Method for identifying connection position of glucosidic bond in oligosaccharide glucosidic bond isomer

Abstract

The invention provides a method for identifying a connection position of a glucosidic bond in an oligosaccharide glucosidic bond isomer, and belongs to the technical field of analysis and detection of saccharide isomers. According to the method for identifying the connection position of the glucosidic bond in the oligosaccharide glucosidic bond isomer, oligosaccharide compounds in a to-be-detected sample are separated at the same time on the basis of a derivatization reagent with a structure as shown in a formula I by utilizing a high performance liquid chromatography-tandem mass spectrometry technology; structural specificity diagnosis ions are generated based on an electron collision excitation collision mode of organic matter ions to identify the types of glycosidic bonds of disaccharide, trisaccharide, tetrasaccharide, pentasaccharide, hexasaccharide and heptasaccharide isomers of different glycosidic bonds. The method is high in identification efficiency and accurate in identification, and can meet the requirements of glycogen storage disease diagnosis biomarker research. And the formula I of the # imgabs0 # is shown in the specification.

Description

Technical Field

[0001] The present invention belongs to the technical field of analysis and detection of carbohydrate isomers, and particularly relates to a method for identifying the glycosidic bond linkage position in oligosaccharide glycosidic bond isomers. Background Art

[0002] Glycans, nucleic acids, proteins, and lipids are all very abundant and important biopolymers in organisms. The synthesis, degradation, and metabolism of glycans play very important roles in many physiological and pathological processes. Among them, oligosaccharides or oligosaccharides are straight-chain or branched-chain carbohydrate compounds formed by linking 2-10 identical or different monosaccharide units through glycosidic bonds. According to the number of monosaccharide units, they are divided into disaccharides, trisaccharides, tetrasaccharides, pentasaccharides, etc. In organisms, oligosaccharides are generally synthesized step by step from monosaccharides or produced by the degradation of polysaccharides. Oligosaccharides are a new type of functional sugar source and are widely used in the fields of food, health products, beverages, medicine, etc. At the same time, oligosaccharides can be used as specific biomarkers for some rare diseases related to sugar metabolism, such as glucose tetramer 6-α-D-glucopyranosyl-maltotriose (6-α-D-glucopyranosyl-maltotriose, Glc 4 ) can be used as a specific biomarker for Pompe disease.

[0003] However, compared with other biopolymers, due to the structural complexity of oligosaccharides, different oligosaccharide isomers may exhibit different biological functions. For example, Glc 4 can be used as a specific biomarker for Pompe disease screening, while its glycosidic bond linkage position isomer maltotetraose (M4) is not. The structural complexity of oligosaccharides lies not only in different monosaccharide building blocks, but also in different glycosidic bonds, variable conformations, and complex branching, which makes their analysis extremely challenging. Therefore, the current understanding of the biological functions of oligosaccharides is still very limited.

[0004] The main means for the analysis and detection of oligosaccharides include high performance liquid chromatography (HPLC) and nuclear magnetic resonance spectroscopy (NMR), etc. However, these methods have problems such as the need for reference substances, long analysis cycles, complex sample pretreatment steps, and great difficulty in the separation and identification of multiple isomers. Mass spectrometry (MS) is an analytical technique that simultaneously has high sensitivity, good specificity, and fast response speed among many analytical techniques. Especially high-resolution mass spectrometry shows absolute advantages in terms of sensitivity and anti-interference ability for samples with complex matrices and is becoming an important development trend in carbohydrate detection methods.

[0005] If a prior art discloses a method for simultaneously quantitatively analyzing 14 saccharide compounds by using a high performance liquid chromatography-tandem mass spectrometry device, it only discloses that 2,4-bis(diethylamino)-6-hydrazino-1,3,5-triazine (T3) reagent is used for labeling polysaccharides (specifically, polyglucose) in capillary electrophoresis-mass spectrometry technology and effectively solves chromatographic retention and ionization efficiency in liquid chromatography-tandem mass spectrometry technology, improving the separation degree of monosaccharides and disaccharides. However, the detailed structures of oligosaccharide isomers with the same mass-to-charge ratio still cannot be identified with high resolution. Summary of the Invention

[0006] The object of the present invention is to provide a method for identifying the glycosidic bond connection position in oligosaccharide glycosidic bond isomers, which can identify the glycosidic bond connection position in oligosaccharide glycosidic bond isomers with high resolution.

[0007] In order to achieve the object of the present invention, the present invention provides the following technical solutions: A method for identifying the glycosidic bond connection position in oligosaccharide glycosidic bond isomers, comprising the following steps: Performing a derivatization reaction on a sample to be tested and a derivatization reagent having the structure shown in Formula I to obtain a solution containing derivatized saccharide compounds; Formula I; Performing high performance liquid chromatography-tandem mass spectrometry detection on the solution containing derivatized saccharide compounds to obtain a mass chromatogram; the mass spectrometry detection is performed by using an electron impact excitation collision mode of organic ions to generate structure-specific diagnostic ions for detection: Analyzing the mass chromatogram to identify the type and connection order of glycosidic bonds in oligosaccharide glycosidic bond isomers; The analysis comprises the following steps: Based on the mechanism for differentiating the type and order of oligosaccharide glycosidic bond isomers by structure-specific diagnostic ions; a. Calculating the total number of monosaccharide units based on the mass-to-charge ratio of the oligosaccharide parent ion; b. Differentiating the type and order of oligosaccharide glycosidic bond isomers by using specific structure-specific diagnostic ions; c. Compared with the parent ion, using the ion losing C 6 H 12 O 6 +[(C 6 H 10 O 5 )×(n - 1)]+CH 2 O to determine whether the glycosidic bond between the nth monosaccharide and the (n + 1)th monosaccharide unit counted from the reducing end of the sugar is a 1,4-glycosidic bond connection; d. Compared with the parent ion, using the ion losing C 6 H 12 O6 +[(C 6 H 10 O 5 )×(n - 1)]+CH 2 ions to determine whether there is a 1,6-glycosidic bond connection between the nth monosaccharide and the (n + 1)th monosaccharide unit counted from the reducing end of the sugar; e. If there is no 1,4- and 1,6-glycosidic bond connection, use the "series of diagnostic ions of losing C 6 H 12 O 6 +[(C 6 H 10 O 5 )×(n - 2)]+C 3 H 4 O 2 " to determine whether there is a 1,3-glycosidic bond connection between the nth monosaccharide and the (n + 1)th monosaccharide unit counted from the reducing end of the sugar; f. If there is no 1,4-, 1,6- and 1,3-glycosidic bond connection, then there is a 1,2-glycosidic bond connection between any two monosaccharide units.

[0008] Preferably, the detection conditions of the high performance liquid chromatography include: the mobile phase is mobile phase A and mobile phase B, and the elution program is gradient elution; The mobile phase A is an aqueous-methanol-isopropanol solution of ammonium formate, the mass concentration of ammonium formate is 1 - 20 mmol / L, the volume ratio of water, methanol and isopropanol in the mobile phase A is 1 - 4:1:1, and the mobile phase B is acetonitrile; The program of the gradient elution is as follows: 1) From 0.00 to 2.00 minutes, maintain 75% by volume of mobile phase B; 2) From 2.00 to 58.00 minutes, 75% to 50% by volume of mobile phase B; 3) From 58.00 to 58.10 minutes, 50% to 75% by volume of mobile phase B; 4) From 58.10 to 75.00 minutes, maintain 75% by volume of mobile phase B.

[0009] Preferably, the pH value of the mobile phase A is 4.0 - 6.0.

[0010] Preferably, the chromatographic column in the high performance liquid chromatography is an amide chromatographic column.

[0011] Preferably, the detection conditions of the mass spectrometry include: Electrospray ionization source; Monitoring: MRM mode positive ion mode scanning; Fragmentation method: Electron excitation cleavage; The pressure of gas 1 is 50 - 70 psi; The pressure of gas 2 is 50 - 70 psi; The pressure of curtain gas is 35 - 50 psi; The pressure of collision gas is 7 - 10 psi; Gas 1, gas 2, collision gas and curtain gas are nitrogen; The ion source temperature is 550 - 600 °C; The spray voltage is 5500 - 6500 V; The declustering voltage is 50 - 80 V; The TOF MS collision voltage is 10 - 20 V; The TOF MSMS collision voltage is 12 - 16 V; The electron kinetic energy of the irradiated electron beam is 10 - 22 eV.

[0012] Preferably, the mass ratio of the oligosaccharide compound to the derivatization reagent in the sample to be tested is 1:50 - 1:2000.

[0013] Preferably, the temperature of the derivatization reaction is 30 - 45 °C and the time is 0.5 - 6 h.

[0014] Preferably, the sample to be tested is from the urine or plasma of patients with different subtypes of glycogen storage disease.

[0015] Preferably, the application of two hexasaccharides and two heptasaccharides in the urine of patients with different subtypes of glycogen storage disease as diagnostic biomarkers for GSD-II; The glycosidic bond linkage positions in the two hexasaccharides are 1,4-1,4-1,6-1,4-1,6 reducing end → non-reducing end and 1,4-1,4-1,4-1,6-1,6 reducing end → non-reducing end respectively; The glycosidic bond linkage positions in the two heptasaccharides are 1,4-1,4-1,4-1,4-1,4-1,6 reducing end → non-reducing end and 1,4-1,4-1,6-1,4-1,4-1,6 reducing end → non-reducing end respectively.

[0016] The present invention provides a method for identifying the glycosidic bond linkage position in oligosaccharide glycosidic bond isomers, comprising the following steps: performing a derivatization reaction on a sample to be tested and a derivatization reagent having the structure shown in Formula I to obtain a solution containing derivatized carbohydrate compounds; performing high performance liquid chromatography-tandem mass spectrometry detection on the solution containing derivatized carbohydrate compounds to obtain a mass chromatogram; the mass spectrometry detection is performed by using the electron impact excitation collision mode of organic ions to generate structure-specific diagnostic ions for detection: analyzing the mass chromatogram to identify the type and connection order of glycosidic bonds in oligosaccharide glycosidic bond isomers; the analysis comprises the following steps: based on the mechanism for differentiating the type and order of oligosaccharide glycosidic bond isomers by structure-specific diagnostic ions; a. calculating the total number of monosaccharide units based on the mass-to-charge ratio of the oligosaccharide parent ion; b. differentiating the type and order of oligosaccharide glycosidic bond isomers by using specific structure-specific diagnostic ions; c. compared with the parent ion, using the ion losing C 6 H 12 O 6 +[(C 6 H 10 O 5 )×(n-1)]+CH 2 O to determine whether the nth monosaccharide and the (n + 1)th monosaccharide unit from the reducing end of the sugar are connected by a 1,4-glycosidic bond; d. compared with the parent ion, using the ion losing C 6 H 12 O 6 +[(C 6 H 10 O 5 )×(n-1)]+CH 2 to determine whether the nth monosaccharide and the (n + 1)th monosaccharide unit from the reducing end of the sugar are connected by a 1,6-glycosidic bond; e. if there is no 1,4 and 1,6-glycosidic bond connection, using the "series of diagnostic ions losing C 6 H 12 O 6 +[C 6 H 10 O 5 ×(n-2)]+C 3 H 4 O 2 " to determine whether the nth monosaccharide and the (n + 1)th monosaccharide unit from the reducing end of the sugar are connected by a 1,3-glycosidic bond; f. if there is no 1,4, 1,6 and 1,3-glycosidic bond connection, then any two monosaccharide units are connected by a 1,2-glycosidic bond.

[0017] Compared with the prior art, the derivatizing reagent based on the structure shown in Formula I of the present invention uses high performance liquid chromatography-tandem mass spectrometry to simultaneously separate oligosaccharide compounds in biological samples, and identifies the glycosidic bond types of disaccharide, trisaccharide, tetrasaccharide, pentasaccharide, hexasaccharide and heptasaccharide isomers with different glycosidic bonds based on the electron impact excitation collision mode of organic ions to generate structure-specific diagnostic ions (EIEIO). The identification method of the present invention has high sensitivity and good resolution, can specifically identify different glycosidic bond types without relying on standards, and has high practicability and reliability. Moreover, based on the identification method of the glycosidic bond connection position in the oligosaccharide glycosidic bond isomers provided by the present invention, it is found that two hexasaccharides and two heptasaccharides in the urine of patients with different subtypes of glycogen storage disease can be used as GSD-II diagnostic urine oligosaccharide biomarkers.

[0018] In addition, the identification efficiency of the present invention is high and the identification is accurate, which can meet the needs of glycogen storage disease biomarker research. BRIEF DESCRIPTION OF THE DRAWINGS

[0019] In order to more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for use in the embodiments. Obviously, the drawings in the following description are only some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can be obtained based on these drawings.

[0020] Figure 1 It is the chromatogram of disaccharide, tetrasaccharide and pentasaccharide isomers with different glycosidic bonds in Example 1; Figure 2 and Figure 3 It is the daughter ion chromatogram of EIEIO of 7 disaccharide isomers with different glycosidic bonds in Example 1; Figure 4 It is the daughter ion chromatogram of EIEIO of 3 tetrasaccharide isomers with different glycosidic bonds in Example 1; Figure 5 It is the chromatogram of different oligosaccharide markers in the urine of children with glycogen storage disease in Example 2; Figure 6 and Figure 7 It is the daughter ion analysis chromatogram of glycosidic bonds of specific tetrasaccharide, hexasaccharide and heptasaccharide markers of glycogen storage disease in Example 2; Figure 8 and Figure 9 It is the box plot of oligosaccharide isomers with different glycosidic bond connection positions in the urine of children with different subtypes of glycogen storage disease. DETAILED DESCRIPTION OF THE INVENTION

[0021] The present invention provides a method for identifying the glycosidic bond connection position in oligosaccharide glycosidic bond isomers, including the following steps: Derivatize the sample to be tested and a derivatizing reagent with the structure shown in Formula I to obtain a solution containing derivatized saccharide compounds; Formula I; Perform high performance liquid chromatography-tandem mass spectrometry detection on the solution containing derivatized saccharide compounds to obtain a mass chromatogram; the mass spectrometry detection is carried out by using the electron impact excitation collision mode of organic ions to generate structure-specific diagnostic ions for detection: Analyze the mass chromatogram to identify the type and connection order of glycosidic bonds in oligosaccharide glycosidic bond isomers; The analysis includes the following steps: Oligosaccharide glycosidic bond isomer type and order discrimination mechanism based on structure-specific diagnostic ions; a. Calculate the total number of monosaccharide units based on the mass-to-charge ratio of the oligosaccharide parent ion; b. Identify the type and order of oligosaccharide glycosidic bond isomers according to specific structure-specific diagnostic ions; c. Compared with the parent ion, use the ion losing C 6 H 12 O 6 +[(C 6 H 10 O 5 )×(n - 1)]+CH 2 O to determine whether the nth monosaccharide and the (n + 1)th monosaccharide unit from the reducing end of the sugar are connected by a 1,4-glycosidic bond; d. Compared with the parent ion, use the ion losing C 6 H 12 O 6 +[(C 6 H 10 O 5 )×(n - 1)]+CH 2 to determine whether the nth monosaccharide and the (n + 1)th monosaccharide unit from the reducing end of the sugar are connected by a 1,6-glycosidic bond; e. If there is no 1,4 and 1,6-glycosidic bond connection, use "the series of diagnostic ions losing C 6 H 12 O 6 +[(C 6 H 10 O 5 )×(n - 2)]+C 3 H 4 O 2 " to determine whether the nth monosaccharide and the (n + 1)th monosaccharide unit from the reducing end of the sugar are connected by a 1,3-glycosidic bond; f. If there is no 1,4, 1,6 and 1,3-glycosidic bond connection, then any two monosaccharide units are connected by a 1,2-glycosidic bond.

[0022] In the present invention, unless otherwise specified, all raw materials are commercially available products well-known to those skilled in the art.

[0023] In the present invention, the sample to be tested is derived from the plasma or urine of patients with different subtypes of glycogen storage disease. In a specific embodiment, it can be the urine of patients with different subtypes of glycogen storage disease; the oligosaccharide compounds in the sample to be tested include disaccharides, trisaccharides, tetrasaccharides, pentasaccharides, hexasaccharides, and heptasaccharide compounds; the disaccharide compounds include one or more of maltose, isomaltose, lactose, melibiose, 3-O-α-D-mannopyranosyl-D-mannose, cellobiose, and gentiobiose; the tetrasaccharide includes maltotetraose or isomaltotetraose or glucose tetramer 6-α-D-glucopyranosyl-maltotriose (Glc 4 ); the pentasaccharide includes maltopentaose or isomaltopentaose.

[0024] In the present invention, the solvent in the sample to be tested is methanol and water, and the volume ratio of methanol to water is 1:1; the concentration of the oligosaccharide compounds in the sample to be tested is 0.5 - 3 μg / mL. In a specific embodiment, it can be 1 μg / mL or 2 μg / mL; the derivatization reagent having the structure shown in Formula I is used in the form of a derivatization reagent solution; the solvent of the derivatization reagent solution is a methanol-water mixture of formic acid; the volume fraction of formic acid is 1 - 3%; the volume ratio of methanol to water is 7 - 9:1; the concentration of the derivatization reagent in the derivatization reagent solution is 1 - 5 mg / mL. In a specific embodiment, it can be 2 or 3 mg / mL.

[0025] In the present invention, the mass ratio of the oligosaccharide compounds to the derivatization reagent in the sample to be tested is 1:50 - 1:2000. In a specific embodiment, it can be 1:100, 1:500, or 1:1000.

[0026] In the present invention, the temperature of the derivatization reaction is 30 - 45 °C. In a specific embodiment, it can be 33, 37, or 40 °C, and the time is 0.5 - 6 h. In a specific embodiment, it can be 1, 2, or 4 h.

[0027] In the present invention, after the derivatization reaction, it further includes washing, drying, and re-dissolving in sequence; the washing includes water washing and extraction washing; the reagent for extraction washing is dichloromethane; the number of washing times is 1 - 3 times; each time during washing, the bottom organic phase is removed and the aqueous phase is retained; the drying is by vacuum concentrator drying; the reagent used for re-dissolving is an acetonitrile-water mixture, and the volume ratio of acetonitrile to water is 2:1.

[0028] In a specific embodiment of the present invention, the preparation of the solution containing derivatized carbohydrate compounds includes the following steps: Add 300 μL of the derivatization reagent with a concentration of 2 mg / mL to 60 μL of plasma or urine from a glycogen storage disease patient or a healthy subject, and mix well. After reacting at 37 °C for 4 hours, add 300 μL of water and mix well. Extract and wash twice with 600 μL of dichloromethane. Remove the bottom organic phase each time and keep the aqueous phase. Dry 600 μL of the upper layer with a vacuum concentrator and redissolve it in 50 μL of an acetonitrile-water mixture (volume ratio 2:1) before analysis.

[0029] In the present invention, the detection conditions of the high-performance liquid chromatography include: the mobile phase is mobile phase A and mobile phase B, and the elution program is gradient elution; mobile phase A is an aqueous-methanol-isopropanol solution of ammonium formate, the concentration of ammonium formate in mobile phase A is 1 - 20 mmol / L, and in specific embodiments, it can be 5 mmol / L, 10 mmol / L, or 15 mmol / L; the volume ratio of water, methanol, and isopropanol in mobile phase A is 1 - 4:1:1, and in specific embodiments, it can be 1.5:1:1 or 2:1:1; the pH value of mobile phase A is 4.0 - 6.0, and in specific embodiments, it can be 4.5 or 5; if the pH value of mobile phase A in the present invention is not within the above range, the present invention also includes adjusting the pH with formic acid; mobile phase B is acetonitrile; the flow rate of the mixed mobile phase is 0.1 - 0.5 mL / min, and in specific embodiments, it can be 0.3 mL / min.

[0030] In the present invention, the gradient elution program is as follows: 1) From 0.00 to 2.00 minutes, maintain 75% by volume of mobile phase B; 2) From 2.00 to 58.00 minutes, 75% to 50% by volume of mobile phase B; 3) From 58.00 to 58.10 minutes, 50% to 75% by volume of mobile phase B; 4) From 58.10 to 75.00 minutes, maintain 75% by volume of mobile phase B; where the total amount of mobile phase A and mobile phase B is 100% by volume; In the present invention, the chromatographic column in the high-performance liquid chromatography is an amide chromatographic column, and in specific embodiments, it can be a chromatographic column filled with ethyl-bridged hybrid particle technology; the column temperature is 25 - 50 °C, and in specific embodiments, it can be 35 °C or 40 °C.

[0031] In the present invention, the conditions for mass spectrometry detection include: Electrospray ionization source; Monitoring: Scanning in positive ion mode in MRM (Multiple reaction monitoring) mode; Fragmentation method: Electron impact dissociation; The pressure of Gas 1 (Ion source gas 1) is 50 - 70 psi; The pressure of Gas 2 (Ionsource gas 2) is 50 - 70 psi; The pressure of curtain gas is 35 - 50 psi; The pressure of CAD gas (Collision gas) is 7 - 10 psi; Gas 1, Gas 2, collision gas and curtain gas are independently nitrogen; The ion source temperature (Temperature) is 550 - 600 °C; The spray voltage is 5500 - 6500 V; The declustering potential is 50 - 80 V; The TOF MS (Collision energy) collision voltage is preferably 10 - 20 V; The TOF MSMS (Collision energy) collision voltage is 12 - 16 V; The electron kinetic energy (ElectronKE) of the irradiated electron beam is 10 - 22 eV, and in a specific embodiment, it can be 14 or 18 eV.

[0032] To further illustrate the present invention, the method for identifying the glycosidic bond linkage position in the oligosaccharide glycosidic bond isomers provided by the present invention will be described in detail below in conjunction with the accompanying drawings and embodiments, but they should not be construed as limiting the protection scope of the present invention.

[0033] In the following embodiments of the present invention, the derivatizing reagent having the structure of Formula I is synthesized according to the method described in Chinese Patent CN102584728A; The sources, purities, glycosidic bond types, structural formulas, etc. of the disaccharide, tetrasaccharide and pentasaccharide standards in the embodiments are shown as follows.

[0034] 1. Maltose: sourced from the National Institutes for Food and Drug Control, with a purity of 93.7%, glycosidic bond type of glucose-α1,4-glucose, derivatized structural formula of Formula 1-1, and non-derivatized structural formula of Formula 1-2; Formula 1-1; Formula 1-2.

[0035] 2. Isomaltose: sourced from Tianmo Quality Inspection Standard Substance Center, with a purity of 99.9%, glycosidic bond type of glucose-α1,6-glucose, derivatized structural formula of Formula 2-1, and non-derivatized structural formula of Formula 2-2; Formula 2-1; Formula 2-2.

[0036] 3. Lactose: sourced from the National Institutes for Food and Drug Control, China, with a purity of 99.3%, glycosidic bond type of galactose-1,4-glucose, derivatized structural formula as Formula 3-1, and non-derivatized structural formula as Formula 3-2; Formula 3-1; Formula 3-2.

[0037] 4. Melibiose: sourced from Sigma-Aldrich, with a purity ≥ 98%, glycosidic bond type of galactosyl-α1,6-glucose, derivatized structural formula as Formula 4-1, and non-derivatized structural formula as Formula 4-2; Formula 4-1; Formula 4-2.

[0038] 5. 3-O-α-D-Mannopyranosyl D-Mannose: sourced from Toronto Research Chemicals, with a purity > 95%, glycosidic bond type of mannopyranosyl-α1,3-mannose, derivatized structural formula as Formula 5-1, and non-derivatized structural formula as Formula 5-2; Formula 5-1; Formula 5-2.

[0039] 6. Cellobiose: sourced from Sigma-Aldrich, with a purity ≥ 98%, glycosidic bond type of glucose-β1,4-glucose, derivatized structural formula as Formula 6-1, and non-derivatized structural formula as Formula 6-2; Formula 6-1; Formula 6-2.

[0040] 7. Gentiobiose: sourced from Macklin, with a purity of 95%, glycosidic bond type of glucopyranosyl-β1,6-glucose, derivatized structural formula as Formula 7-1, and non-derivatized structural formula as Formula 7-2; Formula 7-1; Formula 7-2.

[0041] 8. Maltotetraose: sourced from Aladdin, with a purity of 97%, glycosidic bond type of glucose-α1,4-glucose-α1,4-glucose-α1,4-glucose, derivatized structural formula as Formula 8-1, and non-derivatized structural formula as Formula 8-2; Formula 8-1; Formula 8-2

[0042] 9. Isomaltotetraose: sourced from Aladdin, with a purity of 95%, glycosidic bond type is glucose-α1,6-glucose-α1,6-glucose-α1,6-glucose, the derivatized structural formula is Formula 9-1, and the non-derivatized structural formula is Formula 9-2; Formula 9-1; Formula 9-2

[0043] 10. Glucose tetramer 6-α-D-glucopyranosyl-maltotriose (Glc 4 ): sourced from Toronto Research Chemicals, purity > 95%, glycosidic bond type is glucose-α1,6-glucose-α1,4-glucose-α1,4-glucose, the derivatized structural formula is Formula 10-1, and the non-derivatized structural formula is Formula 10-2; Formula 10-1; Formula 10-2

[0044] 11. Maltopentaose: sourced from Aladdin, with a purity of 95%, glycosidic bond type is glucose-α1,4-glucose-α1,4-glucose-α1,4-glucose-α1,4-glucose, the derivatized structural formula is Formula 11-1, and the non-derivatized structural formula is Formula 11-2; Formula 11-1; Formula 11-2

[0045] 12. Isomaltopentaose: sourced from Aladdin, with a purity of 95%, glycosidic bond type is glucose-α1,6-glucose-α1,6-glucose-α1,6-glucose-α1,6-glucose, the derivatized structural formula is Formula 12-1, and the non-derivatized structural formula is Formula 12-2; Formula 12-1; Formula 12-2

[0046] Methanol, acetonitrile (HPLC grade, Honeywell, USA), formic acid (analytical grade, Sigma, USA), ammonium formate (chemical grade, Sigma-Aldrich, USA) were all obtained commercially; Unless otherwise specified, the purified water used in the experiment was prepared by a Milli-Q water purification system (Millipore Corporation, Mosheim, France).

[0047] The instruments used in the examples are as follows: LC-30AD high-performance liquid chromatography system (Shimadzu Corporation, Japan); The tandem mass spectrometer was a 7600 ZenoTOF system (Sciex Corporation, USA).

[0048] Example 1 (1) The conditions for reverse-phase liquid chromatography detection include: The mobile phase system consists of mobile phase A and mobile phase B. Mobile phase A is a water-methanol-isopropanol solution of 10 mmol / L ammonium formate (the volume ratio of water, methanol, and isopropanol is 2:1:1, adjusted to pH 5.0 with formic acid); mobile phase B is acetonitrile; the flow rate of the mobile phase system is 0.3 mL / min; the elution method is gradient elution; The program for gradient elution is as follows: 1) From 0.0 to 2.0 minutes, 75% to 75% mobile phase B; 2) From 2.00 to 58.00 minutes, 75% to 50% mobile phase B; 3) From 58.00 to 58.10 minutes, 50% to 75% mobile phase B; 4) From 58.10 to 75.00 minutes, 75% to 75% mobile phase B; where the total amount of mobile phase A and mobile phase B is 100%; The column temperature is 40 °C, the temperature of the auto-sampler is 10 °C, and the injection volume is 5 μL; The working parameters of the tandem mass spectrometry are shown in Table 1: Table 1 Working parameters of tandem mass spectrometry

[0049] (2) Separation and identification of oligosaccharide standards with different glycosidic bond linkages Analyze oligosaccharide standards maltose, isomaltose, maltotetraose, isomaltotetraose, glucose tetramer 6-α-D-glucopyranosyl-maltotriose, maltopentaose, isomaltopentaose. The glycosidic bond positions of the above standards are as shown above.

[0050] Solution preparation: Weigh accurately the above-mentioned 7 oligosaccharide compounds respectively, and prepare oligosaccharide compound solutions with a concentration of 1 mg / mL. Then use a diluent (volume ratio of methanol to water = 1:1) to dilute the above-mentioned 1 mg / mL oligosaccharide compound solutions to 1 μg / mL test solutions containing oligosaccharide compounds; Weigh accurately the derivatization reagent with the structure of Formula I, and dissolve it with a methanol-water mixture containing 1% formic acid by volume (volume ratio of methanol to water = 9:1) to prepare a derivatization reagent solution with a concentration of 2 mg / mL; Derivatization process: Add 200 μL of the derivatization reagent solution to 20 μL of the above-prepared 1 μg / mL test solution containing oligosaccharide compounds. After vortex mixing for 30 s, react at 37 °C for 4 h. Then add 200 μL of ultrapure water, extract and wash twice with dichloromethane (leave the supernatant each time), and mix the supernatant with a total volume of 100 μL with 200 μL of acetonitrile to obtain a derivatized saccharide compound solution.

[0051] Detect the derivatized saccharide compound solution according to the conditions described in step (1), and the results are as Figure 1 shown. Figure 1 It is the chromatogram of disaccharide, tetrasaccharide and pentasaccharide isomers with different glycosidic bonds in Example 1. It can be seen from Figure 1 that disaccharides (maltose, isomaltose), tetrasaccharides (maltotetrose, isomaltotetrose, glucose tetramer 6-α-D-glucopyranosyl-maltotriose) and pentasaccharides (maltopentaose, isomaltopentaose) with different glycosidic bond connection positions are well separated under the detection conditions of the present invention.

[0052] Oligosaccharide glycosidic bond isomer type and sequence identification mechanism based on structure-specific diagnostic ions; a. Calculate the total number of monosaccharide units based on the mass-to-charge ratio of the oligosaccharide parent ion; b. Identify the oligosaccharide glycosidic bond isomer type and sequence according to specific EIEIO; c. Compared with the parent ion, use the ion losing C 6 H 12 O 6 +[(C 6 H 10 O 5 )(n - 1)] + CH 2 O to determine whether the nth monosaccharide and the (n + 1)th monosaccharide unit from the sugar reducing end are connected by a 1,4-glycosidic bond; d. Compared with the parent ion, use the ion losing C 6 H 12 O 6 +[(C 6 H 10 O 5 )(n - 1)] + CH2 For the ions, determine whether there is a 1,6-glycosidic bond connection between the n-th monosaccharide and the (n + 1)-th monosaccharide unit counted from the reducing end of the sugar; e. If there is no 1,4- and 1,6-glycosidic bond connection, use the "series of diagnostic ions of losing C 6 H 12 O 6 +[C 6 H 10 O 5 ×(n - 2)]+C 3 H 4 O 2 to determine whether there is a 1,3-glycosidic bond connection between the n-th monosaccharide and the (n + 1)-th monosaccharide unit counted from the reducing end of the sugar; f. If there is no 1,4-, 1,6- and 1,3-glycosidic bond connection, then the connection between any two monosaccharide units is a 1,2-glycosidic bond.

[0053] Figure 2 and Figure 3 are the daughter ion spectra of 7 different glycosidic bond disaccharide isomers EIEIO in Example 1. From Figure 2 and Figure 3 it can be seen that the characteristic ions of EIEIO can specifically distinguish disaccharide isomers with different glycosidic bond connections.

[0054] Figure 4 are the daughter ion spectra of 3 different glycosidic bond tetrasaccharide isomers EIEIO in Example 1. From Figure 4 it can be seen that the above rule is still feasible when extrapolated from disaccharide compounds to oligosaccharide compounds.

[0055] Example 2 Apply the identification method described in Example 1 to the study of biomarkers for subtypes of glycogen storage disease, and analyze the positional isomers of different glycosidic bond connections of oligosaccharides in the plasma and urine of children with different subtypes of glycogen storage disease. After the protocol was approved by the Ethics Committee (K24C2687), urine samples of 39 patients with glycogen storage disease and Gaucher's disease who were outpatients, inpatients, and under long-term follow-up were collected. All patients signed the informed consent form. After collection by enzyme or gene testing and clinical symptom identification, they were placed in a -80 °C refrigerator; Sample preparation: Add 300 μL of the derivatization reagent described in Example 1 with a concentration of 2 mg / mL to 60 μL of urine from a glycogen storage disease patient or a healthy subject, and mix evenly. After reacting at 37 °C for 4 h, add 300 μL of water and mix evenly. Extract and wash twice with 600 μL of dichloromethane, remove the bottom organic phase each time, and leave the aqueous phase. Dry the 600 μL upper layer with a vacuum concentrator and redissolve it in 50 μL of an acetonitrile-water mixture (volume ratio 2:1) before analysis. Measure according to the method described in Example 1, and the results are shown below.

[0056] Figure 5 It is the chromatogram of different oligosaccharide markers in the urine of a child with glycogen storage disease in Example 2; Figure 6 and Figure 7 is the glycosidic bond daughter ion resolution diagram of the specific tetrasaccharide, hexasaccharide, and heptasaccharide markers for glycogen storage disease in Example 2; As can be seen from Figures 5 - 7 it, using the identification method provided by the present invention, based on the new derivatization reagent, the oligosaccharide glycosidic bond isomers in the urine of patients with different subtypes of glycogen storage disease are separated and identified by the analysis method of high performance liquid chromatography-tandem mass spectrometry, and the glycosidic bond connection type and position of the oligosaccharide compounds are identified. Explore the possibility of these oligosaccharide compounds as biomarkers for glycogen storage disease typing, so as to possibly provide a basis for the pharmacodynamics evaluation of glycogen storage disease.

[0057] Figure 8 and Figure 9 are box plots of oligosaccharide isomers with different glycosidic bond connection positions in the urine of children with different subtypes of glycogen storage disease. As can be seen from Figure 8 and Figure 9 it, it shows that there are significant differences in different isomers in different subtypes of glycogen storage disease, and they can be used as biomarkers for the diagnosis of GSD-II. Further analysis shows that two hexasaccharide isomers, "hexasaccharide marker-1" (1,4-1,4-1,6-1,4-1,6 reducing end → non-reducing end) and "hexasaccharide marker-2" (1,4-1,4-1,4-1,6-1,6 reducing end → non-reducing end), and two heptasaccharide isomers, "heptasaccharide marker-1" (1,4-1,4-1,4-1,4-1,4-1,6 reducing end → non-reducing end) and "heptasaccharide marker-2" (1,4-1,4-1,6-1,4-1,4-1,6 reducing end → non-reducing end), can better distinguish Pompe disease from other subtypes of glycogen storage disease than the existing Glc 4 and have the potential to be specific biomarkers for Pompe disease. The method provided by the present invention helps to efficiently, sensitively, and accurately identify oligosaccharide isomers with different glycosidic bond connection positions, and further promotes the further research on the biological functions of oligosaccharides.

[0058] Although the above embodiments have described the present invention in detail, they are only a part of the embodiments of the present invention, rather than all embodiments. People can also obtain other embodiments based on this embodiment without creative efforts, and these embodiments all fall within the protection scope of the present invention.

Claims

1. A method for identifying the glycosidic bond connection position in oligosaccharide glycosidic bond isomers, characterized in that: The following steps are involved: Carrying out a derivatization reaction between the sample to be tested and a derivatization reagent having a structure shown in Formula I to obtain a solution containing a derivatized saccharide compound; Formula I; The derivatized sugar compound-containing solution is subjected to high performance liquid chromatography-tandem mass spectrometry detection to obtain a mass chromatogram; the mass spectrometry detection uses an electron collision excitation collision mode of organic ions to generate structure-specific diagnostic ions for detection: Analyzing the mass chromatogram to identify the type and connection order of glycosidic bonds in the oligosaccharide glycosidic bond isomers; The analysis includes the following steps: The mechanism for distinguishing the type and order of oligosaccharide glycosidic bond isomers based on structure-specific diagnostic ions; a. Calculate the total number of monosaccharide units based on the mass-to-charge ratio of the oligosaccharide parent ion; b. Identify the type and order of oligosaccharide glycosidic bond isomers based on specific structure-specific diagnostic ions; c. Compared with the parent ion, the loss of C6H 12 O6+[(C6H 10 O5)×(n-1)]+CH2O ions, determine whether the nth monosaccharide and the n+1th monosaccharide unit from the reducing end of the sugar are connected by a 1,4 glycosidic bond; d. Compared with the parent ion, the loss of C6H 12 O6+[(C6H 10 O5)×(n-1)]+CH2 ions to determine whether the nth monosaccharide and the n+1th monosaccharide unit from the reducing end of the sugar are connected by a 1,6 glycosidic bond; e. If there are no 1,4 and 1,6 glycosidic bonds, use "missing C6H 12 O6+[(C6H 10 O5)×(n-2)]+C3H4O2 series diagnostic ions", to determine whether the nth monosaccharide and the n+1th monosaccharide unit from the sugar reducing end are connected by a 1,3 glycosidic bond; f. If there are no 1,4, 1,6 and 1,3 glycosidic bonds, any two monosaccharide units are connected by 1,2 glycosidic bonds.

2. The identification method according to claim 1, characterized in that: The detection conditions of the high performance liquid chromatography include: the mobile phase is mobile phase A and mobile phase B, and the elution procedure is gradient elution; The mobile phase A is a water-methanol-isopropanol solution of ammonium formate, the mass concentration of the ammonium formate is 1-20 mmol / L, the volume ratio of water, methanol and isopropanol in the mobile phase A is 1-4:1:1, and the mobile phase B is acetonitrile; The procedure of the gradient elution is: 1) 0.00 to 2.00 min, maintaining 75 vol% mobile phase B; 2) 2.00 to 58.00 min, 75 vol% to 50 vol% mobile phase B; 3) 58.00 to 58.10 minutes, 50% to 75% by volume mobile phase B; 4) 58.10 to 75.00 minutes, maintaining 75 vol% mobile phase B.

3. The identification method according to claim 2, characterized in that: The pH value of the mobile phase A is 4.0-6.

0.

4. The identification method according to claim 2, characterized in that: The chromatographic column in the high performance liquid chromatography is an amide chromatographic column.

5. The identification method according to claim 1, characterized in that: The detection conditions of the mass spectrometry include: Electrospray ion source; monitoring: MRM mode positive ion mode scanning; fragmentation mode: electron excitation fragmentation; gas 1 pressure is 50~70 psi; gas 2 pressure is 50~70 psi; curtain gas pressure is 35~50 psi; collision gas pressure is 7~10psi; gas 1, gas 2, collision gas and curtain gas are nitrogen; ion source temperature is 550~600 ℃; spray voltage is 5500~6500 V; declustering voltage is 50~80 V; TOF MS collision voltage is 10~20 V; TOF MSMS collision voltage is 12~16V; electron kinetic energy of the irradiating electron beam is 10~22 eV.

6. The identification method according to claim 1, characterized in that: The mass ratio of the oligosaccharide compound to the derivatization reagent in the sample to be tested is 1:50-1:2000.

7. The identification method according to claim 1 or 6, characterized in that: The temperature of the derivatization reaction is 30-45° C., and the time is 0.5-6 h.

8. The identification method according to claim 1, characterized in that: The samples to be tested are derived from urine or plasma of patients with different subtypes of glycogen storage disease.

9. The identification method according to claim 8, characterized in that: The use of two hexasaccharides and two heptasaccharides in the urine of patients with different subtypes of glycogen storage disease as diagnostic biomarkers for GSD-II; The glycosidic bond connection positions of the two hexasaccharides are 1,4-1,4-1,6-1,4-1,6 reducing end→non-reducing end and 1,4-1,4-1,4-1,6-1,6 reducing end→non-reducing end respectively; The glycosidic bond connection positions in the two heptasaccharides are 1,4-1,4-1,4-1,4-1,4-1,6 reducing end→non-reducing end and 1,4-1,4-1,6-1,4-1,4-1,6 reducing end→non-reducing end respectively.

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