JP4931332B2 - How to remove sugar - Google Patents

Description

  The present invention relates to a method for analyzing a high molecular weight substance in a sample containing a high molecular weight substance having a sugar chain and a low molecular weight saccharide. In particular, the present invention relates to a method for measuring the structure of a sugar chain of only a high molecular weight substance with high accuracy while eliminating the adverse effects of low molecular weight sugars. In one embodiment, the present invention relates to a method for analyzing a sugar chain in a compound having a sugar chain contained in serum or plasma.

  Conventionally, research on the structure of various sugar chains has been carried out, but since there was no attempt to capture sugar chains by sugar chain capture molecules using serum as a sample, what kind of influence is caused by free low molecular weight sugars such as glucose? It was unknown whether to give. In addition, when sugar chains were purified by conventional methods such as gel filtration and ion exchange, labeled, and analyzed by HPLC, there was no influence of glucose.

For example, Non-Patent Document 1 describes a method for analyzing a sugar chain by HPLC, but in such a method, it was not necessary to remove glucose.
Nakagawa, H .; , Kawamura, Y .; Kato, K .; , Shimada, I .; and Takahashi, N .; : Identification of Neutral and Sialyl N-Linked Oligosaccharide Structures from Human Serum Glycoproteins USING Three Kinds of Life-Perform. Analytical Biochem. , 226, 130-138 (1995)

  The present invention will be described below.

An object of the present invention is to provide a method for removing low molecular weight sugars (for example, glucose, galactose, etc.) in a sample (serum) . In particular, an object of the present invention is to provide a method for removing glucose in blood without impairing the sugar chain pattern in the sample.

  Throughout this specification, it should be understood that the singular forms also include the plural concept unless specifically stated otherwise. In addition, it is to be understood that the terms used in the present specification are used in the meaning normally used in the art unless otherwise specified.

  According to the present invention, the following method is provided, which solves the above problems.

(1) In a pretreatment for capturing a sugar chain of a sample containing a high molecular weight substance having a sugar chain and a low molecular weight saccharide with a capture resin for the sugar chain, the low molecular weight saccharide is removed from the sample by ultrafiltration of the sample. ,
The sample is serum, the high molecular weight substance having a sugar chain is a glycoprotein having a sugar chain, and the upper limit of the molecular weight that can pass through the filter used for the ultrafiltration is between 3000 and 10,000. A method for removing low molecular weight sugars from a sample, characterized.

(2) The following steps:
1) a step of removing low molecular weight saccharides from the sample having a sugar chain of (1), and 2) a step of performing a sugar chain analysis using a sample from which low molecular weight saccharides have been removed, A method for analyzing a sugar chain from a sample containing a high molecular weight substance having a sugar chain and a low molecular weight sugar.

  According to the present invention, there is provided a method for capturing a sugar chain cut out from a glycoprotein in blood and removing low-molecular-weight sugars (for example, glucose) in blood that adversely affect the accuracy of analysis when the sugar chain is analyzed. Provided.

  According to the method of the present invention, even after removing low molecular weight sugars in a sample, the sugar chain pattern is not impaired, and sugar chains can be analyzed with high accuracy.

The method of the present invention is a method for removing low molecular weight saccharides from a sample (serum) containing a high molecular weight substance having a sugar chain and low molecular weight saccharides.

(High molecular weight substance with sugar chain)
The method of the present invention is applied to sugar chain analysis of a high molecular weight substance having a sugar chain.

  The sugar chain is a general term including glucose, galactose, mannose, fucose, xylose, N-acetylglucosamine, N-acetylgalactosamine, sialic acid, molecules in which monosaccharides of these derivatives are linked by glycosidic bonds, and the like. Sugar chains are very diverse and are substances that are involved in various functions of naturally occurring organisms.

  In the present specification, the “sugar chain” refers to a compound formed by connecting two or more unit sugars (monosaccharide and / or a derivative thereof). Each unit sugar is bonded by dehydration condensation by a glycosidic bond. Examples of such sugar chains include polysaccharides (glucose, galactose, mannose, fucose, xylose, N-acetylglucosamine, N-acetylgalactosamine, sialic acid, and complexes and derivatives thereof) contained in the living body. Other examples include, but are not limited to, a wide range of sugar chains that are decomposed or derived from complex biomolecules such as degraded polysaccharides, glycoproteins, proteoglycans, glycosaminoglycans, glycolipids, and the like. Therefore, in the present specification, the sugar chain can be used interchangeably with “polysaccharide”, “carbohydrate”, and “carbohydrate”. Further, unless otherwise specified, the “sugar chain” in the present specification may include both sugar chains and sugar chain-containing substances.

  In the present specification, the “high molecular weight substance having a sugar chain” refers to a substance containing a sugar chain and a substance other than the sugar chain. Such a “high molecular weight substance having a sugar chain” is often found in the living body. For example, in addition to polysaccharides contained in the living body, degraded polysaccharides, glycoproteins, proteoglycans, glycosaminoglycans, Examples include, but are not limited to, a wide range of sugar chains that are decomposed or derived from complex biomolecules such as glycolipids.

  In the present specification, examples of the “glycoprotein” include, but are not limited to, enzymes, hormones, cytokines, antibodies, vaccines, receptors, serum proteins and the like.

(Low molecular weight sugars)
In the present specification, the low molecular weight saccharide refers to a monosaccharide or oligosaccharide having a molecular weight smaller than that of the above-described high molecular weight substance. The upper limit of the molecular weight is not particularly limited as long as it is smaller than the high molecular weight substance to be analyzed, but preferably has a number average molecular weight of about 3000, for example.

  In one embodiment, the low molecular weight saccharides to be removed by the method of the present invention are glucose and galactose. Glucose, in particular, is present in large amounts in the blood, and when it is captured and purified by the sugar chain capture molecule after releasing the sugar chain of the high molecular weight substance in the blood, the analysis accuracy of the high molecular weight substance is significantly reduced. According to the present invention, it is possible to significantly improve the analysis accuracy by removing glucose.

The "monosaccharide" in the present specification, not hydrolyzed in simpler molecules which refers to a compound represented by the general formula C _n H _2n O _n. Here, those in which n = 2, 3, 4, 5, 6, 7, 8, 9 and 10 are referred to as diose, triose, tetrose, pentose, hexose, heptose, octose, nonose and decourse, respectively. Generally, it corresponds to an aldehyde or ketone of a chain polyhydric alcohol. The former is called aldose and the latter is called ketose. The n is 10 or less as in the above general formula _C n _H 2n _{O n,} which is a typical example of a low molecular weight saccharides. In the present specification, “aldehyde” is a generic term for a substance containing —CHO, which is a characteristic group.

  In the present specification, the “monosaccharide derivative” refers to a substance in which one or more hydroxyl groups on the monosaccharide are substituted with another substituent and the resulting substance is not within the range of the monosaccharide. Examples of such monosaccharide derivatives include sugars having a carboxyl group (for example, aldonic acid in which C-1 position is oxidized to carboxylic acid (for example, D-gluconic acid in which D-glucose is oxidized), terminal Uronic acid in which C atom of carboxylic acid becomes carboxylic acid (D-glucuronic acid in which D-glucose is oxidized), sugar having amino group or amino group derivative (for example, acetylated amino group) (for example, N- Acetyl-D-glucosamine, N-acetyl-D-galactosamine, etc.), sugars having both amino groups and carboxyl groups (for example, N-acetylneuraminic acid (sialic acid), N-acetylmuramic acid, etc.), deoxy Such as, but not limited to, glycated sugars (eg, 2-deoxy-D-ribose), sulfated sugars containing sulfate groups, phosphorylated sugars containing phosphate groups, etc. NOT. Alternatively, the sugars form a hemiacetal structure, glycosides also reacted with an acetal structure and an alcohol, in the range of monosaccharide derivatives.

  In the present invention, such a monosaccharide derivative is also included in the low molecular weight saccharide.

(Functional group capable of reacting with aldehyde group)
In the present specification, the “functional group capable of reacting with an aldehyde group” refers to a functional group having a property of reacting with an aldehyde group of a sugar chain to form a specific and stable bond. Such functional groups include, but are not limited to, oxyamino groups, N-alkyloxyamino groups, hydrazide groups and thiosemicarbazide groups and derivatives thereof, and cysteine derivatives. More preferably, such functional groups are oxyamino groups and cysteine derivatives. This is because the connection mode (oxime bond) between an oxyamino group and a sugar is particularly weak in acidity and has an advantage that a step of cutting out a sugar chain from a sugar chain-trapping carrier can be easily performed.

(Glycan analysis)
In this specification, the sugar chain analysis refers to analyzing the structure of a sugar chain of a high molecular weight substance having a sugar chain. For example, a spectroscopic analyzer, preferably an infrared spectrophotometer, ultraviolet spectrophotometer, visible spectrophotometer, mass spectrometer, gas chromatography, liquid chromatography, NMR, X-ray analysis, elemental analysis, etc. , Chemical analysis by observing chemical specific reaction, biochemical analysis by determining substrate specificity of enzyme, etc., or judging reaction of organism (for example, microorganisms such as bacteria) But not limited thereto. For example, infrared spectrum analysis, ultraviolet spectrum analysis, visible light spectrum analysis, mass spectrometry, gas chromatography, liquid chromatography, NMR, X-ray analysis, physical analysis such as elemental analysis, observation of chemical specific reaction, etc. Such as chemical analysis by biochemistry, biochemical analysis by determining the substrate specificity of the enzyme, or biological analysis by determining the reaction of organisms (eg, microorganisms such as bacteria) It is not limited to. A physical analysis method is preferred, and a physical analysis method using an analytical instrument is more preferred. Further preferred is chromatography, spectroscopic analysis or mass spectrometry, and further preferred is mass spectrometry or liquid chromatography. Particularly preferred is mass spectrometry or HPLC.

  For mass spectrometry in the method of the present invention, any ionization technique (for example, electrospray (ESI) method, ultrasonic spray (SSI) method, matrix-assisted laser desorption ionization (MALDI) method)) is used in this field. Any mass spectrometric method utilized in can be used. Any mass separation method (for example, time-of-flight type (TOF), quadrupole type, magnetic field type) can be used for mass spectrometry in the method of the present invention. In preferred embodiments, mass spectrometry is performed by ESI-magnetic MS or MALDI-TOF MS.

In this specification, “MALDI-TOF (MS)” is an abbreviation of Matrix Assisted Laser Deposition Ionization-Time-of-Flight (Mass Spectrometer). MALDI is a technique found by Tanaka et al. And developed by Hillenkamp et al. (Karas M., Hillenkamp, F., Anal. Chem. 1988, 60, 2299-2301). In this method, the sample and the matrix solution are mixed at a molar ratio of (10 ^{−2 to} 5 × 10 ⁻⁴ ): 1, and then the mixed solution is dried on the target to be in a crystalline state. Large energy is given to the matrix by pulse laser irradiation, and sample-derived ions such as (M + H) ⁺ and (M + Na) ⁺ and matrix-derived ions are desorbed. Even if it is contaminated with a small amount of phosphate buffer, Tris buffer, guanidine, etc., it can be analyzed. MALDI-TOF (MS) measures mass based on time of flight using MALDI. When an ion is accelerated at a constant acceleration voltage V, the ion mass is m, the ion velocity is v, the ion charge number is z, the elementary charge is e, and the ion flight time is t. m / z is
m / z = 2eVt ² / L ²
Can be expressed as For such MALDI-TOF measurement, Shimadzu / Kratos's KOMPACT MALDI II / III can be used. In the measurement, a pamphlet created by the manufacturer can be referred to.

  For the various analysis methods described above, various conventionally known analysis devices according to the analysis method can be used.

(Low molecular weight sugar removal process)
In the step of removing the low molecular weight saccharide, any method can be used as long as the low molecular weight saccharide can be removed without affecting the structure of the sugar chain portion of the high molecular weight substance.

(Ultrafiltration method)
In one embodiment of the present invention, by the sample (serum) ultrafiltration, low molecular weight saccharides in a sample (e.g., glucose) is filtered. Moreover, although some glycoproteins were also removed by ultrafiltration and there was a possibility that the sugar chain pattern might change, it was confirmed that the sugar chain pattern did not change under these conditions.

  A conventionally known ultrafiltration method can be used as the ultrafiltration method.

  Preferably, the ultrafiltration conditions are set so as to remove sugars having a molecular weight of 3000 or less.

  As an apparatus used for the ultrafiltration, any conventionally known ultrafiltration apparatus can be used.

  As the filter, any filtration membrane that can be used for ultrafiltration can be used. Therefore, an ultrafiltration membrane can be used, and a dialysis membrane can also be used.

  Any conventionally known material can be used as a filter material used for ultrafiltration. For example, regenerated cellulose, cellulose acetate, polyethersulfone, polyacrylonitrile and the like can be used. Most preferred is a regenerated cellulose ultrafiltration membrane.

The upper limit of the molecular weight that can pass through the filter can be appropriately selected in consideration of the molecular weight of the high molecular weight substance to be analyzed and the molecular weight of the low molecular weight saccharide to be removed. For example, the lower limit of the molecular weight of the high molecular weight substance to be analyzed can be set as the upper limit of the molecular weight that can pass through the filter. In this invention, it is a filter from which the upper limit of the molecular weight which can pass is 1000-10000.

(Low molecular weight saccharide-degrading enzyme)
Any enzyme capable of degrading low molecular weight saccharides can be used as the low molecular weight saccharide degrading enzyme. Glucose oxidase is preferable. If glucose oxidase is used, glucose can be oxidized to lactone.

  In addition, glucose oxidase and catalase may be used in combination. If catalase is used in combination, hydrogen peroxide produced by the reaction of glucose oxidase can be decomposed, and the reaction of glucose oxidase can be further promoted.

(Glucose oxidase)
Glucose oxidase (EC 1.1.3.4) has the following reaction:
β-D-glucose + O ₂ → D-glucono-δ-lactone + H ₂ O ₂
It is an enzyme that catalyzes.

  As the glucose oxidase, commercially available enzyme preparations such as deoxyn (Penicillium amagasakienase) [Nagase Sangyo] and Hyderase 15 (Asp. Nigar) [Amano Pharmaceutical]; and the like can be used.

(Catalase)
Catalase (EC 1.11.1.6) is a hydrogen peroxide decomposition reaction:
H ₂ O ₂ + H ₂ O ₂ → O ₂ + 2H ₂ O
It is an enzyme that catalyzes. As the catalase, any commercially available enzyme preparation can be used.

(Enzyme usage conditions)
As conditions for allowing the enzyme to act on the sample, any known conditions corresponding to the enzyme can be used. About temperature, in one embodiment, it is 35 to 40 degreeC, and is 37 degreeC in a preferable embodiment. Although reaction time is not specifically limited, Preferably, it is 30 seconds or more, More preferably, it is 1 minute or more, More preferably, it is 2 minutes or more. The reaction time is preferably 3 days or less. In one embodiment, it is 1 hour to 3 days, and in a preferred embodiment, it is 12 hours to 2 days. If the reaction time is too long, the overall working time becomes longer. Conversely, if the reaction time is too short, the low molecular weight saccharide may not be sufficiently decomposed.

(Low molecular weight sugar capture reagent)
Any reagent capable of capturing low molecular weight saccharides can be used as the low molecular weight saccharide capturing reagent. Examples of such a reagent include a reagent having a group that can react with an aldehyde group of a sugar (for example, an oxyamino group, an N-alkyloxyamino group, a hydrazide group, and a thiosemicarbazide group). For example, L-cysteine ethyl ester hydrochloride or sugar chain capture resin can be used. A resin having an oxyamino group at the terminal is preferable.

  The sugar chain-trapping resin can be produced by introducing a group capable of reacting with an aldehyde group into a conventionally known resin. Examples of resins that can be used include polyethylene, ethylene, polypropylene, polyisobutylene, polyethylene terephthalate, unsaturated polyester, fluorine-containing resin, polyvinyl chloride, polyvinylidene chloride, polyvinyl acetate, polyvinyl alcohol, polyvinyl acetal, acrylic resin, poly Acrylonitrile, polystyrene, acetal resin, polycarbonate, polyamide, phenol resin, urea resin, epoxy resin, melamine resin, styrene / acrylonitrile copolymer, acrylonitrile butadiene styrene copolymer, silicone resin, polyphenylene oxide, polysulfone, etc. can be used. . These resins may be cross-linked as necessary.

  As a method for introducing a group capable of reacting with an aldehyde group, any known method can be used as a method for introducing a substituent into the resin. For example, a method such as a method of copolymerizing an ethylenically unsaturated monomer having a group capable of reacting with an aldehyde group with an unsaturated monomer of the above various resins is possible.

  The obtained sugar chain-trapping resin can be used in any form. In one embodiment, it can be in the form of particles. The particles of the sugar chain-trapping resin are preferably dispersed in water or an organic solvent. The average particle size of the sugar chain-trapping resin is not particularly limited, but is preferably 0.001 to 1 mm, more preferably 0.1 to 500 microns.

(Conditions for using low molecular weight saccharide capture reagent)
As conditions for allowing the low-molecular-weight saccharide capturing reagent to act on the sample, any known conditions according to the reagent can be used. About temperature, in one embodiment, it is 35 to 40 degreeC, and is 37 degreeC in a preferable embodiment. Although reaction time is not specifically limited, Preferably, it is 30 seconds or more, More preferably, it is 1 minute or more, More preferably, it is 2 minutes or more. The reaction time is preferably 3 days or less. In one embodiment, it is 1 hour to 3 days, and in a preferred embodiment, it is 12 hours to 2 days.

  The amount of low molecular weight saccharide capture reagent used can be calculated stoichiometrically. For example, it is preferable to mix so that the low molecular weight saccharide in a sample may be 0.1-1 mol with respect to 1 mol of groups (for example, oxyamino group) which can react with the aldehyde group which the reagent has. More preferably, it mix | blends so that it may become 0.2-0.8 mol.

  The used saccharide capture reagent can be reused as necessary. For example, when a saccharide-capturing reagent having an oxyamino group is used, it is possible to treat with an acid by utilizing the property that the oxyamino group-sugar linkage mode (oxime bond) is weakly acidic. The saccharide capture reagent can be easily reused by cutting out the saccharide from the saccharide capture reagent.

(Measurement after removing low molecular weight sugars)
For samples from which low-molecular-weight sugars have been removed, sugar chain analysis can be performed using various methods. As the method for sugar chain analysis, any of the methods described above for the term “sugar chain analysis” may be used.

  When the high-molecular-weight substance having a sugar chain is a compound in which a sugar chain part and a part other than a sugar chain are combined, the sugar chain analysis is performed after decomposing the part other than the sugar chain to release only the sugar chain. It is preferable to carry out.

  For example, when the high molecular weight substance having a sugar chain is a glycoprotein, the low molecular weight sugar is removed, the protein portion of the glycoprotein is decomposed, the sugar chain is cut out and released, and then the sugar chain capture resin It is preferable to capture and analyze the released sugar chain.

  As a method for degrading the protein portion of glycoprotein, any method can be used. For example, an enzyme degrading protein can be used. Any peptidase can be used as an enzyme that degrades protein, and specific examples include trypsin and chymotrypsin. As a procedure of the enzyme reaction, for example, an enzyme is added to an appropriate solvent (for example, a Tris buffer or ammonium bicarbonate solution having a pH of 8.0), and a temperature suitable for the reaction of the enzyme (for example, 37 ° C.). ) In a suitable time that the enzyme can react sufficiently (for example, preferably 1 minute to 3 days, or 1 hour to 3 days in the case of a reaction system in which the reaction rate is not so fast). Can be disassembled.

  As a method for cutting out and releasing a sugar chain, any method for releasing an aldehyde group of a sugar chain in a sample is possible.

  In this specification, “releasing an aldehyde group of a sugar chain” means that an aldehyde group in a sugar chain or a sugar chain-containing substance is exposed. Moreover, the production | generation of the galactose residue and 6th-position aldehyde group by galactose oxidase, and the production | generation of the aldehyde group by periodate decomposition of a diol group are also included. By releasing the aldehyde group in the sample, the interaction reaction between the substance that specifically interacts with the sugar chain of the present invention and the sugar chain or the sugar chain-containing substance becomes easier. . Alternatively, by exposing the sample to conditions that liberate aldehyde groups in the sample, it is possible to separate, concentrate, purify, or analyze only the sugar chains in the sugar chain-containing substance, and such a state is desired. May be advantageous.

  Examples of conditions for liberating aldehyde groups in such a sample include, but are not limited to, proton treatment by enzyme treatment and / or chemical methods. Examples of such enzyme treatment include treatment with a sugar chain free enzyme such as N-glycosidase (for example, an enzyme derived from Flavobacterium meningosepticum expressed in E. coli), glycopeptidase A (almond) and the like. Is not limited to this. Such enzymes used in the present invention include, but are not limited to, glucosidases derived from plants, yeasts and fungi, preferably N-glucosidases derived from Flavobacterium. Examples of the chemical method include, but are not limited to, hydrazine decomposition (liquid phase or gas phase). In hydrazine degradation, for example, a sample (for example, a sample containing 200 to 1000 μg of glycoprotein) is lyophilized (using a screw cap vial or a screw cap test tube), and anhydrous hydrazine (for example, 100 to 200 μl is added at 100 ° C. Heat for several hours to several tens of hours (eg, using a drive lock heater or oven), then add a few drops of toluene, place the sample vial in a desiccator, and depressurize with a vacuum pump with a cooling trap for several hours to remove hydrazine. The azeotropic distillation is repeated several times, and the hydrazine decomposition is achieved by completely distilling off the hydrazine, and the desired sugar chain is separated.

  As a specific procedure, for example, an appropriate enzyme (for example, N-glycosidase) is added to a sample and reacted at an appropriate temperature (for example, 37 degrees) for an appropriate time (for example, 30 seconds to 1 day). Sugar chains are cut out.

  The sugar chain thus cut out and released may be directly analyzed by an analysis device (for example, liquid chromatography and mass spectrometry). For example, it is preferably analyzed after being captured by a sugar chain capturing resin. As the sugar chain-trapping resin, any sugar chain-trapping resin described for the low molecular weight saccharide-trapping reagent can be used.

(General techniques used in this specification)
Unless otherwise specifically stated, the techniques used in this specification are within the technical scope of the relevant field, such as microfluidics, microfabrication, organic chemistry, biochemistry, genetic engineering, molecular biology. Well known and conventional techniques in science, microbiology, genetics and related fields may be used. Such techniques are well described, for example, in the documents listed below and in references cited elsewhere herein.

  Molecular biological techniques, biochemical techniques, and microbiological techniques used herein are well known and commonly used in the art, and are described in, for example, Maniatis, T. et al. et al. (1989). Molecular Cloning: A Laboratory Manual, Cold Spring Harbor and its 3rd Ed. (2001); Ausubel, F .; M.M. , Et al. eds, Current Protocols in Molecular Biology, John Wiley & Sons Inc. NY, 10158 (2000); Innis, M .; A. (1990). PCR Protocols: A Guide to Methods and Applications, Academic Press; A. et al. (1995). PCR Strategies, Academic Press; Sinsky, J. et al. J. et al. et al. (1999). PCR Applications: Protocols for Functional Genomics, Academic Press; Gait, M .; J. et al. (1985). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Gait, M .; J. et al. (1990). Oligonucleotide Synthesis: A Practical Approach, IRL Press; Eckstein, F .; (1991). Oligonucleotides and Analogues: A Practical Approc, IRL Press; Adams, R .; L. et al. (1992). The Biochemistry of the Nucleic Acids, Chapman &Hall; Shabarova, Z. et al. et al. (1994). Advanced Organic Chemistry of Nucleic Acids, Weinheim; Blackburn, G .; M.M. et al. (1996). Nucleic Acids in Chemistry and Biology, Oxford University Press; Hermanson, G .; T.A. (1996). Bioconjugate Technologies, Academic Press; Method in Enzymology 230, 242, 247, Academic Press, 1994; Separate Experimental Medicine “Methods for Gene Introduction & Expression Analysis” Yodosha, 1997; Hatanaka, Nishimura et al., Carbohydrate Science and Engineering, Kodansha Scientific, 1997; Design and Physiological Function of Glycomolecules, is described in the Chemical Society of Japan, Japan Society for Publication Press, 2001, etc., and these are relevant for reference (may be all) in this specification. Incorporated.

<Example 1>
Human serum was diluted 10-fold with 100 mM NH ₄ HCO ₃ and ultrafiltered using a centrifugal filter unit (Microcon YM-10 Centrifugal Filter Device, molecular weight cut-off 10,000). The fraction of unpermeated material remaining without filtration (Retentate) was collected, and the amount of glucose was measured using a glucose measurement kit. It was 3.4% before filtration, and glucose was removed by ultrafiltration. It was confirmed that

  For the Retentate fraction, a sugar chain was cut out from the glycoprotein by enzyme treatment, and a sugar chain capturing experiment was performed using sugar chain capturing particles.

  The sugar chain-trapping particles were prepared as follows. That is, 15 mg of a sugar chain-trapping molecule and 45.0 mg of PCPDA were dissolved in 10 mL of chloroform and mixed. After concentration, 40 mL of water was added, heated at 85 ° C. for 10 minutes, and then irradiated with ultrasonic waves for 40 minutes to be hexylated. After cooling on ice for 10 minutes, the mixture was filtered with a filter having a pore size of 0.45 μm and polymerized by irradiating with UV (254 nm) on ice for 2 hours while flowing argon gas. This was filtered with a filter having a pore size of 0.45 μm, and 36 mL was centrifuged at 55 ° C. for 3 hours to obtain 400 μL.

  As a control, the same procedure was performed for unfiltered serum. When the captured sugar chain was released and MS was measured, no peak of MS was observed for the unfiltered sample, but in the sample after ultrafiltration, as shown in FIG. An MS peak derived from a sugar chain was observed. Therefore, it was confirmed that removal of glucose is effective in capturing glycoprotein sugar chains in serum.

  Similarly, the sugar chain pattern of glycoprotein was analyzed for serum from which glucose was removed by ultrafiltration. First, in order to confirm that there is no protein leakage or adsorption by ultrafiltration, SDS-PAGE was performed on the Retentate fraction and the filtered Filrate fraction. The results are shown in FIG. The Retentate fraction showed the same migration pattern as that of the serum before ultrafiltration, and no protein was observed in the Filtrate fraction. Next, for each fraction, a sugar chain was cut out from the glycoprotein by enzyme treatment, the sugar chain was purified by gel filtration, fluorescently labeled with 2-aminopyridine, and subjected to HPLC analysis using an ODS column. The obtained chromatograms are shown in FIGS. In addition, FIG. 4 shows a comparison of sugar chain patterns (area ratio of each peak to all peaks) for ultrafiltration-untreated serum and the retentate fraction after ultrafiltration. No sugar chain was detected in the Filtrate fraction, and no change in sugar chain pattern due to ultrafiltration was observed.

  From the above, removal of glucose in serum by ultrafiltration increased the efficiency of sugar chain capture without affecting the sugar chain pattern derived from glycoprotein.

< Reference Example 1 >
A sample in which glucose oxidase and catalase were added to human serum and a sample in which only glucose oxidase was added to human serum were prepared and incubated at 37 ° C. With respect to the reaction solution after 0 to 30 minutes, the sugar was quantified using the orcinol-sulfuric acid method, and the residual ratio of glucose was determined. The results are shown in FIG. It was confirmed that serum glucose was decreased by glucose oxidase.

< Reference Example 2 >
3 mL of L-cysteine ethyl ester hydrochloride was added per 1 mL of human serum, and the mixture was shaken overnight at 37 ° C. The amount of glucose before and after the reaction was measured using a glucose measurement kit, and the residual rate was determined. The results are shown in Table 1.

It was confirmed that serum glucose decreased by reacting with L-cysteine ethyl ester.

< Reference Example 3 >
1 g of a sugar chain-trapping resin was added to 1 mL of a 10 mg / mL glucose solution and shaken overnight at 37 ° C. The amount of glucose before and after the reaction was measured using a glucose measurement kit, and the residual rate was determined. Table 2 shows the structure and results of the resins used.

In addition, the base resin portion of the sugar chain-trapping resin of Example 4A, that is, the R ¹ portion is a copolymer of styrene and 1% divinylbenzene, and has a functional group of 0.50 to 1.30 mmol per 1 g of the resin. Yes. The R ² part in Example 4B is a copolymer of polystyrene and polyethylene glycol having a low degree of crosslinking, and 0.20 to 0.30 mmol of functional groups are attached per 1 g of resin.

  It was confirmed that glucose reacted with the sugar chain-trapping resin and decreased.

  Since the present invention has the above-described configuration, it is very effective for analyzing a high molecular weight substance having a sugar chain in a sample such as serum.

  As mentioned above, although this invention has been illustrated using preferable embodiment of this invention, this invention should not be limited and limited to this embodiment. It is understood that the scope of the present invention should be construed only by the claims. It is understood that those skilled in the art can implement an equivalent range based on the description of the present invention and the common general technical knowledge from the description of specific preferred embodiments of the present invention. Patents, patent applications, and documents cited herein should be incorporated by reference in their entirety, as if the contents themselves were specifically described herein. Understood.

MS spectrum after sugar chain capture of ultrafiltration sample Serum SDS-PAGE before and after ultrafiltration Chromatogram of unfiltered serum after ultrafiltration Chromatogram of Retentate fraction after ultrafiltration Chromatogram of Filrate fraction after ultrafiltration Comparison of sugar chain patterns between unfiltered serum after ultrafiltration and Filrate fraction after ultrafiltration Changes in serum glucose level by glucose oxidase

Claims (2)

In the pretreatment of capturing a sugar chain of a sample containing a high molecular weight substance having a sugar chain and a low molecular weight saccharide with a capture resin for the sugar chain , removing the low molecular weight saccharide from the sample by ultrafiltration of the sample,
The sample is serum, the high molecular weight substance having a sugar chain is a glycoprotein having a sugar chain, and the upper limit of the molecular weight that can pass through the filter used for the ultrafiltration is between 3000 and 10,000. A method for removing low molecular weight sugars from a sample, characterized. The following steps:
1) a step of removing low molecular weight saccharides from a sample having a sugar chain according to claim 1, and 2) a step of performing a sugar chain analysis using a sample from which low molecular weight saccharides have been removed, A method for analyzing a sugar chain from a sample containing a high molecular weight substance having a sugar chain and a low molecular weight sugar.