Method for in-vitro detection of mucopolysaccharide and pompare disease related biomarkers

Abstract

The invention discloses a method for in-vitro detection of mucopolysaccharide and pompare disease related biomarkers, and belongs to the technical field of detection. The method comprises the following steps: 1) carrying out enzymolysis treatment on a sample containing GAG by adopting GAG enzyme, purifying and separating to obtain a to-be-detected solution containing 23 oligosaccharides; 2) adding an internal standard substance into the solution to be detected, and performing quantitative detection by adopting a UPLC-MS / MS method; wherein a mobile phase A of liquid chromatography is an acetonitrile-water mixed solution of ammonium formate, a mobile phase B is acetonitrile, and gradient elution is carried out; the mass spectrum adopts a multi-ion reaction monitoring mode of negative ion electrospray ionization. The method realizes simultaneous detection of oligosaccharides such as an internal disaccharide (dp2) repeating unit containing a main chain structure, non-reducing end biomarkers (monosaccharide and disaccharide) and glucotetraose (Glc4), not only has sensitivity and specificity for detecting MPS subtypes, but also can provide feasible and accurate detection for MPS and PD.

Description

Technical Field

[0001] The present invention relates to a detection method, in particular to a method for detecting mucopolysaccharide and Pompe disease related biomarkers in vitro, belonging to the technical field of analysis and detection. Background Art

[0002] Current laboratory diagnosis of mucopolysaccharidosis (MPS) is based on the detection of storage products GAG, enzyme activity levels, and DNA levels. The colorimetric test using the dimethyl methylene blue (DMB) reaction to quantify GAG is low-cost and simple to operate, but has poor specificity, insufficient sensitivity, and a high false negative rate. Both MS / MS and fluorescence assays for enzyme activity have found that most newborns with activity below the cutoff are false positives. Many NBS laboratories perform DNA sequencing of related genes when DBS enzyme activity is low, but the understanding of the genotype / phenotype relationship is insufficient, showing a large number of inconclusive results.

[0003] GAGs accumulated in MPS patients are copolymers containing similar repeating units—iduronic acid (IdoA) or glucuronic acid (GlcA), N-acetylgalactosamine GalNAc, and N-acetylglucosamine (GlcNAc), including chondroitin sulfate (CS), dermatan sulfate (DS), heparan sulfate (HS), keratin sulfate (KS), and hyaluronic acid (HA). These polymers can be degraded by chemical and enzymatic degradation to produce simple oligosaccharide molecules containing repeating subunits. Chemical degradation decomposes DS, HS, and KS into disaccharide structures with the same repeating units, respectively, while the structural information of the characteristic non-reducing terminal carbohydrates is lost in this process. The specific GAG substructure information corresponds to the relevant defective enzyme, which affects the interpretation and subsequent treatment of different subtypes of MPS. GAG depolymerization using bacterial lytic enzymes can produce unsaturated internal disaccharide biomarkers ("internal disaccharide method") and characteristic non-reducing terminal residue biomarker GAG-NRE ("endogenous biomarker method"), which have excellent sensitivity and specificity compared with chemical degradation.

[0004] Acid alpha-glucosidase (GAA) enzyme activity is the mainstay of diagnostic testing for Pompe disease (PD). Enzyme replacement therapy (ERT) is currently a proven effective treatment for Pompe disease, but enzyme diagnostics are not useful for monitoring disease progression once ERT is initiated. In this setting, it is more effective to use a biomarker produced by digestion of glycogen and other branched glucose polymers, namely tetrasaccharide glucose (Glc4), which can be used for secondary sorting of samples after NBS based on primary enzyme activity and monitoring of enzyme replacement therapy. Summary of the invention

[0005] It should be noted that the present invention is not aimed at disease diagnosis, but is mainly an improvement proposed for the technical problems existing in the above-mentioned prior art. The purpose of the present invention is to provide a method for in vitro detection of mucopolysaccharides and Pompe disease-related biomarkers, which can not only detect mucopolysaccharides (MPS) and Pompe disease (PD) at the same time, but also depolymerize GAG ​​by bacterial lyase, combine the joint detection of unsaturated internal disaccharide biomarkers ("internal disaccharide method") and characteristic non-reducing terminal residue biomarker GAG-NRE ("endogenous biomarker method"), and provide a combination and use of oligosaccharide biomarkers for detecting one or more of multiple subtypes of MPS and PD. On this basis, standard products and internal standards are introduced to prepare standard curves for quantification of oligosaccharides, so that the detection method not only has the sensitivity and specificity of detecting different subtypes of MPS, but also can provide feasible and accurate diagnosis, prognosis and monitoring of treatment effects for different subtypes of MPS and Pompe disease.

[0006] In order to achieve the above technical objectives, the present invention provides a method for detecting mucopolysaccharide and Pompe disease related biomarkers in vitro, which comprises the following steps:

[0007] 1) The GAG-containing sample is enzymatically treated with GAG enzyme, and then purified and separated to obtain a test solution containing 23 oligosaccharides;

[0008] 2) An internal standard is added to the solution to be tested, and a UPLC-MS / MS method is used for quantitative detection; wherein the mobile phase A of the liquid chromatography is a mixed solution of ammonium formate-acetonitrile-water, and B is acetonitrile, with gradient elution; and the mass spectrometry adopts a multiple ion reaction monitoring mode of negative ion electrospray ionization.

[0009] As a preferred scheme, the GAG ​​enzyme includes Hep-I&Hep-II&Hep-III, Ch-ABC and K-II; wherein, Hep-I&Hep-II&Hep-III is composed of Hep-I, Hep-II and Hep-III according to the enzyme activity concentration ratio of 0.8-1.2:0.8-1.2:0.8-1.2, and the enzyme activity concentration ratio of Hep-I&Hep-II&Hep-III to Ch-ABC and K-II is 90:10-15:1-2; the addition amount of the GAG ​​enzyme relative to the GAG-containing sample is 25-40 mU / sample.

[0010] As a preferred solution, the conditions for the enzymatic hydrolysis treatment are: reaction at a temperature of 35 to 39° C. for 8 to 24 hours.

[0011] As a preferred solution, the purification and separation process is: adding CAN and EDTA solution to the enzymatic hydrolysis reaction solution, vortexing thoroughly, centrifuging and settling, and taking the supernatant.

[0012] As a preferred embodiment, the 23 oligosaccharides are: ΔUA–GlcNS (IV-S), ΔUA–GlcNAc (IV-A), ΔUA–GalNAc4S (ΔUA–4S), ΔUA2S–GlcNS6S (IS), ΔUA2S–GlcNS (III-S), ΔUA2S–GlcNAc6S (IA), ΔUA–GlcNS6S (II-S), ΔUA2S–GlcNAc (III-A), ΔUA–GlcNAc6S (II-A), ΔUA–GalNAc, ΔUA–

[0013] GalNAc6S(ΔUA–6S), ΔUA–GalNAc4S6S(ΔUA–diSE), ΔUA2S–

[0014] GalNAc4S (ΔDi–diSB), ΔUA2S–GalNAc6S (ΔDi–diSD), ΔUA2S–GalNAc (ΔDi–UA2S), Glcalpha1-6Glcalpha1-4G lcalpha1-4Glc, IdoAGlcNS, IdoA2SGlcNS, IdoAGlcNS(6S), IdoA2SGlcNS(6S), Galβ1-4GlcNAc(6S), Gal(6S )β1-4GlcNAc(6S), GalNAc4S, GalNAc6S, the corresponding structure codes (DSC) are: a4, a6, D0S0, D0A0, D0a4, D2S6, D2S0, D2A6, D0S6, D2A0, D0A6, D0a0, D0a6, D0a10, D2a4, D2a6, D2a0, Glc4, I0S0, I2S0, I0S6, I2S6, KS1 (KS-SO4), KS2 (KS-2SO4).

[0015] As a preferred embodiment, the internal standard substances are: chondroitin (IS-CS), ML-13C6-glucose tetrasaccharide (IS-Glc4), GlcNCOEt-6S (IP).

[0016] As a preferred embodiment, the liquid chromatography conditions are as follows: chromatographic column: ACQUITY UPLC BEH Amide Column, 1.7 μm, 2.1 mm×50 mm, column temperature: 40°C; mobile phase A: water containing 10 mM ammonium formate + 50% acetonitrile, mobile phase B: acetonitrile; gradient elution conditions: 0-0.5 min, 95% B-75% B, 0.5-1.25 min, 75% B, 1.25-2 min, 75% B-70% B, 2-3.25 min, 70% B-

[0017] 40% B, 3.25-3.75min, 40%-38.5% B, 3.75-4.25min, 38.5% B, 4.25-4.5min, 38.5%-0% B, 4.5-5min, 0% B, 5-5.1min, 0%-95% B, 5.1-7min, 95% B; flow rate 0.35mL / min; injection volume: 10μL.

[0018] The specific gradient elution conditions are:

[0019]

[0020]

[0021] As a preferred scheme, the mass spectrometry conditions are: multiple ion reaction monitoring mode of negative ion electrospray ionization, nebulizing gas: 50 kPa, heating gas: 50 kPa, curtain gas: 30 kPa, spray voltage: -4500 V, desolvation temperature: 500°C.

[0022] As a preferred solution, the mass spectrometry parameters of each oligosaccharide and internal standard are:

[0023] a4: Q1, 300; Q3, 198.9; DP, -96; CE, -27;

[0024] a6: Q1, 299.8; Q3, 198.7; DP, -107; CE, -24;

[0025] D0S0: ​​Q1, 415.9; Q3, 137.9; DP, -15; CE-26;

[0026] D0A0+D0a0: Q1, 378; Q3, 174.9; DP, -75; CE, -14;

[0027] D0a4: Q1, 457.9; Q3, 300.1; DP, -40; CE, -30;

[0028] D2S6: Q1, 576; Q3, 416; DP, -100; CE, -33;

[0029] D2S0+D0S6: Q1, 496; Q3, 416; DP, -150; CE, -26;

[0030] D2A6+D0a10+D2a4+D2a6: Q1, 457.9; Q3, 156.7; DP, -100; CE, -32;

[0031] D2A0+D0A6+D0a6+D2a0: Q1, 538; Q3, 457.9; DP, -150; CE, -21;

[0032] GLC4: Q1, 665.1; Q3, 503; DP, -132; CE, -19;

[0033] I0S0: Q1, 434.1; Q3, 312.8; DP, -20; CE, -18;

[0034] I2S0+I0S6: Q1, 514.1; Q3, 434; DP, -200; CE, -27;

[0035] I2S6: Q1, 594; Q3, 434; DP, -20; CE-20;

[0036] KS-SO4: Q1, 461.9; Q3, 360.9; DP, -200; CE, -33;

[0037] KS-2SO4: Q1, 542; Q3, 462; DP, -200; CE, -28;

[0038] IS-CS: Q1, 354.1; Q3, 192.9; DP, -124; CE, -21;

[0039] GLC4-IS: Q1, 671.1; Q3, 509.2; DP, -140; CE, -17;

[0040] IS-disaccharide: Q1, 471.8; Q3, 96.9; DP, -230; CE, -70.

[0041] As a preferred solution, the quantitative detection process is as follows: a standard curve is prepared from a mixed standard solution of 7 concentrations, with the concentration of the standard and internal standard solution as the X-axis and the peak area of ​​the standard and internal standard as the Y-axis; a linear regression analysis is performed to obtain a regression equation, the corresponding oligosaccharide peak area is substituted into the regression equation, and the concentration of the oligosaccharides in the sample is calculated.

[0042] As a more preferred scheme, the mixed standard solutions of the 7 concentrations are prepared by adding the calibration line matrix to the mixed standard working solution for dilution, the calibration line matrix is ​​a substitute matrix for urine, and the dilution multiples corresponding to the 7 concentrations of the mixed standard solutions are 2, 5, 10, 25, 50, and 100 times, respectively; in the mixed standard working solution, the concentrations of a4, a6, D0S0, D0A0+D0a0, D0a4, D2S6, D2S0+D0S6, D2A6+D0a10+D2a4+D2a6, D2A0+D0A6+D0a6+D2a0, and Glc4 are 50000, 200000, 10000, 50000, 200000, 50000, 50000, 25000, 100000, and 250000 ng / mL, respectively.

[0043] The specific preparation scheme of the mixed standard working solution is as follows:

[0044]

[0045] The concentration of the calibration line is designed as follows:

[0046]

[0047]

[0048] The above method includes quality control products: quality control urine containing three concentration levels (low, medium and high), the quality control products are prepared by adding mixed oligosaccharide working solution to normal urine, amniotic fluid, saliva and serum / plasma samples, the concentration of each oligosaccharide in the low, medium and high quality control is distributed in seven gradient concentrations of the mixed standard solution, and the target value is determined by detection.

[0049] The GAG-containing samples applicable to the present invention include but are not limited to: urine, amniotic fluid, saliva, serum / plasma, etc.

[0050] The test solution of the present invention is obtained by the following method (using urine as an example for illustration):

[0051] (1) Enzymatic hydrolysis of samples:

[0052] To the urine sample, add 9 μL, 12 μL, and 7.5 μL of 10 U / mL Hep-I & Hep-II & Hep-III, 5 U / mL Ch-ABC, and 0.2 U / mL K-II lyase solutions, respectively, add 41.5 μL of 50 mM Tris-Hcl (containing 2 mM CaCl2) buffer, vortex to mix, place in a constant temperature water bath, and allow the enzymatic reaction to proceed overnight at 37°C.

[0053] (2) Protein precipitation:

[0054] The next morning, the tube was taken out and 100 μL ACN, 5 μL IS working solution, and 2 μL 100 mM EDTA solution were added to the centrifuge tube. The tube was vortexed thoroughly and centrifuged at 10°C and 14,000 rpm for 10 min.

[0055] (3) Loading:

[0056] After centrifugation, take the supernatant to obtain the sample to be tested.

[0057] The mixed internal standard of the present invention is prepared into a mixed internal standard solution by methanol-water solution, wherein the internal standard mixed solution contains chondroitin (IS-CS), ML-13C6-glucose tetrasaccharide (IS-Glc4), The concentrations of GlcNCOEt-6S(IP) were 250, 250, and 100 μg / mL, respectively.

[0058] Compared with the prior art, the beneficial technical effects of the technical solution of the present invention are:

[0059] The present invention establishes a liquid chromatography-mass spectrometry method for detecting the content of mucopolysaccharides (MPS) and Pompe disease (PD) related biomarkers in urine by optimizing the sample pretreatment method and ultra-high performance liquid chromatography-mass spectrometry conditions. The present invention can simultaneously detect 23 oligosaccharides including a4, a6, D0S0, D0A0, D0a4, D2S6, D2S0, D2A6, D0S6, D2A0, D0A6, D0a0, D0a6, D0a10, D2a4, D2a6, D2a0, Glc4, I0S0, I2S0, I0S6, I2S6, KS1 (KS-SO4), KS2 (KS-2SO4), and chondroitin (IS-CS), ML-13C6-glucose tetrasaccharide (IS-Glc4), GlcNCOEt-6S (IP) and three non-physiological disaccharide internal standards. The LC-MS / MS method used to analyze oligosaccharides not only has the sensitivity and specificity to detect all MPS isoforms, but also meets the requirements for the quantification of relevant biomarkers.

[0060] The method of the present invention uses a specific mucopolysaccharide lyase to cleave the GAG ​​polymer, uses a non-physiological disaccharide as an internal standard, and simultaneously quantifies the internal disaccharide and the characteristic non-reducing end biomarker, retains the structural characteristic information, and performs LC-MS / MS detection. The quantitative evaluation of each biomarker is performed through the normalization strategy of creatinine in urine samples. At the same time, the method uses three levels of quality control products, low, medium, and high, to examine the accuracy and effectiveness of the method to ensure the accuracy of the test results.

[0061] The present invention realizes for the first time the purpose of simultaneously detecting 23 oligosaccharides and 3 non-physiological disaccharide internal standards in a sample by using liquid chromatography-tandem mass spectrometry technology. This method is easy to operate, not only has the sensitivity and specificity to detect MPS subtypes, but also can provide feasible and accurate detection for MPS and PD. BRIEF DESCRIPTION OF THE DRAWINGS

[0062] The accompanying drawings, which constitute a part of the specification of the present invention, are used to provide a further understanding of the contents of the present invention. The exemplary embodiments of the present invention and their descriptions are used to explain the present invention and do not constitute improper limitations on the present invention.

[0063] Figure 1 This is the TIC diagram of the quality control obtained in Example 1.

[0064] Figure 2 This is a comparison chart of the negative sample and the MPS-I positive TIC obtained in Example 1. The biomarkers of MPS-I are significantly increased.

[0065] Figure 3 This is a comparison chart of the negative sample and the MPS-II positive TIC obtained in Example 1. The biomarkers of MPS-II are significantly increased.

[0066] Figure 4 This is a comparison chart of the negative sample and the MPS-IV positive TIC obtained in Example 1. The biomarkers of MPS-IV are significantly increased.

[0067] Figure 5 This is a comparison chart of the TIC of the negative sample and the MPS-VI positive sample obtained in Example 1. The biomarkers of MPS-VI are significantly increased. DETAILED DESCRIPTION

[0068] It should be noted that the following detailed descriptions are illustrative and are intended to provide further explanation of the present application. Unless otherwise specified, all technical and scientific terms used herein have the same meanings as those commonly understood by those skilled in the art to which the present application belongs.

[0069] It should be noted that the terms used herein are only for describing specific embodiments and are not intended to limit the exemplary embodiments according to the present application. As used herein, unless the context clearly indicates otherwise, the singular form is also intended to include the plural form. In addition, it should be understood that when the terms "comprise" and / or "include" are used in this specification, it indicates the presence of features, steps, operations, devices, components and / or combinations thereof.

[0070] Embodiment 1:

[0071] (1) Preparation of samples to be tested:

[0072] ① Dissolution of lyase:

[0073]

[0074] ② Enzymatic hydrolysis of samples:

[0075] Pipette 30 μL of urine sample into a 1.5 mL centrifuge tube, add the lytic enzyme solution dissolved in the above table - 9 μL, 12 μL, and 7.5 μL of Hep-I & Hep-II & Hep-III, Ch-ABC, and K-II, respectively, add 41.5 μL of 50 mM Tris-Hcl (containing 2 mM CaCl2) buffer, vortex to mix, place in a constant temperature water bath, and perform enzymatic reaction at 37.5°C overnight.

[0076] ③Protein precipitation:

[0077] The next morning, the tube was taken out and 100 μL ACN, 5 μL IS working solution, and 2 μL 100 mM EDTA solution were added to the centrifuge tube. The tube was vortexed thoroughly and centrifuged at 10°C and 14,000 rpm for 10 min.

[0078] ④ Loading:

[0079] After centrifugation, take the supernatant to obtain the sample to be tested.

[0080] (2) Ultra-high performance liquid chromatography-mass spectrometry detection:

[0081] The sample to be tested enters the chromatographic column for separation through gradient elution mode. The reference conditions of liquid chromatography are:

[0082] Column: Waters BEH Amide 50×2.1 mm, 1.7 μm;

[0083] Mobile phase A: 10 mM ammonium formate 50% acetonitrile water;

[0084] Mobile phase B: acetonitrile;

[0085] Column temperature: 40°C;

[0086] Injection volume: 10 μL;

[0087] Elution gradient:

[0088] Time(min) Flow(mL / min) ％A ％B Curve Initial 0.35 5 95 Initial 0.5 0.35 25 75 6 1.25 0.35 25 75 6 2 0.35 30 70 6 3.25 0.35 60 40 6 3.75 0.35 61.5 38.5 6 4.25 0.35 61.5 38.5 6 4.5 0.35 100 0 6 5 0.35 100 0 6 5.1 0.35 5 95 6 7 0.35 5 95 6

[0089] Mass spectrometry parameter reference conditions:

[0090] The multiple ion reaction monitoring mode of negative ion electrospray ionization was adopted, with nebulizing gas: 50 kPa, heating gas: 50 kPa, curtain gas: 30 kPa, spray voltage: -4500 V, and desolvation temperature: 500°C.

[0091]

[0092]

[0093] (3) Calculation results

[0094] Preparation of standard working solution: The concentrations of mixed standard working solutions of a4, a6, D0S0, D0A0+D0a0, D0a4, D2S6, D2S0+D0S6, D2A6+D0a10+D2a4+D2a6, D2A0+D0A6+D0a6+D2a0, and Glc4 are 50000, 200000, 10000, 50000, 200000, 50000, 50000, 25000, 100000, and 250000 ng / mL respectively. The preparation scheme of mixed standard working solution is:

[0095]

[0096] The mixed internal standard substance was prepared into a mixed internal standard solution by methanol-water solution, wherein the internal standard mixed solution contained chondroitin (IS-CS), ML-13C6-glucose tetrasaccharide (IS-Glc4), The concentrations of GlcNCOEt-6S(IP) were 250, 250, and 100 μg / mL, respectively.

[0097] The matrix of the calibration line is a urine substitute matrix exclusively synthesized by our laboratory, which is an aqueous solution including 0.15% g / mL creatinine, 0.05% g / mL serum protein, 0.01% g / mL glucose, 0.01% g / mL uric acid, 0.001% g / mL ascorbic acid, 1.8% g / mL urea, 0.02% g / mL citrulline, 0.1% g / mL disodium hydrogen phosphate, 0.1% g / mL anhydrous sodium sulfate, 0.4% g / mL sodium chloride, 0.2% g / mL potassium chloride, and 0.107% g / mL ammonium chloride.

[0098] The calibration line uses seven concentrations of mixed standard solution to prepare a standard curve, which is prepared by adding mixed oligosaccharide working solution to the surrogate matrix, and the calibration line concentration is shown above. The above working solution and calibration line matrix are used to prepare a standard curve, with the concentration of the standard and internal standard solution as the X-axis and the peak area of ​​the standard and internal standard as the Y-axis; linear regression analysis is performed to obtain a regression equation, and the corresponding oligosaccharide peak area is substituted into the regression equation to calculate the concentration of oligosaccharides in the sample;

[0099] ng / mL D1 D2 D3 D4 D5 D6 D7 a4 500 1000 2000 5000 10000 25000 50000 a6 2000 4000 8000 20000 40000 100000 200000 D0S0 100 200 400 1000 2000 5000 10000 D0A0+D0a0 500 1000 2000 5000 10000 25000 50000 D0a4 2000 4000 8000 20000 40000 100000 200000 D2S6 500 1000 2000 5000 10000 25000 50000 D2S0+D0S6 500 1000 2000 5000 10000 25000 50000 D2A6+D0a10+D2a4+D2a6 250 500 1000 2500 5000 12500 25000 D2A0+D0A6+D0a6+D2a0 1000 2000 4000 10000 20000 50000 100000 Glc4 2500 5000 10000 25000 50000 125000 250000

[0100] Data analysis: The linearity measured by the method of the present invention is as follows:

[0101]

[0102]

[0103] The quality control products include quality control urine at three concentration levels (low, medium and high). The quality control products are prepared by adding mixed oligosaccharide working solution to normal urine, amniotic fluid, saliva and serum / plasma samples. The concentration of each oligosaccharide in the low, medium and high quality control is distributed in seven gradient concentrations of the mixed standard solution, and the target value is determined by detection.

[0104] Example 2

[0105] The accurate values ​​of each biomarker of normal samples and MPS subtype positive samples were obtained by the method of the present invention. When the influence of different urine excretion rates was considered for correct interpretation, the urine oligosaccharide analysis was normalized to the urine creatinine concentration. The values ​​normalized with urine creatinine are as follows:

[0106]

[0107]

[0108] As shown in the table above, we have performed sample validation on disease samples of four different subtypes: MPS-I, MPS-II, MPS-IV, and MPS-VI. For different MPS subtypes, the method of the present invention sensitively detected the increase of specific biomarkers in disease samples, and quantified the content of biomarkers by urine creatinine normalization.

[0109] The method of the present invention detected internal disaccharides and characteristic non-reducing terminal residue biomarkers of dermatan sulfate (DS) and heparan sulfate (HS) in MPS-I type - D0S0, D0A0+D0a0, D0a4, D2S6, D2S0+D0S6, D2A6+D0a10+D2a4+D2a6, D2A0+D0A6+D0a6+D2a0, I0S0, I2S0+I0S6;

[0110] The method of the present invention detected internal disaccharides and characteristic non-reducing terminal residue biomarkers of dermatan sulfate (DS) and heparan sulfate (HS) in MPS-II type - D0S0, D0A0+D0a0, D0a4, D2S6, D2S0+D0S6, D2A6+D0a10+D2a4+D2a6, D2A0+D0A6+D0a6+D2a0, I2S0+I0S6, I2S6;

[0111] The method of the present invention detected oligosaccharides specific to keratan sulfate (KS) and chondroitin sulfate (CS) in MPS-IV type—a6, D0A0+D0a0, D0a4, D2A6+D0a10+D2a4+D2a6, KS-SO4, KS-2SO4;

[0112] The method of the present invention detected dermatan sulfate (DS) and chondroitin sulfate (CS) specific oligosaccharides in MPS-VI type - a4, D0A0+D0a0, D0a4, D2A6+D0a10+D2a4+D2a6, D2A0+D0A6+D0a6+D2a0.

[0113] Example 3

[0114] The present invention provides one or more oligosaccharide biomarker combinations and uses thereof in detecting multiple subtypes of MPS and PD in subjects, thereby improving the rapid and efficient diagnosis capability of related lysosomal storage diseases.

[0115]

[0116] Condition optimization example:

[0117] The present invention selects a combination of five biological enzymes, namely Hep-I, Hep-II, Hep-III, Ch-ABC and K-II, to respectively cleave the mucopolysaccharides accumulated in the body, namely dermatan sulfate (DS), heparan sulfate (HS), keratan sulfate (KS) and chondroitin sulfate (CS). These enzymes act through a β-elimination mechanism, resulting in the breaking of the bond between the hexosamine residue and the uronic acid, and producing a disaccharide containing Δ4,5-unsaturated uronic acid, which is connected to N-acetylated / N-sulfated hexosamine. KS is depolymerized by keratan sulfate enzymes, and disaccharides containing different sulfated galactose and N-acetylglucosamine residues are produced by hydrolysis. Biological enzymes are selective and specific for cutting specific sites, and the combined use of Hep-I, Hep-II, Hep-III, Ch-ABC and K-II is indispensable.

[0118] In the present invention, the amounts of the five enzymes were also optimized, and the following four solutions, A, B, C, and D, were compared:

[0119] μL A B C-Method of the Invention D Hep-I 0.75 1.5 3 7.5 Hep-II 0.75 1.5 3 7.5 Hep-III 0.75 1.5 3 7.5 ChB 3 6 12 30 KII 1.875 3.75 7.5 18.75

[0120] The peak areas were compared by injecting samples of group B as the comparison group:

[0121]

[0122] Comparing the three schemes A, B, and C, the response of each substance increases with the increase of the concentration of each bacterial enzyme. The response of each substance in group D is basically consistent with the increase of the concentration of each bacterial enzyme. The method of the present invention simultaneously meets the enzymatic hydrolysis effect and cost-effectiveness of the pretreatment.

[0123] This study used the LC-MS / MS method to simultaneously determine urine mucopolysaccharides (MPS) and Pompe disease (PD). The pre-treatment method of depolymerizing GAG by bacterial lysing enzymes combined the detection of internal unsaturated disaccharide biomarkers and characteristic non-reducing terminal residue biomarkers GAG-NRE, making the detection method sensitive and specific for detecting different subtypes of MPS. On this basis, standard products and internal standards were introduced to prepare standard curves for quantification of oligosaccharides, and the increase of specific biomarkers was sensitively detected in disease samples. The biomarkers were quantified by urine creatinine normalization, which can provide feasible and accurate diagnosis, prognosis and monitoring treatment for the disease.

[0124] The present invention is not limited to the above-mentioned optimal implementation mode. Anyone can derive other various forms of products under the inspiration of the present invention. However, no matter what changes are made in the shape or structure, all technical solutions that are the same or similar to those of the present application fall within the protection scope of the present invention.

[0125] The above description is only the preferred embodiment of the present application and is not intended to limit the present application. For those skilled in the art, the present application may have various modifications and variations. Any modification, equivalent replacement, improvement, etc. made within the spirit and principle of the present application shall be included in the protection scope of the present application.

Claims

1. A method for in vitro detection of mucopolysaccharides and Pompe disease-related biomarkers, characterized in that: The following steps are involved: 1) The GAG-containing sample is enzymatically treated with GAG enzyme, and then purified and separated to obtain a test solution containing 23 oligosaccharides; 2) An internal standard is added to the solution to be tested, and a UPLC-MS / MS method is used for quantitative detection; wherein the mobile phase A of the liquid chromatography is a mixed solution of ammonium formate-acetonitrile-water, and B is acetonitrile, with gradient elution; and the mass spectrometry adopts a multiple ion reaction monitoring mode of negative ion electrospray ionization.

2. The method for in vitro detection of mucopolysaccharides and Pompe disease-related biomarkers according to claim 1, characterized in that: The GAG ​​enzymes include Hep-I&Hep-II&Hep-III, Ch-ABC and K-II; wherein, Hep-I&Hep-II&Hep-III is composed of Hep-I, Hep-II and Hep-III. According to the enzyme activity concentration ratio of 0.8~1.2:0.8~1.2:0.8~1.2, the enzyme activity concentration ratio of Hep-I&Hep-II&Hep-III to Ch-ABC and K-II is 90:10~15:1~2; The amount of the GAG ​​enzyme added to the GAG-containing sample is 25-40 mU / sample; The conditions of the enzymatic hydrolysis treatment are: reaction at a temperature of 35 to 39° C. for 8 to 24 hours.

3. The method for in vitro detection of mucopolysaccharides and Pompe disease-related biomarkers according to claim 1, characterized in that: The purification and separation process is as follows: adding acetonitrile and EDTA solution to the enzymatic hydrolysis reaction solution, fully vortexing, centrifuging and settling, and taking the supernatant.

4. The method for in vitro detection of mucopolysaccharides and Pompe disease-related biomarkers according to claim 1, characterized in that: The 23 oligosaccharides are: ΔUA–GlcNS, ΔUA–GlcNAc, ΔUA–GalNAc4S, ΔUA2S–GlcNS6S, ΔUA2S–GlcNS, ΔUA2S–GlcNAc6S, ΔUA–GlcNS6S, ΔUA2S–GlcNAc, ΔUA–GlcNAc6S, ΔUA–GalNAc, ΔUA–GalNAc6S, ΔUA–GalNAc4S6S, ΔUA2S–GalNAc S, ΔUA2S–GalNAc6S, ΔUA2S–GalNAc, Glcalpha1-6Glcalpha1-4Glcalpha1-4Glc, IdoAGlcNS, IdoA2SGlcNS , IdoAGlcNS(6S), IdoA2SGlcNS(6S), Galβ1-4GlcNAc(6S), Gal(6S)β1-4GlcNAc(6S), GalNAc4S and GalNAc6S; The internal standards are: chondroitin, ML-13C6-glucose tetrasaccharide, ΔUA-2S GlcNCOEt-6S.

5. The method for in vitro detection of mucopolysaccharides and Pompe disease-related biomarkers according to claim 1, characterized in that: The conditions of the liquid chromatography are as follows: chromatographic column: ACQUITY UPLC BEH Amide Column, 1.7 μm, 2.1 mm×50 mm, column temperature: 40° C.; mobile phase A: water containing 10 mM ammonium formate + 50% acetonitrile, mobile phase B: acetonitrile; Gradient elution conditions: 0-0.5min, 95% B-75% B, 0.5-1.25min, 75% B, 1.25-2min, 75% B-70% B, 2-3.25min, 70% B-40% B, 3.25-3.75min, 40%-38.5% B, 3.75-4.25min, 38.5% B, 4.25-4.5min, 38.5%-0% B, 4.5-5min, 0% B, 5-5.1min, 0%-95% B, 5.1-7min, 95% B; flow rate 0.35mL / min; injection volume: 10μL.

6. The method for in vitro detection of mucopolysaccharides and Pompe disease-related biomarkers according to claim 1, characterized in that: The mass spectrometry conditions are: multiple ion reaction monitoring mode using negative ion electrospray ionization, nebulizing gas: 50 kPa, heating gas: 50 kPa, curtain gas: 30 kPa, spray voltage: -4500 V, desolvation temperature: 500°C.

7. The method for in vitro detection of mucopolysaccharide and Pompe disease-related biomarkers according to any one of claims 1 to 4, characterized in that: The quantitative detection process is as follows: a standard curve is prepared from a mixed standard solution of 7 concentrations, with the concentration of the standard and internal standard solution as the X-axis and the peak area of ​​the standard and internal standard as the Y-axis; a linear regression analysis is performed to obtain a regression equation, the corresponding oligosaccharide peak area is substituted into the regression equation, and the concentration of the oligosaccharide in the sample is calculated.

8. The method for in vitro detection of mucopolysaccharides and Pompe disease-related biomarkers according to claim 7, characterized in that: The 7 concentrations of mixed standard solutions are prepared by adding the calibration line matrix to the mixed standard working solution for dilution, the calibration line matrix is ​​a substitute matrix for urine, and the dilution multiples corresponding to the 7 concentrations of the mixed standard solutions are 2, 5, 10, 25, 50, and 100 times, respectively; in the mixed standard working solution, the concentrations of a4, a6, D0S0, D0A0+D0a0, D0a4, D2S6, D2S0+D0S6, D2A6+D0a10+D2a4+D2a6, D2A0+D0A6+D0a6+D2a0, and Glc4 are 50000, 200000, 10000, and 10000, respectively. 50000, 200000, 50000, 50000, 25000, 100000, 250000ng / mL.

9. The method for in vitro detection of mucopolysaccharides and Pompe disease-related biomarkers according to claim 6, characterized in that: The mass spectrometry parameters of each oligosaccharide and internal standard are: a4: Q1, 300; Q3, 198.9; DP, -96; CE, -27; a6: Q1, 299.8; Q3, 198.7; DP, -107; CE, -24; D0S0: ​​Q1, 415.9; Q3, 137.9; DP, -15; CE-26; D0A0+D0a0: Q1, 378; Q3, 174.9; DP, -75; CE, -14; D0a4: Q1, 457.9; Q3, 300.1; DP, -40; CE, -30; D2S6: Q1, 576; Q3, 416; DP, -100; CE, -33; D2S0+D0S6: Q1, 496; Q3, 416; DP, -150; CE, -26; D2A6+D0a10+D2a4+D2a6:Q1,457.9;Q3,156.7;DP,-100;CE,-32;D2A0+D0A6+D0a6+D2a0:Q1,538;Q3,457.9;DP,-150;CE,-21;GLC4:Q1,665.1;Q3,503;DP,-132;CE,-19; I0S0:Q1,434.1;Q3,312.8;DP,-20;CE,-18; I2S0+I0S6:Q1,514.1;Q3,434;DP,-200;CE,-27; I2S6:Q1,594;Q3,434;DP,-20;CE,-20; KS-SO4:Q1,461.9;Q3,360.9;DP,-200; CE,-33; KS-2SO4:Q1,542;Q3,462;DP,-200;CE,-28; IS-CS:Q1,354.1;Q3,192.9;DP,-124;CE,-21; GLC4-IS:Q1,671.1;Q3,509.2;DP,-140;CE,-17; IS-disaccharide:Q1,471.8;Q3,96.9;DP,-230;CE,-70。

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