JP6499580B2 - Method for releasing and labeling modifying group, method for analyzing modifying group, and method for labeling free sugar chain - Google Patents

Description

  The present invention relates to a method and kit for releasing and labeling a modifying group, a method and kit for analyzing a modifying group, and a method for labeling a free sugar chain.

  In vivo, serine and threonine are phosphorylated, sulfated, sugar chain modified (O-GalNAc type, O-GlcNAc type, O-Fuc type, O-Man type, O-Xyl type, O-Gal type, O -Glc type) and other post-translational modifications, and these post-translational modifications play an important role in the regulation of higher-order functions of various life such as signal transduction and differentiation control.

  There is a movement to use such post-translational modifications in the medical field. For example, drugs that inhibit kinases responsible for phosphorylation of specific proteins have already been marketed as pharmaceuticals, and various diseases can be diagnosed by examining the qualitative and quantitative fluctuations of O-linked sugar chains. I know it is possible.

  Among the post-translational modifications of serine and threonine, O-phosphorylation, O-sulfation, and O-GlcNAcation are those in which a phosphate group, a sulfate group, and an O-GlcNAc group are combined independently, On the other hand, generally O-linked sugar chain modifications such as O-GalNAcation, O-Fucation, O-Manylation, and O-Xylation have sugar chains further extended to have various structures.

  As one method for analyzing phosphorylated binding sites of phosphorylated serine and threonine, a β elimination / Michael addition method (BEMA method) is known. This method applies that the phosphate group is β-eliminated under basic conditions to produce an unsaturated carbonyl, which becomes a Michael reaction acceptor (Patent Documents 1 and 2, Non-Patent Document 1). ). Several applications of the BEMA method have been reported. For example, a compound having a thiol group, such as dithiothreitol, acts as a Michael donor to label the phosphorylation site, purify peptides, turn on beads, Used for fluorescent labeling on a chip (Patent Document 3).

  Although the BEMA method contributes to research related to protein phosphorylation and has become a widely used method, other post-translational modifications including serine and threonine, including O-GlcNAcation, are also released by the BEMA method. Therefore, in this method, it is impossible to distinguish what kind of post-translational modification was present in serine and threonine. Further, as the Michael donor in the BEMA method, a compound having a thiol group is generally used, and there have been almost no reports of application examples of other Michael donors.

  Analyzes various structures of the sugar chain of serine and threonine (O-GalNAc type, O-GlcNAc type, O-Fuc type, O-Man type, O-Xyl type, O-Gal type, O-Glc type) In this case, since an effective enzyme that efficiently cleaves sugar chains having various structures from proteins has not been discovered, a chemical method based on a β-elimination reaction under a strong alkali (pH of about 11.5 to 12.5) Widely used. In this case, since sugar chains released from glycoproteins and glycopeptides can also undergo degradation (peeling reaction) by sequentially receiving β elimination from the reducing end side of the sugar chain, adding a high concentration of reducing agent, A method has been developed to reduce the reducing end to sugar alcohol simultaneously with the elimination reaction. This method was reported by Carlson in 1968 and is still a standard method for releasing O-GalNAc type sugar chains (Non-patent Document 2). However, in this method, (1) β-elimination in the presence of a reducing agent causes degradation of the peptide portion, making it difficult to analyze the protein portion. (2) The reducing end of the sugar chain is reduced by reduction. Since it is lost, it has two disadvantages that it is difficult to separate sugar chains and to make derivatization for high sensitivity analysis.

  In order to overcome these problems, a method of coexisting a labeling agent such as a pyrazolone reagent (BEP method) when releasing post-translational modification of serine and threonine by β-elimination reaction, the present inventors, Reported by other research groups (Patent Documents 4 and 5, Non-Patent Documents 3-5). According to this BEP method, the released sugar chain can quickly react with the labeling agent (pyrazolone derivative), and the decomposition of the released sugar chain or peptide portion can be almost completely avoided.

  On the other hand, Reinhold et al. Can shorten the reaction time of β elimination reaction by a modified method of Carlson method using organic amine (microwave irradiation, pH condition: pH about 12.5 (additional test), reaction temperature: 70 ° C.). (Non-Patent Document 6).

Special table 2007-515625 gazette US Pat. No. 7,803,751 JP 2009-19002 A International Publication No. 2012/111775 International Publication No. 2011/038874

“Enrichment analysis of phosphoproteins as a tool for probing the phosphoprotein”, Nature Biotechnology (2001), 19 (4), 379-382. Don M. Carlson and Charles Blackwell, “Structures and Immunochemical Properties of Oligosaccharides Isolated from Pig Pigmentary Mucins”, J. Am. Biol. Chem. 1968, 243: 616-626. Jun-ichi Furukawa et al., “A Versatile Method for Analysis of Serine / Threone Posttranslational Modulation by β-Elimination in the Presence of the Presence. Chem. 2011, 83: 9060-9067. Zauner G et al, “Mass spectrometric O-glycan analysis after combined O-glycan release by beta-elimination and 1-phenyl-3-methyl-5-pyrazoleone. 2012, 1820, 1420-1428. Wang C et al, “One-pot nonreductive O-glycan release and labeling with 1-phenyl-3-methyl-5-pyrazolone followed by ESI-MS analysis.”, Prote. 2011, 11, 4229-4242. Stephanie Maniatis et al, “Rapid De-O-Glycosylation Concomitant with Peptide Labeling Using Microwave Radiation and an Alkyl Amine Base”. Chem. 2010, 82: 2421-2425.

  However, it requires a long reaction time (10 to 24 hours) to release post-translational modification groups based on β-elimination reactions, including the Carlson method and BEP method, limiting large-scale analysis of post-translational modification groups. It was one factor to do. In addition, no microwave irradiation in the BEP method has been reported so far.

  The present invention has been made in view of the above circumstances, and an object of the present invention is to provide a method and a kit capable of releasing and labeling a modifying group efficiently and in a short time. It is another object of the present invention to provide a method and kit for analyzing a modifying group using the above method. Another object of the present invention is to provide a method capable of labeling a free sugar chain efficiently and in a short time.

In order to achieve the above object, a method for releasing and labeling a modifying group from at least one of a serine residue and a threonine residue of a modified glycoprotein in a sample according to the first aspect of the present invention is as follows. ,
Irradiating microwaves to a solution having a pH of 6.0 to 10.0 containing the modified glycoprotein and a labeling agent that is at least one of a pyrazolone derivative, an isoxazolone derivative, a hydantoin derivative, a rhodanine derivative, and a maleimide derivative. Process.

  The pH of the solution may be pH 7.5 to pH 9.0.

  At least one of the serine residue and the threonine residue from which the modifying group is released may be labeled.

  The labeling agent may be a pyrazolone derivative.

  A thiol compound may be used together with the labeling agent.

  The modifying group may be a post-translational modifying group.

  The post-translational modification group may be an O-linked sugar chain.

The method for analyzing a modifying group according to the second aspect of the present invention is free from at least one of a serine residue and a threonine residue of a glycoprotein modified in a sample by the method according to the first aspect of the present invention. Analyzing the labeled modifying group,
It is characterized by that.

  The kit for the method according to the first aspect of the present invention according to the third aspect of the present invention is a labeling agent that is at least one of a pyrazolone derivative, an isoxazolone derivative, a hydantoin derivative, a rhodanine derivative, and a maleimide derivative A solution comprising

  A kit for analyzing a modifying group according to the fourth aspect of the present invention comprises the kit according to the third aspect of the present invention.

  The free sugar chain labeling method according to the fifth aspect of the present invention includes a free sugar chain and a labeling agent that is at least one of a pyrazolone derivative, an isoxazolone derivative, a hydantoin derivative, a rhodanine derivative, and a maleimide derivative. a step of irradiating a solution having a pH of 6.0 to 10.0 with microwaves.

  ADVANTAGE OF THE INVENTION According to this invention, the method and kit which can release | eliminate and label a modification group efficiently and in a short time can be provided. Moreover, according to this invention, the analysis method and kit of a modifying group using the said method can be provided. Moreover, according to this invention, the method which can label a free sugar chain efficiently and in a short time can be provided.

It is the figure which examined about the influence which the pH of the reaction solution has with respect to reaction by the pyrazolone derivative of a free sugar chain. (A) is a reaction solution (pH 8.0) with 0.3 M sodium hydroxide added, (b) is a reaction solution (pH 8.3) with 0.4 M sodium hydroxide added, (c) is 0.5 M water. The reaction solution (pH 11.4) to which sodium oxide was added and (d) are the results for the reaction solution (pH 11.8) to which 0.6 M sodium hydroxide was added. It is the figure which examined the influence which the presence or absence of the heating by microwave irradiation has with respect to reaction by the pyrazolone derivative of a free sugar chain. (A) is the comparative example heated with the heat block, (b) is the spectrum of the Example heated with the microwave generator. It is the figure of the graph which compared the collection amount of the labeled sugar_chain | carbohydrate with the Example heated with the microwave generator, and the comparative example heated with the heat block. It is the figure which examined about the influence which the presence or absence of the heating by microwave irradiation has in the free labeling reaction by the pyrazolone derivative of the glycoprotein which has O-linked sugar chain. (A) is the comparative example heated with the heat block, (b) is the spectrum of the Example heated with the microwave generator. It is the figure of the graph which compared the collection amount of the free-labeled sugar_chain | carbohydrate with the Example heated with the microwave generator, and the comparative example heated with the heat block. It is the figure which examined about the influence which the kind of reaction solvent gives in the free labeling reaction by the pyrazolone derivative of the glycoprotein which has O-linked sugar chain. (A) is a case where dimethyl sulfoxide (DMSO) is used, and (b) is a case where methanol is used. It is the figure which examined whether peptide decomposition | disassembly was seen in the free labeling reaction by the pyrazolone derivative of the glycopeptide which has an O-GalNAc type sugar chain. It is the figure which examined the influence which the difference in the heating temperature by microwave irradiation has on the reaction efficiency in the free labeling reaction by the pyrazolone derivative of the glycoprotein which has O-linked sugar chain. (A) is heating temperature 85 ° C, (b) is heating temperature 100 ° C, (c) is heating temperature 110 ° C, (d) is heating temperature 120 ° C, (e) is heating temperature 130 ° C, (f) is heating The temperature is 140 ° C. It is the figure of the graph which compared the collection amount of the free-labeled sugar_chain | carbohydrate in reaction of each temperature. FIG. 3 is a graph showing the evaluation of changes over time in the total amount of free-labeled sugar chains in the free labeling reaction of porcine stomach mucin, a glycoprotein, with a pyrazolone derivative. It is the figure of the spectrum which examined whether O-linked sugar chain could be detected by free labeling reaction using a pyrazolone derivative using a cell pellet.

  Hereinafter, embodiments of the present invention will be described in detail.

  A method for releasing and labeling a modifying group from at least one of a serine residue and a threonine residue of a modified glycoprotein in a sample according to the present invention (hereinafter referred to as “free labeling method” in the present specification). This reaction is referred to as “free labeling reaction”) and includes a step of irradiating a solution having a pH of 6.0 to 10.0 containing a modified glycoprotein and a labeling agent with microwaves.

  In the method for free labeling according to the present invention, a modifying group bonded through the oxygen atom of a serine residue and a threonine residue in a (modified) glycoprotein is released from the protein (hereinafter, this reaction is referred to as “free reaction”). Labeling with a labeling agent (hereinafter, this reaction is referred to as “labeling reaction”).

  The above-mentioned “sample” is not particularly limited as long as it contains a glycoprotein described later, and may be a sample derived from chemical synthesis or a sample derived from a living body. In the case of a sample derived from a living body, for example, a sample derived from an animal such as a human, rat, mouse, guinea pig, marmoset, rabbit, dog, cat, sheep, pig, chimpanzee, or these immunodeficient animals can be mentioned. The sample may be derived from a plant.

  The above-mentioned “modified glycoprotein” may be a glycoprotein that has undergone post-translational modification in vivo or may be a glycoprotein derived from synthesis, but preferably a sugar that has undergone post-translational modification. It is a protein. More specifically, a phosphate group, a sulfate group, a glycan, or the like, or a glycoprotein to which one or more of these groups are bonded as a modifying group via the oxygen atom of a serine residue and a threonine residue is “modified”. Called "glycoprotein". In the case of a glycoprotein that has undergone post-translational modification, in this specification, a phosphate group bonded through an oxygen atom as a post-translational modification group described later is referred to as an “O-phosphate group”. A sulfate group bonded through an oxygen atom is referred to as “O-sulfate group”, and a glycan bonded through an oxygen atom as a post-translational modification group is referred to as “O-linked sugar chain”. Moreover, in this specification, glycoprotein is understood as including a glycopeptide. In other words, in the present specification, a glycoprotein is understood to be at least one of a glycoprotein and a glycopeptide.

  In the above-mentioned “O-linked sugar chain”, the sugar that binds to the oxygen atom of the serine residue and threonine residue starts from any of N-acetylgactosamine, mannose, N-acetylglucosamine, xylose, and glucose. It may start from any one of N-acetylgactosamine, mannose, fucose, xylose, and glucose, and may be elongated by binding of sugar.

  The above-mentioned “release and label the modifying group” means that at least one of a serine residue and a threonine residue of a glycoprotein, a modifying group (a phosphate group, a sulfate group, a sugar chain, etc., or one of these) Alternatively, it means that two or more groups) are released by β elimination reaction, and the double bond formed when liberated by β elimination reaction is labeled with a labeling agent described later by Michael addition reaction. When a glycoprotein that has undergone post-translational modification is used, the modifying group may be a post-translational modifying group (O-phosphate group, O-sulfate group, O-linked sugar chain, etc., or one or more of these groups). Group). The modifying group is labeled with a labeling agent simultaneously with or subsequent to release from the glycoprotein.

  In the free labeling method of the present invention, at least one of the serine residue and threonine residue of the glycoprotein from which the modifying group is released may be labeled. The labeling is performed by a labeling agent in the same manner as the labeling to the modifying group.

  The following shows an example in which both a reaction in which an O-linked glycan is released from a glycoprotein and labeled as a post-translational modification group and a reaction in which the glycoprotein side is modified are generated (as a labeling agent, A pyrazolone derivative described later (3-methyl-1-phenyl-5-pyrazolone (PMP) is used).

  The aforementioned “labeling agent” is at least one of pyrazolone derivatives, isoxazolone derivatives, hydantoin derivatives, rhodanine derivatives, and maleimide derivatives. The labeling agent may be one of these, or two or more. The pyrazolone derivative used for the labeling agent can be appropriately selected as long as it is a compound having a pyrazolone group. For example, 3-methyl-1-phenyl-5-pyrazolone (PMP), 1,3-dimethyl-pyrazolone (DP) 3-methyl-1-p-tolyl-5-pyrazolone (MTP), 3-methyl-1- (quinolin-8-yl) -1H-pyrazol-5 (4H) -one, and the like. Examples of the isoxazolone derivative used for the labeling agent include 3 (2H) -isoxazolone, 5-methyl-3 (2H) -isoxazolone, 2,5-dimethyl-3 (2H) -isoxazolone, and the like. Examples of the hydantoin derivative used for the labeling agent include 2,4-imidazolidinedione, 3-methyl-2,4-imidazolidinedione, 3- (2-propyn-1-yl) -2,4- And imidazolidinedione. Examples of the rhodanine derivative used for the labeling agent include 2-thioxo-4-thiazolidinone and 3-methyl-2-thioxo-4-thiazolidinone. In the free labeling method of the present invention, a pyrazolone derivative can be suitably used.

  In the free labeling method of the present invention, a thiol compound may be used together with the labeling agent. As this thiol compound, any compound having a thiol group can be used without particular limitation. For example, dithiothreitol (DTT), 2-aminoethanethiol, thiocholine, 2- (pyridine-4- Yl) ethanethiol and the like. For example, when an O-linked sugar chain is released from a glycoprotein and labeled as a post-translational modification group, and a thiol compound is used together with the labeling agent, for example, the O-linked sugar chain side is labeled with the labeling agent, For example, the protein side can be labeled with a thiol compound, which may be advantageous for analysis of the O-linked sugar chain side.

  The solution containing the glycoprotein and the labeling agent used in the free labeling method according to the present invention may be in a state where the glycoprotein and the labeling agent are dissolved in the solvent, or the glycoprotein and the labeling agent are dispersed in the solvent. It may be in a state of being allowed. The free labeling method according to the present invention is preferably performed in a state in which the glycoprotein and the labeling agent are dissolved in a solvent from the viewpoint of proceeding the reaction efficiently. As the solvent, for example, water, methanol, tetrahydrofuran (THF), acetonitrile, dimethylformamide (DMF), dioxane, dimethyl sulfoxide (DMSO), propanol or the like can be used. Any solvent that exhibits the effects of the present invention can be selected as appropriate.

  The pH of the aforementioned solution is in the range of pH 6.0 to pH 10.0. This pH range is milder (lower pH) than pH conditions (pH 11.5 to 12.5) of β elimination reaction by the conventional method (including Carlson's method), and pH 6.0 to pH 10.0. In this range, the modification group can be efficiently released and labeled while reducing degradation of the glycoprotein (peptide degradation). The pH of the solution is more preferably pH 7.5 to pH 9.0, and even more preferably pH 8.0 to pH 9.0. The pH of the solution can be adjusted, for example, by adding a sodium hydroxide solution to the solution.

  The free labeling method according to the present invention includes a step of irradiating the aforementioned solution with microwaves.

  The above-mentioned “irradiating with microwaves” means irradiating the solution with microwaves having a wavelength of 1 mm to 1 m and a frequency of 300 MHz to 300 GHz. The microwave irradiation can be performed using a known microwave generator. For example, a microwave generator capable of obtaining a microwave output with a frequency of about 1000 to 5000 MHz by magnetron oscillation can be used. As the microwave generator, for example, Monowave 300 (Anton Paar Co., Ltd.) capable of obtaining a microwave output of a maximum of 850 W at a frequency of 2455 MHz and a maximum pressure of 30 bar by magnetron oscillation can be used.

  The temperature of the solution in the aforementioned “microwave irradiation step” is preferably 4 ° C. to 135 ° C. In the range of 4 ° C. to 135 ° C., the modifying group can be released and labeled efficiently, and glycoprotein degradation (peptide degradation) can be reduced. In addition, the temperature of a solution becomes like this. More preferably, it is 35 to 135 degreeC, More preferably, it is 85 to 130 degreeC.

  The amount of the modifying group released from the glycoprotein by the free labeling method according to the present invention is determined according to the modified group that has been released and labeled with the labeling agent (hereinafter referred to as “labeled modification”). It can be confirmed by analyzing the “group”) by an analysis method described later.

  According to the free labeling method of the present invention, the reaction rate of the β-elimination reaction can be improved and the reaction rate of the modification of the modifying group by the modifying agent can be improved. Can be released and labeled. Accordingly, the amount of labeled modifying group recovered can be improved.

  Moreover, in the free labeling method of the present invention, the reaction is carried out under weakly alkaline conditions rather than strong alkaline conditions such as the pH conditions (pH 11.5 to pH 12.5) in the conventional β elimination reaction. Degradation (peptide degradation) can be reduced.

  Next, a kit for releasing and labeling a modifying group according to the present invention will be described.

  The above-described kit of the present invention comprises a solution containing the above-described labeling agent and is used for performing a free labeling method. The labeling agent, pH conditions (the pH of the solution is adjusted to pH 6.0 to pH 10.0), microwave irradiation, temperature conditions, and the like are all the same as described above. The kit includes a container containing a solution containing the labeling agent described above. A sample containing glycoprotein (for example, a sample obtained by purifying a cell disruption solution) is placed in the container and mixed with the solution, and the pH of the mixed solution is adjusted to pH 6.0 to pH 10.0 by adding, for example, sodium hydroxide solution. The container may be directly irradiated with microwaves, or the solution in the container may be transferred from the container to another container to produce a mixture with glycoprotein (adjusted to pH 6.0 to pH 10.0). ), Or microwave irradiation. Therefore, as the material of the container, in the former case, a material that does not adversely affect the solution in the container even when irradiated with microwaves (for example, plastic such as polypropylene, glass) is used. In the latter case, the material is stable. In particular, any material that can store a solution can be used without particular limitation. According to the kit of the present invention described above, the modifying group can be released and labeled efficiently and in a short time.

  Next, the method for analyzing a modifying group according to the present invention will be described in detail.

  According to the method for analyzing a modifying group according to the present invention, a labeled modifying group (labeled modification) released from at least one of serine residues and threonine residues of a glycoprotein modified in a sample by the aforementioned free labeling method. Group) is analyzed.

  The analysis of the labeling modifying group can be performed using liquid chromatography or high performance liquid chromatography. In addition, it can be appropriately selected according to the type of target molecule. For example, mass spectrometry (MS) (electrospray ionization (ESI), etc.), nuclear magnetic resonance (NMR), ultraviolet absorptiometer (UV), evaporator A reactive light scattering detector (ELS), an electrochemical detector, or the like can be used. You may analyze by any method and may analyze combining 2 or more. Any analysis method that exhibits the effects of the present invention can be selected as appropriate. In addition, when analyzing using the glycoprotein in a biological sample, after extracting and refine | purifying glycoprotein by a well-known method, it can use for an analysis.

  In the above-described free labeling method, a labeling modification group can be obtained efficiently and in a short time. Therefore, the analysis method of the present invention by analyzing the labeling modification group obtained by the free labeling method is more rapid. In addition, quantitative and qualitative analysis of the modifying group in the sample can be performed, and more samples can be analyzed efficiently.

  Further, in the above-described free labeling method, the recovery amount of the labeling modification group can be improved. Therefore, in the analysis method of the present invention by analyzing the labeling modification group obtained by the free labeling method, the amount of the labeling modification group is higher. Sensitivity can be analyzed for modifying groups. For example, the modifying group can be subjected to structural analysis and quantitative analysis with high sensitivity.

  When analyzing an O-linked sugar chain in a sample, according to the analysis method of the present invention, an O-linked sugar chain contained in a cell or tissue can be analyzed with high sensitivity. Comprehensive analysis of O-linked glycans in cell lysates, which has been difficult, becomes possible. In addition, for example, it is possible to analyze living-derived sugar chains that exist only in trace amounts, such as O-glycome in cells.

  In addition, it has recently been found that O-linked sugar chains can be used as markers for stem cells including iPS cells. Therefore, according to the analysis method of the present invention, for example, in the field of regenerative medicine, in iPS cell-derived tissue used for transplantation, whether or not iPS cells have been differentiated or whether there are any remaining iPS cells remains. The check can be performed efficiently and in a short time. As described above, according to the analysis method of the present invention, more and more samples can be analyzed for regenerative medicine, which can greatly contribute to the determination of a regenerative medicine treatment strategy.

  Next, a kit for analyzing a modifying group according to the present invention will be described.

  The kit for analyzing a modifying group according to the present invention includes a kit for the aforementioned free labeling method. With this kit, the modifying group can be analyzed efficiently and in a short time, in the same manner as the above-described modifying group analysis method.

  Next, the method for labeling free sugar chains according to the present invention will be described in detail.

  The method for labeling a free sugar chain according to the present invention includes a step of irradiating a solution of pH 6.0 to pH 10.0 containing a free sugar chain and a labeling agent with microwaves.

  The above-mentioned “free sugar chain” is one starting from any of N-acetylgactosamine, mannose, N-acetylglucosamine, xylose, glucose, or any of N-acetylgactosamine, mannose, fucose, xylose, glucose. It may start and be further extended by binding of sugar. The details of the labeling agent, pH condition, temperature condition, and microwave irradiation are the same as described above.

  Hereinafter, an example of a labeling reaction of a free sugar chain when a pyrazolone derivative (3-methyl-1-phenyl-5-pyrazolone (PMP)) is used as a labeling agent is shown.

  According to the method for labeling a free sugar chain of the present invention, the reaction rate of the modification of the modifying group by the modifying agent can be improved, so that the free sugar chain can be labeled efficiently and in a short time.

  Hereinafter, the present invention will be specifically described with reference to examples. However, the present invention is not limited to these examples.

Example 1
The influence of the pH of the reaction solution on the reaction with the free sugar chain labeling agent (pyrazolone derivative) was examined.

  To maltotetraose (Tokyo Chemical Industry, 1 mmol / mL, 10 μL), 20 μL of 0.3 M, 0.4 M, 0.5 M and 0.6 M sodium hydroxide and 20 μL of 0.5 M 3-methyl-1- A methanol solution of phenyl-5-pyrazolone (PMP) was added (reaction solvent: water), maintained at a heating temperature of 85 ° C. with a heat block, reacted for 60 minutes, and maltotetraose was labeled with PMP. Regarding the pH of the reaction solution, the reaction solution added with 0.3M sodium hydroxide was pH 8.0, the reaction solution added with 0.4M sodium hydroxide was pH 8.3, the reaction solution added with 0.5M sodium hydroxide. The pH of the reaction solution added with 0.6M sodium hydroxide was pH 11.8 (substantially the same as the pH conditions for the β elimination reaction by the conventional method (including Carlson's method)).

  After completion of the reaction, 10 nmol of bisPMP-labeled chitotetraose prepared by chitotetraose (Seikagaku Corporation) was added, then mixed with 2,5-dihydrobenzoic acid (10 mg / mL), and MALDI-TOF (/ TOF) ( (UltraFlex II, Bruker).

  The reaction formula for labeling maltotetraose with PMP is shown below.

  FIG. 1 shows a MALDI-TOF spectrum of labeled maltotetraose labeled with PMP. FIG. 1 (a) shows a reaction solution (pH 8.0) with 0.3M sodium hydroxide added, FIG. 1 (b) shows a reaction solution (pH 8.3) with 0.4M sodium hydroxide added, FIG. 1 (c). ) Shows the results for the reaction solution (pH 11.4) to which 0.5M sodium hydroxide was added, and FIG. 1 (d) shows the results for the reaction solution (pH 11.8) to which 0.6M sodium hydroxide was added. When 0.6M sodium hydroxide is added (pH 11.8 (FIG. 1 (d))), the pH conditions are the same as those of conventional β-elimination reaction methods such as the Carlson method. Tetraose signal did not appear. On the other hand, when 0.3M, 0.4M and 0.5M sodium hydroxide were added (pH 8.0 (FIG. 1 (a)), pH 8.3 (FIG. 1 (b)), and pH 11.4 (FIG. 1 (c))), a labeled maltotetraose signal appears, and when 0.4M sodium hydroxide is added (pH 8.3 (FIG. 1 (b))), the highest labeled maltotetraose A signal appeared.

  From the above, in the reaction with maltotetraose pyrazolone derivatives, unlike the conventional β elimination reaction method such as the Carlson method, when the pH condition of the reaction solution is pH 8.3, The reaction efficiency was shown to be high.

(Example 2)
The effect of heating by microwave irradiation on the labeling reaction of free sugar chains with pyrazolone derivatives was investigated.

  To maltohexaose (Tokyo Chemical Industry, 1 mmol / mL, 100 μL) was added 200 μL of 0.4 M NaOH and 200 μL of a 0.5 M 3-methyl-1-phenyl-5-pyrazolone (PMP) methanol solution (reaction) Solvent: water), in the examples, maintained at a heating temperature of 85 ° C. by a microwave generator (Monowave 300, Anton Paar), and in the comparative example, maintained at a heating temperature of 85 ° C. by a heat block, 10 minutes, 20 minutes, 30 Let stand for 60 minutes. The pH of each reaction solution was 8.3.

  After completion of the reaction, 500 pmol of bisPMP-labeled chitotetraose (similar to Example 1) was added, and then mixed with 2,5-dihydrobenzoic acid (10 mg / mL), and MALDI-TOF (/ TOF) (Example 1). The same as above.

  FIG. 2 shows a MALDI-TOF spectrum of labeled maltohexaose labeled with PMP. FIG. 2A is a spectrum according to a comparative example heated by a heat block, and FIG. 2B is a spectrum according to an example heated by a microwave generator. In all cases, a labeled maltohexaose signal appeared.

  FIG. 3 shows the result of comparison of the recovered amount of labeled maltohexaose using an external standard. In the comparative example (heated to 85 ° C. with a heat block), the rise of the labeling of maltohexaose was slow, and a moderate increase in the recovered amount was observed even 60 minutes after the start of the reaction. On the other hand, in the example (heating to 85 ° C. by microwave irradiation), labeling of maltohexaose was almost completed 10 minutes after the start of the reaction, and the reaction efficiency 60 minutes after the start of the reaction was compared with the comparative example (microwave irradiation). 20% higher than (none).

  From the above, it was shown that the reaction efficiency of labeling with a pyrazolone derivative of maltohexaose was improved by heating by microwave irradiation, and the recovery amount of labeled maltohexaose was increased.

(Example 3)
The effect of heating by microwave irradiation on the free labeling reaction of a glycoprotein having an O-linked sugar chain with a pyrazolone derivative was examined.

  Porcine gastric mucin (Sigma Aldrich, 1 mg / mL, 100 μL), a glycoprotein having an O-linked sugar chain, 200 μL of 0.4 M sodium hydroxide, and 200 μL of 0.5 M 3-methyl-1-phenyl- A methanol solution of 5-pyrazolone (PMP) was added (reaction solvent: water). In the examples, the heating temperature was maintained at 85 ° C. by a microwave generator (same as in Example 2), and in the comparative examples, the heating temperature was 85 by a heat block. The temperature was maintained at ° C. and left for 2 hours. The pH of each reaction solution was 8.3.

  After completion of the reaction, 100 pmol of bisPMP-labeled chitotetraose (similar to Example 1) was added, neutralized with 1M hydrochloric acid, and the reaction solution was purified with an Iatrobead column (Iatron), and 2,5-dihydrode It mixed with benzoic acid (10 mg / mL) and analyzed by MALDI-TOF (/ TOF) (similar to Example 1).

  The reaction formula for free labeling of mucin with PMP is shown below.

  FIG. 4 shows a MALDI spectrum of the detected sugar chain. FIG. 4A shows a spectrum of a comparative example heated by a heat block, and FIG. 4B shows a spectrum of an example heated by a microwave generator. In either case, the peak of the protein degradation product was not detected, and a signal of a sugar chain labeled with PMP appeared.

  FIG. 5 shows a histogram of detected sugar chains. As a result of comparison of the recovered amount with an external standard, in the example (heated to 85 ° C. by microwave irradiation), the labeled O-linked sugar chain was compared with the comparative example (heated to 85 ° C. by heat block). The total amount of was increased about 4 times.

  As described above, the irradiation efficiency of microwave irradiation improves the reaction efficiency of the β-elimination reaction of O-linked sugar chains and the labeling reaction of free sugar chains with pyrazolone derivatives, and labeled O-linked sugar chains. Was recovered with high efficiency.

Example 4
The influence of the difference in the type of reaction solvent on the free labeling reaction of a glycoprotein having an O-linked sugar chain with a pyrazolone derivative was examined.

  Porcine gastric mucin (as in Example 3) (1 mg / mL, 100 μL), a glycoprotein having an O-linked sugar chain, using DMSO or methanol as a reaction solvent, 200 μL of 0.4 M NaOH, and 200 μL Of 0.5M 3-methyl-1-phenyl-5-pyrazolone (PMP) was added, and the mixture was allowed to stand at a heating temperature of 120 ° C. for 2 hours with a microwave generator (similar to Example 2). The pH of each reaction solution was 8.3.

  After completion of the reaction, 100 pmol of bisPMP-labeled chitotetraose (same as in Example 1) was added, neutralized with 1M hydrochloric acid, washed with chloroform, and then mixed with 2,5-dihydrobenzoic acid (10 mg / mL). And it analyzed by MALDI-TOF (/ TOF) (same as Example 1).

  FIG. 6 shows a MALDI spectrum of the detected sugar chain. The same degree of sugar chain recovery was confirmed when the reaction solvent was DMSO (FIG. 6 (a)) or methanol (FIG. 6 (b)).

  From the above, it was shown that DMSO and methanol can be used as the reaction solvent for the free labeling reaction with the pyrazolone derivative of mucin.

(Example 5)
Whether or not peptide degradation was observed in a free labeling reaction of a glycopeptide having an O-GalNAc type sugar chain with a pyrazolone derivative was examined.

  To a synthetic glycopeptide having an O-GalNAc type sugar chain (20 μg / 100 μL), 200 μL of 0.4 M NaOH and 200 μL of 0.5 M 3-methyl-1-phenyl-5-pyrazolone (PMP) in methanol are added ( Reaction solvent: water) and a microwave generator (similar to Example 2) were maintained at a heating temperature of 120 ° C. and allowed to stand for 2 hours. The pH of the reaction solution was 8.3.

  After completion of the reaction, the reaction mixture was neutralized with 1M hydrochloric acid, the reaction solution was washed with chloroform, mixed with 2,5-dihydrobenzoic acid (10 mg / mL), and MALDI-TOF (/ TOF) (same as Example 1). Analysis was performed.

  FIG. 7 shows a MALDI spectrum after free labeling reaction of a glycopeptide having an O-GalNAc type sugar chain. Signals of sugar chain and peptide labeled with PMP appeared, and no peptide-degraded peak was detected.

As described above, in the free labeling reaction of the glycopeptide having an O-GalNAc type sugar chain with the pyrazolone derivative, almost no peptide degradation was detected, indicating that the reaction was completed in 2 hours. This is because the free labeling reaction is carried out under pH conditions (> 10 ³ at a hydrogen ion concentration) different from those of conventional β-elimination reactions such as the Carlson method, and the peptide degradation is suppressed. It is considered that the decrease in reactivity was compensated by microwave irradiation.

(Example 6)
In the free labeling reaction of a glycoprotein having an O-linked sugar chain with a pyrazolone derivative, the influence of the difference in heating temperature by microwave irradiation on the reaction efficiency was examined.

  Porcine gastric mucin (as in Example 3) (1 mg / mL, 100 μL), a glycoprotein having an O-linked sugar chain, 200 μL of 0.45 M NaOH, and 200 μL of 0.5 M 3-methyl-1- A methanol solution of phenyl-5-pyrazolone (PMP) was added (reaction solvent: water), and the heating temperature was 85 ° C., 100 ° C., 110 ° C., 120 ° C., 130 ° C. and 140 ° C. using a microwave generator (same as in Example 2). It left still at 2 degreeC for 2 hours. The pH of the reaction solution was 8.3.

  After completion of the reaction, 100 pmol of bisPMP-labeled chitotetraose (similar to Example 1) was added, neutralized with 1M hydrochloric acid, and the reaction solution was purified with an iatrobead column (same as Example 3). The mixture was mixed with 5-dihydrobenzoic acid (10 mg / mL) and analyzed by MALDI-TOF (/ TOF) (same as in Example 1).

  FIG. 8 shows a MALDI spectrum of a glycoprotein sugar chain. 8A is a heating temperature of 85 ° C., FIG. 8B is a heating temperature of 100 ° C., FIG. 8C is a heating temperature of 110 ° C., FIG. 8D is a heating temperature of 120 ° C., and FIG. The heating temperature is 130 ° C., and FIG. 8 (f) is the heating temperature 140 ° C. At the heating temperature of 120 ° C., the recovery amount of sugar chains labeled with PMP was the largest (FIG. 8 (d)). On the other hand, at a heating temperature of 140 ° C., a peak that seems to be a protein degradation product was detected (FIG. 8F).

  FIG. 9 shows a histogram of the detected sugar chains (recovery amount was compared by an external standard). At the heating temperature of 120 ° C. by microwave irradiation, the recovery amount of the O-linked sugar chain labeled with the pyrazolone derivative was the largest.

  From the above, it was shown that in the free labeling reaction of mucin with a pyrazolone derivative, the reaction efficiency was highest at a heating temperature of 120 ° C. by microwave irradiation.

(Example 7)
In the free labeling reaction of porcine gastric mucin, a glycoprotein, with a pyrazolone derivative, the change over time in the total amount of free-labeled sugar chains was evaluated.

  Porcine gastric mucin (as in Example 3) (1 mg / mL, 100 μL), a glycoprotein having an O-linked sugar chain, 200 μL of 0.4 M NaOH, and 200 μL of 0.5 M 3-methyl-1- A methanol solution of phenyl-5-pyrazolone (PMP) was added (reaction solvent: water) and heated at 120 ° C. with a microwave generator (similar to Example 2) for 0.5 hour, 1 hour, 2 hours, And left for 4 hours. The pH of the reaction solution was 8.3.

  In the comparative example, 20 μL of 0.4 M NaOH and 20 μL of 0.5 M PMP methanol solution were added to porcine gastric mucin (10 mg / mL, 10 μL) as described above (reaction solvent: water) and heated by a thermal cycler. It left still at the temperature of 85 degreeC for 2 hours, 4 hours, 8 hours, and 16 hours. The pH of the reaction solution was 8.3.

  After completion of the reaction, 100 pmol of bisPMP-labeled chitotetraose (similar to Example 1) was added, neutralized with 1M hydrochloric acid, and the reaction solution was purified with an iatrobead column (same as Example 3). The mixture was mixed with 5-dihydrobenzoic acid (10 mg / mL) and analyzed by MALDI-TOF (/ TOF) (same as in Example 1).

  FIG. 10 shows changes over time in the total amount of sugar chains released from porcine gastric mucin and labeled with PMP (Example (heated to 120 ° C. by microwave irradiation) and comparative example (heated to 85 ° C. by a thermal cycler)) . In the comparative example (heated to 85 ° C. by a thermal cycler), the rise was slow, and the recovered amount was less than 1000 pmol even 16 hours after the start of the reaction. On the other hand, in the example (heated to 120 ° C. by microwave irradiation), the recovery amount became maximum 2 hours after the start of the reaction, and about 7 times the recovery amount was obtained as compared with the comparative example.

  From the above, it was shown that in the free labeling reaction of mucin with a pyrazolone derivative, free labeled sugar chains can be recovered efficiently and in a short time by heating by microwave irradiation.

(Example 8)
It was investigated whether or not O-linked sugar chains could be detected by free labeling reaction with pyrazolone derivatives using cell pellets.

Cell pellets (1 × 10 ⁶ cells) of human iPS cells (HiPS-RIKEN-1A, RIKEN) were sonicated in tris acetate buffer (100 μL) containing 2% sodium dodecyl sulfate (irradiated for 5 seconds). +10 second intervals). 2 μL of 0.5 M tris (2-carboxyethyl) phosphine (final concentration of about 100 mM) and 200 mM of iodoacetamide (final concentration of about 20 mM) were added to the solubilized cell lysate to reductively alkylate the disulfide bond of the protein. (Room temperature, 30 minutes). 472 μL (4 times volume) ice-cold ethanol was added to the reaction solution, ethanol precipitation (−30 ° C., 2 hours) was performed, and centrifugation (20000 × g, 10 minutes, 4 ° C.) gave a precipitate (protein fraction). And the supernatant. The precipitate was subjected to O-linked sugar chain analysis. Ultrapure water was added to the precipitate to dissolve it, and Amicon (Millipore) was used to obtain a glycoprotein concentrated fraction having a molecular weight of 3000 or more.

  To 100 μL of the collected glycoprotein-enriched fraction, 200 μL of 0.4 M sodium hydroxide and 200 μL of a 0.5 M 3-methyl-1-phenyl-5-pyrazolone (PMP) methanol solution were added (reaction solvent: water), The mixture was allowed to stand at a heating temperature of 120 ° C. for 2 hours using a microwave generator (similar to Example 2). The pH of the reaction solution was 8.3.

  After completion of the reaction, 100 pmol of bisPMP-labeled chitotetraose (same as in Example 1) was added, neutralized with 1M HCl, excess reagent was removed by washing with chloroform, graphite carbon and iatrobeads (Example 3), the sugar chain part was purified. The purified sample was mixed with 2,5-dihydrobenzoic acid (10 mg / mL) and analyzed by MALDI-TOF (/ TOF) (same as in Example 1).

  FIG. 11 shows a mass spectrum of the detected O-linked sugar chain. Various O-linked sugar chains were detected with high sensitivity from the cell pellet.

  From the above, it was shown that O-linked sugar chains can be analyzed with high sensitivity from cell pellets by utilizing a free labeling reaction with pyrazolone derivatives.

  It should be noted that the present invention can be variously modified and modified without departing from the broad spirit and scope of the present invention. Further, the above-described embodiment is for explaining the present invention, and does not limit the scope of the present invention. That is, the scope of the present invention is shown not by the embodiments but by the claims. Various modifications within the scope of the claims and within the scope of the equivalent invention are considered to be within the scope of the present invention.

  The present invention is based on Japanese Patent Application No. 2013-115782 filed on May 31, 2013. The specification, claims, and entire drawings of Japanese Patent Application No. 2013-115782 are incorporated herein by reference.

  According to the present invention, quantitative and qualitative analysis of a modifying group in a sample can be performed efficiently and in a short time, and highly sensitive structural analysis and quantitative analysis of the modifying group are also possible. Thus, the present invention enables comprehensive analysis of modifying groups including O-linked sugar chains, and is expected to contribute to biology and medicine.

Claims (9)

100-130 ° C. in a pH 6.0-pH 10.0 solution containing the modified glycoprotein and a labeling agent that is at least one of pyrazolone derivatives, isoxazolone derivatives, hydantoin derivatives, rhodanine derivatives, and maleimide derivatives . Including a step of irradiating microwaves at a temperature ,
A method for releasing and labeling a modifying group from at least one of a serine residue and a threonine residue of a modified glycoprotein in a sample. The pH of the solution is pH 7.5 to pH 9.0.
The method according to claim 1. At least one of a serine residue and a threonine residue from which the modifying group is released is labeled;
The method according to claim 1 or 2, characterized in that The labeling agent is a pyrazolone derivative.
The method according to any one of claims 1 to 3, characterized in that: Using a thiol compound with a labeling agent,
The method according to any one of claims 1 to 4, characterized in that: The modifying group is a post-translational modifying group,
6. A method according to any one of claims 1 to 5, characterized in that The post-translational modification group is an O-linked sugar chain.
The method according to claim 6. Analyzing a labeled modifying group released from at least one of serine and threonine residues of a modified glycoprotein in a sample by the method according to any one of claims 1 to 7.
And a method for analyzing the modifying group. A solution of pH 6.0 to pH 10.0 containing a free sugar chain and a labeling agent that is at least one of a pyrazolone derivative, an isoxazolone derivative, a hydantoin derivative, a rhodanine derivative, and a maleimide derivative at a temperature of 100 to 130 ° C. Including a step of irradiating microwaves,
A method for labeling free sugar chains.