JP2015230262A - Mass analysis data analysis method and device - Google Patents

Abstract

PROBLEM TO BE SOLVED: To identify a peptide and a modification site with high accuracy even for a peptide that is subjected to posttranslational modification that cannot easily be identified in an ordinary database retrieval.SOLUTION: De novo sequencing is executed on a MSspectrum derived from a target peptide in a low m/z direction using as the origin a plurality of peaks where ion intensity is relative large, and a plurality of tag candidates are extracted by estimating an amino acid sequence (S1-S3). The tag candidates whose peaks as the origin differ are compared, those having an amino acid sequence in common are detected assuming that their reliability is high, and a sequence tag based on the amino acid sequence is created (S4). A posttranslational modification molecule is estimated from a difference between the start m/z's and between the finish m/z's of the amino acid sequence in the plurality of detected tag candidates (S5). A target peptide is identified by executing a sequence tag search using the sequence tag and modification molecule information as search conditions (S6-S7).

Description

The present invention relates to a mass spectrometry data analysis method and apparatus for performing analysis processing on data collected by performing MS ⁿ analysis on biological compounds such as peptides and identifying the polymer compounds, and in particular, translation The present invention relates to a mass spectrometry data analysis method and apparatus for identifying a post-modification peptide or 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, database search methods are well known as peptide analysis methods using mass spectrometers such as MALDI-IT-TOFMS (Matrix Assisted Laser Desorption / Ionization Ion Trap Time-of-Flight Mass Spectrometer). Yes. In the database search method, the peak list that lists the mass-to-charge ratios of the peaks that appear in the MS ⁿ spectrum obtained by MS ⁿ analysis is checked against the mass-to-charge ratio calculated from the protein registered in the database, and the degree of coincidence The amino acid sequence of the peptide is determined using as a clue. In addition, a method has been developed for identifying a highly probable peptide and its modification site by specifying the type of modification received by the peptide as a search condition when searching the database.

For example, Mascot provided by Matrix Science, Inc., UK, includes a search engine named MS / MS ion search. As a search parameter, the type of digestive enzyme used for protein degradation (Enzyme) ), The type of modification (modification), the allowable value of accuracy of mass spectrometry (MS / MS tol.), And the like can be input and set by the user (see Non-Patent Document 1). In the MS / MS ion search method, a database search according to a search condition is performed on a plurality of MS / MS (MS ⁿ ) spectra, and peptides having a high matching score (score) are listed.

As described above, in the conventional database search method, the type of post-translational modification can be specified as one of the search conditions. Even if a database search technique such as the / MS ion search method is applied, it is quite difficult to identify a peptide or modification site with a reasonable score. The main reason is that the MS ⁿ spectrum obtained by MS ⁿ analysis using MALDI-IT-TOFMS is a mixture of modified product ions and product ions (neutral loss) from which modified molecules are eliminated. This is because the peak list becomes quite complicated.

As an example, database search using MS / MS ion search method for peak list created based on MS ² spectrum with peptide ion derived from human transferrin modified with one HexNAc sugar chain fragment as precursor ion The results are shown in FIG. As shown in FIG. 6, in this example, the peptide and modification site could not be identified with a reasonable score. FIG. 7 is a diagram showing the MS ³ spectrum that is the basis of the search of FIG. 6 and the attribution result. FIG. 8 is a diagram showing the degree of coincidence between the MS ³ spectrum shown in FIG. 7 and the theoretical product ion of human transferrin peptide.

In FIG. 7, y _n + ■ is an assignment result under the condition that one sugar chain is added. As shown in FIG. 7, it can be seen that HexNAc adduct ions (y ions with a black square in the figure) and ions (y ions) generated by the neutral loss of HexNAc are mixed. When FIG. 7 and FIG. 8 are compared, it can be seen that ions having relatively large intensity, such as y (8), y (11), etc. generated by the HexNAc neutral loss are not assigned as product ions at the time of database search. I understand. These ion peaks are considered to have been treated as noise, and it is expected that the identification score will be lowered.

On the other hand, there is a method called sequence tag search as one method of database search for peptide identification. In sequence tag search, a tag indicating a continuous amino acid sequence that may exist in a peptide or protein (sequence tag) is used, and in the same way as MS / MS ion search, a virtual CID spectrum of a protein registered in a database is searched. Attempt peptide identification by matching. As a tool for executing a sequence tag search, for example, “Mascot Sequence Query” provided by Matrix Science, UK is well known (see Non-Patent Document 2). The database search setting screen in “Mascot Sequence Query” is the same search setting screen as in “Mascot MS / MS ion search”, except that the sequence tag is input as data instead of the peak list collected from the MS ² spectrum. Are different (see Non-Patent Document 3).

The sequence tag search has a feature that a correct peptide can be uniquely identified if the given sequence tag is accurate and the corresponding peptide is registered in the database. Therefore, when using a sequence tag search, it is very important to obtain a sequence tag with high accuracy. Since it is generally to search for sequence tags from MS ⁿ spectrum de novo (de novo) sequencing is utilized, MS ⁿ spectra for peptides that have undergone post-translational as described above modification is quite complicated, It is difficult to find an accurate sequence tag by simply executing de novo sequencing.

JP 2012-220365 A

"Matrix Science Mascot MS / MS Ions Search", [online], UK Matrix Science Ltd., [May 23, 2014 search] , Internet <URL: http://www.matrixscience.com/cgi/search_form.pl?FORMVER=2&SEARCH=MIS> "Sequence Query", [online], Matrix Science Ltd., UK [Search May 23, 2014], Internet <URL: http://www.matrixscience.com /help/sq_help.html> "Protein Identification System MASCOT Server Version 2.2 Tutorial", [online], Matrix Science Ltd., UK (searched on May 23, 2014), Internet <URL: http: //www.matrixscience. jp / pdf / jap_2.2_tutorial.pdf>

The present invention has been made in view of the above problems, and the object of the present invention is to receive post-translational modifications that cannot be identified with sufficient reliability by conventional database search methods due to neutral loss. To provide a mass spectrometric data analysis method and apparatus capable of identifying the type and site of post-translational modification and the amino acid sequence of a peptide with high accuracy using the MS ⁿ spectrum of the peptide.

A first invention made to solve the above problems uses a data collected by performing mass spectrometry on a test sample, and uses a peptide subjected to post-translational modification contained in the test sample. A mass spectrometry data analysis method for identifying,
a) Same mass-to-charge ratio starting from multiple different ion peaks for MS ⁿ spectra obtained by performing MS ⁿ analysis (n is an integer of 2 or more) using ions derived from the target peptide as precursor ions A tag candidate extraction step in which de novo sequencing is performed in the axial direction, and partial amino acid sequences for different ion series are respectively determined as tag candidates;
b) For one or a plurality of tag candidates for each ion series obtained in the tag candidate extraction step, a tag including a common amino acid sequence in the same mass-to-charge ratio axis direction with respect to different ion series is found. A tag selection step for generating a sequence tag as a high tag;
c) a database search step for performing a sequence tag search using a database in which amino acid sequence information of proteins is stored and using the sequence tag generated in the tag selection step as a search condition;
It is characterized by having.

A second invention made to solve the above problems is an apparatus for performing the mass spectrometry data analysis method according to the first invention, which is collected by executing mass analysis on a test sample. In a mass spectrometry data analysis apparatus for identifying a peptide having undergone post-translational modification contained in the test sample,
a) Same mass-to-charge ratio starting from multiple different ion peaks for MS ⁿ spectra obtained by performing MS ⁿ analysis (n is an integer of 2 or more) using ions derived from the target peptide as precursor ions Tag candidate extraction means for performing de novo sequencing in the axial direction and obtaining partial amino acid sequences for different ion sequences as tag candidates,
b) For one or a plurality of tag candidates for each ion series obtained by the tag candidate extraction means, a tag including a common amino acid sequence in the same mass-to-charge ratio axis direction with respect to different ion series is found and reliable. Tag selection means for generating a sequence tag as a high tag,
c) a database storing protein amino acid sequence information;
d) database search means for performing a sequence tag search using the database and the sequence tag generated by the tag selection means as a search condition;
It is characterized by having.

  Here, not only treats b-series ions derived from the N-terminus of peptides and y-series ions derived from the C-terminus as different ion sequences, but also ions with post-translational modifications added to, for example, b-series ions are eliminated. It is treated as an ion series different from the selected ion.

For example, in the b-series (or y-series) fragment ion from which the modifying molecule is detached and the b-series (or y-series) fragment ion to which the modifying molecule is added (remaining), the latter is a mass fraction corresponding to the modifying molecule. Should have a large mass-to-charge ratio. That is, in the MS ⁿ spectrum, the fragment ion peak derived from the latter should appear at a position shifted on the mass-to-charge ratio axis by a mass corresponding to the modifying molecule from the fragment ion peak derived from the former. Therefore, de novo sequencing is performed in the same mass-to-charge ratio axis direction starting from the b-series (or y-series) fragment ion from which the modifying molecule is detached and the b-series (or y-series) fragment ion to which the modifying molecule is added. When executed, the amino acid sequences at least from the origin to a certain position (the position of the smallest ion in which the modifying molecule is included in the latter) are identical. In other words, even if the ion sequence of the starting point for performing de novo sequencing is unknown, if the amino acid sequences obtained by executing de novo sequencing from two different starting points match, The sequence estimation result can be regarded as correct.

Therefore, in the mass spectrometry data analysis method according to the first invention, the MS ⁿ spectrum from test sample is obtained, first, in the tag candidate extracting step in MS ⁿ spectra, for example, a large plurality of peaks relatively intensity As a starting point, de novo sequencing is performed in the same mass-to-charge ratio axis direction. Thereby, one or a plurality of partial amino acid sequences are obtained for a plurality of different ion series. In the tag selection step, for one or a plurality of tag candidates for each ion series, a tag including an amino acid sequence common in the same mass-to-charge ratio axis direction for different ion series is detected. As described above, since the detected tag can be regarded as having high reliability, the sequence tag is generated based on this. In the database search step, a target peptide is identified by executing a sequence tag search using the above sequence tag as a search condition using a database storing protein amino acid sequence information.

  When performing a sequence tag search, it is necessary to provide the post-translational modification molecule type, modification site, modification molecule mass, and the like as one of the search conditions. If the type of modifying molecule is known, such known information may be given as a search condition. On the other hand, even when the type of post-translational modification molecule is unknown, it can be easily estimated from the tag candidates having high reliability obtained as described above.

  That is, in the mass spectrometric data analysis method according to the first aspect of the invention, it is preferable that the target peptide is translated by comparing a plurality of tags for different ion series confirmed to contain a common amino acid sequence in the tag selection step. A modification estimation step for estimating a post-modification molecule may be further included, and the post-translational modification molecule estimated in the modification estimation step in the database search step may be one of the search conditions. This makes it possible to identify the target peptide even if the type of post-translational modification received by the target peptide is unknown.

  As a database search means for executing a sequence tag search, known software such as “Mascot Sequence Query” described above can be used.

According to the mass spectrometry data analysis method according to the first invention and the mass spectrometry data analysis apparatus according to the second invention, a highly reliable sequence tag can be obtained from a complex MS ⁿ spectrum derived from a peptide subjected to post-translational modification. As a result, it is possible to identify the amino acid sequence of the post-translationally modified peptide and the modified site with high reliability while using a conventional database search.

BRIEF DESCRIPTION OF THE DRAWINGS The whole block diagram of one Example of the peptide analysis system containing the mass spectrometry data analyzer which concerns on this invention. The flowchart which shows an example of the data analysis process procedure implemented in the peptide analysis system of a present Example. It shows a pseudo MS ³ spectra of acrylamide labeled monovalent HexNAc additional peptide ions obtained by actual measurement from two-branched sugar chain binding glycopeptides of human transferrin (m / z 1694). Figure pseudo MS ³ ionic strength on the spectrum of Figure 3 is running de novo sequencer sequencing starting high peak shows the result of estimating the amino acid sequence. The figure which shows the result of having performed the sequence tag search using the sequence tag (EA [IL] [PV | VP], start m / z 741.4, end m / z 1250.6). It shows the results of a database search by MS / MS ion search method to MS ² spectra of peptide ions derived from the human transferrin and precursor ion. It shows the assignment result and the search for the original 6 since MS ³ spectrum. FIG. 8 is a diagram showing the degree of agreement between the MS ³ spectrum shown in FIG. 7 and the theoretical product ion of human transferrin peptide.

  Hereinafter, an embodiment of a peptide analysis system including a mass spectrometry data analysis apparatus according to the present invention will be described with reference to the accompanying drawings. FIG. 1 is an overall configuration diagram of the peptide analysis system of this example.

  The peptide analysis system according to the present embodiment is roughly divided into a mass analysis unit 1 and a control / processing unit 2 configured mainly by a computer. The mass spectrometer 1 is a matrix-assisted laser desorption ionization quadrupole ion trap time-of-flight mass spectrometer (MALDI-QIT-TOFMS). A three-dimensional quadrupole ion trap 11 capable of temporarily capturing generated ions and performing ion selection according to the mass-to-charge ratio m / z and ion cleavage by CID; A time-of-flight mass analyzer 12 that separates and detects the various emitted ions according to the mass-to-charge ratio. The time-of-flight mass analyzer 12 is a flight space 13 in which ions are turned back by an electric field generated by a reflectron electrode, and ions temporally separated according to a mass-to-charge ratio while flying in the flight space 13. And an ion detector 14 for detecting sequentially.

The control / processing unit 2 includes an analysis control unit 28 that controls each unit of the mass analysis unit 1, a data collection unit 21 that digitizes detection signals obtained from the ion detector 14 and stores them in an internal storage unit, and collected data Analysis unit 22 for generating MS spectrum and MS ⁿ spectrum from the same, tag candidate extraction unit 23 for performing partial de novo sequencing on MS ⁿ spectrum data to obtain partial amino acid sequences as tag candidates, identical MS ⁿ spectrum A tag selection unit 24 that generates a highly reliable sequence tag based on a plurality of tag candidates obtained based on the tag, a modified product determination unit 25 that obtains a post-translationally modified molecule based on a highly reliable tag, a sequence tag Sequence tag search that identifies target peptides in a test sample by database search by setting search conditions such as the type of molecule or modified molecule Line unit 26, and comprises a identification database (DB) 27 which has been previously registered identification information for estimating the amino acid sequence of the protein (peptide). For the function of the sequence tag search execution unit 26, for example, existing search engine software such as “Mascot Sequence Query” described above can be used.

  The entity of the control / processing unit 2 is a computer, and the above-described various functions are achieved by the operation of dedicated control / processing software installed in the computer. Specifically, the input unit 3 connected to the control / processing unit 2 is a pointing device such as a keyboard and a mouse connected to the computer, and various kinds of input necessary for the user to input and set search conditions and for spectrum analysis. It is for performing operations. Similarly, the display unit 4 connected to the control / processing unit 2 is specifically a monitor display for displaying a search condition input setting screen or displaying an identification result.

  A characteristic data analysis process in the peptide analysis system of the present embodiment will be described with reference to the flowchart showing the peptide identification process procedure shown in FIG.

The analyst prepares a test sample containing a peptide (modified peptide) subjected to post-translational modification by digesting the target protein with an appropriate enzyme (for example, trypsin enzyme). When MS ¹ analysis is performed on the test sample by the mass analysis unit 1 under the control of the analysis control unit 28, the spectrum analysis unit 22 calculates the MS ¹ spectrum based on the data collected by the data collection unit 21. create. The spectrum analysis unit 22 extracts an ion peak derived from the target peptide from the MS ¹ spectrum and selects it as a precursor ion for MS ² analysis (step S1).

Subsequently, under the control of the analysis control unit 28, the mass analysis unit 1 performs MS ² analysis on the precursor ion on the test sample. More specifically, various ions generated from the test sample by laser light irradiation in the ionization unit 10 are once captured by the ion trap 11, and only ions having a mass-to-charge ratio of precursor ions are selected in the ion trap 11. Later, dissociation by CID is performed. At this time, the binding of the amino acid sequence of the target single peptide is broken at various sites, and various amino acid residues are captured by the ion trap 11 as product ions. The product ions are simultaneously emitted from the ion trap 11 and subjected to mass analysis by the time-of-flight mass analyzer 12. The data collection unit 21 collects data obtained by MS ² analysis, and the spectrum analysis unit 22 collects the data. An MS ² spectrum is created based on the data (step S2).

In step S2, the operation of selecting a precursor ion from ion peaks on the MS ² spectrum and executing MS ³ analysis is repeated one or more times to create an MS ⁿ spectrum where n is 3 or more. Also good. In order to execute the processing described later, product ions from which post-translational modifications are desorbed and product ions from which post-translational modifications are not desorbed must be observed in the MS ⁿ spectrum. If it is easy to release, it may be n = 2 (however, it is MS ² analysis that is actually executed as described later, but it can be said that it is a pseudo MS ³ analysis). When the molecule is difficult to desorb, it is desirable that n is 3 or more.

Next, the tag candidate extraction unit 23 selects a plurality of peaks having relatively high ionic strength in the MS ² spectrum (or MS ⁿ spectrum), and executes de novo sequencing from each peak toward the low mass-to-charge ratio side. . That is, the amino acid sequence is estimated by sequentially searching for a peak that matches the mass of a known amino acid from the starting peak toward the low mass to charge ratio side. Then, the obtained amino acid sequence is extracted as a sequence tag candidate (step S3). When cleaved ions derived from the modified peptide, the MS ⁿ spectrum contains a mixture of product ion peaks of the ion series from which the post-translationally modified molecule is eliminated and product ion peaks of the ion series to which the post-translationally modified molecule is added. It appears quite complicated. Therefore, as shown in an example described later, when de novo sequencing is attempted from a certain peak, a plurality of different amino acid sequences are listed, and a plurality of sequence tag candidates are extracted.

  Subsequently, the tag selection unit 24 compares the amino acid sequences of the sequence tag candidates obtained by de novo sequencing in the same mass-to-charge ratio direction starting from different peaks, and extracts tags having a common amino acid sequence. In general, the peak of high ionic strength, which is the starting point of de novo sequencing, has different ion series (here, the same b-series ion and y-series ion are different series depending on whether or not there is a post-translationally modified molecule. )). When ion peaks belonging to the same ion series are accurately de novo sequenced, for example, the amino acid sequence obtained for b-series ions with post-translational modification molecules and b-series ions without post-translational modification molecules are obtained. The amino acid sequence should be almost identical. Therefore, it is considered that the reliability of the portion having a certain amino acid sequence in common (amino acid residues of about 4 or more) is high. Therefore, a tag candidate having the longest amino acid sequence in common is selected, and a sequence tag used for the search is created based on the common amino acid sequence (step S4).

  The difference in the start mass-to-charge ratio and the end mass-to-charge ratio of the common amino acid sequences in the tag candidates selected in step S4 is likely to be the mass of the post-translationally modified molecule. Therefore, the modified product determination unit 25 obtains the mass of the post-translational modification molecule from the above difference and obtains the type of post-translational modification molecule corresponding to this (step S5).

  Next, the sequence tag search execution unit 26 sets search conditions based on the highly reliable sequence tag obtained in step S4 and the type of post-translationally modified molecule obtained in step S5 (step S6). A sequence tag search for collating the information (amino acid sequence, starting mass-to-charge ratio, ending mass-to-charge ratio) indicated by the sequence tag with information such as the amino acid sequence and mass of the protein registered in the identification database 27 To identify the peptide of interest. For example, when the above-described “Mascot Sequence Query” is used as the sequence tag search execution unit 26, a confidence index value such as a score or an expected value indicating the reliability of the search is attached to the peptide hit by the search. Therefore, even when a plurality of peptides are hit, if the reliability index value of one peptide is significantly higher than the reliability index values of other peptides, only that peptide may be listed as an identified peptide. . On the other hand, if a unique peptide cannot be identified, a plurality of possible peptides may be selected as candidates together with the reliability index value. The peptide or peptide candidate thus identified is displayed on the display unit 4 to inform the analyst (step S7).

  In the sequence tag search, if the target protein is registered in the identification database 27 and the given sequence tag is accurate, a search result including a correct peptide is derived. That is, it does not occur that a search result that does not include a correct answer in the identification database 27 is obtained. Since the sequence tag obtained by the above-described procedure is highly reliable, it is guaranteed that the peptide identification result obtained based on the sequence tag is also highly reliable.

  A specific example of peptide identification by the above-described characteristic mass spectrometry data analysis method will be described.

FIG. 3 is a pseudo MS ³ spectrum of a monovalent HexNAc-added peptide ion (m / z 1694) obtained from a bi-branched sugar chain-linked glycopeptide of acrylamide-labeled human transferrin. In the N-linked glycopeptide, sugar chains are easily detached. For this reason, when MS ¹ analysis of glycopeptides is performed, by ring cleavage of the peptide HexNAc, which is a peptide ion from which all sugar chains have been removed, in addition to the glycopeptide ions (or sialic acid-desorbed glycopeptide ions) to be analyzed by MS ² The resulting ^0,2 X (83 Da) -added peptide ion and three peaks (triplet peaks) corresponding to the HexNAc-added peptide ion may be observed on the MS ¹ spectrum. Therefore, MS triplet peaks appearing in ¹ spectrum, when the MS ² analyte ions having the same mass-to-charge ratio and the observed triplet peaks on MS ² spectra obtained by MS ² analysis of the triplet peaks Any one or more are selected as precursor ions for pseudo MS ³ analysis, and MS ² analysis is performed on the precursor ions. Thereby, a pseudo MS ³ spectrum can be acquired while actually performing MS ² analysis.

As described above, when comparing the mass-to-charge ratio between the triplet peak on the MS ¹ spectrum and the triplet peak on the MS ² spectrum, the valences are the same if the valences of the triplet ions are different. It is necessary to compare the mass to charge ratio after correcting the mass to charge ratio. As described in Patent Document 1, in an N-linked glycopeptide in which a specific amino acid residue such as glutamine, glutamic acid, carbamidomethylated cysteine or the like is located at the N-terminus, the N-terminal specific amino acid residue in the process of CID Is preferentially generated, and therefore, neutral loss of NH ₃ and H ₂ O is likely to occur. In such a case, the ion peak on the MS ² spectrum may have a mass shift due to the above neutral loss. Therefore, when such a mass shift is detected, the mass shift is performed on the ion peak on the MS ² spectrum. The mass-to-charge ratio between the triplet peaks is compared.

In the pseudo MS ³ spectrum shown in FIG. 3, an ion peak caused by the neutral loss of HexNAc appears as a product ion for both the b-series ion added with HexNAc and the y-series ion added with HexNAc. A database search using the conventional MS / MS ion search method was performed on the peak list obtained from this pseudo MS ³ spectrum, but homology to the HexNAc-added peptide was obtained, but the peptide and modification site were identified. A score as high as possible could not be obtained (specifically, score: 20, Expected value: 0.86).

FIG. 4 is a diagram illustrating a result of performing the above-described processing of step S3 on the pseudo MS ³ spectrum illustrated in FIG. 3, that is, the amino acid sequence estimation by de novo sequencing. When the maximum intensity ion peak (m / z 1491.1) in the pseudo MS ³ spectrum was used as the starting point, proper sequencing was not possible, but the second strongest ion peak (m / z 1250.6) was used as the starting point. Then, five types of amino acid sequence estimation results as shown in FIG. 4 (a) were obtained. These are tag candidates obtained starting from the ion peak at m / z 1250.6. On the other hand, when the ion peak with the third strongest intensity (m / z 1047.4) is used as the starting point, five types of amino acid sequence estimation results as shown in FIG. 4B were obtained. These are tag candidates obtained starting from an ion peak at m / z 1047.4. When an amino acid sequence common to both FIG. 4 (a) and FIG. 4 (b) was searched, amino acid sequences EA “I / L” and “PV | VP” were found (in FIG. 4, amino acids surrounded by a one-dot chain line). Array). The number of amino acid residues in the common amino acid sequence is 4 or more, and its reliability is considered high. Therefore, from the mass-to-charge ratio of both ends of the amino acid sequence and the HexNAc-added peptide ion sequence, a sequence tag (amino acid sequence: EA [IL] [PV | VP], start mass-to-charge ratio 741.4, end mass-to-charge ratio) 1250.6).

Moreover, when the start mass-to-charge ratio and end mass-to-charge ratio of this common amino acid sequence are compared, it can be seen that there is a difference corresponding to HexNAc (203 Da). From this, it can be confirmed that the pseudo MS ³ spectrum being analyzed is highly likely to be a spectrum of a peptide modified with HexNAc.

FIG. 5 shows the result of performing a sequence tag search by “Mascot Sequence Query” with the sequence tag and post-translationally modified molecule information set as search conditions. As shown in FIG. 5, the target peptide and the HexNAc modification site are identified with a high score (score: 62) and a low expected value (Expect: 8.3 × 10 ⁻⁶ ). This is a much better result than the score and expected value obtained by the conventional method described above, and it can be confirmed that the data analysis method according to the present invention is useful for identifying a peptide having undergone post-translational modification.

In the above example, a highly reliable sequence tag was created based on the de novo sequencing result obtained from one MS ⁿ spectrum derived from the target peptide (MS ³ pseudo spectrum in the above example). When a plurality of MS ⁿ spectra are obtained, a highly reliable sequence tag may be created based on a de novo sequencing result obtained from the plurality of MS ⁿ spectra).

  Further, the reliability of the sequence tag may be quantified based on information such as peak intensity and mass difference accuracy. Thereby, for example, when there are a plurality of types of common amino acid sequences, the tag used for the sequence tag search can be determined based on the quantified reliability index value of the sequence tag.

  Further, the above-described embodiment is merely an example of the present invention, and it is obvious that the present invention is encompassed in the scope of the claims of the present application even if appropriate modifications, corrections, additions, etc. are made within the scope of the present invention.

DESCRIPTION OF SYMBOLS 1 ... Mass analysis part 10 ... Ionization part 11 ... Ion trap 12 ... Time-of-flight mass analyzer 13 ... Flight space 14 ... Ion detector 2 ... Control and processing part 20 ... Analysis control part 21 ... Data collection part 22 ... Spectral analysis Unit 23 ... Tag candidate extraction unit 24 ... Tag selection unit 25 ... Modified product determination unit 26 ... Sequence tag search execution unit 27 ... Identification database 28 ... Analysis control unit 3 ... Input unit 4 ... Display unit

Claims (4)

A mass spectrometry data analysis method for identifying a peptide subjected to post-translational modification contained in a test sample using data collected by performing mass spectrometry on the test sample,
a) Same mass-to-charge ratio starting from multiple different ion peaks for MS ⁿ spectra obtained by performing MS ⁿ analysis (n is an integer of 2 or more) using ions derived from the target peptide as precursor ions A tag candidate extraction step in which de novo sequencing is performed in the axial direction, and partial amino acid sequences for different ion series are respectively determined as tag candidates;
b) For one or a plurality of tag candidates for each ion series obtained in the tag candidate extraction step, a tag including a common amino acid sequence in the same mass-to-charge ratio axis direction with respect to different ion series is found. A tag selection step for generating a sequence tag as a high tag;
c) a database search step for performing a sequence tag search using a database in which amino acid sequence information of proteins is stored and using the sequence tag generated in the tag selection step as a search condition;
A method for analyzing mass spectrometry data, comprising: The mass spectrometry data analysis method according to claim 1,
The database search further comprises a modification estimation step of estimating post-translational modified molecules of the target peptide by comparing a plurality of tags for different ion series confirmed to contain a common amino acid sequence in the tag selection step In the step, the post-translationally modified molecule estimated in the modification estimation step is used as one of the search conditions. In a mass spectrometry data analyzer for identifying a peptide subjected to post-translational modification contained in the test sample, using data collected by performing mass spectrometry on the test sample,
a) Same mass-to-charge ratio starting from multiple different ion peaks for MS ⁿ spectra obtained by performing MS ⁿ analysis (n is an integer of 2 or more) using ions derived from the target peptide as precursor ions Tag candidate extraction means for performing de novo sequencing in the axial direction and obtaining partial amino acid sequences for different ion sequences as tag candidates,
b) For one or a plurality of tag candidates for each ion series obtained by the tag candidate extraction means, a tag including a common amino acid sequence in the same mass-to-charge ratio axis direction with respect to different ion series is found and reliable. Tag selection means for generating a sequence tag as a high tag,
c) a database storing protein amino acid sequence information;
d) database search means for performing a sequence tag search using the database and the sequence tag generated by the tag selection means as a search condition;
A mass spectrometry data analysis device comprising: The mass spectrometry data analysis device according to claim 3,
The database search means further comprises modification estimation means for estimating post-translationally modified molecules of the target peptide by comparing a plurality of tags for different ion series confirmed to contain a common amino acid sequence by the tag selection means. Is a post-translationally modified molecule estimated by the modification estimating means as one of the search conditions.

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