JP7185232B2 - Methods for analyzing glycosaminoglycans - Google Patents

Description

The present invention relates to a method for analyzing glycosaminoglycans.

Glycosaminoglycans (hereinafter abbreviated as “GAG”) are linear polysaccharides having a structure in which disaccharides composed of uronic acid or galactose and hexosamine are repeatedly arranged as constitutional units. It is classified into hyaluronic acid, chondroitin sulfate, dermatan sulfate, keratan sulfate, heparan sulfate, etc. according to the disaccharide composition. GAG is known as a polysaccharide that exists in vivo in a protein-bound or non-bound form on cell membranes and extracellular matrices, and is a substance that has attracted attention in the fields of biochemistry, biology, pharmacy, medicine, and the like.

For example, mucopolysaccharidoses are one of the diseases caused by congenital disorders (lowered activity) or deficiency of enzymes involved in the degradation metabolism of GAG. In mucopolysaccharidosis, specific GAGs accumulate in living tissues depending on the type of enzyme that is damaged or deficient. Therefore, mucopolysaccharidoses can be diagnosed by analyzing the types of GAGs contained in biological samples such as blood.

Conventionally, GAG contained in a biological sample is decomposed into disaccharides by a degrading enzyme, separated by liquid chromatography, and analyzed by measuring light intensity such as absorbance and fluorescence intensity. However, since the disaccharides cannot be sufficiently separated by liquid chromatography, it has been difficult to measure multiple types of GAGs simultaneously.

On the other hand, Patent Document 1 and Non-Patent Document 1 disclose a method of simultaneously analyzing multiple types of GAGs contained in a biological sample by using liquid chromatography mass spectrometry. In the method described in Patent Document 1, a reversed-phase column or a Hypercarb (registered trademark) column is used as the liquid chromatography column, and in the method described in Non-Patent Document 1, a Hypercarb column is used. .

Japanese Patent Application Laid-Open No. 2008-102114

Shunji Tomatsu, et al., "Establishment of Glycosaminoglycan Assays for Mucopolysaccharidoses" Metabolites 2014, 4, 655-679; doi:10.3390/metabo4030655

Although the reversed-phase column is commonly used in liquid chromatography, it is difficult to stably separate and analyze highly polar compounds such as disaccharides that constitute GAGs due to their weak retention. On the other hand, the hypercurve column is a column filled with a base material made of porous graphite carbon, and has the property of being excellent in retention and separation of highly polar compounds. However, there are variations in retention performance, separation performance, etc. between product lots of hypercurve columns, and there is a problem that the reproducibility of analysis is lowered depending on the magnitude of the variation.

The problem to be solved by the present invention is to provide a method capable of stably and highly reproducibly analyzing disaccharides derived from glycosaminoglycans.

A first aspect of the present invention, which has been made to solve the above problems,
a first step of adding multiple types of glycosaminoglycan-specific enzymes to a biological sample to generate multiple types of disaccharides derived from glycosaminoglycans contained in the biological sample;
A second step of separating and analyzing the plurality of types of disaccharides by liquid chromatography mass spectrometry,
The present invention relates to a method for analyzing glycosaminoglycans, wherein the column used in liquid chromatography in the liquid chromatography-mass spectrometry method is a column filled with a solid-phase carrier to which an amide group (carbamoyl group) as a functional group is bound.

In addition, the second aspect of the present invention, which was made to solve the above problems,
a first step of adding multiple types of glycosaminoglycan-specific enzymes to a biological sample to generate multiple types of disaccharides derived from glycosaminoglycans contained in the biological sample;
A second step of separating and analyzing the plurality of types of disaccharides by liquid chromatography mass spectrometry,
The present invention relates to a method for analyzing glycosaminoglycans, wherein the column for liquid chromatography in the liquid chromatography-mass spectrometry method is a column filled with a solid-phase carrier to which an adamantyl group as a functional group is bound.

In the method for analyzing glycosaminoglycans according to the present invention, glycosaminoglycans in a biological sample are enzymatically degraded to produce a plurality of types of disaccharides, and then these disaccharides are analyzed using liquid chromatography mass spectrometry. Separate and analyze. The disaccharides that constitute glycosaminoglycans are highly negatively charged, highly polar compounds with numerous sulfate and carboxyl groups. In the present invention, liquid chromatography in liquid chromatography-mass spectrometry uses a column packed with a solid-phase carrier to which an amide group or an adamantyl group, which is a functional group that strongly binds to a highly polar compound, is bonded. can be analyzed stably and with high reproducibility.

A combination of precursor ion m/z and product ion m/z of each of five disaccharides and one internal standard substance (chondrosin) was set as an MRM transition, and LC/MS/MS analysis was performed. Resulting MRM chromatogram. A combination of precursor ion m/z and product ion m/z for each of the six disaccharides and one internal standard substance (chondrosin) was set as the MRM transition and analyzed by LC/MS/MS. Resulting MRM chromatogram. An MRM chromatogram obtained by analyzing glycosaminoglycans contained in a blood (whole blood) sample using a liquid chromatography mass spectrometer. An MRM chromatogram obtained by analyzing glycosaminoglycans contained in a blood (serum) sample using a liquid chromatography mass spectrometer.

EXAMPLES The present invention will be described in more detail below based on examples, but these are not intended to limit the present invention in any way.

[Example 1]
First, it was examined whether analysis of glycosaminoglycans (GAG) contained in biological samples is possible by MRM (multiple reaction monitoring) measurement using a liquid chromatograph mass spectrometer (LC/MS/MS).
<Sample preparation>
A 1 μmol/L aqueous solution of known compounds (including an internal standard substance (chondrosin) for quantitative analysis) shown in Table 1 below was prepared and used as a sample. All the compounds shown in Table 1 were purchased from Seikagaku Corporation.

In Table 1, "CS" is an abbreviation for chondroitin sulfate, "DS" for dermatan sulfate, "HS" for heparan sulfate, and "KS" for keratan sulfate.
In addition, ΔDi-4S and Di-0S are disaccharides obtained by degrading (digesting) chondroitin sulfate and dermatan sulfate with chondroitinase B, respectively, and ΔDiHS-0S, ΔDiHS-NS, and ΔDiHS-6S are disaccharides obtained by decomposing (digesting) heparan sulfate into heparitinase. Gal-GlcNac(6S) and Gal(6S)-GlcNac(6S) are disaccharides produced by the decomposition of keratan sulfate with keratanase II.

In addition, the equipment names and analysis conditions used for liquid chromatography mass spectrometry are as follows.
<Equipment>
Liquid chromatograph: Ultra high performance liquid chromatograph Nexera X2 (manufactured by Shimadzu Corporation)
Mass spectrometer: Ultrafast triple quadrupole mass spectrometer LCMS-8050 (manufactured by Shimadzu Corporation)

<LC analysis conditions>
Column: Inert Sustain Amide Column (GL Sciences Inc.)
(2.1mm I.D. × 100mmL, 3.0μm)
Mobile phase: Mobile phase A 5mmol/L ammonium formate-acetonitrile
Mobile phase B ammonium formate - ultrapure water gradient: B concentration 5% by weight (0-1min) → 30% by weight (4min) → 90% by weight (5-8min) → 5% by weight (8.01-13min)
Flow rate: 0.3mL/min
Column temperature: 40℃
Injection volume: 1 μL

<MS analysis conditions>
Ionization mode: ESI (negative)
Analysis mode: MRM
Nebulizer gas flow rate: 3.0L/min
Drying gas flow rate: 10.0mL/min
Interface gas flow rate: 10.0L/min
Interface temperature: 300℃
Desolvation tube (DL) temperature: 250°C
Heat block temperature: 400℃

Table 2 shows the m/z (this combination is called MRM transition) and CE of the precursor ions and product ions of the disaccharides shown in Table 1, excluding ΔDiHs-6S. The values shown in Table 2 were set in consideration of disaccharide detection sensitivity and separation from isobaric substances. However, ΔDi-4S, which has the same precursor ion as ΔDiHs-6S, produces the same product ion (m/z 300.10) as ΔDiHs-6S by mass spectrometry (MS/MS), so both are separated by MS. cannot be detected by In addition, ΔDiHS-0S, which has the same precursor ion as ΔDi-0S, generates the same product ion (m/z 175.10) as ΔDi-0S by MS/MS, so both are separated by MS and detected. I can't.

FIG. 1 shows the MRM chromatogram obtained as a result of analysis by LC/MS/MS. Figure 1 shows, from top to bottom, the m/z and product of precursor ions of ΔDi-4S, ΔDiHs-0S, ΔDiHS-NS, Gal-GlcNac(6S), Gal(6S)-GlcNac(6S), and chondrosin. MRM chromatograms obtained as a result of LC/MS/MS analysis by setting m/z combinations of ions as MRM transitions are shown. As shown in the MRM chromatogram at the top of FIG. 1, when the MRM transition is a combination of the precursor ion m/z and the product ion m/z of ΔDi-4S, the ΔDi-4S peak has ΔDiHS-6S overlapped, and the two could not be separated by either LC or MS. This means that ΔDi-4S and ΔDiHS-6S have equivalent elution times in liquid chromatography.

When multiple types of disaccharides that cannot be separated by either liquid chromatography or mass spectrometry are to be analyzed, an enzyme that produces one of the multiple types of disaccharides is added to the biological sample. A sample obtained by adding an enzyme that produces another type of disaccharide to a biological sample, and a sample that an enzyme that produces another type of disaccharide is added to a biological sample are prepared in order, and these samples are prepared. are sequentially analyzed by liquid chromatography mass spectrometry. This allows individual analysis of multiple types of disaccharides.

[Example 2]
The same sample as in Example 1 was subjected to MRM measurement using LC/MS by changing the column type in the LC/MS/MS liquid chromatography. LC analysis conditions and MS analysis conditions are as follows.

<LC analysis conditions>
Column: Capsule Pack Inert Adme Column (Osaka Soda Co., Ltd.)
(2.0mm I.D. × 150mmL, 3.0μm)
Mobile phase: Mobile phase A 0.1% formic acid - ultrapure water
Mobile phase B 0.1% formic acid-acetonitrile isocratic: B concentration 2% by weight
Flow rate: 0.3mL/min
Column temperature: 50°C
Injection volume: 1 μL

<MS analysis conditions>
Ionization mode: ESI (negative)
Analysis mode: MRM
Nebulizer gas flow rate: 3.0L/min
Drying gas flow rate: 10.0 mL/min
Heating gas flow rate: 10.0 mL/min
Interface temperature: 150℃
Desolvation tube (DL) temperature: 150°C
Heat block temperature: 400℃

The conditions of MRM measurement (MRM transition and CE) are the same as in Example 1 (Table 2).

FIG. 2 shows the MRM chromatogram obtained as a result of analysis by LC/MS/MS. In FIG. 2, m A combination of /z and product ion m/z was set as an MRM transition, and an MRM chromatogram obtained as a result of LC/MS/MS analysis is shown. However, as shown in FIG. 2, the top MRM chromatogram shows a ΔDi-4S peak and a ΔDiHs-6S peak, and the second MRM chromatogram from the top shows a ΔDi-0S peak and a ΔDi-0S peak. A peak of ΔDiHS-0S is observed. In addition, two peaks of Gal-GlcNAc(6S) are observed in the fifth MRM chromatogram from the top.

Disaccharides with two peaks in one MRM chromatogram are considered to have undergone anomeric separation during liquid chromatography-mass spectrometry. For example, the MRM chromatogram at the top of FIG. 2 shows three peaks. Some show ΔDiHS-6S peaks. Anomer separation occurs in both ΔDi-4S and ΔDiHS-6S. Three peaks appeared in the MRM chromatogram as a result of the combination of the two peaks as a result of the close retention times. In addition, two peaks can be seen in the second MRM chromatogram from the top of FIG. indicates the peak of ΔDiHS-0S. This is because anomeric separation occurred in ΔDi-0S, and as a result of combining the peak of the anomer with strong retention with the peak of ΔDiHS-0S, two peaks appeared in the MRM chromatogram.

A peak in which multiple types of disaccharide peaks are combined cannot be used for quantification of these disaccharides. Therefore, in such cases, the peak not combined with other disaccharides is used to quantify the disaccharide. Specifically, for example, in the MRM chromatogram at the top of FIG. 2, ΔDi-4S is quantified using the peak on the left with a short retention time, and ΔDiHS-6S is quantified using the peak on the right with a long retention time. and the middle peak is not used for quantification of either ΔDi-4S or ΔDiHS-6S.

[Example 3]
As a biological sample, blood (whole blood) added with a mixture of GAG-specific enzymes chondroitinase B, heparitinase, and keratanase II was soaked in filter paper and dried. Using the (DBS)) ) sample, the recovered product containing the disaccharide extracted from the DBS sample was introduced into LC/MS/MS for quantitative analysis of GAGs. LC analysis conditions, MS analysis conditions, and MRM measurement conditions were set to the same conditions as in Example 2. In this example, 10 DBS samples (DBS1-10) were prepared. The procedure for obtaining the recovered product from the DBS sample and introducing it into the LC/MS/MS is as follows.

<Recovery of Disaccharide and Introduction to LC/MS/MS>
1. A blood-impregnated portion of each DBS sample was cut out with a DBS puncher (PerkinElmer (registered trademark), PerkinElmer Japan Co., Ltd.) to obtain a disk (3.3 mm in diameter ).
2. The disc was placed in each well of a 96-well filter plate (Omega 10K, Nippon Pall Co., Ltd.) containing 100 μL of 0.1% BSA.
3. GAG-specific enzymes (chondroitinase B, heparitinase and keratanase II) were added to each well and incubated overnight at 37°C.
4. The resulting GAG digest was filtered through a filter plate, and the resulting filtrate was centrifuged at 2500 xg for 15 minutes, after which the harvest was introduced into LC/MS/MS.

<Analysis results>
FIG. 3 shows the MRM chromatogram obtained as a result of analysis by LC/MS/MS. Figure 3 shows m/z of precursor ions and product ions of ΔDi-4S, ΔDiHS-0S, ΔDiHs-NS, Gal-GlcNAc(6S), Gal(6S)-GlcNAc(6S), and chondrosin, respectively. MRM chromatograms obtained as a result of LC/MS/MS analysis by setting m/z combinations as MRM transitions are shown.

Table 3 shows the quantitative results of disaccharides contained in each sample. Quantitative values were calculated by one-point calibration (internal standard method) using the analysis results of a sample in which 1 μmol/L of the chondrosin solution (standard solution) shown in Table 2 was added to sample DBS4.

Table 4 below examines the influence of the matrix (contaminants) contained in the sample on the measurement. The influence of the matrix was calculated from the results of a sample obtained by adding 1 μmol/L of a standard solution to sample DBS4, a standard solution of 1 μmol/L, and sample DBS4.

[Example 4]
As a biological sample, a blood (serum) sample to which a mixture of GAG-specific enzymes chondroitinase B, heparitinase, and keratanase II was added was used, and the disaccharide-containing collected material extracted from each serum sample was subjected to LC/MS/ Quantitative analysis of GAG was performed by introducing into MS. LC analysis conditions, MS analysis conditions, and MRM measurement conditions were set to the same conditions as in Example 2. In this example, 10 serum samples (Serum 2, 4 , 6, 8, 10, 12, 14, 16, 18, 20) were prepared. The procedure for obtaining a harvest from a serum sample and introducing it into the LC/MS/MS is as follows.

<Recovery of Disaccharide and Introduction to LC/MS/MS>
1. 10 μL of serum sample and 90 μL of 50 mM Tris-HCl buffer (pH 7.0) were placed in wells of a 96-well filter plate (Omega 10K, Nippon Pall Co., Ltd.).
2. GAG-specific enzymes (chondroitinase B, heparitinase and keratanase II) were added to each well, and 60 μL of 50 mM Tris-HCl buffer (pH 7.0) was added and incubated overnight at 37°C.
3. After centrifuging the 96-well filter plate at 2200 xg for 15 minutes, the harvest was introduced into the LC/MS/MS.

<Analysis results>
FIG. 4 shows the MRM chromatogram obtained as a result of analysis by LC/MS/MS. Figure 4 shows m/z of precursor ions and product ions of ΔDi-4S, ΔDiHS-0S, ΔDiHs-NS, Gal-GlcNAc(6S), Gal(6S)-GlcNAc(6S), and chondrosin, respectively. MRM chromatograms obtained as a result of LC/MS/MS analysis by setting m/z combinations as MRM transitions are shown.

Table 5 shows the quantitative results of disaccharides contained in each sample. Quantitative values were calculated by one-point calibration (internal standard method) using the results of analyzing a sample obtained by adding 1 μmol/L of standard solution to serum sample Serum4.

Table 6 below examines the influence of the matrix. The effect of the matrix was calculated from the results of a sample obtained by adding 1 μmol/L of the standard solution to the serum sample Serum4, the standard solution of 1 μmol/L, and the serum sample Serum4.

Although the embodiments of the present invention have been described in detail above, those skilled in the art will understand that the embodiments are specific examples of the following aspects.

A first aspect of the present invention is
a first step of adding multiple types of glycosaminoglycan-specific enzymes to a biological sample to generate multiple types of disaccharides derived from glycosaminoglycans contained in the biological sample;
A second step of separating and analyzing the plurality of types of disaccharides by liquid chromatography mass spectrometry,
The analysis method, wherein the column used in liquid chromatography in the liquid chromatography-mass spectrometry method is a column packed with a solid-phase carrier to which an amide group as a functional group is bound.

Moreover, the second aspect of the present invention is
a first step of adding multiple types of glycosaminoglycan-specific enzymes to a biological sample to generate multiple types of disaccharides derived from glycosaminoglycans contained in the biological sample;
A second step of separating and analyzing the plurality of types of disaccharides by liquid chromatography mass spectrometry,
In the analysis method, the column used in liquid chromatography in the liquid chromatography-mass spectrometry method is a column packed with a solid phase carrier to which an adamantyl group as a functional group is bound.

In the method for analyzing glycosaminoglycans of the first aspect and the second aspect of the present invention, the liquid chromatography column in the liquid chromatography mass spectrometry method is a solid phase support to which an amide group and an adamantyl group as functional groups are bonded. A packed column was used. Both amide and adamantyl groups hold highly polar compounds such as the disaccharides that make up glycosaminoglycans due to strong interactions between them. Therefore, glycosaminoglycans can be analyzed stably and with high reproducibility.

The biological sample in the method for analyzing glycosaminoglycans (hereinafter abbreviated as "GAG") of the first and second aspects of the present invention contains GAGs, and separates GAGs by liquid chromatography mass spectrometry, Anything that can be analyzed can be used, and specific examples include blood (whole blood, serum, plasma), urine, saliva, and living tissue. Blood is preferable, and plasma or serum is particularly preferable, considering the ease of collection of biological samples and the high content of GAGs.

As the GAG-specific enzyme used in the GAG analysis method of the present invention, any enzyme can be used as long as it specifically decomposes GAG. In mucopolysaccharidosis, it is known that mainly keratan sulfate, heparan sulfate, and dermatan sulfate accumulate in living tissues. Therefore, when the results obtained by the GAG analysis method of the present invention are used to diagnose mucopolysaccharidosis, enzymes that specifically decompose chondroitin sulfate, dermatan sulfate, keratan sulfate, and heparan sulfate can be used. preferable. Moreover, in the present invention, two or more enzymes can be used in combination. Commercially available GAG-specific enzymes include, for example, keratanase, keratanase II, heparitinase, heparitinase I, heparitinase II, heparinase, chondroitinase B, and the like.

As described above, in the first aspect of the present invention, a column in which an amide group (carbamoyl group) as a functional group is bonded to a solid support is used in liquid chromatography in liquid chromatography mass spectrometry. Commercially available products of such columns include, for example, InertSustain (registered trademark) Amide analytical columns (GL Sciences Inc.). This is a HILIC (Hydrophilic Interaction Chromatography) column and is suitable for analysis of polar compounds.

Further, in the second aspect of the present invention, a column in which an adamantyl group as a functional group is bound to a solid support is used in liquid chromatography in liquid chromatography-mass spectrometry. Commercially available products of such a column include, for example, CAPCELL PAK INERT ADME column (Osaka Soda Co., Ltd.). Although this is a reversed-phase column, it has a better balance between hydrophobicity and surface polarity on the solid-phase surface than conventional reversed-phase columns, and can stably separate highly polar compounds.

In both the first aspect and the second aspect of the present invention, analysis conditions such as column size (inner diameter, length), solid phase carrier size, mobile phase properties (pH), mobile phase flow rate, column temperature, etc. The retention time of the disaccharide on the column varies depending on the Therefore, in the present invention, the analysis conditions are appropriately set so that the disaccharide retained in the column is eluted from the column at a time (retention time) appropriate for mass spectrometry.

In particular, in the methods for analyzing glycosaminoglycans of the first and second aspects of the present invention, a column in which a characteristic functional group is bound to a solid phase carrier is used as a liquid chromatography column, and an appropriate mobile phase is selected. By doing so, the separation performance of the disaccharides constituting the glycosaminoglycan can be enhanced by the column.

A third aspect of the present invention is the method for analyzing glycosaminoglycans according to the first aspect, wherein in the first step, among the plurality of types of glycosaminoglycan-specific enzymes, a glycosaminoglycan contained in the biological sample is adding to the biological sample a first enzyme that produces at least one of a plurality of types of disaccharides derived from saminoglycan; adding a second enzyme that produces one or more types of disaccharides derived from glycosaminoglycans other than the disaccharide produced by one enzyme,
In the second step, the biological sample to which the first enzyme has been added and the biological sample to which the second enzyme has been added are subjected to liquid chromatography mass spectrometry.

According to the method for analyzing glycosaminoglycans of the third aspect, it is possible to analyze disaccharides that have equivalent retention times in liquid chromatography and cannot be separated and detected by mass spectrometry. Examples of such disaccharides include ΔDi-4S and ΔDiHS-6S, ΔDi-0S and ΔDiHS-0S.

Therefore, a fourth aspect of the present invention is the method for analyzing glycosaminoglycans of the third aspect, wherein the disaccharide produced by the first enzyme contains ΔDi-4S, and the disaccharide produced by the second enzyme is the disaccharide contains ΔDiHS-6S, or the disaccharide produced by the first enzyme contains ΔDi-0S, and the disaccharide produced by the second enzyme contains ΔDiHS-0S. There is.

A fifth aspect of the present invention is the method for analyzing glycosaminoglycans of the second aspect, wherein in the second step, the MRM chromatogram obtained as a result of liquid chromatography mass spectrometry with one MRM transition When a gram contains 3 or more peaks, the peak with the shortest elution time or the peak with the longest elution time is used to quantify the disaccharide.
In the fifth aspect of the present invention, even when a disaccharide constituting a glycosaminoglycan produced by a glycosaminoglycan-specific enzyme is anomerically separated during liquid chromatography mass spectrometry, the disaccharide is It can be analyzed quantitatively.

The results obtained by the glycosaminoglycan analysis method of the first aspect or the second aspect of the present invention can be used to test for the presence or absence of mucopolysaccharidoses in subjects from whom biological samples were collected. Thus, another aspect of the first aspect of the present invention is
obtaining a biological sample from a subject;
adding a plurality of types of glycosaminoglycan-specific enzymes to the biological sample to generate a plurality of types of disaccharides derived from the glycosaminoglycans contained in the biological sample;
A step of separating and analyzing the plurality of types of disaccharides by liquid chromatography mass spectrometry;
A step of examining the presence or absence of mucopolysaccharidosis in the subject based on the analysis result,
The method for testing mucopolysaccharidosis, wherein the column used in liquid chromatography in the liquid chromatography-mass spectrometry method is a column filled with a solid phase carrier to which an amide group as a functional group is bound.

Also, another aspect of the second aspect of the present invention is
obtaining a biological sample from a subject;
adding a plurality of types of glycosaminoglycan-specific enzymes to the biological sample to generate a plurality of types of disaccharides derived from the glycosaminoglycans contained in the biological sample;
A step of separating and analyzing the plurality of types of disaccharides by liquid chromatography mass spectrometry;
A step of examining the presence or absence of mucopolysaccharidosis in the subject based on the analysis result,
The liquid chromatography column in the liquid chromatography-mass spectrometry method is a column filled with a solid-phase carrier to which an adamantyl group as a functional group is bound.

Claims (5)

a first step of adding multiple types of glycosaminoglycan-specific enzymes to a biological sample to generate multiple types of disaccharides derived from glycosaminoglycans contained in the biological sample;
A second step of separating and analyzing the plurality of types of disaccharides by liquid chromatography mass spectrometry,
The second step includes a separation step of separating the plurality of types of disaccharides by liquid chromatography, and a solution containing the separated plurality of types of disaccharides is introduced into a mass spectrometer to detect the plurality of types of disaccharides. and a detection step for
The column used in the liquid chromatography in the separation step is a column filled with a solid-phase carrier to which an amide group as a functional group is bound,
Simultaneously analyzing at least ΔDi-4S or ΔDiHS-6S, ΔDiHS-0S, ΔDiHS-NS, Gal-GlcAc(6S), Gal(6S)-GlcNAc(6S), and chondrosin among the plurality of types of disaccharides, A method for analyzing saminoglycans. a first step of adding multiple types of glycosaminoglycan-specific enzymes to a biological sample to generate multiple types of disaccharides derived from glycosaminoglycans contained in the biological sample;
a second step of separating and analyzing the plurality of types of disaccharides by liquid chromatography mass spectrometry;
with
The column used in liquid chromatography in the liquid chromatography-mass spectrometry method is a column filled with a solid-phase carrier to which an amide group as a functional group is bound,
In the first step, among the plurality of types of glycosaminoglycan-specific enzymes, the first step of generating at least one disaccharide among the plurality of types of disaccharides derived from glycosaminoglycans contained in the biological sample. a step of adding an enzyme to the biological sample; and one or more types of disaccharide derived from a glycosaminoglycan other than the disaccharide produced by the first enzyme among the plurality of types of glycosaminoglycan-specific enzymes. and adding a second enzyme that produces
The method for analyzing glycosaminoglycans, wherein in the second step, the biological sample to which the first enzyme has been added and the biological sample to which the second enzyme has been added are sequentially subjected to liquid chromatography mass spectrometry. The disaccharide produced by the first enzyme contains ΔDi-4S and the disaccharide produced by the second enzyme contains ΔDiHS-6S, or the disaccharide produced by the first enzyme contains ΔDi-0S, and the disaccharide produced by said second enzyme contains ΔDiHS-0S. a first step of adding multiple types of glycosaminoglycan-specific enzymes to a biological sample to generate multiple types of disaccharides derived from glycosaminoglycans contained in the biological sample;
A second step of separating and analyzing the plurality of types of disaccharides by liquid chromatography mass spectrometry,
A method for analyzing glycosaminoglycans, wherein the column used in liquid chromatography in the liquid chromatography-mass spectrometry method is a column filled with a solid phase carrier to which an adamantyl group as a functional group is bound. In the second step, when the MRM chromatogram obtained as a result of performing liquid chromatography mass spectrometry with one MRM transition contains three or more peaks, the peak with the shortest elution time or the peak with the longest elution time The method for analyzing glycosaminoglycans according to claim 4, wherein the disaccharide is quantified using

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