JP5817980B2 - Peptide detection method - Google Patents

Description

  The present invention relates to a method for detecting a peptide. The present invention is particularly useful for the detection and quantification of collagen-derived peptides.

  Peptides are very important biomolecules that not only play a central role in life phenomena as hormones and signaling substances, but are also used as therapeutic drugs and disease markers. Peptide detection / quantification is generally performed by a combination of separation by high performance liquid chromatography (HPLC) and absorbance measurement at a UV wavelength of 210 to 300 nm. Detection by absorbance depends on the amount of peptide bonds and aromatic substituents contained in the peptide, so the sensitivity is low and the detection limit is about nanomolar. Since the fluorescence method has higher sensitivity than the absorbance method, detection and quantification of the peptide from a very small amount of biological sample is facilitated if only the peptide can be selectively fluorescently derivatized.

  For fluorescence detection of conventional peptides, o-phthalaldehyde reagent and fluorescamine reagent that react with primary amine to give fluorescence, and benzoin that reacts with amidino group of arginine in peptide chain to give fluorescence Fluorescent derivatization reagents for several peptides have been developed, such as reagents and hydroxylamine reagents that specifically make peptides containing tyrosine at the N-terminus phosphor. However, since these reagents react with biological components other than peptides such as amino acids present in large amounts in living organisms to give fluorescence, they have the disadvantage of low selectivity for peptides in biological samples.

  On the other hand, the present inventors have developed a peptide quantification method using reverse phase liquid chromatography and a phosphor formation reaction at the peptide N-terminal site (see Non-Patent Document 1). In this reaction, the peptide and the catechol are heated to a high temperature around 100 ° C. in the presence of an oxidizing agent such as sodium periodate in a borate buffer solution to derivatize the peptide.

By the way, collagen is an important protein for maintaining the structure of tissues such as organs and bones, and occupies about 30% of the total protein present in the human body. Because the amount of collagen is closely related to aging, the development of simple and specific detection of collagen contained in biological samples and the development of simple and specific measurement of collagen amount is intended to examine the health condition of humans. Also important. Furthermore, since it is necessary to obtain a collagen sample from a subject surgically, it is also necessary to measure the amount of collagen with high sensitivity from a very small amount of sample so as to minimize the burden on the subject.
At present, techniques based on ELISA, colorimetry, fluorescence using o-phthalaldehyde, and the like are known as known collagen quantification methods, and kits based on these techniques are commercially available. However, since the ELISA method depends on the antigen-antibody reaction, it is an expensive kit. In addition, the colorimetric method has disadvantages such as low sensitivity for measuring changes in absorption based on the interaction between collagen and pigment, and the need for many samples and low specificity. . In addition, in the method using o-phthalaldehyde (see Non-Patent Document 2), a protein or peptide other than collagen (or a peptide derived from collagen) contained in a sample, or a peptide having an amino group, is characterized by detecting the amino terminus. The side chain was also detected, and there was a drawback that the blank was very high.

Kabashima T. et al. et al. , Peptides 29 (2008) pp. 356-363 J. et al. Biochemical and biophysical methods 70, pp. 878-882 (2008)

  There is a need for methods and reagents that can selectively derivatize peptides under mild conditions. In particular, a method capable of simply and selectively detecting and quantifying a collagen-derived peptide is desired.

As a result of screening the reactivity of various catechol-related compounds to peptides this time, the present inventors changed the reactivity depending on the substituent of the benzene ring of catechol, and in particular, according to several catechol-related compounds, It was clarified that the peptide can be selectively converted into a fluorescent derivative under mild conditions around 20-50 ° C., most preferably around 37 ° C.
Furthermore, according to a specific catechol-related compound, it has also been clarified that a peptide derived from collagen can be selectively converted into a fluorescent derivative under the above conditions.
As a result of further intensive studies based on these findings, the present inventors have completed the present invention.

That is, the present invention relates to, for example, the following.
[1] Peptide and formula (I)

(Wherein R represents a halogen atom, carboxy C _1-6 alkyl or carboxyl), and a phosphor is produced by reacting in a boric acid solution at 20 to 50 ° C. in the presence of an oxidizing agent. A method for detecting a peptide, comprising:
[2] The method according to [1], wherein the reaction is performed at pH 7 to 8;
[3] The method according to [1] or [2], wherein the oxidizing agent is sodium periodate;
[4] Any of [1] to [3], wherein the compound represented by the formula (I) is selected from 3,4-dihydroxyphenylacetic acid, 3,4-dihydroxybenzoic acid, and 4-halogenated catechol A method according to one;
[5] The compound represented by the formula (I) is 3,4-dihydroxybenzoic acid, and the peptide is a peptide whose N-terminal is proline, [1] to [4] the method of;
[6] The method according to any one of [1] to [4], wherein the compound represented by the formula (I) is 3,4-dihydroxyphenylacetic acid, and the peptide is a peptide containing glycine;
[7] The compound represented by the formula (I) is 3,4-dihydroxyphenylacetic acid, and the peptide is a peptide obtained by treating a sample with collagenase, according to any one of [1] to [4] Described method;
[8] The method according to [7], which is a method for detecting collagen in a sample;
[9] The method according to [7], which is a method for quantifying collagen in a sample;
[10] Formula (I)

A peptide detection reagent comprising a compound represented by the formula (wherein R represents a halogen atom, carboxy C _1-6 alkyl or carboxyl), an oxidizing agent and a boric acid solution;
[11] The reagent according to [10], wherein the oxidizing agent is sodium periodate;
[12] The compound represented by formula (I) is selected from 3,4-dihydroxyphenylacetic acid, 3,4-dihydroxybenzoic acid and 4-halogenated catechol, according to [10] or [11] reagent;
[13] The compound represented by formula (I) is 3,4-dihydroxybenzoic acid, and the peptide is a peptide having N-terminal proline as described in any one of [10] to [12]. Reagents of;
[14] The reagent according to any one of [10] to [12], wherein the compound represented by the formula (I) is 3,4-dihydroxyphenylacetic acid, and the peptide is a peptide containing glycine;
[15] Any one of [10] to [12], wherein the compound represented by the formula (I) is 3,4-dihydroxyphenylacetic acid, and the peptide is a peptide obtained by collagenase treatment of a sample. The described reagents;
[16] The reagent according to [15], which is a collagen detection reagent;
[17] Peptide and formula (I)

(Wherein R represents a halogen atom, carboxy C _1-6 alkyl or carboxyl) and is reacted in a boric acid solution at 20 to 50 ° C. in the presence of an oxidizing agent. A method for fluorescent labeling of peptides;
[18] The method according to [17], wherein the reaction is performed at pH 7 to 8;
[19] The method according to [17] or [18], wherein the oxidizing agent is sodium periodate;
[20] Any of [17] to [19], wherein the compound represented by the formula (I) is selected from 3,4-dihydroxyphenylacetic acid, 3,4-dihydroxybenzoic acid and 4-halogenated catechol The method according to 1.

The peptide detection method of the present invention is characterized in that a peptide is selectively fluorescently derivatized under mild temperature conditions. Therefore, it does not react with biological components other than the peptide to give fluorescence, and the peptide can be fluorescentized at a high rate regardless of mild conditions.
In particular, the peptide detection method of the present invention recognizes the types of amino acids and adjacent peptide bonds, and highly selectively fluoresces specific peptides (for example, peptides whose N-terminal is proline or peptides containing glycine). can do. In particular, detection of a peptide containing glycine is useful in that it can be applied as a collagen detection method in combination with the use of collagenase.
Further, according to the collagen detection method, it is possible to quantitatively measure collagen, and it is also possible to diagnose a disease using collagen as a biomarker.
Furthermore, since the peptide detection method of the present invention is capable of highly selective fluorescence detection of peptides in a short time under milder temperature conditions than the conventional peptide fluorophore formation reaction, a method for measuring enzyme activities of various proteases, etc. It can be applied to the fields of biochemistry and pathological examination.

It is a figure which shows the fluorescence intensity of the peptide fluorinated using dihydroxyphenylacetic acid. It is a figure which shows the fluorescence intensity of the peptide fluorescentized using dihydroxy benzoic acid. It is a figure which shows the fluorescence intensity of the peptide fluorescentized using 4-chlorocatechol. It is a figure which shows having detected the collagen selectively by fluorescentizing the peptide using dihydroxyphenylacetic acid. It is a figure which shows the relationship between the amount of collagenase-treated human collagen and bovine collagen and the fluorescence intensity of the fluorinated peptide.

  The present invention relates to peptides and formula (I)

(Wherein R represents a halogen atom, carboxy C _1-6 alkyl or carboxyl) (hereinafter abbreviated as compound (I)) in a boric acid solution in the presence of an oxidizing agent, Provided is a method for detecting a peptide, which comprises producing a phosphor by reacting at 30 to 40 ° C.

  The method for detecting a peptide of the present invention is divided into a step of making a peptide a fluorescent substance and a step of detecting the obtained fluorescent substance. In the following, each process will be described separately.

Step (1): Step of Phosphorizing a Peptide “Peptide” in the present invention is not particularly limited in sequence as long as it is fluorescentized by a phosphor forming reaction described later. For example, a peptide having 2 or more amino acids Is mentioned.

In the phosphor-forming reaction described later, when compound (I) is a compound in which R is carboxyl (3,4-dihydrobenzoic acid) or a compound in which R is a halogen atom (4-halogenated catechol), N of the peptide The terminal is preferably proline. That is, in the method of the present invention, when compound (I) is 3,4-dihydrobenzoic acid, it is possible to selectively fluoresce and detect a peptide having N-terminal proline among peptides. On the other hand, when the compound (I) is a compound in which R is carboxyl-C _1-6 alkyl (for example, 3,4-dihydroxyphenylacetic acid), fluorescence is emitted to a peptide having a random amino acid sequence. However, among peptides, peptides containing glycine can be selectively fluorescentized and detected.

  The peptide in the phosphor-forming reaction described later is usually a single peptide, but may be a plurality of two or more peptides. By using a plurality of peptides, the obtained peptide phosphors increase, and the resulting fluorescence intensity changes. Therefore, according to the method of the present invention, it is possible not only to detect peptides but also to predict the existence of various peptides by creating a database of the obtained fluorescence intensity data.

Alternatively, the peptide to be detected may be produced by any method, and those skilled in the art can select an appropriate production method. For example, a person skilled in the art can produce a peptide using a peptide synthesis method known per se, or a commercially available product may be purchased and used. Moreover, the peptide fragmented using enzymes, such as protease, may be sufficient. The protease is not particularly limited, and those skilled in the art can appropriately select a protease in accordance with the final purpose. For example, when the peptide detection method of the present invention is applied to collagen detection / quantification, a peptide may be obtained using collagenase.
Those skilled in the art can appropriately select the reaction conditions and the amount of collagenase when using collagenase. The type of collagenase can be appropriately selected according to the type of collagen to be detected and quantified.

  The sample containing the peptide of the present invention may be the peptide of the present invention itself, but may contain any substance as long as it does not interfere with the phosphor formation reaction described later. For example, examples of the sample containing the peptide of the present invention include not only aqueous peptide solutions and peptide buffers, but also various biological samples containing peptides: blood, serum, lymph, cell extract, cell culture solution, tissue extract, Tissue culture medium and the like. Therefore, for example, when quantifying collagen in blood, the blood itself may be treated with collagenase, and this may be used as it is as a sample containing a peptide derived from collagen. In addition, these samples may be further processed so as not to interfere with the phosphor formation reaction described later. Those skilled in the art can appropriately select and execute the processing method.

  The phosphor is produced by reacting the peptide and compound (I) in a boric acid solution in the presence of an oxidizing agent at 20 to 50 ° C.

The “oxidizing agent” in this step is not particularly limited, and examples thereof include oxidizing agents such as hydrogen peroxide, sodium periodate, sodium perborate, sodium perchlorate, and m-chloroperbenzoic acid. . Of these, sodium periodate is more preferable.
The addition amount of the oxidizing agent is not particularly limited as long as the phosphor forming reaction described later proceeds to form a phosphor that emits light at a specific wavelength. However, when there is a substance that reacts with the oxidant in the reaction system, or when a substance that prevents phosphor formation described later is present, the oxidant is consumed and the process (1) is substantially performed. Since the amount of oxidant used is reduced, it is necessary to increase the amount of oxidant. For example, when the sample used for detection is blood or serum, the oxidizing agent may be consumed by substances in the blood or serum in this step. In such a case, this process can be advanced by increasing the amount of oxidizing agent than usual.
Those skilled in the art can appropriately determine the amount of such an oxidizing agent.

Compound (I) of the present invention is preferably 3,4-dihydroxyphenylacetic acid, 3,4-dihydroxybenzoic acid or 4-halogenated catechol.
In particular, in the phosphor forming reaction described later, when a compound (3,4-dihydroxybenzoic acid) in which R is carboxyl or a compound (4-halogenated catechol) in which R is a halogen atom is used as compound (I), Peptides whose N-terminal is proline can be highly selectively fluorescent.
On the other hand, when a compound (for example, 3,4-dihydroxyphenylacetic acid) in which R is carboxyl-C _1-6 alkyl is used as compound (I), it gives fluorescence to a peptide having a random amino acid sequence. As shown in Examples 1 and 2 to be described later, fluorescence is given particularly to peptides containing glycine (see FIG. 1).

  In this step (1), the amino group at the N-terminal of the peptide reacts with compound (I) in the presence of the oxidizing agent described above, and this reacts with boron in the boric acid solution to form a phosphor. I think that. A reaction formula relating to the synthesis of a peptide phosphor estimated when 3,4-dihydroxyphenylacetic acid (3,4-DHPAA) is used as the compound (I) is shown below.

  The amount of compound (I) that reacts with the peptide and the amount of boric acid (boron) in the boric acid solution are not particularly limited as long as this step proceeds, and those skilled in the art can appropriately adjust an appropriate amount. Is possible.

  The composition of the boric acid solution is not particularly limited, and any solvent may be used as long as it does not inhibit the reaction in this step, and any compound other than boric acid (boron) is included. It doesn't matter. The boric acid solution in this step is preferably an aqueous boric acid solution or a boric acid buffer. The pH when the boric acid solution is a borate buffer is not particularly limited, but is preferably near neutral (pH 6-8), more preferably pH 7-8.

  The reaction temperature in this step is usually 20 ° C. to 50 ° C. in view of the feature that the present invention is carried out under mild conditions, but the reaction in this step proceeds even at a high temperature of about 100 ° C. However, the reaction temperature in this step is preferably 30 ° C to 40 ° C, more preferably 35 ° C to 38 ° C, and most preferably 37 ° C. If the reaction temperature is lower than 20 ° C. (preferably 30 ° C., more preferably 35 ° C.), it becomes difficult to form a phosphor even with the compound (I) of the present invention, and the reaction temperature is 50 ° C. (preferably 40 ° C.). Even if the temperature is higher than 38 ° C. (preferably 38 ° C.), it is difficult to form a phosphor due to degradation of the peptide or increase of side reactions.

  The present invention is different from the conventionally known peptide quantification methods according to Non-Patent Document 1, and can fluoresce peptides under milder conditions. Therefore, the field of biochemistry and pathological examinations such as a method for measuring enzyme activities of various proteases. It can also be applied to. In addition, the present invention has advantages such as reduction of the possibility of peptide degradation under severe reaction conditions near 100 ° C. and simplification of operation by omitting the heating operation as compared with conventionally known methods.

  In addition, although reaction temperature in this process is as above-mentioned, it does not necessarily mean that this invention cannot be implemented unless it is in this range. The present invention is based on the discovery of a compound (I) that can fluorinate a peptide under milder conditions than conventional peptide fluorination methods, and the reaction temperature is slightly outside the scope of the present invention. Even so, the present invention can be carried out by appropriately adjusting the components of the present invention such as the compound (I), oxide, boric acid solution, and the reaction time described later.

  The reaction time is usually 3 to 15 minutes, preferably 5 to 13 minutes, more preferably 10 minutes.

  By this step, a phosphor having an excitation wavelength of about 370 to 450 nm and a fluorescence wavelength of about 465 to 540 nm is manufactured. The obtained phosphor can be subjected to the next step (2) without isolation.

  By the way, the detection method of the present invention can be applied particularly to detection and quantification of collagen. Prior to the step (2), a phosphor manufacturing process according to a collagen detection method and a quantification method using the detection method of the present invention will be described.

The “collagen” that is the target of collagen detection is not particularly limited, and examples include type I collagen, type II collagen, type III collagen, type IV collagen, and the like. The type of collagen to be detected depends on the type of collagenase used.
Collagen has a primary structure called glycine-amino acid (X) -amino acid (Y) in which glycine is repeated every 3 residues, and can be decomposed into peptide fragments containing glycine at the N-terminus by various collagenases. . The collagenase may be selected in accordance with the type of collagen to be detected. Therefore, for example, when detecting type I collagen, a sample containing collagen may be digested with type I collagenase (collagenase that digests type I collagen). Good. Since the obtained peptide fragment is derived from type I collagen, type I collagen is specifically detected by detecting this peptide fragment. The reaction conditions and amount of collagenase can be appropriately selected by those skilled in the art.

As described above, this peptide fragment containing glycine can be specifically fluorinated by the compound (I) in which R is carboxy C _1-6 alkyl. In particular, as shown in FIG. 1, which will be described later, it can be seen that the compound (I) of the present invention is very reactive to sequences GP and GPP (G: glycine, P: proline). In the primary structure of collagen, the amino acid (X) is often proline and the amino acid (Y) is often hydroxyproline. As a result, the compound (I) of the present invention is particularly a peptide derived from collagen. The fragments will be selectively phosphorylated.
Therefore, collagen present in the sample can be detected only by applying the phosphor obtained through the above steps to a fluorescence spectrometer or the like in step (2) described later and measuring the fluorescence intensity.

In the collagen detection method of the present invention, the compound (I) is not particularly limited, but a compound in which R is carboxy C _1-6 alkyl is desirable, and 3,4-dihydroxyphenylacetic acid in which R is carboxymethyl is more preferable.

  Although it does not specifically limit as a sample containing collagen, A biological sample, for example, blood, serum, urine etc. are mentioned. Also included are foods, beverages, cosmetics, laboratory reagents and the like. In the collagen detection method of the present invention, these samples containing collagen may contain components other than collagen as long as they do not inhibit collagen peptide fragmentation by collagenase.

  Conventional detection of collagen has been performed by ELISA, colorimetry, or fluorescence using o-phthalaldehyde. The collagen detection method of the present invention detects many peptide fragments produced by cleaving collagen contained in a sample with collagenase by a fluorescent reaction specific to a peptide containing glycine. Since the sensitivity of this method increases as the number of peptide fragments increases, the signal increases, so according to the collagen detection method of the present invention, even a very small amount of collagen can be detected with high sensitivity.

  By the way, in the collagen detection method of this invention, as FIG. 4 mentioned later shows, linearity is recognized between collagen amount and fluorescence intensity. Therefore, the amount of collagen can be quantified by creating a calibration curve for each detection method. Therefore, the collagen detection method of the present invention can also be a collagen quantification method.

Furthermore, since this fluorescent reaction proceeds even under mild conditions of 20 to 50 ° C., it is possible to simultaneously add collagenase and a fluorescent reagent to a sample, and simultaneously perform peptide fragmentation with collagenase and fluorescentization with a fluorescent reagent. . As a result, the immune reaction and the washing operation required in the conventional collagen detection method are omitted, so that the collagen in the sample can be detected and quantified by a simple operation while greatly reducing the detection time.
At present, a collagen measurement kit based on such a fluorescence method is not commercially available, and the present invention can be a simple and highly sensitive method for detecting and quantifying fluorescence of collagen.

Moreover, blood collagen (especially blood type IV collagen) is a liver fibrosis marker that correlates with liver fibrosis, and urine collagen (particularly urine type IV collagen) is a kidney disease caused by diabetes or nephritis. It is known to be a marker that correlates with glomerular collapse.
Therefore, according to the collagen detection / quantification method of the present invention, these diseases can be discovered and diagnosed.

Step (2): Step of detecting phosphor This step is a step of detecting a peptide present in the sample by detecting the phosphor obtained in step (1).

  The detection method of the fluorescent substance is not particularly limited, but it is desirable to detect the fluorescent substance with a fluorescence spectrometer or a microplate reader in view of convenience, necessity of analyzing a large number of peptides, presence of contaminants, and the like. Separation by HPLC or microchip electrophoresis is also possible. By using HPLC or microchip electrophoresis, a large number of peptides can be separated and analyzed. As HPLC conditions, the excitation wavelength is set to around 320 to 500 nm, preferably around 370 to 450 nm, and the fluorescence is set around 400 to 590 nm, preferably around 465 to 540 nm. A person skilled in the art can appropriately set optimum conditions each time depending on the compound contained in the compound (I). Other HPLC conditions are not particularly limited, and those skilled in the art can appropriately set conditions.

When detecting the phosphor, the sample containing the phosphor may contain other components. Therefore, in the detection method of the present invention, in step (1), compound (I), oxidizing agent, boric acid solution or protease is added to the sample to produce a phosphor, and then a mixture containing the phosphor is added to step (2). Peptides and collagen can be detected by a very simple operation of measuring fluorescence as it is.
In addition, when contaminants hinder fluorescence measurement, an operation of removing components other than the phosphor may be performed by a method known per se, or an operation of separating the phosphor such as the HPLC may be performed. Such methods are known to those skilled in the art.

  The present invention also provides a peptide detection reagent comprising compound (I), an oxidizing agent and a boric acid solution. Compound (I), oxidizing agent and boric acid solution contained in the peptide detection reagent of the present invention are as described above.

  By adding the peptide detection reagent of the present invention to a target sample, the peptide contained in the sample can be converted into a fluorescent substance. At this time, the amount of each component added to the sample can be appropriately determined by those skilled in the art.

  The present invention will be described more specifically with reference to the following examples. The following examples are merely examples of the present invention, and the present invention is not limited to this range.

Example 1: Conversion to phosphor and detection of phosphor by spectrometer In 250 μL of each 40 μM peptide solution, 375 μL of 0.5 mM Compound (I), 312 μL of 100 mM H ₃ BO ₃ -NaOH (pH 8.0), 63 μL of 5 mM NaIO ₄ was added (the final concentration was 10 μM peptide, 31 mM Na ₃ BO _3, 315 μM NaIO ₄ ), and the reaction was allowed to proceed at 37 ° C. for 10 minutes. Thereafter, the reaction solution was ice-cooled for 10 minutes, and the fluorescence intensity was measured with a fluorescence spectrometer (excitation wavelength: 370 nm, fluorescence wavelength: 465 nm). The results are shown in FIGS.
Fluorometric analysis:
Model: JASCO FP-6300 Spectrofluorometer
Slit width: 10nm, 10nm
Sensitivity: medium
Response: medium

Example 2: Detection of phosphor by HPLC The reaction solution obtained in Example 1 was analyzed by HPLC.
HPLC conditions: mobile phase; 15-80% methanol _{+ 5% 0.25M pH7.0 H 3 BO} 3 -NaOH (0-40min linear gradient), excitation wavelength 370～450Nm, fluorescence wavelength 465～540Nm, column ODS-80Ts ( 150 mm × 4.6 mm id, pore size 5 mm, Tosoh).

  When 3,4-dihydroxyphenylacetic acid (3,4-DHPAA) is used as compound (I) (FIG. 1), a peptide having a random amino acid sequence, particularly a peptide containing glycine, can be fluorescentized. Thus, it was found that 3,4-dihydroxybenzoic acid (FIG. 2) and 4-halogenated catechol (FIG. 3) highly selectively fluoresce a peptide whose N-terminal is proline.

Example 3 Detection of Collagen Each protein containing collagen (20 nM) or HeLa cell extract (total protein amount 15.2 μg / tube; 4 × 10 ⁵ cells / tube) was added to 50 mM borate buffer containing 5 mM CaCl _2. (PH 7.5) was reacted with a microorganism-derived collagenase (2.0 μg / tube, manufactured by Nacalai Tesque) at 37 ° C. for 1 hour. Then, in the presence of 315 μM NaIO ₄ and 31.2 mM borate buffer, it was reacted with 187 μM 3,4-DHPAA at 37 ° C. for 10 minutes to produce a phosphor. Thereafter, the reaction solution was ice-cooled for 10 minutes, and the fluorescence intensity of the solution containing the obtained phosphor was measured with a fluorescence spectrometer (see Example 1) at an excitation wavelength of 370 nm and a fluorescence wavelength of 465 nm. The results are shown in FIG.
According to the collagen detection method of the present invention, it was revealed that collagen can be specifically detected.

Example 4: Quantification of Collagen Human or bovine collagen-I (manufactured by Sigma) in a 50 mM borate buffer solution (pH 7.5) containing 5 mM CaCl ₂ (2.0 μg / tube, Nacalai Tesque) And made at 37 ° C. for 1 hour. Then, in the presence of 315 μM NaIO ₄ and 31.2 mM borate buffer, it was reacted with 187 μM 3,4-DHPAA at 37 ° C. for 10 minutes to produce a phosphor. Thereafter, the reaction solution was ice-cooled for 10 minutes, and the fluorescence intensity of the solution containing the obtained phosphor was measured with a fluorescence spectrometer (see Example 1) at an excitation wavelength of 370 nm and a fluorescence wavelength of 465 nm. The obtained results were plotted in relation to the amount of human or bovine collagen-I to create a calibration curve. The results are shown in FIG.

The peptide detection method of the present invention is characterized in that a peptide is selectively fluorescently derivatized under mild temperature conditions. Therefore, it does not react with biological components other than the peptide to give fluorescence, and the peptide can be fluorescentized at a high rate regardless of mild conditions.
In particular, the peptide detection method of the present invention recognizes the types of amino acids and adjacent peptide bonds, and highly selectively fluoresces specific peptides (for example, peptides whose N-terminal is proline or peptides containing glycine). can do. In particular, detection of a peptide containing glycine is useful in that it can be applied as a collagen detection method in combination with the use of collagenase.
Further, according to the collagen detection method, it is possible to quantitatively measure collagen, and it is also possible to diagnose a disease using collagen as a biomarker.
Furthermore, since the peptide detection method of the present invention is capable of highly selective fluorescence detection of peptides in a short time under milder temperature conditions than the conventional peptide fluorophore formation reaction, a method for measuring enzyme activities of various proteases, etc. It can be applied to the fields of biochemistry and pathological examination.

  This application is based on a patent application No. 2011-054991 (filing date: March 2, 2011), the contents of which are incorporated in full herein.

Claims (25)

Peptide detection characterized by producing a phosphor by reacting a peptide whose N-terminal is glycine with 3,4-dihydroxyphenylacetic acid in a boric acid solution in the presence of an oxidizing agent at 20 to 50 ° C. Method.   The process according to claim 1, wherein the reaction is carried out at a pH of 7-8.   The method according to claim 1 or 2, wherein the oxidizing agent is sodium periodate. Peptide is a peptide obtained by collagenase treatment of the sample, the method according to any one of claims 1 to 3. The method according to claim 4 , which is a method for detecting collagen in a sample. The method according to claim 4 , which is a method for quantifying collagen in a sample. A reagent for detecting a peptide whose N-terminus is glycine, comprising 3,4-dihydroxyphenylacetic acid , an oxidizing agent and a boric acid solution. The reagent according to claim 7 , wherein the oxidizing agent is sodium periodate. Peptide is a peptide obtained by collagenase treatment of the sample, reagent according to claim 7 or 8. The reagent according to claim 9 , which is a detection reagent for collagen. A peptide fluorescent labeling method comprising reacting a peptide having N-terminal glycine with 3,4-dihydroxyphenylacetic acid in a boric acid solution in the presence of an oxidizing agent at 20 to 50 ° C. The reaction is carried out in pH 7-8, the method of claim 1 1. Oxidizing agent is sodium periodate method of claim 1 1 or 1 2. A peptide comprising a peptide comprising an amino acid sequence of proline-glycine and 3,4-dihydroxybenzoic acid in a boric acid solution in the presence of an oxidizing agent at 20 to 50 ° C. to produce a phosphor. Detection method. The process according to claim 14, wherein the reaction is carried out at pH 7-8 and the oxidizing agent is sodium periodate. A reagent for detecting a peptide consisting of an amino acid sequence of proline-glycine, comprising 3,4-dihydroxybenzoic acid, an oxidizing agent, and a boric acid solution. The reagent according to claim 16, wherein the oxidizing agent is sodium periodate. A method for fluorescent labeling of a peptide, comprising reacting a peptide having an amino acid sequence of proline-glycine and 3,4-dihydroxybenzoic acid in a boric acid solution in the presence of an oxidizing agent at 20 to 50 ° C. The process according to claim 18, wherein the reaction is carried out at pH 7-8 and the oxidizing agent is sodium periodate. Characterized in that a phosphor comprising a peptide having an amino acid sequence of proline-glycine or proline-alanine and 4-chlorocatechol in a boric acid solution in the presence of an oxidizing agent at 20 to 50 ° C., Peptide detection method. 21. The method of claim 20, wherein the reaction is carried out at pH 7-8 and the oxidizing agent is sodium periodate. A reagent for detecting a peptide comprising an amino acid sequence of proline-glycine or proline-alanine, comprising 4-chlorocatechol, an oxidizing agent, and a boric acid solution. The reagent according to claim 22, wherein the oxidizing agent is sodium periodate. A method for fluorescently labeling a peptide, comprising reacting a peptide comprising an amino acid sequence of proline-glycine or proline-alanine with 4-chlorocatechol in a boric acid solution in the presence of an oxidizing agent at 20 to 50 ° C. 25. A process according to claim 24, wherein the reaction is carried out at pH 7-8 and the oxidizing agent is sodium periodate.