WO2014192923A1 - Procédé de libération et de marquage de groupe de modification, nécessaire pour ledit procédé, procédé d'analyse de groupe de modification, nécessaire pour l'analyse et procédé de marquage de chaîne sucre libre - Google Patents

Procédé de libération et de marquage de groupe de modification, nécessaire pour ledit procédé, procédé d'analyse de groupe de modification, nécessaire pour l'analyse et procédé de marquage de chaîne sucre libre Download PDF

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WO2014192923A1
WO2014192923A1 PCT/JP2014/064436 JP2014064436W WO2014192923A1 WO 2014192923 A1 WO2014192923 A1 WO 2014192923A1 JP 2014064436 W JP2014064436 W JP 2014064436W WO 2014192923 A1 WO2014192923 A1 WO 2014192923A1
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labeling
reaction
derivative
sugar chain
kit
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PCT/JP2014/064436
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English (en)
Japanese (ja)
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康郎 篠原
潤一 古川
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国立大学法人北海道大学
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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/107—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides
    • C07K1/1072—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups
    • C07K1/1077—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length by chemical modification of precursor peptides by covalent attachment of residues or functional groups by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/13—Labelling of peptides

Definitions

  • the present invention relates to a method and kit for releasing and labeling a modifying group, a modifying group analysis method and kit, and a method for labeling a free sugar chain.
  • 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 various higher-order functions of life such as signal transduction and differentiation control.
  • 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
  • O-linked sugar chain modifications such as O-GalNAc, O-Fuc, O-Man, O-Xyl, etc. generally take various structures by further extending the sugar chain.
  • BEMA method As one method for analyzing phosphorylated binding sites of phosphorylated serine and threonine, the ⁇ elimination / Michael addition method (BEMA method) is known. This method applies that the phosphate group is ⁇ -eliminated under basic conditions to produce an unsaturated carbonyl, which becomes a Michael reaction acceptor (Patent Documents 1 and 2, Non-Patent Document 1). ).
  • 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).
  • a method of coexisting a labeling agent such as a pyrazolone reagent (BEP method) when releasing post-translational modification of serine and threonine by ⁇ -elimination reaction
  • BEP method a labeling agent
  • the present inventors Reported by other research groups (Patent Documents 4 and 5, Non-Patent Documents 3-5).
  • 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.
  • Zauna G et al “Mass spectrometric O-glycan analysis after combined O-glycan release by beta-elimination and 1-phenyl-3-methyla-bylamine. 2012, 1820, 1420-1428. Wang C et al, “One-pot nonreductive O-glycan release and labeling with 1-phenyl-3-methyl-5-pillarone flowed by ESI-MS analysis.” 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.
  • 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.
  • 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 the solution with a pH of 6.0 to 10.0 containing a 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
  • the kit for analyzing the modifying group according to the fourth aspect of the present invention includes the kit according to the third aspect of the present invention.
  • the free sugar chain labeling method 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. irradiating the solution of pH 6.0 to pH 10.0 with microwaves.
  • the present invention it is possible to provide a method and a kit capable of releasing and labeling a modifying group efficiently and in a short time. 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.
  • FIG. 3 is a diagram illustrating the influence of heating by microwave irradiation in a free labeling reaction of a glycoprotein having an O-linked sugar chain with a pyrazolone derivative.
  • (A) is the comparative example heated with the heat block
  • (b) is the spectrum of the Example heated with the microwave generator.
  • FIG. 3 is a diagram for examining the influence of different types of reaction solvents in a free labeling reaction of a glycoprotein having an O-linked sugar chain with a pyrazolone derivative.
  • A is a case where dimethyl sulfoxide (DMSO) is used, and
  • b) is a case where methanol is used.
  • FIG. 3 is a diagram showing whether peptide degradation is observed in a free labeling reaction of a glycopeptide having an O-GalNAc type sugar chain with a pyrazolone derivative.
  • DMSO dimethyl sulfoxide
  • FIG. 2 is a diagram illustrating the influence of the difference in heating temperature due to microwave irradiation on the reaction efficiency in a free labeling reaction of a glycoprotein having an O-linked sugar chain with a pyrazolone derivative.
  • 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
  • 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.
  • FIG. 6 is a spectrum diagram in which it was examined whether an O-linked sugar chain can be detected by free labeling reaction using a pyrazolone derivative using a cell pellet.
  • 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 called “free labeling reaction”) and includes a step of irradiating a solution of pH 6.0 to pH 10.0 containing a modified glycoprotein and a labeling agent with microwaves.
  • labeling reaction 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”).
  • 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.
  • 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.
  • 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".
  • 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.
  • the sugar that binds to the oxygen atom of the serine residue and the threonine residue starts from any of N-acetylgactosamine, mannose, N-acetylglucosamine, xylose, and glucose. Or one starting from any one of N-acetylgactosamine, mannose, fucose, xylose, and glucose, and further linked with sugar and elongated.
  • 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 ⁇ elimination reaction, and the double bond formed when liberated by ⁇ elimination reaction is labeled with a labeling agent described later by Michael addition reaction.
  • 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.
  • 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 aforementioned “labeling agent” is at least one of a pyrazolone derivative, an isoxazolone derivative, a hydantoin derivative, a rhodanine derivative, and a maleimide derivative.
  • 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.
  • 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 PMP
  • 1,3-dimethyl-pyrazolone DP
  • MTP 3-methyl-1-p-tolyl-5-pyrazolone
  • 3-methyl-1- (quinolin-8-yl) -1H-pyrazol-5 (4H) -one PMP
  • 1,3-dimethyl-pyrazolone DP
  • MTP 3-methyl-1-p-tolyl-5-pyrazolone
  • 3-methyl-1- (quinolin-8-yl) -1H-pyrazol-5 (4H) -one 4-methyl-1- (quinolin-8-yl) -1H-pyrazol-5 (4H) -one, and the like.
  • Examples of hydantoin derivatives used as labeling agents 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.
  • a pyrazolone derivative can be suitably used.
  • a thiol compound may be used together with the labeling agent.
  • the thiol compound any compound having a thiol group can be used without particular limitation.
  • dithiothreitol (DTT) 2-aminoethanethiol
  • thiocholine 2- (pyridine-4- Yl) ethanethiol and the like.
  • the O-linked sugar chain side is labeled with the labeling agent
  • 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.
  • 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 above 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 ⁇ 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 still 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 microwaves” means irradiating a 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.
  • a microwave generator capable of obtaining a microwave output with a frequency of about 1000 to 5000 MHz by magnetron oscillation can be used.
  • 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.
  • the temperature of the solution is more preferably 35 ° C. to 135 ° C., and still more preferably 85 ° C. to 130 ° C.
  • 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.
  • the reaction rate of the ⁇ -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.
  • the reaction is carried out under a weakly alkaline condition rather than a strong alkaline condition as in the conventional pH elimination reaction (pH 11.5 to pH12.5).
  • Degradation peptide degradation
  • the above-described kit of the present invention comprises a solution containing the above-mentioned 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 a glycoprotein eg, a sample obtained by purifying a cell disruption solution
  • the pH of the mixed solution is adjusted to pH 6.0 to pH 10.0 by adding, for example, a 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 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.
  • 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 labeled 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
  • 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.
  • 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.
  • the modifying group can be subjected to structural analysis and quantitative analysis with high sensitivity.
  • an O-linked sugar chain contained in a cell or tissue can be analyzed with high sensitivity.
  • 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.
  • 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.
  • 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 aforementioned “free sugar chain” is one starting from any of N-acetylgactosamine, mannose, N-acetylglucosamine, xylose and glucose, or any of N-acetylgactosamine, mannose, fucose, xylose and 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.
  • the following is an example of a labeling reaction for free sugar chains when a pyrazolone derivative (3-methyl-1-phenyl-5-pyrazolone (PMP)) is used as a labeling agent.
  • a pyrazolone derivative (3-methyl-1-phenyl-5-pyrazolone (PMP)) is used as a labeling agent.
  • the reaction rate of the modification with the modifier of the modifying group can be improved, so that the free sugar chain can be labeled efficiently and in a short time.
  • 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.
  • 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 was added with 0.6M 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 ⁇ elimination reaction by the conventional method (including Carlson's method)).
  • 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
  • FIG. 1 (d) shows the results for the reaction solution (pH 11.8) to which 0.6M sodium hydroxide was added.
  • 0.6M sodium hydroxide is added (pH 11.8 (FIG.
  • Example 2 The effect of heating by microwave irradiation on the labeling reaction of free sugar chains with pyrazolone derivatives was investigated.
  • reaction To maltohexaose (Tokyo Chemical Industry, 1 mmol / mL, 100 ⁇ L), 200 ⁇ L of 0.4 M NaOH and 200 ⁇ L of 0.5 M 3-methyl-1-phenyl-5-pyrazolone (PMP) in methanol are added (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.
  • 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
  • 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.
  • 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.
  • 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).
  • Example 3 The effect of heating by microwave irradiation on the free labeling reaction of glycoproteins with O-linked sugar chains by pyrazolone derivatives was examined.
  • Porcine gastric mucin (Sigma Aldrich, 1 mg / mL, 100 ⁇ L), a glycoprotein having an O-linked sugar chain, 200 ⁇ L of 0.4 M sodium hydroxide, and 200 ⁇ L of 0.5 M 3-methyl-1-phenyl-
  • reaction solvent water
  • the heating temperature was maintained at 85 ° C. by a microwave generator (similar to that in Example 2). The temperature was maintained at ° C. and left for 2 hours.
  • the pH of each reaction solution was 8.3.
  • 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
  • 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 the detected sugar chains.
  • the irradiation efficiency of microwave irradiation improves the reaction efficiency of the ⁇ -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 effect of different types of reaction solvents on the free labeling reaction of glycoproteins with O-linked sugar chains by pyrazolone derivatives was investigated.
  • Porcine gastric mucin (as in Example 3) (1 mg / mL, 100 ⁇ L), a glycoprotein having an O-linked sugar chain, using DMSO or methanol as a reaction solvent, 200 ⁇ L of 0.4 M NaOH, and 200 ⁇ L Of 0.5M 3-methyl-1-phenyl-5-pyrazolone (PMP) was added and 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.
  • 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)).
  • Example 5 It was examined whether peptide degradation was observed in the free labeling reaction of a glycopeptide having an O-GalNAc type sugar chain with a pyrazolone derivative.
  • reaction solution 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 a 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.
  • Porcine gastric mucin (as in Example 3) (1 mg / mL, 100 ⁇ L), a glycoprotein having an O-linked sugar chain, 200 ⁇ L of 0.45 M NaOH, and 200 ⁇ L of 0.5 M 3-methyl-1-
  • a methanol solution of phenyl-5-pyrazolone (PMP) was added (reaction solvent: water) and heated at 85 ° C., 100 ° C., 110 ° C., 120 ° C., 130 ° C. and 140 ° C. using a microwave generator (similar to Example 2). It left still at 2 degreeC for 2 hours.
  • the pH of the reaction solution was 8.3.
  • 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.
  • FIG. The heating temperature is 130 ° C.
  • FIG. 8 (f) is the heating temperature 140 ° C.
  • the recovery amount of sugar chains labeled with PMP was the largest (FIG. 8 (d)).
  • 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 (recovered amounts were 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.
  • 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 ⁇ L), a glycoprotein having an O-linked sugar chain, 200 ⁇ L of 0.4 M NaOH, and 200 ⁇ L 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 (same as in Example 2) for 0.5 hour, 1 hour, 2 hours, And left for 4 hours.
  • the pH of the reaction solution was 8.3.
  • 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)) .
  • 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.
  • 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.
  • Cell pellets (1 ⁇ 10 6 cells) of human iPS cells (HiPS-RIKEN-1A, RIKEN) were sonicated in tris acetate buffer (100 ⁇ L) containing 2% sodium dodecyl sulfate (irradiated for 5 seconds) +10 second intervals).
  • 2 ⁇ L 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 reduce and alkylate the disulfide bond of the protein. (Room temperature, 30 minutes).
  • 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.
  • 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.
  • O-linked sugar chains can be analyzed with high sensitivity from cell pellets by utilizing a free labeling reaction with a pyrazolone derivative.
  • the present invention it is possible to perform a quantitative and qualitative analysis of a modifying group in a sample efficiently and in a short time, and highly sensitive structural analysis and quantitative analysis of the modifying group are also possible.
  • the present invention enables comprehensive analysis of modifying groups including O-linked sugar chains, and is expected to contribute to biology and medicine.

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  • Medicinal Chemistry (AREA)
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Abstract

L'invention concerne un procédé de libération d'un groupe de modification de résidus de sérine et/ou de résidus de thréonine d'une glycoprotéine modifiée contenue dans un échantillon et de marquage du groupe de modification, le procédé comprenant une étape dans laquelle une solution ayant un pH de 6,0 à 10,0 et contenant une glycoprotéine modifiée et un agent de marquage qui est au moins l'un parmi des dérivés de pyrazolone, d'isoxazolone, d'hydantoïne, de rhodanine et de maléimide est irradiée par des micro-ondes.
PCT/JP2014/064436 2013-05-31 2014-05-30 Procédé de libération et de marquage de groupe de modification, nécessaire pour ledit procédé, procédé d'analyse de groupe de modification, nécessaire pour l'analyse et procédé de marquage de chaîne sucre libre WO2014192923A1 (fr)

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CN107796889A (zh) * 2017-10-13 2018-03-13 西北大学 还原性糖链和糖蛋白o‑糖链的氨基吡唑啉酮类异二官能团试剂衍生化及分离分析方法
CN109824740A (zh) * 2019-03-05 2019-05-31 西北大学 还原性糖的不同吡唑啉酮试剂衍生物之间相互转化的方法
WO2020058277A1 (fr) * 2018-09-19 2020-03-26 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Protéines et peptides de sérine adp-ribosylée spécifiques à un site et leur procédé de production

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Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN107796889A (zh) * 2017-10-13 2018-03-13 西北大学 还原性糖链和糖蛋白o‑糖链的氨基吡唑啉酮类异二官能团试剂衍生化及分离分析方法
WO2020058277A1 (fr) * 2018-09-19 2020-03-26 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Protéines et peptides de sérine adp-ribosylée spécifiques à un site et leur procédé de production
CN109824740A (zh) * 2019-03-05 2019-05-31 西北大学 还原性糖的不同吡唑啉酮试剂衍生物之间相互转化的方法
CN109824740B (zh) * 2019-03-05 2023-03-10 西北大学 还原性糖的不同吡唑啉酮试剂衍生物之间相互转化的方法

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