CN107560920B

CN107560920B - Novel material for enriching and separating sialylated glycopeptides and application thereof

Info

Publication number: CN107560920B
Application number: CN201610506699.8A
Authority: CN
Inventors: 李智立; 张沫
Original assignee: Institute of Basic Medical Sciences of CAMS
Current assignee: Institute of Basic Medical Sciences of CAMS
Priority date: 2016-07-01
Filing date: 2016-07-01
Publication date: 2020-06-19
Anticipated expiration: 2036-07-01
Also published as: CN107560920A

Abstract

The invention discloses a specific enriched salivaMethods for acidifying glycopeptides. The invention claims g-C₃N₄The application in the enrichment of sialylglycopeptide and/or sialylated glycan and/or sialylated glycoside. The invention provides a method for enriching sialylated glycopeptides, which comprises the following steps: (1) g to C₃N₄Adding the dispersion liquid into a digestion product of the sialylated glycoprotein, and oscillating to enrich the sialylated glycopeptide; (2) centrifuging the enriched solution to g-C₃N₄Adding eluent into the precipitate to obtain sialylated glycopeptide solution. The method provided by the invention is based on g-C which is environment-friendly, simple and convenient to prepare and low in cost₃N₄The material has mild enrichment condition and high specificity, and keeps the structural information of the sialylated sugar chain to the maximum extent. The method can truly reflect the natural glycosylation state of the protein, and has important application value in the aspects of detection of sialylated glycopeptide in a complex sample and screening of related tumor markers.

Description

Novel material for enriching and separating sialylated glycopeptides and application thereof

Technical Field

The invention relates to a new material and a new method for enriching and separating sialylated glycopeptides. Based on graphite-phase carbon nitride (g-C)₃N₄) The material has the characteristic that a large number of N atoms in a triazine ring structure can form hydrogen bonds with sialylated sugar chains O-H and the characteristic pi interaction adsorption performance of a conjugated structure, and realizes efficient, low-cost and specific enrichment and separation of sialylated glycopeptides, sialylated glycans or sialylated glycosides on the premise of not damaging glycoprotein structures. Provides a new idea and a new method for detecting sialylglycopeptide in a complex sample, screening related tumor markers and the like.

Background

In the life activities of organisms, sialylated sugar chains play a role in specific recognition and mediation, and have a very important influence on the structure and function of proteins. It has been found that sialic acid expression in humans is very closely related to many pathological conditions, such as autoimmune diseases, cardiovascular diseases, tumorigenesis and tumor progression, bacterial and viral infections, neurological diseases, and endocrine disorders. The sialylated glycoprotein is an important protein posttranslational modification, can be used as a disease marker, and has very important significance for deeply researching the occurrence mechanism of diseases, diagnosing and treating and the like. The abundance of sialic acid in complex biological sample systems varies widely in dynamic, and a considerable portion of low-abundance sialylated glycopeptides, sialylated glycans, or sialylated glycosides are easily inhibited by other components. The first problem faced in the study of sialic acid is therefore the efficient isolation and enrichment thereof.

There are many methods for enriching sialylated glycopeptides, mainly including lectin method, chemical reaction method, hydrophilic material chromatography, etc. The lectin affinity method is the most widely applied method in the sialic acid glycoprotein enrichment research at present. The lectin can specifically enrich sialic acid, but has high cost and weak binding force, and is not beneficial to the screening of high-flux tumor markers and other works. Common chemical reaction enrichment methods comprise a boric acid chemical reaction method, a hydrazide chemical reaction method and the like, and are well applied to the identification of high-flux glycosylation sites, particularly N-type glycosylation sites. However, such methods have many chemical reaction steps, the conditions are not easy to control, and the chemical reaction process destroys the structure of the sugar chain, which is not favorable for further identification and analysis of the type and structure of the sugar chain. The zwitterionic hydrophilic chromatography and the strong ion exchange chromatography can nonselectively enrich various types of glycopeptides, but the specificity is poor, the enrichment efficiency needs to be improved, and sialic acid information is easily lost.

Disclosure of Invention

The invention aims to provide a new material for enriching sialylated glycopeptide and a new enrichment method thereof.

The invention claims the application of carbon nitride material in the enrichment of sialylated glycopeptides.

The invention also provides a method for enriching sialylated glycopeptides, which comprises the following steps:

(1) g to C₃N₄Adding the dispersion liquid into a digestion product of the sialylated glycoprotein, and oscillating to enrich the sialylated glycopeptide;

(2) centrifuging the enrichment solution, and adding sialylated glycopeptide-enriched g-C₃N₄Adding eluent into the precipitate to obtain sialylated glycopeptide solution.

In the above enrichment method, the enzyme-cleaved product is a product obtained by subjecting sialylated glycoprotein to enzyme cleavage. The enzyme digestion is specifically trypsin enzyme digestion.

In the above enrichment method, in step (1), g-C₃N₄The material is prepared by calcining dicyandiamide or melamine or urea. The amount of the calcined precursor is 1-10 g, such as 5 g. g-C obtained₃N₄The powder may be pre-treated as follows: g-C₃N₄And (6) washing. The specific washing method comprises the following steps: ultrasonic washing with 0.1M hydrochloric acid, ultrapure water and 80% acetonitrile in water, respectively (ultrasonic parameters)Specifically 20KHz, 5 min).

In the above enrichment method, in step (1), g-C₃N₄The dispersion is a mixed solution of acetonitrile/water solution, and the volume fraction of the acetonitrile is 70-80%, such as 80%.

In the enrichment method, in the step (1), the shaking enrichment time is 0.5-4 hours, such as 1 hour.

In the enrichment method, in the step (2), the centrifugal force of the enriched solution is 13500-21000 g, such as 13500 g.

In the enrichment method, in the step (2), the eluent is an aqueous solution with a pH of 5.5-12, such as 0.025% ammonia solution (pH 11.13).

In the enrichment method, in the step (2), the shaking elution time is 0.5-4 hours, such as 45 min.

In the enrichment method, in the step (2), after the elution is finished, the centrifugal method is adopted to obtain the aqueous solution of the sialylated glycopeptide, and the centrifugal force is 13500-21000 g, such as 13500 g.

In the above enrichment method, the sialylated glycoprotein may be bovine fetuin, transferrin, and a mixture of bovine fetuin, transferrin, albumin and ribonuclease B.

g-C in the invention₃N₄Has good enrichment effect on sialylglycopeptide and/or sialylated glycan and/or sialylated glycoside, the purity of the enriched product is close to 100 percent, and the recovery rate is 100 percent. Compared with a titanium dioxide enrichment method, the method provided by the invention has the advantages that the conditions are mild (sialic acid is prevented from falling off), the materials are cheap and easy to obtain, the enrichment specificity is high, and the effect is obvious. The enrichment by adopting the method provided by the invention can keep the structural information of sialylated sugar chains and/or glycans, retain complete specific peptide fragment information, is beneficial to subsequent mass spectrometry, reflects the natural glycosylation state of the protein, and has important significance for biomarker screening research, early warning, process detection, prognosis evaluation and the like of major diseases.

Drawings

FIG. 1 is a mass spectrum diagram of fetuin enzymolysis liquid (a), enriched liquid (b), enriched residual liquid (c), enriched liquid after sialidase treatment (d) and enriched liquid after PNGase enzyme treatment (e), wherein mass/charge refers to the ratio of molecular mass to charge number, ◆ shows that the difference between sialylated glycopeptide mass spectrum peaks is 291 Da.

FIG. 2 is a mass spectrogram of transferrin enzymolysis solution (a), enriched solution (b), enriched residual solution (c), enriched solution after sialidase treatment (d) and enriched solution after PNGase enzyme treatment (e), wherein mass/charge refers to the ratio of molecular mass to charge number, ◆ shows that the difference between sialylated glycopeptide mass spectrum peaks is 291 Da.

FIG. 3 is g-C₃N₄And (3) an enrichment effect mass spectrum of the sialylated glycopeptide of the fetuin with different loading amounts, wherein the mass/charge refers to the ratio of the molecular mass to the charge number.

FIG. 4 is a graph of different elution conditions for g-C₃N₄Enrichment for the effects of fetuin sialylated glycopeptides, mass/charge refers to the ratio of molecular mass to number of charges.

FIG. 5 is a mass spectrum of titanium dioxide enriched fetuin sialylated glycopeptide,

FIG. 6 shows g-C₃N₄Scanning electron micrographs of the materials.

FIG. 7 is g-C₃N₄Transmission electron micrographs of the materials (a) and (b).

FIG. 8 is g-C₃N₄The structure diagram of the material (a inset), the X-ray photoelectron spectrum (a) and the infrared spectrum (b).

Detailed Description

The following examples are given to facilitate a better understanding of the invention, but do not limit the invention. The experimental procedures in the following examples are conventional unless otherwise specified. The test materials used in the following examples were purchased from a conventional biochemical reagent store unless otherwise specified. The test results in the following examples are set up in triplicate and averaged. In the following examples, "%" represents a volume percentage unless otherwise specified.

In the long-term test process, the inventor of the invention finds that the g-C is₃N₄The sialylated glycopeptide can be separated and enriched efficiently, specifically and mildly as an enrichment material.

Pipette tips (tips) (maximum sample volume of 200. mu.L) were purchased from Axygen Scientific, Inc. under catalog number T-200-Y. Bovine fetuin, transferrin, bovine albumin, ammonium bicarbonate, iodoacetamide, formic acid, 2, 5-dimethylbenzoic acid and titanium dioxide were purchased from Sigma-Aldrich company under the product catalog numbers F3004, T3309, A5503, 09830, V900355, 14265, 85707 and 798517 in sequence. Dithiothreitol is available from Merck under catalog number 8011. Acetonitrile was purchased from Thermo Fisher under catalog number a 998. Trifluoroacetic acid was purchased from Tedia under catalog number TS 4295. Trypsin was purchased from Roche under the catalog number 11418025001. Sialidases, PNGase F enzymes were purchased from NewEnglan Biolabs under catalog numbers P0720S, P0704S, respectively. Ultrapure water was obtained from Merck millipore synergy ultrapure water machine.

Example 1, use of g-C₃N₄Enrichment of sialylated glycopeptides

Mono, g-C₃N₄Step of enriching sialylated glycopeptides

Step one, g-C₃N₄Preparation of the Material

Grinding the precursor (dicyandiamide or melamine or urea), adding into a crucible, placing into a muffle furnace, covering and calcining at 550 ℃ for 4 hours, taking out the yellowish product, and grinding to obtain g-C₃N₄And (3) powder.

Step two, preparation of enzymolysis sample solution

20 μ L of 1 μ g/μ L protein (which may be bovine fetuin or transferrin or a mixture of bovine fetuin, transferrin and bovine albumin) was added to 2.2 μ L of 250mM ammonium bicarbonate and 0.6 μ L of 1M dithiothreitol (formulated with 25mM ammonium bicarbonate), incubated at 56 deg.C for 60 minutes (reduction reaction to open disulfide bonds), then 5.6 μ L of iodoacetamide (formulated with 25mM ammonium bicarbonate) was added, mixed well, and then reacted at 25 deg.C (room temperature) for 45 minutes in the absence of light (to block the disulfide bonds that were opened during reduction reaction), then 1.5 μ L of 100mM dithiothreitol (formulated with 25mM ammonium bicarbonate) was added to terminate the reaction, and the system was freeze-dried and stored at-80 deg.C until use.

Step three, enriching sialylated glycopeptides from sample solution

1. Taking g-C₃N₄The powder was washed with 0.1M hydrochloric acid, ultrapure water and 80% acetonitrile in water (ultrasound parameters specifically 20KHz, 5 min).

2.2 mg of g-C₃N₄Dispersing in 1mL acetonitrile/water solution to obtain g-C₃N₄And (3) dispersing the mixture. The volume fraction of acetonitrile/water solution acetonitrile was 80%.

3. Subpackaging the enzymolysis sample, collecting 2 μ g of proteolysis, freeze drying, and adding 60 μ L g-C₃N₄And (3) dispersing the mixture. Continuously shaking to enrich sialylated glycopeptides. The shaking enrichment time was 1 hour.

4. After step 3 is completed, the enriched g-C is added₃N₄The dispersion was centrifuged at a centrifugal force of 13500 g. And taking out supernatant after the centrifugation is finished, namely, enriching residual liquid. To obtain g-C₃N₄Precipitation, i.e. adsorption of the enriched material of sialylated glycopeptides.

5. After completion of step 4, g-C of sialylglycopeptide is adsorbed₃N₄Adding ammonia water eluent into the precipitate, and continuously oscillating to adsorb at g-C₃N₄The sialylated glycopeptides on the surface were eluted. The concentration of the eluent ammonia water is 0.025%. The volume of the eluate was 60. mu.L. The elution time was 45 minutes with shaking.

6. After completion of step 5, the solution after completion of elution was centrifuged at a centrifugal force of 13500 g. And taking out supernatant after the centrifugation is finished, namely the eluent rich in the sialylated glycopeptide.

7. After completion of step 6, the eluate was freeze dried and subjected to mass spectrometry.

Comparative test

1. In the second step, the enzymolysis stock solution of the bovine fetuin is desalted by a C18 column (1.7 μm).

2. The rich retentate from step 4 was desalted using a C18 column (1.7 μm).

3. And step three, obtaining the enrichment liquid from step three 1-6.

4. The bovine fetuin enzymolysis stock solution obtained in the second step is re-dissolved with 16. mu.L of water per 20. mu.g of protein, added with 2. mu.L of 10 XG 1 reaction buffer and 2. mu.L of sialidase, and incubated at 37 ℃ for 16 h. And after the enzymolysis is finished, enriching according to 1-6 in the step two.

5. The bovine fetuin enzymolysis stock solution obtained in step two was reconstituted with 16. mu.L of water per 20. mu.g of protein, added with 2. mu.L of 10 Xreaction buffer and 2. mu.L of 10 XNP-40, 0.5. mu.L of PNGase enzyme, and incubated at 37 ℃ for 16 h. And after the enzymolysis is finished, enriching according to 1-6 in the step two.

Third, limit test (different sample concentration)

Steps 1 to 2 are the same as steps 1 to 2 of step three.

3. Subpackaging the enzymolysis sample, respectively taking 0.2 μ g, 0.5 μ g, 1 μ g, 2 μ g, 4 μ g and 8 μ g of the proteolysis product, adding g-C₃N₄And (3) dispersing the mixture. Continuously shaking to enrich sialylated glycopeptides.

Steps 4 to 7 are the same as steps 5 to 7 of step three.

Control test (elution Condition selection)

Steps 1 to 4 are the same as steps 1 to 4 of step three.

5. After the step 4 is completed, respectively eluting with 0.1% trifluoroacetic acid, 0.2% formic acid, ultrapure water, 25mM ammonium bicarbonate and 0.025% ammonia water solution, respectively collecting effluent liquid to obtain five different enrichment solutions, and performing mass spectrometry.

Fifth, control test

Titanium dioxide enrichment

1. Column assembling: the titanium dioxide is dispersed in 100% acetonitrile and filled into the Tip to a filling height of about 4-5mm, preferably 4mm, so as to obtain the titanium dioxide Tip column.

2. And (3) activating the column: adding 50 mu L of acetonitrile, and centrifuging for 5-10 min at 2500 g; adding 50 mu L of ultrapure water, and centrifuging for 10-20 min at 2500 g; mu.L of the loading buffer (100mg of DHB in an aqueous solution of 80% acetonitrile/5% trifluoroacetic acid to prepare a loading buffer solution with a concentration of 100 mg/mL) was added, and centrifuged at 2500g for 10 min.

3. Loading: taking a bovine fetuin enzymolysis sample of 2 mu g, re-dissolving the sample by using 20 mu L of loading buffer solution, adding the re-dissolved sample into a Tip column, centrifuging for 10min at 1500g, re-loading the effluent liquid, and repeating the process for three times.

4. Rinse (2 replicates each): mu.L of an aqueous solution containing 80% acetonitrile and 2% trifluoroacetic acid (2500g, centrifuge for 20min, discard run-off) was added, and 80. mu.L of an aqueous solution containing 20% acetonitrile and 0.1% trifluoroacetic acid (2500g, centrifuge for 20min, discard run-off) was added.

5. After completion of step 4, 80. mu.L of 0.8% phosphate buffer was added, and the mixture was centrifuged at 2500g for 20min, and the effluent was collected, i.e., the eluate containing sialylated glycopeptide.

6. The eluate was freeze-dried and analyzed by mass spectrometry.

Sixthly, comparison of enrichment effects

The mass spectrum is shown in figure 1. FIG. 1a is a mass spectrum of bovine fetuin enzymolysis solution after desalting with C18 column (1.7 μm), wherein 11 mass spectrum peaks of sialylated glycopeptide can be detected in the mass spectrum of bovine fetuin enzymolysis solution, and the peak intensity of glycopeptide is weak. FIG. 1b is g-C₃N₄Mass spectrum of eluate enriched with fetuin, g-C₃N₄45 sialylated glycopeptide mass spectrum peaks are found in the enriched eluate, the glycopeptide peaks are very strong, and the interference of non-glycopeptide is hardly generated in the graph. FIG. 1C is g-C₃N₄Enriched retentate after enrichment, g-C₃N₄The solution remaining after enrichment contained only the signals for glycopeptides and non-glycopeptides that did not contain sialic acid, indicating g-C₃N₄Sialylated glycopeptides can be specifically enriched. FIG. 1d shows the g-C of bovine fetuin after enzymatic cleavage and subsequent treatment with sialidase₃N₄And (4) enriching. After sialidase treatment, the sialic acid at the glycopeptide ends is cleaved and the original sialylated glycopeptide peaks disappear, leaving only some glycopeptide and non-glycopeptide peaks that do not contain sialic acid. FIG. 1E shows the enzymatic digestion of fetuin followed by treatment with PNGase enzyme for g-C₃N₄And (4) enriching. The PNGase enzyme was cleaved off the N-sugar, and all signals were derived from non-glycopeptides. Comparing FIG. 1d with FIG. 1e, it was found that they have partial homogeneous/charged peaks, indicating thatThese peaks are non-glycopeptide peaks. Whereas the peaks shown in FIG. 1d, which are more abundant than those shown in FIG. 1e, should be some glycopeptide peaks that are not sialylated. Comparing FIGS. 1b, 1d and 1e, it was found that the non-sialylated glycopeptide was not a sialylated glycopeptide from sialidase treatment, indicating g-C₃N₄The enrichment effect on non-sialylated glycopeptides and non-glycopeptides is not obvious. When sialoglycopeptide, non-sialoglycopeptide and non-glycopeptide are present together, the bound sialoglycopeptide is preferentially enriched, further indicating that g-C₃N₄High specificity for sialylated glycopeptide enrichment.

Fig. 2a is a mass spectrum of an enzymolysis stock solution of transferrin after desalting with a C18 column, wherein 9 mass spectrum peaks of sialylated glycopeptide can be detected in the mass spectrum of the enzymolysis stock solution of transferrin, and the peak intensity of glycopeptide is weak. FIG. 2b is g-C₃N₄Mass spectrum of eluate enriched with fetuin, g-C₃N₄46 sialylated glycopeptide mass spectrum peaks are found in the enriched eluate, and the glycopeptide peaks are very strong and have almost no interference of non-glycopeptide. FIG. 2C is g-C₃N₄Enriched retentate after enrichment, g-C₃N₄The solution remaining after enrichment contained only signals for glycopeptides and non-glycopeptides that did not contain sialic acid. FIG. 2d shows g-C of transferrin after enzymatic cleavage and treatment with sialidase₃N₄And (4) enriching. The original sialylated glycopeptide peaks disappeared, leaving only some peaks of glycopeptides and non-glycopeptides that do not contain sialic acid. FIG. 2e shows g-C of transferrin treated with PNGase enzyme after enzymatic hydrolysis₃N₄And (4) enriching, wherein the obtained signals are all from non-glycopeptides. Similar to the enrichment law for fetuin in FIG. 1, g-C₃N₄Can also specifically enrich transferrin sialylated glycopeptides. Indicating less modification of the sialylation of transferrin, g-C₃N₄Still has strong enrichment capacity to sialylated glycopeptide.

Comparison of g to C₃N₄Enrichment mass spectra of various amounts of fetuin enzymatic hydrolysate (fig. 3) can find that: no information on sialylglycopeptides was seen at 20fmol bovine fetuin. At 50fmol, information on sialylated glycopeptides appears, but it is believed thatThe noise ratio and peak intensity are not too high. Whereas 0.4pmol had already been rich in sialylated glycopeptide information, the glycopeptide peak reached very high intensity at 0.8 pmol. And the amount of the protein enzymolysis liquid is continuously increased, and the peak intensity and the number of the peak of the sialylated glycopeptide are not obviously changed compared with the peak intensity and the number of the peak of the sialylated glycopeptide after 0.8pmol of the protease enzymolysis liquid is enriched. Indicates g-C₃N₄The lowest detection limit for enrichment was up to 50fmol, with 0.8pmol being the optimal protein loading.

Comparison of elution of g-C with different elution solutions₃N₄The mass spectrum of sialylated glycopeptide above (FIG. 4) shows that: the sialylated glycopeptide cannot be eluted by the strong acid eluent, the obtained sialylated glycopeptide peaks are more and more in number and gradually increased along with the increase of the pH value of the eluent, and the optimal condition is 0.025% ammonia water solution for elution. Indicating that the weakly alkaline environment is favorable for elution of sialylated glycopeptides.

In the mass spectrogram of the titanium dioxide method enriched bovine fetuin enzymolysis liquid (figure 5), it can be found that the sialylated glycopeptides enriched by titanium dioxide are mainly concentrated at 3000-5000 Da, and the efficiency of enriching glycopeptides at a high molecular weight end is not good. And the titanium dioxide enrichment condition is not mild enough, and molecular dehydration occurs, so that the mass spectrogram is more complicated. For example, m/z 4729 corresponds to the signal peak m/z 4711 for one out of one water molecule. In contrast, g-C₃N₄A large amount of sialylated glycopeptides can be enriched (the main distribution area is 3000-7000 m/z), the condition is mild, no dehydration peak appears, and the effect is obviously superior to that of the conventional titanium dioxide enrichment method.

Example 2, g-C₃N₄Morphology and structural characterization of enriched materials

In this example, for g-C₃N₄The enriched material is subjected to morphology and structure characterization, and g-C is further presumed₃N₄Mechanism for enriching sialylated glycopeptides.

FIG. 6 is g-C₃N₄The scanning electron micrograph of (a) shows that g-C₃N₄In order to have a lamellar stacking micro-scale bulk structure, a lamellar structure of 50nm or less can be observed at some positions. FIG. 7a and FIG. 7b are g-C, respectively₃N₄Transmission electron microscope andhigh resolution transmission electron microscopy. As shown in the figure, g-C₃N₄Is formed by amorphous single-layer accumulation.

FIG. 8a is a drawing g-C₃N₄The theoretical structure diagram of (A) shows that the structure has a large number of triazine rings and N-H bonds. FIG. 8a is g-C₃N₄The peaks at 286ev and 397ev correspond to the binding energies of C1s and N1s, indicating that the prepared material is mainly composed of C and N atoms, consistent with its theoretical structure. FIG. 8b is g-C₃N₄The infrared spectrogram of (1), wherein the infrared spectrogram is 1200-1650 cm^-1The absorption peak in the wavenumber range corresponds to the typical stretching vibration of the C-N heterocycle, and 807cm^-1Corresponds to a simple vibration of the triazine structure. 3000-3600 cm^-1The absorption peaks between the two correspond to the stretching vibration of N-H and O-H, which respectively come from unpolymerized amino at the edge of the material and a small amount of oxygen defects in the structure. A large number of triazine rings and N-H structures in the material can form hydrogen bonds with sialic acid, and the specific enrichment of the sialic acid is facilitated.

Claims

1.g-C₃N₄The application in the enrichment and separation of sialylated glycopeptides is characterized in that the method for enriching the sialylated glycopeptides comprises the following steps:

(2) centrifuging the enrichment solution, and adding sialylated glycopeptide-enriched g-C₃N₄Adding eluent into the precipitate to obtain sialylated glycopeptide solution;

in step (1), g to C₃N₄The dispersion liquid is mixed solution of acetonitrile/water, wherein the volume fraction of the acetonitrile is 70-80 percent, and g-C₃N₄The concentration of (A) is 0.5-4 mg/mL;

in the step (2), 0.025% ammonia water solution is used as eluent.

2. A method for enriching sialylated glycopeptides, comprising the steps of:

in the step (2), 0.025% ammonia water solution is used as eluent.

3. The method of claim 2, wherein: and (3) adopting a method of oscillation enrichment and oscillation elution, wherein the oscillation time is 0.5-4 hours.

4. The method of claim 2, wherein: centrifugation to obtain g-C enriched with sialylated glycopeptides₃N₄Precipitating with a centrifugal force of 13500-21000 g.

5. The method of claim 2, wherein: and obtaining the aqueous solution of the sialylated glycopeptide by a centrifugal method, wherein the centrifugal force is 13500-21000 g.