Mucin capturing agent and use thereof

Abstract

To provide a capturing agent for mucin, a mucin fragment, or a mucin-derived glycopeptide or glycan, and a recovery method therefor.SOLUTION: A capturing agent for mucin, mucin fragments, or mucin-derived glycopeptides or glycans, the capturing agent comprising a compound having a betaine structure, the betaine structure including an anionic site, a cationic site, and a linker, the anionic site being a group selected from the group consisting of a phosphate group, a carboxyl group, and a sulfonic acid group, the cationic site being a quaternary ammonium group, and the linker connecting the anionic site and the cationic site being an alkylene group having 1 to 4 carbon atoms.SELECTED DRAWING: None

Description

[Technical Field]

[0001] The present invention relates to a capturing agent for mucin, mucin fragment, or mucin-derived glycopeptide or sugar chain, and a method for recovering the same. [Background technology]

[0002] Mucins are large glycoproteins covered with numerous O-linked glycans. Many tumor markers, such as CA19-9 and CA125, are carbohydrate antigens, and these carbohydrate antigens are thought to be sugar chains on mucins. Therefore, mucins are considered promising targets for the search for new disease markers.

[0003] On the other hand, while marker discovery using proteomics technology has become popular in recent years, mucin is resistant to proteases such as trypsin, making it difficult to fragment into peptides, making it difficult to apply this technology. Therefore, analytical techniques different from conventional proteomics are thought to be necessary.

[0004] For example, Patent Document 1 describes an invention in which mucin-like glycoproteins, human salivary mucins, etc. are separated by cellulose acetate membrane electrophoresis and the sugar chains on the mucins are analyzed. While membrane electrophoresis enables high-level separation and analysis of mucins, there are issues with throughput, and therefore it would be useful to establish a simpler method for purifying mucins. [Prior art documents] [Patent documents]

[0005] [Patent Document 1] Patent Publication No. 2009-265078 Summary of the Invention [Problem to be solved by the invention]

[0006] An object of the present invention is to provide a capture agent and a capture carrier for capturing or recovering mucin, mucin fragments, or mucin-derived glycopeptides or sugar chains, and a method thereof. Another object of the present invention is to provide a capture agent and a capture carrier for capturing or recovering mucin, mucin fragments, or mucin-derived glycopeptides or sugar chains, and a method thereof, which can be used simply and in a short time with a high recovery rate, comprehensively and specifically, with good reproducibility, or in a scalable manner. [Means for solving the problem]

[0007] As a result of intensive research to solve the above problems, the inventors discovered that mucin, mucin fragments, or mucin-derived glycopeptides or glycans can be captured or recovered by using a capture agent containing a compound having a betaine structure, and thus completed the invention. That is, the present invention includes the following aspects. [1] A capture agent for mucin, mucin fragment, or mucin-derived glycopeptide or sugar chain, comprising: The scavenger comprises a compound having a betaine structure, The betaine structure has an anionic moiety, a cationic moiety, and a linker, the anionic moiety is a group selected from the group consisting of a phosphate group, a carboxyl group, and a sulfonic acid group; The cationic moiety is a quaternary ammonium group, A scavenger, wherein the linker connecting the anion moiety and the cation moiety is an alkylene group having 1 to 4 carbon atoms. [2] A carrier for capturing mucin, mucin fragments, or mucin-derived glycopeptides or sugar chains, in which the capturing agent according to [1] is immobilized on an insoluble support. [3] The capture carrier according to [2], wherein the capture carrier is a particle. [4] The capture carrier according to [3], wherein the particles have pores with an average pore diameter of 1 nm or more. [5] A method for recovering mucin, a mucin fragment, or a mucin-derived glycopeptide or sugar chain, the method comprising: a) contacting a sample containing mucin, mucin fragment, glycopeptide, or sugar chain with the capture agent according to [1] or the capture carrier according to any one of [2] to [4] to capture the mucin, mucin fragment, glycopeptide, or sugar chain; and b) washing the capture agent or capture carrier that has captured mucin, mucin fragment, glycopeptide, or sugar chain with solvent 1 to remove substances other than mucin, mucin fragment, and mucin-derived glycopeptides and sugar chains; c) a step of eluting the mucin, mucin fragment, glycopeptide, or sugar chain with solvent 2 from the capture agent or capture carrier that has captured the mucin, mucin fragment, glycopeptide, or sugar chain A method comprising: [6] The method according to [5], wherein the solvent 1 in step b) is a mixed solution of an organic solvent and water or a mixed solution of an organic solvent, water and an organic acid, and contains 10% or more by volume of water. [7] The method according to [6], wherein the organic solvent in step b) comprises one or more selected from alcohol and acetonitrile. [8] The method according to any one of [5] to [7], wherein the solvent 2 in the step c) contains 40% by volume or more of water. [9] The method according to any one of [5] to [8], wherein the solvent 2 in the step c) is a weakly alkaline solvent.

[10] The method according to any one of [5] to [9], wherein the sample containing a mucin fragment or a mucin-derived glycopeptide is a mucin degradation product. [Effects of the Invention]

[0008] According to the present invention, mucin, mucin fragment, or mucin-derived glycopeptide or glycan can be captured or recovered. Furthermore, according to the present invention, mucin, mucin fragment, or mucin-derived glycopeptide or glycan can be detected, captured, recovered, or concentrated simply and in a short time with a high recovery rate, comprehensively and specifically, with good reproducibility, or by a method that can be scaled up. [Brief explanation of the drawings]

[0009] [Figure 1] FIG. 1 is a diagram schematically illustrating an example of a capture carrier according to the present embodiment. [Figure 2] The results of Example 3 are shown, which evaluated the effect of bead pore size on the recovery of mucin-derived glycopeptides. The pore sizes of the beads in each chart are (1) 70 Å, (2) 300 Å, and (3) 1000 Å. In the figure, the peaks at the times indicated by the arrows are mucin-derived peaks. [Figure 3] Example 4 shows the results of evaluating the effect of various bead washing solutions on the recovery of mucin-derived glycopeptides. Each chart shows (1) an aqueous solution containing 75% acetonitrile and 0.1% formic acid, (2) an aqueous solution containing 50% acetonitrile and 0.1% formic acid, (3) an aqueous solution containing 70% acetonitrile and 0.1% formic acid, and (4) an aqueous solution containing 80% acetonitrile and 0.1% formic acid. In the figure, the peak at the time indicated by the arrow is the mucin-derived peak. DETAILED DESCRIPTION OF THE INVENTION

[0010] As used herein, "mucin" refers to a high molecular weight glycoprotein composed of a core protein rich in threonine, serine, and proline, to which numerous glycans are attached. As known in the art, mucins are the main component of mucus, which coats the lining of animal cavities such as the digestive tract and respiratory tract. At least 20 different mucins are known, including MUC1, MUC2, MUC3A, MUC3B, MUC4, MUC5AC, MUC5B, MUC6, MUC7, MUC8, MUC9, MUC10, MUC11, MUC12, MUC13, MUC14, MUC15, MUC16, MUC17, MUC18, MUC19, and MUC20. Mucins can be classified into two structurally and functionally distinct classes: secretory mucins produced by epithelial cells and membrane-bound mucins, which have hydrophobic transmembrane domains and exist bound to the cell membrane. As used herein, the term "mucin fragment" refers to a portion of mucin obtained by fragmenting the glycoprotein mucin with a protease or the like.

[0011] As used herein, "glycopeptide" refers to a peptide to which a sugar chain is attached. As used herein, "sugar chain" refers to a compound formed by a chain of one or more sugar units (monosaccharides and / or their derivatives), and when two or more sugar units are attached, the sugar units are bonded to each other by dehydration condensation via a glycosidic bond. As used herein, "sugar chain" also includes derivatives of sugar chains. Therefore, in this specification, a "mucin-derived glycopeptide" refers to a peptide having a sugar chain attached thereto, which is derived from mucin, and a "mucin-derived sugar chain" refers to a compound formed by a chain of one or more unit sugars (monosaccharides and / or their derivatives), which is derived from mucin.

[0012] As used herein, "mucin, mucin fragment, or mucin-derived glycopeptide or glycochain" includes N-glycans or O-glycans, and is preferably rich in O-glycans. "N-linked glycans" refer to glycans that are bound to the nitrogen atom of the amide group in the side chain of an asparagine residue in a protein. N-linked glycans include those that form branches with mannose as the base, such as di-branched, tri-branched, and quadruple-branched chains. N-linked glycans are also classified into basic, high-mannose, hybrid, and complex types depending on their structure. N-linked glycans are also called "N-linked glycans" or "N-linked glycans." "O-linked glycans" refer to glycans bound to the -OH group contained in each amino acid side chain of serine (Ser) or threonine (Thr) amino acid residues in proteins. O-linked glycans are further classified into 1 to 8 types depending on the core structure. O-linked glycans are also called "O-linked glycans" or "O-linked glycans." Furthermore, the region rich in O-glycans in "mucin, mucin fragment, or mucin-derived glycopeptide or sugar chain" herein is also referred to as a mucin domain.

[0013] (Capturing agents and capture carriers) In one embodiment, the present invention provides a capture agent for mucin, a mucin fragment, or a glycopeptide or sugar chain derived from mucin, comprising: The scavenger comprises a compound having a betaine structure, The betaine structure has an anionic moiety, a cationic moiety, and a linker, the anionic moiety is a group selected from the group consisting of a phosphate group, a carboxyl group, and a sulfonic acid group; The cationic moiety is a quaternary ammonium group, The linker connecting the anionic and cationic moieties contains a scavenger, which is an alkylene group having 1 to 4 carbon atoms.

[0014] The term "capture agent" as used herein includes compounds having a betaine structure, and can be referred to as a capture agent for capturing mucin, mucin fragments, or mucin-derived glycopeptides or sugar chains. The composition, shape, state, etc. of the scavenger are not particularly limited. The scavenger may contain one or more compounds having a betaine structure, and may be a mixture of compounds not having a betaine structure, as long as the scavenger contains a compound having a betaine structure.

[0015] The betaine structure may have the structure shown in formula (1) or (2): -ZLA (1) -ALZ (2) [In formulas (1) and (2), Z represents a cationic group selected from the group consisting of a secondary amino group, a tertiary amino group, a quaternary ammonium group, and an imino group; L represents an alkylene group having 1 to 10 carbon atoms; and A represents an anionic group selected from the group consisting of a phosphate group, a carboxyl group, a phosphonate group, a phosphinate group, a sulfonic acid group, a sulfine group, a sulfene group, a hydroxyl group, a thiol group, and a boronic acid group.]

[0016] Preferably, the betaine structure is having an anionic moiety, a cationic moiety, and a linker; the anionic moiety is a group selected from the group consisting of a phosphate group, a carboxyl group, and a sulfonic acid group; The cationic moiety is a quaternary ammonium group, The linker connecting the anionic moiety and the cationic moiety is an alkylene group having 1 to 4 carbon atoms.

[0017] The compound having a betaine structure may be betaine, or may be a polymer in which a side chain having a betaine structure is bonded to the main chain.

[0018] As used herein, the term "main chain" refers to the longest carbon chain in the polymer structure, and the structure branching from the main chain is called a "side chain." Furthermore, as used herein, the term "sugar chain" includes monosaccharides.

[0019] The capture agent may be immobilized on an insoluble support to form a capture carrier. In this specification, the capture carrier may be referred to as a "carrier for capturing mucin, mucin fragment, or mucin-derived glycopeptide or sugar chain" or simply as a "carrier." In one embodiment, the capture agent may comprise a polymer having a side chain having a betaine structure attached to its backbone, and the polymer may be immobilized on an insoluble support. The betaine structure significantly enhances hydrophilicity, allowing the capture agent to strongly retain highly hydrophilic glycopeptides or sugar chains through hydrophilic interactions.

[0020] In one embodiment, the capture carrier is an insoluble support having a polymer immobilized thereon, the polymer having a side chain having a betaine structure bonded to the main chain, and preferably the polymer covers all or part of the surface of the insoluble support to form a polymer layer. Here, "covering" means that the polymer is attached to the surface of the insoluble support. Figure 1 shows a schematic representation of an example of the "capture carrier." The "capture carrier" in Figure 1 has a polymer layer having a betaine structure formed on the surface of the insoluble support.

[0021] The polymer layer may contain a polymer having no betaine structure in addition to a polymer having a side chain having a betaine structure attached to its main chain. Therefore, as long as a betaine structure is present on the surface of the capture carrier, the capture carrier can capture mucin, mucin fragments, or mucin-derived glycopeptides or sugar chains.

[0022] The insoluble support is a base material insoluble in water and organic solvents used in the recovery process of mucin, mucin fragments, or mucin-derived glycopeptides or sugar chains. It is not particularly limited as long as it can immobilize a polymer having a side chain with a betaine structure attached to its main chain, and any known base material can be used. The material of the insoluble support may be either inorganic or organic, or a composite material combining these. Examples of inorganic materials include silicon compounds such as silica, glass such as silicate glass, oxides such as iron oxide (ferrite, magnetite, etc.), alumina, titania, and zirconia, metals and alloys thereof such as iron, copper, gold, silver, platinum, cobalt, aluminum, palladium, iridium, and rhodium, and carbon materials such as graphite. These materials may be used alone or in combination. Examples of organic substances include synthetic polymers such as cross-linked polyvinyl alcohol, cross-linked polyacrylate, cross-linked polyacrylamide, and cross-linked polystyrene, and polysaccharides such as cross-linked sepharose, crystalline cellulose, cross-linked cellulose, cross-linked amylose, cross-linked agarose, and cross-linked dextran. These may be used alone or in combination of two or more. Furthermore, a polymer having a side chain having a betaine structure attached to its main chain may itself form an insoluble support.

[0023] The insoluble support is preferably an inorganic substance, particularly preferably a silicon compound, and even more preferably silica. Generally, organic substances that can serve as insoluble supports have a specific gravity of around 1, which is small compared to samples containing mucin, mucin fragments, or mucin-derived glycopeptides or sugar chains. Solid-liquid separation is often complicated due to the small difference in specific gravity between the organic substance and samples containing mucin, mucin fragments, or mucin-derived glycopeptides or sugar chains. The use of an inorganic substance facilitates solid-liquid separation, particularly when a capture carrier is used to capture mucin, mucin fragments, or mucin-derived glycopeptides or sugar chains. This effectively separates the mucin, mucin fragments, or mucin-derived glycopeptides or sugar chains adsorbed on the carrier from contaminants other than mucin, mucin fragments, and mucin-derived glycopeptides and sugar chains, such as free proteins, peptide fragments, lipids, and salts. This contributes to improved recovery efficiency. Furthermore, the inorganic substance can impart appropriate strength to the carrier.

[0024] The insoluble support may be a porous or hollow body having voids. For example, the porous body may have an average particle size of 0.1 μm to 100 μm, and the average pore size can be independently controlled within a range of, for example, 1 nm or more, 3 nm or more, 7 nm or more, or 30 nm or more, and an upper limit of 200 nm or less, 120 nm or less, or 100 nm or less, and preferably 1 nm or more to 200 nm or less, 3 nm or more to 120 nm or less, 7 nm or more to 100 nm or less, or 30 nm or more to 100 nm or less. Monolithic silica may also be used. Monolithic silica is a porous silica structure having micrometer-sized three-dimensional network pores (macropores) and nanometer-sized pores (mesopores) in the silica skeleton forming the three-dimensional network structure. The average pore size of the macropores is, for example, 1 μm to 100 μm, preferably 1 μm to 50 μm. The average pore size of the mesopores can be independently controlled, for example, with a lower limit of 1 nm or more, 3 nm or more, 7 nm or more, or 30 nm or more, and an upper limit of 200 nm or less, 120 nm or less, or 100 nm or less, preferably 1 nm to 200 nm, 3 nm to 120 nm, 7 nm to 100 nm, or 30 nm to 100 nm. The average pore size can be measured, for example, by pore size distribution measurement using gas adsorption, gas permeation, mercury intrusion, or bubble point method. By using an insoluble support having such voids, the capture carrier has a larger specific surface area, thereby increasing the amount of capture agent that can be immobilized on the surface of the insoluble support. As a result, when the capture carrier disclosed herein is used to recover mucin, mucin fragments, or mucin-derived glycopeptides or sugar chains, the contact efficiency with mucin, mucin fragments, or mucin-derived glycopeptides or sugar chains is improved, allowing mucin, mucin fragments, or mucin-derived glycopeptides or sugar chains to be efficiently captured, contributing to improved recovery efficiency. It can also be used to adjust the specific gravity of the insoluble support, as described below.

[0025] In one embodiment, the scavenger may be in the form of a polymer in which a side chain having a betaine structure is bonded to the main chain. When the scavenger is in the form of a polymer, the polymer may be a polymer of a polymerizable monomer. The polymerizable monomer is not particularly limited as long as it is a monomer capable of forming a polymer by polymerization reaction. Preferably, the polymerizable monomer is a (meth)acrylic compound having a (meth)acryloyl group, such as a (meth)acrylic acid ester and its derivatives. Further examples include, but are not limited to, compounds having a vinyl group, an allyl group, an α-alkoxymethylacryloyl group, a maleic acid residue, a fumaric acid residue, an itaconic acid residue, a crotonic acid residue, an isocrotonic acid residue, and a citraconic acid residue, and their derivatives. The polymerizable monomer may be used alone or in combination of two or more types. The term "(meth)acryloyl group" refers to an "acryloyl group" or a "methacryloyl group," and the term "(meth)acrylic" refers to "acrylic" or "methacrylic."

[0026] In one embodiment in which a capture agent is immobilized on an insoluble support to form a capture support, a polymer having a side chain having the above-mentioned betaine structure attached to its main chain may be immobilized on the insoluble support. The side chain of the polymer immobilized on the insoluble support is a molecular chain branched from a main chain composed of a polymer of the above-mentioned polymerizable monomer, and has a betaine structure in part or in whole.

[0027] As used herein, the term "betaine structure" refers to a structure having a cationic moiety and an anionic moiety at separate, non-adjacent positions within the same molecule. In one embodiment, the betaine structure has an anionic moiety, a cationic moiety, and a linker.

[0028] The cationic moiety is a positively charged atomic group, which means a so-called cationic group. Examples of cationic groups include a primary amino group, a secondary amino group (-NHR), a tertiary amino group (-NR2), a quaternary ammonium group (-NR3 +), and imino group, etc., but are not limited to these. R in the secondary amino group, tertiary amino group, and quaternary ammonium group is an alkyl group or an aryl group, and when one group has multiple R, they may be the same or different from one another, and examples thereof include, but are not limited to, a methyl group, an ethyl group, and a propyl group. A quaternary ammonium group is preferred, and a trimethylammonium group is particularly preferred. Cationic groups also include salts formed with fluoride ions, chloride ions, bromide ions, iodide ions, hydrochloride ions, acetate ions, sulfate ions, hydrofluoric acid ions, carbonate ions, etc.

[0029] The anionic moiety is a negatively charged atomic group, which is a so-called anionic group. Examples of anionic groups include, but are not limited to, phosphate groups, phosphonate groups, phosphinate groups, sulfonic acid groups, sulfine groups, sulfene groups, carboxyl groups, hydroxyl groups, thiol groups, and boronic acid groups. Preferably, the anionic moiety is selected from the group consisting of phosphate groups, carboxyl groups, and sulfonic acid groups, and more preferably, a phosphate group. In addition, salt forms formed with alkali metal ions such as sodium ions and potassium ions, and alkaline earth metal ions such as calcium ions, are also included in the anionic group.

[0030] The betaine structure is not particularly limited as long as it has the above-mentioned cationic moiety and anionic moiety, and the combination of the cationic moiety and the anionic moiety is not particularly limited. Preferably, the cationic moiety is a quaternary ammonium group, and the anionic moiety is a group selected from the group consisting of a phosphate group, a carboxyl group, and a sulfonic acid group. More preferably, the cationic moiety is a quaternary ammonium group, and the anionic moiety is a phosphate group.

[0031] The polymer to be immobilized on the insoluble support is not particularly limited as long as it has a side chain having a betaine structure bonded to a main chain composed of a polymer of a polymerizable monomer. Therefore, the polymer may be a homopolymer of a polymerizable monomer having a betaine structure, or a copolymer of a polymerizable monomer having a cationic moiety and a polymerizable monomer having an anionic moiety. Furthermore, the polymer may be a copolymer containing an uncharged polymerizable monomer. The inclusion of such a polymerizable monomer can control the solubility of the polymer in water. The copolymer refers to a polymer obtained from two or more types of monomers and may be an alternating copolymer, a block copolymer, a random copolymer, a graft copolymer, or the like. Therefore, the betaine structure may be introduced into each monomer unit of the polymer, into a fixed number of monomer units, or randomly.

[0032] Preferably, the polymer side chain is a homopolymer of a polymerizable monomer having a betaine structure. In this case, the polymerizable monomer having a betaine structure has an anionic moiety and a cationic moiety in the same molecular chain. The linker connecting the two is not particularly limited as long as it has a divalent or higher group, and known linkers can be used. Preferred is an alkylene linker, and examples of the alkylene linker include alkylene linkers having 1 to 10 carbon atoms, preferably 1 to 4 or 2 to 5 carbon atoms.

[0033] Examples of such polymerizable monomers having a betaine structure include, but are not limited to, phosphobetaine monomers having a phosphobetaine group such as a phosphorylcholine group, carboxybetaine monomers having a carboxybetaine group, sulfobetaine monomers having a sulfobetaine group, etc. Preferred are phosphobetaine monomers, and more preferred are phosphobetaine monomers having a phosphorylcholine group.

[0034] As the phosphobetaine monomer, a polymerizable monomer having a phosphorylcholine group is preferred, such as 2-(meth)acryloyloxyethyl phosphorylcholine, 2-(meth)acryloyloxyethoxyethyl phosphorylcholine, 6-(meth)acryloyloxyhexyl phosphorylcholine, 10-(meth)acryloyloxyethoxynonyl phosphorylcholine, 2-(meth)acryloyloxypropyl phosphorylcholine, 2-(meth)acryloyloxybutyl phosphorylcholine, etc. Among these, 2-(meth)acryloyloxyethyl phosphorylcholine is particularly preferred because of its easy availability. Further, examples of the phosphobetaine monomer include dimethyl(2-methacryloyloxyethyl)(2-phosphonatoethyl)aminium, dimethyl(2-acryloyloxyethyl)(2-phosphonatoethyl)aminium, dimethyl(2-methacryloyloxyethyl)(3-phosphonatopropyl)aminium, dimethyl(2-acryloyloxyethyl)(3-phosphonatopropyl)aminium, dimethyl(2-methacryloyloxyethyl)(4-phosphonatobutyl)aminium, dimethyl(2-acryloyloxyethyl)(4-phosphonatobutyl)aminium, dimethyl(2-methacryloyloxyethyl)(phosphonatomethyl)aminium, and dimethyl(2-acryloyloxyethyl)(phosphonatomethyl)aminium.

[0035] Examples of carboxybetaine monomers include dimethyl(2-methacryloyloxyethyl)(2-carboxylatoethyl)aminium, dimethyl(2-acryloyloxyethyl)(2-carboxylatoethyl)aminium, dimethyl(2-methacryloyloxyethyl)(3-carboxylatopropyl)aminium, dimethyl(2-acryloyloxyethyl)(3-carboxylatopropyl)aminium, dimethyl(2-methacryloyloxyethyl)(4-carboxylatobutyl)aminium, dimethyl(2-acryloyloxyethyl)(4-carboxylatobutyl)aminium, dimethyl(2-methacryloyloxyethyl)(carboxylatomethyl)aminium, and dimethyl(2-acryloyloxyethyl)(carboxylatomethyl)aminium.

[0036] Examples of sulfobetaine monomers include dimethyl(2-methacryloyloxyethyl)(2-sulfonatoethyl)aminium, dimethyl(2-acryloyloxyethyl)(2-sulfonatoethyl)aminium, dimethyl(2-methacryloyloxyethyl)(3-sulfonatopropyl)aminium, dimethyl(2-acryloyloxyethyl)(3-sulfonatopropyl)aminium, dimethyl(2-methacryloyloxyethyl)(4-sulfonatobutyl)aminium, dimethyl(2-acryloyloxyethyl)(4-sulfonatobutyl)aminium, dimethyl(2-methacryloyloxyethyl)(sulfonatomethyl)aminium, and dimethyl(2-acryloyloxyethyl)(sulfonatomethyl)aminium.

[0037] The weight of the polymer bound to the insoluble support is determined by the unit surface area (m 2 The polymer weight per unit surface area is preferably about 0.5 mg to 1.5 mg, particularly preferably 0.6 mg to 1.3 mg, and even more preferably 0.7 mg to 1.2 mg. When the polymer weight per unit surface area is within the above range, handling during polymer synthesis is improved, and good contact efficiency with mucin, mucin fragments, or mucin-derived glycopeptides or sugar chains can be ensured, allowing efficient capture of mucin, mucin fragments, or mucin-derived glycopeptides or sugar chains.

[0038] The capture carrier preferably has a specific gravity of approximately 1.05 to 3.00, particularly 1.1 to 2.7, and even more preferably 1.5 to 2.5. If the specific gravity is below the lower limit, sedimentation properties decrease, while if it exceeds the upper limit, dispersibility deteriorates, resulting in poor operability in either case. Therefore, when the capture carrier has a specific gravity within the above range, when used to capture mucin, mucin fragments, or mucin-derived glycopeptides or sugar chains, the capture carrier exhibits good sedimentation properties, allowing for easy and simple solid-liquid separation by gravity-induced natural sedimentation or centrifugation, etc., and the mucin, mucin fragments, or mucin-derived glycopeptides or sugar chains captured on the carrier can be effectively separated from contaminants such as free proteins and peptide fragments, other substances than mucin, mucin fragments, and mucin-derived glycopeptides and sugar chains. Furthermore, the good dispersibility improves the contact efficiency with mucin, mucin fragments, and mucin-derived glycopeptides and sugar chains, enabling efficient capture of mucin, mucin fragments, or mucin-derived glycopeptides or sugar chains. Therefore, a carrier excellent in operability can be provided, and when used for capturing mucin, mucin fragments, or mucin-derived glycopeptides or sugar chains, a carrier excellent in separation from contaminants such as free proteins and peptide fragments other than mucin, mucin fragments, and mucin-derived glycopeptides and sugar chains, and in recovery efficiency of mucin, mucin fragments, or mucin-derived glycopeptides or sugar chains can be provided.

[0039] The shape of the capture carrier is not particularly limited and may be any known shape. Examples include particulate shapes such as beads, plates such as substrates and multiwell plates, membranes such as sheets, films, and membranes, and fibers. The carrier can also be referred to as a solid phase. Preferably, the carrier is in a particle or similar shape. When the capture carrier is particulate, the average particle size is preferably approximately 0.5 μm to 100 μm, particularly preferably 1 μm to 50 μm, or 1 μm to 10 μm. An average particle size of 3 μm to 10 μm is particularly preferred. If the average particle size is below the lower limit, recovery of the carrier by centrifugation or filtration becomes difficult. Furthermore, when the carrier is packed in a column or the like and used, the liquid permeability is poor, requiring the application of a large pressure for liquid passage. On the other hand, if the average particle size exceeds the upper limit, the contact area between the carrier and the sample solution decreases, resulting in a decrease in the adsorption efficiency of mucin, mucin fragments, or mucin-derived glycopeptides or sugar chains, resulting in a decrease in recovery efficiency. Therefore, when the average particle size of the "capture carrier" is within the above range, a carrier excellent in operability can be provided, and when used to capture mucin, mucin fragments, or mucin-derived glycopeptides or sugar chains, a carrier excellent in separation efficiency from substances other than mucin, mucin fragments, and mucin-derived glycopeptides and sugar chains, such as free peptide fragments, and recovery efficiency of sugar chains can be provided. The average particle size can be measured, for example, with a particle size distribution analyzer.

[0040] Furthermore, the capture carrier may be a particle having pores. The capture carrier may have the same average pore size as the insoluble support forming the carrier. The average pore size may be, for example, 1 nm (10 Å) or more, 7 nm (70 Å) or more, 30 nm (300 Å) or more, or 100 nm (1000 Å) or more, and 200 nm (2000 Å) or less, 120 nm (1200 Å) or less, or 100 nm (1000 Å) or less, preferably 1 nm or more to 200 nm or less (10 Å or more to 2000 Å or less), 3 nm or more to 120 nm or less (30 Å or more to 1200 Å or less), 7 nm or more to 100 nm or less (70 Å or more to 1000 Å or less), or 30 nm or more to 100 nm or less (300 Å or more to 1000 Å or less), more preferably 7 nm or more to 100 nm or less (70 Å or more to 1000 Å or less). The average pore size can be measured, for example, by measuring the pore size distribution by a gas adsorption method.

[0041] The capture carrier may be in a form filled in a container such as a filter cup of a spin column or the like, each well of a multi-well plate, each well of a filter plate, or a microtube.

[0042] The polymer can be obtained by polymerizing the above-mentioned polymerizable monomers, but the polymerization method for the polymer is not particularly limited and can be appropriately selected depending on the type of polymerizable monomer, etc. Radical polymerization is preferred.

[0043] The immobilization of the polymer on the insoluble support may be carried out by either physical adsorption or chemical bonding. Chemical bonding is preferred from the viewpoint of stability, as it can prevent the polymer from eluting from the insoluble support. Alternatively, the polymer may be immobilized on the surface of the insoluble support by polymerizing a polymerizable monomer on the surface of the insoluble support, or a prepolymerized polymer may be immobilized on the surface of the insoluble support.

[0044] When immobilizing a polymer on the surface of an insoluble support by polymerizing a polymerizable monomer on the surface of the insoluble support, for example, polymerization initiation sites can be introduced onto the surface of the insoluble support, and the insoluble support with the introduced polymerization initiation sites can be immersed in a polymerizable monomer solution and a polymerization initiator added to grow the polymer from the polymerization initiation sites. This allows the polymer to be immobilized on the surface of the insoluble support by chemical bonding. Polymerizable functional groups, chain transfer groups, dormant species in living radical polymerization, etc. can be used as the polymerization initiation sites.

[0045] Polymerizable functional groups include vinyl groups, allyl groups (2-propenyl groups), (meth)acryloyl groups, Examples of the chain transfer group include a mercapto group, an amino group, etc., with the mercapto group being preferred because of its excellent reactivity.

[0046] The method for introducing a polymerizable functional group or a chain transfer group onto the surface of the insoluble support is not particularly limited, but it is preferable to use a silane coupling agent having a polymerizable functional group or a chain transfer group.

[0047] Examples of silane coupling agents having a polymerizable functional group include (3-methacryloxypropyl)dimethylmethoxysilane, (3-methacryloxypropyl)diethylmethoxysilane, (3-methacryloxypropyl)dimethylethoxysilane, (3-methacryloxypropyl)diethylethoxysilane, (3-methacryloxypropyl)methyldimethoxysilane, (3-methacryloxypropyl)ethyldimethoxysilane, (3-methacryloxypropyl)methyldiethoxysilane, (3-methacryloxypropyl)ethyldiethoxysilane, (3-methacryloxypropyl)trimethoxysilane, and (3-methacryloxypropyl)triethoxysilane. From the viewpoints of reactivity and availability, (3-methacryloxypropyl)trimethoxysilane and (3-methacryloxypropyl)triethoxysilane are preferred. These silane coupling agents can be used alone or in combination of two or more.

[0048] Examples of silane coupling agents having a chain transfer group include (3-mercaptopropyl)trimethoxysilane, (3-mercaptopropyl)methyldimethoxysilane, (3-mercaptopropyl)dimethylmethoxysilane, (3-mercaptopropyl)triethoxysilane, (3-mercaptopropyl)methyldiethoxysilane, (3-mercaptopropyl)dimethylethoxysilane, (mercaptomethyl)trimethoxysilane, (mercaptomethyl)methyldimethoxysilane, (mercaptomethyl)dimethylmethoxysilane, (mercaptomethyl)triethoxysilane, (mercaptomethyl)methyldiethoxysilane, (mercaptomethyl)dimethylethoxysilane, etc., but (3-mercaptopropyl)trimethoxysilane and (3-mercaptopropyl)triethoxysilane are preferred due to their availability.These silane coupling agents can be used alone or in combination of two or more.

[0049] The introduction of polymerizable functional groups or chain transfer groups into an insoluble support using a silane coupling agent having a polymerizable functional group or chain transfer group can be carried out, for example, by forming a covalent bond between the silane coupling agent and the functional group on the surface of the insoluble support.For example, when using alkoxysilanes such as trimethoxysilanes or triethoxysilanes as the silane coupling agent, the silanol groups generated by hydrolysis can be dehydrated and condensed with hydroxyl groups, amino groups, carbonyl groups, silanol groups, etc. on the surface of the insoluble support to form a covalent bond.

[0050] After introducing a polymerizable functional group or a chain transfer group onto the surface of an insoluble support, the insoluble support is mixed with a polymerizable monomer to allow a polymerization reaction to proceed, thereby forming a polymer layer on the surface of the insoluble support. The polymerization reaction is carried out, but is not limited to, by, for example, placing the insoluble support in a solvent containing the polymerizable monomer and a polymerization initiator, and heating the mixture with stirring at a temperature of 0°C to 80°C for 1 hour to 30 hours. The insoluble support is then filtered under reduced pressure, washed, and dried.

[0051] The proportions of the insoluble support, polymerizable monomer, and polymerization initiator used are not particularly limited, but typically, the polymerizable monomer and the polymerization initiator are used in a proportion of 0.1 mmol to 10 mmol per 1 g of insoluble support.

[0052] The solvent may be any solvent that dissolves each polymerizable monomer, and examples thereof include alcohols such as methanol, ethanol, isopropanol, n-butanol, t-butyl alcohol, and n-pentanol, benzene, toluene, tetrahydrofuran, dioxane, dichloromethane, chloroform, cyclohexanone, N,N-dimethylformamide, dimethyl sulfoxide, methyl acetate, ethyl acetate, butyl acetate, methyl ethyl ketone, methyl butyl ketone, ethylene glycol monoethyl ether, ethylene glycol monomethyl ether, and ethylene glycol monobutyl ether. These solvents may be used alone or in combination of two or more.

[0053] The polymerization initiator is not particularly limited, but examples thereof include azo compounds such as 2,2′-azobisisobutylnitrile (hereinafter sometimes abbreviated as “AIBN”) and 1,1′-azobis(cyclohexane-1-carbonitrile), organic peroxides such as benzoyl peroxide, lauryl peroxide, and tert-butyl peroxide, and redox initiators such as hydrogen peroxide-ferrous ion.

[0054] On the other hand, when immobilizing a prepolymerized polymer on the surface of an insoluble support, methods include physically adsorbing or chemically bonding the prepolymerized polymer to the insoluble support. Preferably, a component that is easily adsorbed to the insoluble support or a component having a functional group that can react with a reactive functional group present on the surface of the insoluble support is incorporated into the polymer as a copolymer during polymerization. For example, as a functional group that can react with a reactive functional group present on the surface of the insoluble support, for example, a silanol group obtained by hydrolyzing a silane coupling agent is preferred because it has high reactivity, and can form a covalent bond by dehydration condensation with a hydroxyl group, amino group, carbonyl group, silanol group, etc. on the surface of the solid support. The polymerization reaction of the polymerizable monomer can be carried out in a similar manner to the above.

[0055] By applying the above polymer to the surface of an insoluble support, the polymer can be adsorbed or chemically bonded to the surface of the insoluble support. Examples of application methods include preparing a polymer solution and then immersing or spraying the solution. After application, the solution is preferably dried at room temperature or under heating. When chemical bonding is used, the reaction conditions should be appropriate for each application. This results in the formation of a polymer layer on the surface of the insoluble support.

[0056] The capture carrier is prepared by introducing chain transfer groups such as mercapto groups onto the surface of inorganic particles having hydroxyl groups on the surface of an insoluble support such as silica beads, and synthesizing a polymer layer containing a betaine structure. First, chain transfer groups are introduced onto the surface of the inorganic particles using a silane coupling agent having chain transfer groups. At this time, silanol groups generated by hydrolysis of hydrolyzable groups such as alkoxy groups of the silane coupling agent undergo dehydration condensation with hydroxyl groups on the surface of the inorganic particles to form covalent bonds, thereby introducing the chain transfer groups. Subsequently, the inorganic particles having the chain transfer groups introduced therein and (meth)acrylic monomers, at least some of which have a betaine structure, are radically polymerized in a suitable solvent by adding a polymerization initiator. The chain transfer groups introduced onto the inorganic particles serve as polymerization initiation points, and the inorganic particle surfaces are coated with a polymer layer having a betaine structure to form a polymer layer.

[0057] (Method for recovering mucin, mucin fragment, or mucin-derived glycopeptide or sugar chain) Furthermore, one embodiment of the present invention is a method for recovering mucin, a mucin fragment, or a glycopeptide or sugar chain derived from mucin, the method comprising: a) contacting a sample containing mucin, mucin fragment, glycopeptide, or sugar chain with the capture agent or capture carrier to capture the mucin, mucin fragment, glycopeptide, or sugar chain; and b) washing the capture agent or capture carrier that has captured mucin, mucin fragment, glycopeptide, or sugar chain with solvent 1 to remove substances other than mucin, mucin fragment, and mucin-derived glycopeptides and sugar chains; c) a step of eluting the mucin, mucin fragment, glycopeptide, or sugar chain with solvent 2 from the capture agent or capture carrier that has captured the mucin, mucin fragment, glycopeptide, or sugar chain The present invention includes a method comprising the steps of:

[0058] The sample containing mucin, mucin fragment, glycopeptide, or sugar chain is not particularly limited as long as it can contain mucin, mucin fragment, glycopeptide, or sugar chain. For example, the sample containing mucin fragment or glycopeptide may be a mucin degradation product. The sample containing mucin, mucin fragment, glycopeptide, or sugar chain may be a biological sample or an environmental sample. Examples of biological samples include body fluids, extracts obtained from cells or tissues, cell or tissue secretions, and feces. Examples of body fluids include blood, urine, serum, serous fluid, plasma, lymph, cerebrospinal fluid, saliva, mucosal secretions, vaginal secretions, ascites, pleural fluid, pericardial fluid, peritoneal fluid, and sweat. As used herein, the term "blood sample" refers to a sample containing at least a portion of a blood component, and may be, for example, whole blood, serum, or plasma, or a diluted version of these. The blood sample is preferably serum or plasma, more preferably serum. Blood samples can be prepared by known methods.

[0059] The mucin degradation products can be obtained by decomposing mucin. The decomposition treatment of mucin is not particularly limited as long as it can decompose mucin into mucin fragments or mucin-derived glycopeptides, and examples of the treatment include chemical treatment, protease treatment, and the like. Examples of proteases include, but are not limited to, trypsin, chymotrypsin, pepsin, V8 protease, pronase, proteinase K, lysyl endoprotease, bromelain, thermolysin, ficin, caspase, and subtilisin. Chemical treatments include, for example, denaturation or reduction of mucin. The chemical treatment is preferably carried out before the protease treatment. Denaturants include, for example, surfactants and chaotropic agents, and reducing agents include, but are not limited to, guanidine, dithiothreitol, iodoacetamide, β-mercaptoethanol, glutathione, tris-2-carboxyethylphosphine, tributylphosphine, or salts thereof.

[0060] Step a) is a step of contacting the sample with the capture agent or capture carrier to capture mucin, mucin fragments, or mucin-derived glycopeptides or glycans contained in the sample onto the capture agent or capture carrier. Therefore, the step is not particularly limited as long as the capture agent or capture carrier is under conditions that allow the capture agent or capture carrier to capture mucin, mucin fragments, or mucin-derived glycopeptides or glycans. For example, the step can be performed using a known batch method or spin column method. The capture agent or capture carrier can specifically capture mucin, mucin fragments, or mucin-derived glycopeptides or glycans, while substances present in the sample other than mucin, mucin fragments, and mucin-derived glycopeptides and glycans, such as contaminants such as free proteins, peptide fragments, lipids, and salts, or glycopeptides or glycans not derived from mucin, are not captured by the capture agent or capture carrier and remain in a free state.

[0061] A solvent may be used in step a) above, such as an organic solvent or a mixture of an organic solvent and water. The solvent may be appropriately selected depending on the type of mucin, mucin fragment, or mucin-derived glycopeptide or sugar chain to be captured. The organic solvent is not particularly limited as long as it can dissolve mucin, mucin fragment, or mucin-derived glycopeptide or sugar chain, and may be, for example, one or more selected from alcohol and acetonitrile. Preferred organic solvents include acetonitrile, tetrahydrofuran, acetone, dioxane, pyridine, methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, etc., more preferably acetonitrile, 1-butanol, ethanol, or a combination thereof, and even more preferably ethanol. The solvent in step a) may further contain an organic acid in addition to an organic solvent or a mixed solution of an organic solvent and water. The organic acid can be used to adjust the pH of the solvent in step a). Examples of the organic acid include formic acid, acetic acid, propionic acid, butyric acid, and trifluoroacetic acid, and formic acid is preferred. Therefore, in one embodiment, the solvent in step a) may be a mixed solution containing an alcohol or acetonitrile and water, for example, a mixed solution containing ethanol and water, or a mixed solution containing an organic acid in addition to alcohol or acetonitrile and water, for example, a mixed solution containing formic acid in addition to ethanol and water.

[0062] When the solvent in step a) contains an organic solvent and water, the mixing ratio of water is, for example, 1% by volume or more, 5% by volume or more, 10% by volume or more, 15% by volume or more, or 20% by volume or more as its lower limit, and 99% by volume or less, 70% by volume or less, 60% by volume or less, 50% by volume or less, 40% by volume or less, or 30% by volume or less as its upper limit, and is preferably 1 to 99% by volume, 1 to 70% by volume, 5 to 60% by volume, 10 to 50% by volume, 15 to 40% by volume, or 20 to 30% by volume. Furthermore, when the solvent in step a) contains an organic acid, the mixing ratio of the organic acid is, for example, 0.01 vol% or more, 0.02 vol% or more, or 0.05 vol% or more as its lower limit, and 1 vol% or less, 0.8 vol% or less, 0.5 vol% or less, 0.2 vol% or less, or 0.1 vol% or less as its upper limit, and is preferably 0.01 to 1 vol%, 0.02 to 0.5 vol%, or 0.05 to 0.2 vol%.

[0063] The above-mentioned step b) is a step in which the capture agent or capture carrier that captured mucin, mucin fragment, glycopeptide, or sugar chain in the above-mentioned step a) is washed with solvent 1 to remove substances other than mucin, mucin fragment, and mucin-derived glycopeptides and sugar chains. Therefore, the step is not particularly limited as long as it is carried out under conditions that allow removal of substances other than mucin, mucin fragment, and mucin-derived glycopeptides and sugar chains. For example, the step can be carried out by a known batch method or spin column method. As used herein, "substances other than mucin, mucin fragments, and mucin-derived glycopeptides and sugar chains" include, for example, contaminants such as free proteins, peptide fragments, lipids, and salts, or glycopeptides or sugar chains that are not derived from mucin.

[0064] Solvent 1 in step b) above can be, for example, an organic solvent or a mixed solution of an organic solvent and water. Solvent 1 can also be referred to as a washing solution. Solvent 1 can be appropriately selected depending on the type of mucin, mucin fragment, or mucin-derived glycopeptide or sugar chain to be captured. The organic solvent is not particularly limited as long as it can dissolve mucin, mucin fragment, or mucin-derived glycopeptide or sugar chain, and can be, for example, one or more selected from alcohol and acetonitrile. Preferred organic solvents include acetonitrile, tetrahydrofuran, acetone, dioxane, pyridine, methanol, ethanol, 1-propanol, 2-propanol, 1-butanol, etc., more preferably acetonitrile, 1-butanol, ethanol, or a combination thereof, and even more preferably ethanol. In step b), solvent 1 may further contain an organic acid in addition to an organic solvent or a mixed solution of an organic solvent and water. The organic acid can be used to adjust the pH of solvent 1 in step b). Examples of organic acids include formic acid, acetic acid, propionic acid, butyric acid, and trifluoroacetic acid, with formic acid being preferred. Therefore, in one embodiment, solvent 1 in step b) may be a mixed solution containing an alcohol or acetonitrile and water, for example, a mixed solution containing ethanol and water, or a mixed solution containing an organic acid in addition to alcohol or acetonitrile and water, for example, a mixed solution containing formic acid in addition to ethanol and water.

[0065] When solvent 1 in step b) contains an organic solvent and water, the mixing ratio of water is, for example, 1% by volume or more, 5% by volume or more, 10% by volume or more, 15% by volume or more, or 20% by volume or more as its lower limit, and 99% by volume or less, 70% by volume or less, 60% by volume or less, 50% by volume or less, 40% by volume or less, or 30% by volume or less as its upper limit, and is preferably 1 to 99% by volume, 1 to 70% by volume, 5 to 60% by volume, 10 to 50% by volume, 15 to 40% by volume, or 20 to 30% by volume. Furthermore, when the solvent in step b) contains an organic acid, the mixing ratio of the organic acid is, for example, a lower limit of 0.01 vol% or more, 0.02 vol% or more, or 0.05 vol% or more, and an upper limit of 1 vol% or less, 0.8 vol% or less, 0.5 vol% or less, 0.2 vol% or less, or 0.1 vol% or less, and preferably 0.01 to 1 vol%, 0.02 to 0.5 vol%, or 0.05 to 0.2 vol%.

[0066] The above step c) is a step of eluting mucin, mucin fragment, glycopeptide, or sugar chain from the capture agent or capture carrier that has captured mucin, mucin fragment, glycopeptide, or sugar chain with solvent 2 after washing in the above step b). Therefore, the step is not particularly limited as long as it is performed under conditions that allow elution of mucin, mucin fragment, glycopeptide, or sugar chain. For example, the step can be performed by a known batch method or spin column method.

[0067] Solvent 2 in step c) above can be, for example, an organic solvent, a mixed solution of an organic solvent and water, or an aqueous solution. Solvent 2 can also be referred to as an eluent. Solvent 2 can be appropriately selected depending on the type of mucin, mucin fragment, or mucin-derived glycopeptide or sugar chain to be captured. The organic solvent is not particularly limited as long as it can dissolve mucin, mucin fragment, or mucin-derived glycopeptide or sugar chain, and examples include acetonitrile, tetrahydrofuran, acetone, dioxane, pyridine, methanol, ethanol, 1-propanol, 2-propanol, and 1-butanol. Solvent 2 in step c) may contain a buffer to adjust the pH. The buffer is not particularly limited as long as it is capable of adjusting the pH of a solution containing mucin, a mucin fragment, or a mucin-derived glycopeptide or sugar chain, and examples thereof include ammonium carbonate, ammonium bicarbonate (ammonium hydrogen carbonate), ammonium chloride, diammonium hydrogen citrate, and ammonium carbamate, with ammonium bicarbonate (ammonium hydrogen carbonate) being preferred. Thus, in one embodiment, solvent 2 in step c) may be an aqueous solution containing a buffer, preferably an aqueous ammonium bicarbonate solution.

[0068] When solvent 2 in step c) contains an organic solvent and water, the mixing ratio of water can be, for example, the ratio described above. When solvent 2 is an aqueous solution containing a buffer, the mixing ratio of water can be, for example, 20% by volume or more, 40% by volume or more, 60% by volume or more, 80% by volume or more, 90% by volume or more, or 99% by volume or more as its lower limit, and 99.9% by volume or less, 99.5% by volume or less, or 90% by volume or less as its upper limit, and preferably 20 to 99.9% by volume, 40 to 99.9% by volume, 60 to 99.9% by volume, or 80 to 99.5% by volume. In one embodiment, the solvent 2 may be alkaline, preferably weakly alkaline. In this specification, "weakly alkaline" refers to a pH in the neutral range to a slightly alkaline range, for example, a pH in the range of about 8.0 to 11.0.

[0069] (batch method) In the case of batch recovery, a sample containing mucin, mucin fragments, glycopeptides, or sugar chains is contacted with the capture agent or capture carrier in an appropriate container (e.g., a microtube, centrifuge tube, microplate, etc.), and the capture agent or capture carrier captures the mucin, mucin fragments, glycopeptides, or sugar chains (step a). Preferably, the capture agent is immobilized on an insoluble support to form a capture carrier. Subsequently, the carrier-mucin, mucin fragment, glycopeptide, or sugar chain complex is subjected to solid-liquid separation, and the liquid phase containing contaminants other than mucin, mucin fragments, and mucin-derived glycopeptides and sugar chains, such as proteins, peptide fragments, lipids, and salts, is removed, and only the complex is recovered. Solid-liquid separation can be performed by gravity-induced sedimentation or centrifugation, and the liquid phase can be removed by suction or other methods. Alternatively, solid-liquid separation can be performed by incorporating a magnetic material, such as ferrite, into the insoluble support of the carrier, thereby allowing the complex to accumulate using magnetic force. In this case, centrifugation or other methods are not necessary. Alternatively, solid-liquid separation may be carried out by filtration through a filter, and in this case, the separation may be carried out under reduced pressure or under pressure.

[0070] Next, the capture agent or capture carrier that has captured mucin, mucin fragment, glycopeptide, or sugar chain is washed (step b). Washing can remove contaminants such as proteins and peptide fragments of substances other than mucin, mucin fragments, and mucin-derived glycopeptides and sugar chains captured by the capture agent or capture carrier. Washing can be performed by immersing the capture agent or capture carrier that has captured mucin, mucin fragment, glycopeptide, or sugar chain in a washing solution in an appropriate container and repeatedly changing the washing solution. For example, washing can be performed by placing the capture agent or capture carrier that has captured mucin, mucin fragment, glycopeptide, or sugar chain in an appropriate container, adding a washing solution, shaking or stirring, and then repeatedly removing the liquid phase by solid-liquid separation. Solid-liquid separation can be performed as described above.

[0071] After washing, the mucin, mucin fragment, glycopeptide, or sugar chain is eluted from the capture agent or capture carrier that has captured the mucin, mucin fragment, glycopeptide, or sugar chain (step c). The mucin, mucin fragment, glycopeptide, or sugar chain can be eluted by immersing the capture agent or capture carrier in an elution solution. For example, after thoroughly removing the washing solution, an appropriate amount of elution solution is added to the capture agent or capture carrier that has captured the mucin, mucin fragment, glycopeptide, or sugar chain, and the mixture is shaken or stirred. The capture agent or capture carrier is then recovered by solid-liquid separation, and the eluate is collected in a new appropriate container (e.g., a collection tube, collection plate, etc.). Solid-liquid separation can be performed as described above. If necessary, the eluate can be evaporated to recover the mucin, mucin fragment, glycopeptide, or sugar chain.

[0072] (Spin column method) Recovery by the spin column method can be performed using a container with a built-in filter, such as a filter cup. For example, a filter cup with openings at the top and bottom, the bottom opening of which is covered with a filter, can be used. When using a filter cup, a sample containing mucin, mucin fragments, glycopeptides, or sugar chains is placed in the filter cup filled with the capture agent or capture carrier, and the capture agent or capture carrier is contacted with the sample in a reaction solution by passing the liquid through the filter cup. The capture agent or capture carrier is preferably immobilized on an insoluble support to form a capture carrier. The liquid may be passed through by gravity, by centrifugation, or under reduced or increased pressure. After passing the liquid through the capture agent or capture carrier, the effluent containing impurities such as free proteins, peptide fragments, lipids, and salts other than the mucin, mucin fragments, and mucin-derived glycopeptides and sugar chains is removed.

[0073] Subsequently, the capture agent or capture carrier that has captured mucin, mucin fragments, glycopeptides, or sugar chains is washed. Washing can remove contaminants other than the mucin, mucin fragments, and mucin-derived glycopeptides and sugar chains captured on the capture agent or capture carrier, such as proteins, peptide fragments, lipids, and salts. Washing can be performed by passing a wash solution through the capture agent or capture carrier in the filter cup, allowing washing to be performed consecutively after the capture of mucin, mucin fragments, glycopeptides, or sugar chains. Fluid passing can be performed as described above.

[0074] After washing, the mucin, mucin fragment, glycopeptide, or sugar chain is eluted from the capture agent or capture carrier that has captured the mucin, mucin fragment, glycopeptide, or sugar chain. Elution of the mucin, mucin fragment, glycopeptide, or sugar chain can be carried out by passing the eluate through the capture agent or capture carrier in the filter cup, and can be carried out continuously after the capture of the mucin, mucin fragment, glycopeptide, or sugar chain and the washing procedure. After passing the eluate through the capture agent or capture carrier, the eluate is collected in an appropriate container (e.g., a collection tube or collection plate). The eluate can be passed as described above. If necessary, the mucin, mucin fragment, glycopeptide, or sugar chain can be recovered by distilling off the eluate.

[0075] The mucin, mucin fragment, glycopeptide, or glycan eluted in step c) above can be labeled as necessary and then qualitatively and / or quantitatively analyzed by known methods such as mass spectrometry (e.g., LC-IT-TOF MS or MALDI-TOF MS), chromatography (e.g., high-performance liquid chromatography or HPAE-PAD chromatography), or electrophoresis (e.g., capillary electrophoresis). The labeling reagent used in labeling can be a known labeling reagent for mucins, mucin fragments, glycopeptides, or glycans (e.g., EZGlyco O-Glycan Prep Kit, etc.). Labeling methods include, for example, a method in which labeling is performed by an exchange reaction from a hydrazone bond to an oxime bond, or a method in which free O-linked glycans are contacted with 2-aminobenzamide (2-AB). Furthermore, various databases (for example, GlycoMod, Glycosuite, SimGlycan (registered trademark), etc.) can be used in the analysis of sugar chains. [Example]

[0076] Example 1 Preparation of Mucin Capturing Beads (1) 1) Introduction of chain transfer groups into silica beads 5 g of (3-mercaptopropyl)trimethoxysilane, a silane coupling agent having a chain transfer group, was added to a mixture of 50 mL of acetic acid aqueous solution at pH 3.0 and 50 mL of ethanol, and the mixture was stirred at room temperature for 1 hour to hydrolyze the silane coupling agent. 5 g of silica beads (average particle size 5 μm, pore size 70 Å, manufactured by Fuji Silysia Chemical Ltd., SMB70-5), an example of an insoluble support, was then added and stirred at 70°C for 2 hours. The silica beads were then recovered from the reaction solution by suction filtration and heated at 100°C for 1 hour. The mixture was then dispersed in ethanol and shaken well, after which the supernatant was removed by centrifugation and the mixture was dried.

[0077] 2) Polymer synthesis 2-Methacryloyloxyethyl phosphorylcholine, the structural unit of the polymer, was dissolved in ethanol to prepare 20 mL of a 0.8 mol / L monomer solution. 2,2'-Azobisisobutylnitrile (AIBN) was added to the solution to a concentration of 0.027 mol / L and stirred until homogeneous. 4 g of silica beads treated with methacryloxypropyldimethylmethoxysilane were then added and reacted for 6 hours at 70 °C under an argon gas atmosphere. The silica beads were then recovered from the reaction solution by centrifugation, dispersed in ethanol, shaken thoroughly, and then recovered by suction filtration. The resulting beads were then dried to obtain mucin-capturing beads (1) (average particle size 5 μm, pore size 70 Å) in which a polymer containing structural units derived from 2-methacryloyloxyethyl phosphorylcholine was immobilized on the silica beads.

[0078] Example 2: Preparation of Mucin Capturing Beads (2) and (3) Mucin-capturing beads (2) (silica beads: average particle size 5 μm, pore size 300 Å) and mucin-capturing beads (3) (silica beads: average particle size 5 μm, pore size 1000 Å) were produced using the same method as in Example 1, except that the silica beads in the method shown in Example 1 were changed to silica beads having an average particle size of 5 μm and a pore size of 300 Å (manufactured by Fuji Silysia Chemical Ltd., SMB300-5) or silica beads having an average particle size of 5 μm and a pore size of 1000 Å (manufactured by Fuji Silysia Chemical Ltd., SMB1000-5).

[0079] Example 3 Recovery of mucin 1. (Dissolution of the sample) A mixture of bovine fetuin, bovine submaxillary mucin (BSM), and porcine stomach mucin (PSM) was dissolved in a solvent. This mixture is a mixture of glycoprotein and mucin samples. 2. (Denaturation treatment) The protein was denatured by reductive alkylation in the presence of guanidine hydrochloride. 3. (Trypsin treatment) Trypsin was added to decompose bovine fetuin, bovine submandibular gland mucin, and porcine stomach mucin into glycopeptides. 4. (Sample dilution) The sample was diluted with acetonitrile containing 0.1% formic acid so that the glycopeptide concentration in the sample was 10%. 5. (Application to beads) 2 mg of mucin-capturing beads with one of the pore sizes listed below were placed in a spin tube (Mobicol, MoBiTec) with a filter column. A 10 mM formic acid solution, followed by a column wash (80% ethanol solution), was passed through the column by centrifugation (1000 × g, 1 minute) in a centrifuge to activate the beads. The sample prepared in 4. above was then added to the column in several portions, each time centrifuged (300 × g, 1 minute) in a centrifuge, allowing glycopeptides or glycans (target substances) derived from bovine fetuin, bovine submandibular gland mucin, and porcine stomach mucin to be adsorbed onto the beads. The filtrate was discarded each time. 6. (Washing) 300 μL of an aqueous solution containing 80% ethanol and 0.1% formic acid was added to the spin column, and the solution was removed by centrifugation. This procedure was repeated three times. 7. (Elution) 50 μL of 50 mM ammonium bicarbonate water was added to the spin column, and the solution containing the target substance was collected by centrifugation. 8. (Concentration to dryness) The recovered solution was dried using a centrifugal dryer. 9. (Labeling of O-glycans) O-glycans were labeled using 2AB labeling solution (EZGlyco O-Glycan Prep Kit, Sumitomo Bakelite Co., Ltd.). 10. (LC-MS analysis) O-glycans were measured using LC-IT-TOF MS (Shimadzu Corporation). To identify the glycans, the sugar composition was estimated from the observed m / z. [Pore size of mucin capture beads] (1)70Å, (2)300Å, (3)1000Å

[0080] The results are shown in Figure 2. For all pore sizes, mucin-derived glycopeptides were preferentially recovered, while fetuin-derived glycopeptides were removed. The best recovery was observed with beads having a pore size of (2) 300 Å.

[0081] Example 4 Recovery of mucin-derived glycopeptides 1. (Dissolution of the sample) A mixture of bovine fetuin, bovine submaxillary mucin (BSM), and porcine stomach mucin (PSM) was dissolved in a solvent. This mixture is a mixture of glycoprotein and mucin samples. 2. (Denaturation treatment) The protein was denatured by reductive alkylation in the presence of guanidine hydrochloride. 3. (Trypsin treatment) Trypsin was added to decompose bovine fetuin, bovine submandibular gland mucin, and porcine stomach mucin into glycopeptides. 4. (Sample dilution) The sample was diluted with acetonitrile containing 0.1% formic acid so that the glycopeptide concentration in the sample was 10%. 5. (Application to beads) 2 mg of mucin capture beads (pore size: 300 Å) were placed in a spin tube with a filter column (Mobicol, MoBiTec). A 10 mM formic acid solution, followed by a column wash (80% ethanol solution), was passed through the column by centrifugation (1000 × g, 1 minute) to activate the beads. The sample prepared in step 4 above was then added to the column in several batches, each time centrifuged (300 × g, 1 minute) to adsorb glycopeptides or glycans (target substances) derived from bovine fetuin, bovine submandibular gland mucin, and porcine stomach mucin onto the beads. The filtrate was discarded each time. 6. (Washing) 300 μL of any of the following washing solutions was added to the spin column, and the solution was removed by centrifugation. This procedure was repeated three times. 7. (Elution) 50 μL of 50 mM ammonium bicarbonate water was added to the spin column, and the solution containing the target substance was collected by centrifugation. 8. (Concentration to dryness) The recovered solution was dried using a centrifugal dryer. 9. (Labeling of O-glycans) O-glycans were labeled using 2AB labeling solution (EZGlyco O-Glycan Prep Kit, Sumitomo Bakelite Co., Ltd.). 10. (LC-MS analysis) O-glycans were measured using LC-IT-TOF MS (Shimadzu Corporation). To identify the glycans, the sugar composition was estimated from the observed m / z. [Cleaning solution] (1) Aqueous solution containing 75% acetonitrile and 0.1% formic acid (2) Aqueous solution containing 50% acetonitrile and 0.1% formic acid (3) Aqueous solution containing 70% ethanol and 0.1% formic acid (4) Aqueous solution containing 80% ethanol and 0.1% formic acid

[0082] The results are shown in Figure 3. When any of the washing solutions was used, preferential recovery of mucin-derived glycopeptides was observed. In particular, (1) an aqueous solution containing 75% acetonitrile and 0.1% formic acid was suitable for recovering all glycopeptides, and (4) an aqueous solution containing 80% ethanol and 0.1% formic acid was suitable for recovering mucin domains. It was confirmed that fetuin-derived glycopeptides could be preferentially removed when ethanol was used as a washing solution. [Industrial Applicability]

[0083] The present invention relates to a capture agent for mucin, mucin fragment, or mucin-derived glycopeptide or sugar chain, and a recovery method thereof. Accordingly, the present invention can be used in fields requiring simple and specific capture or recovery of mucin, mucin fragment, or mucin-derived glycopeptide or sugar chain, such as technical fields such as proteomics using mucin domains.

Claims

1. A capture agent for mucin, a mucin fragment, or a glycopeptide or sugar chain derived from mucin, comprising: The scavenger comprises a compound having a betaine structure, The betaine structure has an anionic moiety, a cationic moiety, and a linker, the anionic moiety is a group selected from the group consisting of a phosphate group, a carboxyl group, and a sulfonic acid group; The cationic moiety is a quaternary ammonium group, A scavenger, wherein the linker connecting the anionic moiety and the cationic moiety is an alkylene group having 1 to 4 carbon atoms.

2. A carrier for capturing mucin, mucin fragment, or mucin-derived glycopeptide or sugar chain, comprising the capturing agent according to claim 1 immobilized on an insoluble support.

3. The capturing carrier according to claim 2, wherein the capturing carrier is a particle.

4. 4. The capture carrier according to claim 3, wherein the particles have pores with an average pore size of 1 nm or more.

5. A method for recovering mucin, a mucin fragment, or a mucin-derived glycopeptide or sugar chain, the method comprising: a) contacting a sample containing mucin, mucin fragment, glycopeptide, or sugar chain with the capture agent according to claim 1 or the capture carrier according to any one of claims 2 to 4 to capture the mucin, mucin fragment, glycopeptide, or sugar chain; and b) washing the capture agent or capture carrier that has captured mucin, mucin fragment, glycopeptide, or sugar chain with solvent 1 to remove substances other than mucin, mucin fragment, and mucin-derived glycopeptides and sugar chains; c) a step of eluting the mucin, mucin fragment, glycopeptide, or sugar chain with solvent 2 from the capture agent or capture carrier that has captured the mucin, mucin fragment, glycopeptide, or sugar chain A method comprising:

6. 6. The method according to claim 5, wherein the solvent 1 in step b) is a mixed solution of an organic solvent and water or a mixed solution of an organic solvent, water and an organic acid, and contains 10% or more by volume of water.

7. 7. The method according to claim 6, wherein the organic solvent in step b) comprises one or more selected from an alcohol and acetonitrile.

8. The method according to claim 5, wherein the solvent 2 in step c) contains at least 40% by volume of water.

9. The method according to claim 5, wherein the solvent 2 in step c) is a weakly alkaline solvent.

10. The method according to claim 5, wherein the sample containing mucin fragments or mucin-derived glycopeptides is a mucin degradation product.

Images