Method for measuring proteoglycan

Abstract

The present invention provides a method for measuring proteoglycan, which is capable of detecting proteoglycan with its C-terminus and / or N-terminus maintained. The method for measuring proteoglycan of the present disclosure includes a cleavage step of cleaving a disulfide bond of the proteoglycan, a modification step of modifying the thiol groups of the obtained proteoglycan under alkaline conditions; and a detection step of detecting at least one of the C-terminal domain and the N-terminal domain of the proteoglycan by fluorescence.

Description

[Technical field]

[0001] The present disclosure relates to a method for measuring proteoglycan. [Background technology]

[0002] Proteoglycans are molecules that constitute the extracellular matrix together with collagen, hyaluronic acid, etc. In recent years, purification of proteoglycans by extraction with guanidine hydrochloride, extraction with acetic acid, etc. has been reported (Patent Document 1 and Non-Patent Document 1). [Prior art documents] [Patent documents]

[0003] [Patent Document 1] Patent No. 3731150 [Non-patent literature]

[0004] [Non-Patent Document 1] Kakizaki I, Tatara Y, Majima M, Kato Y, Endo M. Identification of proteoglycan from nasal salmon cartilage. Arch Biochem Biophys. 2011 Feb 1;506(1):58-65. doi: 10.1016 / j.abb.2010.10.025. Epub 2010 Nov 5. PMID: 21056541. Summary of the Invention [Problem to be solved by the invention]

[0005] The proteoglycan exists in the living body in a state bound to hyaluronic acid, etc. Therefore, the proteoglycan in a state capable of binding to hyaluronic acid, etc. may be used in regenerative medicine. However, the proteoglycan may be extracted in a state in which the C-terminal domain (hereinafter also referred to as "C-terminus") and / or the N-terminal domain (hereinafter also referred to as "N-terminus") of the proteoglycan is cut depending on the extraction method. Therefore, it is necessary to evaluate the state of the proteoglycan, more specifically, the presence or absence of the C-terminus and / or the N-terminus of the proteoglycan.

[0006] Therefore, an object of the present disclosure is to provide, for example, a method for measuring proteoglycan that can detect proteoglycan with its C-terminus and / or N-terminus maintained. [Means for solving the problem]

[0007] In order to achieve the above object, the method for measuring proteoglycan of the present disclosure (hereinafter also referred to as the "measurement method") includes a cleavage step of cleaving disulfide bonds of the proteoglycan, a modification step of modifying the thiol groups of the obtained proteoglycan under alkaline conditions; and a detection step of detecting at least one of the C-terminal domain and the N-terminal domain of the proteoglycan by fluorescence. Effect of the Invention

[0008] According to the present disclosure, for example, it is possible to provide a method for measuring proteoglycan that can detect proteoglycan with its C-terminus and / or N-terminus maintained. [Brief description of the drawings]

[0009] [Figure 1] FIG. 1 shows the main disaccharide structures of each chondroitin sulfate. [Diagram 2] FIG. 2 shows the structures of various proteoglycans. [Diagram 3]FIG. 3 is a photograph showing the results of detection of the C-terminal domain in proteoglycan in Example 1. [Figure 4] FIG. 4 is a photograph showing the results of detection of the N-terminal domain in proteoglycan in Example 1. [Diagram 5] FIG. 5 is a photograph showing the results of detection of the C-terminal domain in proteoglycan in Example 1. [Figure 6] FIG. 6 is a photograph showing the results of detection of the C-terminal domain in proteoglycan in Example 2. [Figure 7] FIG. 7 is a photograph showing the results of decorin detection in Example 3. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0010] The present disclosure will be specifically described below with reference to examples. Unless otherwise specified, each disclosure may incorporate the description of the other disclosures.

[0011] <Definition> As used herein, "proteoglycan" refers to a molecule (glycoprotein) in which a protein (core protein) and glycosaminoglycan (GAG, also called "polysaccharide" or "sugar chain") are covalently bonded. The proteoglycan exists as an extracellular matrix in, for example, skin, organs, cartilage, and the like. The glycosaminoglycan is generally known as a sugar chain having a long chain structure without a branched structure. Examples of the proteoglycan include the following. Aggrecan family (also called lectican family or hyalectan family): Aggrecan, Versican, Neurocan, Brevican, etc. Small Leucine Rich Proteoglycans (SLRPs) family: Biglycan, Decorin, Fibromodulin, Lumican, PG-Lb (Epiphycan), Keratocan, Mimecan, etc. Basement membrane proteoglycans: Perlecan, Agrin, Permacan, etc. Others: Testican, Biglycan, Serglycin, Syndecan, Dystroglycan, Claustrin, Glypican, Keratocan, etc. The proteoglycans can also be classified into, for example, chondroitin sulfate proteoglycans, dermatan sulfate proteoglycans, heparan sulfate proteoglycans, or keratan sulfate proteoglycans depending on the type of GAG bound to the protein.

[0012] Examples of the GAG ​​include chondroitin, chondroitin sulfate (CS), dermatan sulfate (DS, chondroitin sulfate B), heparan sulfate, heparin, and keratan sulfate. Examples of the chondroitin include an O-type glycan having a disaccharide structure of glucuronic acid and acetylgalactosamine as the main disaccharide structure, and an iO-type glycan having a disaccharide structure of iduronic acid and acetylgalactosamine as the main structure (hereinafter, also referred to as "chondroitin sulfate O" and "chondroitin sulfate iO", respectively). The chondroitin sulfate (CS) has a structure in which a sulfate group is added to a glycan having a repeating disaccharide structure of glucuronic acid and acetylgalactosamine. Examples of the chondroitin sulfate include chondroitin sulfate A (A type) having a disaccharide structure of glucuronic acid and acetylgalactosamine 4-sulfate as the main disaccharide structure, and chondroitin sulfate C (C type) having a disaccharide structure of glucuronic acid and acetylgalactosamine 6-sulfate as the main disaccharide structure. The dermatan sulfate (DS) has a structure in which a sulfate group is added to a sugar chain in which disaccharides of iduronic acid and acetylgalactosamine are repeated. Examples of the dermatan sulfate include chondroitin sulfate iA (iA type) having a disaccharide structure of iduronic acid and acetylgalactosamine 4-sulfate as the main disaccharide structure, and chondroitin sulfate iC (iC type) having a disaccharide structure of iduronic acid and acetylgalactosamine 6-sulfate as the main disaccharide structure. Each chondroitin sulfate has, for example, the disaccharide structure shown in FIG. 1 as the main disaccharide structure. In FIG. 1, the sulfate group (sulfo group) is bonded to a hydrogen atom, but the present disclosure is not limited to this, and the sulfate group of the GAG ​​may, for example, be ionized by loss of a hydrogen atom, or may form a salt.

[0013] As used herein, the term "domain" refers to a region in a protein that is structured or functionally organized. An example of a structure including each domain of aggrecan, versican, neurocan, brevican, and decorin is shown in FIG. 2.

[0014] In the present specification, the "N-terminal domain (N-terminus)" refers to, for example, the G1 domain, IGD domain (interglobular domain), G2 domain and / or KS-rich domain of the proteoglycan when the proteoglycan is a lectican family such as aggrecan. The "N-terminal domain" refers to, for example, the domain on the N-terminal side of the leucine-rich repeat domain of the proteoglycan when the proteoglycan is a small leucine-rich proteoglycan such as decorin. The G1 domain is known to bind to hyaluronic acid, link protein, and the like. When the proteoglycan is decorin, the N-terminal domain has chondroitin sulfate or dermatan sulfate in the side chain and has four cysteine ​​residues.

[0015] As used herein, the term "C-terminal domain (C-terminus)" refers to the C-terminal domain including the G3 domain of a proteoglycan, for example, when the proteoglycan is a member of the lectican family such as aggrecan. The term "C-terminal domain" refers to the domain on the C-terminal side of the leucine-rich repeat domain of a proteoglycan, for example, when the proteoglycan is a small leucine-rich proteoglycan such as decorin. The G3 domain is known to bind to tenascin, etc. When the proteoglycan is decorin, the C-terminal domain has two cysteine ​​residues.

[0016] As used herein, the term "intermediate domain" refers to a domain between the N-terminal domain and the C-terminal domain of a proteoglycan, for example, when the proteoglycan is a lectican family proteoglycan, and includes at least one glycosaminoglycan-binding domain (GAG). When the proteoglycan is a small leucine-rich proteoglycan such as decorin, the term "intermediate domain" refers to a domain between the N-terminal domain and the C-terminal domain of the proteoglycan, and includes a leucine-rich repeat.

[0017] As used herein, "alkaline" refers to an aqueous solution with a pH greater than 7. As used herein, aqueous solutions of interest are classified as acidic, neutral, and alkaline when their pH is less than 7, 7, and greater than 7, respectively.

[0018] As used herein, the term "disulfide bond" (SS bond) refers to a bond between sulfur atoms formed between two thiol groups (-SH).

[0019] As used herein, "reducing agent" refers to a substance that can reduce (donate electrons to) another substance in an oxidation-reduction reaction.

[0020] As used herein, the term "modification" refers to chemical modification of a specific functional group of an amino acid residue constituting a protein.

[0021] In this specification, "alkylation" refers to converting a hydrogen atom constituting an organic compound into an alkyl group (C n H 2n+1 The chemical reaction that causes this alkylation is called an alkylating agent.

[0022] As used herein, "purification" refers to identifying, separating, recovering from components in the natural state, the state of being identified and separated, and / or the state of being recovered from components in the natural state. The "purification" can be performed, for example, by obtaining at least one purification step. The purification can also be referred to as isolation.

[0023] In this specification, "extraction" means taking out a specific component from a target and / or a state in which a specific component is taken out from a target. The extraction can be carried out, for example, using an extracting liquid such as a solvent. The "extraction" can be carried out, for example, by obtaining at least one extraction process. In this specification, a substance containing a component taken out from the target is called an extract. Also, a liquid containing a component taken out from the target is called an extracting liquid. The extract and the extracting liquid may contain, for example, the proteoglycan. The extract or the extracting liquid may contain, for example, components other than the proteoglycan derived from the animal tissue used for the extraction (other components). The other components include, for example, lipids, nucleic acids, proteins, and the like.

[0024] The present disclosure will be described below with examples, but the present disclosure is not limited to the following examples and can be modified as desired. The present disclosure and the descriptions in each embodiment can be used interchangeably unless otherwise specified. In this specification, the expression "~" is used to mean the numerical or physical values ​​before and after it. In this specification, the expression "A and / or B" includes "A only," "B only," and "both A and B."

[0025] <Method of measuring proteoglycan> In one embodiment, the present disclosure provides a method for measuring proteoglycan. The method for measuring proteoglycan includes a cleavage step of cleaving disulfide bonds of proteoglycan, a modification step of modifying thiol groups of the obtained proteoglycan under alkaline conditions, and a detection step of detecting at least one of the C-terminal domain and the N-terminal domain of the proteoglycan by fluorescence. According to the measurement method of the present disclosure, proteoglycan with the C-terminus and / or N-terminus maintained can be detected. In addition, according to the measurement method of the present disclosure, the N-terminus and C-terminus of the proteoglycan can be measured simultaneously, so that, for example, the number of proteoglycan samples used for measurement can be reduced.

[0026] As a result of intensive research, the present inventors have found that proteoglycan is extracted in a state in which the C-terminus and / or N-terminus of the proteoglycan is maintained or cut, depending on the type of extract, etc. Therefore, as a result of further research, the present inventors have found that the C-terminus and / or N-terminus of the proteoglycan can be detected by a fluorescence detection method by cutting the disulfide bond of the purified proteoglycan and modifying the thiol group of the proteoglycan under alkaline conditions to inhibit the proteoglycan from forming a complex with hyaluronic acid, etc., and have established the present disclosure. Therefore, the measurement method of the present disclosure can detect proteoglycan with the C-terminus and / or N-terminus maintained.

[0027] In the cleavage step, disulfide bonds of the proteoglycan are cleaved. As a result, for example, the three-dimensional structure of the proteoglycan can be disrupted in the cleavage step, and when the proteoglycan has a C-terminus and / or an N-terminus, the formation of a complex formed between the proteoglycan and the hyaluronic acid or the like can be eliminated. Therefore, when the proteoglycan has a C-terminus and / or an N-terminus, the cleavage step can expose the C-terminus and / or the N-terminus of the proteoglycan, that is, make it detectable.

[0028] The cleavage step can be carried out, for example, by a reaction system (cleavage system) containing the proteoglycan and a component capable of cleaving the disulfide bond of the proteoglycan. The cleavage system is, for example, a liquid system such as an aqueous solution. Specifically, in the cleavage step, for example, the proteoglycan is contacted with a component capable of cleaving a disulfide bond to cleave the disulfide bond present in the proteoglycan. The contact between the proteoglycan and the component capable of cleaving the disulfide bond can be carried out, for example, by a known contact method between a solid and a liquid, specifically, by mixing the proteoglycan with a solution containing a component capable of cleaving the disulfide bond.

[0029] An example of the component capable of cleaving a disulfide bond is a reducing agent. When the reducing agent is used in the cleavage step, the cleavage can also be referred to as reduction to a thiol group by the reducing agent. The reducing agent is not particularly limited, and is, for example, a compound capable of reducing a disulfide bond to a free thiol group. Examples of the reducing agent include dithiothreitol (DL-dithiothreitol, DTT), β-mercaptoethanol (BME), tris(2-carboxyethyl)phosphine (TCEP), glutathione, and the like.

[0030] The cleavage conditions (temperature, time, etc.) in the cleavage step are not particularly limited and can be set, for example, under conditions under which the disulfide bond is sufficiently cleaved into the thiol group. The cleavage conditions can be set as appropriate, for example, depending on the type of the reducing agent, etc. The reaction temperature in the cleavage is, for example, preferably 45 to 100°C, more preferably 50 to 60°C. The reaction time in the cleavage is, for example, preferably 3 minutes to 6 hours, more preferably 5 minutes to 3 hours.

[0031] The proteoglycan may be, for example, an animal-derived proteoglycan. The animal is not particularly limited, and may be, for example, fish such as salmonid fish such as chum salmon and Atlantic salmon, flatfish fish such as platypus; avian animals (birds) such as chickens; mammalian animals (mammals) such as pigs. The animal may preferably be salmon, chicken, pig, or flatfish.

[0032] The proteoglycan may be, for example, a proteoglycan derived from a tissue containing proteoglycan. Examples of the tissue include epithelial tissue such as skin, cartilage tissue such as cartilage, digestive organs, circulatory organs, respiratory organs, and placenta. Specific examples of the tissue include cartilage, fins, digestive organs, circulatory organs, respiratory organs, and ears.

[0033] Examples of the sugar chain of the proteoglycan include chondroitin, chondroitin sulfate, dermatan sulfate (chondroitin sulfate B), heparan sulfate, heparin, and keratan sulfate. The sugar chain is preferably chondroitin sulfate.

[0034] The proteoglycan used in the measurement method of the present disclosure may be a self-prepared proteoglycan or a commercially available proteoglycan. In the former case, the measurement method of the present disclosure may include, for example, a step of extracting and / or purifying proteoglycan from an animal or animal tissue prior to the cutting step. When the self-prepared proteoglycan is used, the proteoglycan may be a proteoglycan purified by acetic acid extraction or a proteoglycan purified by guanidine extraction. When the acetic acid extraction is performed, the proteoglycan can be purified with reference to the method of Japanese Patent No. 3731150. When the guanidine extraction is performed, the proteoglycan can be purified with reference to the methods of References 1 to 2 below. Reference 1: Kakizaki I, Tatara Y, Majima M, Kato Y, Endo M. Identification of proteoglycan from salmon nasal cartilage. Arch Biochem Biophys. 2011 Feb 1;506(1):58-65. doi: 10.1016 / j.abb.2010.10.025. Epub 2010 Nov 5. PMID: 21056541. Reference 2: Kakizaki I, Mineta T, Sasaki M, Tatara Y, Makino E, Kato Y. Biochemical and atomic force microscopic characterization of salmon nasal cartilage proteoglycan. Carbohydr Polym. 2014 Mar 15;103:538-49. doi: 10.1016 / j.carbpol.2013.12.083. Epub 2014 Jan 7. Erratum in: Carbohydr Polym. 2015 Jan 22;115:805. PMID: 24528764.

[0035] The molecular weight of the proteoglycan may be measured before the cleavage step. The molecular weight of the proteoglycan means a peak top molecular weight measured by GPC (Gel Permeation Chromatography). The molecular weight of the proteoglycan can be measured using the GPC method or the like. The GPC method can be performed, for example, under the following conditions, and the standard samples (molecular weight markers, pullulan) are individually injected into an HPLC system to calculate the molecular weight calibration curve.

[0036] (Conditions for measuring peak top molecular weight (Mp)) HPLC system: LC-20AD (Shimadzu Corporation) Column: TSKgel G5000-PWXL φ7.8mm×300mm (Tosoh Corporation) Elution solution: pH 6.8 phosphate buffer Flow rate: 0.5mL / min Column temperature: 40℃ Detector: Differential refractive index detector (RID-20A, Shimadzu Corporation) Injection volume: 50μL Molecular weight marker: Shodex STANDARD P-82 (pullulan) The peak top molecular weight (Mp) and weight average molecular weight (Mw) / number average molecular weight (Mn) of the molecular weight markers are as follows. STD P-800:Mp:739,000, Mw / Mn:1.24 STD P-400:Mp:348,000, Mw / Mn:1.33 STD P-200:Mp:216,000, Mw / Mn:1.22 STD P-100:Mp:107,000, Mw / Mn:1.12 STD P-50: Mp:49,400, Mw / Mn:1.08 STD P-20:Mp:22,000, Mw / Mn:1.08 STD P-10: Mp:9,800, Mw / Mn:1.07 STD P-5: Mp: 6,300, Mw / Mn: 1.09

[0037] The peak top molecular weight of the proteoglycan subjected to the measurement is, for example, 300,000 to 1,300,000, or 400,000 to 1,200,000.

[0038] Next, in the modification step, the free thiol groups of the proteoglycan obtained in the cleavage step are modified under alkaline conditions. As a result, in the modification step, for example, the free thiol groups of the proteoglycan are modified, and the reformation of disulfide bonds between the thiol groups can be inhibited. Therefore, in the case where the proteoglycan has a C-terminus and / or an N-terminus, the modification step can maintain the C-terminus and / or the N-terminus of the proteoglycan in an exposed state, that is, in a detectable state. Therefore, in the measurement method of the present disclosure, the modification step can maintain the C-terminus and / or the N-terminus of the proteoglycan in a detectable state, and therefore the C-terminus and / or the N-terminus can be detected in the detection step described below.

[0039] The modification step can be carried out, for example, in a reaction system (modification system) containing the proteoglycan obtained in the cleavage step and a modifying agent used for the modification. The reaction system is, for example, a liquid system such as an aqueous solution. The reaction system can be prepared, for example, by mixing the proteoglycan and the modifying agent.

[0040] The modification step is preferably carried out under alkaline conditions. The pH in the modification step (modification pH) is, for example, preferably pH 7.5 to pH 8.5, and more preferably pH 7.8 to pH 8.2. The modification step can be carried out, for example, at pH 7.5 or higher, to suitably modify the thiol group, thereby suitably detecting the C-terminus and / or N-terminus of the proteoglycan. In the modification step, the pH of the reaction system can be adjusted using, for example, an alkali such as sodium hydroxide, potassium hydroxide, potassium hydroxide, sodium bicarbonate, or sodium borate; or an acid such as hydrochloric acid, sulfuric acid, nitric acid, citric acid, or phosphoric acid.

[0041] The modification may be, for example, a chemical modification such as alkylation, glutathionylation, sulfonation, sulfenation or sulfination, or nitrosation (nitrosylation). The modification may be carried out, for example, by using a modifying agent (reactant) containing a modifying group imparted by the modification. As a specific example, the alkylation may be carried out, for example, by using an alkylating agent. The alkylating agent is, for example, a compound capable of reacting with the thiol group to alkylate the thiol group. The alkylating agent may be, for example, an organic iodide salt such as iodoacetamide, methyl iodide, or iodoacetic acid, or an alkylene oxide such as ethylene oxide. The alkylating agent may be, for example, one type, or a combination of two or more types. The sulfenation or sulfination may be carried out, for example, by using a compound capable of oxidizing the thiol group. The compound capable of oxidizing the thiol group may be, for example, an oxidizing agent. The nitrosation can be carried out, for example, using a compound capable of donating nitric oxide (NO), specifically, S-nitrosoglutathione, etc.

[0042] The modification conditions (temperature, time, etc.) in the modification step are not particularly limited and can be set to conditions that allow the thiol group to be sufficiently modified, for example. The reaction temperature in the modification is, for example, preferably 15 to 40° C. The reaction time in the modification is, for example, preferably 5 minutes or more, more preferably 5 minutes or more to 16 hours.

[0043] In the modification step, the modification may include, for example, a component other than the modifying agent. Examples of the other component include a buffering agent such as a buffer, a pH adjusting agent, and the like.

[0044] The measurement method of the present disclosure may optionally include a solid-phasing step of solidifying the proteoglycan on a carrier such as a substrate after the modification step and prior to the detection step. The solid-phasing method can be carried out by, for example, a known method for solidifying a protein, specifically, a covalent bond method, an ionic bond method, a physical adsorption method, a hydrophobic interaction method, or the like.

[0045] Examples of the carrier include plates such as microwell plates, microarray substrates, membranes (e.g., polyvinylidene fluoride (PVDF) membranes, nitrocellulose membranes, polystyrene nonwoven fabrics, etc.), and low-fluorescence carriers (e.g., low-fluorescence plates, low-fluorescence membranes, etc.).

[0046] In the measurement method of the present disclosure, for example, after the solid-phase step, blocking may be performed on the carrier so that components such as antibodies used in the detection step described below do not bind nonspecifically. Examples of blocking agents used in the blocking include albumin such as bovine serum albumin (BSA), skim milk, and commercially available blocking reagents.

[0047] Next, in the detection step, at least one of the C-terminal domain and the N-terminal domain of the modified proteoglycan is detected by fluorescence. In this way, in the detection step, for example, the presence or absence of the C-terminus and / or the N-terminus of the proteoglycan can be detected. Specifically, the detection method can be carried out by contacting the modified proteoglycan with a probe and detecting the probe bound to the proteoglycan by fluorescence. In the detection, a substrate or the like having a probe or proteoglycan immobilized thereon may be used.

[0048] In the detection step, either one or both of the C-terminal domain and the N-terminal domain of the modified proteoglycan may be detected. In the latter case, it is preferable that, for example, the C-terminal domain and the N-terminal domain of the proteoglycan are detected simultaneously by fluorescence in the detection step.

[0049] The N-terminal domain of the proteoglycan to be detected may be, for example, the G1 domain, the IGD domain, or the G2 domain, and is preferably the G1 domain. In the detection, for example, one, two, or three of the N-terminal domains may be detected. The C-terminal domain of the proteoglycan to be detected may be, for example, the G3 domain.

[0050] The detection can be performed, for example, by detecting the target component by fluorescence, and can be performed, for example, by immunoassay. Examples of the immunoassay include a direct method for detecting the binding between a probe and a proteoglycan; an indirect method for binding a first probe to a proteoglycan and detecting the first probe bound to the proteoglycan using a second probe; a sandwich method for binding a first probe to a proteoglycan and detecting the proteoglycan bound to the first probe using a second probe; a Western blotting method, an immunoprecipitation method, and an immunohistochemical staining method. The immunoassay can be a quantitative method or a qualitative method.

[0051] The probe may be, for example, a target-specific protein such as a polyclonal antibody, a monoclonal antibody, or an antigen-binding fragment; a target-specific nucleic acid molecule such as an aptamer; or the like. The antibody may be, for example, an antibody of any isotype among immunoglobulins selected from IgG, IgM, IgA, IgD, IgE, and IgY. The antibody fragment may be, for example, an antibody fragment such as F(ab')2, Fab', Fab, and Fv.

[0052] The probe may be, for example, labeled. As a specific example, the antibody may be, for example, an antibody labeled for detection. The label may be, for example, a fluorescent dye label, an enzyme label, or a radioactive label. When the fluorescence detection is performed by a direct method, the probe is, for example, preferably a fluorescently labeled probe. When the fluorescence detection is performed by an indirect method, the second probe is, for example, preferably a fluorescently labeled probe.

[0053] The fluorescence detection can be carried out, for example, using a fluorescence detector. EXAMPLES

[0054] Next, examples of the present disclosure will be described. However, the present disclosure is not limited by the following examples. Commercially available reagents were used according to their protocols unless otherwise indicated. In addition, "mol / l" may be abbreviated as "M".

[0055] [Example 1] The measurement method of the present disclosure made it possible to detect the C-terminus and N-terminus of proteoglycan.

[0056] (1) Purification of proteoglycans by guanidine extraction As raw materials, 100 to 150 g each of nasal cartilage of chum salmon, breast cartilage of chicken, bronchial cartilage of pig, and fin of golden porcine flounder were prepared. Five times the amount (W / V) of 4M guanidine hydrochloride aqueous solution was added to the raw materials. After the addition, immersion extraction was performed under conditions of 4°C for one week. After the extraction, filtration was performed using a 60 mesh sieve and filter paper (3 μm). After the filtration, three times the amount (v / v) of ethanol was added. After the addition, the mixture was left to stand at 4°C overnight. After the standing, filtration was performed using filter paper (3 μm) to collect the precipitate. After the collection, phosphate buffer (pH 6.8) was added to dissolve the precipitate. After the dissolution, fractionation and purification were performed using a molecular weight fractionation membrane (MW 50,000, manufactured by Asahi Kasei Corporation). After the fractionation and purification, a proteoglycan fraction was collected. After the recovery, the proteoglycan fraction was freeze-dried to obtain proteoglycan by guanidine extraction.

[0057] (2) Purification of proteoglycans by acetic acid extraction As a raw material, 100 to 150 g of nasal cartilage of chum salmon was prepared. Purification of proteoglycan by acetic acid extraction was performed with reference to the method described in Reference 1 below. By the above purification, proteoglycan by acetic acid extraction was obtained. Reference 1: Patent No. 6317053

[0058] (3) Detection of the C-terminal domain of proteoglycan derived from salmon nasal cartilage The C-terminal domain of the proteoglycan extracted with guanidine and the proteoglycan extracted with acetic acid obtained in Example 1(1) or Example 1(2) was detected. Specifically, 0.1 ml of PBS (pH 8.0) containing 20 mmol / l dithiothreitol (DTT) was added to 20 μg of each of the proteoglycans derived from the nasal cartilage of chum salmon. After the addition, the mixture was left to stand for 10 minutes to cleave the disulfide bond. After the cleavage, 40 mmol / l iodoacetamide was added, and alkylation was performed for 30 minutes under the condition of pH 8.0. After the alkylation, 10 μl, 50 μl, or 100 μl of the proteoglycan (0.5 mg / ml, hereinafter, the same concentration when used for immobilization) or 50 μl of 0.5 mg / ml BSA (control) was immobilized on the membrane. The solid-phase (hydrophobic interaction) was performed using Biodot (manufactured by BioRad) and a low-fluorescence PVDF membrane (Immobilon-FL membrane, manufactured by Merck). After the solid-phase, a primary antibody (anti-aggrecan G3 domain antibody, Anti-Aggrecan Antibody clone 1-C-6, MABT84, manufactured by Merck) diluted 1 / 1000 was added, and a primary reaction was performed under conditions of 4°C and 16 hours. After the primary reaction, the sample was washed with TBS (Tris buffer) containing 0.05% Tween-20. After the washing, a secondary antibody (goat anti-mouse IgG StarBright Blue 700, 12004159, manufactured by BioRad) diluted 1 / 2500 was added, and a secondary reaction was performed under conditions of 25°C and 1 hour. After the secondary reaction, fluorescence was detected using a multiplex fluorescence detector (ChemiDoc Touch MP, manufactured by BioRad). These results are shown in FIG. 3. In the comparative example (non-reduced), detection of the C-terminus was carried out in the same manner, except that neither cleavage of the disulfide bond nor alkylation was carried out.

[0059] FIG. 3 is a photograph showing the detection results of the C-terminal domain in proteoglycan. The upper part of FIG. 3 shows the results when disulfide bonds were cleaved and alkylated, and the lower part of FIG. 3 shows the results when disulfide bonds were not cleaved and alkylated. In FIG. 3, "light" indicates 10 μl of each proteoglycan, "medium" indicates 50 μl of each proteoglycan, and "high" indicates 100 μl of each proteoglycan. As shown in FIG. 3, when disulfide bonds were not cleaved and alkylated, the signal of the C-terminal domain was weakly detected in all samples regardless of the extraction method. On the other hand, when disulfide bonds were cleaved and alkylated in proteoglycan extracted with acetic acid, the signal of the C-terminal domain was strongly detected. In addition, when disulfide bonds were cleaved and alkylated in proteoglycan extracted with guanidine, the signal of the C-terminal domain was weakly detected compared to proteoglycan extracted with acetic acid, but the signal of the C-terminal domain could be detected. From these results, it was found that, by using the measurement method of the present invention, the C-terminal domain is preserved in the proteoglycan extracted with guanidine and the proteoglycan extracted with acetic acid.

[0060] (4) Detection of N-terminal domains of various proteoglycans The N-terminal domains of the proteoglycans extracted with guanidine and the proteoglycans extracted with acetic acid obtained in Example 1(1) or Example 1(2) were detected. Specifically, the detection was performed in the same manner as in Example 1(3) except that the proteoglycan used was a proteoglycan derived from the nasal cartilage of chum salmon, a proteoglycan derived from chicken sternal cartilage, a proteoglycan derived from bronchial cartilage of pig, or a proteoglycan derived from the fin of Japanese flounder, 10 μl or 50 μl of the proteoglycan was used for immobilization, and an anti-aggrecan IGD domain antibody (Aggrecan Antibody, DF7561, epitope: V355-V375, manufactured by Affinity Biosciences) diluted 1 / 1000 times was used as the primary antibody, and a goat anti-rabbit IgG StarBright Blue 700 (12004162, manufactured by BioRad) diluted 1 / 2500 times was used as the secondary antibody. These results are shown in FIG.

[0061] FIG. 4 is a photograph showing the detection results of the N-terminal domain in proteoglycan. The upper row (low concentration) of FIG. 4 shows the results of 10 μl of each proteoglycan, and the lower row (high concentration) of FIG. 4 shows the results of 50 μl of each proteoglycan. As shown in FIG. 4, the N-terminal domain could not be detected in the proteoglycan derived from nasal cartilage of chum salmon extracted with acetic acid. On the other hand, the N-terminal domain could be detected in the proteoglycan derived from nasal cartilage of chum salmon extracted with guanidine, the proteoglycan derived from chicken breast cartilage, the proteoglycan derived from porcine bronchial cartilage, and the proteoglycan derived from the fin of Japanese flounder. From these results, it was found that, by using the measurement method of the present invention, the N-terminus is not preserved in the proteoglycan derived from nasal cartilage of chum salmon extracted with acetic acid, and the N-terminal domain is preserved in the proteoglycans derived from various animals extracted with guanidine.

[0062] (5) Detection of C-terminal domains of various proteoglycans The C-terminal domain of the proteoglycan extracted with guanidine and the proteoglycan extracted with acetic acid obtained in Example 1(1) or Example 1(2) was detected. Specifically, the detection was performed in the same manner as in Example 1(3) except that the proteoglycan was derived from chum salmon nasal cartilage, chicken sternal cartilage, or pig bronchial cartilage, and 10 μl or 50 μl of the proteoglycan was used for immobilization. The results are shown in FIG. 5.

[0063] FIG. 5 is a photograph showing the detection results of the C-terminal domain in proteoglycan. The upper row of FIG. 5 shows the results of 10 μl of each proteoglycan, and the lower row of FIG. 5 shows the results of 50 μl of each proteoglycan. As shown in FIG. 5, the C-terminal domain could be detected in the proteoglycan derived from nasal cartilage of chum salmon extracted with acetic acid, as well as in the proteoglycan derived from nasal cartilage of chum salmon extracted with guanidine, the proteoglycan derived from chicken breast cartilage, and the proteoglycan derived from porcine bronchial cartilage. From these results, it was found that the C-terminal domain is preserved in the proteoglycan derived from nasal cartilage of chum salmon extracted with acetic acid and the proteoglycans derived from various animals extracted with guanidine by using the measurement method of the present invention.

[0064] (6) Detection of various domains of various proteoglycans Various domains of the proteoglycan extracted with guanidine and the proteoglycan extracted with acetic acid obtained in Example 1(1) or Example 1(2) were detected. The various domains were N-terminal domain 1 (epitope: Val20-Gly675, G1-IGD-G2 domain), N-terminal domain 2 (epitope: aa50-150, G1 domain), C-terminal domain 1 (epitope: aa2358-2481, G3 Lectin-BD domain), and C-terminal domain 2 (epitope: aa2255-2280). Specifically, the detection was performed in the same manner as in Example 1(3) except that an antibody capable of detecting the various domains was used as the primary antibody and an antibody capable of detecting the primary antibody was used as the secondary antibody.

[0065] The primary antibodies are shown in the following Table 1. The primary antibody for detecting the N-terminal domain 1 was used at a 500-fold dilution, the primary antibody for detecting the N-terminal domain 2 was used at a 900-fold dilution, the primary antibody for detecting the C-terminal domain 1 was used at a 30-fold dilution, and the primary antibody for detecting the C-terminal domain 2 was used at a 100-fold dilution.

[0066] The secondary antibody used was goat anti-rabbit IgG StarBright Blue 700 (12004162, BioRad) diluted 1 / 2500 or goat anti-mouse IgG StarBright Blue 700 (12004159, BioRad) diluted 1 / 2500.

[0067] [Table 1]

[0068] In the proteoglycan derived from nasal cartilage of chum salmon extracted with acetic acid, the N-terminal domain was hardly detected, and the C-terminal domain was detected for a part of G3, but the lectin domain was not detected. In the proteoglycan derived from nasal cartilage of chum salmon extracted with guanidine, the proteoglycan derived from chicken breast cartilage, and the proteoglycan derived from porcine bronchial cartilage, all domains were detected for the N-terminal domain, and all domains of the G3 domain were detected for the C-terminal domain. From these results, it was found that by using the measurement method of the present invention, only the G3 domain of the C-terminal domain is preserved in the proteoglycan derived from nasal cartilage of chum salmon extracted with acetic acid, and all domains of the N-terminus and all G3 domains of the C-terminus are preserved in the proteoglycans derived from various animals extracted with guanidine.

[0069] [Example 2] The measurement method of the present disclosure made it possible to detect the C-terminus and N-terminus of proteoglycan.

[0070] (1) Detection of various domains of various proteoglycans Detection of various domains was performed on the proteoglycan (sCSPG-Gdm) derived from nasal cartilage of chum salmon by guanidine extraction obtained in Example 1(1), the proteoglycan (sCSPG-AcOH) derived from nasal cartilage of chum salmon by acetic acid extraction obtained in Example 1(2), the proteoglycan (cCSPG) derived from chicken breast cartilage by guanidine extraction obtained in Example 1(1), and the proteoglycan (pCSPG) derived from porcine bronchial cartilage by guanidine extraction obtained in Example 1(1). The various domains were the G1 domain, the IGD domain, the G1-IGD-G2 domain, the G3 domain 1, the G3 domain 2 (epitope: aa2255-2280), and the G3 lectin domain. Specifically, detection was performed in the same manner as in Example 1(3), except that an antibody capable of detecting the various domains was used as the primary antibody, and an antibody capable of detecting the primary antibody was used as the secondary antibody. These results are shown in FIG.

[0071] The primary antibodies are shown in Table 2 below. The primary antibody detecting the G1 domain was used at a 900-fold dilution, the primary antibody detecting the IGD domain was used at a 1000-fold dilution, the primary antibody detecting the G1-IGD-G2 domain was used at a 500-fold dilution, the primary antibody detecting the G3 domain 1 was used at a 1000-fold dilution, the primary antibody detecting the G3 domain 2 (epitope: aa2255-2280) was used at a 100-fold dilution, and the primary antibody detecting the G3 lectin domain was used at a 30-fold dilution.

[0072] The secondary antibody used was goat anti-rabbit IgG StarBright Blue 700 (12004162, BioRad) diluted 1 / 2500 or goat anti-mouse IgG StarBright Blue 700 (12004159, BioRad) diluted 1 / 2500.

[0073] Furthermore, the peak top molecular weights of the various proteoglycans were measured. Specifically, the various proteoglycans were subjected to HPLC analysis under the following measurement conditions. The results are shown in FIG.

[0074] (HPLC measurement conditions) HPLC system: LC-10AD Column: TSKgel G5000-PWXL Φ7.8mm×300mm (Tosoh Corporation) Elution solution: pH 6.8 phosphate buffer Flow rate: 0.5ml / min Column temperature: 40℃ Detector: Differential refractive index detector (RID-10A, Shimadzu Corporation) Injection volume: 50μl Molecular weight marker: Shodex STANDARD P-82 (pullulan)

[0075] [Table 2]

[0076] FIG. 6 is a photograph showing the results of detection of C-terminal domains in proteoglycans. From the top to the bottom, FIG. 6 shows the results of G1 domain, IGD domain, G1-IGD-G2 domain, G3 domain 1, G3 domain 2 (epitope: aa2255-2280), and G3 lectin domain. In addition, from the left to the right, FIG. 6 shows the results of BSA (1), acetic acid-extracted proteoglycan derived from nasal cartilage of chum salmon (2) (sCSPG-AcOH), guanidine-extracted proteoglycan derived from nasal cartilage of chum salmon (3) (sCSPG-Gdm), guanidine-extracted proteoglycan derived from chicken breast cartilage (4) (cCSPG), and guanidine-extracted proteoglycan derived from porcine bronchial cartilage (5) (pCSPG). As shown in Figure 6, the N-terminal domain was hardly detected in the acetic acid-extracted proteoglycan, and the C-terminal domain was detected in part of G3. On the other hand, both the N-terminal and C-terminal domains were detected in the acetic acid-extracted proteoglycan, the guanidine-extracted proteoglycan, cCSPG, and pCSPG. The peak top molecular weights of the acetic acid-extracted proteoglycan, the guanidine-extracted proteoglycan, cCSPG, and pCSPG were 480,000, 680,000, 630,000, and 640,000, respectively.

[0077] (2) Analysis of GAGs in each proteoglycan GAG was analyzed for the various proteoglycans used in Example 2(1). Specifically, GAG was purified from the various proteoglycans used in Example 2(1). 10 g of the sCSPG-Gdm, the cCSPG, or the pCSPG was treated with 80 ml of boiling water for 10 minutes. After the treatment, 100 ml of 0.5 mol / l borate buffer (pH 7.0) was added. After the addition, 0.67 g of Protease N Amano G (manufactured by Amano Enzyme Co., Ltd.) was added, and the mixture was incubated at 55°C for 7 days. After the incubation, quenching was performed with 5% trichloroacetic acid. After the quenching, insoluble matter was filtered and precipitated with 80% ethanol containing 1.25% NaOAc (sodium acetate). After the precipitation, the mixture was centrifuged at 0°C, and the resulting precipitate was dried in vacuum to obtain crude sugar chains. Next, 100 mg of the crude glycan was diluted with 0.05 mol / l acetic acid (pH 4.0) containing 0.15 mol / l LiCl. After the dilution, the solution was applied to a DEAE-cellulose column (φ2.2×15 cm, Wako Pure Chemical Industries, Ltd.). After the application, the column was washed stepwise with 160 ml of buffer containing 0.15, 0.5, 1.0, or 2.0 mol / l LiCl. After the washing, the carbazole-positive fraction was collected and dialyzed, and the dialyzed sample was desalted with a gel permeation column (LH-20, H2O, φ1.1×80 cm) to obtain GAG. Then, 50 μg of the GAG ​​was diluted with 50 μg of H2O. After the dilution, HPLC analysis was performed using linearly coupled size exclusion columns (OHpak SB-G 6B 100×4.6 mm and SB-805 HQ 250×4.6 mm).

[0078] (HPLC measurement conditions) HPLC system: LC-10AD (Shimadzu Corporation) Columns: OHpak SB-G 6B 100×4.6mm and SB-805 HQ 250×4.6mm Eluent: 0.1mol / l NaNO3 Flow rate: 1mL / min

[0079] As a result, the peak top molecular weight (Mp) of guanidine-extracted GAG (sGAG) derived from salmon nasal cartilage was 84,000, the peak top molecular weight (Mp) of guanidine-extracted GAG (cGAG) derived from chicken sternal cartilage was 77,000, and the peak top molecular weight (Mp) of guanidine-extracted GAG (pGAG) derived from pig bronchial cartilage was 51,000.

[0080] (3) Disaccharide structure of each proteoglycan The disaccharide structure of each proteoglycan was analyzed for each of the various GAGs obtained in Example 2(2). Specifically, 50 μg of each of the GAGs obtained in Example 2(2) was diluted with 50 μl H2O, BSA solution (0.05 mg / 5 μl), and 10 μl of 250 mmol / l Tris-HCl (AcOH) solution (pH 8.0). After the dilution, the resulting diluted solution was digested with chondroitinase ABC (25 mU / 25 μl, Sigma Aldrich) or chondroitinase AC (25 mU / 25 μl, Sigma Aldrich) at 37° C. or 30° C. for 8 hours. After the digestion, the unsaturated disaccharides generated by the digestion were subjected to HPLC analysis using a PA-03 (PA-G) column. In the analysis, the content ratio (constituent ratio) of each disaccharide structure was calculated based on the HPLC peak area of ​​the standard unsaturated disaccharide. The results are shown in Table 3 below.

[0081] [Table 3]

[0082] As shown in Table 3 above, the contents of disaccharide structures were as follows: Guanidine-extracted GAG (sGAG) from salmon nasal cartilage Chondroitin sulfate O: 11%, chondroitin sulfate iO: 0%, chondroitin sulfate A: 27%, chondroitin sulfate iA: 0%, chondroitin sulfate C: 61%, chondroitin sulfate iC: 0% Guanidine-extracted chicken breast cartilage derived GAG (cGAG) Chondroitin sulfate O: 5.2%, chondroitin sulfate iO: 0%, chondroitin sulfate A: 63%, chondroitin sulfate iA: 6.5%, chondroitin sulfate C: 24%, chondroitin sulfate iC: 0.3% Guanidine-extracted porcine bronchial cartilage-derived GAG (pGAG) Chondroitin sulfate O: 6.1%, chondroitin sulfate iO: 0%, chondroitin sulfate A: 52%, chondroitin sulfate iA: 20%, chondroitin sulfate C: 19%, chondroitin sulfate iC: 2.1%

[0083] [Example 3] Next, we investigated whether decorin, a proteoglycan, could be detected instead of aggrecan by the measurement method of the present disclosure. Decorin is a small leucine-rich proteoglycan that has a single GAG ​​chain in its core protein.

[0084] First, bovine proteoglycan (dermatan sulfate proteoglycan: DSPG) was prepared. As a raw material, about 824 g of the abomasum of a bovine (Holstein) was prepared. The abomasum was washed and chopped into about 2 cm cubes while removing as much fat as possible. After the chopping, 4 times the amount of ethanol (3288 ml) was added, and the mixture was left to stand for 10 minutes, and this was repeated once. Then, 4 times the amount of ethanol (3288 ml) was added, and the mixture was left to stand overnight. After the standing, the ethanol was removed and the mixture was air-dried. After the air-drying, the mixture was dried under reduced pressure to obtain 173 g of a defatted and dried product. Next, the defatted and dried product was subjected to purification of proteoglycan by guanidine extraction. Specifically, 87 g of the defatted and dried product of Example 2 (1-1) was added with 350 ml of 4 M guanidine hydrochloride aqueous solution, and immersion extraction was performed at 4° C. for 3 days. After the above-mentioned immersion extraction, 347 ml of 4 M guanidine hydrochloride aqueous solution was added, and further immersion extraction was carried out under the condition of 4° C. for 3 days. After the above-mentioned immersion extraction, suction filtration and dialysis were carried out. After the above-mentioned dialysis, freeze-drying was carried out to obtain 9.93 g of proteoglycan solid.

[0085] The proteoglycan was subjected to salt exchange with a neutral buffer (pH 7.5). Specifically, the salt exchange with the neutral buffer (pH 7.5) was carried out by adding 60 ml of a neutral buffer (pH 7.5) to the bovine stomach-derived proteoglycan. After the addition, the mixture was subjected to ultrasonic disruption and allowed to stand overnight. After the standing, the mixture was centrifuged to obtain a supernatant. The supernatant was subjected to dialysis and freeze-drying.

[0086] Next, reduction and alkylation of proteoglycan was performed using FOCUS Protein Reduction-Alkylation (786-231, manufactured by G-Biosciences). 2.5 μl of Reductant Buffer was added to 500 μl of 1 mg / ml proteoglycan after salt exchange with the neutral buffer (pH 7.5) and stirred for 10 seconds. After the stirring, 10 ml of FOCUS Protein Reductant was added and left to stand at 55° C. for 1 hour. After the standing, 2.5 ml of Alkylation Buffer was added and stirred for 10 seconds. After the stirring, 0.4 mol / l Iodoacetamide was added and left to stand under light-shielded conditions at room temperature (about 25° C., the same applies below) for 1 hour to perform alkylation of the sample. Next, a PVDF membrane (Immobilon-FL membrane, manufactured by Merck) was treated with methanol for 1 minute to make it hydrophilic. After the hydrophilization, the PVDF membrane was replaced with TBS for 5 minutes. After the replacement, the PVDF membrane was set in a filtration blotting device (manufactured by BioRad). After the setting, the alkylated sample was applied to the well and aspirated. After the aspirating, the PVDF membrane was collected from the filtration blotting device and the PVDF membrane was left to dry at room temperature. After the static drying, the PVDF membrane was washed with TBS (Tris buffer, TBS-T) containing 0.05% Tween-20. After the washing, the PVDF membrane was blocked for 5 minutes using EveryBlot blocking buffer (manufactured by BioRad). After the blocking, a primary antibody (anti-Decorin antibody, Cat No: 14667-1-AP, manufactured by Proteintech) diluted 1 / 1000 times was added, and the primary reaction was carried out at 4°C for 18 hours. After the primary reaction, the membrane was washed five times for 5 minutes with TBS-T. After the washing, StarBright Blue (manufactured by BioRad) diluted 1 / 2500 times and 0.05% SDS were added, and the secondary reaction was carried out at room temperature for 1 hour. After the secondary reaction, the membrane was washed six times for 5 minutes with TBS-T. After the washing, the PVDF membrane was washed with ultrapure water and replaced with ultrapure water.After the substitution, the fluorescence of the PVDF membrane was detected using a multiplex fluorescence detector (ChemiDoc Touch MP, BioRad). The results are shown in FIG.

[0087] Figure 7 is a photograph showing the results of decorin detection. In Figure 7, the left column shows the results for BSA, and the right column shows the results for bovine-derived DSPG. As shown in Figure 7, a signal could be detected in bovine-derived DSPG. These results demonstrate that decorin is preserved as a core protein in bovine proteoglycan that has been extracted with guanidine and salt-substituted with a neutral buffer (pH 7.5) by using the measurement method of the present disclosure.

[0088] Although the present disclosure has been described above with reference to the embodiments and examples, the present disclosure is not limited to the above-mentioned embodiments and examples. Various modifications that can be understood by a person skilled in the art can be made to the configuration and details of the present disclosure within the scope of the present disclosure.

[0089] This application claims priority based on Japanese Patent Application No. 2023-078729, filed on May 11, 2023, and Japanese Patent Application No. 2023-206830, filed on December 7, 2023, the disclosures of which are incorporated herein in their entirety.

[0090] <Additional Notes> Some or all of the above-described embodiments and examples may be described as follows, but are not limited to the following: <Method of measuring proteoglycan> (Appendix 1) A method for measuring proteoglycan, comprising: A cleavage step of cleaving disulfide bonds of the proteoglycan; a modification step of modifying the thiol groups of the obtained proteoglycan under alkaline conditions; a detection step of detecting at least one of the C-terminal domain and the N-terminal domain of the proteoglycan by fluorescence; A method for measuring proteoglycan, comprising: (Appendix 2) The measurement method according to claim 1, wherein the detection step comprises simultaneously detecting the C-terminal domain and the N-terminal domain of the proteoglycan by fluorescence. (Appendix 3) The measurement method according to claim 1 or 2, wherein the cleavage is reduction to a thiol group by a reducing agent. (Appendix 4) The method for measuring according to any one of claims 1 to 3, wherein the alkalinity is pH 7.5 to pH 8.5. (Appendix 5) The measurement method according to any one of Appendices 1 to 4, wherein in the modification step, the formation of a disulfide bond is inhibited by modifying the thiol group. (Appendix 6) The measurement method according to any one of claims 1 to 5, wherein in the modification step, the thiol group is modified by alkylation. (Appendix 7) The measurement method according to any one of claims 1 to 6, further comprising a step of solidifying the proteoglycan prior to the detection step. (Appendix 8) The measurement method according to any one of appendix 1 to 7, wherein the N-terminal domain is a G1 domain, an IGD domain, and / or a G2 domain. (Appendix 9) The measurement method according to any one of claims 1 to 7, wherein the C-terminal domain is a G3 domain. (Appendix 10) The method according to any one of claims 1 to 9, wherein the detection step comprises detecting the antibody by an immunological assay. (Appendix 11) The method according to any one of claims 1 to 10, wherein the peak top molecular weight of the proteoglycan is 400,000 to 1,200,000. (Appendix 12) The method for measuring the proteoglycan according to any one of claims 1 to 11, wherein the proteoglycan is derived from an animal. (Appendix 13) The method of claim 12, wherein the proteoglycan is a proteoglycan of the lectican family. (Appendix 14) The measurement method according to claim 12 or 13, wherein the proteoglycan is aggrecan. (Appendix 15) 15. The method of any one of claims 12 to 14, wherein the animal is selected from the group consisting of salmon, chicken, pig, and flounder. [Industrial Applicability]

[0091] As described above, according to the present disclosure, for example, it is possible to provide a method for measuring proteoglycan capable of detecting proteoglycan with its C-terminus and / or N-terminus maintained, etc. Therefore, the present disclosure can be said to be extremely useful, for example, in the field of regenerative medicine.

Claims

1. A method for measuring proteoglycan, comprising: A cleavage step of cleaving disulfide bonds of the proteoglycan; a modification step of modifying the thiol groups of the obtained proteoglycan under alkaline conditions; and detecting the C-terminal domain and the N-terminal domain of the proteoglycan simultaneously by fluorescence. Methods for measuring proteoglycans.

2. A method for measuring proteoglycan, comprising: A cleavage step of cleaving disulfide bonds of the proteoglycan; a modification step of modifying the thiol groups of the obtained proteoglycan under alkaline conditions; and detecting at least one of the C-terminal domain and the N-terminal domain of the proteoglycan by fluorescence, A method for measuring proteoglycan, wherein the alkalinity is pH 7.5 or higher.

3. The method according to claim 2 , wherein the detection step comprises simultaneously detecting the C-terminal domain and the N-terminal domain of the proteoglycan by fluorescence.

4. The method according to claim 1 , wherein the cleavage is reduction to a thiol group by a reducing agent.

5. The method according to any one of claims 1 to 3, wherein the alkalinity is pH 7.5 to pH 8.

5.

6. The method according to claim 1 , wherein in the modification step, the thiol group is modified to inhibit formation of a disulfide bond.

7. The method according to claim 1 , wherein the modification step comprises alkylating the thiol group.

8. The method according to claim 1 , further comprising a step of solidifying the proteoglycan prior to the detection step.

9. The method according to claim 1 , wherein the N-terminal domain is a G1 domain, an IGD domain, and / or a G2 domain.

10. The method according to claim 1 , wherein the C-terminal domain is a G3 domain.

11. The method according to claim 1 , wherein the detection step comprises detecting the antibody by an immunological assay.

12. The method according to any one of claims 1 to 3, wherein the peak top molecular weight of the proteoglycan is 400,000 to 1,200,000.

13. The method according to claim 1 , wherein the proteoglycan is derived from an animal.

14. The method according to claim 13, wherein the proteoglycan is a proteoglycan of the lectican family.

15. The method according to claim 13, wherein the proteoglycan is aggrecan.

16. The method of claim 13, wherein the animal is selected from the group consisting of salmon, chicken, pig, and flounder.

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