JP4182219B2 - Method for analyzing and quantifying phosphorylated saccharide - Google Patents

Description

  The present invention relates to an analysis method including quantitative analysis of phosphorylated saccharide contained in a sample.

  It has been clarified that proteins existing in the natural world do not show their original biological activity when their sugar chains are removed (Kiyoyo, Protein Nucleic Acid Enzyme, 36, 775-788 (1991)). Although sugar chains are expected to play an important role in the expression of biological activity, the correlation between the structure of the sugar chain and the biological activity is not always clear, so the structure of the sugar chain added to the protein part (sugar It is necessary to develop a technology that can freely modify and control the type, bond position, chain length, etc.).

  The sugar chains of glycoproteins are roughly classified into Asn-linked, mucin, O-GlcNAc, GPI anchor, and proteoglycan.

The biosynthesis of Asn-linked glycans involves the synthesis of a precursor consisting of N-acetylglucosamine, mannose, and glucose on a lipid carrier intermediate, followed by a specific sequence of glycoprotein (Asn- X-Ser or Thr). Next, processing (cleavage of a glucose residue and a specific mannose residue) is performed to synthesize an M8 high mannose type sugar chain (Man ₈ GlcNAc ₂ ) composed of ₈ mannose residues and 2 N-acetylglucosamine residues. The protein containing this high mannose-type sugar chain is transported to the Golgi apparatus and undergoes various modifications. The modification in the Golgi apparatus differs greatly between yeast and mammals (Kukuruzinska et al., Annu. Rev. Biochem., 56, 915-944 (1987)).

Mammalian cells follow the following three pathways that vary depending on the type of protein undergoing glycosylation. 1) When the above core sugar chain is not changed at all, 2) N-acetylglucosamine-1-phosphate part (GlcNAc-1-P) of UDP-N-acetylglucosamine (UDP-GlcNAc) is the core sugar chain When it is added to the 6-position of Man to form Man-6-P-1-GlcNAc, and only this GlcNAc part is removed and converted to a glycoprotein with an acidic sugar chain, 3) Core sugar chain 5 molecules of Man are sequentially removed from this to become Man ₃ GlcNAc ₂ , and GlcNAc, galactose (Gal), N-acetylneuraminic acid, also known as sialic acid (NeuNAc), etc. are added in succession and variously. There are three types of cases in which complex mixed and complex sugar chains are produced as a mixture [R. Kornfeld and S. Kornfeld, Ann. Rev. Biochem., Vol. 54, p.631-664 (1985)]. It can be said that mammalian sugar chains have a wide variety of structures, and these structures are greatly related to the functions of glycoproteins.

On the other hand, yeast produces a mannan-type sugar chain, a so-called outer chain, in which mannose is added to several to 100 residues of the above core sugar chain (Man ₈ GlcNAc ₂ ). It has been found that acidic sugar chains in which mannose-1-phosphate is added to the chain part and the sugar outer chain part are also produced. Unlike animal cells, this modification has been reported not to function as a sorting signal for vacuolar (organelle equivalent to lysosomes in animal cells) localized glycoproteins in yeast. Therefore, the physiological function of this phosphorylated sugar chain in yeast remains unclear [Kukuruzinska et al, Ann. Rev. Biochem., Vol. 56, p915 (1987)].

By the way, lysosomes, which are one of the organelles in the cell, have many acid hydrolases and are responsible for degrading substances taken into the lysosome from inside and outside the cell. Many of the enzymes that are localized in human lysosomes are biosynthesized and transported to the Golgi apparatus. A phosphate group is added to the 6-position of the mannose residue at the non-reducing end of the high mannose-type sugar chain. Is converted into a glycoprotein having a lysosomal enzyme-specific recognition marker. And after binding to its high-affinity receptor, mannose-6-phosphate receptor (MPR), it is selected from other proteins, transported to endosomes, and dissociated from MPR under acidic conditions. It is transported to the lysosome (von Figura and Hasilik, Annu. Rev. Biochem., 54, 167-193 (1984)). In order to bind to mannose-6-phosphate receptor (M6PR), it is necessary to contain one or more molecules of mannose-6-phosphate in one sugar chain.

Due to genetic defects in these lysosomal enzymes, enzymes involved in the aforementioned phosphate group addition reaction, and factors involved in enzyme activation and stabilization, the enzyme reaction does not proceed and the substrate accumulates in the cell. A series of diseases that occur is called "lysosomal disease" (Leroy and DeMars, Science, 157, 804-806 (1967)). Regarding lysosomal diseases, more than 40 types are known in humans and are one of the important disease groups in pediatrics and internal medicine. Bone marrow transplantation, gene therapy, and the like have been attempted for the development of fundamental treatments for these diseases. In addition, enzyme replacement therapy using lysosomal enzymes has been tried for a long time, but uptake into target organs has become a major obstacle at the time, and for Gaucher disease and Fabry disease at present, No good results other than enzyme replacement therapy. The reason is that many other lysosomal diseases other than Gaucher's disease have a mannose-6-phosphate receptor-mediated transport of the defective enzyme to the lysosome, which is used for enzyme replacement therapy. The enzyme must have a sugar chain containing mannose-6-phosphate, which is a lysosome translocation signal necessary for binding to mannose-6-phosphate receptor (MPR). Therefore, the addition of mannose-6-phosphate to the sugar chain of these lysosomal enzymes is the key.

Currently, methods for purification from placenta, production methods using cultured cells such as fibroblasts and melanoma cells, recombinant methods using cultured cells such as insect cells and Chinese hamster ovary (CHO) cells, milk of transgenic rabbits 1) Poor efficiency of incorporation into lysosomes due to low content of lysosomal enzymes having sugar chains to which phosphate has been added 2) Low productivity and high culture costs. Therefore, there are problems such as 1) large-scale administration, 2) concern about antigenicity, and 3) cost of treatment. Therefore, there is a demand for an enzyme having high lysosomal uptake activity containing mannose-6-phosphate in the sugar chain. One solution is to increase the mannose-6-phosphate content of such enzymes. The present inventors have already disclosed a method for producing an enzyme having an increased mannose-6-phosphate content that can be used for enzyme replacement therapy using yeast (Japanese Patent Application Laid-Open No. 2002-369692; Patent) Reference 1).

Now, measuring the sugar content of such an enzyme is one of the means to evaluate whether it is an enzyme with good uptake efficiency, but it also has a uniform sugar content when commercialized as a therapeutic agent. It is also important in terms of quality control. Until now, the analysis of phosphorylated sugar has mainly been performed by high pH anion exchange chromatography with pulsed amperometric detection (HPAE / PAD) (NM Dahms et al., J. Biol. Chem., 264 (1989) 12115-12118 ) Has been used. This method is a method in which a monosaccharide obtained by hydrolyzing a glycoprotein is subjected to an anion exchange column under strongly alkaline conditions, separated by its structure, and detected by the pulse amperometry principle.

JP 2002-369692 A

  However, the detection sensitivity of the HPAE / PAD method is low, so it was not possible to analyze only the phosphorylated saccharide at the same time as other neutral saccharides and N-acetylated saccharides. Therefore, in order to detect phosphorylated saccharide by the HPAE / PAD method, for example, it is necessary to divide one sample into two and analyze each sample.

  However, in such a method, analysis of phosphorylated saccharide takes time, and the amount of glycoprotein initially required is about several hundred micrograms. As described above, the above-described method has a problem that it is impossible to analyze phosphorylated saccharide with excellent sensitivity.

Furthermore, in the HPAE / PAD method, a strongly alkaline solvent easily reacts with CO ₂ in the air, and the pH changes immediately. In this case, there is a problem that the reproducibility of the analysis result of phosphorylated saccharide cannot be ensured. Further, particularly strong alkaline solvents are dangerous in handling.

  Thus, with the conventional method, it is impossible to analyze phosphorylated saccharide contained in a sample simply and quickly, and it is impossible to analyze phosphorylated saccharide with excellent sensitivity. Therefore, in view of the above situation, the present invention has an object to provide a method for analyzing phosphorylated saccharides that is simple and quick in analyzing a phosphorylated saccharide contained in a sample and that is excellent in sensitivity. .

The present invention that has achieved the above-described object includes the following.
(1) A step of binding the phosphorylated saccharide and the fluorescent labeling substance in a solution containing the phosphorylated saccharide and the fluorescent labeling substance, and the phosphorylation bound to the fluorescent labeling substance contained in the solution A method for analyzing phosphorylated saccharide, comprising a step of detecting saccharide.

(2) The method for analyzing a phosphorylated saccharide according to (1), wherein a phosphorylated saccharide to be analyzed is prepared by chemically or enzymatically cleaving a sugar chain bound to a glycoprotein.

(3) The method for analyzing a phosphorylated saccharide according to (1), wherein in the step of detecting the phosphorylated saccharide, the phosphorylated saccharide and other sugar chains are separated by anion exchange column or capillary electrophoresis. .

(4) In the step of detecting the phosphorylated saccharide, the phosphorylated saccharide contained in the solution is quantified based on the amount of fluorescence derived from the fluorescent labeling substance bound to the phosphorylated saccharide. Analysis method of sugar.

(5) The fluorescent labeling substance is 8-aminopyrene-1,3,6-trisulfonate and / or 2-
The method for analyzing phosphorylated saccharide according to (1), which is aminopyridine.

  In the method for analyzing phosphorylated saccharide according to the invention, phosphorylated saccharide can be analyzed quickly and easily by binding a fluorescent labeling substance to phosphorylated saccharide, and phosphorylated saccharide can be analyzed with excellent sensitivity. Can be analyzed. Moreover, according to the method for quantifying phosphorylated saccharide according to the present invention, phosphorylated saccharide can be quantified quickly and easily, and phosphorylated saccharide can be quantified with excellent sensitivity.

Hereinafter, the present invention will be described in detail.
The present invention can be applied, for example, when analyzing a phosphorylated saccharide bound to a protein. The present invention can also be applied to the evaluation of the phosphorylated saccharide contained in the glycoprotein in a pharmaceutical composition containing the glycoprotein as a main component.

In the method for analyzing phosphorylated saccharide according to the present invention, first, glycoprotein is separated and purified, and the sugar chain of the separated and purified glycoprotein is cleaved. The cleaved sugar chain includes a phosphorylated sugar and other sugar chains that are analyzed by the present method. Here, the other sugar chains are meant to include sugar chains such as Asn-linked, mucin, O-GlcNAc, GPI anchor, and proteoglycan.

  A technique for cleaving a sugar chain of a glycoprotein is not particularly limited, and examples thereof include a chemical method and an enzymatic method. The chemical method means that an acid hydrolysis reaction is caused by various conditions in a solution containing glycoprotein. Specific examples of the chemical method include hydrazine decomposition. The enzymatic method means that an enzyme is present in a solution containing glycoprotein and an acid hydrolysis reaction is caused by the activity of the enzyme. As the enzyme having acid hydrolysis activity, for example, glycopeptidase A (almond) and N-glycanase (Flavobacterium meningosepticum) can be used.

  When cleaving a sugar chain of a glycoprotein, it is desirable to use mild acid hydrolysis conditions so that phosphorylation does not cause dephosphorylation. For example, the glycoprotein is freeze-dried, 2N trifluoroacetic acid is added and sealed, and acid hydrolysis is performed at 100 ° C. for 2 hours. Moreover, since acid hydrolysis causes deacetylation of amino sugars (glucosamine, galactosamine, mannosamine, etc.), N-acetylated amino sugars can be isolated by chemical reacetylation.

  In addition, in this method, it is possible to obtain a standard product with a uniform and clear weight by purchasing commercially available phosphorylated sugar and other sugar chains, and phosphorylated sugar and other sugars. It is also possible to prepare a uniform sample by adjusting the chain by chemical synthesis. That is, the method for analyzing phosphorylated saccharide according to the present invention is not limited to analyzing phosphorylated saccharide bound to glycoprotein, and can be widely applied to analyzing phosphorylated saccharide contained in a solution.

Next, in the method for analyzing phosphorylated saccharide according to the present invention, a solution containing a cleaved sugar chain and a fluorescent labeling substance is prepared. The fluorescent labeling substance is not particularly limited as long as it can bind to the reducing end of the cleaved sugar chain, and any substance may be used. Specific examples of fluorescent labeling substances include 2-aminopyridine, 2-aminobenzamide, 2-aminobenzoic acid, 8-aminopyrene-1,3,6-trisulfonate, and the like. These fluorescent labeling substances can be covalently bonded to the phosphorylated saccharide contained in the cleaved sugar chain by a reductive amination reaction. As will be described in detail later, when analyzing monosaccharides by capillary electrophoresis, a fluorescently labeled substance having a negative charge is excellent in resolution, so 8-aminopyrene-1, 3, 6-trisulfonate is fluorescent. It is preferable to use it as a labeling substance.

  The solution prepared in this step is prepared so that the binding reaction proceeds between the cleaved sugar chain and the fluorescent labeling substance. The pH when the fluorescent labeling substance is reacted is not particularly limited as long as it is on the acidic side where the reductive amination reaction of the sugar chain occurs. However, if a strong acid such as 0.1 N hydrochloric acid is used, dephosphorylation may occur. Therefore, it is desirable to prepare a solvent with a weak acid such as citric acid or acetic acid solution.

  The reaction temperature for labeling with a fluorescent labeling substance is not particularly limited as long as the reaction reaches saturation, but dephosphorylation may occur in phosphorylated saccharide when performed at a high temperature. desirable.

  Next, in the method for analyzing phosphorylated saccharide according to the present invention, the glycan contained in the solution is separated, and the phosphorylated saccharide is detected based on the fluorescent labeling substance. Prior to separation of the sugar chain, extraction with an organic solvent such as gel filtration, paper chromatography, cellulose column, or phenol may be performed to remove unreacted fluorescent labeling substances contained in the solution. .

Fluorescently labeled phosphorylated saccharides can be separated together with other neutral sugars and amino sugars by high performance liquid chromatography (HPLC) equipped with an anion exchange column or electrophoresis including capillary electrophoresis. After separation, when HPLC is used, a fluorescence detector can be connected to the system, or a predetermined amount of the separation liquid eluted from the column can be collected and analyzed by fluorescence measurement. In addition, when capillary electrophoresis is used, rapid analysis can be performed by connecting a fluorescence detector to the system. In the case of other electrophoresis methods, analysis can be performed by applying excitation light to the equipment (gel, filter paper, thin layer chromatography, etc.) used for separation and detecting fluorescence.

In this method, when the fluorescently labeled phosphorylated saccharide contained in the solution is separated on an anion exchange column together with other neutral sugars and amino sugars, the salt is contained so that the salt concentration becomes 0.1 M or more. Separate with a borate buffer (pH 9.0 or higher). When the salt concentration of the borate buffer solution is less than 0.1 M or the pH of the borate buffer solution is less than 9.0, the phosphorylated saccharide may not be separated by the anion exchange column.

  EXAMPLES Hereinafter, although this invention is demonstrated in detail using an Example, the technical scope of this invention is not limited to a following example.

Example 1 Mannos-6-phosphate (hereinafter referred to as Man-6-P) 8-aminopyrene-1, 3,
Fluorescent labeling with 6-trisulfonate (hereinafter referred to as APTS)
Man-6-P was purchased from Sigma. APTS labeling reagent kits were purchased from Beckman Coulter.

1 nmol of Man-6-P was dried under reduced pressure in a 1.5 ml sampling tube. 100 mM APTS (prepared by adding 436 μL of 0.9 M citric acid solution to a vial containing 20 mg of APTS-M (P / N 72709-0 Beckman Coulter)) 2 μL and 1 M NaBH ₃ CN in 1 μL of THF (manufactured by Aldrich) was added, and the labeling reaction was carried out in a heat block at 55 ° C. for 2 hours while shielding from light. On the way, centrifugation was performed after 30 minutes and 60 minutes, and the liquid was collected at the bottom of the tube, and stirred to be redissolved. Next, 200 μL of filtered cooling water was added to stop the reaction. When not proceeding to the next step immediately, it was stored at −20 ° C. in this state.

The reaction solution was diluted 10 times and used for analysis. The apparatus used was a P / ACE ^™ MDQ manufactured by Beckman Coulter, Inc. with a laser-induced fluorescence detector attached. A capillary with an inner diameter of 20 μm and a length of 30 cm (effective length of 20 cm) was used. The electrophoresis conditions were an electrophoresis voltage of 25 kV, a temperature of 25 ° C., and detection was performed at an excitation wavelength of 488 nm and a fluorescence wavelength of 520 nm. . As the running buffer, 120 mM borate buffer (pH 10.2) was used. The results of separation by capillary electrophoresis are shown in the upper part of FIG. Since a single peak was detected around 14 minutes, it was expected that APTS-Man-6-P could be separated by capillary electrophoresis.

Next, in order to confirm that this peak was derived from Man-6-P, alkaline phosphatase treatment was performed as follows. 30 U alkaline phosphatase in 5 mM MgCl ₂ , 50
In addition to APTS-Man-6-P dissolved in mM Tris-HCl (pH 9.0), the mixture was incubated at 37 ° C. for 30 min. After inactivating the enzyme by incubating at 65 ° C. for 30 min, the filtrate was diluted 20-fold through a 0.22 μm filter and analyzed under the above conditions. As a result, the peak after the alkaline phosphatase reaction was eluted at around 6.6 minutes (lower row in FIG. 1). Since this elution time coincided with that of APTS-labeled mannose (APTS-Man), it was confirmed that the peak observed at around 14 minutes was APTS-Man-6-P.

The labeling efficiency when the initial concentration of Man-6-P was changed was investigated. Labeling was performed by the method described above. As a result, there was a correlation between the peak area and the concentration in the range of 50 pmol to 10 nmol at the square of the correlation coefficient r ² = 0.999 (FIG. 2).

  As described above, it was confirmed from the results of Example 1 that the phosphorylated saccharide can be labeled with the fluorescent labeling substance, and the phosphorylated saccharide labeled with the fluorescent labeling substance can be separated and quantified by capillary electrophoresis.

[Example 2] Simultaneous analysis with neutral sugar and amino sugar As sugar chain samples used in Example 2, N-acetylgalactosamine (GalNAc), N-acetylglucosamine (GlcNAc), rhamnose (Rha), mannose (Man ), Glucose (Glc), fucose (Fuc), galactose (Gal), and mannose-6-phosphate (Man-6-P) were mixed to prepare 0.8 nmol each. In this example, the sugar chain sample was labeled with APTS in the same manner as in the method shown in Example 1, and the detection was performed in the same manner as in the method shown in Example 1.

The result is shown in FIG. The seven peaks excluding mannose-6-phosphate have almost the same elution time as the monosaccharide labeled with APTS, and peaks 1 to 7 are APTS-GalNAc, APTS-GlcNAc, APTS-Rha and APTS-, respectively. Man, APTS-Glc, APTS-Fuc and APTS-Gal were confirmed. For peak 8, the elution time was the same as APTS-Man-6-P shown in Example 1. From the above, Man-6-P can be fluorescently labeled simultaneously with other neutral sugars and amino sugars, and APTS-Man-6-P can be labeled with other neutral sugars and amino sugars by capillary electrophoresis. It proved possible to analyze based on the difference of elution time with amino sugar.

[Example 3] Fluorescence labeling of mannose-6-phosphate with 2-aminopyridine In this example, conditions for separating phosphorylated sugars from other sugar chains using an anion exchange column were examined. Mannose-6-phosphate was purchased commercially from Sigma. A kit for labeling with 2-aminopyridine (hereinafter referred to as PA) was purchased from TaKaRa-Bio.

A glycan sample (mannose (Man) or mannose-6-phosphate (Man-6-P), 10 nmol) is dried in a glass tube (TaKaRa-Bio PAL-Station) under reduced pressure, 0.7 M 2- 10 μL of Aminopyridine / acetic acid-MeOH (5: 6) solution was added and reacted at 65 ° C. for 90 min. The reagent was dried at 60 ° C. for 20 min while blowing nitrogen gas. Next, 10 μL of 1 M Borane-dimetylamine complex was added, and a reduction reaction was performed at 60 ° C. for 100 min. 20 μL of methanol and 40 μL of toluene were added and dried at 50 ° C. for 10 min while blowing nitrogen gas. The above operation was repeated, and further 50 μL of toluene was added and dried in the presence of nitrogen gas at 50 ° C. for 10 min. This was redissolved in an appropriate amount of distilled water and analyzed on the column.

  First, separation conditions were examined. The column temperature was 65 ° C., and the attached fluorescence detector was measured with an excitation wavelength of 310 nm and a fluorescence wavelength of 380 nm.

First, using Cosmosyl C18-5P (Nacalai Tesque: 4.6 mmφ × 150 mm) as the column separation mode, 0.2 M potassium borate buffer (pH 8.5) / acetonitrile = 3: 7
Separation was attempted at a flow rate of 0.3 mL / min. However, Man-6-P-PA was eluted in the flow-through fraction around 1 minute, and it was concluded that it was difficult to discuss the quantitativeness under these conditions.

Next, separation was attempted at a flow rate of 0.3 mL / min using a solvent of 0.7 M borate buffer (pH 9.0) / acetonitrile = 9: 1 which is a condition for normal monosaccharide analysis. Analysis is performed on a column for monosaccharide analysis (PALPAK
This was performed using TypeA (TaKaRa) or TSK-GEL SUGAR AXI (TOSOH; 4.6 mmφ × 250 mm). Man-PA was eluted at approximately 70 minutes, but Man-6-P-PA was not eluted under these conditions.

Next, elution was attempted by changing the ionic strength of the solvent. The flow rate was 0.3 mL / min. When using a solvent of 0.25 M borate buffer (pH 8.5) / acetonitrile = 9: 1, Man-PA
Even it was not eluted. When 1 M borate buffer (pH 8.5) / acetonitrile = 9: 1 solvent was used, Man-PA had an elution time of 53 minutes, but Man-6-P-PA was not eluted. It was.

Furthermore, elution was examined by increasing the salt concentration of the solvent. Elution was attempted with 0.7 M borate buffer (pH 8.0) containing 0.3 M KCl, but Man-6-P-PA was not eluted even after 2 hours.
Therefore, when the pH was further adjusted to 9.0, it was eluted in 40 minutes, and separation with an anion exchange column of Man-6-P-PA became possible (FIG. 4).

  From the above results, it was found that, when an anion exchange column was used, it was impossible to separate phosphorylated saccharide from other glycans by examining normal conditions. In order to separate phosphorylated saccharide from other sugar chains using an anion exchange column, it is clear that a boric acid buffer prepared to have a predetermined pH and a predetermined salt concentration must be used. It became.

[Example 4] Simultaneous analysis of mannose-6-phosphate with other neutral sugars and amino sugars using a fluorescent labeling method with 2-aminopyridine N-acetylgalactosamine as a sugar chain sample used in Example 4 (GalNAc), N-acetylglucosamine (GlcNAc), mannose (Man), fucose (Fuc), galactose (Gal) and mannose-6-phosphate (Man-6-P) are mixed to 10 nmol each. The prepared solution was prepared. next,
The prepared sugar chain sample was dried under reduced pressure in a glass tube (for TaKaRa-Bio PAL-Station), followed by fluorescent labeling in the same manner as in Example 3.

The solvent was used at a flow rate of 0.3 mL / min, 0.7 M borate buffer (pH 9.0) containing 0.05 M KCl from 0 to 120 minutes, and 0.7 M borate buffer containing 0.3 M KCl (120 to 80 minutes). pH 9.0), 180-240
The minutes were separated with 0.7 M borate buffer (pH 9.0) containing 0.05 M KCl.

The result is shown in FIG. The five peaks excluding mannose-6-phosphate have almost the same elution time as the commercially available 2-aminopyridine-labeled monosaccharide, and peaks 1 to 5 are PA-GalNAc, PA-GlcNAc, It was confirmed as PA-Man, PA-Fuc, and PA-Gal. For peak 6, as in Example 1, this was fractionated and reacted with alkaline phosphatase and then electrophoresed again under the same conditions. As a result, it was eluted at the position of PA-Man. Proven to be -P. From the above results, Man-6-P can be fluorescently labeled simultaneously with other neutral sugars and amino sugars, and APTS-Man-6-P can be labeled with other neutral sugars and It proved possible to analyze based on the difference of elution time with amino sugar.

In this example, Man-6-P was labeled with 2-aminopyridine at an initial monosaccharide amount of 0.1 nmol, 0.3 nmol, 0.5 nmol or 1 nmol. The result is shown in FIG. As can be seen from FIG. 6, there was a correlation between the peak area and the concentration of Man-6-P in the range where the initial monosaccharide amount was 0.1 to 1 nmol. From this result, it was confirmed that phosphorylated saccharide labeled with a fluorescent labeling substance could be quantified while being separated by an anion exchange column.

It is a characteristic view which shows the result of the separation by capillary electrophoresis, the upper stage shows the result before the alkaline phosphatase treatment, and the lower stage shows the result after the alkaline phosphatase treatment. It is a characteristic figure which shows the labeling efficiency at the time of changing the initial concentration of Man-6-P. It is a characteristic view which shows the result of having labeled with the fluorescent labeling substance with respect to the sugar_chain | carbohydrate sample containing phosphorylated sugar, neutral sugar, and amino sugar, and isolate | separated by capillary electrophoresis. It is a characteristic view which shows the result of isolation | separation of the phosphorylated saccharide labeled with the fluorescent labeling substance by an anion exchange column. It is a characteristic view which shows the result of having labeled with the fluorescent labeling substance with respect to the sugar_chain | carbohydrate sample containing phosphorylated sugar, neutral sugar, and amino sugar, and isolate | separated with the anion exchange column. It is a characteristic figure which shows the labeling efficiency at the time of changing the initial concentration of Man-6-P.

Claims (6)

The phosphorylated saccharide, a mixture containing a neutral sugar other than phosphorylated saccharide and an amino sugar, and a fluorescent labeling substance, and the phosphorylated saccharide , the other neutral sugar and amino sugar, and the fluorescently labeled substance And a step of combining
Separating the phosphorylated saccharide bound to the fluorescent labeling substance contained in the solution from the other neutral sugar and the amino sugar bound to the fluorescent labeling substance, and detecting the phosphorylated saccharide Analysis method. The analysis of phosphorylated saccharide according to claim 1, wherein the sugar chain bonded to the glycoprotein is chemically or enzymatically cleaved, and the phosphorylated saccharide contained in the obtained sugar chain is analyzed. Method. 2. The phosphorylation according to claim 1, wherein in the step of detecting the phosphorylated saccharide, the phosphorylated saccharide is separated from the other neutral saccharide and amino sugar by an anion exchange column or capillary electrophoresis. Analysis method of sugar.   2. The phosphorylated saccharide analysis according to claim 1, wherein in the step of detecting the phosphorylated saccharide, the phosphorylated saccharide contained in the solution is quantified based on a fluorescence amount derived from a fluorescent labeling substance bound to the phosphorylated saccharide. Method.   2. The method for analyzing phosphorylated saccharide according to claim 1, wherein the fluorescent labeling substance is 8-aminopyrene-1,3,6-trisulfonate and / or 2-aminopyridine. In the step of detecting the phosphorylated saccharide, a borate buffer solution having a salt concentration of 0.1 M or more and a pH of 9.0 or more.
The method for analyzing phosphorylated saccharide according to claim 1, wherein:

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