Method for detecting N sugar on bird's nest glycoprotein

Abstract

The invention relates to the technical field of N sugar chain detection, in particular to a method for detecting N sugar on bird's nest glycoprotein, which comprises the following steps: acquiring the length of a plurality of N sugar chains in historical data; determining the rotating speed of a centrifugal machine in the buffer solution cleaning process; outputting a purified glycoprotein solution; determining whether the bird's nest glycoprotein is broken or not; adjusting the time interval between the purification end time of the cubilose protein and the adding time of the hydrolase, or determining the pH value of the denaturation liquid; cutting and releasing an N sugar chain; outputting the marked N sugar chain solution, and outputting a purified N sugar chain solution; determining sample loading voltage in the electrophoretic separation process; performing electrophoretic separation on the purified N sugar chain solution to obtain an electrophoretogram, and analyzing the electrophoretogram to obtain N sugar chain structure information of the bird's nest glycoprotein. According to the method, the detection accuracy of the N sugar chain in the bird's nest glycoprotein is improved.

Description

Technical Field

[0001] The present invention relates to the technical field of N-glycan detection, and particularly to a method for detecting N-glycans on glycoproteins in bird's nest. Background Art

[0002] In the prior art, glycosylation modification of glycoproteins is one of the most important and crucial post-translational modifications of proteins, which has a profound impact on the structural stability and biological activity of glycoproteins. It is also a key indicator for studying and evaluating the quality of bird's nest. Bird's nest is a nest built by swiftlets of the family Apodidae during the breeding season using saliva and its down feathers, and has rich nutritional and medicinal values. Bird's nest contains abundant glycoproteins, including both N-glycoproteins and O-glycoproteins, with the N-linkage form being the main one. The diversity and uniqueness of N-glycan chains are not only directly related to its medicinal value and nutritional and health care functions, but also an important mark for identifying authenticity and evaluating quality.

[0003] Chinese Patent Publication No.: CN103675085A discloses a method for detecting the N-glycan chain structure of immunoglobulin in human serum, including the following steps: purification of immunoglobulin in human serum, release of N-glycans on immunoglobulin, purification of the released N-glycans, and analysis of glycan structure information by mass spectrometry technology. It can be seen that the method for detecting the N-glycan chain structure of immunoglobulin in human serum has problems of reduced efficiency due to the limitations of long time consumption and low throughput of mass spectrometry technology, and reduced accuracy of N-glycan purification due to the fact that the purification conditions do not meet the adaptation conditions of glycoproteins during the purification process of N-glycans, resulting in a decrease in the tightness of N-glycan linkage. Summary of the Invention

[0004] Therefore, the present invention provides a method for detecting N-glycans on glycoproteins in bird's nest to overcome the problems of reduced efficiency due to the limitations of long time consumption and low throughput of mass spectrometry technology in the prior art, and reduced accuracy of N-glycan purification due to the fact that the purification conditions do not meet the adaptation conditions of glycoproteins during the purification process of N-glycans, resulting in a decrease in the tightness of N-glycan linkage.

[0005] To achieve the above object, the present invention provides a method for detecting N-glycans on glycoproteins in bird's nest, including: Obtaining the lengths of several N-glycan chains in historical data; Comparing the lengths of the several N-glycan chains with the lengths of the same N-glycan chains in a database to determine the centrifuge speed during the buffer washing process; Extracting and purifying the glycoproteins in bird's nest in sequence according to the speed to output a purified glycoprotein solution; Determining whether the glycoproteins in bird's nest are broken according to the comparison result between the structure of N-glycan chains and the molecular arrangement structure of glycoproteins in bird's nest in the database. If it is determined that the bird's nest glycoprotein is broken, a protein break adjustment method is executed, including adjusting the interval duration between the end time of the purification of the bird's nest protein and the addition time of the hydrolase. Or, determine the pH value of the denaturing solution according to the fitting degree between the standard deviation of the length of the N-glycan and the pH value of the hydrolase. If it is determined that the bird's nest glycoprotein is not broken, the protein break adjustment method is not executed, and the hydrolase is added to the purified glycoprotein solution to cleave and release the N-glycans in the bird's nest glycoprotein. Use a labeling solution to label the N-glycans to output a labeled N-glycan solution, and purify the N-glycans in the labeled N-glycan solution by magnetic bead suspension centrifugation to output a purified N-glycan solution. Collect the concentration of the purified N-glycan solution, and determine the loading voltage for the electrophoresis separation process according to the concentration and the fitting degree. Perform electrophoresis separation on the purified N-glycan solution according to the loading voltage to obtain an electrophoresis pattern, and analyze the electrophoresis pattern to obtain the N-glycan structure information of the bird's nest glycoprotein.

[0006] Further, determining the centrifuge speed for the buffer washing process includes: Obtain the lengths of the several N-glycans and calculate the average length. Obtain the lengths of the same type of N-glycans in the database and calculate the standard average length. Calculate the average length difference according to the average length and the standard average length. If the average length difference is greater than the preset average length difference, it is determined that the N-glycans are not broken, and the standard centrifuge speed for the buffer washing process is adopted. If the average length difference is less than or equal to the preset average length difference, it is determined that the N-glycans are broken, and the centrifuge speed for the buffer washing process is reduced. Wherein, the average length difference is the absolute value of the difference between the average length and the standard average length; the standard average length is the average value of the lengths of the same type of N-glycans in the database.

[0007] Further, the reduction amplitude of the centrifuge speed for the buffer washing process is determined according to the difference between the preset average length difference and the average length difference.

[0008] Further, adjusting the interval duration between the end time of the purification of the bird's nest protein and the addition time of the hydrolase includes: Obtain the structure of the N-glycans and compare it with the molecular arrangement structure of the bird's nest glycoprotein in the database. If there is a structure on the protein that is the same as the structure of the N-glycan, it is determined that the bird's nest glycoprotein is broken, and the interval duration at the time of addition is increased. Among them, the interval duration is proportional to the number of N-glycans with the same structure.

[0009] Furthermore, determining the pH value of the denaturing solution includes: Obtaining the length of the N-glycan and calculating the standard deviation from the average length; Obtaining the pH value of the solution of the hydrolase; Calculating the linear fitting degree between the standard deviation and the pH value; Comparing the linear fitting degree with a preset second fitting degree; If the linear fitting degree is greater than or equal to the preset second fitting degree, it is determined that the interference degree of the pH value of the hydrolase on the N-glycan hydrolysis reaction does not meet the requirements, and the actual pH value of the solution of the hydrolase is obtained; Adjusting the pH value of the denaturing solution according to the actual pH value of the solution of the hydrolase. Among them, the adjustment direction of the pH value of the denaturing solution is determined according to the pH value of the solution of the hydrolase.

[0010] Furthermore, determining the sample loading voltage in the electrophoresis separation process includes: If the linear fitting degree is greater than or equal to the preset first fitting degree and less than the preset second fitting degree, it is preliminarily determined that the hydrolysis reaction degree of the bird's nest glycoprotein does not meet the requirements; Collecting the concentration of the purified N-glycan solution; If the concentration of the purified N-glycan solution is greater than the preset concentration, it is determined again that the hydrolysis reaction degree of the bird's nest glycoprotein does not meet the requirements, and the sample loading voltage in the electrophoresis separation process is increased. Among them, the sample loading voltage is proportional to the concentration of the purified N-glycan solution.

[0011] Furthermore, the structural information of the N-glycan includes the composition of the N-glycan, the number of isomers of the N-glycan, the molecular weight of the N-glycan, and the mobility of the N-glycan.

[0012] Furthermore, purifying the N-glycans in the labeled N-glycan solution by magnetic bead suspension centrifugation to output a purified N-glycan solution includes: Taking out the magnetic beads and equilibrating them at room temperature for 10 min, and vortexing and mixing the magnetic bead suspension; Adding 5 μl of the magnetic bead suspension to a centrifuge tube and then adding 30 μl of the labeled N-glycan solution; Adding 200 μl of 100% acetonitrile, mixing well, placing it on a magnetic rack, standing for 1 min, and discarding the supernatant; Add 100 μl of 96% acetonitrile, mix well, place on a magnetic stand, let stand for 1 min, discard the supernatant, and repeat this step three times and then dry to obtain purified N-glycans; Add 50 μl of distilled water to the dried purified N-glycans to form a purified N-glycan solution, mix well, place on a magnetic stand, let stand for 1 min, and take the supernatant to a new centrifuge tube.

[0013] Further, electrophoretically separate the purified N-glycan solution according to the loading voltage to obtain an electrophoretogram, including: Add the 2 μl of purified N-glycan solution to a 96-well reaction plate and then add 8 μl of HD formamide mixture; Perform capillary gel electrophoresis on the glycan analyzer according to the adjusted loading voltage and obtain the electrophoretogram, wherein, the mixture and mixing ratio of each 1 μl of formamide mixture is 1 μl of OR600LIZ and 160 μl of HD formamide.

[0014] Further, the hydrolase is PNGase F enzyme.

[0015] Compared with the prior art, the beneficial effects of the present invention are as follows. The method of the present invention determines the cleavage situation of N-glycans. During the purification process of N-glycans, due to too high or too low temperature of the purification environment or pH value of the buffer solution, the obtained result of N-glycans is inaccurate. By adjusting the rotation speed of centrifugal washing of the buffer solution, the damage of centrifugal force to N-glycans is reduced; due to the high temperature during centrifugation, the sugar chains are broken, and the position where the sugar chains are broken is the part where N-glycans pass through the protein. By determining whether there is a break in the glycoprotein, since the sugar chains are prone to break due to the weakening of the glycoprotein, the glycoprotein structure is damaged and hydrolyzed, resulting in the presence of the structure on the glycoprotein in the purified N-glycans, and the accuracy of the detection result of N-glycans on the glycoprotein is decreased. By adjusting the interval time between the completion time of glycoprotein purification and the addition time of the hydrolase, the damage of purification temperature to the glycoprotein structure is reduced, and the improvement of detection accuracy is achieved; due to the decrease in the stability of hydrolase activity during the hydrolysis reaction, there is an error in the recognition and cleavage of N-glycans, resulting in the cleavage of N-glycans. By adjusting the pH value of the denaturing solution in the hydrolysis reaction, the hydrolysis reaction environment is improved, and further the influence of enzyme activity on the incorrect cleavage of N-glycans is reduced, and the improvement of monitoring accuracy and detection efficiency is achieved.

[0016] Furthermore, in the method of the present invention, by determining the cleavage situation of N-glycans, since N-glycans are affected by the purification environment, such as high purification temperature, the solution pH value not meeting the requirements, excessive centrifugation rate or glycoprotein weakening resulting in easy cleavage of the N-glycan linkage position, which further leads to an increase in the error of the detection result. By reducing the rotation speed of the centrifugal cleaning of the buffer solution of the easily cleavable N-glycans, the damage of the centrifugal force to the N-glycans is reduced, and the stability of the detection process is improved.

[0017] Furthermore, in the method of the present invention, by determining whether the glycoprotein is cleaved, since the glycans are prone to cleavage due to glycoprotein weakening, new N-glycans are generated at the cleavage position of the glycoprotein, which further reduces the accuracy of the detection result. By increasing the interval duration between the completion time of purification and the addition time of the hydrolase, and reducing the temperature during the purification process, the damage to the glycoprotein structure is reduced, and the stability of the glycoprotein before hydrolysis is increased, and the accuracy of the recognition and cleavage of N-glycans by the enzyme is improved.

[0018] Furthermore, in the method of the present invention, by determining the pH value of the denaturing solution, since the high-speed centrifugation process will cause the temperature of the PBS buffer solution to rise, resulting in the volatilization of some liquid components, and thus the concentration of the buffer solution becomes larger. When the concentration of the buffer solution becomes larger, the cleaning effect on the ultrafiltration tube will decrease, which may lead to impurities in the dissolved glycoprotein, and thus errors will occur in the hydrolysis position of the dissolved glycoprotein by the enzyme recognition of the impurities during the hydrolase digestion process. Since the pH value of the solution changes during the hydrolysis reaction, the stability of the hydrolase decreases and the ability to recognize the hydrolysis position of N-glycans decreases, resulting in incomplete hydrolysis of N-glycans or an increase in the hydrolysis error amount, which further leads to a decrease in the purity and yield of N-glycans and an increase in the difficulty of subsequent operations. By adjusting the pH value of the denaturing solution, the influence of the pH value change during the hydrolysis process on the enzyme activity is reduced, and the stability of the enzyme activity is increased.

[0019] Furthermore, in the method of the present invention, by determining whether the hydrolysis reaction degree of the bird's nest glycoprotein meets the requirements, since the pH value of the reaction solution changes during the hydrolysis reaction, the activity of the hydrolase is unstable, and the release amount of N-glycans increases. The high concentration of N-glycans will increase the viscosity of the electrophoresis buffer solution, resulting in the aggregation and precipitation of N-glycans. By increasing the loading voltage and increasing the current density to accelerate the electrophoresis speed, the deposition phenomenon is reduced, and the accuracy of the detection result is increased.

[0020] Furthermore, in the method of the present invention, the N-glycans are detected by obtaining an electrophoresis map, and the N-glycans are accurately identified by comparing with a database. Compared with the limitations of traditional detection methods such as high-performance liquid chromatography and mass spectrometry analysis, the structural information of N-glycans is determined by the electrophoresis detection method, and then the composition type of the known N-glycans is determined, and the detection efficiency is improved. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 This is the overall flowchart of the detection method for N-glycans on the edible bird's nest glycoprotein in the embodiments of the present invention; Figure 2 This is the flowchart for determining whether there is a break in the edible bird's nest glycoprotein in the detection method for N-glycans on the edible bird's nest glycoprotein in the embodiments of the present invention; Figure 3 This is the flowchart for determining the pH value of the denaturing solution in the detection method for N-glycans on the edible bird's nest glycoprotein in the embodiments of the present invention; Figure 4 This is the electrophoresis pattern of the detection method for N-glycans on the edible bird's nest glycoprotein in the embodiments of the present invention. Detailed implementation manners

[0022] In order to make the objectives and advantages of the present invention more clear and understandable, the present invention will be further described below in conjunction with embodiments; it should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0023] The preferred implementation manners of the present invention will be described below with reference to the accompanying drawings. Those skilled in the art should understand that these implementation manners are only used to explain the technical principle of the present invention and do not limit the protection scope of the present invention.

[0024] It should be noted that in the description of the present invention, the terms indicating directions or positional relationships such as "upper", "lower", "left", "right", "inner", "outer", etc. are based on the directions or positional relationships shown in the drawings. This is only for convenience of description and does not indicate or imply that the device or element must have a specific orientation, be constructed and operated in a specific orientation, and therefore cannot be understood as a limitation to the present invention.

[0025] In addition, it should be noted that in the description of the present invention, unless otherwise clearly specified and limited, the terms "installation", "connection", and "connection" should be understood in a broad sense. For example, it can be a fixed connection, a detachable connection, or an integral connection; it can be a mechanical connection or an electrical connection; it can be directly connected or indirectly connected through an intermediate medium, and it can be the communication inside two elements. For those skilled in the art, the specific meanings of the above terms in the present invention can be understood according to specific situations.

[0026] Please refer to Figure 1 、 Figure 2 、 Figure 3 as well as Figure 4 shown, which are respectively the overall flowchart of the detection method for N-glycans on the edible bird's nest glycoprotein in the embodiments of the present invention, the flowchart for determining whether there is a break in the edible bird's nest glycoprotein, the flowchart for determining the pH value of the denaturing solution, and the electrophoresis pattern. A detection method for N-glycans on an edible bird's nest glycoprotein of the present invention includes: Obtain the lengths of several N-glycans in historical data; Compare the lengths of the several N-glycans with the lengths of the same N-glycans in the database to determine the centrifuge speed during the buffer washing process; Extract and purify the bird's nest glycoprotein in sequence according to the speed to output a purified glycoprotein solution; Determine whether the bird's nest glycoprotein is broken according to the comparison result between the structure of the N-glycan and the molecular arrangement structure of the bird's nest glycoprotein in the database; If it is determined that the bird's nest glycoprotein is broken, perform a protein break adjustment method, including adjusting the interval duration between the end time of the purification of the bird's nest protein and the addition time of the hydrolase, Or, determine the pH value of the denaturing solution according to the fitting degree between the standard deviation of the length of the N-glycan and the pH value of the hydrolase; If it is determined that the bird's nest glycoprotein is not broken, do not perform the protein break adjustment method, and add the hydrolase to the purified glycoprotein solution to cleave and release the N-glycans in the bird's nest glycoprotein; Use a labeling solution to label the N-glycans to output a labeled N-glycan solution, and purify the N-glycans in the labeled N-glycan solution by magnetic bead suspension centrifugation to output a purified N-glycan solution; Collect the concentration of the purified N-glycan solution, and determine the loading voltage during the electrophoresis separation according to the concentration and the fitting degree; Perform electrophoresis separation on the purified N-glycan solution according to the loading voltage to obtain an electrophoresis pattern, and analyze the electrophoresis pattern to obtain the N-glycan structure information of the bird's nest glycoprotein.

[0027] Specifically, the process of extracting and purifying the bird's nest glycoprotein to output a purified glycoprotein includes: Grind the bird's nest sample into powder in liquid nitrogen, weigh 0.2 g of the bird's nest powder, add 4 mL of distilled water and mix evenly, extract at 60 °C for 6 h, centrifuge at the standard centrifuge speed of 9000 rpm during the buffer washing process for 20 min to take the supernatant, repeat the extraction three times, combine the extracts, and freeze-dry to obtain the crude bird's nest glycoprotein. Take 1 ml of PBS (phosphate buffer solution) to dissolve the crude glycoprotein product; Prepare a 3KD ultrafiltration tube, pour out the PBS previously stored in the ultrafiltration tube, add new PBS, centrifuge and wash the ultrafiltration tube at 7500 rpm, and pour out the PBS in the ultrafiltration tube; Transfer the dissolved crude protein into the ultrafiltration tube, centrifuge at 4 °C and 10000 rpm for 5 min, and discard the effluent; Add 1 ml of 2 M (molar concentration) Urea / 0.1 M NH4CO3 solution and centrifuge at 4°C and 10,000 rpm for 5 min. Discard the supernatant and repeat the above steps once. Add 1 ml of PBS and centrifuge at 10,000 rpm for 5 min. Transfer the remaining 100 - 200 μl of purified glycoprotein solution to a new EP tube. Among them, the meaning of the 3KD ultrafiltration tube is that the molecular retention rate of the ultrafiltration tube is 3KD.

[0028] Specifically, the process of adding hydrolase to the purified glycoprotein solution to cleave and release the N-glycans in the bird's nest glycoprotein includes: Take 10 μl of the purified glycoprotein solution, add 4 μl of denaturing solution and mix well. The mixing conditions are that the temperature of the denaturing solution is 60°C and the mixing time is 10 min. After cooling, add 3 μl of PNGase F enzyme reaction solution for hydrolysis. The hydrolysis conditions are that the temperature of the reaction solution is 37°C and the reaction time is 3 h. Take 5 μl of the N-glycan sample after enzymatic digestion and dry it at 65°C for 20 - 40 min. Among them, the denaturing solution is an aqueous solution of urea and guanidine hydrochloride, and the concentration range of the denaturing solution is 4 - 8 mol / L.

[0029] Specifically, the process of labeling N-glycans with a labeling solution to output a labeled N-glycan solution includes: Add APTS solution and 2 μl of DMSO (dimethyl sulfoxide) solution containing 1 M NaCNBH3 to the dried N-glycan sample, mix well for 30 s, and centrifuge for 10 s. Heat at a temperature of 37°C for a labeling time of 16 h. After labeling is completed, add 50 μl of distilled water to obtain a labeled N-glycan solution. Among them, the APTS solution is a mixture of 20 M APTS (3-aminopropyltriethoxysilane) and 1.2 M citrate buffer.

[0030] In implementation, the method of the present invention determines the cleavage of N-glycans. During the purification process of N-glycans, due to the too high or too low temperature of the purification environment or the pH value of the buffer solution, the obtained result of N-glycans is inaccurate. By adjusting the rotation speed of the centrifugal washing of the buffer solution, the damage to N-glycans caused by the centrifugal force is reduced; due to the high temperature during the centrifugation process, the sugar chains are cleaved, and the position of the sugar chain cleavage is in the part where the N-glycan passes through the protein. By determining whether the glycoprotein is cleaved, since the sugar chains are prone to cleavage due to the weakening of the glycoprotein, the glycoprotein structure is damaged and hydrolyzed, resulting in the presence of the structure on the glycoprotein in the purified N-glycans, and the accuracy of the detection result of the N-glycans on the glycoprotein is decreased. By adjusting the time interval between the completion time of the purification of the glycoprotein and the addition time of the hydrolase, the damage to the glycoprotein structure caused by the purification temperature is reduced, and the improvement of the detection accuracy is achieved; during the hydrolysis reaction process, the stability of the hydrolase activity is reduced, and there is an error in the recognition and cleavage of N-glycans, resulting in the cleavage of N-glycans. By adjusting the pH value of the denaturing solution in the hydrolysis reaction, the hydrolysis reaction environment is improved, and the influence of enzyme activity on the incorrect cleavage of N-glycans is reduced, and the improvement of the monitoring accuracy and the improvement of the detection efficiency are achieved.

[0031] Specifically, determining the rotation speed of the centrifuge during the buffer solution washing process includes: Obtaining the lengths of the several N-glycans and calculating the average length; Obtaining the lengths of the same type of N-glycans in the database and calculating the standard average length; Calculating the average length difference according to the average length and the standard average length; If the average length difference is greater than the preset average length difference, it is determined that the N-glycans are not cleaved, and the standard centrifuge rotation speed during the buffer solution washing process is adopted; If the average length difference is less than or equal to the preset average length difference, it is determined that the N-glycans are cleaved, and the rotation speed of the centrifuge during the buffer solution washing process is reduced; Wherein, the average length difference is the absolute value of the difference between the average length and the standard average length; the standard average length is the average value of the lengths of the same type of N-glycans in the database.

[0032] Specifically, the preset average length difference is 2 sugar units.

[0033] Specifically, the length of the N-glycan is detected by capillary electrophoresis.

[0034] Specifically, the reduction amplitude of the rotation speed of the centrifuge during the buffer solution washing process is determined according to the difference between the preset average length difference and the average length difference.

[0035] Specifically, the centrifuge rotation speed starts to decrease to the standard centrifuge rotation speed.

[0036] In implementation, for every 1 sugar unit exceeded by the average length difference, the centrifuge speed is reduced by 300 rpm. For example, if the average length difference is 3 sugar units, the centrifuge speed is reduced to 9000 rpm - 300 rpm × 3 = 8100 rpm.

[0037] In implementation, in the method of the present invention, by determining the cleavage situation of N-glycans, since N-glycans are affected by the purification environment, such as high purification temperature, unqualified solution pH value, excessive centrifugation rate or weakening of glycoproteins resulting in easy cleavage of the N-glycan linkage position, which in turn increases the error of the detection result. By reducing the rotation speed of the centrifugal cleaning of the buffer solution of the easily cleavable N-glycans, the damage of centrifugal force to N-glycans is reduced, and the stability of the detection process is improved.

[0038] Specifically, adjusting the interval duration between the end time of the purification of the bird's nest protein and the addition time of the hydrolase includes: Obtaining the structure of the N-glycan and comparing it with the molecular arrangement structure of the bird's nest glycoprotein in the database; If there is a structure on the protein structure that is the same as the structure of the N-glycan, it is determined that the bird's nest glycoprotein is cleaved, and the interval duration of the addition time is increased. Wherein, the interval duration is proportional to the number of N-glycans with the same structure.

[0039] Specifically, the proportional relationship that the interval duration is proportional to the number of N-glycans with the same structure is that the interval duration is the product of the number of N-glycans with the same structure and the proportionality coefficient plus the current interval duration. Wherein, the proportionality coefficient is 1.5.

[0040] In implementation, for example, if the number of N-glycans with the same structure is 4 and the current interval duration is 20 s, the interval duration is increased to 4 × 1.5 + 20 s = 26 s.

[0041] In implementation, in the method of the present invention, by determining whether the glycoprotein is cleaved, since the sugar chains prone to cleavage due to the weakening of the glycoprotein result in the generation of new N-glycans at the cleavage position of the glycoprotein, which in turn reduces the accuracy of the detection result. By increasing the interval duration between the completion time of purification and the addition time of the hydrolase, and reducing the temperature during the purification process, the damage to the glycoprotein structure is reduced, and the stability of the glycoprotein before hydrolysis is increased, and the accuracy of the recognition and cleavage of N-glycans by the enzyme is improved.

[0042] Specifically, determining the pH value of the denaturing solution includes: Obtaining the length of the N-glycan and calculating the standard deviation of its length from the average length; Obtain the pH value of the solution of the hydrolase; Calculate the linear fitting degree of the standard deviation and the pH value; Compare the linear fitting degree with a preset second fitting degree; If the linear fitting degree is greater than or equal to the preset second fitting degree, determine that the interference degree of the pH value of the hydrolase on the N-glycan hydrolysis reaction does not meet the requirements, and obtain the actual pH value of the solution of the hydrolase; Adjust the pH value of the denaturing solution according to the actual pH value of the solution of the hydrolase, wherein, the adjustment direction of the pH value of the denaturing solution is determined according to the pH value of the solution of the hydrolase.

[0043] Specifically, the hydrolase is PNGase F enzyme, and the suitable pH value of PNGase F enzyme is 8.0 - 8.5.

[0044] Specifically, the pH value of the denaturing solution is 8.2.

[0045] Specifically, the linear fitting degree of the standard deviation and the pH value is the R² value of the standard deviation and the pH value. The linear fitting degree is a conventional technical means familiar to those skilled in the art, so the calculation process of the R² value of the standard deviation and the pH value will not be elaborated here.

[0046] Specifically, the general value range of the preset second fitting degree is [0.662, 0.679].

[0047] Preferably, the preferred embodiment of the preset second fitting degree is 0.67.

[0048] Specifically, if the pH value of the solution of the hydrolase exceeds 8.5, for every 0.1 exceeded, the pH value of the denaturing solution is reduced by 0.1; if the pH value of the solution of the hydrolase is less than 8, for every 0.1 less, the pH value of the denaturing solution is increased by 0.1. For example, if the pH value of the solution of the hydrolase is 8.6, the pH value of the denaturing solution is reduced to 8.2 - 0.1 = 8.1; if the pH value of the solution of the hydrolase is 7.8, the pH value of the denaturing solution is increased to 8.2 + 0.1 + 0.1 = 8.4.

[0049] In implementation, in the method of the present invention, by determining the pH value of the denaturing solution, since the high-speed centrifugation process will cause the temperature of the PBS buffer solution to rise, resulting in the volatilization of some liquid components, and thus the concentration of the buffer solution becomes larger. When the concentration of the buffer solution becomes larger, the cleaning effect on the ultrafiltration tube will decrease, and further impurities may exist in the dissolved glycoprotein. Therefore, during the hydrolysis enzymolysis process, the enzyme will recognize the impurities and cause an error in the hydrolysis position of the dissolved glycoprotein. Since the pH value of the solution changes during the hydrolysis reaction, the stability of the hydrolysis enzyme decreases and the recognition ability of the hydrolysis position of the N-glycan decreases, resulting in incomplete hydrolysis of the N-glycan or an increase in the hydrolysis error amount, and further leading to a decrease in the purity and yield of the N-glycan, and increasing the difficulty of subsequent operations. By adjusting the pH value of the denaturing solution, the influence of the pH value change during the hydrolysis process on the enzyme activity is reduced, and the stability of the enzyme activity is increased.

[0050] Specifically, determining the loading voltage in the electrophoresis separation process includes: If the linear fitting degree is greater than or equal to the preset first fitting degree and less than the preset second fitting degree, it is preliminarily determined that the hydrolysis reaction degree of the bird's nest glycoprotein does not meet the requirements; Collect the concentration of the purified N-glycan solution; If the concentration of the purified N-glycan solution is greater than the preset concentration, it is secondarily determined that the hydrolysis reaction degree of the bird's nest glycoprotein does not meet the requirements, and the loading voltage in the electrophoresis separation process is increased. Wherein, the loading voltage is proportional to the concentration of the purified N-glycan solution.

[0051] Specifically, the concentration of the purified N-glycan solution is collected by a carbohydrate analysis reagent.

[0052] Specifically, the general value range of the preset first fitting degree is [0.412, 0.443].

[0053] Preferably, the preferred embodiment of the preset first fitting degree is 0.42.

[0054] Specifically, the general value range of the preset concentration is [0.8%, 1.4%].

[0055] Preferably, the preferred embodiment of the preset concentration is 1.2%.

[0056] In implementation, when the difference between the concentration of the purified N-glycan solution and the preset concentration is within 0.1%, the loading voltage is increased to 1.1 times the original. When the difference between the concentration of the purified N-glycan solution and the preset concentration exceeds 0.1%, for every 0.1% exceeded, the loading voltage is increased by 0.1 kv. For example, if the concentration of the purified N-glycan solution is 1.4% and the current loading voltage is 2.2 kv, then the loading voltage is increased to 2.2 kv×1.1 + 0.1 kv = 2.52 kv.

[0057] In implementation, the method of the present invention determines whether the degree of hydrolysis reaction of the bird's nest glycoprotein meets the requirements. Due to the change in the pH value of the reaction solution during the hydrolysis reaction, the activity of the hydrolase is unstable, the release amount of N-glycans increases, and the high concentration of N-glycans will increase the viscosity of the electrophoresis buffer, resulting in the aggregation and precipitation of N-glycans. By increasing the loading voltage and increasing the current density to accelerate the electrophoresis speed and thus reduce the deposition phenomenon, the accuracy of the detection result is increased.

[0058] Specifically, the structural information of the N-glycan includes the composition of the N-glycan, the number of N-glycan isomers, the molecular weight of the N-glycan, and the mobility of the N-glycan.

[0059] Specifically, the N-glycans in the labeled N-glycan solution are purified by magnetic bead suspension centrifugation to output a purified N-glycan solution, including: Take out the magnetic beads and equilibrate them at room temperature for 10 min, and vortex and mix the magnetic bead suspension; Add 5 μl of the magnetic bead suspension to a centrifuge tube, and then add 30 μl of the labeled N-glycan solution; Add 200 μl of 100% acetonitrile, mix well, place it on a magnetic stand, let it stand for 1 min, and discard the supernatant; Add 100 μl of 96% acetonitrile, mix well, place it on a magnetic stand, let it stand for 1 min, discard the supernatant, and repeat this step three times and then dry it to obtain purified N-glycans; Add 50 μl of distilled water to the dried purified N-glycans to obtain a purified N-glycan solution, mix well, place it on a magnetic stand, let it stand for 1 min, and take the supernatant to a new centrifuge tube.

[0060] Specifically, electrophoresis separation of the purified N-glycan solution is carried out according to the loading voltage to obtain an electrophoresis map, including: Add the 2 μl purified N-glycan solution to a 96-well reaction plate, and then add 8 μl of HD formamide mixture; Perform capillary gel electrophoresis on a glycan analyzer according to the adjusted loading voltage, and obtain the electrophoresis map, Among them, the mixture of each 1 μl of formamide mixture and the mixing ratio are 1 μl of OR600LIZ and 160 μl of HD formamide.

[0061] Specifically, the parameters of capillary gel electrophoresis are set as follows: the temperature of the incubator is 30 °C; the pre-electrophoresis voltage is 15 kV; the pre-electrophoresis time is 180 s; the range of the loading voltage is 2 - 6 kV; the loading time is 20 s; the electrophoresis voltage is 15 kV; the electrophoresis time is 2800 s.

[0062] Specifically, the hydrolase is PNGase F enzyme.

[0063] In implementation, the method of the present invention detects N-glycans by obtaining the distribution peak map of electrophoresis, and accurately identifies N-glycans by comparing with a database. Compared with the limitations of traditional detection methods such as high performance liquid chromatography and mass spectrometry analysis, the structural information of N-glycans is determined by the detection method of electrophoresis, and then the composition types of known N-glycans are determined, achieving an improvement in detection efficiency.

[0064] So far, the technical solution of the present invention has been described in conjunction with the preferred embodiments shown in the drawings. However, it is easy for those skilled in the art to understand that the protection scope of the present invention is obviously not limited to these specific embodiments. Without departing from the principle of the present invention, those skilled in the art can make equivalent changes or substitutions to the relevant technical features, and the technical solutions after these changes or substitutions will fall within the protection scope of the present invention.

Claims

1. A method for detecting N-sugar on bird's nest glycoprotein, characterized in that: include: Get the lengths of several N-glycans in historical data; comparing the lengths of the plurality of N-glycans with the lengths of the same N-glycans in a database to determine a centrifuge speed for a buffer washing process; Extracting and purifying the bird's nest glycoprotein in sequence according to the rotation speed to output a purified glycoprotein solution; Determine whether the bird's nest glycoprotein is broken according to the comparison result of the structure of the N sugar chain and the molecular arrangement structure of the bird's nest glycoprotein in the database; If it is determined that the bird's nest glycoprotein is broken, a protein breakage adjustment method is implemented, including adjusting the interval between the end of the purification of the bird's nest protein and the addition of the hydrolase. Or, the pH value of the denaturing solution is determined based on the standard deviation of the length of the N-sugar chain and the pH value of the hydrolase; If it is determined that the bird's nest glycoprotein does not have a cleavage, the protein cleavage adjustment method is not performed, and the hydrolase is added to the purified glycoprotein solution to cut and release the N sugar chain in the bird's nest glycoprotein; Using a labeling solution to label the N-sugar chains to output a labeled N-sugar chain solution, and using magnetic bead suspension centrifugation to purify the N-sugar chains in the labeled N-sugar chain solution to output a purified N-sugar chain solution; collecting the concentration of the purified N-glycan solution, and determining the loading voltage of the electrophoresis separation process according to the concentration and the fit; The purified N-sugar chain solution is subjected to electrophoretic separation according to the loading voltage to obtain an electrophoretic pattern, and the electrophoretic pattern is analyzed to obtain the N-sugar chain structural information of the bird's nest glycoprotein.

2. The method for detecting N-sugar on bird's nest glycoprotein according to claim 1, characterized in that: Determine the centrifuge speed for the buffer wash process, including: Obtaining the lengths of the plurality of N-glycans and calculating an average length; Obtaining the length of the same N-glycan in the database and calculating the standard average length; Calculate the average length difference according to the average length and the standard average length; If the average length difference is greater than a preset average length difference, it is determined that the N-glycans are not broken, and a standard centrifuge speed for a buffer wash process is used; If the average length difference is less than or equal to a preset average length difference, it is determined that the N sugar chain is broken, and the centrifuge speed during the buffer washing process is reduced; The average length difference is the absolute value of the difference between the average length and the standard average length; the standard average length is the average value of the lengths of the same N-sugar chains in the database.

3. The method for detecting N-sugar on bird's nest glycoprotein according to claim 2, characterized in that: The reduction range of the centrifuge speed in the buffer washing process is determined according to the difference between the preset average length difference and the average length difference.

4. The method for detecting N-sugar on bird's nest glycoprotein according to claim 3, characterized in that: The time interval between the end of the purification of the bird's nest protein and the addition of the hydrolase is adjusted, including: Obtaining the structure of the N-glycan and comparing it with the molecular arrangement structure of bird's nest glycoprotein in the database; If there is a structure identical to the structure of the N sugar chain in the structure of the protein, it is determined that the bird's nest glycoprotein is broken, and the interval time of the addition time is increased. The length of the interval is proportional to the number of N sugar chains with the same structure.

5. The method for detecting N-sugar on bird's nest glycoprotein according to claim 4, characterized in that: Determine the pH of the denaturing solution, including: Obtaining the length of the N-glycan and calculating the standard deviation thereof from the average length; Obtaining the pH value of the solution of the hydrolase; Calculate the linear fit between the standard deviation and the pH value; Comparing the linear fit with a preset second fit; If the linear fit is greater than or equal to the preset second fit, it is determined that the interference degree of the pH value of the hydrolase on the N-sugar chain hydrolysis reaction does not meet the requirement, and the actual pH value of the solution of the hydrolase is obtained; According to the actual pH value of the solution of the hydrolase, the pH value of the denaturing solution is adjusted, Wherein, the adjustment direction of the pH value of the denaturing solution is determined according to the pH value of the solution of the hydrolase.

6. The method for detecting N-sugar on bird's nest glycoprotein according to claim 5, characterized in that: Determine the sample loading voltage for electrophoresis separation, including: If the linear fit is greater than or equal to the preset first fit and less than the preset second fit, it is preliminarily determined that the degree of hydrolysis reaction of the bird's nest glycoprotein does not meet the requirements; collecting the concentration of the purified N-glycan solution; If the concentration of the purified N-glycan solution is greater than the preset concentration, it is determined that the hydrolysis reaction degree of the bird's nest glycoprotein does not meet the requirements, and the sample loading voltage of the electrophoresis separation process is increased. Wherein, the loading voltage is proportional to the concentration of the purified N-sugar chain solution.

7. The method for detecting N-sugar on bird's nest glycoprotein according to claim 6, characterized in that: The structural information of the N-sugar chain includes the composition of the N-sugar chain, the number of isomers of the N-sugar chain, the molecular weight of the N-sugar chain and the mobility of the N-sugar chain.

8. The method for detecting N-sugar on bird's nest glycoprotein according to claim 7, characterized in that: Purifying the N-glycans in the labeled N-glycan solution by magnetic bead suspension centrifugation to output a purified N-glycan solution, comprising: Take out the magnetic beads and equilibrate them at room temperature for 10 minutes, and vortex the magnetic bead suspension to mix well; Add 5ul of the magnetic bead suspension to the centrifuge tube and then add 30ul of the labeled N-sugar chain solution; Add 200ul 100% acetonitrile, mix well and place on a magnetic rack, let stand for 1min, and discard the supernatant; Add 100ul 96% acetonitrile, mix well and place on a magnetic rack, let stand for 1min, discard the supernatant, repeat this step three times and air dry to obtain purified N-glycans; Add 50ul of distilled water to the purified N-glycan solution after drying, mix well and place on a magnetic rack, let stand for 1min, and take the supernatant to a new centrifuge tube.

9. The method for detecting N-sugar on bird's nest glycoprotein according to claim 8, characterized in that: The purified N-glycan solution is subjected to electrophoretic separation according to the loading voltage to obtain an electrophoretic pattern, comprising: Add 2ul of the purified N-glycan solution into a 96-well reaction plate and then add 8ul of HD formamide mixed solution; Performing capillary gel electrophoresis on the glycomic analyzer according to the adjusted loading voltage, and obtaining the electrophoresis pattern, Wherein, the mixture of each 1ul of the formamide mixture and the mixing ratio are 1ul of OR600LIZ and 160ul of HD formamide.

10. The method for detecting N-sugar on bird's nest glycoprotein according to claim 9, characterized in that: The hydrolase is PNGase F enzyme.

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