Method for evaluating the accuracy of results of glycosylation analysis methods

Abstract

The embodiment of the present application relates to a method for evaluating the accuracy of a glycosylation analysis method, the method comprising: performing optical analysis on a glycoconjugate having optical properties to obtain a first proportion of different sugar chains in the glycoconjugate; performing glycosylation analysis on the glycoconjugate to obtain a second proportion of different sugar chains in the glycoconjugate; and comparing the second proportion with the first proportion to determine the accuracy of the glycosylation analysis method, wherein the glycoconjugate is formed by a non-sugar substance and a sugar, and each molecule of the non-sugar substance carries one molecule of the sugar chain; and the glycosylation analysis comprises: releasing different sugar chains from the glycoconjugate. The method for evaluating the accuracy of the glycosylation analysis method according to the embodiment of the present application can be widely applicable, and can completely evaluate the entire glycosylation analysis process.

Description

Technical Field

[0001] This invention relates to the field of glycosylation analysis technology, and in particular to a method for evaluating the accuracy of glycosylation analysis results. Background Technology

[0002] Glycosylation is the process by which non-carbohydrate biomolecules such as proteins, lipids, peptides, and nucleic acids are attached with carbohydrates under the control of enzymes. Through this process, carbohydrates and non-carbohydrate biomolecules are covalently bonded to form glycoconjugates. Glycosylation analysis of glycoconjugates is an essential means of unlocking their secrets. Currently, a variety of methods have been used for the glycosylation analysis of glycoconjugates.

[0003] However, there is still a need for a widely applicable method that can comprehensively evaluate the entire glycosylation analysis process to assess the accuracy of the results of glycosylation analysis methods. Summary of the Invention

[0004] To address the aforementioned technical problems, embodiments of the present invention provide a method for evaluating the accuracy of glycosylation analysis results.

[0005] To achieve the above objectives, one aspect of the present invention relates to a method for evaluating the accuracy of glycosylation analysis results, the method comprising:

[0006] Optical analysis was performed on glycoconjugates with optical properties to obtain the first proportion of different sugar chains in the glycoconjugates;

[0007] Glycosylation analysis was performed on the glycoconjugates to obtain a second proportion of different sugar chains in the glycoconjugates; and

[0008] The second ratio was compared with the first ratio to determine the accuracy of the glycosylation analysis method.

[0009] Among them, glycoconjugates are formed by non-sugar substances and sugars, with each molecule of non-sugar substance carrying one sugar chain molecule; and

[0010] Glycosylation analysis includes releasing the glycan chains from the glycoconjugates to release different glycan chains.

[0011] In some embodiments, the non-carbohydrate substance includes at least one of peptides, proteins, lipids, nucleic acids, and small molecule aglycones.

[0012] In some embodiments, the glycoconjugate includes glycoconjugates that inherently contain optically active components and / or glycoconjugates labeled with optical markers.

[0013] In some embodiments, the optical property components include at least one of phenylalanine, tyrosine, and tryptophan.

[0014] In some embodiments, the optical marker includes 2,5-dioxopyrrolidine-1-ylpyrene-1-carboxylate (DPC).

[0015] In some embodiments, optical analysis includes online separation and / or fluorescence detector detection (FLR) and / or ultraviolet detector detection (UV).

[0016] In some embodiments, online separation includes high-performance liquid chromatography (HPLC) and / or capillary electrophoresis.

[0017] In some embodiments, glycosylation includes N-glycosylation and / or O-glycosylation and / or C-glycosylation and / or S-glycosylation, and glycans include acidic glycans and / or neutral glycans and / or basic glycans, as well as reducing glycans and / or glycoamines.

[0018] In some embodiments, glycan release is carried out by enzymatic and / or chemical methods.

[0019] In some embodiments, the glycosylation analysis further includes pretreatment of the released different sugar chains.

[0020] In some embodiments, pretreatment includes at least one of reduction, labeling, and purification.

[0021] In some embodiments, glycosylation analysis further includes obtaining a second proportion of different sugar chains in the glycoconjugate by at least one of chromatography, capillary electrophoresis, mass spectrometry, spectroscopy, and nuclear magnetic resonance.

[0022] Another aspect of the present invention relates to the application of a method for evaluating the accuracy of glycosylation analysis results according to the present application in the analysis of glycoconjugates.

[0023] Another aspect of the present invention relates to a kit for evaluating the accuracy of the results of the glycosylation analysis method according to the present application.

[0024] The technical solutions of this application are widely applicable and can fully evaluate the entire glycosylation analysis process.

[0025] Where technical conditions permit, the technical solutions of the various embodiments in this application can be combined in any way.

[0026] The present application will be further described below with reference to the accompanying drawings. The same or similar reference numerals may be used in the drawings to refer to the same or similar elements, devices, shapes, structures, and steps in different embodiments, and descriptions of the same or similar elements, devices, shapes, structures, steps, features, and effects in different embodiments, as well as descriptions of elements, devices, shapes, structures, steps, features, and effects that are the same or similar to those in the prior art, may be omitted. Attached Figure Description

[0027] Figure 1 This is a flowchart illustrating a method for evaluating the accuracy of glycosylation analysis results according to an embodiment of the present invention.

[0028] Figure 2 The graphs show the fluorescence response of several different optical markers eluted with acetonitrile at different ratios.

[0029] Figure 3 A graph showing the temperature variation of DPC and 7-methoxy-2-oxo-2H-benzopyran-3-carboxylic acid-2,5-dioxopyrrolidine-1-yl ester (MOCM) labeled aminotriacetic acid solutions.

[0030] Figure 4 LC-FLR analysis chromatograms of three N-glycopeptide standards containing different N-glycan chains labeled with three different DPCs;

[0031] Figure 5 Obtained for glycosylation analysis Figure 4 LC-FLR analysis of the three N-glycan chains in three N-glycopeptides;

[0032] Figure 6 LC-FLR chromatograms of two DPC-labeled N-glycopeptide standards containing different N-glycan chains; and

[0033] Figure 7 Obtained for glycosylation analysis Figure 6 LC-FLR analysis of two N-glycan chains in two N-glycopeptides. Detailed Implementation

[0034] Figure 1 This is a schematic flowchart illustrating a method for evaluating the accuracy of glycosylation analysis results according to an embodiment of the present invention. Figure 1 As shown, one aspect of this invention relates to a method 100 for evaluating the accuracy of glycosylation analysis results, the method 100 comprising:

[0035] Step 102. Perform optical analysis on the glycoconjugate with optical properties to obtain the first proportion of different sugar chains in the glycoconjugate;

[0036] Step 104. Perform glycosylation analysis on the glycoconjugate to obtain a second proportion of different sugar chains in the glycoconjugate; and

[0037] Step 106. Compare the second ratio with the first ratio to determine the accuracy of the glycosylation analysis method.

[0038] Among them, glycoconjugates are formed by non-sugar substances and sugars, with each molecule of non-sugar substance carrying one sugar chain molecule; and

[0039] Glycosylation analysis includes releasing the glycan chains from the glycoconjugates to release different glycan chains.

[0040] The method 100 for evaluating the accuracy of the glycosylation analysis results in this application embodiment can be implemented using only common, conventional instruments, such as fluorescence detectors and ultraviolet detectors, without requiring the use of nationally controlled or restricted equipment such as radioactive detectors. Furthermore, the reference standards involved in method 100 are readily available, and their preparation or use is not subject to relevant regulatory restrictions. In addition, method 100 is not only applicable to reducing glycans but also to evaluating other glycan forms, such as the glycoamine form N-glycans released by PNGase F enzyme. Therefore, method 100 in this application embodiment has broad applicability.

[0041] In addition to examining the labeling process, the method 100 in this application embodiment also covers the entire glycosylation analysis process, including glycan release, and can comprehensively evaluate the entire glycosylation analysis process.

[0042] Glycoconjugates are formed by non-sugar substances and sugars, with each molecule of non-sugar substance carrying one sugar chain. Thus, by analyzing the proportion of different molecular chains of the non-sugar substance using methods such as LC-FLR, the proportion of different sugar chains can be characterized.

[0043] Glycosylation analysis involves the release of glycans from glycoconjugates to reveal different glycan chains. Specifically, during glycosylation analysis, the glycan chains of the glycoconjugate are released, and the different glycan chains within the glycoconjugate are then identified using glycosylation detection methods. In other words, glycosylation analysis of glycoconjugates yields a second proportion of different glycan chains at the free sugar level.

[0044] In the embodiments of this application, "glycoconjugate" refers to a complex formed by the covalent bonding of carbohydrates with proteins, lipids, peptides, nucleic acids or other non-carbohydrate substances.

[0045] In the embodiments of this application, "glycosylation" refers to the process by which non-carbohydrate biomolecules such as proteins, lipids, peptides, and nucleic acids are attached with sugars under the control of enzymes.

[0046] In the embodiments of this application, "glycosylation analysis" refers to the process of obtaining the proportion of different sugar chains in the glycoconjugate.

[0047] In the embodiments of this application, the "optical properties" of the glycoconjugate refer to the characteristics that make the glycoconjugate suitable for optical analysis, such as optical absorption, like fluorescence absorption or ultraviolet absorption.

[0048] In some embodiments, the non-carbohydrate substance includes at least one of peptides, proteins, lipids, nucleic acids, and small molecule aglycones.

[0049] In this embodiment, there are no particular limitations on the sugar conjugate, as long as it is suitable for the method 100 of the present invention.

[0050] In some embodiments, the glycoconjugate includes glycoconjugates that inherently contain optically active components and / or glycoconjugates labeled with optical markers.

[0051] Since glycoconjugates themselves contain optically active components, they can be directly analyzed optically to obtain the initial proportions of different sugar chains within them. Glycoconjugates are optically labeled glycoconjugates, meaning that the optical label imparts optical properties to them. Before optical labeling, glycoconjugates may not possess these optical properties, making it impossible to directly obtain the initial proportions of different sugar chains through optical analysis.

[0052] In some embodiments, the optical property components include at least one of phenylalanine, tyrosine, and tryptophan.

[0053] In the embodiments of this application, the "optical property component" can be any part of the glycoconjugate that has optical properties, and there are no particular restrictions on this, as long as it is applicable to the present invention.

[0054] In some embodiments, the optical marker includes 2,5-dioxopyrrolidine-1-ylpyrene-1-carboxylate (DPC).

[0055] The optical marker can be any substance suitable for optically marking glycoconjugates, as long as it is applicable to this invention. For example, 2,5-dioxopyrrolidine-1-ylpyrene-1-carboxylic acid ester (DPC), a fluorescent marker with a stable fluorescence response, can be used as an optical marker. The optical marker can also be an ultraviolet marker with other optical properties, such as an ultraviolet marker with an ultraviolet response.

[0056] In some embodiments, optical analysis includes online separation and / or fluorescence detector detection and / or ultraviolet detector detection.

[0057] Online separation can improve the purity of glycoconjugates and obtain an accurate first ratio.

[0058] If the glycoconjugate has a fluorescent response, the first proportion of different sugar chains in the glycoconjugate can be detected by a fluorescence detector.

[0059] If the glycoconjugate has ultraviolet absorption, the first proportion of different sugar chains in the glycoconjugate can be detected by an ultraviolet detector.

[0060] In some embodiments, online separation includes high-performance liquid chromatography and / or capillary electrophoresis.

[0061] Online separation can be performed by any suitable means, as long as it is applicable to this invention.

[0062] In some embodiments, glycosylation includes N-glycosylation and / or O-glycosylation and / or C-glycosylation and / or S-glycosylation, and glycans include acidic glycans and / or neutral glycans and / or basic glycans, as well as reducing glycans and / or glycoamines.

[0063] The method 100 of this invention is applicable to glycoconjugates formed by various glycosylation forms such as N-glycosylation and / or O-glycosylation and / or C-glycosylation and / or S-glycosylation, and is applicable to acidic sugar chains and / or neutral sugar chains and / or basic sugar chains, as well as reducing sugar chains and / or glycoamines, and has wide applicability.

[0064] In some embodiments, glycan release is carried out by enzymatic and / or chemical methods.

[0065] The release of the sugar chain can be carried out in any suitable manner, as long as it is suitable for the present invention.

[0066] In some embodiments, the glycosylation analysis further includes pretreatment of the released different sugar chains.

[0067] The different sugar chains released from the glycoconjugate can be directly subjected to glycosylation detection to obtain a second proportion of different sugar chains, or they can be subjected to glycosylation detection after pretreatment to obtain a second proportion of different sugar chains.

[0068] In some embodiments, pretreatment includes at least one of reduction, labeling, and purification.

[0069] The different sugar chains released from the glycoconjugate can be reduced, optically labeled, purified, and then glycosylated to obtain a second ratio of different sugar chains.

[0070] In some embodiments, glycosylation analysis further includes obtaining a second proportion of different sugar chains in the glycoconjugate by at least one of chromatography, capillary electrophoresis, mass spectrometry, spectroscopy, and nuclear magnetic resonance.

[0071] The glycosylation detection of different sugar chains released from glycoconjugates can be performed by any method such as chromatography, capillary electrophoresis, mass spectrometry, spectroscopy, nuclear magnetic resonance, etc., as long as it is suitable for this invention.

[0072] In this embodiment of the invention, optical analysis is performed on glycoconjugates with optical properties to obtain a first proportion of different sugar chains in the glycoconjugates. Simultaneously, glycosylation analysis is performed on the glycoconjugates with optical properties to obtain a second proportion of different sugar chains in the glycoconjugates. The second proportion is compared with the first proportion; for example, the ratio of the second proportion to the first proportion is obtained, thereby determining the accuracy of the glycosylation analysis method.

[0073] Another aspect of the present invention relates to the application of a method for evaluating the accuracy of the glycosylation analysis results described in this application in the analysis of glycoconjugates.

[0074] The method for evaluating the accuracy of glycosylation analysis results described in this application can be used to obtain the accuracy of glycosylation analysis results when analyzing glycoconjugates. Furthermore, as mentioned above, the method for evaluating the accuracy of glycosylation analysis results described in this application is widely applicable and can comprehensively evaluate the entire glycosylation analysis process, and can be widely used in the analysis of glycoconjugates.

[0075] Another aspect of the present invention relates to a kit for evaluating the accuracy of the results of the glycosylation analysis method according to the present application.

[0076] The kits of this application can be used to implement the accuracy assessment method for the glycosylation analysis method described in this application, in order to assess the accuracy of the glycosylation analysis method. As mentioned above, this helps to provide a widely applicable assessment method that can comprehensively evaluate the accuracy of the glycosylation analysis method results throughout the entire glycosylation analysis process.

[0077] Example

[0078] Example 1. Screening of optical markers

[0079] Ammoniated triacetic acid (CAS No.: 556-33-2) was labeled with different fluorescent markers listed in Table 1. The fluorescently labeled triacetic acid was then dissolved in 60% acetonitrile (acetonitrile:aqueous phase = 60:40 (v:v)) and injected into an HPLC-FLR column. Isocratic elution was performed using acetonitrile solutions with different ratios (55% (acetonitrile:aqueous phase = 55:45 (v:v)), 65% (acetonitrile:aqueous phase = 65:35 (v:v)), and 75% (acetonitrile:aqueous phase = 75:25 (v:v)) as the mobile phase (aqueous phase was 50 mM ammonium formate aqueous solution, pH 4.4). The results are shown below. Figure 2 As shown. Figure 2 In the figure, the vertical axis represents the relative values ​​of fluorescence responses obtained by elution with different proportions of acetonitrile, with the response value obtained by elution with 75% acetonitrile as 100, and the horizontal axis represents different fluorescent labels.

[0080] from Figure 2 It can be seen that the fluorescence response of DPC or MOCM labeled aminotriacetic acid is basically the same under different proportions of acetonitrile, which shows that DPC and MOCM can be used as optical markers to more easily obtain the actual proportion of the labeled substance.

[0081] Table 1. Different fluorescent markers

[0082]

[0083] Example 2. Stability detection of optical markers

[0084] Prepare freshly prepared solutions of DPC and MOCM-labeled nitric acid solutions separately, and heat at 37°C. o The solution after being placed at C for 2 hours and at 70 o Solutions were left to stand for 2 hours (C), and then analyzed by HPLC-FLR. The degradation status of the solutions was determined based on the peak area of ​​the main peak. Results are shown below. Figure 3 . Figure 3 The vertical axis represents the relative peak areas of the main peak in different solutions, with the peak area of ​​the main peak in the freshly prepared solution taken as 100.

[0085] from Figure 3 It can be seen that the MOCM-labeled nitric acid solution exhibits significant degradation with increasing temperature; while the DPC-labeled nitric acid solution shows degradation at 37°C. o C and 70 o C has good stability.

[0086] Combining the detection results of Examples 1 and 2, it can be determined that DPC maintains a stable fluorescence response at different acetonitrile concentrations and temperatures, which can bring great convenience as an optical marker for glycoconjugates.

[0087] Example 3

[0088] 1) Obtaining high-purity N-glycopeptide reference standard: Dissolve the N-glycan chain in a supersaturated ammonium bicarbonate solution at 40 °C. o After stirring at C for 3 days, the mixture was filtered and then freeze-dried to convert the reduced sugar chains into glycoamines. The DPC-labeled peptide chain and the freeze-dried N-glycan were dissolved in dimethyl sulfoxide, followed by the addition of (3H-1,2,3-triazolo[4,5-b]pyridine-3-oxy)tri-1-pyrrolidinyl hexafluorophosphate and N,N-diisopropylethylamine. The mixture was stirred at room temperature for 30 minutes. The reaction solution was purified using a Hilic column to obtain high-purity fluorescently labeled N-glycopeptide (glycoconjugate).

[0089] 2) Optical analysis of the glycoconjugates: The three DPC-labeled N-glycopeptide standards (G0F-Peptide, G2-Peptide, and G2S2F-Peptide, with the amino acid sequence of GAGNVSG) obtained in step 1) were mixed and analyzed by LC-FLR. A Hilic column was used with gradient elution of acetonitrile-50 mM ammonium formate aqueous solution (pH 4.4) to obtain the ratio of the three N-glycans in the glycopeptide mixture as G0F / G2 / G2S2F: 100 / 47.8 / 54.3 (see...). Figure 4 ).

[0090] 3) Glycosylation analysis of glycoconjugates: An appropriate amount of glycopeptide mixture was taken, and N-glycan chains were released from the peptides using PNGase F enzyme. The mixture was then purified, concentrated, and dried using a graphite carbon column (GCB). 2AB (2-aminobenzamide) labeling solution was added, and the glycan chains were fluorescently labeled using the principle of reductive amination. The labeled glycan chains were purified using a Hilic SPE column and then analyzed by LC-FLR. Using a Hilic column, gradient elution with acetonitrile-50 mM ammonium formate aqueous solution (pH 4.4) yielded three N-glycan chains with a ratio of G0F / G2 / G2S2F: 100 / 45.0 / 56.6 (see...). Figure 5 Meanwhile, a small amount of G2S2F sialic acid degradation product G2S1F (accounting for about 2% of G2S2F) could also be observed.

[0091] 4) Compare the proportions of the three glycans obtained in steps 2) and 3) before and after to evaluate the accuracy of the results of the above glycosylation analysis method. That is, using G0F as a control, the result of G2 is lower than the actual situation (about 6% lower), while the result of G2S2F is slightly higher than the actual situation (about 4% higher). At the same time, the degradation of sialic acid during the glycosylation analysis process can be evaluated.

[0092] Example 4

[0093] 1) Two high-purity DPC-labeled N-glycopeptides were obtained as N-glycopeptide references (glycoconjugates) in the same manner as in step 1) of Example 3.

[0094] 2) Optical analysis of the glycoconjugates: The two DPC-labeled N-glycopeptide standards (G0F-Peptide and G2S2F-Peptide, amino acid sequence of the peptide: GAGNVSG) obtained in step 1) were mixed and analyzed by LC-FLR; using a Hilic column, gradient elution with acetonitrile-50 mM ammonium formate aqueous solution (pH 4.4), the ratio of the two N-glycans in the glycopeptide mixture was G0F / G2S2F: 100 / 63.1 (see...). Figure 6 ).

[0095] 3) Glycosylation analysis of glycoconjugates: An appropriate amount of glycopeptide mixture was taken, and N-glycans were released from the peptides using PNGase F enzyme. The released glycoamine N-glycans were then rapidly labeled with ProA-Osu. After purification using a Hilic SPE column, LC-FLR analysis was performed. A Hilic column was used with gradient elution of acetonitrile-50 mM ammonium formate aqueous solution (pH 4.4). The ratio of the two N-glycans obtained was G0F / G2S2F: 100 / 74.8 (see...). Figure 7 ).

[0096] 4) Compare the two glycan ratios obtained in steps 2) and 3) before and after to evaluate the accuracy of the above glycosylation analysis results. That is, using G0F as a control, the result of G2S2F is higher than the actual value (about 18.5%).

[0097] Example 5

[0098] 1) Two high-purity N-glycopeptide reference standards (glycoconjugates) were obtained in the same manner as in step 1) of Example 3.

[0099] 2) Optical analysis of glycoconjugates: The two DPC-labeled N-glycopeptide standards (G0F-Peptide and Man5-Peptide, amino acid sequence of peptide: GAGNVSG) obtained in step 1) were mixed and analyzed by LC-FLR; using a Hilic column, gradient elution with acetonitrile-50 mM ammonium formate aqueous solution (pH 4.4) was used to obtain the ratio of the two N-glycan chains in the glycopeptide mixture (see Tables 2 and 3 below).

[0100] 3) Glycosylation analysis of glycoconjugates: Take appropriate amounts of glycopeptide mixture solution and etanercept solution. Use PNGase F enzyme to release N-glycan chains from peptides in both samples. Then, purify, concentrate and dry using a graphite carbon column (GCB). Add 2-aminobenzamide labeling solution to fluorescently label the glycan chains. After purification using a Hilic SPE column, perform LC-FLR analysis. Use a Hilic column with gradient elution of acetonitrile-50 mM ammonium formate aqueous solution (pH 4.4). The ratio of G0F and Man5 N-glycan chains obtained is shown in Tables 2 and 3 below.

[0101] Example 6

[0102] The accuracy of the glycosylation analysis results was evaluated in the same manner as in Example 5, except that in step 3) of the glycosylation analysis of the glycoconjugates: appropriate amounts of glycopeptide mixture solution and etanercept solution were taken, and the N-glycan chains of the two samples were released from the peptides using PNGase F enzyme. Procainamine labeling solution was added to fluorescently label the glycan chains. After purification by Hilic SPE column, the labeled glycan chains were analyzed by LC-FLR. Hilic column was used with gradient elution of acetonitrile-50mM ammonium formate aqueous solution (pH 4.4). The ratio of the two N-glycan chains, G0F and Man5, was obtained (see Tables 2 and 3 below).

[0103] Table 2.

[0104]

[0105] Table 3.

[0106]

[0107] As can be seen from the glycosylation analysis results of the glycopeptide reference standards in Tables 2 and 3 above, the Man5 ratio measured in Example 5 is close to the actual value, but the Man5 result measured in Example 6 is significantly lower than the actual value. This data indicates that the accuracy of the glycosylation analysis results in Example 5 is better than that in Example 6. As can be seen from the glycosylation analysis results of the fusion protein etanercept in Table 2, the difference in Man5 results measured by the two glycosylation analysis methods in Examples 5 and 6 is basically consistent with the difference in the two glycosylation analysis results of the glycopeptide reference standards. The Man5 result measured by the glycosylation analysis in Example 6 is significantly lower than that measured by the glycosylation analysis in Example 5. This data indicates that if the glycosylation analysis method in Example 6 is used to test the relevant glycoprotein samples, the Man5 content in the samples may be seriously underestimated, which also demonstrates the practical value of this accuracy assessment method.

[0108] The various specific embodiments described above and shown in the accompanying drawings are for illustrative purposes only and do not represent the entirety of the invention. Any modifications made by those skilled in the art within the scope of the basic technical concept of this invention are within the protection scope of this invention.

Claims

1. A method for evaluating the accuracy of glycosylation analysis results, characterized in that, The method includes: Optical analysis was performed on the glycoconjugate with optical properties to obtain the first proportion of different sugar chains in the glycoconjugate; The glycosylation analysis of the glycoconjugate is performed to obtain a second ratio of different sugar chains in the glycoconjugate; and The second ratio is compared with the first ratio to determine the accuracy of the glycosylation analysis method. The glycoconjugate is formed from a non-sugar substance and a sugar, wherein each molecule of the non-sugar substance carries one molecule of the sugar chain; and The glycosylation analysis includes: releasing the different sugar chains from the glycoconjugate; The glycoconjugates include glycoconjugates that inherently contain optically active components and / or glycoconjugates labeled with optical markers; The optical properties of the glycoconjugate exhibit a consistent response in the mobile phase system used for the optical analysis.

2. The method for evaluating the accuracy of the glycosylation analysis results according to claim 1, characterized in that, The non-carbohydrate substances include at least one of peptides, proteins, lipids, nucleic acids, and small molecule aglycones.

3. The method for evaluating the accuracy of the glycosylation analysis results according to claim 1, characterized in that, The optical properties include at least one of phenylalanine, tyrosine, and tryptophan.

4. The method for evaluating the accuracy of the glycosylation analysis results according to claim 1, characterized in that, The optical markers include 2,5-dioxopyrrolidine-1-ylpyrene-1-carboxylic acid ester.

5. The method for evaluating the accuracy of the glycosylation analysis results according to claim 1, characterized in that, The optical analysis includes online separation and / or fluorescence detector detection and / or ultraviolet detector detection.

6. The method for evaluating the accuracy of the glycosylation analysis results according to claim 5, characterized in that, The online separation includes high-performance liquid chromatography and / or capillary electrophoresis.

7. The method for evaluating the accuracy of the glycosylation analysis results according to claim 1, characterized in that, The glycosylation includes N-glycosylation and / or O-glycosylation and / or C-glycosylation and / or S-glycosylation, and the glycans include acidic glycans and / or neutral glycans and / or basic glycans, as well as reducing glycans and / or glycoamines.

8. The method for evaluating the accuracy of the glycosylation analysis results according to claim 1, characterized in that, The release of the sugar chains is carried out by enzymatic release and / or chemical release methods.

9. The method for evaluating the accuracy of the glycosylation analysis results according to claim 1, characterized in that, The glycosylation analysis also includes: The released different sugar chains are pretreated.

10. The method for evaluating the accuracy of the glycosylation analysis results according to claim 9, characterized in that, The pretreatment includes at least one of reduction, labeling, and purification.

11. The method for evaluating the accuracy of the glycosylation analysis results according to claim 1, characterized in that, The glycosylation analysis also includes: The second proportion of the different sugar chains in the glycoconjugate is obtained by at least one of chromatography, capillary electrophoresis, mass spectrometry, spectroscopy, and nuclear magnetic resonance.

12. The application of a method for evaluating the accuracy of glycosylation analysis results according to any one of claims 1 to 11 in the analysis of glycoconjugates.

13. A reagent kit, characterized in that, A method for evaluating the accuracy of results obtained from implementing the glycosylation analysis method according to any one of claims 1 to 11.

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