JPH09101310A - Method for analyzing amino acid sequence of peptide - Google Patents

Abstract

PROBLEM TO BE SOLVED: To provide a method for analyzing amino acid sequence of peptide by a mass analysis method with improved sensitivity using an N terminal label reagent and a kit using the same. SOLUTION: In the method for analyzing amino acid sequence of peptide by a mass analysis method combining an N terminal label reagent and an impact activation splitting method, the N terminal mark reagent uses a compound containing a pyridine ring having at least chlorine or bromine bonded thereto while allowing product ions to hold an electric load easily. This also provides a kit for the peptide amino acid sequence analysis by a mass analysis method containing the compound as N terminal label reagent.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

TECHNICAL FIELD The present invention relates to a method for analyzing an amino acid sequence of a peptide by mass spectrometry and a kit used therefor.

[0002]

2. Description of the Related Art Obtaining information on an amino acid sequence of a peptide or protein is important for understanding its function and the like. In recent years, the development of the soft ionization method in the field of mass spectrometry has made it possible to analyze the molecular weight and structure of unstable biopolymers such as proteins, which were almost impossible to measure until then. Furthermore, in mass spectrometry, it has become possible to determine the amino acid sequence of peptides by using the collision activation cleavage method. Mass spectrometry is particularly excellent for structural analysis of peptides / proteins having amino acid substitutions or modifications. Amino acid sequence analysis by mass spectrometry is performed using mass data of parent ions and mass data of product ions generated by the collision activation cleavage method. Among the product ions generated, those derived from the N terminus and C It is not easy to determine the sequence only from the mass data, since some of them originate from the ends and some of them do not belong to any of them. To solve this problem,
Isotope distribution among product ions generated by collision activation cleavage method by labeling C-terminal carboxyl group by hydrolyzing peptide or protein with protease in a reaction solution containing H ₂ ¹⁸ O at high concentration (40 atm%) Has been proposed to identify the product ion derived from the unique C-terminal of RAPID COMMUNICATION IN MASS S
PECTROMETRY), Volume 5, Pages 312-315 (199
1)]. In addition, a method has been reported in which the N-terminal amino group of a peptide is modified with 4-bromophenyl isothiocyanate and this product is identified by utilizing the natural isotope distribution ratio of bromine [European Journal of Biochemistry (European Journal of Biochemistry), Volume 20,
P. 72 (1971)]. The method can also be used in the collision-activated cleavage method to identify the product ion derived from the N terminus from the product ions generated. However, the peptide modified with bromophenyl isothiocyanate has a problem that it is difficult to have a charge.

[0003]

The N-terminal amino group of a peptide is reacted with bromophenyl isothiocyanate,
In order to facilitate the amino acid sequence analysis by mass spectrometry by identifying the product ion derived from the N terminus from the complex product ions generated by the collision activation cleavage method using the natural isotope distribution ratio of bromine, When the peptide is labeled with phenylisothiocyanate, there is a problem that the product ion derived from the N-terminal generated by the collision activation cleavage method is less likely to have a charge, and as a result, the sensitivity is lowered. An object of the present invention is to provide a method for analyzing an amino acid sequence of a peptide by mass spectrometry, which uses a compound (N-terminal labeling reagent) whose product ion easily has a charge, and a kit used for the method. is there.

[0004]

Means for Solving the Problems To outline the present invention, the first invention of the present invention relates to a method for analyzing an amino acid sequence of a peptide, wherein an N-terminal labeling reagent and a collision activation cleavage method are used in combination. In the method for analyzing an amino acid sequence of a peptide by mass spectrometry, a compound containing a pyridine ring to which at least chlorine or bromine is bound and having a product ion easily charged is used as the N-terminal labeling reagent. A second invention of the present invention relates to a kit for analyzing an amino acid sequence of a peptide used in the method of the first invention, wherein the kit used in the method of analyzing the amino acid sequence of the peptide by mass spectrometry contains at least chlorine. Alternatively, a compound containing a pyridine ring to which bromine is bonded and having a product ion easily charged is contained as an N-terminal labeling reagent.

[0005]

BEST MODE FOR CARRYING OUT THE INVENTION The present invention will be specifically described below. The reagent for labeling the N-terminal of the peptide used in the method of the present invention is a compound containing at least a pyridine ring to which chlorine or bromine is bonded and having a product ion easily having a charge. In other words, it is a compound having a functional group capable of undergoing a condensation reaction with the N-terminal amino group of a peptide, having a pyridine ring, and further containing chlorine or bromine introduced into the peptide after binding. Examples include carboxylic acids of bromine (or chlorine) substituted pyridines, their reactive derivatives such as esters,
Examples thereof include acid halides, acid anhydrides or salts, or isothiocyanates containing the pyridine ring. As a specific example, the following formula (Formula 1):

[0006]

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N-succinimidyl 5-bromo-3-pyridinecarboxylate represented by the following formula (Formula 2):

[0008]

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There is 5-bromo-2-pyridyl isothiocyanate represented by The labeled peptide may be purified by an operation such as high performance liquid chromatography, but if a volatile reagent or solvent is used, the solvent can be simply distilled off and used for mass spectrometry including the collision activation cleavage method. You can

The collision-activated cleavage method for a labeled peptide can be carried out according to a conventional method, like the collision-activated cleavage method for an unlabeled peptide. That is, the generated ions are expelled with an accelerating voltage and are made to fly in one direction,
Introduce neutral gas such as helium and argon,
It suffices to collide these neutral gas with ions having kinetic energy.

In the analysis of the results of the obtained collision activation cleavage method, first, the product ion derived from the N terminus is selected.
For example, the product ion derived from the N terminus has a characteristic isotope distribution as shown in FIG. 1, and thus can be easily selected. That is, FIG. 1 is a diagram showing an example of isotope distribution of product ions derived from the N-terminal, where the vertical axis represents relative intensity (%), the horizontal axis represents m / z, and the straw mass / charge number. To do. The vertical axis and the horizontal axis in each of FIGS. 2 and subsequent figures shown in the following embodiments have the same meaning as in FIG.

In the amino acid sequence analysis, the product ion data derived from the N terminus is used for analysis, and the product ion data not derived from the N terminus is determined based on the result. Fragmentation of the acid amide bond in peptides is generally quite straightforward, with only regular cleavage on both sides of the carbonyl group. In most amino acids, the mass of each fragment ion differs depending on the difference in the α-substituent. Therefore, the amino acid can be identified by reading the mass difference of the main peaks.

It is convenient to prepare a kit as a set of each reagent for use in the above-described analysis method according to the present invention. As described above, the kit of the present invention may include at least the above-mentioned specific N-terminal labeling reagent. Other reagents may be set in the kit of the present invention as necessary. For example, a solvent used in the N-terminal labeling reaction such as dimethylformamide, pyridine, a reaction buffer such as N-ethylmorpholine and the like can be mentioned. Further, an internal labeling reagent commonly used for mass spectrum measurement, or known 4-bromophenyl isothiocyanate or the like may be set in order to contrast with the conventional method.

[0014]

EXAMPLES Examples of the present invention will be given below, but the present invention is not limited to these examples.

Example 1 (1) Synthesis of N-succinimidyl 5-bromo-3-pyridinecarboxylate 2.02 g of 5-bromonicotinic acid and 1.27 g of N-hydroxysuccinimide were dissolved in 25 ml of tetrahydrofuran and kept at 0 ° C. . To this, 2.27 g of N, N'-dicyclohexylcarbodiimide is added and reacted at 5 ° C for 20 hours. The reaction solution is filtered to remove N, N'-dicyclohexylurea. The filtrate is dried and recrystallized to give N-succinimidyl 5-bromo-3-pyridinecarboxylate.

(2) N-terminal labeling of leucine enkephalin with N-succinimidyl 5-bromo-3-pyridinecarboxylate 10 nmol of leucine enkephalin represented by SEQ ID NO: 1 in the sequence listing and N-succinimidyl 5-bromo-3
-Pyridine carboxylate (1 µmol) was dissolved in dimethylformamide (160 µl), and 50% N-ethylmorpholine (40 µl) was added thereto. After reacting at 37 ° C. for 3 hours, the solvent used for the reaction was removed by a concentrator.

(3) N-terminal labeling of leucine enkephalin with 4-bromophenyl isothiocyanate 10 nmol of leucine enkephalin and 1 μmol of 4-bromophenyl isothiocyanate were added to 50% pyridine 2
It was dissolved in 00 μl and reacted at 50 ° C. for 3 hours. After the reaction, the solvent used for the reaction was removed with a concentrator.

(4) Mass spectrometry of N-terminal labeled leucine enkephalin Mass spectrometry of leucine enkephalin labeled with N-succinimidyl 5-bromo-3-pyridinecarboxylate and mass spectrometry using the collision activation cleavage method were performed.
At the same time, as a control experiment, mass spectrometry of leucine enkephalin labeled with 4-bromophenyl isothiocyanate and mass spectrometry using a collision activation cleavage method were performed. As a mass spectrometer, high-performance quadrupole triple mass spectrometer API
・ III [Perkin-Elmer Cyex (Perkin-E
lmer Sciex)] was used. The results of mass spectrometry are shown in FIGS. 2 and 3, respectively. From FIG. 2, leucine enkephalin labeled with N-succinimidyl 5-bromo-3-pyridinecarboxylate showed a measured value of 739.4 and was labeled with N-succinimidyl 5-bromo-3-pyridinecarboxylate [ Theoretical value: 739.21 (monoisotopic mass)] was confirmed. From FIG. 3, the measured value of leucine enkephalin labeled with 4-bromophenyl isothiocyanate was 7
69.3, and labeled with 4-bromophenyl isothiocyanate [theoretical value: 769.20]
(Monoisotopic mass)] was confirmed. Next, N
The results of mass spectrometry of the leucine enkephalin N-terminally labeled with -succinimidyl 5-bromo-3-pyridinecarboxylate and 4-bromophenylisothiocyanate using the collision activation cleavage method are shown in FIGS. 4 and 5, respectively. When labeled with N-succinimidyl 5-bromo-3-pyridinecarboxylate, as can be seen from the results in FIG. 4, all product ions labeled with the N-terminus were detected, and the amino acid sequence of leucine enkephalin was easily determined. On the other hand, in the case of labeling with 4-bromophenyl isothiocyanate, from the results of FIG. 5, only two peaks were detected for the product ion derived from the N terminus, and sufficient information was not obtained. It is speculated that this is because the N-terminal amino group is less likely to have a charge due to the bond of 4-bromophenyl isothiocyanate, which results in a decrease in sensitivity. N-succinimidyl 5-bromo-3
-In the case of pyridinecarboxylate labeling, it is considered that all product ions were detected with high sensitivity because of the presence of a pyridine ring that is easily charged.

Example 2 (1) Synthetic peptide GYTWLE (Gly-Tyr-T
N-terminal labeling of hr-Trp-Leu-Glu) with 5-bromo-2-pyridyl isothiocyanate Synthetic peptide GYTWL represented by SEQ ID NO: 2 in the sequence listing
E10 nmol and 5-bromo-2-pyridylisothiocyanate 1 μmol were dissolved in 50% pyridine 200 μl and reacted at 50 ° C. for 3 hours. After the reaction, the solvent used for the reaction was removed with a concentrator.

(2) N-terminal labeling of synthetic peptide GYTWLE with 4-bromophenyl isothiocyanate 10 nmol of synthetic peptide GYTWLE and 1 μmol of 4-bromophenyl isothiocyanate were dissolved in 200 μl of 50% pyridine and reacted at 50 ° C. for 3 hours. After the reaction, the solvent used for the reaction was removed with a concentrator.

(3) N-terminal labeled synthetic peptide GYTWL
Mass spectrometry of E Mass spectrometry of the synthetic peptide GYTWLE labeled with 5-bromo-2-pyridylisothiocyanate and mass spectrometry using the collision activated cleavage method were performed. At the same time, as a control experiment, mass spectrometry of the synthetic peptide GYTWLE labeled with 4-bromophenyl isothiocyanate and mass spectrometry using the collision activation cleavage method were performed. As a mass spectrometer,
A high performance quadrupole triple mass spectrometer API III (manufactured by Perkin-Elmer SciX) was used. The results of mass spectrometry are shown in FIGS. 6 and 7, respectively. From FIG. 6, 5-
The bromo-2-pyridyl isothiocyanate-labeled synthetic peptide GYTWLE showed a measured value of 982.3 and was labeled with 5-bromo-2-pyridyl isothiocyanate [theoretical value: 982.28 (monoisotopic Mass)] was confirmed. From FIG. 7, the synthetic peptide GYTWLE that has been subjected to the 4-bromophenyl isothiocyanate labeling reaction shows a measured value of 981.3,
It was confirmed that it was labeled with bromophenyl isothiocyanate [theoretical value: 981.28 (monoisotopic mass)].

Next, the results of mass spectrometry of the synthetic peptide GYTWLE N-terminally labeled with 5-bromo-2-pyridylisothiocyanate and 4-bromophenylisothiocyanate using the collision activation cleavage method are shown in FIGS. 8 and 9, respectively.
Shown in 8 and 9, the measured values 711.
The amino acid sequence of the peptide of the product ion derived from the C terminus showing 2 is shown in SEQ ID NO: 3 in the sequence listing. In addition, in FIG. 8 showing a spectrum when labeled with 5-bromo-2-pyridylisothiocyanate according to the present invention, a measured value 72
The amino acid sequence of the N-terminal derived product ion showing 2.1 is shown in SEQ ID NO: 4 in the sequence listing, and the amino acid sequence showing the measured value 835.2 is shown in SEQ ID NO: 5 in the sequence listing. On the other hand,
In FIG. 9, which shows a spectrum in the case of labeling with conventional 4-bromophenyl isothiocyanate, the amino acid sequence of N-terminal derived product ions showing a measured value of 721.1 is shown in SEQ ID NO: 6 of the sequence listing, and the measured value of 83 is the same.
The one showing 4.1 is shown in SEQ ID NO: 7 in the sequence listing. From the comparison between FIG. 8 and FIG. 9, the ratio of the signal intensity of the N-terminal derived product ion to the C-terminal derived product ion is 5
The labeling with -bromo-2-pyridyl isothiocyanate is higher than that with 4-bromophenyl isothiocyanate, and the labeling with 5-bromo-2-pyridyl isothiocyanate having a pyridine ring is more effective by the collision activation cleavage method. It can be seen that the produced product ions are detected with high sensitivity.

Example 3 Kit Composition Example Table 1 shows a peptide N-terminal labeling kit for amino acid sequence analysis (20 times) with an N-terminal labeling reagent, a solvent for dissolving the N-terminal labeling reagent and a reaction buffer as a set. Built as. In addition, N-succinimidyl 5-bromo-3-pyridinecarboxylate as an N-terminal labeling reagent, dimethylformamide as a solvent for dissolving the N-terminal labeling reagent, and 50 mM N-ethylformolin (pH) as a reaction buffer.
7.5: prepared with acetic acid) was used.

[0024]

[Table 1] Table 1 ───────────────────────── N-terminal labeling reagent 1 mg N-terminal labeling reagent dissolving solvent 100 μl Reaction buffer 1 ml ── ──────────────────────

[0025]

Industrial Applicability According to the present invention, the amino acid sequence analysis using mass spectrometry can be performed with high sensitivity. The method of the present invention is useful for structural analysis of proteins / peptides.

[0026]

[Sequence list]

SEQ ID NO: 1 Sequence length: 5 Sequence type: Amino acid Number of chains: Single chain Topology: Linear Sequence type: Peptide Sequence Tyr Gly Gly Phe Leu 1 5

SEQ ID NO: 2 Sequence length: 6 Sequence type: Amino acid Number of chains: Single chain Topology: Linear Sequence type: Peptide sequence Gly Tyr Thr Trp Leu Glu1 5

SEQ ID NO: 3 Sequence length: 5 Sequence type: Amino acid Number of chains: Single chain Topology: Linear Sequence type: Peptide Sequence Tyr Thr Trp Leu Glu 1 5

SEQ ID NO: 4 Sequence length: 4 Sequence type: Amino acid Number of chains: Single chain Topology: Linear Sequence type: Peptide Sequence characteristics: 1st Gly amino group is 5- It is bonded to bromo-2-pyridyl isothiocyanate. Array Gly Tyr Thr Trp 1

SEQ ID NO: 5 Sequence length: 5 Sequence type: Amino acid Number of chains: Single chain Topology: Linear Sequence type: Peptide Sequence characteristics: 1st amino group of Gly is 5- It is bonded to bromo-2-pyridyl isothiocyanate. Sequence Gly Tyr Thr Trp Leu 1 5

SEQ ID NO: 6 Sequence length: 4 Sequence type: Amino acid Number of chains: Single chain Topology: Linear Sequence type: Peptide Sequence characteristics: 1st Gly amino group is 4- It is bound to bromophenyl isothiocyanate. Array Gly Tyr Thr Trp 1

SEQ ID NO: 7 Sequence length: 5 Sequence type: Amino acid Number of chains: Single chain Topology: Linear Sequence type: Peptide Sequence characteristics: 1st Gly amino group is 4- It is bound to bromophenyl isothiocyanate. Sequence Gly Tyr Thr Trp Leu 1 5

[Brief description of the drawings]

FIG. 1 is a diagram showing an example of an isotope distribution of product ions derived from an N terminus.

FIG. 2 is an example of the present invention, N-succinimidyl 5
It is a figure which shows the result of the mass spectrometry of the leucine enkephalin labeled with -bromo-3-pyridine carboxylate.

FIG. 3 is a diagram showing the result of mass spectrometry of leucine enkephalin labeled with 4-bromophenyl isothiocyanate, which is an example of a conventional method.

FIG. 4 is a diagram showing the result of mass spectrometry when the collision activation cleavage method is further applied to the compound of FIG.

FIG. 5 is a diagram showing the result of mass spectrometry when the collision activation cleavage method is further applied to the compound of FIG.

FIG. 6 is a synthetic peptide GYTWL labeled with 5-bromo-2-pyridyl isothiocyanate, which is an example of the present invention.
It is a figure which shows the result of the mass spectrometry of E.

FIG. 7 is a diagram showing the result of mass spectrometry of a synthetic peptide GYTWLE labeled with 4-bromophenyl isothiocyanate, which is an example of a conventional method.

FIG. 8 is a diagram showing the result of mass spectrometry when the collision activation cleavage method is further applied to the compound of FIG.

9 is a diagram showing the result of mass spectrometry when the collision activation cleavage method is further applied to the compound of FIG. 7.

 ─────────────────────────────────────────────────── ─── Continuation of the front page (72) Inventor Katsuhito Hashizume 3-4-1 Seta, Otsu-shi, Shiga Central Research Institute, Mina Shuzo Co., Ltd. (72) Inventor Susumu Tsunazawa 3-4-1 Seta, Otsu, Shiga Prefecture No.Take Sake Brewing Co., Ltd. Central Research Laboratory (72) Inventor Ikuno Susumu 3-4-1 Seta, Otsu City, Shiga Prefecture

Claims (2)

[Claims] 1. A method for analyzing an amino acid sequence of a peptide by mass spectrometry, which uses an N-terminal labeling reagent and a collision activation cleavage method in combination, wherein the N-terminal labeling reagent contains at least a pyridine ring bonded with chlorine or bromine, A method for analyzing an amino acid sequence of a peptide, characterized in that a compound whose product ion is easily charged is used. 2. A kit used in a method for analyzing an amino acid sequence of a peptide by mass spectrometry, wherein a compound containing at least a pyridine ring to which chlorine or bromine is bound and having a product ion easily charged is used as an N-terminal labeling reagent. A kit for analyzing an amino acid sequence of a peptide, which contains the peptide.