JP2002189029A - Determination method for amino acid sequence at n- terminal of protein - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method, in which the amino acid sequence at the N- terminal of a protein is determined efficiently. SOLUTION: The determination method for the amino acid sequence at the N-terminal of the protein, whose N-terminal is modified includes a process (1) wherein the protein whose N-terminal is modified is cut by a chemical or biochemical means; a process (2), in which the amino group at the N-terminal of a peptide obtained by the process (1) is labeled with heavy hydrogen; a process (3), wherein regarding the peptide obtained by the process (2), the mass spectrum of fragment ions is acquired; and a peptide which is not labeled with the heavy hydrogen is specified and a process (4), wherein the amino acid sequence of the specified peptide is determined.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

[0001] The present invention relates to a method for determining the amino acid sequence of the amino terminus of a protein.

[0002]

2. Description of the Related Art Determination of the amino acid sequence of a protein is important as a means for confirming and identifying the structure of a protein. In particular, it is important to determine the amino acid sequence of the amino terminal portion of a protein, for example, to obtain a gene encoding a useful protein. There are two types of naturally-occurring proteins in which the amino terminus (hereinafter referred to as N-terminus) is modified and in which the amino terminus is not modified. In order to determine the N-terminal amino acid sequence of a protein whose N-terminal is not modified (hereinafter referred to as an N-terminal unmodified protein), that is, a protein having no substituent at the N-terminal amino group, for example, the Edman degradation method is conventionally used. However, there are drawbacks such as a relatively large amount of sample required for the method and a need to purify the sample used in the method. In addition, the Edman degradation method is used for determining the N-terminal amino acid sequence of a protein whose N-terminus has been modified (hereinafter referred to as an N-terminal modified protein), that is, a protein in which the hydrogen atom of the terminal amino group has been substituted with an acetyl group or the like. Can not.

On the other hand, recently, attempts have been made to determine the amino acid sequence of a protein using mass spectrometry.
The mass spectrometry has a feature that it can be applied to a mixture sample or a trace sample. In amino acid sequence determination using mass spectrometry, the mass spectrum of fragment ions (hereinafter, fragment fragment) generated by the collision activation dissociation method (hereinafter, referred to as CID method) or Post Source Decay (hereinafter, referred to as PSD method) treatment The MS / MS method for acquiring ions and the mass spectrum of fragment ions
S / MS spectra) are obtained, and the spectra are analyzed to determine the amino acid sequence. Generally, the MS / MS spectrum is obtained by this method because the molecular weight is about 3000 or less. For high molecular weight substances exceeding this, generally, peptides having a molecular weight of about 3000 or less are cleaved by chemical or biochemical means. And then apply. For example, the amino acid sequence of each peptide can be determined by cleaving the protein using a digestive enzyme or the like and subjecting the resulting peptides to tandem mass spectrometry, but it is not known which peptide is derived from the N-terminus as it is.

In order to determine the N-terminal amino acid sequence of an N-terminal modified protein, a peptide derived from the N-terminus of the original protein is specified from peptides generated by cleavage of the protein, and the amino acid sequence of this peptide is determined. You need to decide. As a method for specifying a peptide derived from the N-terminus of the N-terminal modified protein, a peptide generated by cleavage of the protein (hereinafter referred to as a truncated peptide) is Br.
Method of collecting peptides eluted without adsorption by treatment with a CN-activated Sepharose column (Anal. Biochem., 222,
210-216 (1994)) or a method for identifying a peptide derived from the N-terminus of the original protein by acetylating the cleaved peptide and comparing the elution positions in reverse phase chromatography of the sample before and after the acetylation treatment. (JP-A 8-
27180) are known.

[0005] In the case of an N-terminal unmodified protein, the above-mentioned specific method is applied after the N-terminal is modified in advance with an acetyl group or the like. In any case, complicated operations are required, and a large amount of sample is required.

[0006] Furthermore, the peptide is dissociated mainly at the main chain portion by the CID method or the PSD method, and fragment ions containing N-terminal and C-terminal of the peptide are generated (Methods in Enzymolog).
y, 193 (1990)). In the obtained MS / MS spectrum, the peak of the fragment ion containing the N-terminus and the peak of the fragment ion containing the C-terminus were mixed, and there was a problem that a great deal of labor was required for the analysis. As a method for distinguishing a fragment ion containing an N-terminus from a fragment ion containing a C-terminus, for example, the C-terminus of each fragmented peptide is converted to ¹⁸ by hydrolyzing with an enzyme in a solution containing H ₂ ¹⁸ O. labeled with O, ¹⁸ O-labeled peptide and ¹⁸ O-labeled non method of comparing the MS / MS spectra of the peptide has been reported (RAPIDCOMMUNICATION
IN MASS SPECTROMETRY, Vol. 5, pp312-315 (199
1)). However, since fragmentation by hydrolysis is essential in this method, it was necessary to go through complicated steps to determine the sequence of the full length peptide.

[0007]

SUMMARY OF THE INVENTION An object of the present invention is to provide a method for efficiently determining the amino acid sequence of the N-terminal portion of a protein.

[0008]

Under these circumstances, the present inventors have studied a method for determining the N-terminal amino acid sequence of a protein. As a result, prior to obtaining a mass spectrum of a fragment ion, the present inventors have studied the method of determining the N-terminal amino acid sequence of a protein or its cleaved peptide. The present inventors have found that the N-terminal amino acid sequence of a protein can be determined extremely efficiently by labeling the terminal amino group with deuterium, leading to the present invention. That is, the present invention provides (1) a step of cleaving a protein having a modified N-terminus by chemical or biochemical means, and (2) deuterium labeling of the N-terminal amino group of the peptide obtained by the step of (1). (3) (2)
For the peptide obtained in the step of, obtaining a mass spectrum of fragment ions, specifying a peptide that is not deuterium labeled, and (4) determining the amino acid sequence of the specified peptide A method for determining the amino acid sequence of the N-terminus of a protein whose N-terminus has been modified, and (a) a step of deuterium-labeling the N-terminal amino group of the protein whose N-terminus has not been modified; Cleaving the protein obtained by the step by chemical or biochemical means, obtaining the mass spectrum of fragment ions for the peptide obtained by the steps (c) and (b), and specifying the deuterium-labeled peptide And (d) determining the amino acid sequence of the specified peptide. It relates amino acid sequencing methods of the N-terminal.

[0009]

BEST MODE FOR CARRYING OUT THE INVENTION Hereinafter, the present invention will be described in detail.
In the present invention, a protein refers to a polymer compound composed of a polypeptide chain in which α-amino acids are linked by peptide bonds, and usually has a molecular weight of 4000 or more. A peptide refers to a peptide in which two or more α-amino acids are linked by a peptide bond, and usually refers to a peptide having 50 or less amino acids. Modification of the amino terminus (sometimes referred to as N-terminus) means that at least one hydrogen atom of the amino group at the amino terminus of a protein or peptide is substituted with an acyl group or the like.

[0010] 1. Method for determining N-terminal amino acid sequence of N-terminal modified protein (1) Step of cleaving N-terminal modified protein The method of cleaving N-terminal modified protein is not particularly limited. For example, A Practical Guide to Protein and Pep
tide Purification for Microsequencing, Second Edit
ion, Academic Press, (1993) and Methods in Enzymolog
y, 193, 389-412, (1990) and the like, a chemical cleavage method,
Examples include a biochemical cleavage method using a digestive enzyme. Specifically, for example, the chemical cleavage method is BrCN
And a biochemical cleavage method using a digestive enzyme includes digestion using trypsin, chymotrypsin, or the like. Cleavage of the N-terminally modified protein yields a cleaved peptide, usually with a molecular weight of 3000 or less.

When the N-terminal modified protein contains cysteine as an amino acid constituting the protein, the protein is subjected to a reductive S-alkylation treatment with, for example, iodoacetamide or 4-vinylpyridine before cleavage of the protein. Is practically preferable. This reductive S-alkylation treatment itself is known, and is described in, for example, A Practical Guideto.
Protein and Peptide Purification for Microsequenci
ng, Second Edition, Academic Press, (1993) and the like.

(2) Step of deuterium-labeling the N-terminal amino group of the peptide obtained in step (1) The method of deuterium-labeling the N-terminal amino group of the peptide obtained in step (1) is as follows. A method of introducing a deuterium-containing acyl group at the N-terminus by acting a deuterium-containing acylating agent on a peptide, a method of introducing a deuterium-containing carbamoyl group at the N-terminus by acting a deuterium-containing isocyanate on a peptide, A method in which a deuterium-containing isothiocyanate is allowed to act to introduce a deuterium-containing thiocarbamoyl group into the N-terminus can be mentioned. Here, substituents for deuterium labeling such as a deuterium-containing acyl group, a deuterium-containing carbamoyl group, and a deuterium-containing thiocarbamoyl group introduced into the N-terminus of the peptide (hereinafter referred to as deuterium substituents). In the above, it is preferable that the deuterium atom in the deuterium substituent is not easily replaced with hydrogen by a solvent or the like. From this point, it is preferable that the deuterium atom is connected to the carbon atom of the deuterium substituent. Such deuterium substituents include, for example, -C
OCD ₃ group, -COCD ₂ H group, -COCDH ₂ group, -C
ONHCD ₃ groups, -CON (CD _{3) 2} groups, -CONHC
D ₂ H group, -CONHCD ₃ group, can be mentioned ₃ groups like CSNHCD.

In addition, the deuterium labeling of the N-terminus of the peptide is preferably performed not on all the peptides but on a part of the peptides. In the partial deuterium labeling, the N-terminus not labeled with deuterium is preferably used. A substituent not containing deuterium corresponding to the deuterium substituent (hereinafter, referred to as a deuterium substituent).
For example, a -COCD ₃ group, a -COCD ₂ H group and a -COC
-COCH ₃ group corresponding to the DH ₂ groups corresponds to this. ) Is similarly substituted. Therefore, the deuterium labeling of the N-terminus of the peptide is usually performed by allowing a mixture of a deuterium substituent-introducing agent and a light hydrogen substituent-introducing agent to act on the peptide.

A mixture of a deuterium substituent-introducing agent and a deuterium substituent-introducing agent is allowed to act on the peptide to label the N-terminal amino group with deuterium. Two kinds of molecular species of the peptide into which the substituent is introduced are obtained. These differ in mass by the number of deuterium at the N-terminus.

The number of deuterium atoms in the deuterium substituent may be one or more, but is preferably about two to five in terms of analysis. The molar ratio of the deuterium substituent-introducing agent to the light hydrogen substituent-introducing agent is preferably about 1/1 for analysis.

In the step of labeling the N-terminal amino group of the peptide obtained in the step (1) with deuterium, the peptide is treated with a compound represented by the general formula [1] (R ¹ CO) ₂ O [1] at pH 5 or less. R ¹ represents an alkyl group, provided that at least one hydrogen atom in R ¹ is substituted with a deuterium atom.) And a deuterium carboxylic anhydride represented by the general formula [2] (R ² CO) ₂ O (2) (wherein, R ² represents an alkyl group. However, the hydrogen atom in R ² is not substituted with deuterium atoms.) exerting a mixture of protium carboxylic acid anhydride represented by Or
At pH 6 or lower, the general formula [3] R ³ NCO [3] (wherein R ³ is an optionally substituted alkyl group, an optionally substituted phenyl group, an optionally substituted naphthyl group or a substituted Wherein at least one hydrogen atom in R ³ is substituted with deuterium.) And a deuterium isocyanate compound represented by the general formula [4] R ⁴ NCO [4] ( In the formula, R ⁴ represents an optionally substituted alkyl group, an optionally substituted phenyl group, an optionally substituted naphthyl group or an optionally substituted pyridyl group, provided that hydrogen in R ² Atoms are not substituted with deuterium atoms) to form a mixture of deuterated carboxylic acid anhydride [1] and light water. Even if the amount of the mixture of the carboxylic anhydride [2] or the mixture of the deuterated isocyanate compound [3] and the deuterated isocyanate [4] is not strictly controlled, it can be obtained in a relatively wide range in the presence of ε-amino. Can also introduce the target substituent at the N-terminus with very high selectivity.

(2) -2 Step of treating peptide with carboxylic anhydride or isocyanate compound i) Treatment of a mixture of deuterated carboxylic anhydride [1] and deuterated carboxylic anhydride [2] Deuterated carboxylic anhydride [1]
And a mixture of a light hydrogen carboxylic acid anhydride [2] (hereinafter sometimes simply referred to as a carboxylic acid anhydride mixture [1] and [2]) to form a carboxylic acid whose N-terminal is selectively used. A peptide substituted with the acyl group corresponding to the acid anhydride mixture is obtained. Deuterated carboxylic anhydride [1] in carboxylic anhydride mixture [1] [2] to be used
Determines the ratio of the deuterium-containing acyl group in the acyl group. Deuterated carboxylic anhydride [1]
R ¹ is a linear or branched alkyl group, preferably a C1-6 linear or branched alkyl group such as a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, and a hexyl group; Specifically, acetic anhydride,
Examples thereof include propionic anhydride, butyric anhydride, and valeric anhydride. However, at least one of the hydrogen atoms in the linear or branched alkyl group is substituted with deuterium. R ² in the light hydrogen carboxylic anhydride [2] is the same as R ¹ except that the hydrogen atom is not replaced with deuterium.

This treatment is carried out at pH 5 or less, preferably at pH 2
5, and more preferably in the range of pH 3-5. In this treatment, a buffer for maintaining the pH is usually used as a solvent. As the solvent, volatile solvents such as an aqueous acetic acid solution, an aqueous formic acid solution, a pyridine-acetic acid buffer solution, and an aqueous trifluoroacetic acid solution are preferable in terms of simplicity of post-treatment. Usually, this treatment is carried out by adding a solution of the carboxylic anhydride mixture [1] [2] or a solution thereof such as tetrahydrofuran to a solution or suspension composed of the peptide and the solvent. This treatment is preferably performed at a temperature of about 10 ° C. or less from the viewpoint of selectivity and at a temperature within a range where the solvent used does not freeze. The processing time is usually about 1 to 60 minutes.

The concentration of the carboxylic anhydride mixture [1] and [2] in the treatment system is usually the initial concentration in the system. When the treatment is usually performed at pH 5, the treatment is performed at about 10 to 30 mM and pH 3.3. In this case, it is about 60 to 300 mM. After the treatment in this treatment, the N-acylated peptide is recovered from the treatment solution. For example, when a volatile solvent is used as the solvent,
The treatment liquid may be distilled off under reduced pressure. When a solvent containing a nonvolatile salt is used, the solvent may be distilled off under reduced pressure after desalting. By performing this treatment, the carboxylic anhydride mixture [1] in which only the N-terminus is selectively used
An acylated peptide corresponding to [2] is obtained.

Ii) Treatment of a mixture of a deuterated isocyanate compound [3] and a deuterated isocyanate compound [4] The peptide obtained in the step (1) may be treated with a deuterated isocyanate compound [3] and a deuterated isocyanate compound at pH 6 or less. (Hereinafter sometimes simply referred to as a mixture of isocyanate compounds [3] and [4]), whereby a peptide in which the N-terminal amino group is selectively carbamoylated can be obtained. The ratio of the deuterium-containing carbamoyl group in the carbamoyl group is determined by the ratio of the isocyanate compound mixture [3] [4] used. R ³ in deuterium isocyanate compound [3]
Is an optionally substituted alkyl group, an optionally substituted phenyl group, an optionally substituted naphthyl group or an optionally substituted pyridyl group. Examples of the alkyl group which may be substituted include a fluorine atom,
Examples include an alkyl group which may be substituted with a halogen atom such as a chlorine atom, a bromine atom, and an iodine atom. Specific examples include a methyl group, an ethyl group, a propyl group, a butyl group, a pentyl group, and a hexyl group. A C1-6 alkyl group such as
Chloromethyl group, iodomethyl group, dibromomethyl group,
Examples thereof include C1-6 haloalkyl groups such as a trifluoromethyl group and an iodoethyl group. Examples of the optionally substituted phenyl group and the optionally substituted naphthyl group include, for example, phenyl group, p-chlorophenyl group, o-
A halogen-substituted phenyl group such as a bromophenyl group or a 2,4-dichlorophenyl group; a naphthyl group such as a 1-naphthyl group or a 2-naphthyl group; a halogen-substituted naphthyl group such as a 1-iodonaphthyl group or a 2-chloronaphthyl group; Can be mentioned. As the pyridyl group which may be substituted,
For example, a pyridyl group such as a 2-pyridyl group, a 3-pyridyl group, a 4-pyridyl group, a 2-chloro-4-pyridyl group,
Examples thereof include a halogen-substituted pyridyl group such as a 3,5-difluoro-2-pyridyl group. However, in the above-mentioned alkyl group which may be substituted, phenyl group which may be substituted, naphthyl group which may be substituted or pyridyl group which may be substituted, at least one of hydrogen atoms is deuterium. Has been replaced by

Specific examples of the deuterated isocyanate compound [3] include phenyl isocyanate, p-chlorophenyl isocyanate, 1-naphthyl isocyanate,
2-naphthyl isocyanate, butyl isocyanate,
Ethyl isocyanate, 4-pyridyl isocyanate,
Examples thereof include 3-pyridyl isocyanate.
In addition, R ⁴ in the light hydrogen isocyanate compound [4]
Is the same as R ¹ except that the hydrogen atom is not replaced by deuterium. This treatment is performed at a pH of 6 or less.
H is in the range of about 3 to 6, more preferably pH 5 to 6.

In this treatment, a buffer for maintaining the above-mentioned pH is usually used as a solvent. As the solvent, acetic acid aqueous solution, formic acid aqueous solution, pyridine-acetic acid buffer,
A volatile solvent such as an aqueous trifluoroacetic acid solution is preferred in terms of simplicity of post-treatment. Usually, this treatment is carried out by adding a solution of the isocyanate compound mixture [3] [4] or a solution thereof such as tetrahydrofuran to a solution or suspension comprising the peptide and the solvent. The processing temperature may be, for example, in a range from a low temperature at which the solvent used does not freeze to about 50 ° C. The processing time is usually about 1 to 60 minutes. The concentration of the isocyanate compound mixture [3] [4] in the treatment system is usually about 2 to 300 mM when the treatment is performed at pH 6 as the initial concentration in the system.

After this treatment, the N-carbamoylated peptide is recovered from the treatment solution. For example, when a volatile solvent is used as the solvent, the treatment liquid may be distilled off under reduced pressure. When a solvent containing a nonvolatile salt is used, the solvent may be distilled off under reduced pressure after desalting. By performing this treatment, a carbamoylated peptide corresponding to the isocyanate compound mixture [3] [4] in which only the N-terminus is selectively used is obtained.

(3) A mass spectrum of fragment ions is obtained for the peptide obtained in the step (2),
Step of Specifying Peptide Derived from N-Terminal Of the peptides obtained in the step (2), peptides other than the N-terminal are treated with a reagent comprising hydrogen (H;
There are two types of molecular species whose terminal numbers are different and those whose N-terminals are modified with a labeling reagent containing deuterium (D; mass number 2) having different mass numbers. That is, when the mass spectrum of the reaction product is measured, the peptide fragments other than the N-terminus have two peaks having a difference in mass difference by the number of deuterium at the N-terminus. On the other hand, since the N-terminus-derived peptide is originally substituted at the N-terminus, it is not affected by the deuterium labeling in the step (2) and does not produce the two peaks described above.
Perform mass spectrometry of the peptide obtained by the step (2),
By specifying the two non-peaked peptides having a mass difference in the number of deuteriums, these can be regarded as peptides derived from the N-terminal of the N-terminal modified protein.

As a method for acquiring an MS / MS spectrum,
Any method can be used as long as it can analyze a mass spectrum obtained by cleaving the peptide at the main chain, and the type of mass spectrometer, ionization method, and peptide fragmentation method are not particularly limited. For example, the latest mass spectrometry (Chemical Doujin), a mass spectrometer described in Methods in Enzymology, 270, 453-586 (1996), an ionization method, and a peptide fragmentation method can be used. For example, as a mass spectrometer, an ion trap mass spectrometer, a quadrupole mass spectrometer, a magnetic field mass spectrometer, a time-of-flight mass spectrometer,
Examples include a Fourier transform mass spectrometer, and the ionization method is electrospray ionization (ESI
Method), the MALDI method, the FAB method, and the like. Examples of peptide fragmentation methods include CID and PSD methods.

(4) Step of Determining the Amino Acid Sequence of the Specified Peptide The fragment ion of the peptide derived from the N-terminus of the protein thus specified is subjected to, for example, tandem mass spectrometry to obtain an MS / MS spectrum, By analyzing the spectrum data, the amino acid sequence can be determined. As a method of tandem mass spectrometry, a method applicable in the step (3) can be similarly used.

2. Method for determining N-terminal amino acid sequence of N-terminal unmodified protein (a) Step of deuterium labeling of N-terminal α-amino group of N-terminal unmodified protein As a method for deuterium labeling of N-terminal of N-terminal unmodified protein, A method of introducing a deuterium-containing acyl group to the N-terminus by causing a deuterium-containing acylating agent to act on the peptide, a method of introducing a deuterium-containing carbamoyl group to the N-terminus by acting a deuterium-containing isocyanate on the peptide, Examples include a method in which a deuterium-containing isothiocyanate is allowed to act on a peptide to introduce a deuterium-containing thiocarbamoyl group into the N-terminus. In the processing of this step, the above-mentioned 1. (2)
1. Except for using the N-terminal unmodified protein in place of the peptide obtained after the step (1) in 1. The method of the step (2) can be similarly applied.

(B) Cleavage step of the protein obtained in the step (a) The protein obtained in the step (a) is cleaved by chemical or biochemical means, so that each protein is usually cleaved. Is a peptide having a molecular weight of 3000 or less. Regarding the method of cutting the protein,
1. 1. Except that the protein obtained in the step (a) is used instead of the N-terminal modified protein used in the step (1). The method of the step (1) can be similarly applied.

(C) A step of obtaining a fragment ion mass spectrum of the peptide obtained by the step (b) and specifying a deuterium-labeled peptide The step (b) of the peptide obtained by the step (b) By obtaining a mass spectrum of the two peaks having a mass difference in the number of contained deuterium due to deuterium labeling from the obtained data, and deriving this from the N-terminus of the protein. Can be identified as a peptide. The method of acquiring the MS / MS spectrum is as described in 1. The same method as the method described in the step (3) can be applied.

(D) determining the amino acid sequence of the specified peptide; determining the amino acid sequence of the specified peptide by subjecting the fragment ion of the peptide derived from the N-terminus of the protein to, for example, tandem mass spectrometry. Can be. As a method of tandem mass spectrometry, a method applicable in the step (c) can be similarly used.

In the amino acid sequence determination of the peptide derived from the N-terminus of the protein in this step, the N-terminus of the peptide is labeled with deuterium. Only the fragment ions containing the peptide will cause a mass difference by the number of deuteriums in the deuterium substituents contained in the peptide. Can be determined as a fragment ion derived from the N-terminal amino group, and by specifying the peak of the fragment ion having no change in mass, it can be determined as a fragment ion derived from the C-terminal carboxyl group. . Then, the fragment ion data derived from the C-terminal carboxyl group and the fragment ion data derived from the N-terminal amino group of the peptide specified in this way are respectively obtained by, for example, Method
MS / MS by the method described in S. Enzymology, 193 (1990).
By following the order of mass on the spectrum data, the amino acid can be identified and its sequence order can be analyzed from the difference in mass, and the amino acid sequence of the peptide can be determined from these results. Of course, it is possible in many cases to determine the amino acid sequence of the peptide by analyzing only one of them. However, the amino acid sequence determined from the fragment ion data derived from the N-terminal amino group and the fragment derived from the C-terminal carboxyl group can be determined. By analyzing both of the amino acid sequences determined from the ion data, the amino acid sequence of the peptide can be determined more accurately. In particular, since the fragment ion of a specific portion is not detected, the N-terminal amino group-derived C Since the purpose can be more effectively achieved in the case where it is difficult to determine the amino acid sequence by analysis of either one derived from the terminal carboxyl group, the amino acid sequence of the peptide can be reliably determined.

In the method for determining the amino terminal amino acid sequence of the N-terminal modified protein and the method for determining the amino terminal amino acid sequence of the N-terminal unmodified protein according to the present invention, the protein separated by electrophoresis is retained on a gel. It is also possible to apply it in a state where it is set.

The method for determining the amino-terminal amino acid sequence of the N-terminal modified protein and the method for determining the amino-terminal amino acid sequence of the N-terminal unmodified protein of the present invention allow parallel processing of multiple samples. The sample can be quickly measured by mass spectrometry. Furthermore, since these steps can be automated, multiple samples can be processed quickly.

The method described above makes it possible to rapidly determine the N-terminal sequence of many proteins. For example, by comparing with the genomic sequence, the initiation codon can be determined and the translation initiation signal can be quickly identified. .

[0035]

EXAMPLES Hereinafter, the present invention will be described in more detail by reference examples and examples, but the present invention is not limited to these examples. Example 1 (1) 200 μg of equine cytochrome c (manufactured by Sigma), a protein whose N-terminus is modified, is dissolved in 200 μL of a 1% ammonium bicarbonate solution, 4 μL of 1 mg / mL chymotrypsin is added, and the mixture is added at 37 ° C. for 18 hours. Incubate.

(2) Reaction solution 5 of the chymotrypsin digest
Concentrate μL under reduced pressure and add 0.1M pyridine-acetic acid (pH 5.0) to the residue.
Is added. After incubating the sample on ice for 1 minute, 5 μL of a reagent obtained by mixing 1M of 0.1M acetic anhydride / tetrahydrofuran and 0.1M acetic anhydride d-6 / tetrahydrofuran anhydride using a deuterated acetic anhydride at 1: 1 was added. Incubate on ice for 5 minutes. After the reaction, the solvent is distilled off under reduced pressure, and the residue is used as a reaction sample for mass spectrometry. Mass spectrometry is a double focusing mass spectrometer (JEOL, Model JMS-HX
/ HX110A) and use the FAB ionization method. Each sample was dissolved in 2 μL of water / methanol / acetic acid (50 / 50.1), 1 μL of the sample solution was mixed with 1 μL of glycerol / thioglycerol (1.1), and the mixture was subjected to a mass spectrometer.
Perform the measurement. Of the peaks other than the matrix detected in the FAB mass spectrum of the reaction sample, m / z 1162.5
The other peaks are detected as two peaks having a difference of 3 amu, and the peak at m / z 1162.5 indicates a normal isotope distribution.
Therefore, the peak at m / z 1162.5 can be identified as a peptide derived from the N-terminus of equine cytochrome c. The FAB-MS / MS spectrum of this m / z 1162.5 peak was acquired, and the N-terminal structure was Ac-GDVE
KGKKIF, indicating that this N-terminal structure is the already known N-terminal structure of cytochrome c.

Example 2 5 μL of the reaction solution of the chymotrypsin digest obtained in (1) of Example 1 was concentrated under reduced pressure, and 0.1 M pyridine-acetic acid (pH 6.
Add 12 μL of (0). After incubating the sample on ice for 1 minute, 0.1M phenyl isocyanate / anhydrotetrahydrofuran and 0.1M phenyl isocyanate-d / anhydrotetrahydrofuran using a deuterium-substituted phenyl group of isocyanic acid were mixed at a ratio of 1: 1. Add 5 μL of reagent and incubate on ice for 5 minutes. After the reaction, the solvent is distilled off under reduced pressure, and the residue is used as a reaction sample for mass spectrometry. For mass spectrometry, the same apparatus as in Example 1 was used.
Dissolve in acetic acid (50 ・ 50 ・ 1) 2μL, 1μL of sample solution
Is mixed with 1 μL of glycerol / thioglycerol (1.1) and subjected to a mass spectrometer to measure FAB-MS. Among the peaks other than the matrix detected in the FAB-MS spectrum of the sample after the reaction treatment, the peak other than m / z 1162.5 was 5
detected as two peaks with amu difference, m / z 1162.5
Indicates a normal isotope distribution. Therefore, m / z1162.
The peak at 5 can be identified as a peptide derived from the N-terminus of equine cytochrome c.

Example 3 (1) 200 pmol of recombinant human growth hormone (hereinafter, referred to as hGH), which is a protein whose N-terminus is not modified, is added in an amount of 0.2 pmol.
After dissolving in 12 μL of 1M pyridine-acetic acid (pH 5.0) and incubating on ice for 1 minute, 0.1M acetic anhydride / tetrahydrofuran and 0.1M acetic anhydride d-6 / tetrahydrofuran anhydride using a deuterated acetic anhydride were used. Add 5 μL of the reagent mixed at 1: 1 and incubate on ice for 5 minutes.
After the reaction, the solvent is distilled off under reduced pressure.

(2) 200 pm of the reaction-treated hGH obtained in (1)
was dissolved in 50 μL of a 100 mM ammonium bicarbonate solution, and the solution was dissolved in 0.1 ml.
Add 5 μL of 1 mg / mL trypsin and incubate at 37 ° C. for 18 hours. The reaction solution of the trypsin digest is concentrated under reduced pressure to obtain a reaction sample.

(3) Mass spectrometry uses a double-focusing mass spectrometer (JEOL, model JMS-HX / HX110A), and uses the FAB ionization method. The reaction sample was treated with water / methanol / acetic acid (50
・ 50 ・ 1) Dissolve in 4μL, mix 1μL of sample solution with 1μL of glycerol / thioglycerol (1.1), supply to a mass spectrometer, and measure FAB-MS. Of the peaks detected in the FAB mass spectrum of the reaction sample, only the peak at m / z 972.4 is detected as a doublet peak containing a deuterated product. Therefore, the peak at m / z 972.4 is
It can be identified as a terminal-derived peptide. This m / z 972.4 peak coincides with the hGH N-terminal structure FPTIPLSR acetylated.

Example 4 (1) Analysis of the N-terminus of a protein on an electrophoresis gel using a recombinant human growth hormone (hereinafter referred to as hGH), which is a protein whose N-terminus is not modified, as a sample. Do. For electrophoresis, 0.5 μg of an hGH sample is electrophoresed using a MultiphorII system (Pharmacia) and an SDS-PAGE gel ExcelGel SDS gradient 8-18 (Pharmacia). The gel after electrophoresis is Coomassie Brilliant Blue (CBB).
It is written. ) In water / methanol / acetic acid (40 ・ 60 ・ 1)
Dye for 10 minutes with a solution adjusted to 0.1% concentration in
Decolorize by soaking in methanol / acetic acid (40.60.1) for 1 hour. After the CBB staining operation, a band corresponding to hGH is cut out.

(2) The reductive alkylation of cysteine is performed as follows. Place the gel in a 1.5 mL tube and add 50
Add 0.1 μL of 0.1 M ammonium bicarbonate solution and 50 μL of acetonitrile and allow 30 minutes to infiltrate. After repeating this operation three times, 10 mM dithiothreitol / 0.1 M ammonium bicarbonate solution is added, and the mixture is kept at 56 ° C. for 1 hour. After discarding the solution, 55 mM iodoacetamide / 0.1 M ammonium bicarbonate is added and reacted at room temperature for 45 minutes. After discarding the solution, add 50 μL of 0.1 M ammonium bicarbonate solution
The operation of adding μL of acetonitrile and permeating for 30 minutes is repeated three times, and the solvent contained in the gel is distilled off under reduced pressure.

(3) Prepare the reductively alkylated gel obtained in the preceding paragraph and treat it with phenyl isocyanate. The gel subjected to the reductive alkylation treatment was treated with 12 μL of 0.1 M pyridine-acetic acid (pH 6.0) and 0.1 M phenyl isocyanate / tetrahydrofuran anhydride and 0.1 M phenyl isocyanate-d / d using deuterium-substituted phenyl group of isocyanic acid. After adding 5 μL of a reagent obtained by mixing anhydrous tetrahydrofuran at a ratio of 1: 1 and reacting in ice for 10 minutes, the solvent is distilled off under reduced pressure to carry out phenyl isocyanate treatment.

(4) 0.1 mg / mL of trypsin was added to 5 μL and 15 μL of the phenyl isocyanate-treated gel obtained in (3).
The protein is cleaved by adding 0.1 M ammonium bicarbonate and reacting at 37 ° C. for 18 hours. After cutting,
Water / acetonitrile / formic acid (50 ・ 50.5)
Add 50 μL, infiltrate for 40 minutes, and collect supernatant.
Repeatedly, the cleaved peptide is extracted, the solvent is distilled off under reduced pressure, and the residue is subjected to mass spectrometry. Mass spectrometry uses a double focusing mass spectrometer (JEOL, model JMS-HX / HX110A), and uses the FAB ionization method. Glycerol / thioglycerol (1.1) is converted to water / methanol / acetic acid (50
Use a matrix diluted to 20% in 50.1). The sample is water / methanol / acetic acid (50 ・ 50 ・ 1) 1
It is dissolved in μL, 1 μL of the sample solution is mixed with 1 μL of the matrix, and the mixture is supplied to a mass spectrometer to measure FAB-MS. Reaction sample
Of the peaks detected in the FAB mass spectrum, m
Only the peak at /z1049.4 is detected as a doublet peak containing a deuterated product. Therefore, the peak at m / z 1049.4 can be identified as a peptide derived from the N-terminus of hGH. This m
The /z1049.4 peak is consistent with the carbamoylated version of hGH N-terminal structure FPTIPLSR.

[0045]

According to the present invention, a method for efficiently determining the N-terminal amino acid sequence of a protein can be provided.

Claims (7)

[Claims] (1) a step of cleaving a protein having a modified N-terminus by chemical or biochemical means, and (2) deuterium labeling of the N-terminal amino group of the peptide obtained by the step of (1). (3) obtaining a mass spectrum of fragment ions for the peptide obtained in the step (3) and (2), and specifying a peptide not labeled with deuterium, and (4) the amino acid of the specified peptide. A method for determining an N-terminal amino acid sequence of a protein having an N-terminal modified, comprising a step of determining a sequence. (2) deuterium labeling of the N-terminal amino group of the protein whose N-terminal is not modified, and (b) the protein obtained by the step (a) by a chemical or biochemical means. Cleaving, obtaining a mass spectrum of fragment ions for the peptide obtained by the steps (c) and (b), and specifying the deuterium-labeled peptide; and (d) the amino acid of the specified peptide. A method for determining an N-terminal amino acid sequence of a protein whose N-terminal is not modified, comprising a step of determining a sequence. 3. A step of (1) cleaving a protein having a modified N-terminus by chemical or biochemical means, and (2) a deuterium labeling of the N-terminal amino group of the peptide obtained by the step of (1). And a step of obtaining a fragment ion mass spectrum of the peptide obtained by the steps (3) and (2), and identifying a peptide that has not been labeled with deuterium. A method for identifying a peptide derived from the N-terminus of a given protein. (4) deuterium labeling of the N-terminal amino group of the protein whose N-terminal is not modified, and (b) the protein obtained by the step (a) by a chemical or biochemical means. The N-terminal is modified, comprising a step of cleaving, obtaining a mass spectrum of fragment ions for the peptide obtained by the steps (c) and (b), and specifying a deuterium-labeled peptide. Method for identifying a peptide derived from the N-terminus of a non-protein. 5. The method according to claim 5, wherein the step of labeling the N-terminal amino group of the protein or peptide with deuterium is performed by adding a compound represented by the general formula [1] (R ¹ CO) ₂ O [1] (where R ¹ is An alkyl group, provided that at least one hydrogen atom in R ¹ is substituted with a deuterium atom.) And a deuterium carboxylic anhydride represented by the general formula [2] (R ² CO) ₂ O [ 2] (wherein, R ² represents an alkyl group. However, the hydrogen atom in R ² is not substituted with deuterium atoms.) or to apply a mixture of protium carboxylic acid anhydride represented by,
At pH 6 or lower, the general formula [3] R ³ NCO [3] (wherein R ³ is an optionally substituted alkyl group, an optionally substituted phenyl group, an optionally substituted naphthyl group or a substituted Wherein at least one hydrogen atom in R ³ is substituted with deuterium.) And a deuterium isocyanate compound represented by the general formula [4] R ⁴ NCO [4] ( In the formula, R ⁴ represents an optionally substituted alkyl group, an optionally substituted phenyl group, an optionally substituted naphthyl group or an optionally substituted pyridyl group, provided that hydrogen in R ² The method according to any one of claims 1 to 4, wherein the reaction is carried out with a mixture with a deuterium isocyanate compound represented by the formula (1) wherein the atom is not substituted with a deuterium atom. 6. The method according to claim 1, 3 or 5, wherein prior to the step (1), the N-terminal modified protein is subjected to reductive S-alkylation. 7. The method according to claim 2, wherein prior to the step (a), the protein whose N-terminal is not modified is subjected to reductive S-alkylation.
Or the method of 5.