CN107793496A - A kind of general glycoprotein N sugar chain method for releasing - Google Patents

Abstract

The invention belongs to the field of biotechnology, and specifically discloses a general method for releasing glycoprotein N-sugar chains, which comprises dissolving glycoprotein samples in concentrated ammonia water, reacting in a water bath at 55-70°C for 12-20 hours in a closed container, and obtaining the reaction The solution was concentrated and dried under reduced pressure, redissolved with water, and the obtained N-sugar chain crude solution was adjusted to neutral pH, and then purified by C18 solid-phase extraction column and graphite carbon solid-phase extraction column to complete the release of N-sugar chain and preparation. This method has strong versatility and has the following advantages compared with existing methods: it is suitable for neutral N-glycans, acidic N-glycans and N-glycans with core α-1,3-fucose modification; It can be used for both microanalysis of sugar chains and large-scale preparation of sugar chains; the operation steps are simple, fast, and low in cost, and overcome the defects of traditional enzymatic methods and previously reported chemical methods for releasing glycoprotein N-sugar chains; The reducing N-sugars obtained by this method can be analyzed directly or used for analysis after derivatization by various derivatization methods.

Description

A general method for the release of glycoprotein N-glycan chains

technical field

The invention belongs to the field of biotechnology, and in particular relates to a general method for releasing glycoprotein N-sugar chains.

Background technique

Glycosylation is one of the common post-translational modifications of proteins, which plays an important role in the regulation of cell life activities, such as participating in cell adhesion, signal transduction, immune recognition, and the occurrence and development of diseases. The properties and functions of glycoproteins are closely related to the structure of sugar chains. Glycoproteins have complex structures and large molecular weights, making it difficult to conduct direct analysis and research. Generally, the sugar chains need to be released from the protein before analysis. Therefore, the release of sugar chains is a very critical link in the analysis of sugar chains. It is of great significance to analyze the structure and function of glycoprotein sugar chains.

At present, the release methods of N-glycan chains mainly include enzymatic methods and chemical methods. The enzymatic hydrolysis method mainly uses highly specific glycosidase or proteolytic enzymes to perform enzymatic digestion to obtain free sugar chains. The enzymes used mainly include N-glycosidase F (PNGaseF), N-glycosidase A (PNGasA), endoglycosidase H (EndoH), etc. These are specific specific glycosidases that can recognize specific of glycosidic bonds. Among them, PNGaseF enzymatic hydrolysis is the most commonly used method, which can effectively release most of the N-glycan chains, but the enzyme cannot release the core α-1,3-fucose modified sugar chains from plants, lower animals or microorganisms. N-sugar chains. Although the PNGase A enzyme can release α-1,3-fucose-modified N-glycan chains, the reaction efficiency is relatively low and it is not suitable for large-scale use. Moreover, this enzyme is expensive and only suitable for micro-analysis, which is difficult to use. for large-scale glycomic analysis. In addition, N-sugar chains with non-pentasaccharide core structures also widely exist in nature, such as N-sugar chains with one less mannose or one more N-acetylglucosamine on the basis of the five-sugar core, and some with high Mannose is the N-sugar chain whose reducing end is connected to the polypeptide, etc., which cannot be released by the above two enzymes, so there are certain limitations in the enzymatic method.

The common chemical method is the hydrazinolysis method, that is, the glycoprotein reacts in anhydrous hydrazine at 90°C for 4 hours to release sugar chains non-reductively, but anhydrous hydrazine is highly toxic and deacetylation reaction occurs. The study found that the glycoprotein reacted in 1M NaOH-1M NaBH ₄ system at 100°C for 4-6h can dissociate the reducing N-glycan chain, but it cannot be fluorescently derivatized, which is not conducive to subsequent separation and analysis. We have developed a non-reductive release method for N-glycan chains, that is, glycoproteins are reacted in NaOH solution at 50°C for 16 hours, and N-glycan chains without core α-1,3-fucose can be released without by-products , but the core α-1,3-fucose-modified N-glycans will undergo peeling degradation. There is also a method for sodium hypochlorite to release N-glycan chains that can be used in large-scale preparations, but the release efficiency is low, there are many by-products in the reaction, and the background of miscellaneous peaks is high when analyzing N-glycan chains by micro-mass spectrometry. Therefore, there is currently a lack of general chemical methods to release N-glycans.

To sum up, the main problems in the prior art are that the enzymatic method for preparing N-sugar chains has strong specificity and high cost, and the chemical method lacks versatility. Therefore, the development of a new method for the release and dissociation of glycosylated N-glycan chains with strong versatility, easy operation and low cost is of great significance for the analysis, identification and preparation of N-glycan chains.

Contents of the invention

In order to solve the deficiencies in the prior art, the present invention provides a general method for releasing glycoprotein N-sugar chains, which has strong versatility, simple operation and low cost.

The object of the present invention is to provide a method for releasing glycoprotein N-sugar chains, comprising the following steps:

S1, weigh the glycoprotein sample, dissolve it in concentrated ammonia water, and react in a water bath at 55-70°C for 12-20 hours in a closed container to obtain a reaction solution;

S2, concentrating and drying the reaction solution of S1 under reduced pressure, adding water to redissolve it, and obtaining a crude N-sugar chain solution;

S3, adjusting the pH of the crude N-glycan chain solution obtained in S2 to neutral, and sequentially purifying through a C18 solid-phase extraction column and a graphite carbon solid-phase extraction column to obtain a purified N-glycan chain sample.

Preferably, in the above general method for releasing N-sugar chains of glycoproteins, the concentration of the concentrated ammonia water is 25-18g/100g.

Preferably, in the above general glycoprotein N-sugar chain release method, after the glycoprotein sample is dissolved in concentrated ammonia water, the concentration of the glycoprotein sample is 1-20 mg/mL.

Preferably, in the above general method for releasing glycoprotein N-sugar chains, when the quality of the glycoprotein sample is ≥ 1 g, the crude N-sugar chain solution obtained in S2 must go through the steps of savage method combined with isoelectric precipitation to remove proteins, and then Adjust the pH to neutral;

When the mass of the glycoprotein sample is less than 1 g, the crude N-sugar chain solution obtained in S2 is directly adjusted to neutral pH without the steps of savage combined with isoelectric precipitation to remove protein.

Preferably, the above-mentioned general glycoprotein N-sugar chain release method, the purification process of the C18 solid phase extraction column is as follows: the C18 solid phase extraction column is first activated with 3 times the column volume of acetonitrile, then equilibrated with 10 times the column volume of double distilled water, and then Load the sample, and elute the sugar chain with 10 times the column volume of double distilled water;

The purification process of the graphite carbon solid phase extraction column is as follows: the graphite carbon solid phase extraction column is first activated with 3 times the column volume of acetonitrile, and then equilibrated with 10 times the column volume of double distilled water, and then the sample purified by the C18 solid phase extraction column is loaded , after loading the sample, eluted with 10 times the column volume of double distilled water to remove salt, then eluted with 3ml 25ml/100ml acetonitrile aqueous solution, collected the eluate, concentrated and dried under reduced pressure to obtain the purified N-glycan sample.

Preferably, the above general glycoprotein N-sugar chain release method, the specific operation of the savage method combined with isoelectric point precipitation to remove proteins is as follows: first add 1/5 times N- The savage reagent of the volume of the sugar chain crude product solution, mix well, then adjust the pH to the isoelectric point of the protein, centrifuge after stirring for 10 minutes, and collect the supernatant for later use; wherein, the savage reagent is prepared by dichloromethane and n-butanol at a ratio of 4:1 The volume ratio is mixed.

Preferably, in the above-mentioned general glycoprotein N-sugar chain release method, in the purification process of graphite carbon solid-phase extraction column, if the N-sugar chain to be purified is an acidic N-sugar chain, then add three Fluoroacetic acid is made into acetonitrile water-trifluoroacetic acid solution, the volume concentration of trifluoroacetic acid in the acetonitrile water-trifluoroacetic acid solution is 0.01%, and then eluted.

Compared with the prior art, the general glycoprotein N-sugar chain release method of the present invention has the following beneficial effects:

(1) The method provided by the present invention is based on the fact that the N-sugar chain connected to the glycoprotein is broken in concentrated ammonia water, and the amide bond (—CO—NH—) is broken to generate an unstable sugar amine. Because there is a large amount of excess ammonia water in the system, the sugar amine is protected, so as to achieve the purpose of protecting the reducing end of the sugar chain, so that the peeling reaction of the dissociated sugar chain does not occur. When the reaction is finished, the ammonia water is removed to generate reducing N-sugar chains, thereby completing the release of glycoprotein N-sugar chains. This method effectively avoids peeling degradation of N-glycan chains with core α-1,3-fucose.

(2) The N-sugar chains released by the present invention exist in the form of reducing N-sugar chains, have high stability and will not degrade, and can be directly analyzed after preliminary analysis or derivatization, and can be used for different types of N-sugar chains. The release and preparation of sugar chains overcome the defects of traditional enzymatic methods and previously reported chemical release methods. The method of the present invention releases glycoprotein N-sugar chains simply, quickly, with strong versatility and low cost, and is applicable to the modification of neutral N-sugar chains, acidic N-sugar chains and core α-1,3-fucose N-sugar chains, and the release efficiency of the sugar chains are high, can be used for both the microanalysis of the sugar chains and the large-scale preparation of the sugar chains, and provide a feasible method for the large-scale preparation of the N-sugar chains.

Description of drawings

Fig. 1 is the chemical reaction principle of the method for releasing glycoprotein N-sugar chains of the present invention;

Fig. 2 is the experimental result of the reaction temperature optimization of the method for releasing glycoprotein N-sugar chains of the present invention;

Fig. 3 is the experimental result of the reaction time optimization of the method for releasing glycoprotein N-sugar chains of the present invention;

Figure 4 is the ESI-MS spectrum of RiboB N-glycan chains released by different methods;

Wherein, Fig. 4A is the ESI-MS spectrum of the RiboB N-sugar chain released by the PNGaseF enzyme, and Fig. 4B is the ESI-MS spectrum of the RiboB N-sugar chain released by the method of Example 1;

Fig. 5 is the ESI-MS collection of patterns that different methods release the N-sugar chain on the ginkgo seed total protein;

Wherein, Fig. 5A is the ESI-MS spectrum of PNGaseF enzyme releasing the N-sugar chain on the total protein of ginkgo seeds; Fig. 5B is the ESI-MS spectrum of PNGaseA enzyme releasing the N-sugar chain on the total protein of ginkgo seeds; Fig. 5C is the implementation Example 2 method releases the ESI-MS spectrum of the N-sugar chain on the ginkgo seed total protein;

Fig. 6 is the secondary mass spectrometry analysis of the peak of 862 (m/z) when the N-sugar chain on the ginkgo seed total protein of release;

Figure 7 is the MALDI-TOF-MS spectrum of N-glycan chains on chicken albumin released by different methods and derivatized by GP;

Among them, Figure 7A is the MALDI-TOF-MS spectrum of N-glycan chains on chicken albumin released by PNGaseF enzyme and derivatized by GP; -TOF-MS spectrum; Figure 7C is the MALDI-TOF-MS spectrum of the N-glycan chain on the chicken albumin released by the method of Example 3 derivatized by GP;

Fig. 8 is the histogram obtained from the relative abundance ratio of each N-glycan chain of chicken albumin released by the three methods of PNGaseF enzyme, sodium hypochlorite and the method of Example 3 and the internal standard (IS);

Figure 9 is the ESI-MS spectrum of the released fetal bovine serum sialylated N-sugar chain;

Figure 10 is the ESI-MS spectrum of the released egg white N-glycan chain;

Among them, in Figure 4-Figure 7, Figure 9 and Figure 10, the gray circle represents mannose, the black square represents N-acetylglucosamine, the black triangle represents fucose, the white five-pointed star represents xylose, and the white circle represents galactose, Black diamonds represent sialic acid.

Detailed ways

The specific embodiments of the invention will be described in detail below, but it should be understood that the protection scope of the present invention is not limited by the specific embodiments. The test methods for which specific conditions are not indicated in the following examples are usually in accordance with conventional conditions, or in accordance with the conditions suggested by each manufacturer. In the examples described below, unless otherwise indicated, all temperature units are degrees Celsius, and the reaction temperature is room temperature, room temperature refers to 25°C±5°C, and all temperature errors are ±5°C.

In the following examples, bovine pancreatic ribonuclease B (RiboB), egg albumin, and Girard reagent P were all purchased from Sigma-Aldrich; PNGase F was purchased from New England BioLabs; sodium dodecyl sulfate (SDS ), dithiothreitol (DTT), and NP-40 were purchased from Aladdin Industrial Inc; fetal bovine serum (FBS) was purchased from ThermoScientific; Ginkgo seeds were picked on campus, and the total protein was extracted by our laboratory; The phase extraction cartridge Sep-Pak C18 (100mg/1mL) was purchased from Waters; the solid phase extraction cartridge porous graphite carbon column (150mg/4mL) was purchased from Alltech Associates; other reagents were of analytical grade. Concentrated ammonia water is purchased directly, with a concentration of 26% to 28%. Double distilled water is that laboratory is prepared with automatic double pure water distiller; Mass spectrometry identification AXIMA ALDI-TOF-MS mass spectrometer (Japan Shimadu company), ESI-MS and multistage mass spectrometry identification (MS ⁿ ) use electrospray in the present invention Electron linear ion trap mass spectrometry (LTQ XL, Thermo Scientific, USA) detection.

The primary ESI-MS parameters are set as follows: sample volume, 2 μL sample loop control; sample loading mobile phase is methanol/water (50%/50%, v/v); flow rate is 50 μL/min; working voltage is 4kV; The sheath gas flow rate is 20arb; the auxiliary gas flow rate is 10arb; the capillary voltage is 37V; the capillary lens voltage is 250V; the capillary temperature is 300°C; Acquisition was performed using LTQ Tune software.

The detection parameters of multi-stage mass spectrometry (MS ⁿ ) were set as follows: sample volume, 2 μL sample loop control; sample loading mobile phase was 50% methanol aqueous solution with a flow rate of 50 μL/min; working voltage was 4 kV; sheath gas flow rate was 20arb; assist gas flow rate is 10arb; capillary voltage is 37V; capillary lens voltage is 250V; capillary temperature is 300°C; maximum injection time is 1000ms; The ion collision energy is 35% to 45%; the activation charge is 0.25; the activation time is 30ms.

In the first-order mass spectrometry (MS) detection and multi-stage mass spectrometry (MS ⁿ ) detection in the present invention, acidic N-glycan chains in fetal bovine serum are detected in negative ion mode, and the rest are detected in positive ion mode.

The corresponding Chinese of abbreviations among the present invention is as follows:

ACN (acetonitrile), arb arbitrary unit (arbitrary unit, belongs to the pressure unit), DMSO (dimethyl sulfoxide), DTT (dithiothreitol), FBS (fetal bovine serum), MeOH (methanol), MSn (multiple mass spectrometry), mL (milliliters), min (minutes), ms (milliseconds), h (hours), Relative Abundance (relative abundance), SDS (sodium dodecyl sulfate), V (volts), M (mol /mL), IS (internal standard), TFA (trifluoroacetic acid).

The method provided by the invention is based on the fact that the amide bond (—CO—NH—) of the N-sugar chain connected to the glycoprotein is broken in concentrated ammonia water to generate an unstable sugar amine. Because there is a large amount of excess ammonia water in the system, the sugar amine is protected, so as to achieve the purpose of protecting the reducing end of the sugar chain, so that the peeling reaction of the dissociated sugar chain does not occur. When the reaction is finished, the ammonia water is removed to generate reducing N-sugar chains, thereby completing the release of glycoprotein N-sugar chains. This method effectively avoids peeling degradation of N-glycan chains with core α-1,3-fucose. Fig. 1 is the chemical reaction principle of releasing glycoprotein N-sugar chains by ammonia water method. Fig. 2 is the experimental result of the reaction temperature optimization of the ammonia release glycoprotein N-sugar chain; Fig. 3 is the experimental result of the reaction time optimization of the ammonia release glycoprotein N-sugar chain; the five lines in Fig. 2 and Fig. 3 represent the RiboB protein The five sugar chains released in , H represents hexose, N represents N-acetylglucosamine, and the different numbers behind H and N represent the number of hexose and N-acetylglucosamine. It can be seen from Figure 2 and Figure 3 that the optimum reaction temperature for the release of N-sugar chains is 60°C, and the optimum reaction time is 16h (see Example 1 for the specific release method).

List several examples below to specify release method of the present invention:

Example 1

A method for releasing glycoprotein N-sugar chains, the release object is the neutral N-sugar chains on the Ribo B protein, and the specific steps are as follows:

S1, weigh 5mg of Ribo B protein sample, dissolve it in 4ml of concentrated ammonia water, make the concentration of Ribo B protein sample 1.25mg/mL, react in a water bath at 60°C in a closed container for 16h, and obtain a reaction solution; the concentration of concentrated ammonia water is commercially available It is 25-28% (ie 25-28g/100g) concentrated ammonia water;

S2. Concentrate and dry the reaction solution of S1 under reduced pressure (concentration and dry under reduced pressure by conventional methods), add 3 mL of double distilled water to redissolve to obtain the crude N-glycan chain solution; since it is a small amount of Ribo B protein sample experiment, it can be omitted protein removal steps;

S3, adjust the pH of the crude N-glycan chain solution obtained in S2 to neutral to obtain the sample to be purified, and then pass through C18 solid phase extraction column and graphite carbon solid phase extraction column for purification to obtain the purified N-glycan chain Sample, to complete the release of N-glycan chains in the glycoprotein sample;

Among them, the purification process of the C18 solid-phase extraction column is as follows: the C18 solid-phase extraction column is first activated with 3 times the column volume of acetonitrile, then equilibrated with 10 times the column volume of double-distilled water, and then the sample to be purified is loaded, and 10 times the column volume is double-distilled. Water elutes sugar chains.

The purification process of the graphite carbon solid phase extraction column is as follows: the graphite carbon solid phase extraction column is first activated with 3 times the column volume of acetonitrile, and then equilibrated with 10 times the column volume of double distilled water, and then the sample purified by the C18 solid phase extraction column is loaded , after loading the sample, use 10 times of column volume double-distilled water to elute to desalt, then, use 3ml 25ml/100ml acetonitrile aqueous solution (the volume fraction of acetonitrile is 25%, solvent is water) to carry out elution, collect eluate, reduce pressure Concentrate to obtain a purified N-glycan sample.

Comparative example 1

In comparative example 1, PNGase F was used to enzymatically release glycoprotein N-sugar chains, and the release object was the same as in Example 1, which were all neutral N-sugar chains on Ribo B. The specific steps were as follows:

Weigh 5 mg of Ribo B glycoprotein sample, dissolve in 450 μL double distilled water, add 50 μL protein denaturation solution (preparation method of protein denaturation solution: 50 mg SDS and 62 mg DTT dissolved in 1 mL double distilled water), heat denaturation at 100 °C for 10 min. When the sample is cooled to room temperature, add 50 μL of enzymolysis buffer (preparation method of enzymolysis buffer: dissolve 1.9 g of sodium phosphate in 10 mL of double-distilled water, adjust the pH to 7.5 with phosphoric acid), 50 μL of NP-40 (10%, v /v) and 1 μL PNGase F enzyme, react at 37°C for 24h. After the reaction is completed, inactivate at 100°C for 5 minutes, concentrate under reduced pressure, redissolve the sample in 1 mL of double-distilled water, and purify through a C18 solid-phase extraction column and a graphite carbon solid-phase extraction column in turn to obtain a purified N-glycan sample , to complete the release of N-glycan chains in the glycoprotein sample. Wherein, the purification method of the C18 solid-phase extraction column and the graphite carbon solid-phase extraction column is the same as in Example 1.

The purified N-sugar chain samples obtained in Example 1 and Comparative Example 1 were subjected to ESI-MS detection and analysis, and the ESI-MS spectra of RiboB N-sugar chains released by different methods are shown in Figure 4, wherein Figure 4A is The ESI-MS spectrum of the RiboB N-sugar chain released by PNGaseF enzyme, and Figure 4B is the ESI-MS spectrum of the RiboB N-sugar chain released by the method in Example 1 (ammonia water). The sugar chain types of the two are the same. It shows the reliability of the method in Example 1 for the release of neutral N-sugar chains of glycoproteins.

Example 2

A method for releasing N-sugar chains of glycoproteins. The release object is N-sugar chains on the total protein of ginkgo seeds. It is known that the total protein of ginkgo seeds contains N-sugar chains modified by core α-1,3-fucose, specifically Proceed as follows:

S1, weigh 10 mg of ginkgo seed total protein, dissolve it in 4 ml of concentrated ammonia water, make the concentration of ginkgo seed total protein 2.5 mg/mL, react in a water bath at 60°C for 16 hours in a closed container, and obtain a reaction solution; the concentrated ammonia water uses commercially available The concentration is 25-28% (unit is 25-28g/100g) strong ammonia water;

The reaction solutions of S2 and S1 were concentrated and dried under reduced pressure, and re-dissolved with 3 mL of double distilled water to obtain a crude N-sugar chain solution; since it was a small amount of ginkgo seed total protein sample experiment, the step of protein removal could be omitted;

S3, adjust the pH of the crude N-glycan chain solution obtained in S2 to neutral to obtain the sample to be purified, and then pass through C18 solid phase extraction column and graphite carbon solid phase extraction column for purification to obtain the purified N-glycan chain Samples, complete the release and purification of N-sugar chains in glycoprotein samples;

Among them, the purification process of the C18 solid-phase extraction column is as follows: the C18 solid-phase extraction column is first activated with 3 times the column volume of acetonitrile, then equilibrated with 15 times the column volume of double-distilled water, and then the sample to be purified is loaded, and 15 times the column volume is double-distilled. Water elutes sugar chains.

The purification process of the graphite carbon solid phase extraction column is as follows: the graphite carbon solid phase extraction column is first activated with 3 times the column volume of acetonitrile, then equilibrated with 15 times the column volume of double distilled water, and then the sample purified by the C18 solid phase extraction column is loaded , after loading the sample, use 15 times column volume double-distilled water to elute and desalt, then, use 3ml 25ml/100ml acetonitrile aqueous solution (the volume fraction of acetonitrile is 25%, solvent is water) to carry out elution, collect eluate, reduce pressure Concentrate to obtain a purified N-glycan sample.

Comparative example 2-1

Comparative example 2-1 uses PNGase F enzymolysis to release glycoprotein N-sugar chains, and the release object is the same as in Example 2, all of which are N-sugar chains on the total protein of ginkgo seeds. The specific steps are as follows:

Weigh 5 mg of ginkgo seed total protein sample, dissolve it in 450 μL double-distilled water, add 50 μL protein denaturation solution (preparation method of protein denaturation solution: 50 mg SDS and 62 mg DTT are dissolved in 1 mL double-distilled water), and heat denature at 100 °C for 10 min. When the sample is cooled to room temperature, add 50 μL of enzymolysis buffer (preparation method of enzymolysis buffer: dissolve 1.9 g of sodium phosphate in 10 mL of double-distilled water, adjust the pH to 7.5 with phosphoric acid), 50 μL of NP-40 (10%, v /v) and 1 μL PNGase F enzyme, react at 37°C for 24h. After the reaction is completed, inactivate at 100°C for 5 minutes, concentrate under reduced pressure, redissolve the sample in 1 mL of double-distilled water, and purify through a C18 solid-phase extraction column and a graphite carbon solid-phase extraction column in turn to obtain a purified N-glycan sample , to complete the release of N-glycan chains in the glycoprotein sample. Wherein, the purification method of the C18 solid-phase extraction column and the graphite carbon solid-phase extraction column is the same as in Example 2.

Comparative example 2-2

Comparative example 2-2 uses PNGase A enzymolysis to release glycoprotein N-sugar chains, and the release object is the same as in Example 2, all of which are N-sugar chains on the total protein of ginkgo seeds. The specific steps are as follows:

Weigh 5mg of ginkgo seed total protein sample, dissolve in 1.25ml of hydrochloric acid buffer (pH=2) containing 3mg of pepsin, react at 37°C for 16h, and dry heat at 100°C for 5min after the reaction. Dry the sample with nitrogen, add 1mL citric acid buffer (0.1mol/L, pH=5) and 1.25μL PNGase A, react at 37°C for 48h, dry heat at 100°C for 5min after the reaction, then centrifuge, take Concentrate and blow dry. The sample was redissolved in 1 mL of double-distilled water, and then purified first with a C18 column and then with a graphitic carbon column. The purification operation method is the same as in Example 2, and the purified N-glycan chain sample is obtained and stored at -20°C after drying.

The purified N-sugar chain samples obtained in Example 2, Comparative Example 2-1 and Comparative Example 2-2 were respectively subjected to ESI-MS detection and analysis, and different methods released ESI-MS of N-sugar chains on the total protein of ginkgo seeds The spectrum is shown in Figure 5, where Figure 5A is the ESI-MS spectrum of the N-glycan chains on the total protein of ginkgo seeds released by the PNGaseF enzyme, and there is no N-glycan chain modified with core α-1,3-fucose ; Figure 5B is the ESI-MS spectrum of the PNGaseA enzyme releasing the N-sugar chain on the total protein of ginkgo seeds, which contains the N-sugar chain modified by core α-1,3-fucose; Figure 5C is the method of Example 2 (Ammonia water) releases the ESI-MS spectrum of the N-glycan chains on the total protein of ginkgo seeds, in which the type of N-glycan chains modified by the core α-1,3-fucose is consistent with the type of sugar chains released by the PNGaseA enzyme; The results show that the method of Example 2 is reliable for the release of N-sugar chains containing core α-1,3-fucose modification in glycoproteins.

Example 2 (Figure 5C), compared with the spectrum of PNGaseA enzyme release (Figure 5B), there is one more mass spectrum with a molecular weight of 862 (m/z), we have carried out secondary mass spectrometry analysis on it, and the results are shown in Figure 6 The MS/MS spectrum of this peak is shown. Fig. 6 is the result of secondary mass spectrometry analysis of the peak with a molecular weight of 862 (m/z) when the N-sugar chain on the total protein of ginkgo seeds is released. The result proves that this peak may be caused by the peeling degradation of a small part of the N-glycan chain (molecular weight: 1211, m/z) modified by core α-1,3-fucose. This result shows that ammonia releases the core α-1 , 3-Fucose-modified N-sugar chains will have a small amount of peeling degradation, but we have verified through practice that the degree of degradation is very small and does not affect the content of N-sugar chains at all, and the content error is within 0.2%. It does not affect its preparation result.

Example 3

A method for releasing glycoprotein N-sugar chains. The release object is chicken albumin N-sugar chains (neutral N-sugar chains). The specific steps are the same as in Example 1, except that the protein sample is replaced with 5 mg of chicken albumin. After the purified N-glycan samples were obtained, β-cyclodextrin was added as an internal standard (IS), and the target plate was derivatized and labeled with GP for MALDI-TOF-MS mass spectrometry analysis.

Comparative example 3-1

In comparative example 3-1, PNGase F is used to enzymatically release glycoprotein N-sugar chains, and the release object is the same as that of Example 3, all of which are chicken albumin N-sugar chains. The specific steps are as follows:

Weigh 5 mg of chicken albumin protein sample, dissolve it in 450 μL double-distilled water, add 50 μL protein denaturation solution (preparation method of protein denaturation solution: 50 mg SDS and 62 mg DTT are dissolved in 1 mL double-distilled water), and heat denaturation at 100 °C for 10 min. When the sample is cooled to room temperature, add 50 μL of enzymolysis buffer (preparation method of enzymolysis buffer: dissolve 1.9 g of sodium phosphate in 10 mL of double-distilled water, adjust the pH to 7.5 with phosphoric acid), 50 μL of NP-40 (10%, v /v) and 1 μL PNGase F enzyme, react at 37°C for 24h. After the reaction is completed, inactivate at 100°C for 5 minutes, concentrate under reduced pressure, redissolve the sample in 1 mL of double-distilled water, and purify through a C18 solid-phase extraction column and a graphite carbon solid-phase extraction column in turn to obtain a purified N-glycan sample , to complete the release of N-glycan chains in the glycoprotein sample. Wherein, the purification method of C18 solid phase extraction column and graphite carbon solid phase extraction column is the same as embodiment 3. After the purified N-glycan samples were obtained, β-cyclodextrin was added as an internal standard (IS), and the target plate was derivatized and labeled with GP for MALDI-TOF-MS mass spectrometry analysis.

Comparative example 3-2

In comparative example 3-2, sodium hypochlorite is used to release glycoprotein N-sugar chains, and the release object is the same as that in Example 3, all of which are chicken albumin N-sugar chains. The specific steps are as follows:

Weigh 5 mg of chicken albumin, dissolve it in 250 μL of double-distilled water, add 50 μL of 6% sodium hypochlorite, shake for 15 minutes, then add 2.5 μL of formic acid, shake for 5 minutes, centrifuge at 10,000 rpm for 10 minutes, and discard the precipitate. The supernatant was concentrated under reduced pressure and redissolved in 500 μL of double distilled water, and the N-glycan chains were purified by C18 solid phase extraction column and graphite carbon solid phase extraction column respectively. Among them, C18 solid phase extraction column and graphite carbon solid phase extraction The purification method of the column was the same as that in Example 3. After the purified N-glycan sample was obtained, β-cyclodextrin was added as an internal standard (IS), and the target plate was derivatized and labeled with GP for MALDI-TOF-MS mass spectrometry analysis.

The MALDI-TOF-MS mass spectrometry analysis of Example 3, Comparative Example 3-1, and Comparative Example 3-2 are shown in Figure 7. Figure 7 shows the N-glycan chains on chicken albumin released by different methods and derivatized by GP The MALDI-TOF-MS spectrum;

Among them, Figure 7A is the MALDI-TOF-MS spectrum of N-glycan chains on chicken albumin released by PNGaseF enzyme and derivatized by GP; -TOF-MS spectrum; Fig. 7C is the MALDI-TOF-MS spectrum of the N-sugar chain on the chicken albumin released by the method of Example 3 (ammonia) through GP derivatization; use the relative abundance of each N-sugar chain Compared with the relative abundance of the internal standard, the relative abundance ratio is obtained, and the ratio is analyzed to obtain the results shown in Figure 8, Figure 8 is each N-glycan chain of chicken albumin released by PNGaseF enzyme, sodium hypochlorite and the method of Example 3 The histogram obtained from the relative abundance ratio to the internal standard (IS). The results showed that, from the perspective of most N-glycan chains, the release efficiency of the method in Example 3 was higher than that of PNGaseF enzyme and sodium hypochlorite.

Example 4

A method for releasing glycoprotein N-sugar chains, the release object is sialylated N-sugar chains in fetal bovine serum, the specific steps are the same as in Example 1, the difference is that the protein sample is replaced by a 10 mg fetal bovine serum albumin sample, and the concentrated ammonia water The dosage was changed to 8ml. ESI-MS mass spectrometry analysis was carried out after the purified N-glycan chain sample.

Example 5

A method for releasing N-sugar chains of glycoproteins, the release object is or N-sugar chains in egg white, the specific steps are the same as in Example 1, the difference is that the protein sample is replaced by a 10 mg egg white freeze-dried powder sample, concentrated ammonia water The dosage was changed to 8ml.

Figure 9 is the ESI-MS spectrum of fetal bovine serum sialylated N-glycans released by the method of Example 4; Figure 10 is the ESI-MS spectrum of egg white N-glycans released by the method of Example 5. Both can detect N-glycan chains, further proving the feasibility and versatility of using ammonia water to release N-glycan chains of glycoproteins.

Example 6

A method for releasing glycoprotein N-sugar chains. The release object is sialylated N-sugar chains in fetal bovine serum. The specific steps are as follows:

S1, weigh 1g of fetal bovine serum albumin sample, dissolve it in 1000ml concentrated ammonia water, make the concentration of fetal bovine serum albumin be 1mg/mL, react in a water bath at 55°C for 12h in an airtight container, and obtain the reaction liquid; concentrate and dry under reduced pressure, add Redissolve in double distilled water;

The solution of S2 and S1 was deproteinized by savage method combined with isoelectric precipitation, centrifuged at 10,000rpm for 10min to remove a large amount of protein and peptide, concentrated and dried under reduced pressure to remove organic solvent, and re-dissolved with double distilled water to obtain crude N-glycan chain solution;

Among them, the specific operation of the savage method combined with isoelectric point precipitation to remove protein is: first add 1/5 times the volume of savage reagent to the solution dissolved in water after concentration under reduced pressure, mix well, and then adjust the pH to the isoelectric point of the protein, After stirring for 10 minutes, centrifuge, collect the supernatant for later use, and remove a large amount of protein and peptide; among them, the savage reagent is made by mixing dichloromethane and n-butanol in a volume ratio of 4:1.

S3, the operating steps of S3 are the same as in Example 1, the difference is that the eluent adopts 3ml 25% acetonitrile water-trifluoroacetic acid solution (the volume fraction of acetonitrile is 25%, the volume fraction of trifluoroacetic acid is 1%, and the solvent is water) .

Example 7

A method for releasing glycoprotein N-sugar chains. The release object is sialylated N-sugar chains in fetal bovine serum. The specific steps are as follows:

S1, weigh 20mg of fetal bovine serum albumin sample, dissolve it in 1ml of concentrated ammonia water to make the concentration of fetal bovine serum albumin is 20mg/mL, and react in a water bath at 70°C for 20h in a closed container to obtain a reaction solution;

The reaction solutions of S2 and S1 were concentrated and dried under reduced pressure, and redissolved in 3 mL of double-distilled water to obtain the crude N-glycan chain solution; since the experiment is a small amount of Ribo B protein sample, the step of protein removal can be omitted;

The operation steps of S3 and S3 are the same as in Embodiment 1.

It should be noted that when a numerical range is involved in the claims of the present invention, it should be understood that the two endpoints of each numerical range and any value between the two endpoints can be selected. Since the steps and methods adopted are the same as those in the embodiments, To avoid redundancy, the present invention has described preferred embodiments and their effects, but those skilled in the art can make additional changes and modifications to these embodiments once the basic inventive concept is understood. Therefore, it is intended that the appended claims be construed to cover the preferred embodiment as well as all changes and modifications which fall within the scope of the invention.

Obviously, those skilled in the art can make various changes and modifications to the present invention without departing from the spirit and scope of the present invention. Thus, if these modifications and variations of the present invention fall within the scope of the claims of the present invention and their equivalent technologies, the present invention also intends to include these modifications and variations.

Claims (7)

1. A general glycoprotein N-sugar chain releasing method, is characterized in that, comprises the following steps: S1, weigh the glycoprotein sample, dissolve it in concentrated ammonia water, and react in a water bath at 55-70°C for 12-20 hours in a closed container to obtain a reaction solution; S2, concentrating and drying the reaction solution of S1 under reduced pressure, adding water to redissolve it, and obtaining a crude N-sugar chain solution; S3, adjusting the pH of the crude N-glycan chain solution obtained in S2 to neutral, and sequentially purifying through a C18 solid-phase extraction column and a graphite carbon solid-phase extraction column to obtain a purified N-glycan chain sample. 2. The general method for releasing glycoprotein N-sugar chains according to claim 1, characterized in that the concentration of the concentrated ammonia water is 25-28g/100g. 3. The general method for releasing glycoprotein N-sugar chains according to claim 2, characterized in that, after the glycoprotein sample is dissolved in concentrated ammonia water, the concentration of the glycoprotein sample is 1-20 mg/mL. 4. The general method for releasing glycoprotein N-sugar chains according to claim 1, characterized in that, when the quality of the glycoprotein sample ≥ 1g, the crude N-sugar chain solution obtained in S2 must be combined with isoelectricity by the savage method. Point the step of precipitation and protein removal, and then adjust the pH to neutral; When the mass of the glycoprotein sample is less than 1 g, the crude N-sugar chain solution obtained in S2 is directly adjusted to neutral pH without the steps of savage combined with isoelectric precipitation to remove protein. 5. the general glycoprotein N-sugar chain release method according to claim 1, is characterized in that, the C18 solid phase extraction column purification process is: the C18 solid phase extraction column is first activated with 3 times of column volume acetonitrile, then with 10 Double the column volume with double distilled water to equilibrate, then load the sample, 10 times the column volume with double distilled water to elute the sugar chain; The purification process of the graphite carbon solid phase extraction column is as follows: the graphite carbon solid phase extraction column is first activated with 3 times the column volume of acetonitrile, and then equilibrated with 10 times the column volume of double distilled water, and then the sample purified by the C18 solid phase extraction column is loaded , after loading the sample, eluted with 10 times the column volume of double distilled water to remove salt, then eluted with 3ml 25ml/100ml acetonitrile aqueous solution, collected the eluate, concentrated and dried under reduced pressure to obtain the purified N-glycan sample. 6. The general method for releasing glycoprotein N-sugar chains according to claim 4, characterized in that, the specific operation of the savage method combined with the step of isoelectric precipitation to remove proteins is: first add the N-sugar chain crude product solution Add savage reagent equivalent to 1/5 times the volume of the crude N-sugar chain solution, mix well, then adjust the pH to the isoelectric point of the protein, centrifuge after stirring for 10 minutes, and collect the supernatant for later use; wherein, the savage reagent is made of dichloro Methane and n-butanol are mixed in a volume ratio of 4:1. 7. the general glycoprotein N-sugar chain release method according to claim 5, is characterized in that, in the graphite carbon solid-phase extraction column purification process, if the N-sugar chain to be purified is an acidic N-sugar chain, then Add trifluoroacetic acid to the eluent acetonitrile aqueous solution to prepare acetonitrile water-trifluoroacetic acid solution, the volume concentration of trifluoroacetic acid in the acetonitrile water-trifluoroacetic acid solution is 0.01%, and then perform elution.