CN108484688B

CN108484688B - Method for regenerating reducing sugar from hydrazine chromogenic reagent derivative of reducing sugar

Info

Publication number: CN108484688B
Application number: CN201810064494.8A
Authority: CN
Inventors: 王仲孚; 王承键; 郜茜; 龚桂萍; 黄琳娟
Original assignee: Northwestern University
Current assignee: Northwestern University
Priority date: 2018-01-23
Filing date: 2018-01-23
Publication date: 2020-05-08
Anticipated expiration: 2038-01-23
Also published as: CN108484688A

Abstract

The invention discloses a method for regenerating reducing sugar from hydrazine chromogenic reagent derivatives of the reducing sugar, which comprises the following steps: (1) taking reducing sugar, adding a hydrazine chromogenic reagent according to the amount of reducing sugar with the mole number not less than 10 times, dissolving in weak acid aqueous solution of an organic solvent, shaking up, heating for reaction, and concentrating and drying after the reaction is finished; (2) dissolving the sample obtained in the step (1) in water, and then extracting with an organic solvent, or purifying by using a C18 column and/or a PGC solid phase extraction column; (3) separating the sample obtained in the step (2) by chromatography; (4) and (3) drying the single saccharide derivative fraction collected in the step (3), dissolving the dried fraction in a weak acid aqueous solution, removing the hydrazine chromogenic reagent marker under a heating condition, and regenerating the reducing sugar chain. The invention has the advantages that: the operation is simple, and the reaction is mild and rapid; the regenerated reducing sugar chain monomer has no side reaction product and high recovery rate.

Description

Method for regenerating reducing sugar from hydrazine chromogenic reagent derivative of reducing sugar

Technical Field

The invention relates to a method for regenerating reducing sugar, in particular to a method for regenerating reducing sugar monomers from hydrazine chromogenic reagent derivatives of reducing sugar, and belongs to the technical field of biochemistry.

Background

Glycosylation is one of the common post-translational modifications and plays an important role in the regulation of cell life activities. Studies report that sugar chain conjugates such as glycoproteins, glycolipids, proteoglycans, etc. are involved in various intermolecular interactions in the body, and sugar chains, as an information molecule on the conjugates, are not only involved in cell recognition, adhesion, signal transduction and immune response between cells, but also play an important role in inflammatory and autoimmune diseases, aging, abnormal proliferation and metastasis of cancer cells, pathogen infection, etc. Therefore, it is of great significance to sugar chain structure and function analysis. However, since the biosynthesis process of sugar chains has non-templating properties, the structure is highly complex, the sugar chains have diversity in monosaccharide composition, linkage mode, branching site and anomeric configuration, and the sugar chains have micro-heterogeneity, and sugar chains with various structures are often mixed, which poses a great challenge to structural analysis and functional analysis.

In recent years, the development of techniques such as high-sensitivity and high-resolution biological Mass Spectrometry (MS), High Performance Liquid Chromatography (HPLC), and sugar chip provides an important means for sugar chain analysis. The general procedure for sugar chain structure and function analysis is: the sugar chain is released from the glycoconjugate of animal and plant sources by an enzymatic method or a chemical method, then purified, and then subjected to derivatization for mass spectrometry and liquid phase separation detection, and the separated sugar chain derivative is subjected to a de-labeling process (regeneration) to obtain the reducing sugar chain, and the regenerated (recovered) reducing sugar chain can be further subjected to diversified analysis by techniques such as nuclear magnetic resonance, sugar chip and the like. In this process, recovery of the reducing oligosaccharide chains having a single structure from the various sugar chain derivatives obtained by chromatographic separation is a very critical step. The underivatized sugar chain is usually lack of corresponding hydrophobic and chromophoric groups and is difficult to be used for high-sensitivity mass spectrum and chromatographic detection analysis, so that the sugar chain is usually required to be derivatized by a specific derivatization reagent, so that the sugar chain can be provided with the chromophoric groups to facilitate ultraviolet or fluorescence detection, and meanwhile, the sugar chain can be provided with the hydrophobic groups, thereby reducing the polarity of the sugar chain, leading the sugar chain to be better retained on a chromatographic column and being beneficial to separation of the sugar chain. The most commonly used derivatizing agents at present all have primary amino groups, such as 2-aminopyridine (2-AP), 2-aminobenzoic acid (2-AA), 2-aminobenzamide (2-AB), 2-aminoacridone (2-AMAC) and 2-aminobenzonitrile (o-ABN), and the like, and the derivatizing agents can react with carbonyl (aldose or ketose) at the reducing end of a sugar chain to generate Schiff base under acidic conditions through a reductive amination method and further react to generate stable derivatives with secondary amine (-C-NH-) structures under the action of a reducing agent. However, when these sugar chain derivatives are separated by chromatography, it is often difficult to recover the reducing sugar chains by the removal reagent. Because the structure of the secondary amine (-C-NH-) is very stable, the goal of breaking can be achieved by oxidizing the imino group with hydrogen peroxide generally, the reaction condition is harsh, the yield of the by-product is high, and the yield of the recovered reducing sugar chain is low. 1-phenyl-3-methyl-5-pyrazolone (PMP) and 1, 3 substituted pyrazolone compounds such as PMPMP are also commonly used for HPLC pre-column derivatization of sugar chains, and the derivatives with-C-C-or-C-C-structure are generated by condensation reaction of active methylene and aldehyde group at the reducing end of the sugar chain under alkaline condition, and the structure is very stable, so that regeneration of reducing sugar chain is difficult to realize. Therefore, recovery of reducing sugar chains from sugar chain derivatives remains a bottleneck to be solved in sugar chain research.

The hydrazine chromogenic reagent is a derivatization reagent which is less researched, and can also be used for derivatization of reducing sugar chains. The principle is as follows: hydrazino group (-NH-NH) carried by reagent₂) Can perform nucleophilic reaction with aldehyde group at the reducing end of the sugar chain under the weak acid condition to form hydrazone (-C-N-NH-) derivatives with certain stability, and can be used for chromatographic separation and mass spectrometric detection of the sugar chain. Meanwhile, hydrazone substances after chromatographic separation can be subjected to hydrolysis reaction by heating under the weakly acidic condition, so that sugar chains with reducing end aldehyde groups are generated. As the hydrazine reagent, there are Phenylhydrazine (PNH), Girard reagent P (GP), Benzoylhydrazine (BZH) and Benzenesulfonylhydrazine (BSH), and theoretically, they should be all capable of derivatizing the reducing sugar chain and effecting chromatographic separation of the derivative and then label removal, thereby regenerating and reducing the sugar chainAn original sugar chain.

Disclosure of Invention

Based on the foregoing principle, the present invention develops a novel method for regenerating a reducing oligosaccharide chain monomer from a sugar chain derivative after chromatographic separation, which is simple in operation, mild in reaction, rapid, and efficient, and the regenerated reducing oligosaccharide chain after separation can be used for subsequent diversified structural and functional analyses, and is of great significance for the study of sugar substances.

In order to achieve the above object, the present invention adopts the following technical solutions:

a method for regenerating a reducing sugar from a hydrazine chromophoric reagent derivative of the reducing sugar, comprising the steps of:

(1) taking reducing sugar, adding a hydrazine chromogenic reagent according to the amount of reducing sugar with the mole number not less than 10 times, dissolving in weak acid aqueous solution of an organic solvent, shaking up, heating for reaction, and concentrating and drying after the reaction is finished;

(2) dissolving the sample obtained in the step (1) in water, extracting with an organic solvent, centrifuging, removing an organic phase, taking a water phase, repeating the extraction operation for a plurality of times, removing unreacted hydrazine chromogenic reagent, and finally concentrating and drying the obtained water phase;

or, adding water to dissolve the sample obtained in the step (1), and then purifying the sample by using a C18 column and/or a PGC solid phase extraction column;

(3) separating the sample obtained in step (2) by chromatography, and collecting the single saccharide derivative fraction;

(4) and (3) drying the single saccharide derivative fraction collected in the step (3), dissolving the dried fraction in a weak acid aqueous solution, removing the hydrazine chromogenic reagent marker under a heating condition, and regenerating the reducing sugar chain.

The method described above, wherein in step (1), the reducing sugar comprises: saccharides having a reducing terminal aldehyde group directly extracted from a biological sample by various methods, saccharides having a reducing terminal aldehyde group released from glycoconjugates, and various artificially synthesized saccharides having a reducing terminal aldehyde group.

The method described above, wherein in step (1), the hydrazine-based coloring reagent includes: benzenesulfonyl hydrazide, p-toluenesulfonyl hydrazide, benzoyl hydrazide, phenylacetyl hydrazide, Gilard reagent, phenylhydrazine, 3-methyl-2-benzothiazolinone hydrazone, and other chemical reagents having both a hydrazino group and a chromophoric group.

The method described above, wherein in step (3), the chromatography comprises: HILIC-HPLC, RP-HPLC and PGC-HPLC.

The method as described above, wherein, in the step (4), the weak acid aqueous solution includes: a mixed solution of acetic acid and water, a mixed solution of citric acid and water, a mixed solution of formic acid and water, and a mixed solution of hydrochloric acid and water.

The invention has the advantages that:

1. the method is simple to operate, the reaction is mild and rapid, and the obtained reducing sugar can be further used for diversified analysis such as nuclear magnetic resonance, sugar chip and the like;

2. the regenerated reducing sugar monomer has no side reaction products and high recovery rate;

3. is suitable for neutral reducing sugar, acidic reducing sugar and reducing sugar containing fucose modification.

Drawings

FIG. 1 is a schematic representation of the regeneration of reducing sugars from hydrazine-based chromogenic reagent derivatives of reducing sugars;

FIG. 2(a) is an ESI-MS profile of a BSH derivative of maltodextrin;

FIG. 2(b) is an HPLC chromatogram of a BSH derivative of maltodextrin;

FIG. 2(c) is an ESI-MS spectrum of BSH derivative of maltopentaose after HPLC separation of BSH derivative of maltodextrin;

FIG. 2(d) is an ESI-MS profile of unlabeled maltopentaose;

FIG. 3(a) is an ESI-MS spectrum of a TSH derivative of maltodextrin;

FIG. 3(b) is an HPLC chromatogram of a TSH derivative of maltodextrin;

FIG. 3(c) is an ESI-MS spectrum of a TSH derivative of maltopentaose after HPLC separation of a TSH derivative of maltodextrin;

FIG. 3(d) is an ESI-MS profile of unlabeled maltopentaose;

FIG. 4(a) is an ESI-MS spectrum of a BZH derivative of maltodextrin;

FIG. 4(b) is an HPLC chromatogram of a BZH derivative of maltodextrin;

FIG. 4(c) is an ESI-MS spectrum of a BZH derivative of maltopentaose after HPLC separation of the BZH derivative of maltodextrin;

FIG. 4(d) is an ESI-MS profile of unlabeled maltopentaose;

FIG. 5(a) is an ESI-MS spectrum of a PLH derivative of maltodextrin;

FIG. 5(b) is an HPLC chromatogram of a PLH derivative of maltodextrin;

FIG. 5(c) is an ESI-MS spectrum of a PLH derivative of maltopentaose after HPLC separation of a PLH derivative of maltodextrin;

FIG. 5(d) is an ESI-MS profile of unlabeled maltopentaose;

FIG. 6(a) is an ESI-MS spectrum of a GP derivative of maltodextrin;

FIG. 6(b) is an HPLC chromatogram of a GP derivative of maltodextrin;

FIG. 6(c) is an ESI-MS spectrum of a GP derivative of maltopentaose after HPLC separation of a GP derivative of maltodextrin;

FIG. 6(d) is an ESI-MS profile of unlabeled maltopentaose;

FIG. 7(a) is an ESI-MS spectrum of a PNH derivative of maltodextrin;

FIG. 7(b) is an HPLC chromatogram of a PNH derivative of maltodextrin;

FIG. 7(c) is an ESI-MS spectrum of a PNH derivative of maltopentaose after HPLC separation of the PNH derivative of maltodextrin;

FIG. 7(d) is an ESI-MS profile of unlabeled maltopentaose;

FIG. 8(a) is an ESI-MS spectrum of an MBTH derivative of maltodextrin;

FIG. 8(b) is an HPLC chromatogram of an MBTH derivative of maltodextrin;

FIG. 8(c) is an ESI-MS spectrum of an MBTH derivative of maltopentaose after HPLC separation of the MBTH derivative of maltodextrin;

FIG. 8(d) is an ESI-MS profile of unlabeled maltopentaose;

FIG. 9(a) is an HPLC chromatogram of a BSH derivative of the N-sugar chain of ovalbumin;

FIGS. 9(b-1) to 9(b-34) are ESI-MS spectra before and after sample solution demarking of 13 peaks collected after HPLC;

FIG. 10(a) is an HPLC chromatogram of a BSH derivative of human milk oligosaccharide chain;

FIGS. 10(b-1) to 10(b-40) are ESI-MS spectra before and after sample solution demarking of 20 peaks collected after HPLC;

in FIGS. 9(b-1) to 9(b-34), ■ in the structural formula represents N-acetylglucosamine, ● in the structural formula represents high mannose, H in the chemical formula represents high mannose, and N represents N-acetylglucosamine;

in FIGS. 10(b-1) to 10(b-40), ■ in the structural formulae represents N-acetylgalactosamine, ● represents galactose, ○ represents glucose, and,

Represents fucose, ◆ represents sialic acid, H represents hexose, N represents N-acetylglucosamine, and F represents fucose.

Detailed Description

The invention is described in detail below with reference to the figures and the embodiments.

Reaction principle for regenerating reducing sugars (fig. 1): the reducing end aldehyde group of the reducing sugar can react with excessive hydrazine color development reagent to generate a derivative with a carbon-nitrogen double bond (-C ═ N-) structure through hydrazone reaction under the acidic condition, when the excessive reagent in the derivative is removed, the structure is unstable under the acidic heating condition, and the derivative can be converted into the reducing sugar again.

With respect to reducing sugars: the reducing saccharides according to the present invention include saccharides having a reducing terminal aldehyde group directly extracted from a biological sample by various methods, saccharides having a reducing terminal aldehyde group released from glycoconjugates, and various artificially synthesized saccharides having a reducing terminal aldehyde group, for example: maltodextrin, egg albumin N-sugar chains, human milk oligosaccharide chains, and the like.

Regarding the hydrazine-based coloring reagent: any reagent that carries both a hydrazine group and a chromophore group can be used, for example: benzenesulfonyl hydrazide (BSH), p-toluenesulfonyl hydrazide (TSH), benzoyl hydrazide (BZH), phenylacetyl hydrazide (PLH), Girard's reagent P (GP), Phenylhydrazine (PNH), 3-methyl-2-benzothiazolinone hydrazone (MBTH), and the like.

Maltodextrin, egg albumin, PNH, BSH, TSH, BZH, PLH, GP, MBTH were purchased from Sigma-Aldrich; human milk was taken from healthy mother volunteers three days after delivery (second subsidiary hospital of the university of western's transportation); c18 column (solid phase extraction cartridge-Pak C18, 100mg/1mL) purchased from Waters; PGC column (solid phase extraction small column porous graphite carbon column, 150mg/4mL) purchased from Alltech Hasselates; chromatographically pure acetonitrile was purchased from Fisher Scientific; the double distilled water is prepared by an automatic double pure water distiller for a laboratory; other reagents were analytically pure.

Mass spectrometry identification in the present invention was detected using electrospray ionisation linear ion trap mass spectrometry (LTQ XL, Thermoscientific, USA).

The ESI-MS parameters were set as follows: the sample feeding amount is controlled by a 2 mu L sample feeding ring; the sample-carrying mobile phase is methanol/water (50%/50%, v/v); the flow rate is 50 muL/min; the working voltage is 4 kV; the sheath gas flow rate was 20 arb; the assist gas flow rate was 10 arb; the capillary voltage is 37V; capillary lens voltage was 250V; the capillary temperature was 300 ℃; the scanning type is primary full scanning; the maximum injection time is 1000 ms; the micro-scan is 3 times; data collection was performed using LTQ Tune software.

Example 1: regeneration of free reducing maltooligosaccharide monomer from BSH derivative of maltodextrin

The method comprises the following specific steps:

(1) taking 5mg of standard maltodextrin, and mixing according to a molar ratio of not less than 1: 10 BSH (10mg) was taken, maltodextrin and BSH were dissolved in 1mL of weak acid aqueous solution of an organic solvent (1 mL of 500 μ L ethanol +50 μ L acetic acid +450 μ L water, 50% ethanol and 5% acetic acid), shaken well, reacted at 60 ℃ for 45min, and then concentrated and dried.

(2) Dissolving the obtained sample with water, extracting with organic solvent (dichloromethane), centrifuging, collecting supernatant, repeating for 3-5 times, and concentrating and drying the supernatant.

(3) Dissolving the obtained sample with acetonitrile, and then carrying out HILIC-HPLC separation under the liquid phase conditions that: for HILIC-HPLC separation, a 4.6mm × 250mm TSK-GEL Amide-80 column was used, the column temperature was 20 deg.C, the detection wavelength was 227nm, and the flow rate was 0.8 mL/min. The mobile phase A was acetonitrile, B was 10mM ammonium acetate solution (pH4.5), and C was double distilled water. Sample separation conditions: t is 0min, 85% a, 15% B; t is 60min, 50% a, 50% B. And (3) column washing conditions: t is 0min, 50% a, 50% C; t ═ 20min, 25% a, 75% C; t ═ 30min, 25% a, 75% C; t-50 min, 95% a, 5% C.

(4) After HILIC-HPLC separation, each oligosaccharide fraction was dried, dissolved in weak acid aqueous solution (5% glacial acetic acid), and reacted at 70 deg.C for 30min to complete label removal.

The BSH derivative of maltodextrin has ESI-MS pattern shown in figure 2(a), HPLC pattern shown in figure 2(b), ESI-MS pattern of BSH derivative of maltopentaose after HPLC separation shown in figure 2(c), and ESI-MS pattern of maltopentaose after de-labeling shown in figure 2 (d).

Example 2: regeneration of free reduced maltooligosaccharide monomers from TSH derivatives of maltodextrins

The method comprises the following specific steps:

(1) taking 5mg of standard maltodextrin, and mixing according to a molar ratio of not less than 1: 10 ratio of maltodextrin to TSH (10mg) was dissolved in 1mL of weak acid aqueous solution of organic solvent (1 mL of 500 μ L ethanol +50 μ L acetic acid +450 μ L water, 50% ethanol and 5% acetic acid), shaken, reacted at 60 ℃ for 45min, and then concentrated to dryness.

(4) After separation by HILIC-HPLC, each oligosaccharide fraction was dried, dissolved in weak acid aqueous solution (5% glacial acetic acid), and reacted at 70 deg.C for 30min to complete label removal.

The TSH derivative of maltodextrin has ESI-MS pattern shown in figure 3(a), HPLC pattern shown in figure 3(b), ESI-MS pattern of TSH derivative of maltopentaose after HPLC separation shown in figure 3(c), and ESI-MS pattern of maltopentaose after de-labeling shown in figure 3 (d).

Example 3: regeneration of free reducing maltooligosaccharide monomers from BZH derivatives of maltodextrins

The method comprises the following specific steps:

(1) taking 5mg of standard maltodextrin, and mixing according to a molar ratio of not less than 1: 10 ratio of BZH (10mg), maltodextrin and BZH were dissolved in 1mL of weak acid aqueous solution of organic solvent (1 mL of 500. mu.L ethanol + 50. mu.L acetic acid + 450. mu.L water, 50% ethanol and 5% acetic acid), shaken, reacted at 60 ℃ for 45min, and then concentrated and dried.

(2) Dissolving the obtained sample with water, purifying the sample by a C18 and PGC solid phase extraction column, and passing the sample through a C18 column, wherein the specific operation is as follows: activating by 3mL of acetonitrile, balancing by 12mL of water, loading, washing by 0%, 5%, 10% and 15% of acetonitrile every 5% of gradient for 3mL to remove impurities, eluting by 3mL of 20% of acetonitrile for sugar chains, and then passing through a PGC column, wherein the specific operation is as follows: activating by 3mL of acetonitrile, balancing by 12mL of water, loading, washing by 3mL of 0%, 5%, 10%, 15%, 20% and 25% acetonitrile respectively to remove impurities, eluting by 3mL of 30% acetonitrile to elute sugar chains, connecting by tubes by 0.5mL of tubes, and concentrating and drying.

The ESI-MS spectrum of the BZH derivative of maltodextrin is shown in FIG. 4(a), the HPLC spectrum is shown in FIG. 4(b), the ESI-MS spectrum of the BZH derivative of maltopentaose after HPLC separation is shown in FIG. 4(c), and the ESI-MS spectrum of maltopentaose after de-labeling is shown in FIG. 4 (d).

Example 4: regeneration of free reducing maltooligosaccharide monomers from PLH derivatives of maltodextrin

The method comprises the following specific steps:

(1) taking 5mg of standard maltodextrin, and mixing according to a molar ratio of not less than 1: taking PLH (10mg) at a ratio of 10, dissolving maltodextrin and PLH in 1mL of weak acid aqueous solution of an organic solvent (1 mL of 500. mu.L ethanol + 50. mu.L acetic acid + 450. mu.L water, 50% ethanol and 5% acetic acid), shaking, reacting at 60 ℃ for 45min, and concentrating and drying.

(3) Dissolving the obtained sample by using acetonitrile, and then carrying out HILIC-HPLC separation, wherein the liquid phase conditions are as follows: for HILIC-HPLC separation, a TSK-GEL Amide-80 column of 4.6mm × 250mm was used, the column temperature was 20 deg.C, the detection wavelength was 254nm, and the flow rate was 0.8 mL/min. The mobile phase A was acetonitrile, B was 10mM ammonium acetate solution (pH4.5), and C was double distilled water. Sample separation conditions: t is 0min, 85% a, 15% B; t is 60min, 50% a, 50% B. And (3) column washing conditions: t is 0min, 50% a, 50% C; t ═ 20min, 25% a, 75% C; t ═ 30min, 25% a, 75% C; t-50 min, 95% a, 5% C.

The PLH derivative of maltodextrin has ESI-MS spectrum shown in figure 5(a), HPLC spectrum shown in figure 5(b), ESI-MS spectrum shown in figure 5(c) of PLH derivative of maltopentaose after HPLC separation, and ESI-MS spectrum shown in figure 5(d) of maltopentaose after de-labeling.

Example 5: regeneration of free reducing maltooligosaccharide monomer from GP derivative of maltodextrin

The method comprises the following specific steps:

(1) taking 5mg of standard maltodextrin, and mixing according to a molar ratio of not less than 1: 10 ratio taking GP (15mg), dissolving maltodextrin and GP in 1mL weak acid aqueous solution of organic solvent (1 mL of 500. mu.L methanol + 50. mu.L acetic acid + 450. mu.L water, containing 50% methanol and 5% acetic acid), shaking, reacting at 70 ℃ for 90min, then concentrating and drying.

(2) Dissolving the obtained sample with water, and purifying the sample by a PGC solid phase extraction column, wherein the specific operation is as follows: 3mL of acetonitrile is activated, then 12mL of water is balanced, the sample loading is repeated for 3 times, then 6mL of the reagent is removed by water washing, finally 3mL of 25% acetonitrile is used for eluting sugar chains, the mixture is connected in different tubes, each tube is connected with 0.5mL, and then the mixture is concentrated and dried.

(3) Carrying out HILIC-HPLC separation on the purified sample, wherein the liquid phase conditions are as follows: for HILIC-HPLC separation, a 4.6mm X250 mm TSK-GELAmide-80 column was used, the column temperature was 20 ℃, the detection wavelength was 254nm, and the flow rate was 0.8 mL/min. The mobile phase A was acetonitrile, B was 10mM ammonium acetate solution (pH4.5), and C was double distilled water. Sample separation conditions: t is 0min, 75% a, 25% B; t is 60min, 50% a, 50% B. And (3) column washing conditions: t is 0min, 50% a, 50% C; t ═ 20min, 25% a, 75% C; t ═ 30min, 25% a, 75% C; t-50 min, 95% a, 5% C.

(4) After HPLC separation, the oligosaccharide fractions were dissolved in 5% glacial acetic acid and reacted at 70 ℃ for 90min to complete the label removal.

The ESI-MS spectrum of the GP derivative of maltodextrin is shown in figure 6(a), the HPLC spectrum is shown in figure 6(b), the ESI-MS spectrum of the GP derivative of maltopentaose after HPLC separation is shown in figure 6(c), and the ESI-MS spectrum of maltopentaose after de-labeling is shown in figure 6 (d).

Example 6: regeneration of free reduced maltooligosaccharide monomers from PNH derivatives of maltodextrins

The method comprises the following specific steps:

(1) taking 5mg of standard maltodextrin, and mixing according to a molar ratio of not less than 1: 10 proportion PNH (10mg) was taken, maltodextrin and PNH were dissolved in 1mL of weak acid aqueous solution of organic solvent (1 mL of 500. mu.L methanol + 50. mu.L acetic acid + 450. mu.L water, 50% methanol and 5% acetic acid), shaken well, reacted at 70 ℃ for 30min, and then concentrated and dried.

(2) Dissolving the obtained sample with water, and purifying the sample by a PGC column by the following specific operations: 3mL of acetonitrile, then 12mL of water balance, repeat 3 times, sample loading, then water washing 6mL of the removal reagent, and finally 3mL of 25% acetonitrile to elute sugar chains, tube-in-tube, each tube is connected with 0.5mL, then concentrated and dried.

(3) Dissolving the obtained sample with acetonitrile, and then carrying out HILIC-HPLC separation under the liquid phase conditions that: for HILIC-HPLC separation, a TSK-GEL Amide-80 column of 4.6mm × 250mm was used, the column temperature was 20 deg.C, the detection wavelength was 245nm, and the flow rate was 0.8 mL/min. The mobile phase A was acetonitrile, B was 10mM ammonium acetate solution (pH4.5), and C was double distilled water. Sample separation conditions: t is 0min, 90% a, 10% B; t is 60min, 55% a, 45% B. And (3) column washing conditions: t is 0min, 50% a, 50% C; t is 25min, 25% a, 75% C; t-40 min, 95% a, 5% C.

The PNH derivative of maltodextrin has ESI-MS (see FIG. 7 (a)), HPLC (see FIG. 7(b), ESI-MS (see FIG. 7 (c)) after HPLC separation, and ESI-MS (see FIG. 7 (d)) after de-labeling.

Example 7: regeneration of free reducing maltooligosaccharide monomers from MBTH derivatives of maltodextrins

The method comprises the following specific steps:

(1) taking 5mg of standard maltodextrin, and mixing according to a molar ratio of not less than 1: taking MBTH (10mg) according to the proportion of 10, dissolving maltodextrin and MBTH in 1mL of weak acid aqueous solution of organic solvent (1 mL of 500. mu.L ethanol + 50. mu.L acetic acid + 450. mu.L water, 50% ethanol and 5% acetic acid), shaking uniformly, reacting at 60 ℃ for 45min, and then concentrating and drying.

(3) Dissolving the obtained sample with acetonitrile, and then carrying out HILIC-HPLC separation under the liquid phase conditions that: for HILIC-HPLC separation, a TSK-GEL Amide-80 column of 4.6mm × 250mm was used, the column temperature was 20 deg.C, the detection wavelength was 254nm, and the flow rate was 0.8 mL/min. The mobile phase A was acetonitrile, B was 10mM ammonium acetate solution (pH4.5), and C was double distilled water. Sample separation conditions: t is 0min, 85% a, 15% B; t is 60min, 50% a, 50% B. And (3) column washing conditions: t is 0min, 50% a, 50% C; t ═ 20min, 25% a, 75% C; t ═ 30min, 25% a, 75% C; t-50 min, 95% a, 5% C.

The ESI-MS spectrum of MBTH labeled maltodextrin is shown in figure 8(a), the HPLC spectrum is shown in figure 8(b), the ESI-MS spectrum of maltopentaose after HPLC separation is shown in figure 8(c), and the ESI-MS spectrum of maltopentaose after de-labeling is shown in figure 8 (d).

Comparing ESI-MS spectra before and after maltopentaose de-labeling can find that: the method provided by the invention can efficiently recover the reducing oligosaccharide chains labeled by the seven hydrazine reagents.

In addition, the labeling yield of the seven hydrazine color development reagents to the maltooligosaccharide and the recovery yield of the free reduced maltooligosaccharide monomer after chemical de-labeling are counted, and the statistical results are shown in the following table:

hydrazine reagent	Reducing chain	Mark yield	Recovery yield
				BSH	Maltodextrin	91％	93％
TSH	Maltodextrin	82％	97％
				BZH	Maltodextrin	81％	93％
PLH	Maltodextrin	89％	95％
				GP	Maltodextrin	95％	89％
PNH	Maltodextrin	83％	94％
				MBTH	Maltodextrin	61％	81％

From the above table it can be observed that:

MBTH-derived oligosaccharide chains, both in labeling yield (61%) and recovery yield (81%);

the recovery yield of the sugar chains derived by the four reagents of TSH, BZH, PLD and PNH is high, but the marking yield is below 90%;

GP derived oligosaccharide chains, high labeling yield and low recovery yield;

the BSH derived oligosaccharide chain has marking yield and recovery yield over 90%. Considering the marking rate of the seven hydrazine chromogenic reagents on the malto-oligosaccharide and the recovery rate of free reducing sugar monomers after chemical de-marking comprehensively, BSH is selected as a marking reagent (the BSH is insoluble in water and soluble in dichloromethane, excessive reagents can be removed through simple extraction in subsequent treatment to purify a sample, and when the reducing oligosaccharide chains are prepared through large-scale separation, the BSH can be recycled), and the reducing oligosaccharide monomers in two biological samples are regenerated through the method.

Example 8: regeneration of free reduced N-sugar chain monomers from BSH derivatives of the N-sugar chain of chicken protein proteins

The method comprises the following specific steps:

(1) taking 10mg of N-sugar chains released by the chicken protein, and mixing the N-sugar chains according to a molar ratio of not less than 1: 10 BSH (5mg) was taken, and the egg albumin N-sugar chain and BSH were dissolved in 1mL of a weak acid aqueous solution of an organic solvent (1 mL of 500 μ L of ethanol +50 μ L of acetic acid +450 μ L of water, 50% ethanol and 5% acetic acid), shaken, reacted at 60 ℃ for 45min, and then concentrated and dried.

(2) Dissolving the obtained sample with water, extracting with organic solvent (dichloromethane), centrifuging, collecting supernatant, repeating for three times, and concentrating and drying the supernatant.

(3) Dissolving the obtained sample with acetonitrile, and then carrying out HILIC-HPLC separation under the liquid phase conditions that: in the high performance liquid chromatography separation, a TSK-GEL Amide-80 column of 4.6mm × 250mm is used, the column temperature is 20 ℃, the detection wavelength is 245nm, and the flow rate is 0.8 mL/min. The mobile phase A was acetonitrile, B was 10mM ammonium acetate solution (pH6.0), and C was double distilled water. Sample separation conditions: t is 0min, 85% a, 15% B; t is 60min, 50% a, 50% B. And (3) column washing conditions: t is 0min, 50% a, 50% C; t ═ 20min, 25% a, 75% C; t ═ 30min, 25% a, 75% C; t-50 min, 95% a, 5% C.

The BSH derivative of the egg albumin N-sugar chain has an HPLC (high performance liquid chromatography) pattern shown in figure 9(a), 13 peaks are collected, ESI-MS (ESI-MS) patterns before and after the 13 peaks are collected and used for sample solution de-labeling are shown in figures 9(b-1) to 9(b-34), 17 target sugar chains with complete structures (all sodium ion peaks) are detected, and isomers of 4 sugar chains are separated and detected. The m/z of the BSH-labeled N-sugar chains after HPLC separation are 1087.08, 1290.17, 1290.33, 1249.17, 1249.17, 1493.25, 1493.25, 1452.25, 1452.17, 1411.17, 1696.25, 1655.25, 1614.25, 1899.33, 1573.25, 1817.25 and 1858.25 respectively, and the m/z of the corresponding free reducing N-sugar chains after de-labeling are 933.25, 1136.25, 1136.17, 1095.08, 1095.25, 1339.25, 1339.25, 1298.25, 1298.33, 1257.25, 1542.33, 1501.17, 1460.25, 1745.33, 1419.25, 1663.17 and 1704.33 respectively. The average recovery rate of the egg albumin N-sugar chains is more than 85 percent by calculation.

Example 9: regeneration of free reduced oligosaccharide monomers from BSH derivatives of human milk oligosaccharides

The method comprises the following specific steps:

(1) taking 10mg of freeze-dried human milk, and mixing the freeze-dried human milk with the mixture according to a molar ratio of not less than 1: 10 BSH (5mg) was taken, human milk and BSH were dissolved in 1mL of weak acid aqueous solution of organic solvent (1 mL of 500 μ L ethanol +50 μ L acetic acid +450 μ L water, 50% ethanol and 5% acetic acid), shaken well, reacted at 60 ℃ for 45min, and then concentrated and dried.

BSH derivatives of human milk oligosaccharide chains, the HPLC pattern of which is shown in FIG. 10(a), 20 peaks are collected, ESI-MS patterns before and after the sample solution de-labeling of the 20 peaks are shown in FIGS. 10(b-1) to 10(b-40), 20 structurally intact target sugar chains (all sodium ion peaks) are detected, and isomers of four sugar chains are separated and detected. The m/z of BSH labeled human milk oligosaccharide after HPLC separation is 519.08, 665.25, 665.08, 681.08, 681.08, 681.08, 811.08, 810.08, 884.08, 1030.08, 1030.08, 1176.17, 1149.50, 1176.08, 1321.17, 1466.08, 1395.17, 1614.17, 1541.17 and 1687.17 respectively, and the m/z of the corresponding free reduced oligosaccharide monomer after de-labeling is 365.08, 511.25, 511.08, 527.08, 527.08, 527.08, 657.17, 656.17, 730.17, 876.17, 876.17, 1022.17, 1095.17, 1022.17, 1167.17, 1312.17, 1241.17, 1460.17, 1387.25 and 1533.25. These target sugar chains contain oligosaccharides having various structures, including sialic acid-modified and fucose-modified oligosaccharides. By calculation, the average recovery of human milk oligosaccharide chains was greater than 90%.

Therefore, the method provided by the invention can be used for recovering neutral reducing sugar and acidic reducing sugar with complete structures simply, quickly and efficiently.

It should be noted that the above-mentioned embodiments do not limit the present invention in any way, and all technical solutions obtained by using equivalent alternatives or equivalent variations fall within the protection scope of the present invention.

Claims

1. A method for regenerating a reducing sugar from a hydrazine chromophoric reagent derivative of the reducing sugar, comprising the steps of:

(1) taking reducing sugar, and adding a hydrazine color development reagent according to the molar number of not less than 10 times of the reducing sugar, wherein the hydrazine color development reagent is as follows: benzenesulfonyl hydrazide, p-toluenesulfonyl hydrazide, benzoyl hydrazide, phenylacetyl hydrazide, Gilard reagent, phenylhydrazine, 3-methyl-2-benzothiazolinone hydrazone and other chemical reagents simultaneously having a hydrazino group and a chromophoric group are dissolved in a weak acid aqueous solution of an organic solvent, shaken up, heated for reaction, concentrated and dried after the reaction is finished;

2. The method according to claim 1, wherein in step (1), the reducing sugar comprises: saccharides having a reducing terminal aldehyde group directly extracted from a biological sample by various methods, saccharides having a reducing terminal aldehyde group released from glycoconjugates, and various artificially synthesized saccharides having a reducing terminal aldehyde group.

3. The method of claim 1, wherein in step (3), the chromatography comprises: HILIC-HPLC, RP-HPLC and PGC-HPLC.

4. The method according to claim 1, wherein in step (4), the weak acid aqueous solution comprises: a mixed solution of acetic acid and water, a mixed solution of citric acid and water, and a mixed solution of formic acid and water.