28. In this example, “PMF_human” (an originally constructed N-terminal sequence database for PMF) is designated as the database, and trypsin used for digestion is designated as the enzyme selection condition (“Enzyme”). When PMF was executed under these conditions, two types of N-terminal variant AGP hit.

  In addition, since AGP is a mixture of “A1AG1_Human” and “A1G2_Human”, substantially four types of proteins are hit. When this search result is analyzed in detail, the assignment is determined by the amino acid sequence connected to the first amino acid residue at the end of each remaining signal peptide, and Swiss-Prot that does not consider signal peptide loss is used. It was found that the N-terminal of AGP that was not assigned was also assigned correctly. From these results, it is possible to confirm that the terminal peptide can be assigned even by the PMF method and the protein can be identified by using the above-described PMF terminal sequence database.

  In addition, since the terminal sequence databases 26 and 29 used in the above-mentioned examples are databases in which amino acid sequences only on the N-terminal side of proteins are collected or amino acid sequences in which only the N-terminal side is processed are collected, It only supports dropping out. However, as a matter of course, it is possible to create a terminal sequence database in which the same processing is performed on the C-terminal side, so that it is possible to identify a C-terminal peptide from which some amino acids have been dropped. You can also. In that case, the amino acid residue on the N-terminal side of the amino acid sequence recorded in the terminal sequence database may be aligned up to the end based on the base sequence of RNA, but it is not a problem even if a part is missing. It is clear from the explanation. Furthermore, the range of identifiable proteins can be further expanded by using both the terminal sequence database on the N-terminal side and the terminal sequence database on the C-terminal side and estimating the amino acid sequences of the N-terminal part and the C-terminal part, respectively. Can do.

  In the above description, the amino acid sequence having the N-terminal amino acid residue at the position of the methionine residue M is recorded in the reference protein database (for example, protein database based on cDNA) 5 for creating the terminal sequence database 26. However, the N-terminal amino acid residue is not necessarily located at the methionine residue M position. Specifically, the N-terminal amino acid residue of the recorded amino acid sequence is determined from the position of the methionine residue M, that is, the position of the amino acid residue corresponding to the start codon of the coding region on the mRNA encoding the known protein. , Any position between the position of the N-terminal amino acid residue of the protein existing in the living body based on the base sequence information of the coding region on the mRNA encoding the known protein may be used.

  Furthermore, the above-described embodiments and modifications are merely examples of the present invention, and it will be understood that they are included in the scope of the claims of the present application even if appropriate changes, modifications, additions, etc. are made within the scope of the present invention. It is.

DESCRIPTION OF SYMBOLS 1 ... Mass spectrometer 2 ... Data processing part 20 ... MS ⁿ spectrum data collection part 21 ... Peak information extraction part 22 ... Database search part 23 ... Identification protein database 24 ... Known information reading part 25 ... Terminal sequence database creation and management part 26 ... Terminal sequence database 27 ... Mass spectrum data collection unit 28 ... PMF terminal sequence database creation / management unit 29 ... PMF terminal sequence database 3 ... Input unit 4 ... Display unit 5 ... Reference protein database

Claims (10)

A protein that determines the amino acid sequence of the target protein or provides information for determining the amino acid sequence by performing a database search based on mass spectral information obtained by mass spectrometry of a peptide mixture derived from the target protein In the amino acid sequencing method of
a) A database in which amino acid sequences of known proteins are recorded, and the position S of the N-terminal amino acid residue of the amino acid sequence of each known protein is 1 ≦ S ≦ p (provided that on the mRNA encoding the known protein) The first amino acid residue corresponding to the start codon of the coding region of p, and the p-th amino acid residue of the N-terminal protein present in the living body based on the base sequence information of the coding region on the mRNA encoding the known protein On the basis of amino acid sequence information obtained from a database of known proteins, and an amino acid sequence consisting of amino acid residues of a predetermined length from the N-terminal for each known protein, and the predetermined length The predetermined length obtained by sequentially removing amino acid residues in the amino acid sequence from the N-terminal end in order. Seek terminal near the following amino acid sequences in length, and N-terminal sequence database creation step of creating an N-terminal sequence database by the amino acid sequence,
b) Database search for estimating the amino acid sequence of the terminal portion of the target protein by collating the mass spectrum information derived from the target protein with the amino acid sequence recorded in the N-terminal sequence database or information obtained from the sequence. Steps,
A method for determining the amino acid sequence of a protein, comprising: A protein that determines the amino acid sequence of the target protein or provides information for determining the amino acid sequence by performing a database search based on mass spectral information obtained by mass spectrometry of a peptide mixture derived from the target protein In the amino acid sequencing method of
a) A database in which amino acid sequences of known proteins are recorded, where the position T of the C-terminal amino acid residue of each amino acid sequence of each known protein is q ≦ T ≦ L (provided that on the mRNA encoding the known protein) 1st amino acid residue corresponding to the start codon of the coding region, and q-th amino acid residue C-terminal amino acid residue of the protein in the living body based on the base sequence information of the coding region on the mRNA encoding the known protein Obtained from a database of known proteins consisting of an amino acid sequence that is the Lth amino acid residue corresponding to the codon adjacent to the 5 'upstream of the stop codon of the coding region on the mRNA encoding the known protein) Based on the amino acid sequence information obtained, each known protein has a predetermined length from the C-terminus. An amino acid sequence consisting of amino acid residues is extracted, and amino acid residues in the amino acid sequence of a predetermined length are sequentially removed from the most terminal on the C-terminal side to obtain a near-terminal amino acid sequence having a length shorter than the predetermined length. C-terminal sequence database creation step of creating a C-terminal sequence database from the amino acid sequence;
b) Database search for estimating the amino acid sequence of the terminal portion of the target protein by collating the mass spectrum information derived from the target protein with the amino acid sequence recorded in the C-terminal sequence database or information obtained from the sequence Steps,
A method for determining the amino acid sequence of a protein, comprising: A method for determining an amino acid sequence of the protein according to claim 1 or 2,
The method for determining an amino acid sequence of a protein, wherein the known protein database is a protein database created based on genomic data. A method for determining an amino acid sequence of a protein according to any one of claims 1 to 3,
The mass spectrum information is product ion information based on an MS ⁿ analysis result where ⁿ is an integer of 2 or more,
The database search step uses MS / MS ion search to match the product ion information with the product ion information obtained from each amino acid sequence recorded in the terminal sequence database, thereby determining the amino acid at the terminal portion of the target protein. A method for determining an amino acid sequence of a protein, comprising estimating the sequence. A method for determining an amino acid sequence of a protein according to any one of claims 1 to 3,
The predetermined length is the total length of the protein, and the mass spectrum information is ion information based on a mass analysis result using only MS ¹ ions,
The database search step uses peptide mass fingerprinting to match the ion information with the ion information obtained from each amino acid sequence recorded in the terminal sequence database to obtain an amino acid sequence including the terminal portion of the target protein. A method for determining an amino acid sequence of a protein, comprising estimating. A protein that determines the amino acid sequence of the target protein or provides information for determining the amino acid sequence by performing a database search based on mass spectral information obtained by mass spectrometry of a peptide mixture derived from the target protein In the amino acid sequencing apparatus of
a) A database in which amino acid sequences of known proteins are recorded, and the position S of the N-terminal amino acid residue of the amino acid sequence of each known protein is 1 ≦ S ≦ p (provided that on the mRNA encoding the known protein) The first amino acid residue corresponding to the start codon of the coding region of p, and the p-th amino acid residue of the N-terminal protein present in the living body based on the base sequence information of the coding region on the mRNA encoding the known protein The amino acid residue in the amino acid sequence consisting of amino acid residues of a predetermined length from the N-terminus for each known protein, created based on the amino acid sequence information obtained from the database of known proteins consisting of amino acid sequences The amino acids in the vicinity of the end having a length equal to or shorter than the predetermined length in which the groups are sequentially removed from the N-terminal side from the most end. And N-terminal sequence database acid sequence has been recorded,
b) Database search for estimating the amino acid sequence of the terminal portion of the target protein by collating the mass spectrum information derived from the target protein with the amino acid sequence recorded in the N-terminal sequence database or information obtained from the sequence. And
A protein amino acid sequence determination apparatus comprising: A protein that determines the amino acid sequence of the target protein or provides information for determining the amino acid sequence by performing a database search based on mass spectral information obtained by mass spectrometry of a peptide mixture derived from the target protein In the amino acid sequencing apparatus of
a) A database in which amino acid sequences of known proteins are recorded, where the position T of the C-terminal amino acid residue of each amino acid sequence of each known protein is q ≦ T ≦ L (provided that on the mRNA encoding the known protein) 1st amino acid residue corresponding to the start codon of the coding region, and q-th amino acid residue C-terminal amino acid residue of the protein in the living body based on the base sequence information of the coding region on the mRNA encoding the known protein Obtained from a database of known proteins consisting of an amino acid sequence that is the Lth amino acid residue corresponding to the codon adjacent to the 5 'upstream of the stop codon of the coding region on the mRNA encoding the known protein) For each known protein created based on the amino acid sequence information obtained from the C-terminus A C-terminal sequence database in which amino acid residues in the amino acid sequence consisting of amino acid residues of a fixed length are removed in order from the most terminal end on the C-terminal side, and the amino acid sequences near the terminal of the predetermined length or less are recorded. When,
b) Database search for estimating the amino acid sequence of the terminal portion of the target protein by collating the mass spectrum information derived from the target protein with the amino acid sequence recorded in the C-terminal sequence database or information obtained from the sequence And
A protein amino acid sequence determination apparatus comprising: A protein amino acid sequence determination apparatus according to claim 6 or 7,
The amino acid sequence determination apparatus for proteins, wherein the known protein database is a protein database created based on genomic data. An amino acid sequence determination device for a protein according to any one of claims 6 to 8,
The mass spectrum information is product ion information based on an MS ⁿ analysis result where ⁿ is an integer of 2 or more,
The database search unit uses MS / MS ion search to match the product ion information with the product ion information obtained from each amino acid sequence recorded in the terminal sequence database, thereby determining the amino acid at the terminal portion of the target protein. A protein amino acid sequencing apparatus characterized by estimating a sequence. An amino acid sequence determination device for a protein according to any one of claims 6 to 8,
The predetermined length is the total length of the protein, and the mass spectrum information is ion information based on a mass analysis result using only MS ¹ ions,
The database search unit uses peptide mass fingerprinting to check an amino acid sequence including a terminal portion of a target protein by comparing the ion information with ion information obtained from each amino acid sequence recorded in the terminal sequence database. A protein amino acid sequencing apparatus characterized by estimating.

Images