Method for determining amino acid sequence of peptide, and derivatizing agent

Abstract

The present invention provides: a method for determining the amino acid sequence of a peptide without depending on a database; and a derivatizing agent useful for the method. The present invention is a method for determining the amino acid sequence of a peptide, the method involving: a step for performing multiple-stage tandem mass spectrometry (MSn) on a derivatized peptide in which a derivatizing agent having a coumarin skeleton is bound to a peptide to obtain a fragment ion mass spectrum of the derivatized peptide; and a step for determining the amino acid sequence of the peptide on the basis of peaks of fragment ions of the derivatized peptide in which peptide bonds are sequentially cleaved.

Description

Method for determining the amino acid sequence of a peptide and derivatizing agent

[0001] The present invention relates to a method for determining the amino acid sequence of a peptide, and a derivatizing agent. This application claims priority to Japanese Patent Application No. 2024-043164, filed on March 19, 2024, the contents of which are incorporated herein by reference.

[0002] Obtaining information about the amino acid sequences of peptides and proteins is important for understanding their functions, etc. In recent years, amino acid sequence analysis has been performed based on genetic information and fragment ion mass spectra obtained from mass spectrometers. Generally, for peptides from species with extensive databases, such as humans and mice, the sequence can be determined by referencing the database using genetic information and peaks in the fragment ion mass spectrum.

[0003] However, this method is difficult to apply to biological species for which the number of registered data is so small that it does not function as a database, or for which no database exists. For such species, the only option is to predict the sequence from the peaks in the fragment ion mass spectrum by de novo sequencing. In this case, since it is impossible to distinguish which part of the sequence the fragment ion mass spectrum originates from, a large number of amino acid sequence candidates are calculated, making accurate amino acid sequence analysis difficult.

[0004] In response to this, a technique is known in which the N-terminus or C-terminus of a peptide is chemically modified in advance, and peptide fragments containing the modified termini are detected with high sensitivity in mass spectrometry.

[0005] For example, Patent Document 1 discloses a method for determining the amino acid sequence of a peptide by mass spectrometry, in which an amino acid derivative such as N-biotinylcysteic acid, in which the amino group of an amino acid having an acidic group in the side chain is protected with a protecting group, is bound to the N-terminus of a peptide to be analyzed or a peptide fragment obtained by fragmenting the peptide to be analyzed as needed.

[0006] Japanese Patent Application Laid-Open No. 2004-294431

[0007] As techniques for determining the amino acid sequence of peptides continue to advance, there is a demand for the development of a technique that can determine accurate peptide sequences without relying on databases, as opposed to the conventional methods described above.

[0008] The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a method for determining the amino acid sequence of a peptide without relying on a database, and a derivatizing agent useful for said method.

[0009] The present invention includes the following aspects: [1] A method for determining the amino acid sequence of a peptide, comprising the steps of: performing multistage tandem mass spectrometry (MSn) of a derivatized peptide in which a derivatizing agent having a coumarin skeleton is bound to the peptide to obtain a fragment ion mass spectrum of the derivatized peptide; and determining the amino acid sequence of the peptide based on peaks of fragment ions of the derivatized peptide in which the peptide bonds are sequentially cleaved.

[0010] [2] The method according to [1], wherein the derivatized peptide is a compound represented by the following formula (1):

[0011] [In formula (1), R １ is absent or represents a substituent selected from the group consisting of a hydroxyl group, an alkyl group, an aryl group, an alkoxy group, an acyl group, an acyloxy group, an alkylsulfonyloxy group, an arylsulfonyloxy group, a halogen atom, an amino group, a nitro group, a cyano group, and an alkenyl group. n represents an integer of 0 to 5. When n is an integer of 2 or more, a plurality of R １ When n is an integer of 2 or more, multiple R １ may be bonded to each other to form a ring structure. ２ represents a single bond or a divalent linking group. ３ represents the residue obtained by removing the amino group from the peptide.

[0012] [3] The method according to [1] or [2], wherein the compound represented by formula (1) is a compound represented by the following formula (1′):

[0013] [In formula (1'), R １is absent or represents a substituent selected from the group consisting of a hydroxyl group, an alkyl group, an alkoxy group, a halogen atom, and an amino group. n represents an integer of 0 to 5. When n is an integer of 2 or more, a plurality of R １ When n is an integer of 2 or more, multiple R １ may be bonded to each other to form a ring structure. ３ represents the residue obtained by removing the amino group from the peptide.

[0014] [4] The method according to [1] or [2], wherein the compound represented by formula (1) is a compound represented by the following formula (1″):

[0015] [In formula (1''), R １ is absent or represents a substituent selected from the group consisting of a hydroxyl group, an alkyl group, an alkoxy group, a halogen atom, and an amino group. n represents an integer of 0 to 5. When n is an integer of 2 or more, a plurality of R １ When n is an integer of 2 or more, multiple R １ may be bonded to each other to form a ring structure. ２ represents a single bond or a divalent linking group. ３ represents the residue obtained by removing the amino group from the peptide.

[0016] [5] The method according to any one of [1] to [4], wherein the peptide consists of 10 or less amino acid residues.

[0017] [6] A derivatizing agent used for determining the amino acid sequence of a peptide, comprising a compound represented by the following formula (2):

[0018] [In formula (2), R １ is absent or represents a substituent selected from the group consisting of a hydroxyl group, an alkyl group, an aryl group, an alkoxy group, an acyl group, an acyloxy group, an alkylsulfonyloxy group, an arylsulfonyloxy group, a halogen atom, an amino group, a nitro group, a cyano group, and an alkenyl group. n represents an integer of 0 to 5. When n is an integer of 2 or more, a plurality of R １ When n is an integer of 2 or more, multiple R１ may be bonded to each other to form a ring structure. ２ represents a single bond or a divalent linking group. ４ represents an amine-reactive group.

[0019] [7] The derivatizing agent according to [6], wherein the compound represented by formula (2) is a compound represented by the following formula (2'):

[0020] [In formula (2'), R １ is absent or represents a substituent selected from the group consisting of a hydroxyl group, an alkyl group, an alkoxy group, a halogen atom, and an amino group. n represents an integer of 0 to 5. When n is an integer of 2 or more, a plurality of R １ When n is an integer of 2 or more, multiple R １ may be bonded to each other to form a ring structure. ４ represents an amine-reactive group.] [8] The derivatizing agent according to [6], wherein the compound represented by formula (2) is a compound represented by the following formula (2″): [In formula (2''), R １ is absent or represents a substituent selected from the group consisting of a hydroxyl group, an alkyl group, an alkoxy group, a halogen atom, and an amino group. n represents an integer of 0 to 5. When n is an integer of 2 or more, a plurality of R １ When n is an integer of 2 or more, multiple R １ may be bonded to each other to form a ring structure. ２ represents a single bond or a divalent linking group. ４ represents an amine-reactive group.

[0021] According to the present invention, it is possible to provide a method for determining the amino acid sequence of a peptide without relying on a database, and a derivatizing agent useful for said method.

[0022] The left side of FIG. 1A is a chromatogram showing the results of liquid chromatography (LC) of OH-Cou-G-G-G-G in Example 1-1. The right side of FIG. 1A is a fragment ion mass spectrum showing the results of tandem mass spectrometry (MS / MS) of OH-Cou-G-G-G-G in Example 1-1. The left side of FIG. 1B is a chromatogram showing the results of LC of MeO-Cou-G-G-G-G in Example 1-2. The right side of FIG. 1B is a fragment ion mass spectrum showing the results of MS / MS of MeO-Cou-G-G-G-G in Example 1-2. The left side of FIG. 1C is a fragment ion mass spectrum showing the results of MS / MS of (CH ３ CH ２ ) ２ The right side of FIG. 1C shows a chromatogram of N-Cou-GGGG-G (CH ３ CH ２ ) ２FIG. 1D shows a fragment ion mass spectrum showing the results of MS / MS of N-Cou-G-G-G-G. The left side of FIG. 1D is a chromatogram showing the results of LC of G-G-G-G in Comparative Example 1-1. The right side of FIG. 1D is a fragment ion mass spectrum showing the results of MS / MS of G-G-G-G in Comparative Example 1-1. The left side of FIG. 1E is a chromatogram showing the results of LC of (N-succinimidyloxycarbonylmethyl)tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP)-G-G-G in Comparative Example 1-2. The right side of FIG. 1E is a fragment ion mass spectrum showing the results of MS / MS of TMPP-G-G-G-G in Comparative Example 1-2. The left side of FIG. 2A is a fragment ion mass spectrum showing the results of MS / MS of MeO-Cou-Y-P in Example 2-1. The center of Figure 2A is a fragment ion mass spectrum showing the results of MS / MS of MeO-Cou-S-E-L in Example 2-1. The right side of Figure 2A is a fragment ion mass spectrum showing the results of MS / MS of MeO-Cou-S-Y-F-V in Example 2-1. The left side of Figure 2B is a fragment ion mass spectrum showing the results of MS / MS of Y-P in Comparative Example 2-1. The center of Figure 2B is a fragment ion mass spectrum showing the results of MS / MS of S-E-L in Comparative Example 2-1. The right side of Figure 2B is a fragment ion mass spectrum showing the results of MS / MS of S-Y-F-V in Comparative Example 2-1. The left side of Figure 2C is a fragment ion mass spectrum showing the results of MS / MS of 3-aminopyridyl-N-hydroxysuccinimidyl carbamate (APDS)-Y-P in Comparative Example 2-2. The center of Figure 2C is a fragment ion mass spectrum showing the results of MS / MS of APDS-S-E-L in Comparative Example 2-2. The right side of Figure 2C is a fragment ion mass spectrum showing the results of MS / MS of APDS-S-Y-F-V in Comparative Example 2-2. The left side of Figure 2D is a fragment ion mass spectrum showing the results of MS / MS of 2,4,6-trinitrobenzenesulfonic acid (TNP)-Y-P in Comparative Example 2-3.The center of Figure 2D is a fragment ion mass spectrum showing the results of MS / MS of TNP-S-E-L in Comparative Example 2-3. The right side of Figure 2D is a fragment ion mass spectrum showing the results of MS / MS of TNP-S-Y-F-V in Comparative Example 2-3. The first row from the top of Figure 3A is a chromatogram showing the results of LC of MeO-Cou-G-G-G-G in Example 3. The second row from the top of Figure 3A is a chromatogram showing the results of LC of MeO-Cou-G-G-G-G-G in Example 3. The third row from the top of Figure 3A is a chromatogram showing the results of LC of MeO-Cou-G-G-G-G-G-G in Example 3. The fourth row from the top of Figure 3A is a chromatogram showing the results of LC of MeO-Cou-G-G-G-G-G-G-G in Example 3. The fifth row from the top of FIG. 3A is a chromatogram showing the results of LC of MeO-Cou-G-G-G-G-G-G-G-G in Example 3. The sixth row from the top of FIG. 3A is a chromatogram showing the results of LC of MeO-Cou-G-G-G-G-G-G-G-G-G in Example 3. The seventh row from the top of FIG. 3A is a chromatogram showing the results of LC of MeO-Cou-G-G-G-G-G-G-G-G-G-G in Example 3. FIG. 3B is a fragment ion mass spectrum showing the results of MS / MS of MeO-Cou-G-G-G-G-G-G-G-G in Example 3. FIG. 3C is a fragment ion mass spectrum showing the results of MS / MS of MeO-Cou-G-G-G-G-G-G-G-G in Example 3. The left side of FIG. 4A is a chromatogram showing the results of LC of MeO-Cou-G-G-G-G in Example 4. The right side of FIG. 4A is a fragment ion mass spectrum showing the results of MS / MS of MeO-Cou-G-G-G-G in Example 4. The left side of FIG. 4B is a chromatogram showing the results of LC of SPB (succinimidyl-[4-(psoralen-8-yloxy)]-butyrate)-G-G-G in Example 4. The right side of FIG. 4B is a fragment ion mass spectrum showing the results of MS / MS of SPB-G-G-G-G in Example 4.The left side of Figure 5A is a chromatogram showing the results of LC of PBSE (Pacific bruce succinimidyl ester)-S-L-V-P in Example 5. The right side of Figure 5A is a fragment ion mass spectrum showing the results of MS / MS of PBSE-S-L-V-P in Example 5. The left side of Figure 5B is a chromatogram showing the results of LC of PBSE (Pacific bruce succinimidyl ester)-A-V-T-P in Example 5. The right side of Figure 5B is a fragment ion mass spectrum showing the results of MS / MS of PBSE-A-V-T-P in Example 5.

[0023] In the present invention and this specification, the term "comprising" means that components other than the target component may be included. The term "consisting of" means that components other than the target component are not included. The term "consisting essentially of" means that components other than the target component are not included in a form that exerts a special function (such as a form that completely loses the effect of the invention). In this specification, when "comprising" is used, it includes both an embodiment "consisting of" and an embodiment "consisting essentially of."

[0024] As used herein, the terms "amino acid sequence" and "peptide" may include at least two amino acids or amino acid analogs covalently linked by a peptide (amide) bond or peptide bond analog. As used herein, the term "peptide" includes oligomers and polymers of amino acids or amino acid analogs. The term "peptide" also includes molecules commonly referred to as oligopeptides, which contain from about 2 to 20 amino acid residues. The term "peptide" may also include molecules commonly referred to as polypeptides, which often contain more than 20 amino acid residues. The term peptide may also include molecules commonly referred to as proteins, which may contain at least about 20 amino acid residues and a set of defined structural features (e.g., a set of secondary, tertiary, and quaternary structures). The amino acids of a peptide may be L-amino acids or D-amino acids. Peptides, polypeptides, or proteins may be synthetic, recombinant, or natural. Synthetic peptides are peptides produced in vitro by artificial means.

[0025] <Method for Determining Amino Acid Sequence of Peptide> The method for determining the amino acid sequence of a peptide of this embodiment includes the steps of performing multistage tandem mass spectrometry (MSn) of a derivatized peptide in which a derivatizing agent having a coumarin skeleton (hereinafter also referred to as a "coumarin derivatizing agent") is bound to the peptide to obtain a fragment ion mass spectrum of the derivatized peptide, and determining the amino acid sequence of the peptide based on the peaks of fragment ions of the derivatized peptide in which the peptide bonds have been sequentially cleaved.

[0026] <<Derivatized Peptide Having a Coumarin Skeleton>> A derivatized peptide having a coumarin skeleton (hereinafter also referred to as a "coumarin-derivatized peptide") of this embodiment has a coumarin derivatizing agent bound to the peptide.

[0027] The coumarin-derivatized peptide includes a compound represented by the following formula (1):

[0028] [In formula (1), R １is absent or represents a substituent selected from the group consisting of a hydroxyl group, an alkyl group, an aryl group, an alkoxy group, an acyl group, an acyloxy group, an alkylsulfonyloxy group, an arylsulfonyloxy group, a halogen atom, an amino group, a nitro group, a cyano group, and an alkenyl group. n represents an integer of 0 to 5. When n is an integer of 2 or more, a plurality of R １ When n is an integer of 2 or more, multiple R １ may be bonded to each other to form a ring structure. ２ represents a single bond or a divalent linking group. ３ represents the residue obtained by removing the amino group from the peptide.

[0029] In formula (1), R １ Examples of the alkyl group in the formula (I) include linear, branched, and cyclic monovalent saturated hydrocarbon groups. The linear alkyl group preferably has 1 to 10 carbon atoms, more preferably 1 to 5 carbon atoms, and even more preferably 1 to 3 carbon atoms. The branched alkyl group preferably has 3 to 15 carbon atoms, more preferably 3 to 10 carbon atoms, and even more preferably 3 to 5 carbon atoms. The cyclic alkyl group preferably has 3 to 15 carbon atoms, more preferably 4 to 12 carbon atoms, and even more preferably 5 to 10 carbon atoms.

[0030] In formula (1), R １ The aryl group in the formula (I) is an aryl group having 6 to 20 carbon atoms, of which phenyl and naphthyl groups are preferred.

[0031] In formula (1), R １ The alkoxy group in the formula (I) preferably has 1 to 5 carbon atoms, and is more preferably a methoxy group, an ethoxy group, an n-propoxy group, an iso-propoxy group, an n-butoxy group, or a tert-butoxy group, further preferably a methoxy group or an ethoxy group, and most preferably a methoxy group.

[0032] In formula (1), R １The acyl group in the formula (I) preferably has 1 to 6 carbon atoms, more preferably 1 to 5 carbon atoms, still more preferably 1 to 3 carbon atoms, and particularly preferably 1 or 2 carbon atoms. The alkyl group in the acyl group may be linear or branched. Specific examples of the acyl group include an acetyl group and a propionyl group.

[0033] In formula (1), R １ The acyloxy group in the formula (I) preferably has 1 to 6 carbon atoms, more preferably 1 to 5 carbon atoms, still more preferably 1 to 3 carbon atoms, and particularly preferably 1 or 2 carbon atoms. Specific examples of the acyloxy group include an acetoxy group and an ethylcarbonyloxy group.

[0034] In formula (1), R １ Examples of the halogen atom in include fluorine, chlorine, bromine, and iodine, with a fluorine atom being preferred.

[0035] In formula (1), R １ The amino group in may contain the above-mentioned alkyl group in the amino group, and is preferably a monovalent amino group obtained by removing hydrogen from ammonia, a methylamino group, a dimethylamino group, a dimethylamino group, or a diethylamino group, and more preferably a diethylamino group.

[0036] In formula (1), R １ The alkenyl group in the formula (I) includes linear or cyclic alkenyl groups, preferably having 2 to 5 carbon atoms, more preferably having 2 to 4 carbon atoms, and even more preferably having 2 or 3 carbon atoms.

[0037] In formula (1), R １ is preferably a substituent selected from the group consisting of a hydroxyl group, an alkyl group, an alkoxy group, a halogen atom, and an amino group, and more preferably a substituent selected from the group consisting of a hydroxyl group, a methoxy group, a fluorine atom, and a diethylamino group.

[0038] In formula (1), n ​​is preferably an integer of 0 to 4, more preferably an integer of 0 to 3, even more preferably an integer of 1 to 3, and particularly preferably 1 or 2. When n is an integer of 2 or more, a plurality of R１ may be the same as or different from each other, and may be bonded to each other to form a ring structure. １ The ring structure formed by mutual bonding may be a condensed ring formed with the coumarin skeleton, and may be a monocyclic or polycyclic aliphatic ring, or a monocyclic or polycyclic aromatic ring. Among these, a monocyclic saturated aliphatic ring or a monocyclic aromatic ring is preferred, and a monocyclic aromatic ring is more preferred. Some of the carbon atoms constituting the ring structure may be substituted with a divalent linking group containing a heteroatom. Examples of the heteroatom include an oxygen atom, a sulfur atom, and a nitrogen atom. Among these, an oxygen atom is preferred. When multiple R １ The ring structure formed by bonding together may or may not have a substituent. １ The substituents in the above formula (I) are the same as those in the above formula (I). １ More specific examples of the ring structure formed by mutual bonding include ring structures represented by the following formula (r-1) or (r-2) (a condensed ring with a coumarin skeleton), but are not limited to these.

[0039]

[0040] Multiple Rs １ Among the above, the ring structure formed by mutual bonding is preferably a ring structure represented by formula (r-1).

[0041] In formula (1), R ２ Examples of the divalent linking group in formula (1) include a divalent hydrocarbon group and a divalent linking group containing a hetero atom. Among these, a linear or branched alkylene group containing a bond selected from the group consisting of an ether bond -O-, an ester bond (-C(=O)-O-), and an oxycarbonyl bond (-O-C(=O)-) is preferred. ２ is more preferably —C—C—C—O—, —O—C—C—C— or a single bond.

[0042] In formula (1), R ３The peptide obtained by removing an amino group from the peptide in (1) is not particularly limited as long as the amino acid sequence of the coumarin-derivatized peptide can be determined, and examples thereof include dipeptides, tripeptides, and peptides consisting of more amino acids, as well as various proteins. The peptide preferably has 20 or fewer amino acid residues, more preferably 16 or fewer, even more preferably 13 or fewer, and particularly preferably 10 or fewer.

[0043] Further examples of the coumarin-derivatized peptide include compounds represented by the formula (1) and compounds represented by the following formula (1').

[0044] [In formula (1'), R １ is absent or represents a substituent selected from the group consisting of a hydroxyl group, an alkyl group, an alkoxy group, and an amino group. n represents an integer of 0 to 5. When n is an integer of 2 or more, a plurality of R １ When n is an integer of 2 or more, multiple R １ may be bonded to each other to form a ring structure. ３ represents the residue obtained by removing the amino group from the peptide.

[0045] In formula (1'), R １ The alkyl group and alkoxy group in the formula (1) are １ The same can be mentioned.

[0046] In formula (1'), R １ The linear alkyl group in formula (1') preferably has 1 to 10 carbon atoms, more preferably 1 to 5 carbon atoms, and even more preferably 1 to 3 carbon atoms. １ The branched alkyl group in formula (1') preferably has 3 to 15 carbon atoms, more preferably 3 to 10 carbon atoms, and even more preferably 3 to 5 carbon atoms. １ The cyclic alkyl group in the formula (I) preferably has 3 to 15 carbon atoms, more preferably 4 to 12 carbon atoms, and even more preferably 5 to 10 carbon atoms.

[0047] In formula (1'), R １The alkoxy group in the formula (I) preferably has 1 to 5 carbon atoms, and is more preferably a methoxy group, an ethoxy group, an n-propoxy group, an iso-propoxy group, an n-butoxy group, or a tert-butoxy group, further preferably a methoxy group or an ethoxy group, and most preferably a methoxy group.

[0048] In formula (1'), R １ Examples of the halogen atom in include fluorine, chlorine, bromine, and iodine. Of these, a fluorine atom is preferred.

[0049] In formula (1'), R １ The amino group in the formula (1) is １ Among these, a monovalent amino group obtained by removing hydrogen from ammonia, a methylamino group, a dimethylamino group, a dimethylamino group, and a diethylamino group are preferred, and a diethylamino group is more preferred.

[0050] In formula (1'), R １ As the substituent, a substituent selected from the group consisting of a hydroxyl group, an alkyl group, an alkoxy group, and an amino group is preferred, and a substituent selected from the group consisting of a hydroxyl group, a methoxy group, and a diethylamino group is more preferred.

[0051] In formula (1'), n is preferably an integer of 0 to 4, more preferably an integer of 0 to 3, even more preferably an integer of 1 to 3, and even more preferably 1 or 2. When n is an integer of 2 or more, a plurality of R １ may be the same as or different from each other, and may be bonded to each other to form a ring structure. １ The ring structure formed by bonding together includes R １ Among them, a monocyclic saturated aliphatic ring or a monocyclic aromatic ring is preferred, and a monocyclic aromatic ring is more preferred. A part of the carbon atoms constituting the ring structure is the same as R in the formula (2). １ As in the case of R 1 , the heteroatom may be substituted with a divalent linking group containing a heteroatom. The heteroatom is preferably an oxygen atom. １The ring structure formed by bonding together may or may not have a substituent. １ The substituents in the above formula (I) are the same as those in the above formula (I). １ More specifically, the ring structure formed by bonding together includes R １ Among these, the ring structure represented by the above formula (r-1) is preferred.

[0052] In formula (1'), R ３ The peptide obtained by removing the amino group from the peptide in formula (1) is ３ The peptide preferably has 20 or fewer amino acid residues, more preferably 16 or fewer residues, even more preferably 13 or fewer residues, and particularly preferably 10 or fewer residues.

[0053] Further, the coumarin-derivatized peptide includes a compound represented by formula (1) and a compound represented by the following formula (1″).

[0054] [In formula (1''), R １ is absent or represents a substituent selected from the group consisting of a hydroxyl group, an alkyl group, an alkoxy group, a halogen atom, and an amino group. n represents an integer of 0 to 5. When n is an integer of 2 or more, a plurality of R １ When n is an integer of 2 or more, multiple R １ may be bonded to each other to form a ring structure. ２ represents a single bond or a divalent linking group. ３ represents the residue obtained by removing the amino group from the peptide.

[0055] In formula (1''), R １ The alkyl group and alkoxy group in the formula (1) are １ The same can be mentioned.

[0056] In formula (1''), R １The linear alkyl group in formula (1") preferably has 1 to 10 carbon atoms, more preferably 1 to 5 carbon atoms, and even more preferably 1 to 3 carbon atoms. １ The branched alkyl group in formula (1") preferably has 3 to 15 carbon atoms, more preferably 3 to 10 carbon atoms, and even more preferably 3 to 5 carbon atoms. １ The cyclic alkyl group in the formula (I) preferably has 3 to 15 carbon atoms, more preferably 4 to 12 carbon atoms, and even more preferably 5 to 10 carbon atoms.

[0057] In formula (1''), R １ The alkoxy group in the formula (I) preferably has 1 to 5 carbon atoms, and is more preferably a methoxy group, an ethoxy group, an n-propoxy group, an iso-propoxy group, an n-butoxy group, or a tert-butoxy group, further preferably a methoxy group or an ethoxy group, and most preferably a methoxy group.

[0058] In formula (1''), R １ Examples of the halogen atom in include fluorine, chlorine, bromine, and iodine. Of these, a fluorine atom is preferred.

[0059] In formula (1''), R １ The amino group in the formula (1) is １ Among these, a monovalent amino group obtained by removing hydrogen from ammonia, a methylamino group, a dimethylamino group, a dimethylamino group, and a diethylamino group are preferred, and a diethylamino group is more preferred.

[0060] In formula (1''), R １ As the substituent, a substituent selected from the group consisting of a hydroxyl group, an alkyl group, an alkoxy group, and an amino group is preferred, and a substituent selected from the group consisting of a hydroxyl group, a methoxy group, and a diethylamino group is more preferred.

[0061] In formula (1″), n is preferably an integer of 0 to 4, more preferably an integer of 0 to 3, even more preferably an integer of 1 to 3, and even more preferably 1 or 2. When n is an integer of 2 or more, a plurality of R １may be the same as or different from each other, and may be bonded to each other to form a ring structure. １ The ring structure formed by bonding together includes R １ Among them, a monocyclic saturated aliphatic ring or a monocyclic aromatic ring is preferred, and a monocyclic aromatic ring is more preferred. A part of the carbon atoms constituting the ring structure is the same as R in the formula (2). １ As in the case of R 1 , the heteroatom may be substituted with a divalent linking group containing a heteroatom. The heteroatom is preferably an oxygen atom. １ The ring structure formed by bonding together may or may not have a substituent. １ The substituents in the above formula (I) are the same as those in the above formula (I). １ More specifically, the ring structure formed by bonding together includes R １ Among these, the ring structure represented by the above formula (r-1) is preferred.

[0062] In formula (1''), R ２ The divalent linking group in the formula (1) is ２ Among these, a linear or branched alkylene group containing a bond selected from the group consisting of an ether bond (-O-), an ester bond (-C(=O)-O-), and an oxycarbonyl bond (-O-C(=O)-) is preferred. In formula (1"), R ２ is more preferably —C—C—C—O—, —O—C—C—C— or a single bond, and further preferably —O—C—C—C—.

[0063] In formula (1''), R ３ The peptide obtained by removing the amino group from the peptide in formula (1) is ３ The peptide preferably has 20 or fewer amino acid residues, more preferably 16 or fewer residues, even more preferably 13 or fewer residues, and particularly preferably 10 or fewer residues.

[0064] Preferred examples of coumarin-derivatized peptides include compounds represented by any of the following formulas (1-1) to (1-7).

[0065] (In the formula, R ３ represents the residue obtained by removing the amino group from the peptide.

[0066] Among the compounds represented by any one of the above formulas (1-1) to (1-7), the compounds represented by any one of the following formulas (1-1) to (1-5) are more preferred.

[0067] <Step of Obtaining Fragment Ion Mass Spectrum of Coumarin-Derivatized Peptide> Although the coumarin-derivatized peptide does not require prior separation and purification as long as a fragment ion mass spectrum of the peptide to be measured can be obtained, from the viewpoint of more accurate sequence determination, separation and purification by liquid chromatography (LC) is preferred. LC can be appropriately combined with known peptide separation methods, and examples thereof include separation and purification by high-performance liquid chromatography (HPLC).

[0068] [Multistage tandem mass spectrometry] The ionization method and ion separation detection method for coumarin-derivatized peptides are not particularly limited as long as they can provide a fragment ion mass spectrum. Examples of ionization methods for derivatized peptides include matrix-assisted laser desorption / ionization (MALDI) and electrospray ionization (ESI).

[0069] Mass spectrometers for ion separation and detection of coumarin-derivatized peptides include multistage tandem mass spectrometers such as ion trap mass spectrometers, quadrupole mass spectrometers, magnetic field mass spectrometers, time-of-flight (TOF) mass spectrometers, Fourier transform mass spectrometers, and linear trap (quadrupole)-time-of-flight mass spectrometers. In the present invention and this specification, the term "multistage tandem mass spectrometry (MSn)" includes tandem mass spectrometry (MS / MS).

[0070] <Step of Determining the Amino Acid Sequence of a Peptide> The amino acid sequence of a peptide according to this embodiment can be determined based on the peaks of fragment ions of a coumarin-derivatized peptide in which peptide bonds are sequentially cleaved. The peaks of fragment ions of the coumarin-derivatized peptide are detected in the form in which the target peptide is sequentially cleaved from the N-terminus, thereby enabling sequential sequencing from the N-terminus.

[0071] <Derivatizing Agent Used for Determining Amino Acid Sequence of Peptide> The derivatizing agent of this embodiment has a coumarin skeleton. Examples of the coumarin derivatizing agent include a derivatizing agent used for determining the amino acid sequence of a peptide, which is composed of a compound represented by the following formula (2):

[0072] [In formula (2), R １ is absent or represents a substituent selected from the group consisting of a hydroxyl group, an alkyl group, an aryl group, an alkoxy group, an acyl group, an acyloxy group, an alkylsulfonyloxy group, an arylsulfonyloxy group, a halogen atom, an amino group, a nitro group, a cyano group, and an alkenyl group. n represents an integer of 0 to 5. When n is an integer of 2 or more, a plurality of R １ When n is an integer of 2 or more, multiple R １ may be bonded to each other to form a ring structure. ２ represents a single bond or a divalent linking group. ４ represents an amine-reactive group.

[0073] In formula (2), R １ The alkyl group in the formula (1) is １ Examples of the alkyl group include those similar to those listed above. The linear alkyl group preferably has 1 to 10 carbon atoms, more preferably 1 to 5 carbon atoms, and even more preferably 1 to 3 carbon atoms. The branched alkyl group preferably has 3 to 15 carbon atoms, more preferably 3 to 10 carbon atoms, and even more preferably 3 to 5 carbon atoms. The cyclic alkyl group preferably has 3 to 15 carbon atoms, more preferably 4 to 12 carbon atoms, and even more preferably 5 to 10 carbon atoms.

[0074] In formula (2), R １ The aryl group in the formula (I) is an aryl group having 6 to 20 carbon atoms, of which phenyl and naphthyl groups are preferred.

[0075] In formula (2), R １ The alkoxy group in the formula (I) preferably has 1 to 5 carbon atoms, and is more preferably a methoxy group, an ethoxy group, an n-propoxy group, an iso-propoxy group, an n-butoxy group, or a tert-butoxy group, further preferably a methoxy group or an ethoxy group, and most preferably a methoxy group.

[0076] In formula (2), R １ The acyl group in the formula (I) preferably has 1 to 6 carbon atoms, more preferably 1 to 5 carbon atoms, still more preferably 1 to 3 carbon atoms, and particularly preferably 1 or 2 carbon atoms. The alkyl group in the acyl group may be linear or branched. Specific examples of the acyl group include an acetyl group and a propionyl group.

[0077] In formula (2), R １ The acyloxy group in the formula (I) preferably has 1 to 6 carbon atoms, more preferably 1 to 5 carbon atoms, still more preferably 1 to 3 carbon atoms, and particularly preferably 1 or 2 carbon atoms. Specific examples of the acyloxy group include an acetoxy group and an ethylcarbonyloxy group.

[0078] In formula (2), R １ Examples of the halogen atom in include fluorine, chlorine, bromine, and iodine. Of these, a fluorine atom is preferred.

[0079] In formula (2), R １ The amino group in may contain the above-mentioned alkyl group in the amino group, and is preferably a monovalent amino group obtained by removing hydrogen from ammonia, a methylamino group, a dimethylamino group, a dimethylamino group, or a diethylamino group, and more preferably a diethylamino group.

[0080] In formula (2), R １The alkenyl group in the formula (I) includes linear or cyclic alkenyl groups, preferably having 2 to 5 carbon atoms, more preferably having 2 to 4 carbon atoms, and even more preferably having 2 or 3 carbon atoms.

[0081] In formula (2), R １ As the substituent, a substituent selected from the group consisting of a hydroxyl group, an alkyl group, an alkoxy group, and an amino group is preferred, and a substituent selected from the group consisting of a hydroxyl group, a methoxy group, and a diethylamino group is more preferred.

[0082] In formula (2), n is preferably an integer of 0 to 4, more preferably an integer of 0 to 3, even more preferably an integer of 1 to 3, and particularly preferably 1 or 2. When n is an integer of 2 or more, a plurality of R １ may be the same as or different from each other, and may be bonded to each other to form a ring structure. １ The ring structure formed by bonding together includes R １ Among them, a monocyclic saturated aliphatic ring or a monocyclic aromatic ring is preferred, and a monocyclic aromatic ring is more preferred. A part of the carbon atoms constituting the ring structure may be substituted with a divalent linking group containing a heteroatom. Examples of the heteroatom include R in the formula (1). １ Among them, an oxygen atom is preferable. １ The ring structure formed by bonding together may or may not have a substituent. １ The substituents in the above formula (I) are the same as those in the above formula (I). １ More specifically, the ring structure formed by bonding together includes R １ Among these, the ring structure represented by the above formula (r-1) is preferred.

[0083] In formula (2), R ２ The divalent linking group in the formula (1) is ２Among these, a linear or branched alkylene group containing a bond selected from the group consisting of an ether bond (-O-), an ester bond (-C(=O)-O-), and an oxycarbonyl bond (-O-C(=O)-) is preferred. In formula (2), R ２ is more preferably —C—C—C—O—, —O—C—C—C— or a single bond.

[0084] In formula (2), R ４ The amine-reactive group in is not particularly limited as long as it can be transesterified with the N-terminus of the peptide, but is preferably an isocyanate group, a sulfonyl chloride group, an aldehyde group, a carbodiimide group, an acyl azide group, an anhydride group, a fluorophenyl group, a carbonate group, an imidoester group, an epoxy group, a fluorophenyl ester group, or an N-hydroxysuccinimide group, and more preferably an N-hydroxysuccinimide group.

[0085] Further, the coumarin derivatizing agent includes a compound represented by the formula (2) and a compound represented by the following formula (2').

[0086] [In formula (2'), R １ is absent or represents a substituent selected from the group consisting of a hydroxyl group, an alkyl group, an alkoxy group, a halogen atom, and an amino group. n represents an integer of 0 to 5. １ When n is an integer of 2 or more, multiple R １ may be bonded to each other to form a ring structure. ４ represents an amine-reactive group.

[0087] In formula (2'), R １ The alkyl group and alkoxy group in the formula (2) are １ The same can be mentioned.

[0088] In formula (2'), R １ The linear alkyl group in formula (2') preferably has 1 to 10 carbon atoms, more preferably 1 to 5 carbon atoms, and even more preferably 1 to 3 carbon atoms. １The branched alkyl group in formula (2') preferably has 3 to 15 carbon atoms, more preferably 3 to 10 carbon atoms, and even more preferably 3 to 5 carbon atoms. １ The cyclic alkyl group in the formula (I) preferably has 3 to 15 carbon atoms, more preferably 4 to 12 carbon atoms, and even more preferably 5 to 10 carbon atoms.

[0089] In formula (2'), R １ The alkoxy group in the formula (I) preferably has 1 to 5 carbon atoms, and is more preferably a methoxy group, an ethoxy group, an n-propoxy group, an iso-propoxy group, an n-butoxy group, or a tert-butoxy group, further preferably a methoxy group or an ethoxy group, and most preferably a methoxy group.

[0090] In formula (2'), R １ The amino group in the formula (2) is １ Among these, a monovalent amino group obtained by removing hydrogen from ammonia, a methylamino group, a dimethylamino group, a dimethylamino group, and a diethylamino group are preferred, and a diethylamino group is more preferred.

[0091] In formula (2'), R １ As the substituent, a substituent selected from the group consisting of a hydroxyl group, an alkyl group, an alkoxy group, and an amino group is preferred, and a substituent selected from the group consisting of a hydroxyl group, a methoxy group, and a diethylamino group is more preferred.

[0092] In formula (2'), n is preferably an integer of 0 to 4, more preferably an integer of 0 to 3, even more preferably an integer of 1 to 3, and particularly preferably 1 or 2. When n is an integer of 2 or more, a plurality of R １ may be the same as or different from each other, and may be bonded to each other to form a ring structure. １ The ring structure formed by bonding together includes R １ Among them, a monocyclic saturated aliphatic ring or a monocyclic aromatic ring is preferred, and a monocyclic aromatic ring is more preferred. A part of the carbon atoms constituting the ring structure is the same as R in the formula (2). １In the same manner as in the case of R 1 , the hetero atom may be substituted with a divalent linking group containing a hetero atom. Among these, the hetero atom is preferably an oxygen atom. １ The ring structure formed by bonding together may or may not have a substituent. １ The substituents in the above formula (I) are the same as those in the above formula (I). １ More specifically, the ring structure formed by bonding together includes R １ Among these, the ring structure represented by the above formula (r-1) is preferred.

[0093] In formula (2'), R ４ The amine reactive group in the formula (2) is ４ Among these, an isocyanate group, a sulfonyl chloride group, an aldehyde group, a carbodiimide group, an acyl azide group, an anhydride group, a fluorophenyl group, a carbonate group, an imido ester group, an epoxy group, a fluorophenyl ester group, or an N-hydroxysuccinimide group is preferred, and an N-hydroxysuccinimide group is more preferred.

[0094] Further, the coumarin derivatizing agent includes a compound represented by formula (2) and a compound represented by the following formula (2″).

[0095] [In formula (2''), R １ is absent or represents a substituent selected from the group consisting of a hydroxyl group, an alkyl group, an alkoxy group, a halogen atom, and an amino group. n represents an integer of 0 to 5. When n is an integer of 2 or more, a plurality of R １ When n is an integer of 2 or more, multiple R １ may be bonded to each other to form a ring structure. ２ represents a single bond or a divalent linking group. ４ represents an amine-reactive group.

[0096] In formula (2''), R １ The alkyl group and alkoxy group in the formula (2) are １The same can be mentioned.

[0097] In formula (2''), R １ The linear alkyl group in formula (2') preferably has 1 to 10 carbon atoms, more preferably 1 to 5 carbon atoms, and even more preferably 1 to 3 carbon atoms. １ The branched alkyl group in formula (2″) preferably has 3 to 15 carbon atoms, more preferably 3 to 10 carbon atoms, and even more preferably 3 to 5 carbon atoms. １ The cyclic alkyl group in the formula (I) preferably has 3 to 15 carbon atoms, more preferably 4 to 12 carbon atoms, and even more preferably 5 to 10 carbon atoms.

[0098] In formula (2''), R １ The alkoxy group in the formula (I) preferably has 1 to 5 carbon atoms, and is more preferably a methoxy group, an ethoxy group, an n-propoxy group, an iso-propoxy group, an n-butoxy group, or a tert-butoxy group, further preferably a methoxy group or an ethoxy group, and most preferably a methoxy group.

[0099] In formula (2''), R １ The amino group in the formula (2) is １ Among these, a monovalent amino group obtained by removing hydrogen from ammonia, a methylamino group, a dimethylamino group, a dimethylamino group, and a diethylamino group are preferred, and a diethylamino group is more preferred.

[0100] In formula (2''), R １ As the substituent, a substituent selected from the group consisting of a hydroxyl group, an alkyl group, an alkoxy group, and an amino group is preferred, and a substituent selected from the group consisting of a hydroxyl group, a methoxy group, and a diethylamino group is more preferred.

[0101] In formula (2″), n is preferably an integer of 0 to 4, more preferably an integer of 0 to 3, even more preferably an integer of 1 to 3, and particularly preferably 1 or 2. When n is an integer of 2 or more, a plurality of R １may be the same as or different from each other, and may be bonded to each other to form a ring structure. １ The ring structure formed by bonding together includes R １ Among them, a monocyclic saturated aliphatic ring or a monocyclic aromatic ring is preferred, and a monocyclic aromatic ring is more preferred. A part of the carbon atoms constituting the ring structure is the same as R in the formula (2). １ In the same manner as in the case of R 1 , the hetero atom may be substituted with a divalent linking group containing a hetero atom. Among these, the hetero atom is preferably an oxygen atom. １ The ring structure formed by bonding together may or may not have a substituent. １ The substituents in the above formula (I) are the same as those in the above formula (I). １ More specifically, the ring structure formed by bonding together includes R １ Among these, the ring structure represented by the above formula (r-1) is preferred.

[0102] In formula (2''), R ２ The divalent linking group in the formula (1) is ２ Among these, a linear or branched alkylene group containing a bond selected from the group consisting of an ether bond (-O-), an ester bond (-C(=O)-O-), and an oxycarbonyl bond (-O-C(=O)-) is preferred. In formula (1"), R ２ is more preferably —C—C—C—O—, —O—C—C—C— or a single bond, and further preferably —O—C—C—C—.

[0103] In formula (2''), R ４ The amine reactive group in the formula (2) is ４Among these, an isocyanate group, a sulfonyl chloride group, an aldehyde group, a carbodiimide group, an acyl azide group, an anhydride group, a fluorophenyl group, a carbonate group, an imido ester group, an epoxy group, a fluorophenyl ester group, or an N-hydroxysuccinimide group is preferred, and an N-hydroxysuccinimide group is more preferred.

[0104] Preferred examples of the derivatizing agent of this embodiment include derivatizing agents represented by any of the following formulas (2-1) to (2-7).

[0105]

[0106] Among the derivatizing agents represented by any of the above formulas (2-1) to (2-7), the derivatizing agents represented by any of the above formulas (2-1) to (2-5) are more preferred.

[0107] <Method for Producing Coumarin-Derivatized Peptide> The coumarin-skeleton-derivatized peptide of this embodiment can be obtained by dehydration condensation between a coumarin derivatizing agent and the N-terminus of a peptide to be measured. As the raw material for the coumarin derivatizing agent and the coumarin derivative agent, commercially available compounds or synthesized compounds may be used. Examples of commercially available compounds that can be used as coumarin derivatizing agents include N-succinimidyl 7-methoxycoumarin-3-carboxylate (CAS No.: 150321-92-9), N-succinimidyl 7-hydroxycoumarin-3-carboxylate (CAS No.: 134471-24-2), N-succinimidyl 7-(diethylamino)coumarin-3-carboxylate (CAS No.: 139346-57-9), 6,7-methylenedioxy-4-methyl-3-maleimidocoumarin (CAS No.: 97744-90-6), SPB (succinimidyl-[4-(psoralen-8-yloxy)]-butyrate) (CAS No.: 858128-57-1), and Pacific Blue succinimidyl ester (CAS No.: 215868-33-0).

[0108] The peptide to be measured is R ３The peptide preferably has 20 or fewer amino acid residues, more preferably 16 or fewer residues, even more preferably 13 or fewer residues, and particularly preferably 10 or fewer residues.

[0109] The peptide to be measured may also be a partially modified peptide. The modified peptide is not particularly limited as long as the amino acid sequence of the coumarin-derivatized peptide can be determined, but is preferably one selected from the group consisting of phosphorylated peptides, glycopeptides, and nitrated tyrosine-containing peptides.

[0110] The reaction for dehydrating and condensing a coumarin derivatizing agent with the N-terminus of a peptide to be measured is not particularly limited as long as it can dehydrate and condense a coumarin derivatizing agent with a peptide to be measured, and the peptide can be produced by appropriately combining known methods, as in <Synthesis of Coumarin-Derivatized Peptide> shown in [Examples] below.

[0111] <Other Embodiments> In one embodiment, the present invention provides a method for derivatizing a peptide using the compound represented by formula (2) described above. In one embodiment, the present invention provides use of the compound represented by formula (2) described above in the production of a derivatized peptide for determining the amino acid sequence of the peptide.

[0112] The present invention will be described in more detail below with reference to examples, but the present invention is not limited to these examples.

[0113] <Synthesis of Coumarin-Derivatized Peptides> [Synthesis Example 1] 1 μg of a coumarin analog having a hydroxyl group and 4 ng of a peptide with four consecutive glycine residues (G-G-G-G) (SEQ ID NO: 1) were added to 1 M triethylammonium bicarbonate buffer (pH 8.5) and reacted at 25°C for 30 minutes to obtain the following coumarin-derivatized peptide (OH-Cou-G-G-G-G).

[0114]

[0115] Synthesis Example 2 The following coumarin-derivatized peptide (MeO-Cou-GGGG) was obtained in the same manner as in Synthesis Example 1, except that a coumarin analogue having a methoxy group was used.

[0116]

[0117] Synthesis Example 3 Diethylamino ((CH ３ CH ２ ) ２ The following coumarin-derivatized peptide ((CH ３ CH ２ ) ２ N-Cou-GGGG) was obtained.

[0118]

[0119] Synthesis Example 4 The following coumarin-derivatized peptide (SPB-GGGG-G) was obtained in the same manner as in Synthesis Example 1, except that SPB (succinimidyl-[4-(psoralen-8-yloxy)]-butyrate) was used.

[0120]

[0121] Synthesis Example 5 The following coumarin-derivatized peptide (PBSE-S-L-V-P) was obtained in the same manner as in Synthesis Example 1, except that Pacific blue succinimidyl ester (hereinafter also referred to simply as "PBSE") and the four-residue peptide (S-L-V-P) (SEQ ID NO: 9) were used.

[0122]

[0123] <Determination of Amino Acid Sequence of Coumarin-Derivatized Peptide> In Examples 1 to 5, the coumarin-derivatized peptide was subjected to liquid chromatography tandem mass spectrometry (LC-MS / MS) to obtain a fragment ion mass spectrum of the derivatized peptide, and the amino acid sequence was determined based on the peaks of the fragment ions. A compact LC-MS / MS manufactured by Bruker was used.

[0124] Ionization mode: ESI-positive Dry gas: 330°C, 8.0 mL / min Nebulizer gas: 40 psi Collision energy: m / z x 0.0414 eV

[0125] Example 1 Determination of the amino acid sequence of a coumarin-derivatized peptide (a peptide of four consecutive glycine residues (G-G-G-G)) was demonstrated. The peptides of four consecutive glycine residues coumarin-derivatized by Synthesis Examples 1 to 3 (Examples 1-1 to 1-3) were subjected to LC-MS / MS. In addition, an underivatized peptide of four consecutive glycine residues (G-G-G-G) (Comparative Example 1-1) and a peptide of four consecutive glycine residues derivatized with (N-succinimidyloxycarbonylmethyl)tris(2,4,6-trimethoxyphenyl)phosphonium bromide (TMPP-G-G-G-G) (Comparative Example 1-2) were also subjected to LC-MS / MS.

[0126]

[0127] Example 1-1 The left side of Figure 1A is a chromatogram showing the results of LC of OH-Cou-G-G-G-G. The right side of Figure 1A is a fragment ion mass spectrum showing the results of MS / MS of OH-Cou-G-G-G-G. As shown on the right side of Figure 1A, in the MS / MS of OH-Cou-G-G-G-G, fragment ions corresponding to OH-Cou (190 m / z), OH-Cou-G (246 m / z), OH-Cou-G-G (303 m / z), and OH-Cou-G-G-G (360 m / z) were observed. All of these fragment ions had a coumarin tag (203 m / z). This demonstrates that coumarin derivatization enables sequential sequencing of peptides from the N-terminus.

[0128] [Example 1-2] The left side of Figure 1B is a chromatogram showing the LC results of MeO-Cou-G-G-G-G. The right side of Figure 1B is a fragment ion mass spectrum showing the MS / MS results of MeO-Cou-G-G-G-G. As shown on the right side of Figure 1B, in the MS / MS of MeO-Cou-G-G-G-G, fragment ions corresponding to MeO-Cou (203 m / z), MeO-Cou-G (260 m / z), MeO-Cou-G-G (317 m / z), and MeO-Cou-G-G-G (374 m / z) were observed. All of these fragment ions, like OH-Cou-G-G-G, had a coumarin tag. That is, MeO-Cou-GG-GG-G also enabled sequential sequencing from the N-terminus of the peptide by coumarin derivatization, similar to OH-Cou-GG-GG-G. Furthermore, the intensities of each fragment ion were higher than those of OH-Cou-GG-GG-G, demonstrating its usefulness in sequencing.

[0129] [Example 1-3] The left side of FIG. 1C shows (CH ３ CH ２ ) ２ The right side of Figure 1C shows a chromatogram of N-Cou-GGGG-G (CH ３ CH ２ ) ２ 1C shows a fragment ion mass spectrum of N-Cou-GGGG-G as a result of MS / MS. ３ CH ２ ) ２ In MS / MS of N-Cou-GGGG-G, (CH ３ CH ２ ) ２ N-Cou (244m / z), (CH ３ CH ２ ) ２ N-Cou-G (301m / z), (CH ３ CH ２ ) ２ A fragment ion corresponding to N-Cou-GG (358 m / z) was observed. ３ CH ２ ) ２In the case of N-Cou-GGGG, as in Examples 1-1 and 1-2, sequential sequencing from the N-terminal side of the peptide was possible by coumarin derivatization.

[0130] [Comparative Example 1-1] The left side of Figure 1D is a chromatogram showing the results of LC of G-G-G-G. The right side of Figure 1D is a fragment ion mass spectrum showing the results of MS / MS of G-G-G-G. As shown on the right side of Figure 1D, in MS / MS of the peptide of four consecutive glycine residues (G-G-G-G) without derivatization, fragment ions of G-G (115 m / z) and G-G-G (172 m / z) were observed, but it was not possible to identify which part of the original sequence each fragment ion originated from.

[0131] [Comparative Example 1-2] The left side of Figure 1E is a chromatogram showing the results of LC of TMPP-GGGG-G. The right side of Figure 1E is a fragment ion mass spectrum showing the results of MS / MS of TMPP-GGGG-G. As shown on the right side of Figure 1E, no fragment ions were observed for TMPP-GGGG-G, and sequence determination was not possible with this derivatization.

[0132] Example 2 The amino acid sequence determination of coumarin-derivatized 2- to 4-residue peptides (YP, SEL, and SYFV (SEQ ID NO: 2)) was demonstrated. Peptides (MeO-Cou-Peptide) derivatized in the same manner as described in Synthesis Example 2 were used, except that YP, SEL, and SYFV were used as the coumarin-derivatized 2- to 4-residue peptides, respectively. Each was subjected to LC-MS / MS. In addition, an underivatized peptide (Comparative Example 2-1), the following peptide derivatized with 3-aminopyridyl-N-hydroxysuccinimidyl carbamate (APDS-Peptide) (Comparative Example 2-2), and the following peptide derivatized with 2,4,6-trinitrobenzenesulfonic acid (TNP-Peptide) (Comparative Example 2-3) were also subjected to LC-MS / MS in the same manner.

[0133]

[0134]

[0135] Example 2-1 The left side of Figure 2A is a fragment ion mass spectrum showing the results of MS / MS of MeO-Cou-Y-P. The center side of Figure 2A is a fragment ion mass spectrum showing the results of MS / MS of MeO-Cou-S-E-L. The right side of Figure 2A is a fragment ion mass spectrum showing the results of MS / MS of MeO-Cou-S-Y-F-V. As shown in Figure 2A, in the MS / MS of MeO-Cou-Peptide, fragment ions (b0 to b3 in the figure) sequentially cleaved from the N-terminus of the peptide were observed, while retaining the coumarin backbone tag, similar to the fragment ions described in Example 1-2. In other words, coumarin derivatization enabled sequential sequencing of 2- to 4-residue peptides from the N-terminus.

[0136] Comparative Examples 2-1 to 2-3 The left side of FIG. 2B is a fragment ion mass spectrum showing the results of MS / MS of Y-P. The center of FIG. 2B is a fragment ion mass spectrum showing the results of MS / MS of S-E-L. The right side of FIG. 2B is a fragment ion mass spectrum showing the results of MS / MS of S-Y-F-V. The left side of FIG. 2C is a fragment ion mass spectrum showing the results of MS / MS of APDS-Y-P. The center of FIG. 2C is a fragment ion mass spectrum showing the results of MS / MS of APDS-S-E-L. The right side of FIG. 2C is a fragment ion mass spectrum showing the results of MS / MS of APDS-S-Y-F-V. The left side of FIG. 2D is a fragment ion mass spectrum showing the results of MS / MS of TNP-Y-P. The center of FIG. 2D is a fragment ion mass spectrum showing the results of MS / MS of TNP-S-E-L. The right side of FIG. 2D is a fragment ion mass spectrum showing the results of MS / MS of TNP-S-Y-F-V. As shown in Figures 2B to 2D, in MS / MS of the underivatized peptide (Figure 2B, Comparative Example 2-1), APDS-Peptide (Figure 2C, Comparative Example 2-2), and TNP-Peptide (Figure 2D, Comparative Example 2-3), no fragment ions resulting from sequential cleavage from the N-terminus of the peptide were observed, nor was it possible to identify which part of the original sequence each fragment ion originated from. In other words, sequential sequencing of each peptide (YP, SEL, SYFV) from the N-terminus was not possible.

[0137] Example 3 Determination of the amino acid sequences of consecutive 4 to 10 glycine residues (SEQ ID NOS: 1 and 3 to 8) derivatized with a coumarin analogue having a methoxy group was demonstrated. The MeO-Cou-peptides used were those derivatized in the same manner as in Synthesis Example 2, except that peptides with consecutive 4 to 10 glycine residues were used, and each was subjected to LC-MS / MS.

[0138] The first row from the top of FIG. 3A is a chromatogram showing the LC results of MeO-Cou-G-G-G-G. The second row from the top of FIG. 3A is a chromatogram showing the LC results of MeO-Cou-G-G-G-G-G. The third row from the top of FIG. 3A is a chromatogram showing the LC results of MeO-Cou-G-G-G-G-G-G. The fourth row from the top of FIG. 3A is a chromatogram showing the LC results of MeO-Cou-G-G-G-G-G-G-G. The fifth row from the top of FIG. 3A is a chromatogram showing the LC results of MeO-Cou-G-G-G-G-G-G-G. The sixth row from the top of FIG. 3A is a chromatogram showing the LC results of MeO-Cou-G-G-G-G-G-G-G-G. The seventh row from the top of Figure 3A is a chromatogram showing the LC results of MeO-Cou-G-G-G-G-G-G-G-G-G. Figure 3B is a fragment ion mass spectrum showing the MS / MS results of MeO-Cou-G-G-G-G-G-G-G-G-G. Figure 3C is a fragment ion mass spectrum showing the MS / MS results of MeO-Cou-G-G-G-G-G-G-G-G-G-G. As shown in Figures 3A to 3C, in the MS / MS analysis of MeO-Cou-Peptide containing 4 to 10 consecutive glycine residues, fragment ions were observed that were sequentially cleaved from the N-terminus of the peptide while retaining the coumarin tag, similar to the fragment ions described in Examples 1-2 and 2-2. In other words, it was revealed that coumarin derivatization enables sequential sequencing of 4 to 10 residue peptides from the N-terminus.

[0139] Example 4 The coumarin-derivatized four-residue peptide of glycine (SPB-GGGG-G) according to Synthesis Example 4 was subjected to LC-MS / MS. Furthermore, for comparison of fragment ion intensity, MeO-Cou-GGGG-G was subjected to LC-MS / MS in the same manner as in Examples 1-2. The left side of FIG. 4A is a chromatogram showing the LC results of MeO-Cou-GGGG-G. The right side of FIG. 4A is a fragment ion mass spectrum showing the MS / MS results of MeO-Cou-GGGG-G. As shown on the right side of FIG. 4A, in the MS / MS of MeO-Cou-G-G-G, fragment ions corresponding to MeO-Cou- (203 m / z), MeO-Cou-G (260 m / z), MeO-Cou-G-G (317 m / z), and MeO-Cou-G-G-G (374 m / z) were observed, similarly to Example 1-2.

[0140] The left side of Figure 4B is a chromatogram showing the LC results of SPB-G-G-G-G. The right side of Figure 4A is a fragment ion mass spectrum showing the MS / MS results of SPB-G-G-G-G. As shown in the right side of Figure 4B, fragment ions corresponding to SPB- (271 m / z), SPB-G (328 m / z), SPB-G-G (385 m / z), and SPB-G-G-G (442 m / z) were observed in the MS / MS of SPB-G-G-G-G. Like MeO-Cou-G-G-G-G, these fragment ions all contained a coumarin tag. That is, with SPB-G-G-G-G, coumarin derivatization enabled sequential sequencing from the N-terminus of the peptide, as in Examples 1 to 3. Furthermore, the intensity of the fragment ion (b3 in the figure) of SPB-GGGG-G was higher than that of MeO-Cou-GGGG-G, demonstrating its usefulness in terms of sequencing.

[0141] Example 5 The determination of the amino acid sequences of coumarin-derivatized 4-residue peptides (SLVP (SEQ ID NO: 9) and AVTP (SEQ ID NO: 10)) was demonstrated.

[0142] PBSE-SL-V-P synthesized in Synthesis Example 5 was used as the coumarin-derivatized 4-residue peptide and subjected to LC-MS / MS. In addition, a peptide (PBSE-A-V-T-P) derivatized in the same manner as in Synthesis Example 5, except that SLVP was used as the coumarin-derivatized 4-residue peptide, was used as the coumarin-derivatized 4-residue peptide and subjected to LC-MS / MS.

[0143] The left side of Figure 5A is a chromatogram showing the results of LC of PBSE-S-L-V-P. The right side of Figure 5A is a fragment ion mass spectrum showing the results of MS / MS of PBSE-S-L-V-P. As shown on the right side of Figure 5A, fragment ions corresponding to PBSE- (225 m / z), PBSE-S (312 m / z), PBSE-S-L (425 m / z), and PBSE-S-L-V (524 m / z) were observed in the MS / MS of PBSE-S-L-V-P.

[0144] The left side of Figure 5B is a chromatogram showing the results of LC of PBSE-A-V-T-P. The right side of Figure 5B is a fragment ion mass spectrum showing the results of MS / MS of PBSE-A-V-T-P. As shown on the right side of Figure 5B, fragment ions corresponding to PBSE- (225 m / z), PBSE-A (296 m / z), PBSE-A-V (395 m / z), and PBSE-A-V-T (496 m / z) were observed in the MS / MS of PBSE-A-V-T-P.

[0145] In Example 5, all of the observed fragment ions had a coumarin tag, as in Examples 1 to 4. That is, as in Examples 1 to 4, coumarin derivatization enabled sequential sequencing of the peptide from the N-terminus.

[0146] While preferred embodiments of the present invention have been described and illustrated, it should be understood that these are exemplary of the present invention and should not be considered limiting. Additions, omissions, substitutions, and other modifications can be made without departing from the spirit or scope of the present invention. Accordingly, the present invention is not to be deemed limited by the foregoing description, but is limited only by the appended claims.

[0147] According to the present invention, it is possible to provide a method for determining the amino acid sequence of a peptide without relying on a database, and a derivatizing agent useful for said method.

Claims

1. A method for determining the amino acid sequence of a peptide, comprising the steps of: performing multistage tandem mass spectrometry (MSn) of a derivatized peptide in which a derivatizing agent having a coumarin skeleton is bound to the peptide, to obtain a fragment ion mass spectrum of the derivatized peptide; and determining the amino acid sequence of the peptide based on peaks of fragment ions of the derivatized peptide in which peptide bonds have been sequentially cleaved.

2. The method according to claim 1, wherein the derivatized peptide is a compound represented by the following formula (1): [In formula (1), R １ is absent or represents a substituent selected from the group consisting of a hydroxyl group, an alkyl group, an aryl group, an alkoxy group, an acyl group, an acyloxy group, an alkylsulfonyloxy group, an arylsulfonyloxy group, a halogen atom, an amino group, a nitro group, a cyano group, and an alkenyl group. n represents an integer of 0 to 5. When n is an integer of 2 or more, a plurality of R １ When n is an integer of 2 or more, multiple R １ may be bonded to each other to form a ring structure. ２ represents a single bond or a divalent linking group. ３ represents the residue obtained by removing the amino group from the peptide.

3. The method according to claim 2, wherein the compound represented by formula (1) is a compound represented by the following formula (1'): [In formula (1'), R １ is absent or represents a substituent selected from the group consisting of a hydroxyl group, an alkyl group, an alkoxy group, a halogen atom, and an amino group. n represents an integer of 0 to 5. When n is an integer of 2 or more, a plurality of R １ When n is an integer of 2 or more, multiple R １ may be bonded to each other to form a ring structure. ３ represents the residue obtained by removing the amino group from the peptide.

4. The method according to claim 2, wherein the compound represented by formula (1) is a compound represented by the following formula (1″): [In formula (1''), R １ is absent or represents a substituent selected from the group consisting of a hydroxyl group, an alkyl group, an alkoxy group, a halogen atom, and an amino group. n represents an integer of 0 to 5. When n is an integer of 2 or more, a plurality of R １ When n is an integer of 2 or more, multiple R １ may be bonded to each other to form a ring structure. ２ represents a single bond or a divalent linking group. ３ represents the residue obtained by removing the amino group from the peptide.

5. The method according to any one of claims 1 to 4, wherein the peptide consists of 10 or fewer amino acid residues.

6. A derivatizing agent used for determining the amino acid sequence of a peptide, which comprises a compound represented by the following formula (2): [In formula (2), R １ is absent or represents a substituent selected from the group consisting of a hydroxyl group, an alkyl group, an aryl group, an alkoxy group, an acyl group, an acyloxy group, an alkylsulfonyloxy group, an arylsulfonyloxy group, a halogen atom, an amino group, a nitro group, a cyano group, and an alkenyl group. n represents an integer of 0 to 5. When n is an integer of 2 or more, a plurality of R １ When n is an integer of 2 or more, multiple R １ may be bonded to each other to form a ring structure. ２ represents a single bond or a divalent linking group. ４ represents an amine-reactive group.

7. The derivatizing agent according to claim 6, wherein the compound represented by formula (2) is a compound represented by the following formula (2'): [In formula (2'), R １ is absent or represents a substituent selected from the group consisting of a hydroxyl group, an alkyl group, an alkoxy group, a halogen atom, and an amino group. n represents an integer of 0 to 5. When n is an integer of 2 or more, a plurality of R １ When n is an integer of 2 or more, multiple R １ may be bonded to each other to form a ring structure. ４ represents an amine-reactive group.

8. The derivatizing agent according to claim 6, wherein the compound represented by formula (2) is a compound represented by the following formula (2″): [In formula (2''), R １ is absent or represents a substituent selected from the group consisting of a hydroxyl group, an alkyl group, an alkoxy group, a halogen atom, and an amino group. n represents an integer of 0 to 5. When n is an integer of 2 or more, a plurality of R １ When n is an integer of 2 or more, multiple R １ may be bonded to each other to form a ring structure. ２ represents a single bond or a divalent linking group. ４ represents an amine-reactive group.

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