CN106399432B - Method for preparing N-linked glycopeptide from monoclonal antibody and N-linked glycopeptide - Google Patents
Method for preparing N-linked glycopeptide from monoclonal antibody and N-linked glycopeptide Download PDFInfo
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Abstract
The invention provides a high-efficiency separation preparation method for preparing an N-linked glycopeptide component from a monoclonal antibody by using a two-dimensional chromatographic separation technology, which mainly comprises the N-linked glycopeptide component with a peptide segment sequence of EEQYNSTYRN. The process comprises the following steps: and (3) carrying out enzymolysis on the monoclonal antibody by using trypsin, carrying out centrifugal concentration, removing salt, polypeptide and residual protein in the enzymolysis solution from the concentrated solution by using a one-dimensional chromatographic column, and collecting target fraction. And centrifuging and concentrating the collected fractions, and passing the concentrated solution through a two-dimensional chromatographic column to remove residual small peptides by utilizing the orthogonality of two-dimensional chromatographic separation so as to obtain the N-linked glycopeptide component sample with higher purity.
Description
Technical Field
The invention relates to extraction and separation preparation of an N-linked glycopeptide component separated and prepared from a monoclonal antibody, in particular to an N-linked glycopeptide component with 9 amino acid peptide segments, which is prepared and separated from a monoclonal antibody through enzymolysis, wherein glycan in the component consists of 5-15 mannose, galactose, N-acetylglucosamine, fucose or sialic acid, and the molecular weight is 2000-4000.
Background
Protein glycosylation is an important post-translational modification of proteins, with about more than half of proteins being glycosylated in eukaryotes, particularly mammals, and almost 100% of membrane proteins being present in glycosylated form (Apweiler R, Hermjakob H, Sharon N, On the frequency of protein glycosylation, and reduced from analysis of the SWISS-PROT database. Biochim Biophys Acta-Subj, 1999,1473, 4-8). Glycosylation has a major influence on the structure and function of proteins, such as folding, transport and localization of proteins. The sugar chains of glycoproteins play an important role in cell-to-cell recognition, adhesion, communication, immune response, and the like, and in addition, abnormalities in glycosylation of proteins are closely related to the development and diagnosis of diseases such as tumors, development, immunological abnormalities, and infections. At present, diseases such as tumors and rheumatoid diseases are known to cause abnormal changes of sugar chain structures, so that sugar chains are considered as potential disease markers and targets of new vaccines (Ohtsubo K, Marth JD, Glycosylation in cellular mechanisms of health and disease. cell 2006,126, 855-867; Kim EH, Misek DE, Glycoproteomics based identification of cancer specific markers International patent of Proteomics,2011, 1-10). Because glycopeptides only account for a small fraction (2-5%) of all peptide fragments after enzymatic hydrolysis, their mass spectral response is easily inhibited by high-abundance non-glycopeptides. Meanwhile, due to the microscopic heterogeneity of sugar chains, the types of sugar chains at one glycosylation site may be as many as several tens, further reducing the relative amounts of sugar chains to make them difficult to detect, which makes the isolation and preparation of glycoprotein glycopeptides abnormally difficult. Although sugar chains are not easy to obtain, the influence of non-glycopeptides, phosphorylated peptides and other peptide segments in proteins is removed, on the basis of high-purity glycopeptides, the structural information of the sugar chains is researched, and the discovery of some potential diagnostic markers and therapeutic targets becomes simpler, more convenient and more direct, and accurate results can be obtained more easily, so that an efficient and high-flux separation and preparation method is urgently needed for the separation and preparation of the glycopeptides. In order to obtain glycopeptide standard product with a single structure, the glycopeptide mixture is enriched and separated from the enzymolysis liquid, which is the first step.
The two-dimensional liquid chromatography is an emerging technology, and two chromatographic columns with the same or different properties are used for separating complex samples. As an ideal tool for analyzing complex samples, it has been widely used in various fields such as medical and health, food science, environment and agricultural science. In recent years, with the development of two-dimensional liquid chromatography theory and application, two-dimensional preparative liquid chromatography has been gradually developed. Compared with single-dimensional preparative liquid chromatography, two-dimensional preparative liquid chromatography not only has higher peak capacity, but also has different selectivities in two dimensions.
The enzyme method for releasing glycoprotein sugar chains is simple, has mild conditions, can provide information about sugar chain residue composition, arrangement sequence, glycosidic bond configuration and the like, and is widely applied at present, wherein Trypsin (Trypsin) is more widely applied. So far, most studies have focused on the detection of glycopeptides in an enzymatic hydrolysate by a mass spectrometry method, although many studies have been made on the release of tryptsin enzymatic sugar chains and structural characterization of immunoglobulins and monoclonal antibodies in serum. Meanwhile, with the continuous knowledge of the glycopeptide structure, the research on the glycopeptide function is more and more interesting, which makes the acquisition of glycopeptide samples urgent. However, it has not been reported that glycopeptides are enriched and separated from a monoclonal antibody enzymatic hydrolysate and a glycopeptide component with high purity is prepared by two-dimensional liquid chromatography.
Disclosure of Invention
In order to further study the structure and function of glycoproteins and glycopeptides, the acquisition of glycopeptide samples is the most basic and important. However, it is very difficult to obtain a glycopeptide sample by removing non-glycopeptides from proteins in an amount of 95% to 98%. So far, no report of enrichment and separation of glycopeptide from monoclonal antibody enzymolysis liquid by a chromatographic method is available. The invention provides a high-efficiency separation preparation method for preparing an N-linked glycopeptide component from a monoclonal antibody by using a two-dimensional chromatographic separation technology.
In order to achieve the purpose, the invention adopts the technical scheme that:
1) protein denaturation: adding a buffer solution of urea into a monoclonal antibody sample for reaction, adding dithiothreitol into a reaction product for reaction, adding iodoacetic acid into the product, and reacting in a dark place to obtain a denatured protein solution;
2) enzymolysis: adding enzyme into the denatured protein solution obtained in the step (1) to perform enzymolysis reaction to obtain an enzymolysis reaction product;
3) and (3) performing two-dimensional liquid chromatography separation preparation on the enzymolysis reaction product obtained in the step (2), and drying the collected components to obtain the N-linked glycopeptide component.
The conditions for the reaction of the monoclonal antibody sample in the buffer solution of urea in the step (1) are 20-30 ℃ incubation, the pH value of the buffer solution is 7-9, and the reaction time is 1-5 hours; after the dithiothreitol is added, the reaction condition is 35-40 ℃ incubation, and the reaction time is 1-5 hours; the reaction condition after adding the iodoacetic acid is temperature incubation at 20-30 ℃ in a dark place, and the reaction time is 10-60 minutes.
The concentration of urea in the buffer solution in the step (1) is 4-10M; the used buffer solution is ammonium bicarbonate buffer solution with the concentration of 30-80 mM; the mass ratio of the monoclonal antibody to the urea added into the buffer solution is 1:1-1: 40.
The final concentration of the added dithiothreitol in the system is 30-100 mM, and the mass ratio of the initial monoclonal antibody to the dithiothreitol is 14:1-140: 1;
the final concentration of the iodoacetic acid in the system after the iodoacetic acid is added is 30-100 mM, and the mass ratio of the initial monoclonal antibody to the iodoacetic acid is 18:1-180: 1.
Before the enzymolysis reaction in the step (2), diluting the denatured protein solution by 5-10 times with ammonium bicarbonate buffer solution, and then adding enzyme, wherein the concentration of the ammonium bicarbonate buffer solution is 30-80 mM; the pH value of the enzymolysis reaction is 7-9, the temperature is 35-40 ℃, and the reaction time is 10-24 hours; and (3) boiling the enzymolysis liquid obtained after enzymolysis in boiling water for 5-10 minutes to inactivate the enzyme to obtain an enzymolysis reaction product.
In step (2) the enzyme is trypsin; the mass ratio of the enzyme to the initial monoclonal antibody is 1:10-1: 50.
And (3) taking a bonding material taking silica gel as a matrix as chromatographic column packing, taking Uniry C18 or XAqua (Hua Shi Xin Chu Co., Ltd.) as one-dimensional reversed-phase packing, and taking XAmide or XIon (Hua Shi Xin Chu Co., Ltd.) as two-dimensional hydrophilic packing.
The granularity of the silica gel matrix bonding material adopted by the one-dimensional preparation in the two-dimensional liquid chromatography separation preparation in the step (3) is 2-20 microns, the mobile phase adopted by the one-dimensional liquid chromatography preparation is one or two of methanol or acetonitrile and water, wherein the methanol or the acetonitrile is an organic phase and does not contain or contains 0.1 v% of formic acid, and the aqueous phase does not contain or contains 0.1 v% of formic acid; the elution condition is carried out according to the gradient that the volume fraction of the organic phase is 5-30 percent isocratic or 5-50 minutes, and the volume fraction of the organic phase is increased from 5 percent to 40 percent; the column temperature is 25-40 deg.C, the total flow rate of eluent is 0.2-1.0 mL/min, and the fraction with retention time of 10-30min is collected; the granularity of the silica gel matrix bonding material adopted in the two-dimensional preparation is 2-20 microns, the adopted mobile phase is acetonitrile which is used as an organic phase and is mixed with water which is used as a water phase, the elution condition is carried out in a gradient way that the volume fraction of the water phase is increased from 10-15% to 30-60% according to the isocratic degree of 10-50% or the volume fraction of the water phase is increased from 5-30 minutes, the column temperature is 25-40 ℃, the total flow rate of the eluent is 0.2-1.0 mL/min, and the components with the retention time of 10-30 are collected.
In the one-dimensional preparation, the optimal mobile phase is formed by mixing acetonitrile containing or not containing 0.1 v% formic acid with water containing or not containing 0.1 v% formic acid, and the volume ratio of the acetonitrile is 8-15% by adopting an isocratic mode; the optimum mobile phase for two-dimensional preparation is acetonitrile with or without 0.1 v% formic acid mixed with water with or without 0.1 v% formic acid, using gradient conditions, with or without 0.1 v% formic acid at 15% water volume concentration, held for 5 minutes, and increased to 35% -45% over 15-20 minutes.
The main component of the N-linked glycopeptide prepared by the method is an N-linked glycopeptide component with 9 amino acid peptide segments, the peptide segment sequence is EEQYNSTYRN, N-linked glycan is based on a pentasaccharide core consisting of two N-acetylglucosamines and 3 mannoses, and then one or more than two of mannose, galactose, N-acetylglucosamine, fucose or sialic acid with the total number of 0-10 are linked, and the molecular weight of the N-linked glycopeptide is 2000-4000.
THE ADVANTAGES OF THE PRESENT INVENTION
1. By adopting a two-dimensional liquid chromatography separation technology and selecting proper chromatographic columns and chromatographic separation preparation conditions, impurity proteins, polypeptides and non-glycopeptides are removed, and an N-linked glycopeptide component is obtained from the monoclonal antibody.
2. The purity of the obtained N-linked glycopeptide component is high and can reach more than 80%.
3. The method for preparing the high-purity N-linked glycopeptide from the monoclonal antibody has the advantages of high degree of automation of instruments, convenient and simple operation, capability of being carried out at normal temperature and normal pressure, and suitability for the requirement of large-scale preparation.
Detailed Description
The present invention will be further described with reference to examples. The examples are given solely for the purpose of illustration and are not intended to be limiting.
Example 1:
monoclonal antibody protein (Sigma-Aldrich, cat # 527858) was weighed at 1mg, added to 167. mu.L of 4M urea in 30mM ammonium bicarbonate buffer and incubated at 20 ℃ for 1 hour, the pH of the buffer being 7. The denatured monoclonal antibody protein was added to 15. mu.L of 30mM aqueous dithiothreitol solution, incubated at 35 ℃ for 1 hour to reduce the disulfide bond of the denatured monoclonal antibody protein, then added to 10. mu.L of 30mM aqueous iodoacetic acid solution, incubated at 30 ℃ for 10 minutes in the absence of light, and the reduced disulfide bond was alkylated.
Adding the denatured monoclonal antibody protein into 960 mu L ammonium bicarbonate buffer solution with the concentration of 0.1M; adding 0.1mg of Trypsin, wherein the pH value of the enzymolysis buffer solution is 7, the enzymolysis reaction temperature is 35 ℃, the enzymolysis time is 10 hours, and after enzymolysis, the enzymolysis liquid is boiled in boiling water for 5 minutes to inactivate enzyme. The enzymatic hydrolysate was centrifugally concentrated 3 times (program temperature 15 ℃) on a LabConco centrifugal concentrator to obtain an enzymatic concentrate. Packing the column with a Unitry (Wasaburra Innovation Co., Ltd.) filler with the particle size of 2 microns, wherein the column diameter is 3mm, the column length is 250mm, acetonitrile is selected as an organic phase as a mobile phase, water is used as a water phase, 5% of the organic phase is adopted for isocratic elution, the column temperature is 25 ℃, the flow rate of the mobile phase is 0.2mL/min, ultraviolet detection is carried out at 280nm, eluted components for 20-30 minutes are collected, and the components are centrifugally concentrated by 5 times according to the centrifugal concentration conditions to obtain one-dimensional components. XAmide (Hua spectral Xin Chuang Co., Ltd.) with the particle size of 2 microns is used for filling a column, the column diameter is 3mm, the column length is 150mm, the two-dimensional separation chromatographic column is used, acetonitrile (containing 0.1 v% formic acid) is used as an organic phase of a mobile phase, water (containing 0.1 v% formic acid) is used as a water phase, 10% water phase is adopted for isocratic elution, the column temperature is 35 ℃, the flow rate of the mobile phase is 0.2mL/min, 280nm ultraviolet detection is carried out, elution components are collected for 20-30 minutes, the elution components are centrifugally concentrated to dryness according to the centrifugal concentration conditions, and the N-linked glycopeptide component containing 9 amino acids is obtained, and the number of glycopeptides is 6 and the purity is 85% through mass spectrometry. Example 2:
10mg of monoclonal antibody (Sigma-Aldrich, cat # 527858) was weighed into 100. mu.L of 8M urea in 50mM ammonium bicarbonate buffer, and incubated at 30 ℃ for 5 hours, the pH of the buffer being 9. The denatured monoclonal antibody was added to 15. mu.L of a 30mM aqueous solution of dithiothreitol, incubated at 40 ℃ for 5 hours to reduce the disulfide bond of the denatured monoclonal antibody, then added to 6. mu.L of a 50mM aqueous solution of iodoacetic acid, incubated at 20 ℃ for 60 minutes in the absence of light, and the reduced disulfide bond was alkylated.
Adding the denatured monoclonal antibody into 1210 mu L ammonium bicarbonate buffer solution with the concentration of 0.5M, adding 0.2mg Trypsin, wherein the pH value of the enzymolysis buffer solution is 9, the enzymolysis reaction temperature is 40 ℃, the enzymolysis time is 24 hours, and after enzymolysis, the enzymolysis solution is boiled in boiling water for 10 minutes to inactivate enzyme. The enzymatic hydrolysate was centrifugally concentrated 5 times (program temperature 15 ℃) on a LabConco centrifugal concentrator to obtain an enzymatic concentrate.
Packing a column by XAqua (Hua Shi Xin Chuang Co., Ltd.) with the particle size of 20 microns, wherein the column diameter is 4.6mm, the column length is 150mm, the mobile phase selects methanol (containing 0.1% v formic acid) as an organic phase, water (containing 0.1% v formic acid) as an aqueous phase, and adopting a gradient elution mode: increasing the volume concentration of the organic phase from 5% to 40% in 50 min, detecting with ultraviolet rays at 280nm at the column temperature of 40 ℃ and the flow rate of the mobile phase of 1.0mL/min, collecting the eluted components for 15-20 min, and performing centrifugal concentration for 5 times according to the centrifugal concentration conditions to obtain the one-dimensional component. XAmide (Hua spectral Xin Chuang Co., Ltd.) with the particle size of 20 micrometers is used for filling a column, the column diameter is 4.6mm, the column length is 150mm, the two-dimensional separation chromatographic column is used, acetonitrile (containing 0.1% v formic acid) is selected as an organic phase as a mobile phase, water (containing 0.1% v formic acid) is used as a water phase, 50% of organic phase is adopted for isocratic elution, the column temperature is 40 ℃, the flow rate of the mobile phase is 0.7mL/min, 280nm ultraviolet detection is carried out, elution components are collected for 10-15 minutes, the elution components are centrifugally concentrated to dryness according to the centrifugal concentration conditions, the N-linked glycopeptide component containing 9 amino acids is obtained, and the number of the glycopeptides is 6 and the purity is 70% through mass spectrometry.
Example 3:
30mg of monoclonal antibody (Sigma-Aldrich, cat # 527858) was weighed into 100. mu.L of 10M urea in 50mM ammonium bicarbonate buffer, and incubated at 25 ℃ for 2 hours, the pH of the buffer being 7. The denatured monoclonal antibody was added to 10. mu.L of 100mM aqueous dithiothreitol solution, incubated at 37 ℃ for 5 hours to reduce the disulfide bond of the denatured monoclonal antibody, then added to 25. mu.L of 100mM aqueous iodoacetic acid solution, incubated at 30 ℃ for 20 minutes in the absence of light, and the reduced disulfide bond was alkylated.
Adding the denatured monoclonal antibody into 1500 mu L ammonium bicarbonate buffer solution with the concentration of 5M, adding 1mg of Trypsin, wherein the pH value of the enzymolysis buffer solution is 8, the enzymolysis reaction temperature is 37 ℃, the enzymolysis time is 24 hours, and after enzymolysis, boiling the enzymolysis solution in boiling water for 5 minutes to inactivate enzyme. The enzymatic hydrolysate was centrifugally concentrated 7 times (program temperature 15 ℃) on a LabConco centrifugal concentrator to obtain an enzymatic concentrate.
Filling XAqua (Hua spectral New creation Co., Ltd.) with the particle size of 5 microns into a column, wherein the column diameter is 4.6mm, the column length is 250mm, acetonitrile is selected as an organic phase as a mobile phase, water is selected as a water phase, 30% of the organic phase is adopted for isocratic elution, the column temperature is 25 ℃, the flow rate of the mobile phase is 0.8mL/min, ultraviolet detection is carried out at 280nm, elution components for 10-15 minutes are collected, and centrifugal concentration is carried out by 5 times according to the centrifugal concentration conditions to obtain one-dimensional components. Filling a column with XIon (Hua spectral New creation Co., Ltd.) filler with the particle size of 5 microns, wherein the column diameter is 4.6mm, the column length is 250mm, the two-dimensional separation chromatographic column is adopted, acetonitrile is selected as an organic phase as a mobile phase, water is selected as a water phase, and a gradient elution mode is adopted: the concentration of the water phase is increased to 60 percent from 10 percent in 30 minutes, the column temperature is 30 ℃, the flow rate of the mobile phase is 1.0mL/min, ultraviolet detection is carried out at 280nm, elution components for 15-20 minutes are collected and centrifugally concentrated to be dry according to the centrifugal concentration conditions, namely the N-linked glycopeptide component containing 9 amino acids, the number of glycopeptides is 10 and the purity is 90 percent through mass spectrometry analysis and identification.
Example 4:
10mg of monoclonal antibody (Sigma-Aldrich, cat # 527858) was weighed into 200. mu.L of 8M urea in 50mM ammonium bicarbonate buffer, and incubated at 25 ℃ for 2 hours, the pH of the buffer being 8. The denatured monoclonal antibody was added to 40. mu.L of 400mM dithiothreitol 50mM ammonium bicarbonate buffer solution, incubated at 35 ℃ for 1 hour to reduce the disulfide bond of the denatured monoclonal antibody, then 10. mu.L of 100mM iodoacetic acid 50mM ammonium bicarbonate buffer solution was added, and incubated at 25 ℃ for 10 minutes in the absence of light to alkylate the reduced disulfide bond.
Adding 9000 mu L of ammonium bicarbonate buffer solution with the concentration of 0.05M into the denatured monoclonal antibody, adding 0.25mg of Trypsin, wherein the pH value of the enzymolysis buffer solution is 8, the enzymolysis reaction temperature is 37 ℃, the enzymolysis time is 20 hours, and after enzymolysis, the enzymolysis solution is boiled in boiling water for 5 minutes to inactivate enzyme. The enzymatic hydrolysate was centrifugally concentrated 10-fold (program temperature 15 ℃) on a LabConco centrifugal concentrator to obtain an enzymatic concentrate.
Packing the column with a Unitry (Wasaburra Innovation Co., Ltd.) packing material with the particle size of 5 microns, wherein the column diameter is 3.0mm, the column length is 150mm, acetonitrile (containing 0.1% v formic acid) is selected as an organic phase as a mobile phase, water (containing 0.1% v formic acid) is selected as a water phase, 10% of the organic phase is adopted for isocratic elution, the column temperature is 30 ℃, the flow rate of the mobile phase is 0.4mL/min, ultraviolet detection is carried out at 280nm, elution components for 12-15 minutes are collected, and the components are centrifugally concentrated by 5 times according to the centrifugal concentration conditions to obtain one-dimensional components. Packing a column by using an Xion (Hua spectral New creation Co., Ltd.) packing with the particle size of 3 micrometers, wherein the column diameter is 2.1mm, the column length is 150mm, the two-dimensional separation chromatographic column is used, acetonitrile (containing 0.1% v of formic acid) is selected as an organic phase as a mobile phase, water (containing 0.1% v of formic acid) is selected as a water phase, and a gradient elution mode is adopted: the concentration of the water phase is increased to 40% from 15% in 30 minutes, the column temperature is 30 ℃, the flow rate of the mobile phase is 0.2mL/min, ultraviolet detection is carried out at 280nm, elution components for 17-24 minutes are collected, centrifugal concentration is carried out under the centrifugal concentration condition until the elution components are dried, namely the N-linked glycopeptide component containing 9 amino acids, the number of glycopeptides is 8 and the purity is 95% through mass spectrometry analysis and identification.
Claims (5)
1. A method for producing an N-linked glycopeptide from a monoclonal antibody, comprising:
1) protein denaturation: adding a buffer solution of urea into a monoclonal antibody sample for reaction, adding dithiothreitol into a reaction product for reaction, adding iodoacetic acid into the product, and reacting in a dark place to obtain a denatured protein solution;
2) enzymolysis: adding enzyme into the denatured protein solution obtained in the step 1) to perform enzymolysis reaction to obtain an enzymolysis reaction product; before enzymolysis reaction, diluting the denatured protein solution by 5-10 times with ammonium bicarbonate buffer solution, and adding enzyme, wherein the concentration of the ammonium bicarbonate buffer solution is 30-80 mM; the pH value of the enzymolysis reaction is 7-9, the temperature is 35-40 ℃, and the reaction time is 10-24 hours; boiling the enzymolysis liquid obtained after enzymolysis in boiling water for 5-10 minutes to inactivate enzyme to obtain an enzymolysis reaction product; the enzyme is trypsin; the mass ratio of the enzyme to the initial monoclonal antibody is 1:10-1: 50;
3) performing two-dimensional liquid chromatography separation preparation on the enzymolysis reaction product obtained in the step 2), and drying the collected components to obtain N-linked glycopeptide components;
step 3) taking a bonding material with silica gel as a matrix as a chromatographic column filler, taking Uniry C18 or XAqua as a one-dimensional reversed-phase filler, and taking XAmide or XIon as a two-dimensional hydrophilic filler;
the granularity of the silica gel matrix bonding material adopted by the one-dimensional preparation in the two-dimensional liquid chromatography separation preparation in the step 3) is 2-20 microns, the specific size of a chromatographic column is one of column diameter 3mm, column length 150mm, column diameter 3mm, column length 250mm or column diameter 4.6mm and column length 150mm, and the mobile phase adopted by the one-dimensional liquid chromatography preparation is one or two of methanol or acetonitrile mixed with water, wherein the methanol or acetonitrile is an organic phase and does not contain or contains 0.1 v% of formic acid, and the aqueous phase does not contain or contains 0.1 v% of formic acid; the elution condition is carried out according to the gradient that the volume fraction of the organic phase is 5-30 percent isocratic or 5-50 minutes, and the volume fraction of the organic phase is increased from 5 percent to 40 percent; the column temperature is 25-40 deg.C, the total flow rate of eluent is 0.2-1.0 mL/min, and the fraction with retention time of 10-30min is collected; the granularity of the silica gel matrix bonding material adopted in the two-dimensional preparation is 2-20 microns, the specific size of a chromatographic column is one of the column diameter of 2.1mm, the column length of 150mm, the column diameter of 3mm, the column length of 150mm, the column diameter of 4.6mm, the column length of 150mm or the column diameter of 4.6mm and the column length of 250mm, the adopted mobile phase is acetonitrile which is used as an organic phase and water which is used as a water phase to be mixed, the elution condition is carried out in a gradient way that the volume fraction of the water phase is increased from 10-15% to 30-60% after 5-30 minutes, the column temperature is 25-40 ℃, the total flow rate of the eluent is 0.2-1.0 mL/min, and the component with the retention time of 10-30min is collected.
2. The method of claim 1, wherein: the conditions for the reaction of the monoclonal antibody sample in the buffer solution of urea in the step 1) are 20-30 ℃ incubation, the pH value of the buffer solution is 7-9, and the reaction time is 1-5 hours; after the dithiothreitol is added, the reaction condition is 35-40 ℃ incubation, and the reaction time is 1-5 hours; the reaction condition after adding the iodoacetic acid is temperature incubation at 20-30 ℃ in a dark place, and the reaction time is 10-60 minutes.
3. The method of claim 1, wherein: step 1), the concentration of urea in the buffer solution is 4-10M; the used buffer solution is ammonium bicarbonate buffer solution with the concentration of 30-80 mM; the mass ratio of the monoclonal antibody to the urea added into the buffer solution is 1:1-1: 40;
the final concentration of the added dithiothreitol in the system is 30-100 mM, and the mass ratio of the initial monoclonal antibody to the dithiothreitol is 14:1-140: 1;
the final concentration of the iodoacetic acid in the system after the iodoacetic acid is added is 30-100 mM, and the mass ratio of the initial monoclonal antibody to the iodoacetic acid is 18:1-180: 1.
4. The method of claim 1, wherein: in the one-dimensional preparation, acetonitrile containing or not containing 0.1 v% formic acid and water containing or not containing 0.1 v% formic acid are mixed as a mobile phase, and the volume ratio of the acetonitrile is 8-15% by adopting an isocratic mode; the two-dimensional preparation mobile phase is that acetonitrile containing or not containing 0.1 v% formic acid is mixed with water containing or not containing 0.1 v% formic acid, the volume concentration of the water containing or not containing 0.1 v% formic acid is 15% by adopting gradient condition, the water is kept for 5 minutes, and the volume concentration is increased to 35% -45% after 15-20 minutes.
5. An N-linked glycopeptide prepared by the method of any one of claims 1-4, wherein: the main component is N-linked glycopeptide component with 9 amino acid peptide segments, the peptide segment sequence is EEQYNSTYR, and N-linked glycan is based on pentasaccharide core composed of two N-acetylglucosamine and 3 mannoses, and then is linked with one or more than two of mannose, galactose, N-acetylglucosamine, fucose or sialic acid with the total number of 0-10; the molecular weight of N-linked glycopeptides is 2000-4000.
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