JP5299060B2 - Glycopeptide structure analysis method and apparatus - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To estimate the amino acid sequence and sugar chain bonding region of peptide constituting glycopeptide with high reliability by the retrieval of the database based on an MS/MS spectrum wherein the ions originating from target glycopeptide are set as precursor ions. <P>SOLUTION: The ions originating from the glycopeptide are found out from an MS spectrum to obtain the MS/MS spectrum (S1-S3) and the product ions, which revert to peptide wherein all of sugar chains are eliminated from the MS/MS spectrum and revert to peptide receiving the simple modification due to sugar, are extracted to respectively form the lists of peaks having a m/z of the respective product ions (S4 and S5) or below. The modification condition (the kind of sugar) turned clear in S4 is set as a retrieval condition to subject the peak lists to the retrieval of the database and the candidates of peptides with a high degree of coincidence are listed (S6-S8). The retrieval result of the respective peak lists is synthetically determined to display the candidate list of the amino acid sequence and the sugar chain bonding region (S9). <P>COPYRIGHT: (C)2011,JPO&amp;INPIT

Description

  The present invention relates to a glycoprotein or glycopeptide structure analysis method and structure analysis apparatus using mass spectrometry.

  It is said that more than half of proteins constituting a living body are subjected to sugar chain modification, and the sugar chain modification plays an important role in regulating the structure and function of the protein. Recent research has also revealed the relationship between various diseases such as immune diseases and abnormal sugar chain structures and abnormal glycation. For these reasons, structural analysis of glycoproteins and glycopeptides, that is, identification of peptide amino acid sequences and sugar chain binding sites, is very important in various fields such as life science, medicine, and drug development. Although mass spectrometry has become one of the main methods in glycan structure analysis due to the recent improvement in mass spectrometer performance, the glycan structure analysis method using mass spectrometry is still in the process of research. Various methods have been proposed.

  In general, peptide identification methods using a mass spectrometer include a peak list that lists the mass-to-charge ratios of peaks that appear in MS / MS spectra obtained by MS / MS analysis, and proteins that are registered in the database. A so-called database search method is well known in which the calculated mass-to-charge ratio is collated and the amino acid sequence of the peptide is determined using the degree of coincidence 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 in the UK includes a search engine called MS / MS ion search. As search parameters, the type of digestive enzyme used for protein degradation (enzyme) and modification The user can input and set the type (modification), the allowable value of mass spectrometry accuracy (MS / MS tol.), And the like (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 spectra (or MS ⁿ spectra), and peptides having a high matching score (score) are listed.

  However, using a peak list created from an MS / MS spectrum obtained by MS / MS analysis of a glycopeptide modified with a sugar chain and simply performing a database search by the above MS / MS ion search method, etc. is reasonable. It is quite difficult to identify such peptides. This is because, since glycopeptides are generally modified by a plurality of sugars having a complex sugar chain structure, the content of the peak list obtained from the MS / MS spectrum is very complex and unique to glycopeptides. Because it becomes. For example, the inventor of the present application tried to perform a database search using the MS / MS ion search method for a peak list created from an MS / MS spectrum for sialic acid-eliminated ions, which are ions derived from a glycopeptide derived from human transferrin. However, transferrin-derived peptides were not listed, and appropriate peptides and sugar chain binding sites could not be identified.

On the other hand, Patent Document 1, Patent Document 2, Non-Patent Document 2, and the like disclose a method for performing sugar chain structure analysis using a mass spectrometer capable of MS ⁿ analysis (n is an integer of 3 or more). In this method, the structure is estimated using an MS ⁿ spectrum obtained by mass analysis of product ions finely fragmented by performing cleavage operation two or more times. Although such a technique is effective for sugar chain structure analysis, it is necessary to perform MS ⁿ analysis in which n is 3 or more. Mass spectrometers capable of MS / MS analysis are relatively common, while mass spectrometers capable of MS ⁿ analysis with n of 3 or more are quite expensive and not readily available. Therefore, such a structural analysis method is not versatile and cannot be easily adopted.

JP 2005-265697 A JP 2005-300420 A

"Matrix Science-Mascot-MS / MS Ions Search", [online], UK Matrix Science Ltd., [April 2009 Search 13 days], Internet <URL: http://www.matrixscience.com/help/mis_help.html> Yamada, Fukuyama, “Protein Glycosylation Analysis by MALDI-QIT-TOFMS”, Shimazu Review, Shimazu Review Editorial Department, Vol. 63, No. 1, No. 2, September 29, 2006

  The present invention has been made in view of the above-mentioned problems, and its object is to use a MS / MS analysis result for glycoproteins and glycopeptides that have been difficult to identify by conventional database search methods. It is to provide a glycopeptide structure analysis method and an analysis apparatus that can identify the amino acid sequence of a simple peptide and its sugar chain binding site.

A first invention made to solve the above problems is a glycopeptide structure analysis method for identifying a peptide constituting a glycopeptide and a sugar chain binding site using a mass spectrometer capable of MS / MS analysis. ,
a) Find an ion derived from a glycopeptide appearing in an MS spectrum obtained by MS analysis of a test sample, select the ion as a precursor ion, perform MS / MS analysis on the test sample, An analysis execution step of acquiring an MS spectrum;
b) from MS / MS spectra obtained by the analysis execution step, the product ions all sugar chains are attributed to desorbed peptides, caused by cleavage of the N-type sugar chain binding glycopeptides as a simple modification by sugar Search for product ions attributed to peptides modified with only N-acetylhexosamine and modified with a ring-cleavable fragment of N-acetylhexosamine, and each of the plurality of product ions in the MS / MS spectrum. A peak list creation step for creating a peak list listing ion peaks having a mass to charge ratio equal to or lower than the mass to charge ratio of the product ions;
c) A search condition is set as a search condition that can be modified only by N-acetylhexosamine or by a ring-cleavage fragment of N-acetylhexosamine generated by cleavage of the N-type sugar chain-linked glycopeptide, and the peak list is searched for set, and database search executing step for executing a database search using the identified database in accordance with the search condition,
d) a result providing step of outputting information on the amino acid sequence and sugar chain binding site of the peptide from which a result that the degree of coincidence is relatively high with respect to the plurality of peak lists is obtained by the database search;
It is characterized by having.

The second invention is a glycopeptide structure analysis apparatus for carrying out the glycopeptide structure analysis method according to the first invention, wherein a peptide constituting the glycopeptide using a mass spectrometer capable of MS / MS analysis is provided. In a glycopeptide structure analysis apparatus for identifying a sugar chain binding site,
a) Find an ion derived from a glycopeptide appearing in an MS spectrum obtained by MS analysis of a test sample, select the ion as a precursor ion, perform MS / MS analysis on the test sample, Analysis control means for acquiring an MS spectrum;
b) from MS / MS spectrum obtained under the control of the analysis control unit, and product ions all sugar chains are attributed to desorbed peptides, N-type sugar chain binding glycopeptides as a simple modification by sugar The product ions attributed to the peptides that have been modified with only N-acetylhexosamine resulting from the cleavage of N and acetylated with the ring-cleavable fragment of N-acetylhexosamine are searched for, and for each of the plurality of product ions, the MS / A peak list creating means for creating a peak list listing ion peaks having a mass to charge ratio equal to or lower than the mass to charge ratio of the product ion in the MS spectrum;
c) A search condition is set as a search condition that can be modified only by N-acetylhexosamine or by a ring-cleavage fragment of N-acetylhexosamine generated by cleavage of the N-type sugar chain-linked glycopeptide, and the peak list is searched for set, and database search executing means for executing a database search using the identified database in accordance with the search condition,
d) a result providing means for outputting the information on the amino acid sequence and sugar chain binding site of the peptide from which the result that the matching degree is relatively high for the plurality of peak lists is obtained by the database search;
It is characterized by having.

  In the mass spectrometer used in the glycopeptide structure analysis method and apparatus according to the present invention, for example, MALDI (matrix-assisted laser desorption ionization) or ESI (electrospray ionization) is used as an ion source for ionizing various components in a test sample. Can be used. For example, in MALDI mass spectrometry using a MALDI ion source, it is known that acidic sugar chains to which sialic acid or sulfate groups have been added are relatively easy to desorb (Sekiya, et al., “Glycan analysis by mass spectrometry. "Trends in Glycoscience and Glycotechnology, Vol. 20, No. 111 (January 2008), pp. 51-65). Therefore, for example, in an MS spectrum obtained by MALDI mass spectrometry of a peptide modified with a sugar chain having sialic acid, there are an ion peak due to the original glycopeptide and an ion peak where sialic acid is eliminated from the glycopeptide. appear. Therefore, an ion derived from a glycopeptide can be found by finding two peaks having a mass-to-charge ratio difference corresponding to sialic acid. In addition to sialic acid and sulfate groups, including the case where ESI is used, ions derived from glycopeptides can be used by removing or adding groups having a known mass-to-charge ratio or by detaching sugar chains. Can be found. Of course, the mass-to-charge ratio of ions derived from the target glycopeptide may be known in advance.

  In the MS / MS spectrum obtained by MS / MS analysis performed using ions derived from the target glycopeptide as precursor ions, peaks due to product ions generated by cleavage of the glycopeptide in various modes appear. Of course, if there are impurities, the peak derived from it also appears. Therefore, in the peak list creation step performed by the peak list creation means, among the many peaks appearing in the MS / MS spectrum, the peak due to the product ion belonging to the peptide from which all the sugar chains are eliminated, and the sugar Search for peaks due to product ions attributed to simple modified peptides. The former is a product ion belonging to only a peptide, which does not include not only all sugars constituting the sugar chain but also sugar cleaved pieces. The latter is a simple modification with one sugar (ie with one sugar attached) or a simple modification with one sugar cleavage fragment (ie a part of one sugar) This is the peak of the product ion attributed to the peptide.

In the case of a typical N-glycan-linked glycopeptide, since a characteristic triplet (triple) peak having a mass-to-charge ratio difference according to the type of modification (condition of modification) is observed, find the triplet peak, it is the case where a plurality of sets exist that select a set of triplet peaks so as to select from among them shows the choice automatically selected to or user set a maximum for example, strength.

Thus, if a product ion belonging to a peptide from which all sugar chains have been removed and a product ion belonging to a peptide that has undergone simple modification with one sugar are obtained, each of them is below the product ion. A peak list is created by collecting peak information (at least the mass-to-charge ratio) of product ions having the mass-to-charge ratio . If two product ions attributed to the peptide having received a simple modification by sugar (i.e. modification by one of the sugar, two and modification by one open lobe sugar) peak list in three Become.

  In the database search execution step executed by the database search execution means, a database search is performed for each of the plurality of peak lists created as described above according to a predetermined search condition and registered in the database. The peptide is matched. The search conditions include at least the modification conditions assumed when searching for product ions in the peak list creation step. Of course, when there are modification conditions other than sugar chains, it is preferable to set these as search conditions. As the database search method itself, an existing one can be used. For example, the above-described MS / MS ion search method provided by UK Matrix Science can be used, but the method is not limited to this.

  For example, if a database search is performed using three peak lists, three independent search results are obtained. In general, a search result is a list in which a plurality of candidates are listed in order from the amino acid sequence (or amino acid sequence and sugar chain modification) of the peptide having the highest degree of matching (that is, most likely). Therefore, in the result providing step performed by the result providing means, for example, information on the amino acid sequence and sugar chain binding site of the peptide is listed in order of relatively high matching based on the search results for a plurality of peak lists. Output on the display screen.

  As described above, in the glycopeptide structure analysis method and apparatus according to the present invention, all the sugar chains are attributed to peptides that appear on the MS / MS spectrum obtained as an ion derived from the target glycopeptide as a precursor ion. Of ions with lower mass-to-charge ratio than the product product ion peak and product ions attributed to peptides that have undergone simple modification with one sugar (and one sugar fragment) The amino acid sequence and sugar chain binding site of the peptide are identified by subjecting the peak list on the low mass-to-charge ratio side of the peak to database search. As described above, according to the present invention, since the database search results of a plurality of peak lists created using different product ions derived from the same glycopeptide are used for identification, identification by the conventional method is difficult. There is a high possibility that the amino acid sequence and sugar chain binding site of a glycopeptide based on the MS / MS spectrum can be identified.

In addition, in the present invention, since it is not necessary to perform MS ⁿ analysis where n is 3 or more in order to identify the amino acid sequence and sugar chain binding site of the glycopeptide, it can be obtained at a relatively low cost which can be performed only up to MS / MS analysis. Can be used to analyze the structure of glycoproteins and glycopeptides.

BRIEF DESCRIPTION OF THE DRAWINGS The whole block diagram of one Example of the glycopeptide structure-analysis system which enforces the glycopeptide structure-analysis method based on this invention. The flowchart which shows the process sequence of the glycopeptide structure analysis implemented in the glycopeptide structure analysis system of a present Example. The figure which shows an example of the MS spectrum actually obtained by carrying out MS analysis of the sample containing a glycopeptide. The figure which shows an example of the display screen in the case of the triplet peak search in MS / MS spectrum. The figure which shows an example of the setting screen of the search conditions in the case of a database search. The figure which shows an example of the result finally output by using for the database search the peak list created based on FIG. The figure for demonstrating the method to search the peptide from which all the sugar_chain | carbohydrates removed from the MS / MS spectrum with respect to an O-type sugar_chain | carbohydrate coupling | bonding glycopeptide, and the peak derived from the peptide which received the simple sugar chain modification.

  Hereinafter, an embodiment of a glycopeptide structure analysis system for performing a glycopeptide structure analysis method according to the present invention will be described with reference to the accompanying drawings. FIG. 1 is an overall configuration diagram of the glycopeptide structure analysis system of this example, and FIG. 2 is a flowchart showing a processing procedure of glycopeptide structure analysis in this system.

  The glycopeptide structure analysis system according to the present embodiment is broadly divided into a mass analyzer 1 and a control / processing unit 2 mainly configured by a computer. The mass analyzer 1 is a matrix-assisted laser desorption ionization quadrupole ion trap time-of-flight mass spectrometer (MALDI-QIT-TOFMS), which is an ionization unit based on MALDI that ionizes molecules and atoms in a sample to be analyzed. 10 and a three-dimensional quadrupole type that can temporarily capture generated ions and perform ion selection according to mass-to-charge ratio (m / z) and ion cleavage by collision-induced dissociation (CID). An ion trap 11 and a time-of-flight mass analyzer 12 that separates and detects various ions emitted from the ion trap 11 according to a mass-to-charge ratio are provided. 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.

  Here, the ionization unit 10 is a MALDI ion source, but the ionization method is not limited to this, and for example, ESI may be used. However, as in the examples described later, it is derived from the desired glycopeptide on the MS spectrum by utilizing the elimination of sialic acid or sulfate group from the sugar chain by ion source decomposition (ISD = Ion-Source Decoy). When detecting these ions, it is necessary to adopt an ionization method in which ISD or the like is likely to occur. For that purpose, MALDI (or other ionization method using laser irradiation) is suitable.

  The control / processing unit 2 includes an analysis control unit 20 that controls each unit of the mass analysis unit 1, a spectrum creation unit 21 that creates an MS spectrum and an MS / MS spectrum based on a detection signal obtained from the ion detector 14, and an MS spectrum. For example, a spectrum analysis unit 22 that performs data processing such as analyzing a MS / MS spectrum and extracting a specific peak and creating a peak list related to the peak, for identifying an amino acid sequence of a peptide The database includes an identification database (DB) 24 in which information is registered in advance, a database search unit 23 that searches for peptides that are highly likely to match the peak list using the database 24, and the like as functional blocks.

  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. The input unit 28 is used by the user to input and set search conditions and perform various operations necessary for spectrum analysis. Specifically, the input unit 28 is a pointing device such as a keyboard or a mouse connected to a computer. It is. The display unit 29 is used to display a search condition input setting screen or display an identification result.

Next, the structure analysis method of glycopeptide in the glycopeptide structure analysis system of the present embodiment will be described with reference to the flowchart of FIG. Here, AXIMA-Q manufactured by Shimadzu Corporation / Kratos Analytical is used as a mass spectrometer, and N-glycan-linked glycopeptide derived from human transferrin (bibranched N-glycan-binding peptide, [M + H] ⁺ : m / z = 3680) as a test sample, and an explanation will be given with an analysis example for an MS / MS spectrum using the sialic acid-desorbed ions (m / z = 3101) as precursor ions.

  The analyst digests the target protein (in this example, human transferrin) with an appropriate enzyme (in this example, trypsin enzyme) to prepare a test sample containing a glycopeptide. When the MS analysis is performed on the test sample by the mass analyzer 1 under the control of the analysis controller 20, the spectrum generator 21 generates an MS spectrum (step S1). The spectrum analysis unit 22 finds a product ion derived from the target glycopeptide from the created MS spectrum, and selects this ion as a precursor ion (step S2).

  In an example where a trypsin enzyme digest of human transferrin is used as a test sample, an MS spectrum as shown in FIG. 3 is obtained. As shown in FIG. 3, since the sugar chain that modifies the human transferrin-derived N-type sugar chain-linked glycopeptide contains sialic acid (m / z = 291), sialic acid is easily detached by MALDI mass spectrometry. An ion peak of a sialic acid-eliminated glycopeptide ion in which sialic acid is eliminated from the original glycopeptide is observed. Therefore, as a method for selecting a precursor ion in step S2, first, two peaks corresponding to sialic acid, that is, a pair peak, are detected from the product ions on the high mass to charge ratio side. In FIG. 3, two peaks of m / z = 339.77 and m / z = 3100.71 are detected as pair peaks. Further, the peak having the strongest peak intensity is selected as the precursor ion among the peaks derived from the plurality of product ions arranged in the mass-to-charge ratio difference of known sugar chains (sugar chain fragments) on the lower mass to charge ratio side. In the example of FIG. 3, since the intensity of the peak at m / z = 3100.71 is the largest, this is selected as the precursor ion. The mass-to-charge ratio of the sugar chain fragments is known to be 162 [Da] for hexose, 203 [Da] for N-acetylhexosamine (HexNAc), and 146 [Da] for fucose.

  If no peak corresponding to the sialic acid elimination ion is observed, whether or not a peak corresponding to the elimination or addition of another group having a known mass-to-charge ratio, the elimination of a sugar chain, etc. is observed. By examining this, the presence or absence of a peak derived from a glycopeptide can be determined.

  Next, under the control of the analysis control unit 20, MS / MS (MS2) analysis in which the precursor ion selected in step S2 is set by the mass analysis unit 1 is executed (step S3). That is, various ions generated from the test sample in the ionization unit 10 are once trapped in the ion trap 11, and only ions having the above-described mass-to-charge ratio are selected in the ion trap 11, followed by one cleavage operation by CID. The Various product ions generated thereby are emitted simultaneously from the ion trap 11, subjected to mass analysis by the time-of-flight mass analyzer 12, and an MS / MS spectrum is created by the spectrum creation unit 21.

  Subsequently, the spectrum analysis unit 22 analyzes the peak appearing in the MS / MS spectrum, and the product ion belonging to the peptide from which all sugar chains are eliminated and one peptide that has undergone simple modification with sugar. Product ions belonging to the peptide to which the sugar (and one sugar fragment) is added are extracted (step S4). In the case of an N-type sugar chain-linked glycopeptide, a triplet peak consisting of three peaks that are characteristic to each other in mass-to-charge ratio appears. Therefore, based on the mass-to-charge ratio of known sugars and sugar cleavage fragments, A triplet peak candidate is searched by determining the mass-to-charge ratio difference of the peaks. When there are a plurality of triplet peak candidates, the candidates are displayed on the screen of the display unit 29 in descending order of the ion peak intensity. For example, the analyst selects one set of triplet peaks using the input unit 28. To do.

  FIG. 4 is a diagram illustrating an example of the display screen 30 displayed on the display unit 29 during the MS / MS spectrum analysis process. In this example, a mass-to-charge ratio difference obtained by elimination of HexNAc and HexNAc ring-cleavage fragment, which is characteristic as a product ion obtained by cleavage of an N-type sugar chain-linked glycopeptide, is 120 [Da], 83 Three peaks lined up with [Da] are detected, and the set of detected peaks is displayed at the top of the MS / MS spectrum 31 with a symbol 32 indicating that it is a triplet peak candidate. Further, in the candidate display window 33 opened separately, three mass-to-charge ratios constituting the triplet peak are listed as a set in order of peak intensity. The analyst selects and designates an appropriate set of triplet peaks from among them. In general, a triplet peak candidate displayed at the top, that is, a triplet peak giving the maximum peak intensity is selected. Note that a triplet peak that gives the maximum peak intensity may be automatically selected without depending on such an analyzer selection instruction. Here, the highest triplet peak in the candidate display window 33 is m / z = 14726.2470, 15599.2850, and 1679.3051.

  For each product ion extracted in step S4, the spectrum analysis unit 22 has a product ion having a low mass-to-charge ratio less than the mass-to-charge ratio of each product ion in MS / MS spectrum (mostly product ions derived from peptide cleavage). ) Is created (step S5). That is, three product ions, a product ion derived from a peptide from which all sugar chains are eliminated, a product ion derived from a peptide modified with HexNAc, and a product ion derived from a peptide modified with a HexNAc ring-cleavage fragment A total of three peak lists having a mass-to-charge ratio less than or equal to the mass-to-charge ratio of the product ions are created. In general, since any peak list includes product ion peaks derived from peptide cleavage, a considerable number of peaks are common to the three peak lists.

  Next, the database search unit 23 sets search conditions for database search for identification described later based on the three peak lists created in step S5 and the modification conditions obtained in step S4 (step S6). . In this example, as described above, the difference in mass-to-charge ratio between the three peaks constituting the triplet peak is 120 [Da] and 83 [Da]. Therefore, modification with HexNAc and modification with HexNAc ring-cleavage fragments, respectively. This is a modification condition to be set as a search condition. Of course, if it is determined from the difference in mass-to-charge ratio between the peaks constituting the triplet peak that the modification is due to another sugar and a sugar fragment, it may be used as the modification condition. Here, the mascot MS / MS ion search method provided by Matrix Science, Inc. described above is used as the database search. However, the database search method is not limited to this, and other well-known methods may be used. Good.

FIG. 5 is an example of a search condition setting screen in the case of the MS / MS ion search method. Here, since the HexNAc ring-cleavage fragment (83 [Da]) is not registered as a modification condition option in the standard state mascot, the HexNAc ring-cleavage fragment (83 [Da]) is selected by the user as a modification condition option. As a mascot. In this example, “ ^0,2 XHexNAc (N)”, which is a ^0,2 X ion generated by cleavage of HexNAc, is registered as a modification condition corresponding to the HexNAc ring cleavage fragment (83 [Da]). And In addition, even when the ring-cleavage fragment of the sugar to be set as the modification condition is not registered in the mascot, the user may perform additional registration in the mascot in advance. In FIG. 5, ^0,2 XHexNAc (N) (indicated as “02xHexNAc (N)” in FIG. 5) and HexNAc (N) indicating the above-described modification conditions are changed according to the input operation of the analyst. modification). At this time, if the analyst knows in advance that there is a modification other than a sugar chain, the modification other than the sugar chain is also set as a fixed modification or a variable modification. In this example, the analyst knows that the C-terminus of the peptide is carbamidomethylated, so this is set as a fixed modification. In general, the type of modification other than the sugar chain depends on the pretreatment method of the protein as a sample, and this is performed by the analyst himself and is therefore known to the analyst.

  The database search unit 23 performs a database search by matching with the peak pattern of the amino acid sequence of the peptide registered in the identification database 24 in accordance with the search condition set in step S6 (step S7). And in the search, since an index (score) indicating the degree of coincidence of the peak pattern with a known peptide is calculated, those having a high score and receiving the specified modification are selected as candidates, for example, in the order of the score. List up (step S8). That is, a plurality of peptide candidates having a high degree of coincidence are listed for each of the three peak lists, but the order is not necessarily the same. Therefore, the database search unit 23 comprehensively determines the rank or score of each candidate in the three results, lists the peptide amino acid sequence candidates in order from the most probable one, and displays the list on the screen of the display unit 29 And displayed to the analyst (step S9).

  FIG. 6 is a diagram showing a result that is finally output by using the peak list created based on FIG. 4 for database search. Specifically, the peak list created for the triplet peak shown at the top of the candidate display window 33 in FIG. 4 is subjected to database search. Here, the search results are presented in descending order of scores among all the results. In this example, in the result presented at the top, the result retrieved from the peak list for the product ion of m / z = 14766.2470 is ranked first and has the highest score (Score) 35. , [CGLPVPVLAENNYNK] amino acid sequence, that is, human transferrin sequence is hit. In addition, in the result searched from the peak list for the product ion of m / z = 1559.2850, the modification by the amino acid sequence + [HexNAc ring-cleavage fragment of [CGLPVPVLAENYNK] is hit at rank 8. Furthermore, in the results retrieved from the peak list for the product ion of m / z = 1679.305, the modification by [CGLVPVLAENNYNK] with the amino acid sequence + HexNAc is hit in the second rank. From this, the amino acid sequence of [CGLVPVLAENYNK] is comprehensively cited as the most likely candidate. The sugar chain binding site is indicated by the underlined position in the amino acid sequence.

  As described above, according to the glycopeptide structure analysis system of the present example, the amino acid sequence of the peptide constituting the glycopeptide based on the MS / MS spectrum obtained using the target glycopeptide-derived ion as the precursor ion. And the sugar chain binding site can be estimated with high reliability.

  In the above description, an N-type sugar chain-linked glycopeptide has been described as an example, but glycoproteins have O-type sugar chain bonds in addition to N-type sugar chain bonds. In general, in the case of an O-type sugar chain-binding glycopeptide, in order to perform the process of step S4, a triplet based on known sugar chain modification conditions on an MS / MS spectrum using a glycopeptide-derived ion as a precursor ion. The peak cannot be used. Therefore, for example, first, detection of a triplet peak based on known sugar chain modification conditions is attempted, and when the triplet peak cannot be detected, it is determined that there is a possibility of an O-type sugar chain-binding glycopeptide. Extraction of product ions attributed to peptides from which all chains have been eliminated and product ions attributed to peptides that have undergone simple modification with sugars may be attempted.

  FIG. 7 is an MS / MS spectrum for an enzymatic digest of fetuin, an O-type sugar chain-linked glycoprotein. As shown in FIG. 7, from the precursor ion with m / z = 3602.84 to the low mass-to-charge ratio side, the mass-to-charge ratio difference is the mass-to-charge ratio of the known sugar chain as described above (for example, hexose). For example, 162 [Da], and N-acetylhexosamine 203 [Da]) is a product ion-derived peak. It can be inferred that the product ion having the smallest mass-to-charge ratio is a peptide ion derived from a peptide from which all sugar chains have been eliminated. Whether this guess is correct is based on the peak derived from the product ion (obtained by peptide cleavage) obtained by amino acid elimination on the low mass-to-charge ratio side of the selected product ion. This can be confirmed. In other words, the validity of the selected peptide can be evaluated by examining whether or not the interval between the peaks in the range of X2 in the MS / MS spectrum shown in FIG. 7 matches the mass-to-charge ratio of a known amino acid. it can.

  As a result, a peptide from which all sugar chains are eliminated is determined, and a peak at a position separated from the high mass-to-charge ratio side by a known sugar chain modification is attributed to one sugar (or one sugar cleavage fragment). A peak derived from a simple modified peptide may be used. Except for the extraction of such product ions, the amino acid sequence and sugar chain binding site of the O-type sugar chain-binding glycopeptide can be identified by performing the same treatment as the above-mentioned N-type sugar chain-binding glycopeptide. .

  In the above embodiment, for example, a set of triplet peaks having the maximum peak intensity is selected in step S4, and the sugar chain structure is identified from the peak list corresponding to the peak. When present, the triplet peak with the maximum peak intensity is not always appropriate. Therefore, for example, the process of steps S4 to S8 is repeated while selecting triplet peaks one by one in descending order of the peak intensity, that is, a database search based on different triplet peaks is executed, and finally the results of the database search In addition, some of the most relevant results may be presented to the analyst.

  Further, whether or not the triplet peak selected when extracting the product ions in step S4 is appropriate may be checked by performing a sugar chain analysis. As a method of glycan analysis, the mass-to-charge ratio of a known glycan is set on the high mass-to-charge ratio side starting from the peptide-derived peak with the smallest mass-to-charge ratio in the triplet peak, that is, all glycans are eliminated The operation of finding peaks that exist at intervals that match the ratio is repeated, and it is finally examined whether or not a peak that matches the peak derived from the precursor ion can be found. If a peak that matches the peak derived from the precursor ion cannot be found, the starting point set at the beginning may not be appropriate, and the triplet peak including the peak may be excluded from the candidates. In addition, it is possible to estimate the composition of the sugar chain that modifies the precursor ion by repeating the operation of finding a peak that exists at an interval that matches the mass-to-charge ratio of the known sugar chain as described above.

  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 ... Spectrum preparation part 22 ... Spectrum analysis Unit 23 ... Database search unit 24 ... Identification database 28 ... Input unit 29 ... Display unit

Claims (4)

A glycopeptide structure analysis method for identifying a peptide constituting a glycopeptide and a sugar chain binding site using a mass spectrometer capable of MS / MS analysis,
a) Find an ion derived from a glycopeptide appearing in an MS spectrum obtained by MS analysis of a test sample, select the ion as a precursor ion, perform MS / MS analysis on the test sample, An analysis execution step of acquiring an MS spectrum;
b) from MS / MS spectra obtained by the analysis execution step, the product ions all sugar chains are attributed to desorbed peptides, caused by cleavage of the N-type sugar chain binding glycopeptides as a simple modification by sugar Search for product ions attributed to peptides that have been modified only with N-acetylhexosamine and modified with a ring-cleavable fragment of N-acetylhexosamine, and for each of the plurality of product ions in the MS / MS spectrum, A peak list creation step for creating a peak list listing ion peaks having a mass to charge ratio equal to or lower than the mass to charge ratio of the product ions;
c) A search condition is set as a search condition that can be modified only by N-acetylhexosamine or by a ring-cleavage fragment of N-acetylhexosamine generated by cleavage of the N-type sugar chain-linked glycopeptide, and the peak list is searched for set, and database search executing step for executing a database search using the identified database in accordance with the search condition,
d) a result providing step of outputting information on the amino acid sequence and sugar chain binding site of the peptide from which a result that the degree of coincidence is relatively high with respect to the plurality of peak lists is obtained by the database search;
A glycopeptide structure analysis method characterized by comprising: The glycopeptide structure analysis method according to claim 1 ,
In the peak list creation step, the product ion attributed to the peptide from which all sugar chains are eliminated, the modification with only N-acetylhexosamine generated by the cleavage of the N-type sugar chain-linked glycopeptide, and the ring of the N-acetylhexosamine Search for a triplet peak having a characteristic mass-to-charge ratio difference to a product ion attributed to a peptide modified by a cleavage fragment, and select a triplet peak that gives the maximum intensity when multiple triplet peaks exist, Based on the triplet peak, a product ion attributed to a peptide from which all sugar chains are eliminated, a modification with only N-acetylhexosamine generated by cleavage of an N-type sugar chain-linked glycopeptide, and a ring of the N-acetylhexosamine Peptides belonging to peptides modified by cleavage fragments A glycopeptide structural analysis method characterized by obtaining a rhododonic ion. In a glycopeptide structure analysis apparatus for identifying a peptide constituting a glycopeptide and a sugar chain binding site using a mass spectrometer capable of MS / MS analysis,
a) Find an ion derived from a glycopeptide appearing in an MS spectrum obtained by MS analysis of a test sample, select the ion as a precursor ion, perform MS / MS analysis on the test sample, Analysis control means for acquiring an MS spectrum;
b) from MS / MS spectrum obtained under the control of the analysis control unit, and product ions all sugar chains are attributed to desorbed peptides, N-type sugar chain binding glycopeptides as a simple modification by sugar The product ions attributed to the peptides that have been modified with only N-acetylhexosamine resulting from the cleavage of N and acetylated with the ring-cleavable fragment of N-acetylhexosamine are searched for, and for each of the plurality of product ions, the MS / A peak list creating means for creating a peak list listing ion peaks having a mass to charge ratio equal to or lower than the mass to charge ratio of the product ion in the MS spectrum;
c) A search condition is set as a search condition that can be modified only by N-acetylhexosamine or by a ring-cleavage fragment of N-acetylhexosamine generated by cleavage of the N-type sugar chain-linked glycopeptide, and the peak list is searched for set, and database search executing means for executing a database search using the identified database in accordance with the search condition,
d) a result providing means for outputting the information on the amino acid sequence and sugar chain binding site of the peptide from which the result that the matching degree is relatively high for the plurality of peak lists is obtained by the database search;
A glycopeptide structure analyzing apparatus comprising: The glycopeptide structure analyzing apparatus according to claim 3 ,
The peak list creation means includes a product ion attributed to a peptide from which all sugar chains are eliminated, a modification only by N-acetylhexosamine generated by cleavage of an N-type sugar chain-binding glycopeptide, and a ring of the N-acetylhexosamine. Search for product ions attributed to peptides modified by cleavage fragments and triplet peaks with a characteristic mass-to-charge ratio difference, and select the triplet peak that gives the maximum intensity when multiple triplet peaks exist. Based on the triplet peak, the product ion attributed to the peptide from which all sugar chains are eliminated, the modification with only N-acetylhexosamine generated by the cleavage of the N-type sugar chain-binding glycopeptide, and the N-acetylhexosamine A product belonging to a peptide modified by a ring-cleavage fragment A glycopeptide structure analyzing apparatus characterized in that it obtains a cact ion.

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