KR100699437B1 - Apparatus and Method for Analysis of Amino Acid Sequence - Google Patents

Abstract

An apparatus and method are provided for analyzing amino acid sequences contained in proteins.

The present invention extracts a sequence tag list included in the peptide to be analyzed and determines a candidate peptide including the same, and then generates a list of peptides containing a space through binding between the sequence tags, and then applies the mass variation of the blank portion and the candidate peptide. By searching for the amino acid sequence or the post-translational modification of the corresponding blank portion using the amino acid sequence information of the included blank portion, it is possible to minimize the search procedure for the amino acid sequence and the post-translational modification included in the protein.

Protein, amino acid sequence, post-translational modification

Description

Apparatus and Method for Analysis of Amino Acid Sequence

1 is a block diagram of a protein sequence analysis system to which the amino acid sequence analysis device according to the present invention is applied,

2 is a detailed block diagram of the amino acid sequence analysis device shown in FIG.

3 is a flowchart for explaining an amino acid sequence analysis method according to an embodiment of the present invention;

4 is a detailed flowchart of a tag extraction process of the amino acid sequence analysis method according to the present invention;

5 is a detailed flowchart of a peptide search process in amino acid sequence analysis according to the present invention;

6 is a detailed flowchart of a tag placement process in amino acid sequence analysis according to the present invention;

7 is a detailed flow chart of the post-translational amino acid sequence determination process of the amino acid sequence analysis according to the present invention,

8 to 12 are views for explaining an example of the amino acid sequence analysis method of the protein by the amino acid sequence analysis device according to the present invention.

<Description of the symbols for the main parts of the drawings>

10 control unit 20 tandem mass spectrometer

30: amino acid sequence analysis 40: interface unit

50: database 310: peak selector

320: tag extraction unit 322: mass calculation means

324: amino acid matching means 330: peptide search unit

332: peptide comparison means 334: candidate peptide list determination means

340: sequence tag generation unit 342: tag filtering means

344: tag binding means 346: peptide list generation means

348: peptide list filtering means

350: amino acid sequence determination unit 352: blank sequence matching means

354: means for generating and comparing spectra

The present invention relates to a protein sequencing method, and more particularly, to an apparatus and a method for analyzing an amino acid sequence contained in a protein.

In order to elucidate the secrets of the human body, not only the study of the genome with all the genetic information, but also the study of the function of the protein, the expression type of the genetic information, is very important. Because protein is the function that regulates all the functionalization activities of the human body, if you can understand how the protein works, the disease can also identify the causal protein and start developing therapeutics.

A proteome is a species of all proteins that can be made from the genome. It is a dynamic concept that changes according to specific physiological and pathological conditions in a cell or tissue, and proteomics refers to methods and techniques for studying proteome. . In other words, by studying the properties of the protein expression, post-translational modification, binding to other proteins, etc., it refers to a research field to comprehensively understand the intracellular transformation process and network formation process in connection with the disease progression process.

In general, a method using a tandem mass spectrum has been widely used to identify proteins contained in a living body. The tandem mass spectrometry method involves collidation-induced dissociation of a peptide, a fragment produced as a result of digesting the protein using proteolytic enzymes such as trypsin, and then digesting the protein. By ionizing the resulting material and then measuring its mass, the sequence of the peptide is identified and finally used to find the sequence of the protein.

The sequence of peptides produced by digesting the protein with trypsin is characterized by ending with amino acids R or K. For example, if the sequence of the protein is "MEMEKEFEQIDK SGSWAAIYQDIR HEASDFPCRVAK LPKNKNRNRYRDVSPFDHSR K LHQEDNDYINASLIKMEEAQR SYILTQ", then digesting it with trypsin For example, peptide sequences including "SGSWAAIYQDIR", "LPKNKNRNRYRDVSPFDHSR", "LHQEDNDYINASLIKMEEAQR", and the like can be obtained. In addition, collision-induced cleavage of a peptide having the sequence "SGSWAAIYQDIR" breaks the chemical bond constituting the peptide, and particularly, in the case of collision-induced cleavage of the peptide by applying small energy, mainly an amino bond on the peptide is destroyed. Substances beginning with the leading amino acid (prefix, eg S) of the peptide sequence (eg, "S", "SG", "SGSWA", "SGSWAAIYQDI") or trailing amino acids (suffix) of the peptide sequence For example, substances starting with R) (eg, "R", "IR", "DIR", "QDIR", "GSWAAIYQDIR") are produced.

These substances exist in an ionized form, and substances that begin with the leading amino acid are called b ions and substances that begin with the trailing amino acid are called y ions. Various ions such as bH ₂ O, b-NH ₃ , yH ₂ O, and y-NH ₃ , in which a, c, x, z ions, water or ammonia ions are lost, in addition to the b and y ions generated by breaking the amino bond Will be generated. The tandem mass spectrum is a measure of mass by detecting fragments of ionized peptides, and the intensity of how many ions with the same mass are detected. This analysis of tandem mass spectra allows us to infer the mass of the original peptide.

In the tandem mass spectrum, the intensity of y and b ions is relatively higher than that of other ions, and thus, a method of extracting peaks, which are the largest points in the spectrum, and analyzing the differences are widely used. For example, if the intensities of two peaks, measured from the tandem mass spectrum, represent y ions with sequence "DIR" and y ions with sequence "QDIR", respectively, the difference is the mass of amino acid "Q". It can be seen that the peptide sequence of contains Q.

However, in predicting peptide sequences using tandem mass spectrometry, post-translational modifications in which the amino acids are chemically modified to change the mass, mutations such as substitution of amino acids by other amino acids, There are many difficulties in predicting the sequence due to various modification factors such as errors and miscleavage due to poor digestion when the protein is digested with trypsin enzyme.

Especially in the case of post-translational modifications of proteins, the problem of identifying post-translational modifications from proteins itself is important because information about what kind of modifications occur at which positions in the protein's sequence plays a key role in elucidating the biological function of the protein. It is also recognized as a very important task in the field of proteomics. Therefore, even if a large number of various modification factors such as post-translational modification of the protein occurs, it is necessary to identify the sequence of peptides and proteins from the tandem mass spectrum and to find the type and location of the post-translational modification.

There are a number of algorithms for identifying proteins or peptide sequences from tandem mass spectra, including three methods: database search, de novo sequencing, and sequence tagging.

First, the database search technique generates a combination of all possible peptides, including post-translational modifications, from the database where the protein sequences are stored, generates virtual tandem mass spectra for each combination, and then runs the experiment. Compared with the tandem mass spectrum obtained through

Next, the de novo sequencing technique is a method of predicting the total sequence of a peptide including post-translational modifications from tandem mass spectra without using a protein database.

Finally, the sequence tag technique uses a de novo sequencing technique to search for a sequence tag, which is a short length peptide sequence from a tandem mass spectrum, find a peptide containing the sequence tag retrieved from a protein sequence database, and then find the virtual tandem of the found peptide. This method compares the mass spectrum and the tandem mass spectrum obtained through experiments.

However, since the above methods focus on identifying the sequence of the protein, there is a problem in that the sequence of the protein cannot be correctly predicted when there are several modifying elements. In particular, when predicting the sequence of a peptide having a plurality of post-translational modifications, there is a disadvantage that the execution time for this increases exponentially depending on the number and type of post-translational modifications. Therefore, when a plurality of post-translational modifications are present in the peptide, current protein sequencing methods cannot accurately predict the protein sequence, and a lot of time and effort are consumed.

The present invention has been made to solve the above-described problems, the type and location of a plurality of post-translational modifications included in the amino acids or amino acids constituting the protein by using the mass variation of the blank portion of the sequence tag in the sequence analysis of the protein It is an object of the present invention to provide an apparatus and method for quickly and easily searching for an amino acid sequence.

The present invention for achieving the above object is an apparatus for analyzing an amino acid sequence contained in a protein, comprising: a database comprising peptide sequence data and post-translational modification data; When a sequence tag list is generated that does not include post-translational modifications from a plurality of peaks selected through mass spectrometry of the analysis target protein, a candidate peptide including the sequence tag extracted by referring to the peptide sequence data is received. A sequence tag generation unit for generating a sequence tag having a space by combining and arranging all sequence tags and a bindable sequence tag included in the candidate peptide; And referring to the peptide sequence data or the post-translational modification database, replacing the space present in the sequence tag generated by the sequence tag generation unit with an amino acid sequence or an amino acid sequence including post-translational modification to generate the peptide filled with the space. To include; amino acid sequence determination unit.

The present invention finds the sequence tag by mass spectrometry of the protein of interest and uses it to search the peptide sequence database. At this time, the number of proteins to be searched is limited to several dozens, and then the types and positions of post-translational modifications included in the proteins are searched. In addition, the length or number is not limited when searching for a sequence tag, and a suffix array can be used to find a peptide including a sequence tag from a protein database. And even if the protein contains several kinds of post-translational modifications or several post-translational modifications, there is an advantage that the execution time does not increase significantly.

Hereinafter, exemplary embodiments of the present invention will be described in detail with reference to the accompanying drawings.

1 is a block diagram of a protein sequence analysis system to which the amino acid sequence analysis device according to the present invention is applied.

As shown in FIG. 1, the protein sequencing system includes a control unit 10 and a result of a tandem mass spectrometer 20 and a tandem mass spectrometer 20 that generate mass spectra by analyzing masses of proteins in two stages. Once a candidate peptide is determined that includes a sequence tag extracted from the peaks included in the resulting mass spectrum, a peptide list is generated from the sequence tag and the amino acid sequence and / or post-translational modifications included in the undetermined sequence tag portion of the peptide list. Amino acid sequence analysis device 30 to determine the interface, the interface unit 40 and a database 50 for receiving a control command from the user and outputs the processing result. Here, the mass spectrometry of the protein is not limited to the analysis using the tandem mass spectrometer 20, of course, any one of all possible mass spectrometers can be used.

Here, the database 50 includes a peptide sequence database, a post-translational modification database, and the like, and the peptide sequence database is a collection of peptide data that does not include post-translational modifications in the form of a suffix array generated by a computational method from a protein sequence. The post-translational modification database includes the name of the post-translational modification, the type of amino acid that occurs, the mass change of the amino acid, if any, and the conditions that occur. In particular, in the peptide sequence database, a group of peptides having the same mass is stored in a plurality of groups according to mass, each peptide is stored in a suffix form, and information on mass variation before and after the sequence tag included in each peptide is also stored.

The tandem mass spectrometry device 20 cuts any protein with a proteolytic enzyme (eg, trypsin) to produce a plurality of peptide fragments, as described in the art, and then ionizes each peptide. It is a device that fragments through collision with collision gas, and then separates by mass to charge ratio, and generates a mass spectrum for each peptide by using the same. Here, the abscissa of the mass spectrum represents the ratio of mass to the amount of charge, and the ordinate represents the ratio (intensity) of the amount of abundance with respect to the abscissa.

Amino acid sequence analysis device 30 is a device for searching for the amino acid sequence and / or post-translational modifications included in the protein of analysis from the tandem mass spectrometry obtained by the tandem mass spectrometer 20, the specific configuration is as shown in FIG.

FIG. 2 is a detailed configuration diagram of the amino acid sequence analysis apparatus illustrated in FIG. 1. For convenience of description, FIG. 8 is a view illustrating an example of an amino acid sequence analysis method included in a protein by the amino acid sequence analysis apparatus according to the present invention. This will be described with reference to FIG. 12.

As shown, the amino acid sequence analysis device 30 of the present invention is a peak selector for extracting a plurality of relatively high peaks from the raw data of the tandem mass spectrum obtained as a result of the tandem mass spectrometer 20 ( 310), the tag extractor 320 searches for a short length amino acid sequence (ie, a sequence tag) that does not include post-translational modifications included in the spectrum by using the mass difference of the peak extracted by the peak selector 310, The peptide search unit 330 for extracting the candidate peptide including the sequence tag searched by the tag extractor 320 with reference to the peptide sequence data stored in the database 50, and the candidate peptide extracted from the peptide search unit 330 Consecutive sequences of spaces, including all sequence tags included therein and also where multiple sequence tags can be placed simultaneously Search for the sequence tag generator 340 and the post-translation database to generate the sequence tag and replace the blanks in the sequence tag generated by the sequence tag generator 340 with the amino acid sequence or the amino acid sequence including the post-translational modification. And an amino acid sequence determination unit 350 for generating a virtual spectrum of the candidate peptide sequence including the amino acid sequence corresponding to the blank or the post-translational modification and comparing the original spectrum with the original spectrum.

Here, the peak selector 310, the tag extractor 320, and the peptide searcher 330 may have a function of selecting a peak from a protein to be analyzed, a function of extracting a sequence tag, and a function of extracting a candidate peptide. It can be configured using any of the possible devices.

Referring to the amino acid sequence analysis device 30 of the present invention shown in Figure 2 in more detail as follows.

First, the peak selector 310 extracts a plurality of high intensity peaks (for example, 20 to 200) from each mass spectrum data generated by the tandem mass spectrometer 20. If the intensity of peaks near a specific mass is small and important peaks such as y ions or b ions cannot be extracted, change the mass range for peak extraction to add a relatively high peak in that mass range. Extract. In other words, not only the peak having a large intensity as a whole, but also the peak having a large intensity locally.

The tag extracting unit 320 includes a mass calculating means 322 and an amino acid matching means 324, and uses a de novo sequencing method, for example, to search for an amino acid sequence included in a spectrum using a mass difference of peaks. Can be.

First, the mass calculation means 322 calculates the difference in mass represented by each pair of peaks for all pairs of peaks extracted by the peak selector 310, and the amino acid matching means 324 calculates the mass calculation means 322. By comparing the difference in the mass of each peak pair and the mass of each amino acid calculated in the above, it is determined that the peak pair may represent a specific amino acid when the mass difference value and the mass difference of the specific amino acid are within a specified error range. That is, a result is obtained that amino acids having a mass corresponding to the difference in mass of the peak pair are present in the peptide to be analyzed.

In addition, when two pairs of peaks representing one amino acid share one peak, the amino acid matching means 324 determines that three peaks combining two pairs of peaks may represent two amino acids. In a similar manner, all cases where n peaks represent n-1 amino acids are calculated to generate a sequence tag list representing the amino acid n-1 sequence tags and the n peaks constituting the tag.

Referring to Fig. 8, it can be seen that the mass difference between peak pairs consisting of peak numbers 15 and 17 represents amino acid G. Similarly, the mass difference of the peak pair consisting of peak numbers 17 and 20 represents amino acid M, the mass difference of the peak pair consisting of peak numbers 20 and 24 represents amino acid I, and the mass difference of the peak pair consisting of peak numbers 24 and 29 It can be seen that represents the amino acid D. In this case, the sequence tag list is generated as G (15, 17), M (17, 20), I (20, 24), D (24, 39).

In addition, since amino acids G and M share the same peak 17 in FIG. 8, it can be determined that three peaks (15, 17, 20) combining two pairs of peaks representing G and M represent two amino acids. This generates sequence tags such as GM (15, 17, 20). In the same way, five peaks (15, 17, 20, 24, 29) can represent four amino acids, resulting in a sequence tag such as GMIN (15, 17, 20, 24, 29).

Meanwhile, the peptide search unit 330 searches the peptide list including the sequence tag list generated by the tag extractor 320 with reference to the peptide sequence database, and includes a peptide comparison means 332 and a candidate peptide list determination means ( 334). In this case, since it is not possible to determine whether the sequence tag determined by the tag extractor 320 is the b ion direction or the y ion direction, a database search for both the forward and inverse sequences of one sequence tag is performed. Since peptides are stored in the database in the form of suffix arrays, the comparison of the length of the sequence tag only needs to be performed when searching for the sequence tag. Therefore, there is no problem of slowing down the rate of generating and searching more sequence tags than the existing algorithms. .

Subsequently, the candidate peptide list determining means 334 extracts at least one or more peptides including a sequence tag having a specified first length (eg, 3) or more from the comparison result of the peptide comparing means 332 to list the first candidate peptide. Create The second candidate peptide list is generated by extracting a peptide including a sequence tag of a second length (for example, 2), which is again designated for each of the peptides included in the first candidate peptide list. In addition, a list of all sequence tags included in each of the second candidate peptides and information on the direction in which the sequence tags and the second candidate peptides coincide (b ion direction in the forward direction and y ion direction in the reverse direction) are stored.

For example, a list of the sequence tag extracted by the tag extractor 320 is shown in [Table 1], and a part of the peptide sequence list stored in the peptide sequence database is shown in [Table 2]. As follows.

TABLE 1

V [L | I] GAN, AITGADKT,...

WWG, AT [L | I], VSV, TIA, ITG,...

LDA, GMI, NVL,…

VI, VO, GM, MI, ID,...

G, M, I, K,...

TABLE 2

VTAEDKGTGNK

RALSSQHQAR

VYADQRPLTK

ETMEKAVEEK

EFFNGKEPSR

QATKDAGTIAGLNVLR

VEIINQDAGNR

FLPFKVVEKK

：

The peptide comparison means 332 searches a database including a sequence tag list generated as shown in Table 1 and a peptide sequence as shown in Table 2 to search all peptide sequences including the sequence tag list. At this time, since the exact direction of each sequence tag is not known, a search for both forward and reverse directions is performed for each sequence tag. For example, a search for b ion direction (forward direction) is performed for AITGADKT in the sequence tag list, while a search for a tag sequence in the y ion direction (reverse direction) of AITGADKT, that is, tkdagtia is also performed. Herein, a tag sequence represented by an uppercase letter means a forward ion, and a tag sequence represented by a lowercase letter means a reverse ion.

Subsequently, the candidate peptide list determining means 334 extracts a peptide including, for example, three or more sequence tags from the peptide list including the sequence tag, and generates a candidate peptide list including two of the sequence tags. do. In the example shown in FIG. 9, QATKDAGTIAGLNVLR including the reverse ion of AITGADKT and NVL among the peptide sequences stored in the database is determined as a candidate peptide list. Although one candidate peptide list is illustrated in this example, a plurality of candidate peptide lists may be selected.

As such, after the candidate peptide list is determined, the sequence tag generation unit 340 arranges all sequence tags included in the candidate peptide and includes a sequence sequence in which spaces are present, including when multiple sequence tags can be placed at the same time. Create To this end, the sequence tag generation unit 340 includes a tag filtering unit 342, a tag combining unit 344, a peptide list generating unit 346, and a peptide list filtering unit 348.

First, the tag filtering means 342 may perform mother mass filtering and filtering by comparing the front and rear masses of sequence tags, and among them, the parent mass filtering may be omitted. Here, the parent mass refers to the molecular weight of the peptide, and if the peptide contains a modification, it is a value including the molecular weight of the modification. The parent mass filtering compares the mass of each candidate peptide with the mass of the peptide to be analyzed. For example, when the parent mass of the candidate peptide is smaller than the mass of the peptide to be analyzed by a first predetermined value (eg, 100 da) or more, the candidate peptide is excluded from the candidate. The reason why the mass filtering is performed is that when the post-translational modification occurs in the peptide, the mass of the peptide rarely decreases by more than the first set value (for example, 100 da).

Next, in filtering by comparing the front and back masses of the sequence tag, the mass for the position of the first peak or the last peak constituting the sequence tag is set to the second set value (for example, the mass for the position of the peak theoretically calculated from the candidate peptide. For example, when less than 100 da), the corresponding sequence tag is deleted from the sequence tag list. That is, by comparing the difference between the mass of the first or last peak of the sequence tag and the mass of the corresponding sequence tag in the candidate peptide, the corresponding sequence tag is deleted when the mass difference is smaller than the second set value.

When this filtering process is completed, the tag combining means 344 checks the consistency between the sequence tags that match the candidate peptide and combines them if there are two or more sequence tags that can be combined. Whether or not sequence tags can be combined depends on the relative position of the sequence tag, when one of two sequence tags is included in the other, when two sequence tags overlap, when two sequence tags are adjacent, It can be identified by dividing it into the case.

In the case where one sequence tag of the two sequence tags is included in the other, when the two sequence tags overlap and in the case of neighboring, it is determined by combining the difference in the mass variation of the two sequence tags. That is, when the difference in mass variation is close to the first reference value (for example, 0), the direction of the sequence tag is different, and the two sequence tags may be combined. This is because a pair of sequence tags having the same direction and having a difference in the amount of variation may be close to zero. In addition, in the above three cases, when the difference in mass variation is the second reference value (for example, 17) or the third reference value (for example, 18), any one of the ions forming the sequence tag is in the mass spectrometry process. It is possible to combine the two sequence tags because it can be seen that H ₂ 0 or NH ₃ is lost during collision-induced cleavage. In addition, when the difference in mass variation is the fourth reference value (for example, 28), the ions represented by the two sequence tags are ions (a ions) generated by cleaving carbon bonds, not ions produced by amino bond cleavage. As you can see, you can combine two sequence tags. As such, predefine a list of mass variation values when two sequence tags can be combined and combine the two sequence tags if the difference in mass variation of the sequence tag pairs approximates a difference in the list. Do not combine.

On the other hand, if two sequence tags are spaced apart, after calculating the mass variation for the space between the two sequence tags generated when the two sequence tags are combined, the mass variation is within the allowable third setting value (for example, 100 da). If present, combine the two sequence tags.

After performing the sequence tag assembling process as described above, the peptide list generating means 346 generates a peptide list including spaces in each sequence tag and sequence tag combination.

10A and 10B, FIG. 10A illustrates a sequence tag list, and FIG. 10B illustrates a sequence list including a space obtained by combining a combinable sequence tag.

First, FIG. 10A shows that when the candidate peptide is for example QATKDAGTIAGLNVLR and the sequence tag list is tkdagtia, KDA, TKD, nvl, the sequence tag list is arranged to match the candidate peptide and displays the mass variation for the resulting blanks. I can see that. That is, for the sequence tag tkdagtia, the mass shift for the blank [①, ②] is [-17.12, 0.07], for KDA the mass shift for the blank [③, ④] is [-17.00, -0.03], for TKD The mass shift for the blank [⑤, ⑥] is [26.03, -43.07], and nvl indicates that the mass shift for the blank [⑦, ⑧] is [-17.05, 0.03]. Here, the mass shift of the blank is a shift that is the difference between the theoretical position of the blank partial peak and the actual spectral peak position. That is, since the post-translational modifications have their respective masses as well as amino acids, it is possible to check what post-translational modifications have occurred in the amino acids included in the corresponding blanks from the mass variation of the blanks. 0 indicates that no mutation occurred in the space.

Next, FIG. 10B shows that a list of peptides is generated by combining a sequence capable of binding among sequence tags (tkdagtia, KDA, TKD, and nvl). First, a sequence tag containing tkdagtia and nvl is a combination of tkdagtia, KDA, and nvl; a sequence tag containing KDA and nvl is a combination of KDA and nvl; and a sequence tag containing TKD and nvl is TKD. And nvl combined with spaces [⑨, ⑩, ⑪] are [-17.12, 0.07, 0.03], spaces [⑫, ⑬, ⑭] are [-17.00, -0.05, 0.03], spaces [⑮,?, ?] Has a mass variation of [26.03, -43.09, 0.03].

As described above, after generating a peptide list including a space consisting of a sequence of sequence tags matched with the candidate peptide, and after mass variation for each space is calculated, the peptide list filtering means 348 is included in the peptide list. The peptide with less correlation among the plurality of peptides is deleted. For example, as shown in FIG. 11, a normalization is performed by dividing the mass spectrum of the peptide to be analyzed into a plurality of sections, setting the intensity of the largest peak in each section to 100, and adjusting the size of the remaining peaks to be proportional thereto. Then, the score of each free peptide matched to the candidate peptide is calculated to select the highest score among all free peptides obtained from that spectrum for one spectrum, and then the score of the selected peptide. All empty peptides with scores less than or equal to the specified value (eg 1/2 of the score) are deleted from the empty peptide list. Here, the score of the peptide with a blank can be calculated using [Equation 1].

[Equation 1]

Subsequently, the amino acid sequence determiner 350 determines an amino acid sequence to be included in the corresponding blank or an amino acid sequence including post-translational modifications. For this purpose, the amino acid sequence determination unit 350 generates a blank sequence matching means 352 and generates a spectrum and Comparing means 354 is included.

The empty sequence matching means 354 searches the post-translational modification database for the remaining peptides through the above-described process. In order to find the amino acid sequence or post-translational modification of the blank portion, the blank sequence matching means 354 performs a post-translational modification database on the sequence of candidate peptides corresponding to the blank portion and the mass corresponding to the blank portion in the blank peptide list. Query and receive the result of the type and location of the post-translational transformation if a match is found. If the mass variation of the empty portion is close to 0, it is determined that the post-translational modification does not occur in the empty portion, and thus the candidate peptide extracted previously is determined to be the resulting amino acid sequence, and the mass variation of the empty portion is 0. If it is not an approximate value, it is determined that there is a post-translational modification in the amino acid sequence included in the blank portion, and the post-translational modification database is searched.

Referring to FIG. 12, a database is searched using a sequence (QA) and a mass variation (-17.12) corresponding to the first empty portion of the peptide __tkdagtia__nlv_, in which case the mass variation is not close to zero, and thus after translation Search the variant database. In addition, since the mass variation 0.07 for the sequence GL and 0.03 mass variation for the sequence R are close to zero, it is determined that no post-translational modification has occurred. The post-translational modification database contains the mass that changes when a modification occurs, the amino acid that changes, and the type of post-translational modification, so you can see what post-translational modifications are included from the input sequence (change amino acid) and the mass variation. have. In the case of Figure 12 the peptide to be analyzed can be obtained the result of Q AtkdagtiaGLNnvlR, post-translational modification occurred in amino acid Q, it can be seen that the type of post-translational modification is Pyroglutamic acid.

Accordingly, the blank peptide is filled with candidate peptide sequences that include the type and location of the post-translational modification.

Thereafter, the spectrum generating and comparing means 356 generates a mass spectrometry spectrum for each candidate peptide (blank filled candidate peptide) obtained by the blank sequence matching means 354, compares it with the spectrum to be analyzed and outputs the result. do. This is preferably done for all candidate peptides and it is desirable to provide a score for each candidate peptide.

3 is a flowchart illustrating an amino acid sequence analysis method according to an embodiment of the present invention, which will be described below with reference to FIGS. 4 to 7, which are detailed processing flowcharts of each process.

In order to analyze the amino acid sequence included in the protein of interest, a two-step mass spectrometry process is first performed on the protein of interest and mass spectra thereof are generated (S10). Thereafter, the peak selector 310 of the amino acid sequence analysis device 30 extracts a plurality of peaks having relatively high intensity from the raw data of the tandem mass spectrum (S20). In this case, not only the peak having a large intensity as a whole, but also the peak having a large intensity locally, for example, it is preferable to select 20 to 200 peaks.

Thereafter, by using the mass difference of the peak extracted in the peak selection step, the tag extractor 320 extracts an amino acid sequence of short length (ie, sequence tag) that does not include post-translational modifications included in the spectrum of the peptide to be analyzed. do. The tag extraction process S30 will be described in more detail with reference to FIG. 4 as follows. First, the mass difference of all extracted peak pairs is calculated (S310), and the amino acid corresponding to the mass difference value is determined (S320). Thereafter, by comparing the mass difference between the plurality of peaks and the mass difference between the plurality of amino acids (S330), a short sequence tag list including no post-translational modification is generated (S340). The sequence tag list includes at least one sequence tag.

After the sequence tag list is generated as described above, the peptide search unit 330 searches the peptide sequence database to extract candidate peptides including the sequence tag list (S40). In this case, as shown in FIG. 5, the peptide stored in the peptide sequence database is compared with each sequence tag to extract a first candidate peptide having a sequence tag equal to or greater than a first length (m, for example, 3) specified ( S410), a second candidate peptide having a sequence tag of the first candidate peptide and a sequence tag having a specified second length (n, for example, 2) is extracted (S420). Thereafter, a candidate peptide list is generated using the second candidate peptide, in which case the sequence direction and the coincidence direction of the peptide are also stored (S430).

Here, the steps S10 to S40 are not limited to the above-described method, and any one of all possible methods for selecting a peak from an analysis target protein, extracting a sequence tag, and determining a candidate peptide may be applied.

Next, all sequence tags included in the candidate peptide extracted in the peptide search step (S40) are arranged, and when a plurality of sequence tags can be combined, a sequence sequence combining a plurality of sequence tags is arranged to form a continuous sequence in which spaces exist. A tag is generated (S50). More specifically, referring to FIG. 6, before the sequence tag is generated, the mother mass filtering (S510) and the filtering by comparing the front and rear masses of the tag (S520) are first performed, and the mother mass filtering may be omitted. It is possible. In the parental mass filtering step (S510), when the parental mass of each of the candidate peptides is compared with the mass of the peptide to be analyzed, when the parental mass of the candidate peptide is smaller than the mass of the peptide to be analyzed by a first predetermined value (eg, 100 da) or more. The candidate peptide is excluded from the candidate.

Next, in the filtering step (S520) by comparing the front and rear masses of the sequence tag, the mass of the position of the first peak or the last peak of the sequence tag is set to be higher than the mass of the position of the peak theoretically calculated from the candidate peptide. If the value is small (for example, 100 da) or less, the corresponding sequence tag is deleted from the sequence tag list.

Thereafter, consistency between the sequence tags matched to the candidate peptide is checked, and if there are two or more sequence tags that can be combined, they are combined (S530 and S540). When one sequence tag of the two sequence tags is included in the other, when the two sequence tags are overlapped and neighboring, and spaced apart, the combineable sequence tags are combined in consideration of the mass relationship described in FIG. . When the combining of the sequence tag is completed, each peptide generates a list of peptides including a space in the combination of the sequence tag and the sequence tag (S550).

Subsequently, filtering is performed on each of the peptides forming the peptide list including the space (S560). In the peptide list filtering step (S560), a normalization process for each peptide is performed, and a score of each spaced peptide matched to the candidate peptide is calculated, and among all spaced peptides obtained from the spectrum for one spectrum. After selecting the highest score, delete all blank peptides with a score less than the score of the selected peptide (eg, 1/2 of the score) from the blank peptide list. The peptide list filtering step may be omitted.

After the peptide list including the blank is generated in the tag arrangement process (S50) described above, the amino acid sequence determiner 350 uses the sequence and the mass variation of the blank portion to generate amino acids included in the peptide from the post-translational modification database. The position and type of occurrence of the sequence or post-translational modification are searched and the candidate peptide filled with a blank is determined through this (S60). Specifically, referring to FIG. 7, a post-translational modification database is searched for a peptide having a space to identify an amino acid sequence corresponding to a condition or a location, a kind, and the like of a post-translational modification (S610). Subsequently, the candidate peptide list is filled by filling the blank with the amino acid sequence or the post-translational modification identified in step S610 (S620), and generates a mass spectrometry spectrum for each candidate peptide list, and then the spectrum of the peptide to be analyzed. Compared to (S630) and outputs the result (S640).

By comparing the mass spectrometry spectra for the candidate peptides with the spectra for the peptides to be analyzed, the candidate peptides with the best scores can be selected, and the information about the selected peptides (sequence, location and type of post-translational modifications) can be known. Therefore, it can be utilized for protein sequencing.

As such, those skilled in the art will appreciate that the present invention can be implemented in other specific forms without changing the technical spirit or essential features thereof. Therefore, the above-described embodiments are to be understood as illustrative in all respects and not as restrictive. The scope of the present invention is shown by the following claims rather than the detailed description, and all changes or modifications derived from the meaning and scope of the claims and their equivalents should be construed as being included in the scope of the present invention. do.

As described above, according to the present invention, in searching for the amino acid sequence and post-translational modification, which are an important part of protein sequence analysis, the sequence tag included in the peptide to be analyzed is extracted and the candidate peptide including the same is determined, and then the sequence By combining the tags to generate a list of peptides containing spaces, and then searching for the amino acid sequence or post-translational modification of the spaces using the mass variation of the spaces and the amino acid sequence information of the spaces included in the candidate peptide, The search procedure and time for the amino acid sequence and post-translational modifications included in the protein can be minimized.                     

In addition, even when there are a plurality of post-translational modifications in a protein, the location and type of post-translational modifications can be easily determined, thereby further accelerating the development of proteomics technology.

Claims (26)

An apparatus for analyzing an amino acid sequence contained in a protein, Peptide sequence database and post-translational modification database; A peak selector configured to receive a mass spectrometry spectrum from a tandem mass spectrometer that analyzes the mass of the peptide constituting the protein to be analyzed, and extract a plurality of peaks from the mass spectrometry spectra; When a sequence tag list including post-translational modifications is generated from the plurality of peaks extracted by the peak selector, a candidate peptide including the sequence tag extracted by referring to the peptide sequence data is input and included in the candidate peptide. A sequence tag generation unit generating a sequence tag having a space by combining all the sequence tags and the sequence sequence to be combined; And Amino acid sequence determination unit for generating the peptide filled with the blank by replacing the blank present in the sequence tag generated by the sequence tag generation unit with an amino acid sequence or an amino acid sequence including a post-translational modification by referring to the post-translational modification database ; Amino acid sequence analysis device comprising a. delete The method of claim 1, The peak selector amino acid sequence analysis device characterized in that for selecting 20 to 200 peaks. The method of claim 1, The amino acid sequence analysis device further comprises a tag extraction unit for searching for a sequence tag that does not include post-translational modifications from the mass difference of the selected peak, and generates a sequence tag list including the sequence tag. Analysis device. The method of claim 4, wherein The tag extracting unit includes mass calculating means for calculating a difference in mass represented by each pair of peaks with respect to all pairs of peaks extracted by the peak selecting unit; And Comparing the mass difference of each peak pair and the mass of each amino acid calculated by the mass calculation means, searching for an amino acid having a mass equal to the mass difference of the corresponding peak pair to generate a sequence tag list including a plurality of amino acids Amino acid matching means for; Amino acid sequence analysis device comprising a. The method of claim 1, The amino acid sequence analysis device further comprises a peptide search unit for extracting the candidate peptide containing the sequence tag, with reference to the peptide sequence data. The method of claim 6, The peptide search unit may include: peptide comparison means for searching whether the sequence tag is included in the peptide data; And Extracting at least one or more peptides including sequence tags of a first length or more specified from a comparison result of the peptide comparison means, and including a sequence tag of a second length designated for each of the peptides included in the first candidate peptide list Candidate peptide list determination means for extracting a candidate peptide and generating a candidate peptide list using the second candidate peptide; Amino acid sequence analysis device comprising a. The method of claim 7, wherein The peptide comparison means is an amino acid sequence analysis device, characterized in that for performing a forward and reverse comparison to the sequence tag. The method of claim 7, wherein And the candidate peptide list determining means stores a concordance direction between the candidate peptide list and the sequence tag list. The method of claim 1, The sequence tag generation unit compares the mass for the position of the first peak or the last peak included in each sequence tag with the mass for the position of the candidate peptide, and if the mass comparison result is smaller than a specified value, the sequence tag is recalled. Tag filtering means for deleting from the sequence tag list; Tag binding means for confirming binding between sequence tags matched with the candidate peptide, and for binding a sequence tag capable of binding; And Peptide list generating means for generating a peptide list including a space from each sequence tag and the associated sequence tag; Amino acid sequence analysis device comprising a. The method of claim 10, The tag filtering means compares the masses of the candidate peptide and the peptide extracted from the protein to be analyzed before performing the filtering on the front and rear masses of the sequence tag, and selects candidate peptides having a result of the mass comparison that is smaller than or equal to a first predetermined value. Amino acid sequencing device, characterized in that to perform further more mass filtering to remove from the list. The method of claim 10, The tag binding means may be configured to combine the sequence tag when one sequence tag of two sequence tags is included in the other, or when two sequence tags overlap or neighbor, and a difference in mass variation between the two sequence tags is close to a reference value. Amino acid sequence analysis device, characterized in that. The method of claim 10, The tag combining means combines the two sequence tags when the two sequence tags are spaced apart, when the mass variation of the space between the two sequence tags occurring after the two sequence tags is included within a predetermined setting. An amino acid sequence analysis apparatus. The method of claim 10, The sequence tag generation unit normalizes the mass spectrometry that is a result of the mass spectrometry of the analysis target protein, and selects the peptide having the highest peptide score among the peptides having the gap, compared to all peptides having the gap, and selects the selected peptide. And a peptide list filtering means for deleting from the list all empty peptides having a score less than or equal to a score of. The method of claim 14, And the score of the blank peptide is extracted from the number of matched peaks with the spectrum to be analyzed, the sum of the normalized intensities of the matched peaks, and the length of the matched peptides. The method of claim 1, The amino acid sequence determination unit refers to the sequence of the candidate peptide corresponding to the blank portion of the peptide having the blank and the amino acid sequence or the post-translational amino acid from the post-translational modification database by referring to the variation of the mass corresponding to the blank portion. Blank sequence matching means for retrieving the sequence; And After filling the peptide having the blank by the amino acid sequence retrieved from the blank sequence matching means or the amino acid sequence including the post-translational modification, mass spectrometry is generated from the blank-filled peptide and compared with the spectrum to be analyzed. Spectral generation and comparison means; Amino acid sequence analysis device comprising a. The method of claim 1, Peptide having the same mass is grouped in the peptide database, each peptide sequence is stored in the form of a suffix, amino acid sequence analysis device characterized in that it comprises the front and rear mass information of the sequence tag included in each peptide sequence. A method for analyzing amino acid sequences included in proteins using a peptide sequence database and a post-translational modification database, Extracting a plurality of peaks from the tandem mass spectrum by receiving a tandem mass spectrum of each peptide for the protein to be analyzed; Extracting a sequence tag not including post-translational modifications included in the spectrum of the peptide of analysis from the mass difference of the extracted peaks and determining a sequence tag list including the sequence tag; Searching a peptide sequence database to determine candidate peptides that include the sequence tag; Combining and placing all sequence tags included in the candidate peptide and the bindable sequence tags to generate a sequence tag having a space; And The post-translational modification database is searched using the amino acid sequence of the candidate peptide for the blank portion of the sequence tag in which the blank exists and the mass variation of the blank portion, and the amino acid sequence included in the blank portion of the peptide to be analyzed. An amino acid sequencing process for searching for the type or location and type of post-translational modification; Amino acid sequence analysis method comprising a. The method of claim 18, Amino acid sequence analysis method characterized in that the 20 to 200 selected peaks. delete The method of claim 18, The determining of the sequence tag list may include calculating mass differences of all the extracted peak pairs; Determining an amino acid corresponding to a mass difference value on the peak; And Comparing the mass difference between the plurality of peaks and the mass difference between the plurality of amino acids to generate a sequence tag list including no post-translational modifications; Amino acid sequence analysis method comprising a. The method of claim 18, The determining of the candidate peptide list may include extracting a first candidate peptide having a sequence tag equal to or greater than a first length specified by comparing each of the sequence tags with peptides stored in the peptide sequence database; Extracting a second candidate peptide among the first candidate peptides in which the sequence tag matches a sequence tag of a specified second length; And Generating a candidate peptide list using the second candidate peptide, and storing a matching direction between the sequence tag and the peptide; Amino acid sequence analysis method comprising a. The method of claim 18, The process of generating a sequence tag in which the space exists includes comparing the position of the first peak or the last peak of the sequence tag with the position of a peak theoretically calculated from the candidate peptide. Deleting a sequence tag from the sequence tag list; Combining a sequence tag capable of binding among sequence tags included in the sequence tag list; And Generating a list of peptides including a space consisting of the sequence tag and a combination of sequence tags; Amino acid sequence analysis method comprising a. The method of claim 23, Before performing the filtering step by comparing the before and after mass of the sequence tag, the parent peptide of each of the candidate peptides is compared with the mass of the peptide to be analyzed, and the candidate peptide having a value obtained as a result of the comparison is smaller than a specified value. The method of amino acid sequence analysis, characterized in that it further performs the step of filtering out the mass mass from the list. The method of claim 18, The amino acid sequencing process, before searching the database, performs a normalization process for the peptide extracted from the protein to be analyzed, calculates the degree of matching with the peptide present in the gap, so that the gap with poor matching degree The method of amino acid sequence analysis, characterized in that further performing the deletion of the peptide. The method of claim 18, The amino acid sequencing process searches for the amino acid sequence or the post-translational modification, fills the peptide with the amino acid sequence or the post-translational modification with the blank to generate a blank-filled peptide, and performs mass spectrometry on the blank-filled peptide. And generating and comparing the spectrum to be analyzed.

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