JP2012220365A - Sugar peptide analysis method and analysis apparatus - Google Patents

Abstract

PROBLEM TO BE SOLVED: To identify peptide with high precision even with respect to N-bound sugar peptide although peptide identification may become difficult because of m/z displacement since NHor HO is detached by preferential cyclization in the case where N-terminal amino acid residue is glutamine or cysteine of methyl carbamide.SOLUTION: When implementing de novo sequence processing on a peak train indicating a sugar chain composition that appears in Msspectrum, the presence/absence of m/z displacement caused by NHor HO detachment is estimated from a mass charge ratio difference between one of the peaks and MSprecursor (S4, S5). Further, the presence/absence of similar m/z displacement is also estimated from a mass charge ratio difference between a triplet peak characteristic for N-bound sugar peptide on Msspectrum and a triplet peak on MSspectrum (S6, S7). If the presence of cyclization of N-terminal amino acid residue is determined, that is defined as one of search conditions in database search, thereby improving peptide identification precision.

Description

  The present invention relates to an analysis method for analyzing the structure of a protein or peptide subjected to sugar chain modification using mass spectrometry, and an analysis apparatus using the method.

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 has become very important in various fields such as life science, medicine, and drug development. Due to recent performance improvements of mass spectrometers, mass spectrometry, especially MS ⁿ analysis with multi-step dissociation operations, has become one of the major methods in protein analysis, but in general, glycoprotein analysis has been modified. It is much more difficult than the analysis of non-proteins, and the analysis method is still under study.

  The information that is desired to be acquired in the analysis of glycoproteins is mainly the amino acid sequence, sugar chain structure, and sugar chain binding site of the peptide. Conventionally, general methods for analyzing glycoproteins are as follows (see Non-Patent Document 1).

First, a test sample containing a glycopeptide is prepared by enzymatic digestion of the target glycoprotein, and MS (= MS ¹ ) analysis is performed on the test sample to obtain an MS ¹ spectrum. A peak presumed to be derived from a glycopeptide is selected according to a predetermined standard with respect to the peak appearing in the MS ¹ spectrum, and an ion corresponding to the peak is used as a precursor ion to MS / MS (= MS ² for the test sample). ) Perform analysis to obtain MS ² spectra.

In the case of an N-linked glycopeptide, the MS ² spectrum characteristically has three peaks (hereinafter referred to as “triplet peaks”) arranged at predetermined mass-to-charge ratio intervals. Specifically, from the low mass-to-charge ratio side, peptide ions from which all sugar chains have been eliminated, ^0,2 X (83 Da) -added peptide ions generated by ring cleavage of sugar HexNAc, HexNAc (203 Da) -added peptide ions, Are three peaks corresponding to. Therefore, generally, peptide and sugar chain binding sites are identified by performing MS ³ analysis using this triplet ion as a precursor ion. If a triplet peak with a mass-to-charge ratio that matches this triplet peak is found in the MS ¹ spectrum, each matched ion is selected as a pseudo MS ³ precursor ion, and MS ² analysis (pseudo MS ³ analysis) is performed. ) Is also performed for the purpose of highly sensitive and rapid peptide and sugar chain binding site identification.

In addition, for the MS ² spectrum of an N-linked glycopeptide, the product ion sequence obtained by the neutral loss of sugar between the triplet peak and the MS ² precursor ion is assigned by de novo sequencing. Thus, the sugar chain composition of the MS ² precursor ion is also examined. As a specific method, analysis software such as PEAKS ^TM provided by Bioinformatic Solutions of Canada is known.

By the way, according to Non-Patent Documents 2 to 4 and the like, MS ² analysis with collision-induced dissociation (CID) is performed on a peptide in which glutamine (Gln), glutamic acid (Glu), and carbamidomethylated cysteine (Cys) are located at the N-terminus. In a monovalent ion often observed in matrix-assisted desorption laser ionization (MALDI) mass spectrometry, neutral loss of ammonia (NH ³ ) or water (H ₂ O) derived from N-terminal cyclization (− 17Da or -18Da), and this neutral loss is known to occur preferentially compared to the cleavage of any amino acid in the peptide backbone.

In the MS ² spectrum of glycopeptides, NH ₃ and H ₂ O neutral losses from sugars or amino acids are frequently observed, but in general, the location where NH ₃ and H ₂ O neutral losses occur should be identified in advance. It is difficult. Therefore, even if the product ion generated by the neutral loss of NH ₃ or H ₂ O is selected as the precursor ion for MS ³ analysis and the MS ³ spectrum is acquired, it is appropriately modified as a search condition for database search using a protein database. Conditions cannot be set, and it is difficult to increase peptide identification accuracy. In addition, when estimating the sugar chain composition and sugar chain structure based on the product ion generated by the neutral loss of the sugar against the glycopeptide, it is assumed that NH ₃ and H ₂ O are desorbed from the product ion. Errors in estimation of chain composition and sugar chain structure often occur.

Nakaya, "Conjugate Carbohydrate Analysis-Usefulness of Mass Spectrometer", Shimazu Review, Shimazu Review Editorial Department, Vol. 66, No. 3, No. 4, March 31, 2010, p.173-184 Baldwin and 5 others, “Tandem Mass Spectrometry of Peptides with N-Tempem Mass Spectrometry of Peptides with N- Terminal Glutamine Studies on a Prion Protein Peptide ”, J. Am. Soc. Mass Spectrom., 1990, 1, p.258-264 Neta and four others, “Dehydration Versus Deamination of N-Terminal Glutamine in Collision-Induced Dissociation of a Proto-Peptidized ( Dehydration Versus Deamination of N-Terminal Glutamine in Collision-Induced Dissociation of Protonated Peptides) ", Journal of American Society for Mass Spectrom., 2007, 18, p.27-36 Harrison, “Fragmentation reactions of protonated peptides containing glutamine or glutamic acid”, Journal of American Society・ Four Mass Spectrometry (J. Am. Soc. Mass Spectrom.), 2003, 38, p.174-187

The present invention has been made in view of the above problems, and the object of the present invention is that even if a mass-to-charge ratio shift due to cyclization occurs in the N-terminal amino acid residue of a glycopeptide, MS It is intended to provide a glycopeptide analysis method and analysis apparatus that can improve the accuracy of peptide identification based on ⁿ analysis results.

As described above, when it is presumed that sequence-dependent N-terminal cyclization preferentially occurred in MS ² analysis of glycopeptides, a triplet peak in which a mass-to-charge ratio shift occurred in the MS ² spectrum is formed. MS ³ analysis is performed by selecting at least one ion as a precursor ion and MS ³ spectrum is acquired, and glutamic acid (Glu), glutamine (Gln) or carbamidomethylated cysteine (Cys) is present in the N-terminal sequence It is assumed that it is more efficient to perform a database search and identify the peptides. Also, in MS ² spectrum analysis, when it is estimated that cyclization occurred at the N-terminus of the peptide, the product ion generated from the glycopeptide to the neutral loss of the sugar is determined at the N-terminal amino acid residue of the peptide. Increase the possibility of obtaining accurate information on sugar chain composition and sugar chain structure by trying to assign the product ion after assuming that the mass-to-charge ratio shift has occurred due to NH ₃ or H ₂ O desorption. It is considered possible. For this reason, the present inventor determined whether or not sequence-dependent cyclization has occurred from the MS ² analysis result effectively and with high accuracy, and when such specific cyclization has occurred. I came up with the idea of peptide identification and sugar chain structure analysis.

The 1st invention made in order to solve the said subject uses the mass spectrometer which can perform the ^MSn analysis whose n is 1-3, The structure of the N-linked glycopeptide in a test sample A glycopeptide analysis method for analyzing
a) MS ² precursor selection step of selecting, as a precursor ion, an ion derived from a glycopeptide appearing in an MS ¹ spectrum obtained by MS ¹ analysis of a test sample;
and MS ² analysis execution step of obtaining the MS ² spectra b) the running MS ² analysis on precursor ion was selected in the MS ² precursor selection step to a test sample,
c) While applying a de novo sequencing process to product ion peak sequences arranged with a specific mass-to-charge ratio difference on the MS ² spectrum and assigning product ions corresponding to each peak, the de novo sequence By detecting a specific mass-to-charge ratio shift caused by a difference in mass-to-charge ratio between a product ion having the largest mass-to-charge ratio and a precursor ion among peaks assigned by processing, a ring of a specific amino acid residue at the N-terminus An N-terminal cyclization detection step that estimates the presence of cyclization;
d) A triplet peak characteristically observed in the MS ² spectrum of the N-linked glycopeptide is detected on the MS ² spectrum obtained in the MS ² analysis execution step, and at least one ion constituting the triplet peak MS ³ precursor selection step for selecting as a precursor ion for MS ³ analysis,
and MS ³ analysis execution step of obtaining the MS ³ spectrum e) the relative test sample running MS ³ analysis on selected the precursor ions in the MS ³ precursor selection step,
f) When it is presumed that cyclization of the N-terminal specific amino acid residue is present in the N-terminal cyclization detection step, the cyclization of the N-terminal specific amino acid residue is used as a modification condition, and acquired by the MS ³ analysis execution step. An identification processing step of performing a database search based on information of ion peaks detected in the generated MS ³ spectrum to identify a peptide of a target glycopeptide;
It is characterized by having.

A second invention made to solve the above problems is an apparatus that embodies the glycopeptide analysis method according to the first invention, wherein n is a mass capable of performing MS ⁿ analysis of 1 to 3. In a glycopeptide analyzer that analyzes the structure of an N-linked glycopeptide in a test sample using an analyzer,
a) MS ¹ analysis execution control means for controlling the operation of the mass spectrometer to perform MS ¹ analysis on the test sample and acquire an MS ¹ spectrum;
b) MS ² precursor selection means for selecting a glycopeptide-derived ion appearing in the MS ¹ spectrum as a precursor ion;
c) MS ² analysis execution control means for controlling the operation of the mass spectrometer so as to acquire MS ² spectrum by executing MS ² analysis on the precursor ion selected by the MS ² precursor selection means for the test sample. When,
d) Applying de novo sequencing to product ion peak sequences arranged with a difference in specific mass-to-charge ratio on the MS ² spectrum, and assigning product ions corresponding to each peak, the de novo sequence By detecting a specific mass-to-charge ratio shift caused by a difference in mass-to-charge ratio between a product ion having the largest mass-to-charge ratio and a precursor ion among peaks assigned by processing, a ring of a specific amino acid residue at the N-terminus N-terminal cyclization detection means for estimating the presence of cyclization;
at least one ion that e) N-linked glycopeptide of MS ² spectra detected on characteristically observed the MS ² spectra of triplet peaks were obtained for the test samples are to constitute the triplet peaks MS ³ precursor selection means for selecting as a precursor ion for MS ³ analysis,
f) MS ³ analysis execution control means for controlling the operation of the mass spectrometer to perform MS ³ analysis on the precursor ions selected by the MS ³ precursor selection means on the test sample to acquire an MS ³ spectrum. When,
g) When the N-terminal cyclization detection means presumes that cyclization of the N-terminal specific amino acid residue exists, the cyclization of the N-terminal specific amino acid residue is used as a modification condition, and is obtained for the test sample. An identification processing means for performing a database search based on information of ion peaks detected in the MS ³ spectrum and identifying a peptide of a target glycopeptide;
It is characterized by having.

In the glycopeptide analysis method according to the first invention, in the N-terminal cyclization detection step, for example, one of the triplet peaks on the MS ² spectrum, for example, an ion peak having the largest mass-to-charge ratio is used as the starting point. ^{2 With the} precursor ion peak as the end point, sequentially check the difference in mass-to-charge ratio between adjacent peaks in the product ion peak sequence existing between them, and de novo sequencing the sugar desorption component corresponding to the mass-to-charge ratio difference The product ion or neutral loss corresponding to each peak is assigned by mathematically estimating the product ion, and the product ion peak having the largest mass-to-charge ratio among the assigned product ions is obtained. When neutral loss such as NH ₃ or H ₂ O due to N-terminal cyclization occurs during the dissociation operation of CID, etc., the mass charge between the MS ² precursor ion peak and the product ion having the largest mass-to-charge ratio The neutral loss of sugar corresponding to the difference in ratio is not found and cannot be assigned. On the other hand, MS ² attribution of neutral loss of sugar virtual MS ² precursor ions shifted to the low mass to charge ratio side specific mass-to-charge ratio difference Delta] m (17Da or 18 Da) against the precursor ion as an end point of the de novo sequencing process If possible, that is, if the product ion having the largest mass-to-charge ratio can be assigned as the neutral loss of sugar from the virtual MS ² precursor ion, this is not a sugar but a specific amino acid at the N-terminal in the peptide ion. It is presumed that there is a high possibility of a mass-to-charge ratio shift due to residue cyclization.

If it can be estimated with high accuracy that cyclization of the N-terminal specific amino acid residue has occurred in this way, when identifying a peptide by database search using the peak information of the MS ³ spectrum, Cyclization of a specific amino acid residue may be set as one of the modification conditions for the search. Thereby, since the search range of the database search is considerably narrowed, the accuracy of peptide identification can be improved. In the N-terminal cyclization detection step, the assignment of neutral loss assuming the presence of cyclization of the N-terminal specific amino acid residue between the triplet peak and the MS ² precursor ion peak is performed. The estimation accuracy of the sugar chain structure can also be improved.

The triplet peak that is characteristically observed in the MS ² spectrum of the N-linked glycopeptide is typically three peaks lined up with a mass-to-charge ratio difference of 83 Da and 120 Da from the smaller mass-to-charge ratio. It is. These three peaks correspond to a peptide ion from which all sugar chains are eliminated, a ^0,2 X (83 Da) -added peptide ion, and a HexNAc-added peptide ion generated by ring cleavage of the sugar HexNAc.

In addition, when MS ¹ analysis of a glycopeptide is performed, pseudo MS ² analysis is performed by in-source decomposition or the like, and a triplet peak having a specific mass-to-charge ratio difference as described above is observed in the MS ¹ spectrum. Sometimes. In such a case, the intensity of the triplet peak that generally appears in the MS ¹ spectrum is greater than the intensity of the triplet peak that appears in the MS ² spectrum. Therefore, a triplet peak appearing in the MS ¹ spectrum is selected as a pseudo MS ³ precursor ion, and a pseudo MS ³ spectrum is obtained by performing MS ² analysis (pseudo MS ³ analysis) on the precursor ion, and this pseudo MS ³ spectrum is obtained. It is possible that peptide identification with higher accuracy can be performed by subjecting the peak information to the database search in the same manner as the peak information of the normal MS ³ spectrum.

That is, the glycopeptide analysis method according to the first invention preferably has
Discrimination whether or not a triplet peak obtained by adding a specific mass-to-charge ratio shift obtained in the N-terminal cyclization detection step to the triplet peak detected on the MS ² spectrum is detected in the MS ¹ spectrum. and a pseudo-MS ³ precursor selection step of selecting this as pseudo MS ³ precursor ion when triplet peaks were detected,
Further anda pseudo MS ³ analysis execution step of obtaining the pseudo-MS ³ spectra running pseudo MS ³ analysis on selected pseudo MS ³ precursor ion in the pseudo MS ³ precursor selection step to said test sample ,
In the identification processing step, when it is estimated that cyclization of the N-terminal specific amino acid residue is present in the N-terminal cyclization detection step, cyclization of the N-terminal specific amino acid residue is used as a modification condition, and the pseudo MS ³ A database search based on information on ion peaks detected in the pseudo MS ³ spectrum acquired by the analysis execution step may be executed to identify the peptide of the target glycopeptide.

  Moreover, although the estimation process of N-terminal specific amino acid residue cyclization carried out in the glycopeptide analysis method according to the first invention and the glycopeptide analysis apparatus according to the second invention has sufficiently high estimation accuracy, It is possible to further improve the estimation accuracy by adding estimation from another viewpoint.

As a specific embodiment, in the glycopeptide analysis method according to the first invention, the N-terminal cyclization detection step is performed in a specific mass produced by a difference in mass to charge ratio between at least one assigned product ion and a precursor ion. In addition to detecting the mass-to-charge ratio shift, the mass-charge between the triplet peak detected on the MS ² spectrum and the triplet peak detected on the MS ¹ spectrum with the same mass-to-charge difference as the triplet peak. When the difference in the ratio is a specific value, such as 17 Da or 18 Da, it may be assumed that there is cyclization of the specific amino acid residue at the N-terminus. If there are a plurality of sets of similar triplet peaks, for example, the one having the maximum sum of the triplet peak intensities may be selected.

  As another embodiment, in the glycopeptide analysis method according to the first invention, the N-terminal cyclization detection step is carried out using the cyclization of the N-terminal specific amino acid residue performed in the identification processing step as a modification condition. As a result of the database search, when the cyclized peptides match under a predetermined condition, it may be estimated that the cyclization of the specific amino acid residue at the N-terminal exists.

  In the N-terminal cyclization detection step, when it is determined that there is no cyclization of the N-terminal specific amino acid residue, peptide identification and estimation of the sugar chain composition may be performed by a conventionally known method. .

Of course, in the N-terminal cyclization detection step in the glycopeptide analysis method according to the first invention, detecting a specific mass-to-charge ratio shift caused by a difference in mass-to-charge ratio between at least one assigned product ion and a precursor ion instead had to estimate the presence of cyclization of a particular amino acid residues at the N-terminus by a triplet peak detected on MS ² spectra, said with a same mass-to-charge ratio value and the triplet peaks MS ¹ spectrum When the difference in mass-to-charge ratio from the triplet peak detected above is a specific value, for example, 17 Da or 18 Da, it may be assumed that cyclization of a specific amino acid residue at the N-terminus exists.
That is, another aspect related to the first invention is to analyze the structure of an N-linked glycopeptide in a test sample using a mass spectrometer capable of performing MS ⁿ analysis where n is 1 to 3. A glycopeptide analysis method for
a) MS ² precursor selection step of selecting, as a precursor ion, an ion derived from a glycopeptide appearing in an MS ¹ spectrum obtained by MS ¹ analysis of a test sample;
and MS ² analysis execution step of obtaining the MS ² spectra b) the running MS ² analysis on precursor ion was selected in the MS ² precursor selection step to a test sample,
c) A triplet peak characteristically observed in the MS ² spectrum of the N-linked glycopeptide is detected on the MS ² spectrum obtained in the MS ² analysis execution step, and has the same mass-to-charge ratio as the triplet peak. Cyclization of a specific amino acid residue at the N-terminal by detecting a triplet peak having a difference on the MS ¹ spectrum and checking whether the difference between the mass-to-charge ratios of the two triplet peaks is a specific value. An N-terminal cyclization detection step that presumes the presence of
d) MS ³ precursor selection step of selecting at least one ion constituting the triplet peak detected on the MS ² spectrum as a precursor ion for MS ³ analysis;
and MS ³ analysis execution step of obtaining the MS ³ spectrum e) the relative test sample running MS ³ analysis on selected the precursor ions in the MS ³ precursor selection step,
f) When it is presumed that cyclization of the N-terminal specific amino acid residue is present in the N-terminal cyclization detection step, the cyclization of the N-terminal specific amino acid residue is used as a modification condition, and acquired by the MS ³ analysis execution step. An identification processing step of performing a database search based on information of ion peaks detected in the generated MS ³ spectrum to identify a peptide of a target glycopeptide;
It can have. In addition, since the aspect of this analysis method or the apparatus embodying this method can also estimate the presence or absence of N-terminal specific amino acid residue cyclization with sufficiently high accuracy, Almost the same effect can be achieved.

  According to the glycopeptide analysis method according to the first invention and the glycopeptide analysis apparatus according to the second invention, the glycopeptide to be analyzed has a specific amino acid residue such as glutamine, glutamic acid, carbamidomethylated cysteine at the N-terminus Even if a mass-to-charge ratio shift due to cyclization occurs in the amino acid residue due to CID, it is possible to identify peptides with high accuracy and to estimate sugar chain composition and sugar chain structure. it can.

BRIEF DESCRIPTION OF THE DRAWINGS The whole block diagram of one Example of the glycopeptide analysis system which enforces the glycopeptide analysis method based on this invention. The flowchart which shows the glycopeptide analysis process procedure implemented in the glycopeptide analysis system of a present Example. The conceptual diagram for demonstrating the glycopeptide analysis process shown in FIG. The conceptual diagram for demonstrating the glycopeptide analysis process shown in FIG. The figure which shows an example of the search condition setting screen of MS / MS ion search.

  Hereinafter, an embodiment of a glycopeptide analysis system for carrying out a glycopeptide analysis method according to the present invention will be described with reference to the accompanying drawings. FIG. 1 is an overall configuration diagram of a glycopeptide analysis system of the present embodiment, FIG. 2 is a flowchart showing a processing procedure of glycopeptide analysis in this system, and FIGS. 3 and 4 explain the glycopeptide analysis process shown in FIG. It is a conceptual diagram for.

  The glycopeptide analysis system according to the present embodiment is broadly divided into a mass analyzer 1 and a control / processing unit 2 mainly composed of a computer. The mass spectrometer 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 ion trap that can temporarily trap generated ions and perform ion selection according to mass-to-charge ratio m / z and ion dissociation by collision-induced dissociation (CID) 11 and a time-of-flight mass analyzer (TOFMS) 12 that separates and detects various ions emitted from the ion trap 11 according to a 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.

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, when detecting ions of the desired glycopeptide on the MS ¹ spectrum using the elimination of sialic acid or sulfate groups from sugar chains due to insource decomposition, etc. It is necessary to adopt an ionization method that easily occurs, and for that purpose, a MALDI method or other ionization method using laser irradiation is suitable.

The control / processing unit 2 includes an analysis control unit 3 that controls each unit of the mass analysis unit 1, a data processing unit 20 that processes a detection signal obtained from the ion detector 14, and a user who inputs and sets search conditions or performs spectral analysis. Control for controlling the entire system in an integrated manner is connected to an input unit 5 for performing various operations necessary for the purpose and a display unit 6 for displaying a search condition input setting screen and displaying an identification result. Part 4. The data processing unit 20 includes a spectrum generation unit 21 that generates an MS ⁿ spectrum based on the detection signal, a spectrum analysis unit 22 that performs data processing such as extracting a specific peak by analyzing the MS ⁿ spectrum, MS ⁿ A de novo sequence analysis processing unit 23 for performing processing by de novo sequence on the peak information collected from the spectrum, an identification database (DB) 25 in which identification information for estimating the amino acid sequence of the peptide is registered in advance, and the database 25 includes a database search unit 24 that searches for peptides that are highly likely to match the peak list. For example, the database search unit 24 can use a search engine named MS / MS ion search included in Mascot provided by Matrix Science, UK. For the function of the de novo sequence analysis processing unit 23, for example, software such as the above-described PEAKS ^™ can be used.

  Next, a characteristic N-linked glycopeptide analysis method in the glycopeptide analysis system of this example will be described with reference to the flowchart of FIG.

The analyst digests the target protein with an appropriate enzyme (for example, trypsin enzyme) to prepare a test sample containing a glycopeptide. When MS ¹ analysis is performed on the test sample by the mass analysis unit 1 under the control of the analysis control unit 3, the spectrum creation unit 21 creates an MS ¹ spectrum based on the data obtained by the MS ¹ analysis. (Step S1).

The spectrum analysis unit 22 finds the product ion peak expressed by the neutral loss of the sugar chain such as sialic acid from the MS ¹ spectrum created in step S1, and selects this product ion as a precursor ion for MS ² analysis. (Step S2).

Next, when the MS ² analysis for the precursor ion selected in step S2 is performed on the test sample by the mass analysis unit 1 under the control of the analysis control unit 3, the spectrum creation unit 21 performs the MS. ^Two spectra are created (step S3). More specifically, various ions generated from the test sample in the ionization unit 10 are once trapped in the ion trap 11, and only ions having a mass-to-charge ratio of precursor ions are selected in the ion trap 11, and then once by CID. The dissociation operation is performed. Various product ions generated thereby are simultaneously emitted from the ion trap 11 and subjected to mass analysis by the time-of-flight mass analyzer 12. Based on the detection signal obtained thereby, the spectrum creation unit 21 creates an MS ² spectrum. .

In the example shown in FIG. 3, the ion peak of m / z = M1 is selected as the precursor ion on the MS ¹ spectrum shown in (a), and the result of performing MS ² analysis on it is shown in (b). MS ² spectra were obtained. As described above, when the N-linked glycopeptide contained in the test sample has a specific amino acid residue such as glutamine, glutamic acid, or carbamidomethylated cysteine at the N-terminus, the CID of step S3 In the process, the cyclization of a specific amino acid residue at the N-terminus occurs preferentially, and as a result, a neutral loss of NH ₃ or H ₂ O occurs. As a result, each product ion peak generated by the neutral loss of sugar appearing in the MS ² spectrum is shifted in the direction of low mass-to-charge ratio by the amount of NH ₃ or H ₂ O neutral loss, that is, 17 Da or 18 Da. It will be. These preferential cyclizations occur simultaneously with the neutral loss of sugars in glycopeptides, and particularly when the N-terminal amino acid residue is glutamine (Gln), a sugar chain containing ions constituting the triplet peak described above The present inventor has confirmed that a mass-to-charge ratio shift of 17 Da to the low mass-to-charge ratio side occurs in all the product ions obtained with the neutral loss.

Subsequently, the spectrum analysis unit 22 and the de novo sequence analysis processing unit 23 analyze the peak appearing in the MS ² spectrum, and execute the de novo sequence processing based on the peak information, thereby converting the peak in the MS ² spectrum into the CID. It is attributed as a product ion produced by detaching the sugar neutrally (without changing the charge) by the dissociation action such as. Then, it is estimated whether there is a mass-to-charge ratio shift peculiar to the cyclization of the N-terminal amino acid residue using the assignment result (step S4).

Specifically, first, a triplet peak characteristically appearing in the MS ² spectrum of an N-linked glycopeptide (three peaks with a mass-to-charge ratio difference of 83 Da and 120 Da from the low mass-to-charge ratio side) is searched. When there are a plurality of triplet peak candidates, for example, the candidates are displayed on the screen of the display unit 6 in order from the group having the highest ion peak intensity. For example, the analyst can select one triplet peak from the input unit 5. Just choose. If a triplet peak is detected, the peak having the maximum mass-to-charge ratio in the triplet peak is set as the starting point, and the mass-to-charge ratio M1 of the precursor ion in MS ² analysis is set as the ending point. The mass-to-charge ratio difference between two adjacent peaks, that is, the product ions generated by the neutral loss of the sugar chain are sequentially assigned (see FIG. 3B). For the peaks that cannot be assigned, for example, an appropriate algorithm such as skipping the number of peaks can be adopted.

Then, product ions are assigned in order, and a product ion peak having the largest mass-to-charge ratio among the assigned product ions is obtained. The neutral loss of sugar cannot be assigned between the product ion peak having the largest mass-to-charge ratio and the precursor ion of MS ² analysis, and Δm ((() with respect to the mass-to-charge ratio M 1 of the precursor ion of MS ² analysis 17Da or 18Da), assuming that the neutral loss of the sugar could be assigned to a hypothetical MS ² precursor ion assuming a neutral loss of 17 (or a mass to charge ratio of M1-Δm) When it is possible to assign a product ion having a large mass-to-charge ratio as a neutral loss of a sugar from a virtual MS ² precursor ion, there is a mass-to-charge ratio shift peculiar to the cyclization of an N-terminal amino acid residue (Y in step S5).

If a triplet peak cannot be detected on the MS ² spectrum, it is highly likely that the O-linked glycopeptide is not subject to analysis here, but it is desired to estimate the sugar chain composition of the glycopeptide. For this, the glycan composition estimation may be performed by de novo sequencing from the MS ² precursor ion toward the direction in which the mass to charge ratio decreases.

  Moreover, since the process of step S4 is substantially equivalent to estimating a sugar chain composition, it is also possible to estimate a sugar chain composition and a sugar chain structure based on the attribution information obtained here.

If it is estimated in step S5 that the N-terminal amino acid residue is cyclized, the process proceeds to step S6. From a viewpoint different from that in step S4, specifically, a specific observation observed on the MS ² spectrum is performed. Based on the difference in mass-to-charge ratio between the triplet peak and the specific triplet peak observed on the MS ¹ spectrum, the presence or absence of cyclization of the N-terminal amino acid residue is confirmed.

That is, the spectrum analysis unit 22 searches for the triplet peak composed of the three peaks in which the mass-to-charge ratio difference is 83 Da and 120 Da from the low mass-to-charge ratio side in the MS ¹ spectrum acquired in step S1. Then, a difference Δm in mass-to-charge ratio between the triplet peak T1 on the MS ¹ spectrum and the triplet peak T2 on the MS ² spectrum detected in step S4 is obtained, and this difference is used for desorption of NH ₃ or H ₂ O. In the case of corresponding 17 Da or 18 Da, it is presumed that there is a mass-to-charge ratio shift due to cyclization of the N-terminal specific amino acid residue with respect to the peptide ion rather than the sugar chain (Y in step S7). In this case, since the same result is obtained in the two different estimations of step S4 and step S6, it is determined that cyclization of the N-terminal amino acid residue exists at this point (step S8).

  In either step S4 or step S6, if a result that does not match the mass-to-charge ratio shift corresponding to the cyclization of the N-terminal amino acid residue of the peptide is obtained, it is determined as N in steps S5 and S7, Step S8 is passed and it progresses to step S9. Therefore, in this case, it can be concluded that there is no cyclization of the N-terminal amino acid residue of the peptide.

Proceeding to step S9, the spectrum analysis unit 22 converts the MS ² spectrum acquired in step S3 into ions corresponding to the triplet peaks extracted in step S4, that is, peptides from which all sugar chains are eliminated. MS ^{3 represents} one of the product ion derived, the product ion derived from the peptide modified with the HexNAc ring-cleavage fragment to which one sugar chain is added, and the product ion derived from the peptide modified with HexNAc. Select as precursor ion for analysis.

In step S6, a triplet peak is detected on the MS ¹ spectrum as shown in FIG. 4, and the triplet peak on the MS ² spectrum corresponding to the ion intensity of one of the ions constituting the triplet peak is formed. When the ion intensity is larger than one ion, an ion corresponding to a triplet peak on the MS ¹ spectrum may be selected as a precursor ion for pseudo MS ³ analysis. This is because when the in-source decomposition appears remarkably is MS ¹ spectrum was substantially the MS ² spectra equivalent pseudo MS ² spectra, is better to implement the pseudo MS ³ analysis for pseudo MS ³ precursor ion MS ³ there is a high likely that pseudo MS ³ spectra are obtained ionic strength than the spectral.

Information on the selected precursor ion is sent to the analysis control unit 3, and under the control of the analysis control unit 3, MS ³ analysis (or pseudo MS ³ analysis) on the precursor ion is executed in the mass analysis unit 1. Based on the data acquired by the above, the spectrum creation unit 21 creates an MS ³ spectrum (or pseudo MS ³ spectrum) (step S10).

Next, the spectrum analysis unit 22 collects information on peaks from which the significant peaks, that is, noise peaks are removed from the created MS ³ spectrum (or pseudo MS ³ spectrum), and creates a peak list (step S11). Thereafter, the database search unit 24 sets search conditions for database search for peptide identification, which will be described later, based on the peak list created in step S11 and the modification information known or estimated (step S12). If it is determined in step S8 that cyclization of the N-terminal amino acid residue exists, it is set as one of the modification conditions. Specifically, “Gln-> Pyro-Glu (N-term Q)” or the like may be set as a variable modification at the time of MS / MS ion search setting as described later. Further, if it is found from the result of de novo sequence treatment or the like that there is a modification with a specific sugar or a sugar cleavage fragment, it may be added as a modification condition.

  The database search unit 24 performs a database search by collating with the peak pattern of the amino acid sequence of the peptide registered in the identification database 25 according to the search condition set in step S12 (step S13). For example, the MS / MS ion search method can be used as the database search, but the database search method is not limited to this, and other known methods may be used.

  For example, in a database search by the MS / MS ion search method, an index (score) indicating the degree of coincidence of a peak pattern with a known peptide is calculated, so that the score is high and the specified modification is applied. The candidates are listed in order of score, for example. In addition, sugar chain composition candidates estimated by the de novo sequence processing in step S4 are also listed. Then, the list is displayed on the screen of the display unit 6 as an identification result and presented to the analyst (step S14).

In the glycopeptide analysis system of this example, when the N-terminal amino acid residue of the N-linked glycopeptide is glutamine, glutamic acid, or carbamidomethylated cysteine, NH ₃ or H ₃ is obtained by cyclization of the amino acid residue. _{Even if 2} O is desorbed, this preferential desorption is detected at the stage of MS ² spectrum, and a database is provided on the condition that such a specific amino acid residue exists at the N-terminus of the peptide. Peptide identification can be performed by searching. Therefore, the accuracy of peptide identification in N-linked glycopeptides can be improved.

  In the above example, in addition to estimating the presence or absence of cyclization of the N-terminal amino acid residue in step S4, in step S6, estimating the presence or absence of cyclization of the N-terminal amino acid residue by another method, Although the reliability of the estimation is increased, in many cases, according to the study of the present inventor, the presence or absence of cyclization of the N-terminal amino acid residue is determined with a sufficiently high reliability only by the estimation in step S4. be able to. Therefore, even if the processing of steps S6 and S7 is omitted, there is no practical problem.

In addition, instead of omitting the processes of steps S6 and S7, a database search is attempted for the peak information collected from the MS ² spectrum, and a process for determining whether or not the N-terminal amino acid residue is cyclized based on the result is added to the flow. You may do it. Here, MS / MS ion search is used for database search.

  FIG. 5 is an example of a search condition setting screen for MS / MS ion search. The main search conditions to be set are the database used for matching (Database), the type of digestive enzyme used for protein degradation (Enzyme), and the type of modification that occurs deterministically (fixed modification: Fixed modification), types of (non-deterministic) modifications that may occur (variable modification), tolerance of mass spectrometry accuracy (MS / MS tol.), Etc. Cyclization of the N-terminal amino acid residue as described above can be designated as a variable modification. “Gln-> Pyro-Glu (N-term Q)” shown in FIG. 5 indicates that the N-terminal glutamine (Q) residue is cyclized to become pyroglutamic acid (Pyro-Glutamic acid). “Glu-> Pyro-Glu (N-term E)” indicates a case where the N-terminal glutamic acid (E) residue is cyclized to become pyroglutamic acid (“MASCOT Server Modification Definition Method 3”, Internet <URL: (See http://www.matrixscience.jp/pdf/jap_mod_file.pdf>). In addition, “Pyro-carbamidomethyl (N-term C)” is referred to as a variable modifier as a case where N-terminal carbamidomethylated cysteine (C) is cyclized to form pyrocarbamidomethyl cysteine. It can be specified as a citation.

Therefore, the spectrum analysis unit 22 collects peaks from the MS ² spectrum acquired in step S3 and creates a peak list, and the database search unit 24 uses “Gln-> Pyro” as a variable modification for the peak list. -Glu (N-term Q) ","Glu-> Pyro-Glu (N-term E) "," Pyro-carbamidomethyl (N-term C) ", etc. are specified and a database search is executed. As a result, it is confirmed whether or not the cyclized peptide hits under certain conditions. For example, if a peptide with glutamine or glutamic acid cyclized at the N-terminal amino acid residue hit with the highest score, the conditions specified as variable modification are appropriate, ie, N ^{2 in} the MS ² spectrum. It is determined that elimination of NH ₃ or H ₂ O occurs due to cyclization of the terminal amino acid residue. If it is determined as Y in step S5, and it is further determined that the N-terminal amino acid residue is cyclized as a result of the database search for the MS ² spectrum as described above, the process proceeds to step S9 and MS ³ analysis is performed. Should be executed. Even if it does in this way, since the estimation of the presence or absence of the cyclization of an N terminal amino acid residue is made with high reliability, the precision of peptide identification can be improved.

  It should be noted that the above embodiment is merely an example of the present invention, and it will be understood 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 / processing part 20 ... Data processing part 21 ... Spectrum preparation part 22 ... Spectral analysis Unit 23 ... De novo sequence analysis processing unit 24 ... Database search unit 25 ... Identification database 3 ... Analysis control unit 4 ... Control unit 5 ... Input unit 6 ... Display unit

Claims (5)

A glycopeptide analysis method for analyzing the structure of an N-linked glycopeptide in a test sample using a mass spectrometer capable of performing MS ⁿ analysis in which n is 1 to 3,
a) MS ² precursor selection step of selecting, as a precursor ion, an ion derived from a glycopeptide appearing in an MS ¹ spectrum obtained by MS ¹ analysis of a test sample;
and MS ² analysis execution step of obtaining the MS ² spectra b) the running MS ² analysis on precursor ion was selected in the MS ² precursor selection step to a test sample,
c) While applying a de novo sequencing process to product ion peak sequences arranged with a specific mass-to-charge ratio difference on the MS ² spectrum and assigning product ions corresponding to each peak, the de novo sequence By detecting a specific mass-to-charge ratio shift caused by a difference in mass-to-charge ratio between a product ion having the largest mass-to-charge ratio and a precursor ion among peaks assigned by processing, a ring of a specific amino acid residue at the N-terminus An N-terminal cyclization detection step that estimates the presence of cyclization;
d) A triplet peak characteristically observed in the MS ² spectrum of the N-linked glycopeptide is detected on the MS ² spectrum obtained in the MS ² analysis execution step, and at least one ion constituting the triplet peak MS ³ precursor selection step for selecting as a precursor ion for MS ³ analysis,
and MS ³ analysis execution step of obtaining the MS ³ spectrum e) the relative test sample running MS ³ analysis on selected the precursor ions in the MS ³ precursor selection step,
f) When it is presumed that cyclization of the N-terminal specific amino acid residue is present in the N-terminal cyclization detection step, the cyclization of the N-terminal specific amino acid residue is used as a modification condition, and acquired by the MS ³ analysis execution step. An identification processing step of performing a database search based on information of ion peaks detected in the generated MS ³ spectrum to identify a peptide of a target glycopeptide;
A method for analyzing a glycopeptide, comprising: The glycopeptide analysis method according to claim 1,
Discrimination whether or not a triplet peak obtained by adding a specific mass-to-charge ratio shift obtained in the N-terminal cyclization detection step to the triplet peak detected on the MS ² spectrum is detected in the MS ¹ spectrum. and a pseudo-MS ³ precursor selection step of selecting this as pseudo MS ³ precursor ion when triplet peaks were detected,
Further anda pseudo MS ³ analysis execution step of obtaining the pseudo-MS ³ spectra running pseudo MS ³ analysis on selected pseudo MS ³ precursor ion in the pseudo MS ³ precursor selection step to said test sample ,
In the identification processing step, when it is estimated that cyclization of the N-terminal specific amino acid residue is present in the N-terminal cyclization detection step, cyclization of the N-terminal specific amino acid residue is used as a modification condition, and the pseudo MS ³ A glycopeptide analysis method comprising performing a database search based on information on ion peaks detected in a pseudo MS ³ spectrum acquired by an analysis execution step to identify a peptide of a target glycopeptide. The glycopeptide analysis method according to claim 1 or 2,
The N-terminal cyclization detection step is detected on the MS ² spectrum in addition to detecting a specific mass-to-charge ratio shift caused by a difference in mass-to-charge ratio between at least one assigned product ion and a precursor ion. A specific amino acid residue at the N-terminal when the difference in mass to charge ratio between the triplet peak and the triplet peak detected on the MS ¹ spectrum with the same mass to charge ratio difference as the triplet peak is a specific value. A glycopeptide analysis method characterized in that it is presumed that cyclization occurs. The glycopeptide analysis method according to claim 1 or 2,
In the N-terminal cyclization detection step, as a result of database search using the cyclization of the N-terminal specific amino acid residue as a modification condition executed in the identification processing step, the cyclized peptides matched under a predetermined condition. In this case, it is presumed that cyclization of a specific amino acid residue at the N-terminal is present. In a glycopeptide analyzer that analyzes the structure of an N-linked glycopeptide in a test sample using a mass spectrometer capable of performing MS ⁿ analysis in which n is 1 to 3,
a) MS ¹ analysis execution control means for controlling the operation of the mass spectrometer to perform MS ¹ analysis on the test sample and acquire an MS ¹ spectrum;
b) MS ² precursor selection means for selecting a glycopeptide-derived ion appearing in the MS ¹ spectrum as a precursor ion;
c) MS ² analysis execution control means for controlling the operation of the mass spectrometer so as to acquire MS ² spectrum by executing MS ² analysis on the precursor ion selected by the MS ² precursor selection means for the test sample. When,
d) Applying de novo sequencing to product ion peak sequences arranged with a difference in specific mass-to-charge ratio on the MS ² spectrum, and assigning product ions corresponding to each peak, the de novo sequence By detecting a specific mass-to-charge ratio shift caused by a difference in mass-to-charge ratio between a product ion having the largest mass-to-charge ratio and a precursor ion among peaks assigned by processing, a ring of a specific amino acid residue at the N-terminus N-terminal cyclization detection means for estimating the presence of cyclization;
at least one ion that e) N-linked glycopeptide of MS ² spectra detected on characteristically observed the MS ² spectra of triplet peaks were obtained for the test samples are to constitute the triplet peaks MS ³ precursor selection means for selecting as a precursor ion for MS ³ analysis,
f) MS ³ analysis execution control means for controlling the operation of the mass spectrometer to perform MS ³ analysis on the precursor ions selected by the MS ³ precursor selection means on the test sample to acquire an MS ³ spectrum. When,
g) When the N-terminal cyclization detection means presumes that cyclization of the N-terminal specific amino acid residue exists, the cyclization of the N-terminal specific amino acid residue is used as a modification condition, and is obtained for the test sample. An identification processing means for performing a database search based on information of ion peaks detected in the MS ³ spectrum and identifying a peptide of a target glycopeptide;
A glycopeptide analyzing apparatus comprising:

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