JP2015145840A - Analytic method for sugar peptide and program for sugar chain structure analysis - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide an analytic method for sugar chain structure of sugar peptide that can easily identify a detailed sugar chain structure without being affected greatly by an amino acid sequence of a peptide part.SOLUTION: In an analytic method, a sugar chain structure of sugar peptide is analyzed through negative ion mass analysis. An MSprecursor is selected out of fragment ions originating from negative ions of sugar peptide with which N-combined type sugar chain is combined and MSanalysis is taken. As the MSprecursor, a fragment ion is selected which has a mass charge ratio (G+97-z)/z. Here, G is the mass of a sugar chain residue and "z" is a natural number. In this analytic method, structure information on the N-combined type sugar chain can be extracted based upon an MSspectrum of the sugar peptide obtained through the mass analysis.

Description

  The present invention relates to a glycopeptide analysis method and a sugar chain structure analysis program for realizing the analysis method.

  Glycosylation to a peptide chain is one of the most important processes in post-translational modification. Glycoproteins with sugar chains added to peptide chains are involved in various life phenomena. In vivo, it is thought that signal transduction and molecular recognition between cells are controlled by precisely recognizing slight differences in the structure of sugar chains, and structural analysis of glycoproteins and glycopeptides It is expected to contribute greatly to elucidation of life phenomena, drug discovery, biomarker development, etc.

In structural analysis of proteins and DNA, applications of multi-stage mass spectrometry (MS / MS analysis or MS ⁿ analysis where n is 2 or more) using ion cleavage methods such as ISD, CID, IRMPD, ECD, ETD, etc. are progressing. In the structural analysis of sugar chains, the application of multistage mass spectrometry is progressing.

In mass spectrometry of sugar chains, the spectrum of MS ² or MS ⁿ (n is 3 or more) of a sugar chain standard sample is stored as a database, and the mass spectrum of the analysis target is matched with the mass spectrum of the database. A method for identifying a sugar chain structure has been applied. Also, based on the existing glycan structure database, a list of glycan theoretical fragments that can be generated is created as a database, and the glycan structure is estimated by matching the mass spectrum obtained from the analysis target with the theoretical fragment. Has been developed.

  In conventional sugar chain structure analysis by mass spectrometry, analysis of free sugar chains released from glycoproteins or glycopeptides has been the mainstream. Since the free sugar chain does not contain a peptide-derived structure, it is easy to identify the structure by matching with the database as described above. However, analysis of free sugar chains is easy to analyze the structure of sugar chains, but the origin of sugar chains is not clear. For example, the possibility of analyzing sugar chains derived from contaminants cannot be denied. There's a problem.

  On the other hand, in glycopeptide analysis, the origin of a sugar chain is clear, and information on the addition position of a sugar chain in a peptide can be obtained. Glycopeptide analysis is a field that has attracted particular attention in recent years because it allows heteroglycan structure analysis for each additional site and provides information that cannot be obtained by conventional free sugar chain analysis.

In MS ² analysis of glycopeptides, fragments derived from peptides, fragments derived from sugar chains, and fragments containing both peptides and sugar chains can be generated. In general, in MS ² analysis of glycopeptides, the amount of glycan-derived fragments is small, and no glycan-derived fragments are generated at all, which makes it difficult to analyze the glycan structure. In the MS ² analysis of glycopeptides, the amino acid sequence of the peptide part greatly affects the cleavage of the sugar chain part. Therefore, the MS ² or MS ⁿ database of glycopeptides needs to include data on both the sugar chain structure and amino acid sequence of each glycopeptide. Considering the diversity of amino acid sequences, it is extremely difficult to identify structures by matching mass spectra and fragments of glycopeptides into a database and matching them with conventional analysis methods.

In view of such problems related to the structural analysis of glycopeptides, attempts have been made to analyze the structure of sugar chains by adopting an ionization method that preferentially generates sugar chain-derived fragments in MS ⁿ analysis. . For example, Patent Document 1 shows that the primary structure of a sugar chain can be analyzed by positive ion mode MS ⁿ analysis of a metal adduct such as sodium.

  However, in mass spectrometry of positive ion mode, it is often difficult to identify the branched structure of a sugar chain. In sugar chains, all of the hydroxy groups of monosaccharides that are constituent materials can participate in glycosidic bonds, and thus often have a branched structure. For example, an N-linked sugar chain bonded to an asparagine residue has at least one branch. It is also known that fucose is transferred to N-acetylglucosamine (GlcNAc) at the reducing end of the sugar chain by the action of α1,6 fucose transferase (Fut8) and the like. In the fucose transition, the 1-position of fucose is α-bonded to the 6-position of GlcNAc to form a branched structure. Fucose (core fucose) transferred to GlcNAc at the sugar chain reducing end is involved in the control of the functions of many glycoproteins. For example, it has been reported that the presence or absence of core fucose changes during cell adhesion and canceration. ing. In mass spectrometry of the positive ion mode, since core fucose is easily desorbed at the time of cleavage by CID or the like, it is difficult to specify the presence or absence of core fucose.

  On the other hand, in mass spectrometry of glycopeptides in negative ion mode, it has been reported that a fragment derived from a sugar chain that does not contain a peptide moiety is obtained preferentially. Therefore, mass spectrometry in negative ion mode is easy to create a sugar chain mass spectrum and fragment database, and has attracted attention as a method for analyzing the sugar chain structure of glycopeptides.

For example, in Non-Patent Document 1 and Non-Patent Document 2, structure analysis of glycopeptides is performed using both positive ion mode and negative ion mode mass spectrometry, and ^2,4 A generated in negative ion mode MS ² is used. An example of analyzing fragments such as _R by MS ³ has been reported. Incidentally, ^2,4 and A _R is a non-reducing terminal fragment of sugar without the peptide moiety, cleavage at C-C bonds of the 2-3 position and 4-5 of the reducing end monosaccharide sugar chain Is a fragment generated by the occurrence of

  Patent Document 2 also discloses a method for analyzing the structure of a glycopeptide using both a positive ion mode and a negative ion mode, and it is shown that the structure analysis of a sugar chain is possible in the negative ion mode. Has been. Further, in Patent Document 2, mass spectrometry in a negative ion mode of a glycopeptide containing an O-linked sugar chain bonded to a serine residue or a threonine residue hardly causes elimination of core fucose upon cleavage by CID. It has been reported that the presence or absence of core fucose can be specified.

JP 2005-300420 A JP 2008-51552 A

Ito, H. et al., Rapid Commun. Mass Spectrom., Vol. 20, pp. 3357-3565 (2006) Deguchi, K. et al., Rapid Commun. Mass Spectrom., Vol. 20, pp.741-746 (2006)

As described above, several sugar chain structure analysis methods for glycopeptides by multi-stage mass spectrometry in negative ion mode have been proposed. However, as described in Patent Document 2, in MS ² analysis of N-linked sugar chains, core fucose may be detached during CID cleavage, and it is difficult to identify the presence or absence of core fucose. there were. Further, in Non-Patent Documents 1 and 2, ^{2, 4} A _R fragment to be detected in the negative ion mode MS ² has included most of the sugar chain structure, 1,2 GlcNAc in the reducing end in the sugar chains, It does not contain 5th and 6th carbons. Core fucose is to be added to 6-position of GlcNAc, the sugar chains irrespective of whether containing core fucose, ^{2, 4} A _R fragment does not contain core fucose. Therefore, even if further cleaved allowed MS ³ or more multi-stage mass spectrometry the ^{2, 4} A _R fragment, it is impossible to determine the presence or absence of core fucose.

  Thus, in the prior art, it is difficult to obtain a sugar chain-derived fragment reflecting the branched structure of the sugar chain, the presence or absence of core fucose, etc. by multi-stage mass spectrometry of a glycopeptide containing an N-linked sugar chain. . That is, a universal sugar chain structure analysis method that can easily identify a detailed sugar chain structure using matching with a database or the like without being greatly affected by the structure of the peptide portion has not yet been developed. In view of such a current situation, an object of the present invention is to provide a glycopeptide analysis method that can also identify the presence or absence of a complex sugar chain structure or core fucose by multistage mass spectrometry.

The present inventor has obtained many negative ion MS ² spectra of glycopeptides containing N-linked sugar chains and is proceeding with investigations on methods for analyzing sugar chain structures. It was found that an MS ² fragment with m / z) was produced. The [Tokusarizanmoto +96] ^- where the fragments were analyzed in detail MS ^3, includes a complete carbohydrate residue structure, the MS ³ analysis, it was found that the presence or absence of core fucose can also be analyzed. Also, [Tokusarizanmoto +96] ^- fragment, without depending on the amino acid sequence of the peptide portion, it has been found indicating the same MS ³ peaks.

The present invention has been made based on the above findings and relates to a method for analyzing a glycopeptide in which an N-linked sugar chain is bound to asparagine using a mass spectrometer. In the analysis method of the present invention, negative ion MS ⁿ analysis (n is an integer of 2 or more) of glycopeptides is performed. The analysis method of the present invention, MS ⁿ precursor selection step selects a MS ⁿ precursor from the negative ions from the fragment ions of glycopeptides; was cleaved selected the MS ⁿ precursor in and MS ⁿ precursor selection step, MS ⁿ MS ⁿ analysis steps for performing the analysis in this order.

In the analysis method of the present invention, in the MS ⁿ precursor selection step, a fragment ion having a mass-to-charge ratio of (G + 97−z) / z is selected as the MS ⁿ precursor. Here, G is the mass of the sugar chain residue. The mass of sugar chain residues is the sum of the masses of monosaccharide residues constituting the sugar chain. The mass of the monosaccharide residue is a value obtained by subtracting the mass of one molecule of water from the mass of the monosaccharide. z is a natural number and is the number of charges of the MS ⁿ precursor. For example, when the fragment ion obtained by MS ⁿ⁻¹ is a monovalent negative ion, z = 1, and the selected MS ⁿ precursor is m / z = (G + 96).

A monoisotopic mass is preferably used for the mass calculation of the sugar chain residue as a reference. In this case, the MS ⁿ precursor may be selected only for the peak corresponding to (G + 97−z) / z calculated by the monoisotopic mass, or may include the isotope peak, and only the isotope peak may be included. There may be. Depending on the performance (resolution) of the mass spectrometer, the monoisotopic peak and its isotope peak may not be separated. In such a case, the average mass may be used for calculating the mass of the sugar chain residue. The selection range when selecting the MS ⁿ precursor is appropriately selected according to the performance of the mass spectrometer, for example, within a range of ± 10 of the value calculated by monoisotopic mass, preferably within a range of ± 5, more preferably Is in the range of ± 2.

In one form of the analysis method of the present invention, before the MS ⁿ precursor selection step, a MS ¹ analysis step in which a glycopeptide in which an N-linked sugar chain is bound to asparagine is negatively ionized and MS ¹ analysis is performed; and MS The negative ion of the glycopeptide obtained in ^one analysis step or the fragment ion obtained by cleaving the negative ion of the glycopeptide obtained in MS ¹ analysis step is cleaved as an MS ^n-1 precursor, and MS ⁿ⁻ MS ^n-1 analysis steps to perform ^one analysis. Here, n is an integer of 3 or more. For example, when n = 3, MS ² analysis is performed using the negative ion of the glycopeptide obtained in the MS ¹ analysis step as the MS ² precursor (MS ^n-1 precursor), and the saccharide obtained in the MS ² analysis A fragment ion having a mass-to-charge ratio of (G + 97-z) / z is selected as an MS ³ precursor (MS ⁿ precursor) from fragments derived from negative ions of peptides.

In a preferred form of the invention, the MS ^n-1 precursor subjected to the MS ^n-1 analysis step is N-linked to the C-terminal asparagine residue of the peptide or an asparagine residue adjacent to the C-terminal amino acid residue. It is a negative ion derived from a glycopeptide to which a type sugar chain is bound. If negative ions peptides sugar chain bound to asparagine in the vicinity of the C-terminal was ^{MS n-1} analysis as ^{MS n-1} precursor, in ^{MS n-1} spectrum, [Tokusarizanmoto +96] ^- fragment (mass There is a tendency that a strong peak of a fragment ion having a charge ratio of G + 96) appears. Therefore, the amount of the MS ⁿ precursor provided for the MS ⁿ analysis step is increased, and high-precision analysis is possible even with a low concentration sample. For example, in the case of n = 2, before MS ² analysis, negative ions in which sugar chains are bound to asparagine near the C-terminal from among negative ions derived from glycopeptides appearing in the MS ¹ spectrum obtained by MS ¹ analysis. Ions may be selected as MS ² precursors.

In MS ¹ analysis, in order to obtain a negative ion in which a sugar chain is bound to asparagine near the C-terminal, for example, in the sample preparation step before the glycopeptide is subjected to the MS ¹ analysis step, the C of asparagine to which the sugar chain is bound. A protease may be selected so that the peptide bond is cleaved at the terminal, an amino acid residue adjacent to the C-terminal side of asparagine to which a sugar chain is bound. In one form of the invention, both endopeptidase and exopeptidase are used as proteases in the sample preparation step.

In the analysis method of the present invention, a method for selecting a fragment ion having a mass-to-charge ratio of (G + 97−z) / z as the MS ⁿ precursor is not particularly limited. For example, the MS ⁿ precursor can be selected by collating the peak list of the MS ^n-1 spectrum obtained in the MS ^n-1 step with the mass Sx of each sugar chain in any known sugar chain database. In this case, a fragment ion having a mass-to-charge ratio of (Sx + 79−z) / z with respect to the known sugar chain mass Sx may be selected as the MS ⁿ precursor.

Further, two or more peaks are extracted from the peak list of the MS ^n-1 spectrum obtained in the MS ^n-1 analysis step, and the m / z difference of the extracted peaks is determined from a predetermined value set in advance. An MS ⁿ precursor can also be selected from matching peak combinations. The predetermined value set in advance may be one, or two or more. For example, the predetermined values are 240 and 386. In this case, two peaks with a m / z difference of 240 or 386 are extracted, and the fragment ion with the larger m / z is selected as the MS ⁿ precursor.

  In the present specification, the description that the mass-to-charge ratio m / z “matches” or the equal sign (=) does not require that m / z completely match, and the performance of the mass spectrometer Accordingly, an error within a range of ± 1 (preferably within ± 0.5, more preferably within ± 0.1) is allowed.

As described above, when the m / z difference between the two peaks is 240 and 386, the fragment ion having the smaller m / z is expressed as ^2,4 A _R (2-3 of N-acetylglucosamine at the reducing end). And fragment ions on the non-reducing terminal side cleaved at the CC bond at the 4th and 5th positions). The fragment ion having the larger m / z is a fragment ion of m / z = G + 96 (where z = 1), and has a core fucose when the difference between the m / z of the two peaks is 240. If the m / z difference is 386, the core has a fucose.

Even if MS ⁿ analysis is not performed, the difference in mass to charge ratio between a predetermined sugar chain-derived fragment ion and a fragment ion having a mass to charge ratio of (G + 97-z) / z in the MS ^n-1 spectrum. The presence or absence of core fucose can also be determined based on the above. For example, a ^{2, 4} A _R, the fragment ions having a mass to charge ratio of (G + 97-z) / z, and calculates the difference in mass-to-charge ratio, no "core fucose If the difference between the mass to charge ratio of 240 "If the difference in mass to charge ratio is 386, it can be determined that" core fucose is present ".

In the analysis step, extracting structure information of the N- linked sugar chain is carried out, for example, by comparing the MS ⁿ spectrum obtained by MS ⁿ analysis step, the MS ^N database of known sugar chains derived fragment . Here, N is an integer of 2 or more, and may be the same as or different from n. In one form, the MS ^N database of known glycan derived fragment ions is a known MS ^N spectrum or MS ^{N of} fragment ions obtained by cleaving a precursor ion having a mass-to-charge ratio of (Gx + 97-zx) / zx. A database containing a peak list. In another form, the MS ^N database of known sugar chain-derived fragment ions has a mass-to-charge ratio of (Sx + 79−zx) / zx calculated based on the sugar chain structure stored in any sugar chain database. It is a database including a virtual MS ^N spectrum or a virtual MS ^N peak list of fragment ions. Here, Sx is the mass of a known N-linked sugar chain. zx is a natural number and is the number of charges of a fragment ion (precursor).

Furthermore, this invention relates to the mass spectrometer for performing said mass spectrometry. The mass spectrometer of the present invention comprises a mass analyzer; a database; and a data processor that identifies the sugar chain structure of the glycopeptide to be analyzed. The mass spectrometer includes a mass separation unit and an ion cleavage unit, and performs MS ⁿ analysis of the glycopeptide to be analyzed. The data processing unit includes the MS ⁿ spectrum of the glycopeptide to be analyzed obtained by MS ⁿ analysis in the mass spectrometry unit, the MS ^N spectrum or the MS ^N peak list stored in the database unit, or the database unit. The sugar chain structure is identified by collating with the virtual MS ^N spectrum or the virtual MS ^N peak list.

The present invention also provides a glycopeptide by comparing an existing database, a virtual MS ^N spectrum calculated based on the existing database, and the MS ⁿ spectrum obtained by the negative ion MS ⁿ analysis. The present invention relates to a sugar chain structure analysis program for identifying the structure of N-linked sugar chains.

[Definition of terms]
In the present specification, the “mass” is a value expressed by a unified atomic mass unit (u) or Da, and a unit equivalent thereto, and is a dimensionless number. The mass-to-charge ratio (m / z) is obtained by dividing the above-mentioned mass m by the number of charges z of ions.

  In the present specification, the “negative ion of a glycopeptide” is a glycopeptide that is negatively ionized by, for example, elimination of a proton from the glycopeptide. The “fragment ion derived from the negative ion of the glycopeptide” is a fragment ion generated by ion cleavage of the negative ion of the glycopeptide. It should be noted that the fragment ion derived from the negative ion of the glycopeptide does not necessarily have to be generated in the order that the glycopeptide is cleaved after being negatively ionized. For example, even if the glycopeptide is negatively ionized after the cleavage of the glycopeptide, as in some types of in-source decomposition, it may have the same structure as the glycopeptide cleaved after being negatively ionized. For example, it corresponds to “fragment ion derived from negative ion of glycopeptide”.

“MS ⁿ analysis” is an analysis method in which a sample is ionized in the first stage (MS ¹ ), ions are cleaved in the second stage (MS ² ) and subsequent steps, and the cleaved ions (fragment ions) are detected. . In this specification, a mass spectrum obtained by ionizing a sample is referred to as “MS ¹ spectrum”, a product ion spectrum obtained by using a specific ion observed in the MS ¹ spectrum as a precursor is referred to as “MS ² spectrum”, and MS ² spectrum. A product ion spectrum obtained by using the observed specific ions as a precursor is denoted as “MS ³ spectrum”. A product ion spectrum obtained by using a specific ion observed in the MS ⁿ spectrum as a precursor is referred to as “MS ^{n + 1} spectrum”.

In in-source cleavage (ISD), cleavage occurs almost simultaneously with ionization, and fragment ions are generated. In this specification, as in the case of ISD, ionization (n = 1) and cleavage (n = 2) that occur at substantially the same time are considered to be two stages. First stage) and cleavage (second stage) are considered to have occurred. ISD causes ionization (n = 1) and cleavage (n = 2) almost simultaneously, and when fragment ion obtained thereby is used as a precursor and further ion cleavage is performed, at the time of ionization and cleavage by ISD, It can be interpreted that a fragment ion equivalent to MS ² is obtained by mass spectrometry (post-source cleavage). Therefore, when the fragment ion obtained by ionization and cleavage by ISD is used as a precursor ion and further cleaved, it is regarded as MS ³ analysis.

The fragment ion of m / z = (G + 97−z) / z generated when the negative ion of the glycopeptide is cleaved contains the complete structure of the glycopeptide sugar chain, such as the presence or absence of core fucose. Information is reflected. The MS ³ spectrum obtained by cleaving this fragment ion as an MS ³ precursor is not greatly affected by the structure of the peptide moiety, and the same MS ³ peak can be obtained if the sugar chain structure is the same. Therefore, according to the present invention, a detailed sugar chain structure can be easily identified using matching with a database or the like.

Is a flowchart illustrating an example of a method of identifying a sugar chain structure from MS ³ spectra. Is a flowchart illustrating an example of a method of identifying a sugar chain structure from MS ³ spectra. Analysis 1 is a mass spectrum of IgG (without core fucose) derived glycopeptides (Example 1), (A) is ^MS 2, (B1) is ^{2, 4} A _R fragment ions of ^MS 3, (B2) is m / z is the MS ³ spectrum of the fragment ion of sugar chain residue +96. IgG Analysis 2 (Example 1) (core fucose present) is a mass spectrum derived glycopeptides, (A) is ^MS 2, (B1) is ^{2, 4} A _R fragment ions of ^MS 3, (B2) is m / z is the MS ³ spectrum of the fragment ion of sugar chain residue +96. It is a mass spectrum of glycopeptide derived from IgG of Analysis 3 (Example 1) (with core fucose), (A) is MS ² , and (B) is an MS ³ spectrum of a fragment ion of m / z of sugar chain residue +96. is there. It is a MS ² spectrum of the IgG-derived glycopeptide of Analysis 4 (Example 1). It is a mass spectrum of the transferrin-derived glycopeptide of Analysis 5 (Example 2), (A) is MS ² , and (B) is an MS ³ spectrum of a fragment ion whose m / z is sugar chain residue +96. (A) is a mass spectrum of the α1-acid glycoprotein-derived glycopeptide of Analysis 6 (Example 3). (B) and (C) are the mass spectra of Analysis 1 and Analysis 2, respectively. In each of (A) to (C), the upper part is MS ² , and the lower part is an MS ³ spectrum of a fragment ion of m / z of sugar chain residue +96. It is a mass spectrum of the lactoferrin-derived glycopeptide of Analysis 7 (Example 4), (A) is MS ² , and (B) is an MS ³ spectrum of a fragment ion whose m / z is sugar chain residue +96. It is a MS ² spectrum of the lactoferrin-derived glycopeptide of Analysis 8 (Example 4).

  In the glycopeptide analysis method of the present invention, the analysis of the sugar chain structure is carried out by mass spectrometry using a glycopeptide in which an N-linked sugar chain is bound to asparagine (Asn). The glycopeptide is not particularly limited as long as it contains an N-linked sugar chain. The glycopeptide to be analyzed may be prepared as it is. Preferably, sample preparation is performed so as to be suitable for the analysis method of the present invention.

In the following, MS ¹ is used to negatively ionize the glycopeptide, MS ² analysis is performed using the negative ion of the glycopeptide obtained by MS ¹ analysis as an MS ² precursor, and the glycopeptide derived from the negative ion of the glycopeptide obtained by MS ² analysis An embodiment of the present invention will be described focusing on a case where an MS ³ precursor is selected from fragment ions and MS ⁿ analysis (n = 3) is performed.

[Sample preparation]
In general, in the structural analysis of peptides by mass spectrometry, if the number of amino acid residues in the peptide portion is large, ionization at MS ¹ becomes difficult or the number of peaks in the MS ² spectrum increases, making analysis difficult. is there. Therefore, protease digestion and chemical treatment are performed, and analysis is performed using peptide fragments obtained by cleaving the peptide portion to a length (number of amino acid residues) suitable for mass spectrometry. Similarly, in the glycopeptide analysis method of the present invention, it is preferable that a glycopeptide fragment having a length suitable for mass spectrometry is generated by protease digestion or the like in sample preparation before mass spectrometry. The length suitable for mass spectrometry is, for example, that the number of amino acid residues is 30 or less, preferably 20 or less, more preferably 15 or less. On the other hand, from the viewpoint of clarifying the origin of the glycopeptide, the number of amino acid residues in the peptide portion is preferably 2 or more.

  The protease used for sample preparation is not particularly limited, and both endopeptidase (which selectively cleaves specific bonds of specific sequences) and exopeptidase (which cleaves peptide bonds of C-terminal or N-terminal amino acids) are used. Can be used.

  Endopeptidases include trypsin (which cleaves peptides at the C-terminal side of basic amino acid residues (Arg and Lys)), Lys-C (which cleaves peptides at the C-terminal side of Lys), arginine endopeptidase (of Arg). Cleavage peptides at the C-terminal side), chymotrypsin (cleaves peptides at the C-terminal side of aromatic amino acids (Phe, Tyr and Trp)), pepsin (at the N-terminal side of aromatic amino acids (Phe, Tyr and Trp)) Etc.) are widely known. Moreover, asparagine endopeptidase (Asn-C) that cleaves a peptide at the C-terminal side of Asn, such as legmaturin, is also preferably used. In addition to the above, endopeptidases with low specificity such as thermolysin and proteinase K can also be used.

  As the exopeptidase, either an aminopeptidase that sequentially cleaves peptide bonds from the N-terminus of a peptide or a carboxypeptidase that cleaves peptide bonds sequentially from the C-terminus can be used.

As will be described in detail later, in one form of the analysis method of the present invention, MS ² analysis is performed using a negative ion of a glycopeptide having a sugar chain bound to Asn near the C-terminal as an MS ² precursor. In this case, in the MS ² precursor, it is preferable that an N-linked sugar chain is bound to Asn at the C-terminal of the peptide or Asn adjacent to the amino acid residue at the C-terminal. Therefore, in sample preparation by protease digestion, it is preferable that a glycopeptide fragment in which a sugar chain is bound to Asn near the C-terminus is generated. In other words, in the present invention, it is preferable to select a protease so that a glycopeptide in which a sugar chain is bound to Asn near the C-terminus is generated during sample preparation.

  The protease is selected in view of the amino acid sequence of the peptide. For example, by using the above asparagine endopeptidase alone, a glycopeptide in which a sugar chain is bound to the C-terminal Asn can be obtained. In addition, when Arg or Lys is present adjacent to the C-terminal side of Asn to which the sugar chain is bound, the sugar chain is bound to the asparagine residue adjacent to the C-terminal amino acid residue by using trypsin alone. The resulting glycopeptide is obtained.

  Moreover, as a protease used for sample preparation, both endopeptidase and exopeptidase may be used. For example, when it is difficult to obtain a glycopeptide in which a sugar chain is bound to Asn near the C terminus with endopeptidase alone, the sugar chain is attached to Asn near the C terminus by using both endopeptidase and exopeptidase. Bound glycopeptides can be generated. The digestion with endopeptidase and the digestion with exopeptidase may be performed simultaneously or sequentially. It is also possible to use a mixture of exopeptidase and endopeptidase, such as Pronase E.

  The conditions for protease digestion are not particularly limited, and an appropriate protocol according to the protease to be used is adopted. For example, it is preferable to incubate at a temperature of about 37 ° C. for about 4 to 20 hours in a buffer solution prepared near the optimum pH of the protease. Prior to protease digestion, denaturation treatment or alkylation treatment of proteins and peptides in the sample may be performed. The conditions for the modification treatment and the alkylation treatment are not particularly limited, and known conditions are appropriately employed.

  The glycopeptide sample after protease digestion may be subjected to treatments such as purification, desalting, solubilization, concentration, and drying as necessary. These processes can be performed using a known method. In particular, when analyzing a sample containing multiple glycopeptides or a complex sample such as a mixture with other peptides, before performing mass spectrometry, liquid chromatography (LC) or solid phase The glycopeptide is preferably separated and concentrated by extraction (solid-phase extraction: SPE) or the like.

  When the sample is separated by LC, LC / MS provided with LC as a preceding stage of mass spectrometry may be used, and the eluate from LC may be directly ionized and subjected to ion cleavage. Alternatively, the eluate from LC may be collected once and then subjected to mass spectrometry. LC column and SPE carrier are not particularly limited, and a hydrophobic column such as C30, C18, C8, C4 or the like generally used for peptide analysis, a carrier for hydrophilic affinity chromatography, etc. are appropriately selected. Can be used. In addition, a carbon column or a carbon support may be used for the purpose of separating glycopeptide isomers.

[Mass spectrometry]
The glycopeptide sample is subjected to multistage mass spectrometry. The second stage (MS ² ) and subsequent stages of multistage mass spectrometry is an analysis method that causes fragment ion cleavage, and the ion cleavage method is classified into a post-source type and an in-source type. Post source ion cleavage methods include Post Source Decay (PSD), Collision Induced Dissociation (CID), Infrared multiphoton dissociation (IRMPD), Surface Induced Dissociation (surface) -induced dissociation (SID) and photo-induced dissociation (PID). Electron-capture dissociation (EDD), electron-transfer dissociation (EDD), electron-detachment dissociation (EDD), and odd-electron-induced dissociation techniques based on them are also available. It may be used. When the structure of a glycopeptide is analyzed by MS ³ analysis, ion cleavage in MS ² and MS ³ is caused by collision induced dissociation (CID) and infrared multiphoton dissociation (IRMPD). It is preferable that the ion activation method is accompanied by vibration excitation according to the above. As a mass spectrometer capable of performing ion cleavage by CID or IRMPD, a mass spectrometer having a collision chamber or a quadrupole or ion trap having a function of a collision chamber can be given. Specifically, ion trap mass spectrometer, double focusing mass spectrometer, triple quadrupole mass spectrometer, quadrupole time-of-flight mass spectrometer, quadrupole-ion trap mass spectrometer , Quadrupole-Fourier transform mass spectrometer, quadrupole-orbitrap mass spectrometer, ion trap-time-of-flight mass spectrometer, time-of-flight-time-of-flight mass spectrometer, and the like.

In addition, when multi-stage mass spectrometry of MS ⁿ (n is 4 or more) is performed, a cleavage method for specifically cleaving the peptide moiety in the MS at the stage (n-2) or prior thereto is performed. It is also preferable to use it. For example, in MS ² , a sugar chain is bound to Asn near the C-terminal of a peptide by adopting a cleavage method that specifically cleaves the peptide part, such as electron-detachment dissociation (EDD). Fragment ions derived from the negative ions of the glycopeptide obtained are obtained. In this case, fragment ions similar to those obtained when MS ² analysis is performed using a negative ion of a glycopeptide having a sugar chain bound to Asn near the C-terminal as an MS ² precursor can be expected to improve analysis accuracy (details will be described later). ).

  In-source dissociation (ISD) includes a method of cleaving based on interaction with an ionization auxiliary substance (for example, a matrix in the case of MALDI method) used in ionization, a laser used in ionization, Examples include a method of inducing cleavage by increasing the application of voltage, a method of inducing cleavage by collision with a residual gas in the ionization chamber immediately after ionization, and the like. In ISD, fragment ions are generated in one stage during ionization.

Among these, in the MALDI-ISD method in which the matrix used in the MALDI method is an ionization and cleavage auxiliary substance, the peptide portion of the glycopeptide can be specifically cleaved by selecting the matrix. Therefore, in the MALDI-ISD method or the like, a negative ion derived from a glycopeptide in which a sugar chain is bound to Asn near the C terminus by cleavage that occurs almost simultaneously with ionization, that is, a glycopeptide in which the sugar chain is bound to Asn near the C terminus. The same fragment ion as in the case of performing MS ² analysis using a negative ion of MS ^{2 as a} precursor may be obtained, and improvement in analysis accuracy can be expected (details will be described later).

  When performing ISD, in addition to the above-described collision chamber or mass spectrometer having a collision chamber function, the collision chamber function such as a quadrupole type, a time-of-flight mass spectrometer, and a magnetic field type mass spectrometer is provided. You may use the mass spectrometer which does not have.

The fragment ion generated by ISD can be used as a precursor to further cause ion cleavage. In this case, an in-source type and a post-source type may be combined. As described above, when a fragment ion obtained by ionization and cleavage by ISD is used as a precursor ion and this is further cleaved, it is considered to be MS ³ analysis.

<MS ¹ analysis>
In mass spectrometry of glycopeptides, in the MS ¹ analysis step, the glycopeptide is negatively ionized and MS ¹ analysis is performed. Examples of the ionization method of the first mass spectrometry (MS ¹ ) include a matrix-assisted laser desorption ionization (MALDI) method, an electrospray ionization (ESI), and a nanoelectrospray ionization (nano-ESI) method. In particular, the MALDI method is suitable.

<MS ² precursor selection and MS ² analysis>
Next, in MS ² analysis (MS ^n-1 analysis step), the negative ions obtained in MS ¹ are cleaved by CID, IRMPD, etc., and MS ² analysis is executed. In MS ² analysis, all peaks obtained in MS ¹ can be comprehensively subjected to MS ² , but a negative ion derived from a glycopeptide appearing in the MS ¹ spectrum is selected as an MS ² precursor, and the selected negative it is advantageous to subject the selective MS ² ions.

When the mass of the glycopeptide to be analyzed is known, the MS ² precursor can be selected based on the negative ion m / z calculated from the mass. Almost all N-linked sugar chains have the structure of Manα1-6 (Manα1-3) Manβ1-4GlcNAcβ1-4GlcNAc (trimannosyl core) at the reducing end, and the non-reducing end of the trimannosyl core Since the types of monosaccharides added to the are also finite, the mass that can be taken by the N-linked sugar chain is finite. Therefore, even if the mass of the sugar chain moiety is unknown, the m / z candidate that can be taken by the negative ion of the glycopeptide is calculated based on [the mass of the peptide portion + the mass that can be taken by the N-linked sugar chain residue]. By comparing these candidates with the m / z of the peak appearing in the MS ¹ spectrum, the MS ² precursor can be selected. The mass that can be taken by the N-linked sugar chain residue can be calculated using an existing sugar chain database or the like.

In the present invention, a negative ion of a glycopeptide having a sugar chain bound to Asn near the C-terminal is preferably selected as the MS ² precursor. Among them, it is particularly preferable that the selected MS ² precursor has an N-linked sugar chain bonded to Asn at the C-terminal of the peptide or Asn adjacent to the amino acid residue at the C-terminal.

In the negative ion MS ² analysis of a glycopeptide having an N-linked sugar chain, the present inventor has generated a fragment ion having a mass-to-charge ratio of m / z = (G + 97−z) / z. It was found that there is a tendency that the amount of fragment ions of 1, ie, m / z = G + 96, is large. Here, G is the mass of the sugar chain residue, and z is the number of charges of the ion. Furthermore, when MS ³ analysis was performed using the fragment ion of m / z = (G + 97−z) / z as a precursor, the same MS ³ peak was obtained if the sugar chain structure was the same regardless of the structure of the peptide moiety. It was found that an MS ³ spectrum with In addition, the sugar chain skeleton is the same, and the sugar chain in which core fucose is added to N-acetylglucosamine (GlcNAc) at the reducing end and the sugar chain to which core fucose is not added are detected by MS ^2. m The difference in m / z of the fragment ions (where z = 1) of / z = (G + 96) is 146. This difference is the same as the mass of the fucose residue from which water molecules are eliminated from fucose (molecular weight 164), and the fragment ion of m / z = (G + 96) has a complete sugar chain structure including the presence or absence of core fucose. Reflects.

(Inferred structure of fragment)
At this stage, the molecular structure of the fragment ion of m / z = (G + 97−z) / z is not clear, but this fragment ion has a larger mass than the sugar chain residue and affects the amino acid sequence of the peptide part. Therefore, it is considered to be a negative ion containing an N-linked sugar chain residue and a part of asparagine (molecular weight: 132) to which the sugar chain is bonded. Since the molecular weight of asparagine is 132, G + 97 can be expressed as G + Asn-35, and it is considered that the chemical species corresponding to mass 35 is desorbed from Asn. Considering the chemical formula of asparagine, there is a high possibility that ONH ₅ = 35 is desorbed, and the fragment ion of m / z = (G + 97−z) / z is a chemistry in which H ₂ O and NH ₃ are desorbed from asparagine. Presumed to contain structure and sugar chain residues. In the negative fragment ion having the charge number z, since z protons are desorbed, the mass-to-charge ratio m / z of the fragment ion is (G + 97−z) / z, and when z = 1, m / z = G + 96.

  As a molecular structure having a mass of G + 97 (= G + Asn−35), for example, as represented by the following formula, a structure in which a sugar chain residue is added to the nitrogen atom of maleimide is estimated.

  In post-translational modification of proteins, as represented by the following chemical formula, the unpaired electron of the nitrogen atom constituting the peptide bond on the C-terminal side of Asn nucleophilically attacks the side chain of Asn, via a succinimide intermediate. It is known to produce aspartic acid (Asp) (biochemical deamidation).

In the negative ion cleavage of the peptide, as represented by the following chemical formula, preferential cleavage at the CN bond between the asymmetric carbon of Asp and the amino group occurs, and the peptide on the N-terminal side of Asp (described below) It is known that c) of the formula is eliminated. Further, along with this cleavage, unpaired electrons of the nitrogen atom constituting the peptide bond on the C-terminal side of Asp nucleophilically attacks the side chain of Asp, and the z-H ₂ O type having an N-substituted maleimide structure It is known that negative ions are generated.

As described above, the side chains of Asn and Asp are both likely to form a five-membered ring imide structure due to the nucleophilic reaction of the nitrogen atom, and, together with the five-membered ring imide (maleimide) structure during negative ion cleavage, N It is known that cleavage at the terminal side tends to occur. Considering these, the negative ion MS ² of the glycopeptide containing Asn ((g) Asn) to which a sugar chain is added is also represented by the Asn CN bond, as in the negative ion cleavage of the peptide containing Asp. As shown by the following presumed mechanism, it is presumed that a negative ion of z-H ₂ O type is generated.

  As described above, according to the presumed mechanism that cleavage occurs between the asymmetric carbon of Asn and the amino group, in the fragment ion of m / z = (G + 97−z) / z, one nitrogen atom from asparagine Can reasonably explain that the atoms are desorbed. It can also be rationally explained that the same chemical species (negative ion) is generated regardless of the amino acid sequence of the peptide on the N-terminal side of Asn to which a sugar chain is bound.

In the above estimation mechanism, Asn ((g) Asn) to which a sugar chain is added is shown as the C-terminal amino acid of the glycopeptide. According to the study of the present inventor, (g) C of Asn It has been confirmed that a fragment ion of m / z = (G + 97−z) / z is also detected in MS ² of a negative ion of a glycopeptide having an amino acid on the terminal side (see, for example, FIG. 7A). ). In the negative ion cleavage of a peptide, as described above, selective cleavage is likely to occur at the CN bond of Asn, and in addition, cleavage at the peptide bond on the N-terminal side of the C-terminal amino acid of the peptide occurs. The elimination of amino acid residues has also been reported (Journal of Mass Spectrometry, 41 (2006), pp. 939-949). Therefore, (g) when the negative ion of a glycopeptide having an amino acid on the C-terminal side of Asn is an MS ² precursor, the elimination of the amino acid on the C-terminal side occurs first by the negative ion cleavage of MS ^2. The C-terminus becomes (g) Asn, and then a mechanism such as the generation of a fragment ion of m / z = (G + 97−z) / z can be considered as in the above estimation mechanism.

In the above report, it is reported that when the C-terminal amino acid is an acidic amino acid (Glu or Asp) or Ser, the C-terminal elimination is significant. On the other hand, in the mass spectrometric method for glycopeptides of the present invention, as shown in a later example, (g) the amino acid adjacent to the C-terminal side of Asn is Lys, which is a basic amino acid. Desorption has been shown to occur. Therefore, when the MS ² precursor is (g) a negative ion of a glycopeptide having an amino acid on the C-terminal side of Asn, (g) even if the amino acid on the C-terminal side of Asn is other than Glu, Asp, Ser, m / It can be seen that an MS ² peak of z = (G + 97−z) / z is detected.

As described above, (g) Cs-N bond cleavage of the asymmetric carbon and amino group of Asn is regioselective (amino acid selective), and (g) has many amino acids on the N-terminal side of Asn. This cleavage is considered to occur even with peptides. On the other hand, (g) elimination of Asn on the C-terminal side is considered to be sequential elimination from the C-terminal amino acid. Therefore, the MS ² precursor may have (g) a large number of amino acids on the N-terminal side of Asn, but (g) when the number of amino acid residues on the C-terminal side of Asn increases, m / z = ( The MS ² peak intensity of G + 97−z) / z tends to decrease rapidly. Therefore, the MS ² precursor in the analysis method of the present invention preferably has (g) Asn in the vicinity of the C-terminal, and it is particularly preferable that the C-terminal amino acid or the amino acid adjacent to the C-terminal amino acid is (g) Asn. . In addition, when analysis of the sugar chain structure of a glycopeptide is performed by MS ⁿ analysis (n is 4 or more), it is preferable that the MS ^n-1 precursor has (g) Asn in the vicinity of the C-terminus.

<MS ³ precursor selection and MS ³ analysis>
In MS ³ analysis (MS ⁿ analysis step), the negative fragment ion obtained in MS ² is cleaved by CID, IRMPD, or the like, and MS ³ analysis is executed. The MS ³ analysis is performed for the purpose of analyzing a sugar chain structure in a glycopeptide. Therefore, MS ³ analysis is performed after a fragment ion having a mass-to-charge ratio m / z = (G + 97−z) / z is selected as the MS ³ precursor from the MS ² peak.

When the mass (or molecular weight) of the sugar chain is known, G + 96 (when the number of charges z = 1), (G + 95) / 2 (when z = 2), (G + 94) / 3 (when z = 3) MS ³ precursor candidates having m / z such as can be easily selected. When the mass (or molecular weight) of the sugar chain is unknown, the MS ³ precursor candidate can be selected by estimating the possible value of (G + 97−z) / z.

The possible value of (G + 97−z) / z can be calculated from the mass of a known sugar chain, for example. In this case, the MS ² spectrum peak list is compared with the mass Sx of each glycan stored in the known glycan database, and from the MS ² spectrum peak list, (Sx + 79−z) / z Primary fragment ions having a mass to charge ratio can be selected as MS ³ precursor candidates. The mass of the sugar chain residue is the sum of the masses of the monosaccharide residues constituting the sugar chain, and is the value obtained by subtracting the mass of one molecule of water from the mass of the sugar chain. In this case, (G + 97−z) / z = (S + 79−z) / z.

Also, the MS ³ precursor can be selected by extracting two peaks having a predetermined difference in mass-to-charge ratio from the peak list of the MS ² spectrum. It is known that a specific fragment ion tends to be detected with a large peak intensity in a negative ion MS ⁿ (n is 2 or more) of a glycopeptide. Also in the analysis method of the present invention, in the MS ² spectrum, in addition to the fragment ion of m / z = (G + 97−z) / z, a fragment ion derived from the same sugar chain as conventionally known ( ^{2, 4} A _R , ² , ⁴ A _R-1 , _BR-1 etc.) are detected. The structure of the N-linked sugar chain on the non-reducing end side is diverse, but as described above, the reducing end of almost all N-linked sugar chains is a trimannosyl core, and the core fucose Only the presence or absence is different. Therefore, the fragment ion of m / z = (G + 97−z) / z and the m / z of fragment ions derived from a predetermined sugar chain such as ^2,4 A _R , ^2,4 A _R-1 , B _R-1 The value that can be taken by the difference of z is constant.

For example, in the negative ion ^MS n (n is 2 or ^more), it is known that peak strong 2, 4 _{A R} fragment ion appears. ^{2, 4} A case both fragment ions _R fragment ions and m / z = G + 96 is either a monovalent negative ion, the difference between these m / z are perforated core fucose at the reducing end monosaccharide sugar 240 if not, and 386 if it has a core fucose. Therefore, ^two peaks having an m / z difference of 240 or 386 are extracted from the MS ² peak list, and the fragment ion having the larger m / z of these two peaks is m / z = ( G + 97−z) / z (where z = 1), and can be selected as an MS ³ precursor candidate.

In the above, m / z = (G + 97-z) / but MS ³ precursor of z is exemplified a case is a monovalent negative ion (z = 1), MS ³ precursor divalent or more negative ions There may be. Also, sugar chains derived fragment ions ^{2, 4} A _R or the like serving as a reference for MS ³ precursor selected may also be a divalent or more negative ions. When the number of charges of the fragment ions is 2 or more, the “predetermined 1 or more predetermined value” for selecting the MS ³ precursor needs to be set in consideration of the number of charges.

For example, when m / z of two extracted fragment ions are a ₁ and a ₂ respectively, the former charge number is z ₁ and the latter charge number is z ₂ , the negative ionization of each fragment ( When the mass of the chemical species before deprotonation is A ₁ and A ₂ ,
a ₁ = (A ₁ −z ₁ ) / z ₁ (Formula 1)
a ₂ = (A ₂ −z ₂ ) / z ₂ (Formula 2)
It can be expressed as.

These two fragments ions and fragment ions of m / z = (G + 97 -z) / z, when a combination of 2, 4 _{A R} fragment ions, the difference between the _{A 1} and _{A 2,} 240 (without core fucose Case) or 386 (with core fucose) (where A ₁ > A ₂ ).

To summarize (Equation 1) and (Equation 2) above,
A ₁ = a ₁ z ₁ + z ₁ (Formula 1a)
A ₂ = a ₂ z ₂ + z ₂ (Formula 2a)
Because
A ₁ −A ₂ = a ₁ z ₁ + z ₁ −a ₂ z ₂ −z ₂ (Formula 3)
It becomes.

From these relationships, the value of A ₁ -A ₂ (the left side of Equation 3 above) is equal to 240 or 386, indicating that two extracted peaks (m / z = a ₁ and m / z = a ₂ ) , There is a combination of z ₁ and z ₂ (both natural numbers) in which the value of a ₁ z ₁ + z ₁ -a ₂ z ₂ -z ₂ (the right side of the above formula 3) is 240 or 386. It can be said that they are equivalent. That is, it is possible to select an MS ³ precursor candidate based on the presence of a combination of z ₁ and z _{2 in} which the value of a ₁ z ₁ + z ₁ -a ₂ z ₂ -z ₂ is 240 or 386. I understand. When such a combination exists, a fragment ion of m / z = a ₁ = (A ₁ −z ₁ ) / z ₁ can be selected as an MS ³ precursor candidate.

^{Incidentally,} 2,4 for _{A R} fragment ions, most of which is a monovalent negative ion, in order to increase the accuracy of detection and selection, based on the monovalent ^2,4 A _R fragment ions, m / The MS ³ precursor selection is preferably performed depending on whether or not the difference of z is equal to a preset predetermined value of 1 or more. In this case, in the above (Formula 2), since z ₂ = 1, the MS ³ precursor is based on the existence of a natural number z _{1 in} which the value of a ₁ z ₁ + z ₁ −a ₂ is 241 or 387. Candidates can be selected.

When both of the two fragment ions are monovalent negative ions, z ₁ = 1 and z ₂ = 1. Therefore, based on the value of a ₁ -a ₂ being 240 or 386, MS ³ precursor candidate Is selected. In this case, as described above, it is determined that the fragment ion having the larger m / z (m / z = a ₁ ) is m / z = (G + 97−z) / z (where z = 1). Then, the MS ³ precursor candidate is selected.

"Preset one or more predetermined values" for MS ³ precursor selection can also be set with reference to sugar-derived fragment ions other than ^{2, 4} A _R. For example, the difference in m / z between the ^2,4 A _R-1 fragment ion and the fragment ion of m / z = G + 96 (both monovalent negative ions) is 443 when the sugar chain does not have core fucose, 589 when having a core fucose. Therefore, an MS ³ precursor candidate can also be selected by extracting two peaks whose m / z difference matches any of these.

It is also possible to extract three or more peaks from the MS ² spectrum and select or narrow down MS ³ precursor candidates based on the m / z difference of each peak. For example, the difference in m / z of each of the three extracted peaks (all of which are monovalent negative ion fragments) is calculated, and the fragment ion having the largest m / z and the fragment ion having the second largest m / z If the difference in m / z is 240 or 386, the fragment ion having the largest m / z can be extracted as the MS ³ precursor candidate as described above.

Here, the m / z difference between the ^2,4 A _R fragment ion and the ^2,4 A _R-1 fragment ion is 203 regardless of the presence or absence of core fucose. Therefore, the difference in m / z between the fragment ion having the largest m / z and the fragment ion having the second largest m / z among the three extracted peaks is 240 or 386, and m / z If the m / z difference between the fragment ion with the second largest fragment ion and the fragment ion with the smallest m / z is 203, the three peaks are fragment ions with m / z = (G + 97−z) / z, ^{2, It} can be said that the probability corresponding to the combination of ⁴ A _R fragment ions and ^2,4 A _R-1 fragment ions is high. On the other hand, even if two peaks with m / z difference of 240 or 386 are extracted, if there is no third peak with m / z difference of 203, the two extracted peaks are , because there is likely a combination other than fragment ions and ^{2, 4} a _R fragment ions of m / z = (G + 97 -z) / z, can be excluded from the MS ³ precursor candidates.

In this way, by selecting MS ³ precursor candidates based on the difference in m / z of three or more peaks, it is possible to narrow down MS ³ precursor candidates and improve the reliability of the precursor selection. it can. In addition, when selecting the MS ³ precursor based on the difference in m / z of three or more peaks, two of the three or more peaks are extracted and the difference in m / z is calculated. Done. Therefore, this form is included in the form that “MS ³ precursor (MS ⁿ precursor) is selected based on the extraction result of two peaks”.

In selecting the MS ³ precursor, it is not always necessary to narrow the number of precursor candidates to one. When a plurality of MS ³ precursor candidates are selected as a result of selecting an MS ³ precursor by combining a plurality of precursor selection methods as described above alone or in combination, a plurality of MS ³ precursors are sequentially selected as MS ^3. You may use for an analysis.

Whether the selected MS ³ precursor is suitable as a fragment ion of m / z = (G + 97−z) / z can also be confirmed from the MS ³ spectrum of the fragment. Since m / z = (G + 97−z) / z includes the complete structure of the sugar chain, the MS ³ spectrum obtained by cleaving this fragment ion as an MS ³ precursor is ² , ⁴ A _R, etc. Includes a peak of fragment ion derived from sugar chain. Therefore, whether or not the selection of the MS ³ precursor is appropriate can be determined based on whether or not these sugar chain-derived fragments are detected in the expected m / z in the MS ³ spectrum.

For example, when the MS ³ precursor and its fragment ion are both monovalent ions and the sugar chain contains core fucose, the fragment ion ( ^2,4 A _R ) whose m / z is 386 smaller than that of the MS ³ precursor. , MS ³ even m / z 446 smaller fragment ions than the precursor _{(B R-1), MS} 3 intense peak m / z is a like 589 small fragment ions ^(2,4 _{A R-1)} than the precursor is, MS Often detected in ^three spectra. Further, if the sugar chains does not contain core fucose is, MS ³ 240 small fragment ions ^(2,4 _A R) m / z than the precursor, MS ³ m / z 443 small fragment ions than the precursor ^{(2 , 4} A _R-1 ) and the like are often detected in the MS ³ spectrum.

Such a difference in m / z between the MS ⁿ precursor and the MS ⁿ fragment ion may be referred to as “neutral loss”. As described above, the fragment of m / z = (G + 97 -z) / z as MS ³ precursor, executing the MS ^{3 _{analysis, 2,4 A R, 2,4 A R}} -1, B R-1 Neutral loss when generating fragment ions derived from sugar chains, etc. is universal regardless of the difference in sugar chain structure. Therefore, it can be determined whether or not the selection of the MS ³ precursor is appropriate based on whether or not peaks corresponding to these neutral losses are detected in the MS ³ spectrum. When MS ³ analysis is performed using a fragment other than m / z = (G + 97−z) / z as a precursor, these fragments are not detected in the MS ³ spectrum. Alternatively, even if these fragment groups are detected, no peak is observed at m / z obtained by subtracting neutral loss from m / z of (G + 97-z) / z, so that the selection of the MS ³ precursor is inappropriate. It can be judged that it was. In this case, the MS ³ precursor may be selected again and the MS ³ analysis performed. In addition, since bivalent (G + 97-z) / z may produce monovalent ^2,4 A _R , B _R-1 , ^2,4 A _R-1 , the precursor or fragment ion or both When the number of charges is 2 or more, it is necessary to set the value of the neutral loss for determining whether or not the selection of the MS ³ precursor is appropriate in consideration of the number of charges.

[Extraction of glycan structure information]
In the analysis method of the present invention, the structure information of an N-linked sugar chain is extracted based on the obtained mass spectrometry results (MS ¹ , MS ² and MS ³ spectra), and the sugar chain structure is analyzed. Is preferred. The structure information of the extracted sugar chain is not particularly limited.

For example, if the mass of the sugar chain is unknown before analysis, the mass G of the sugar chain residue is determined from the MS ² spectrum based on the m / z value of the fragment ion of m / z = (G + 97−z) / z. Can be calculated. The mass G of the sugar chain residue is an example of the structure information of the N-linked sugar chain. Further, as described above, based on the peak list of the MS ² spectrum, m of fragment ions derived from a predetermined sugar chain (for example, ^2,4 A _R ) and m / z = (G + 97−z) / z. By calculating the / z difference, it is possible to determine whether or not core fucose is added to the sugar chain reducing terminal monosaccharide.

When the fragment ion is divalent or more, as described above with respect to the selection of the MS ³ precursor, the value of a ₁ z ₁ + z ₁ -a ₂ z ₂ -z ₂ (the right side of the above formula 3) is 240 or Whether or not core fucose is added to the sugar chain-reducing terminal monosaccharide can also be determined depending on whether or not a combination of z ₁ and z ₂ that is 386 is present. Also, three or more fragment ions (for example, a fragment ion of m / z = (G + 97−z) / z, ^2,4 A _R fragment ion and ^2,4 A _R-1 fragment ion) are extracted, By calculating the difference in m / z, the presence or absence of core fucose can also be determined. Thus, by performing the determination based on the m / z of 3 or more fragment ions, in addition to the combination of the fragment ions and ^{2, 4} A _R fragment ions of m / z = (G + 97 -z) / z, m Even when there is a combination of peaks having a difference of / z of 240 or 386, misjudgment is suppressed and the reliability of analysis can be improved.

The sugar chain structure information such as the sugar chain (residue) mass and the presence or absence of core fucose can be extracted even when MS ³ analysis is not performed. In addition, as described above, whether or not the fragment ion of m / z = (G + 97−z) / z is correctly selected by performing the MS ³ analysis and collating the MS ² spectrum with the MS ³ spectrum. And the reliability of sugar chain structure information extracted from the MS ² spectrum can be improved.

Various sugar chain structure information can also be extracted from the MS ³ spectrum. Fragment ions MS ² of m / z = (G + 97 -z) / z because it contains the complete sugar residue structure, MS ³ spectra are obtained as a ^precursor, such as 2, 4 _{A R} In many cases, it has more sugar chain structure information (for example, information on the presence or absence of core fucose) than the MS ³ spectrum obtained by using a sugar chain-derived fragment ion as a precursor. Moreover, fragment ions m / z = (G + 97 -z) / z is to include only a small portion of the peptide portion of the glycopeptide (part of asparagine sugar chain is ^bonded), such as 2, 4 _{A R} As in the case of cleaving a sugar chain-derived fragment ion, information on the sugar chain structure can be selectively and accurately extracted.

If a glycopeptide in which an N-linked sugar chain is bound to the C-terminal asparagine residue of the peptide or an asparagine residue adjacent to the C-terminal amino acid residue is selected as the MS ² precursor, MS ² in the spectrum, m / z = G + 96 ( although, z = 1) fragment ions ^of, than the sugar chain-derived fragment ions such as 2, 4 _{a R,} tend to be detected in a large peak intensity. Therefore, from the MS ³ spectrum obtained a fragment of m / z = G + 96 as MS ³ precursor, compared to other sugar-derived fragment ions in MS ³ to precursor, more extraction of precise glycan structure information Can be expected.

<Extraction of glycan structure information by collation with database>
Furthermore, since the fragment ion of m / z = (G + 97−z) / z in MS ² contains only a part of asparagine to which the sugar chain of the peptide part is bound, the structure of the N-linked sugar chain in the glycopeptide Are identical, the chemical structure of this fragment ion is the same regardless of the amino acid sequence of the peptide. Therefore, when MS ³ analysis is performed using this fragment ion as an MS ³ precursor, the same MS ³ spectrum can be obtained if the sugar chain structure is the same. That is, since the MS ³ spectrum obtained by the analysis method of the present invention is not greatly influenced by the structure of the peptide moiety, it is easy to create a database of the MS ³ spectrum and the MS ³ peak list.

For example, using a glycoprotein or glycopeptide with a known sugar chain structure as a sample, MS ^N analysis (N is 2 or more) is performed, and a precursor ion having a mass-to-charge ratio of (Gx + 97-zx) / zx is obtained. The MS ^N spectrum of the fragment ion to be obtained can be acquired, and the MS ^N spectrum and MS ^N peak list can be made into a database. Here, Gx is the mass of a known sugar chain residue. zx is the number of charges of the precursor ion, which is a natural number.

Thus, if the MS ^N spectrum or MS ^N peak list of fragment ions obtained by cleaving a precursor ion having a mass-to-charge ratio of (Gx + 97−zx) / zx is stored in a database, the mass of the glycopeptide to be analyzed By collating the MS ³ spectrum obtained by the analysis with the database, it is possible to extract sugar chain structure information, more specifically, identify the sugar chain structure.

The collation between the MS ³ spectrum (or peak list) of the glycopeptide and the MS ^N spectrum (or peak list) stored in the database may be performed by visual inspection of the experimenter or the like, using a sugar chain structure analysis program. May be executed.

  A sugar chain structure analysis system for analyzing a sugar chain structure includes an analysis data storage unit, a database storage unit, and a data processing unit. When a structure analysis (identification) of an N-linked sugar chain of a glycopeptide is performed using a sugar chain structure analysis program, the program stores the data in the analysis data storage unit and the database storage unit in the computer of the data processing unit. Check against the data. In data collation, for example, two data are compared to determine whether or not they match, or to score both similarities.

The analysis data storage unit stores data including an MS ³ spectrum or an MS ³ peak list of glycopeptides obtained by mass spectrometry. In addition to the MS ³ data, the analysis data storage unit may store information obtained by MS ² analysis such as the mass of sugar chain residues and the presence or absence of core fucose, analysis information, and the like.

In the database storage unit, a precursor ion having a mass-to-charge ratio of (Gx + 97-zx) / zx where Gx is the mass of any known N-linked sugar chain (where zx is the number of charges and is a natural number) The data including the known MS ^N spectrum or MS ^N peak list of fragment ions obtained by cleavage of The data can be obtained from a database of MS ^N spectra or peak lists of fragment ions obtained by cleaving a precursor having a mass-to-charge ratio of (Gx + 97−zx) / zx. The database storage unit may store information such as the mass Gx of the N-linked sugar chain and the presence or absence of core fucose in the MS ^N precursor ion (corresponding to the peak of the MS ^N-1 spectrum).

FIG. 1 is a flowchart showing an example of a method for performing structural analysis of a sugar chain by comparing mass spectrometry data with a database. First, the result of MS ³ obtained by mass spectrometry is stored in the analysis data storage unit (S101). At this time, it is preferable that data related to the mass such as the mass G of the sugar chain residue or the mass S of the sugar chain is also stored in the analysis data storage unit.

Next, the mass Gx of the known sugar chain residues stored in the database is compared with the mass G stored in the analysis data storage unit, and a match is extracted (S102). As described above, by performing the primary determination based on the mass, it is possible to narrow down the target of collation, improve the analysis accuracy, and simplify the data processing. The database storing information such as MS ^N spectrum and mass of known sugar chains may be stored in advance in a computer for executing the program, and is acquired from the outside through a storage medium, a telecommunication line, or the like. May be.

K pieces in which the mass G of sugar chain residues in the analytical data and the mass Gx of known sugar chain residues coincide (that is, candidates for sugar chains (residues) to be verified) are extracted (S103). The data of the MS ^N spectrum or MS ^N peak list of each candidate (k = 1 to K) is stored in the database storage unit. The MS ^N spectrum and MS ^N peak list of known sugar chains may be stored in advance in the analysis data storage unit, and the MS ^N spectrum and MS ^N peak list corresponding to the k th candidate are obtained from the database each time. And you may memorize | store in a database memory | storage part.

In the data processing unit, the MS ³ spectrum or MS ³ peak list of the glycopeptide stored in the analysis data storage unit and the MS ^N spectrum or MS ^N peak list of the k-th sugar chain stored in the database storage unit Is performed (S114). The program of the present invention causes a computer of a data processing unit to execute a comparison of a mass spectrum or a peak list.

  The collation here is, for example, determination of whether or not they match (identity of sugar chains), or scoring of similarity between both (S115). The identity and similarity of the mass spectrum or peak list can be determined based on the m / z value of the peak or the peak intensity ratio, and the method is not particularly limited. Thereafter, a matching result (for example, a score of similarity) is stored in an arbitrary storage unit as necessary (S116).

  After this collation (S114 to S118) is performed for all sugar chain (residue) candidates from k = 1 to K, a display unit such as a display or a print medium, or an appropriate storage unit The collation result is output (S122). The analysis results such as the similarity score are rearranged in the order of the score order or the like as necessary (S121), and then the data is output (S122).

Note that FIG. 1 and the above description show one example of the structure analysis (identification) form of the N-linked sugar chain using the sugar chain structure analysis program. Obtained by performing MS ³ analysis using MS ³ data obtained by mass spectrometry of glycopeptide and a negative fragment ion having a mass-to-charge ratio of (G + 97-z) / z derived from glycopeptide as a precursor. The method for identifying the sugar chain by collating with the MS ^N spectrum or the MS ^N peak list is not limited to the above.

In FIG. 1, the number of sugar chains to be collated is narrowed down based on whether the masses match in advance, but it was obtained by analysis with the MS ^{N of} all sugar chains stored in the database. Contrast with MS ³ may be performed. Further, in FIG. 1, similarity score scoring is performed for all k = 1 to Kth candidates, but the collation is finished when a collation result exceeding a predetermined score is obtained. May be. When collation is performed based on whether or not the sugar chain stored in the database matches the sugar chain to be analyzed, the collation is performed when it is determined that the k = x th candidate matches. You may end.

In the above description, the sugar chain identification method by comparing the MS ³ spectrum obtained by the analysis of the glycopeptide with the MS ^N spectrum of the sugar chain measured in advance has been described. Based on the existing sugar chain structure database, A method of identifying a sugar chain structure from analysis data by collating with a virtual MS ^N spectrum of a theoretical fragment of a sugar chain that can be generated or a virtual MS ^N peak list can also be adopted.

The virtual MS ^N spectrum and the MS ^N peak list can be calculated based on the sugar chain structure stored in an arbitrary sugar chain database. For example, from the structure of an N-linked sugar chain having a mass of Sx, a z ₁ -H ₂ O chemical species in which a sugar chain residue is added to the nitrogen atom of maleimide as described in the above-described presumed mechanism Generate. The virtual negative ion obtained by deprotonation of this species has a mass to charge ratio of (Sx + 79−zx) / zx. Note that zx is the number of charges of virtual negative ions (fragment ions).

The theoretical fragment generated when this virtual negative ion is cleaved as an MS ^N precursor can be calculated by various methods (such as using a theoretical fragment prediction program). From the list of theoretical fragments, a virtual MS ^N spectrum or virtual An MS ^N spectrum can be generated.

The verification of the virtual MS ^N spectrum or the virtual MS ^N peak list and the MS ³ spectrum obtained by the analysis may be executed by visual observation by an experimenter or may be executed using a sugar chain structure analysis program. Good.

When a sugar chain structure analysis program is used, the database storage unit of the sugar chain structure analysis system includes a virtual MS ^N spectrum or a virtual MS ^N peak list of fragment ions having a mass-to-charge ratio of (Sx + 79−zx) / zx Data is stored. The computer of the data processing unit collates the data including the MS ³ spectrum or the MS ³ peak list stored in the analysis data storage unit with the data stored in the database storage unit. The sugar chain structure analysis program causes the computer to execute the above-described verification. The collation in this case is the same as in the example of FIG. 1 described above. For example, by comparing two pieces of data, it is determined whether or not they match, and the similarity between the two is scored.

FIG. 2 is a flowchart showing an example of a method for analyzing the structure of a sugar chain by collating mass spectrometry data with virtual MS ^N using this program. Since the flowchart of FIG. 2 is similar to that of FIG. 1, the description of the parts common to FIGS. 1 and 2 is omitted below.

The result of MS ³ obtained by mass spectrometry is stored in the analysis data storage unit (S201). The mass G of the sugar chain residue is compared with the mass Sx of the sugar chain in the sugar chain database, and the difference between the two is equal to 18 (the mass of the water molecule desorbed by the sugar chain residue). S202). In addition, the mass Gx of the sugar chain residue is calculated based on the sugar chain mass Sx in the sugar chain database, and this is compared with the mass G of the sugar chain residue obtained by mass spectrometry. It may be extracted.

K candidates of sugar chains (residues) having the same mass are extracted (S203), and the virtual MS ^N spectrum or virtual MS ^N peak list data of each candidate (k = 1 to K) is stored in the database storage unit. Is done. In the data processing unit, the MS ³ spectrum or MS ³ peak list of the glycopeptide stored in the analysis data storage unit, and the virtual MS ³ spectrum or virtual MS ³ peak list of the k-th sugar chain stored in the database storage unit Are collated (S213). Prior to collation, a virtual MS ^N spectrum or MS ^N peak list is generated from the virtual negative ions of the sugar chains (S212).

In FIG. 2, the mass Sx extracts a sugar chain to match the G + 18 (S202), from the extracted sugar chain structure, each time, although the flow to generate a virtual MS ^N is shown, virtual MS ^N spectrum The virtual MS ^N peak list may be generated in advance from an arbitrary sugar chain database, and may be made into a database. In the flow of FIG. 2, the data collation method and the flow after the collation are the same as those in FIG.

[Mass spectrometer]
The analysis method of the present invention can also be carried out using a mass spectrometer for analyzing a sugar chain structure of a glycopeptide provided with the above program or other data collating means. The mass spectrometer includes a mass analyzer, an analysis data storage unit that stores analysis data obtained by the mass analyzer, a database storage unit that stores structure information obtained from an existing sugar chain structure database, and an analysis target A data processing unit for identifying the sugar chain structure of the glycopeptide.

The mass analysis unit includes mass separation means and ion cleavage means. The mass separation means separates ions based on the mass to charge ratio. The ion cleaving means cleaves selected ions by a technique such as CID or IRMPD to generate fragment ions. In this mass spectrometer, MS ³ analysis of the glycopeptide to be analyzed is performed.

The data processing unit includes a computer. This computer may be the same as the computer for controlling the analysis in the mass spectrometer. The data processing unit can identify the sugar chain structure by collating the analysis data stored in the analysis data storage unit with the MS ⁿ spectrum or the MS ⁿ peak list stored in the database storage unit. It is configured.

As described above, the program for identifying the sugar chain structure in the data processing unit is a computer that collates the MS ^N spectrum or MS ^N peak list obtained from an existing database with the mass analysis result. To run. The MS ^N spectrum or the MS ^N peak list here may be a virtual MS ^N spectrum or an MS ^N peak list generated by calculation.

The above program, sugar chain database, MS ^N spectrum database of sugar chains, etc. may be provided separately from the mass spectrometer, for example, obtained in a state stored in a storage medium, It can be obtained via communication means.

[MS ⁿ analysis when n is 4 or more]
Above, the fragment negative ionized glycopeptide MS ^1, was cleaved with MS ^2, having from the negative ions from the fragment ions of glycopeptides in MS ² spectra, the mass-to-charge ratio of (G + 97-z) / z The embodiment of the present invention has been described with a focus on MS ⁿ analysis where ions are selected as MS ³ precursors, ie, n = 3. The analysis of glycopeptides by performing MS ⁿ analysis using a fragment ion having a mass-to-charge ratio of (G + 97−z) / z among fragment ions derived from negative ions of glycopeptides is as follows. The present invention can also be applied to the above MS ⁿ analysis.

For example, a fragment ion derived from a negative ion of a glycopeptide obtained by MS ² is cleaved by MS ³ (MS ^n-1 ), and has a mass-to-charge ratio of (G + 97-z) / z obtained by MS ³ MS ⁴ (MS ⁿ ) analysis may be performed using fragment ions as precursors. In this case, as an ion cleavage method MS ^2, as EDD, by employing the cleavage method capable of preferentially cleaving the peptide moiety, as MS ² product ion, binding sugar chain Asn near the C-terminus of the peptide It is also possible to obtain a fragment ion derived from the negative ion of the glycopeptide. In this way, if the peptide portion is cleaved by ion cleavage in mass spectrometry, the peptide preparation C (protease digestion), even if a peptide fragment having a sugar chain bound to asparagine near the C-terminus is not obtained, A fragment ion derived from the negative ion of a glycopeptide having a sugar chain bound to Asn near the end is obtained. By performing MS ^n-1 analysis using this fragment ion as an MS ^n-1 precursor, the amount of fragment ions having a mass-to-charge ratio of (G + 97-z) / z increases, so that the analysis accuracy can be improved. I can expect.

  EXAMPLES The present invention will be specifically described below with reference to examples, but the present invention is not limited to the following examples. In the following, “%” represents “% by weight” unless otherwise specified.

<Production of cellulose microchip>
A small amount of cotton was packed at the tip of a 200 μL micropipette tip, and 1 mg of cellulose powder was added thereon. To the obtained cellulose microchip, 100 μL of water was added from above, air was sent from above with a syringe, and discharged from below (chip tip). This was repeated twice. Further, the same operation was performed twice with 100 μL of 50% acetonitrile (ACN) and 0.1% TFA aqueous solution and twice with 80% ACN and 0.1% TFA aqueous solution to wash and equilibrate the cellulose microchip. .

[Example 1: Analysis of IgG-derived glycopeptide]
<Preparation of glycopeptide for measurement>
Human immunoglobulin G (IgG) purchased from SIGMA was reacted for 45 minutes at room temperature in the presence of urea: 6M, ammonium bicarbonate: 50 mM, and tris (2-carboxyethyl) phosphine hydrochloride (TCEP): 5 mM, Denaturation and reduction were performed. Next, the alkylation was performed by reacting in the presence of iodoacetamide (IAA): 10 mM for 45 minutes under light-shielding conditions at room temperature, and then reacting for 45 minutes in the presence of dithiothreitol (DTT): 10 mM under light-shielding conditions at room temperature. Excess IAA was inactivated. Thereafter, Pronase E was added and reacted overnight at 37 ° C. to perform protease digestion. After digestion, desalting was performed using a carbon column.

  To the IgG digest after desalting, 100 μL of 80% ACN, 0.1% TFA aqueous solution was added and dissolved. This was added to the cellulose microchip after washing and equilibration, air was sent from above with a syringe, and discharged from below. The discharged solution was added again to the cellulose microchip and discharged in the same manner, and this was repeated once more. Finally, 20 μL of 50% ACN, 0.1% TFA aqueous solution was added to elute the glycopeptide from the cellulose microchip. This was repeated twice, and these eluates were combined and dried.

<Mass spectrometry>
The obtained sample was redissolved in water, mixed on a MALDI plate with a liquid matrix (3AQ / CHCA) composed of 3-aminoquinoline and α-cyano-4-hydroxycinnamic acid, and a MALDI-MS analyzer ( Multistage mass spectrometry using negative ion CID was performed by Shimadzu Corporation AXIMA (registered trademark) -Resonance.

<Analysis 1 and Analysis 2>
In Analysis 1 and Analysis 2, the negative ion (deprotonated glycopeptide without core fucose) obtained by MS ¹ with m / z = 1900.7, and the negative ion (core fucose) without m / z = 2046.8 Each of which was cleaved with a negative ion CID and subjected to MS ² analysis to obtain MS ² spectra shown in FIGS. 3 (A) and 4 (A). The deprotonated products obtained by MS ¹ are all deprotonated glycopeptides in which the peptide part has an amino acid sequence of Phe-Asn and a sugar chain is added to asparagine.

(Analysis 1: MS ³ analysis of glycopeptide without core fucose)
Fragment of m / z = 1478.5 obtained by MS ² (FIG. 3 (A)) of a negative ion (m / z = 1900.7) from which one proton is eliminated from a glycopeptide not containing core fucose Each of the ion ( ^2,4 A _R ) and the fragment ion ([Glycan + 96] ⁻ ) of m / z = 1718.5 was used as a precursor, and further cleaved by CID, and MS ³ analysis was performed. FIG. 3 (B1) and FIG. The MS ³ spectrum shown in (B2) was obtained. ^{Incidentally,} a _{2,4 A R [Glycan + 96]} - the difference m / z with is 240.

(Analysis 2: MS ³ analysis of glycopeptides containing core fucose)
Fragment ion of m / z = 1478.5 obtained by MS ² (FIG. 4 (A)) of a negative ion (m / z = 2046.8) from which one proton was eliminated from a glycopeptide containing core fucose Each of ( ^2,4 A _R ) and fragment ion ([Glycan + 96] ⁻ ) of m / z = 1864.7 was used as a precursor, and further cleaved with negative CID, and MS ³ analysis was performed. FIG. 4 (B1) and FIG. The MS ³ spectrum shown in (B2) was obtained. ^{Incidentally,} 2, 4 _{A R} and [Glycan + 96] ^- the difference between the m / z is 386.

<Analysis 3 and Analysis 4>
Each of the negative ion of m / z = 2931.2 obtained by MS ¹ (analysis 3) and the negative ion of m / z = 3119.3 (analysis 4) was cleaved by CID, and MS ² analysis was performed. The MS ² spectrum shown in FIG. 5 (A) and FIG. 6 was obtained. Note that the negative ion at m / z = 2931.2 and the negative ion at m / z = 319.3 are Thr-Lys-Pro-Arg-Glu-Glu-Gln-Tyr-Asn and Thr-Lys, respectively. -Pro-Arg-Glu-Glu-Gln-Tyr-Asn-Ser-Thr, which is a deprotonated glycopeptide in which a sugar chain is added to asparagine. These all have sugar chains having the same structure as the glycopeptide of Analysis 2.

In the MS ² spectra of FIG. 5 (A) and FIG. 6, as in Analysis 2 (FIG. 4 (A)), the fragment ion ( ^2,4 A _R ) and m / z at m / z = 1478.5 = 1864.6 fragment ions ([Glycan + 96] ⁻ ) were obtained. [Glycan + 96] in Analysis 3 (FIG. 5A) was used as a precursor and further cleaved with CID to conduct MS ³ analysis, and an MS ³ spectrum shown in FIG. 5B was obtained.

<Evaluation of analyzes 1 to 4>
The MS ² spectra of FIG. MS ² spectra and FIG. 3 (A) 4 (A) , both _{^{2,4 A R, B R-1}} , 2,4 A R-1, D , etc. fragment ions are the same in has a m / ^z, 2, 4 _{a R} of m / z is 1478.5. In any of FIG. 3 (A) and FIG. 4 _^(A), 2,4 A also peak intensity greater greater than _R [Glycan + 96] ^- fragment ions are detected. In both FIG. 3 (A) and FIG. 4 (A), the [Glycan + 96] ⁻ fragment ion is 182 smaller in m / z than the glycopeptide negative ion [M−H] ⁻ and [M−H] ⁻ It can be seen that the same chemical species are desorbed. The m / z of [Glycan + 96] ⁻ and [M−H] ⁻ is both 146 greater in Analysis 2 (FIG. 4A) than in Analysis 1 (FIG. 3A), and this difference is different from fucose ( This is the same as the water molecule desorbed from the molecular weight 164). These results, FIG. 3 (A) and MS ² spectra differences of FIG. 4 (A) ([Glycan + 96] - the difference in fragment ions m / z) is a reflection of the presence or absence of core fucose in the sugar chain structure It can be said that there is.

^{2, 4} A MS ³ spectra of Figure 3 obtained by CID cleavage of _R and (B1) FIG. 4 (B1) is approximately the same, it can be seen that from both the same sugar chain structure. Also, [Glycan + 96] ^- in Figure 3 obtained by CID cleavage of (B2) and FIG. 4 (B2), in any of the MS ³ spectra, ^{2, 4} A peak of _R with m / z = 1478.5 Or fragment ions such as D, E, ¹ , ³ A ₃ are common to the MS ² spectrum. These results, [Glycan + 96] fragment ions MS ^{2 is,} 2, 4 _A further reducing terminal of the sugar chain structure than _R, i.e., it can be seen that a fragment ion containing the entire structure of Tokusarizanmoto .

In Analysis 2 (FIG. 4), Analysis 3 (FIG. 5), and Analysis 4 (FIG. 6), the sugar chain structures of the glycopeptides are all the same, and only the amino acid sequence of the peptide portion is different. These MS ² analysis, the common ^{2, 4} A _R and [Glycan + 96] ^- is obtained. In addition, the MS ³ spectrum of [Glycan + 96] ⁻ in analysis 2 (FIG. 4 (B2)) and the MS ³ spectrum of [Glycan + 96] ⁻ in analysis 3 (FIG. 5 (B)) are substantially the same, and both are the same. It can be seen that it is derived from the sugar chain structure. On the other hand, the MS ³ spectrum of Analysis 1 in which the sugar chain does not have core fucose (FIG. 3B), although the same fragment ions as those in FIGS. 4B2 and 5B are observed, The pattern of is clearly different.

From these results, the MS ³ spectrum obtained by performing MS ³ analysis using a fragment ion whose m / z is sugar chain residue +96 as a precursor has a sugar chain structure even if the amino acid sequence of the peptide portion is different. If they are the same, it can be seen that they have substantially the same spectral shape. Moreover, since the shape (peak list) of the MS ³ spectrum changes depending on whether or not core fucose is added to GlcNAc at the reducing end of the sugar chain, the presence or absence of core fucose can also be identified from MS ³ .

In MS ² analysis 1-3 related C-terminal glycopeptide asparagine sugar chain added peptides (FIG. 3 (A), the FIGS. 4 (A) and (FIG. 5 ^(A)), from 2, 4 _{A R} greater [Glycan + 96] of the peak intensity ^- fragment ions are detected ^{Therefore, [Glycan + 96] -.} MS 3 analysis and precursor to is feasible even at a low concentration of the sample, is excellent as a method of analyzing glycopeptides It can be said that.

On the other hand, in Analysis 4 using a glycopeptide having 2 amino acid residues (Ser-Thr) on the C-terminal side of the sugar-modified asparagine, the peak of [Glycan + 96] ⁻ in MS ² compared to Analysis 1 to 3 The strength is reduced (FIG. 6). From this result, in the analysis method of the present invention, the negative ion of the glycopeptide in which the asparagine in the vicinity of the C-terminal of the peptide is glycosylated is used as an MS ² precursor, whereby the amount of [Glycan + 96] ⁻ fragment ion generated (peak intensity) It can be said that the analysis accuracy can be improved.

Moreover, glycopeptides used for analysis 3 (FIG. 5) has the 8 amino acid residues at the N-terminal side of asparagine sugar chains are added, as in the analysis 2, etc., MS ² In the spectrum, a strong peak of [Glycan + 96] ⁻ is detected. From this result, in the analysis method of the present invention, it is preferable that the glycopeptide negative ion selected as the MS ² precursor has a sugar chain bonded to asparagine near the C-terminus of the peptide. It is understood that the amino acid sequence on the N-terminal side of the asparagine is not particularly limited and may have 2 or 3 or more amino acids.

[Example 2: Analysis of transferrin-derived glycopeptide]
<Preparation of glycopeptide for measurement>
Human transferrin purchased from SIGMA was digested with trypsin according to Example 1, and then desalted using a carbon column. The transferrin-digested product after desalting was treated in a 0.8% TFA aqueous solution at 80 ° C. for 40 minutes to remove sialic acid at the sugar chain reducing end. In the same manner as in Example 1, multistage mass spectrometry using negative ions CID was performed using a sample purified and concentrated using a cellulose microchip.

<Analysis 5>
Analyzing 5, negative ions of m / z = 3097.3 obtained in ^{MS 1 ([M-H]} -) The MS ² spectra obtained by CID cleaved as precursor (FIG. 7 (A)), and in MS ² MS ³ spectrum (FIG. 7B) obtained by further CID cleavage using [Glycan + 96] ⁻ with m / z = 1718.6 as a precursor was obtained. In addition, the negative ion of m / z = 3097.3 has an amino acid sequence (Cys residue is Cys-Gly-Leu-Val-Pro-Val-Leu-Ala-Glu-Asn-Tyr-Asn-Lys). It is a deprotonated glycopeptide in which a sugar chain is added to the second residue Asn from the C-terminus. In MS ² spectra (FIG. _{^{7 (A)), 2,4 A}} R and [Glycan + 96] ^- the difference between the m / z was 240.

[Example 3: Analysis of α1-acid glycoprotein-derived glycopeptide]
<Preparation of glycopeptide for measurement>
Human α1-acid glycoprotein purchased from SIGMA was digested with pronase E in the same manner as in Example 1, and then desalted with a carbon column and purified and concentrated with a cellulose microchip. And multistage mass spectrometry with negative ion CID was performed.

<Analysis 6>
In Analysis 6, the negative ion of m / z = 2445.0 obtained by MS ¹ was subjected to CID cleavage using MS ² precursor as the MS ² precursor, and MS ² analysis was performed. [Glycan + 96] of m / z = 1718.6 in MS ^{2 -} the fragment ions to further CID cleaved as MS ³ precursor, to obtain a MS ³ spectrum (FIG. 8 (a)). In addition, the negative ion of m / z = 2445.0 has a peptide part having an amino acid sequence of Arg-Asn-Glu-Glu-Tyr-Asn, and is a glycopeptide in which a sugar chain is added to the C-terminal Asn. Deprotonated form. In MS ² spectra (FIG. _{^{8 (A)), 2,4 A}} R and [Glycan + 96] ^- the difference between the m / z was 240.

<Evaluation of Analysis 5 and Analysis 6>
FIG. 8 (A), FIG. 8 (B) and FIG. 8 (C) are mass spectra of analysis 6 (α1-acid glycoprotein), analysis 1 (IgG) and analysis 5 (transferrin), respectively. The upper part is an MS ² spectrum, and the lower part is an MS ³ spectrum obtained using [Glycan + 96] ⁻ fragment ion of m / z = 1718.6 as a precursor. Since each MS ³ spectrum in FIG. 8 is substantially the same, m / z is a sugar as in the case where the sugar chain does not have core fucose (analysis 2 and analysis 3). It can be seen that the MS ³ spectrum obtained using the fragment ion of chain residue +96 as the MS ³ precursor shows the same spectral pattern regardless of the structure of the peptide moiety.

In Analysis 5 (FIGS. 7A, 7B, and 8C), a glycopeptide having 1 amino residue (Lys) on the C-terminal side of the sugar chain-modified asparagine is used. Similar to 1 and Analysis 6, a strong peak of [Glycan + 96] ⁻ fragment ion appears at MS ² with m / z = 1718.5. From this result, the glycopeptide to be subjected to the analysis method of the present invention is not limited to the glycopeptide in which the C-terminal asparagine is sugar chain-modified, and one amino acid residue is added to the C-terminal side of the sugar chain-modified asparagine. It can be seen that even the glycopeptides possessed can be carried out with high analytical accuracy.

[Example 4: Analysis of glycopeptide derived from lactoferrin]
<Preparation of glycopeptide for measurement>
Human lactoferrin purchased from SIGMA was digested with Pronase E in the same manner as in Example 1 and then desalted with a carbon column and purified and concentrated with a cellulose microchip. Using this sample, a multistage mass with negative ions CID was used. Analysis was carried out.

<Analysis 7 and Analysis 8>
Each of the negative ion of m / z = 1619.6 (analysis 7) and the negative ion of m / z = 1819.7 (analysis 8) obtained in MS ¹ was cleaved by CID using MS ² precursor, and MS ² Analysis was performed to obtain an MS ² spectrum shown in FIG. 9 (A) and FIG. In analysis 7, an MS ³ spectrum (FIG. 9B) obtained by cleaved CID using a [Glycan + 96] ⁻ fragment ion of MS ^{2 at} MS / z = 1312.4 as an MS ³ precursor was obtained. The negative ion at m / z = 1619.6 has the amino acid sequence Ser-Gly-Gln-Asn in the peptide part, and the negative ion at m / z = 1819.7 has the Ser-Gly-Gln-Asn in the peptide part. It has an amino acid sequence of Gly-Gln-Asn-Val-Thr, and all are deprotonated glycopeptides in which a sugar chain is added to asparagine. In both analysis 7 and analysis 8, the m / z difference between ^2,4 A _R and [Glycan + 96] ⁻ was 240 in the MS ² spectrum (FIG. 9A and FIG. 10).

<Evaluation of Analysis 7 and Analysis 8>
Both analytical 7 and analysis 8, the MS ² spectra, in addition to ^{2, 4} A _R of m / z = 1072.4, the m / z = 1312.4 [Glycan + 96] - fragment ions is obtained . Further, the MS ³ analysis 7 (FIG. 9 (B)), other peaks ^{2, 4} A _R with ^{_{m / z = 1072.4, B R}} -1, 2,4 A R-1, D, Fragment ions common to the MS ² spectrum such as ^0,3 A _R-1 and C ₂ are obtained. The results, [Glycan + 96] fragment ions MS ^{2 is} found to be a fragment ion with a further reducing terminal of the sugar chain structure than 2, 4 _{A R.} This MS ³ spectrum clearly has a different pattern from the MS ³ spectra of the above analysis 1, ³ , 5, and 6, and it can be seen that the difference in sugar chain structure is reflected.

The glycopeptide derived from lactoferrin used in Example 4 is different in sugar chain structure from the glycopeptide used in Examples 1 to 3, but [Glycan + 96] ⁻ MS ² as in Analysis 1-6 above. Fragment ions are generated. From the above results, it can be seen that the analysis method of the present invention is applicable to glycopeptides having various N-linked sugar chains.

In analysis 8 (FIG. 10) using a glycopeptide having 2 amino acid residues (Val-Thr) on the C-terminal side of the sugar chain-modified asparagine, MS ² is compared to analysis 7 (FIG. 9 (A)). The peak intensity of [Glycan + 96] ⁻ in FIG. Thus, the negative ions of glycopeptides having 2 amino acid residues at the C-terminal side of glycosylated asparagine in the case of the MS ² precursor, [Glycan + 96] ^- the MS ² peak intensities of fragment ions is reduced The result is the same tendency as the analysis 4 (FIG. 6).

From this result, in the analysis method of the present invention, the glycopeptide negative ion selected as the MS ² precursor is preferably linked to the asparagine near the C-terminal of the peptide, regardless of the structure of the sugar chain. However, the amino acid sequence on the N-terminal side of asparagine to which a sugar chain is added is not particularly limited, and it can be seen that it may have 2 or 3 or more amino acids.

Claims (15)

A method for analyzing a glycopeptide in which an N-linked sugar chain is bound to asparagine using mass spectrometry,
MS ⁿ to and cleave said MS ⁿ precursor MS ⁿ precursor that is selected in the selection step, performing a MS ⁿ analysis; MS ⁿ precursor selection step selects a MS ⁿ precursor from the negative ions from the fragment ions of glycopeptides Analysis steps,
In this order,
In the MS ⁿ precursor selection step, a fragment peptide having a mass-to-charge ratio of (G + 97-z) / z is selected as the MS ⁿ precursor (where n is an integer of 2 or more, G Is the mass of the sugar chain residue and z is the charge number of the fragment ion, which is a natural number). In the MS ⁿ precursor selection step,
The peak list of fragment ions derived from the negative ion of the glycopeptide is compared with the mass Sx of each sugar chain in any known sugar chain database,
The analysis method according to claim 1, wherein a fragment ion having a mass-to-charge ratio of (Sx + 79-z) / z is selected as the MS ⁿ precursor from the peak list. In the MS ⁿ precursor selection step,
Two peaks are extracted from the peak list of fragment ions derived from the negative ion of the glycopeptide,
2. The analysis method according to claim 1, wherein the MS ⁿ precursor is selected from a combination of peaks in which a difference in mass-to-charge ratio of extracted peaks coincides with one or more of predetermined values set in advance. The analysis according to claim 3, wherein the one or more predetermined values are 240 and 386, and a fragment ion having a larger mass-to-charge ratio among the two extracted peaks is selected as the MS ⁿ precursor. Method. Before the MS ⁿ precursor selection step,
A glycopeptide N- linked sugar chain asparagine bound by negative ionization, MS ¹ analysis step executes MS ¹ analysis; and the negative ions of the resulting glycopeptide MS ¹ analysis step or the MS ¹ analysis, the fragment ions obtained by cleaving the negative ions of the resulting glycopeptide step, MS ^n-1 precursor is cleaved as, MS ^n-1 to perform analysis MS ^n-1 analysis step, a has the MS ^The MS ⁿ precursor is selected from the fragment ions derived from the negative ions of the glycopeptide obtained in the MS ^n-1 analysis step in the ⁿ precursor selection step. (Where n is an integer of 3 or more). A glycopeptide in which an N-linked sugar chain is bound to asparagine used in the MS ¹ analysis is N-linked to an asparagine residue at the C-terminal of the peptide or an asparagine residue adjacent to the C-terminal amino acid residue. The method for analyzing a glycopeptide according to claim 5, which is a glycopeptide to which a type sugar chain is bound. A sample preparation step of preparing the glycopeptide by protease digestion prior to the MS ¹ analysis;
In the sample preparation step, a glycopeptide in which an N-linked sugar chain is bound to the C-terminal asparagine residue of the peptide or an asparagine residue adjacent to the C-terminal amino acid residue is generated. 4. A method for analyzing a glycopeptide according to 1.   8. The analysis method according to claim 7, wherein in the sample preparation step, both endopeptidase and exopeptidase are used as the protease. On the basis of the MS ⁿ spectrum obtained by MS ⁿ analysis step, analysis step of extracting structure information of N- linked glycan, further comprising the analysis method according to any one of claims 1-8 . In the analysis step,
MS ⁿ spectrum obtained in the MS ⁿ analysis step;
When the mass of any known N-linked sugar chain is Gx, a precursor ion having a mass-to-charge ratio of (Gx + 97-zx) / zx (where zx is a charge number and a natural number) is cleaved. A database of known MS ^N spectra or MS ^N peak lists of fragment ions (where N is an integer greater than or equal to 2),
The analysis method according to claim 9, wherein the structure information is extracted by collating. In the analysis step,
MS ⁿ spectrum obtained in the MS ⁿ analysis step;
A virtual MS ^N spectrum or a virtual MS ^N peak list of fragment ions having a mass-to-charge ratio of (Sx + 79−zx) / zx calculated based on the sugar chain structure stored in an arbitrary sugar chain database (where N Is an integer greater than or equal to 2, Sx is the mass of a known N-linked sugar chain, zx is a charge number and a natural number),
The analysis method according to claim 9, wherein the structure information is extracted by collating. A method for analyzing a glycopeptide in which an N-linked sugar chain is bound to asparagine using mass spectrometry,
Discrimination of the presence or absence of core fucose addition to the reducing terminal monosaccharide of N-linked sugar chain based on the mass spectrum obtained by cleaving the negative ion of the glycopeptide or the fragment ion derived from the negative ion of the glycopeptide as a precursor A determination step for performing
In the discrimination step, the core fucose to the sugar chain reducing terminal monosaccharide is determined based on the difference in mass to charge ratio between a predetermined sugar chain-derived fragment ion and a fragment ion having a mass to charge ratio of (G + 97−z) / z. A method for analyzing glycopeptides, wherein the presence or absence of addition is discriminated. In the discrimination step, the predetermined sugar chain-derived fragment ion is a non-reducing end-side fragment ion cleaved at the C-C bond at positions 2-3 and 4-5 of N-acetylglucosamine,
When the difference in mass to charge ratio between the predetermined sugar chain-derived fragment ion and the fragment ion having a mass to charge ratio of (G + 97-z) / z is 240, no addition of core fucose,
When the difference in mass to charge ratio between the predetermined sugar chain-derived fragment ion and the fragment ion having a mass to charge ratio of (G + 97−z) / z is 386, there is an addition of core fucose,
The method for analyzing a glycopeptide according to claim 12, wherein the determination is performed. An analysis data storage unit for storing analysis data obtained by mass spectrometry; a database storage unit for storing structure information obtained from an existing sugar chain structure database; and data of the analysis data storage unit and of the database storage unit The structure of the N-linked glycan of a glycopeptide in which an N-linked glycan is bound to asparagine is identified using a glycan structure analysis system comprising a data processing unit equipped with a computer for data comparison A sugar chain structure analysis program for
In the analysis data storage unit, an MS ⁿ spectrum obtained by performing MS ⁿ analysis using a negative fragment ion having a mass-to-charge ratio of (G + 97−z) / z derived from a negative ion of a glycopeptide as a precursor. Or data containing the MS ⁿ peak list is stored,
In the database storage unit, a precursor ion having a mass-to-charge ratio of (Gx + 97-zx) / zx where Gx is the mass of any known N-linked sugar chain (where zx is the charge of a negative ion) Data containing a known MS ^N spectrum or MS ^N peak list (where N is an integer greater than or equal to 2) of fragment ions obtained by cleaving the number (natural number)
The computer of the data processing unit comprises the step of collating the data in the analysis data storage unit with the data in the database storage unit to determine whether or not they match or scoring the similarity between the two. A program for analyzing a sugar chain structure, characterized in that the program is executed. An analysis data storage unit for storing analysis data obtained by mass spectrometry; a database storage unit for storing structure information obtained from an existing sugar chain structure database; and data of the analysis data storage unit and of the database storage unit The structure of the N-linked glycan of a glycopeptide in which an N-linked glycan is bound to asparagine is identified using a glycan structure analysis system comprising a data processing unit equipped with a computer for data comparison A sugar chain structure analysis program for
In the analysis data storage unit, an MS ⁿ spectrum obtained by performing MS ⁿ analysis using a negative fragment ion having a mass-to-charge ratio of (G + 97−z) / z derived from a negative ion of a glycopeptide as a precursor. Or data including MS ⁿ peak list is stored,
In the database storage unit, a virtual MS ^N spectrum or virtual MS of fragment ions having a mass-to-charge ratio of (Sx + 79−zx) / zx calculated based on the sugar chain structure stored in an arbitrary sugar chain database Data including an ^N peak list (where Sx is the mass of a known N-linked sugar chain, zx is a charge number and a natural number, and N is an integer of 2 or more),
The step of determining whether or not they match by scoring the data in the analysis data storage unit and the data in the database storage unit or scoring the similarity between the two is performed by the computer of the data processing unit A program for analyzing a sugar chain structure, characterized in that the program is executed.

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