A method for detecting relative quantification of glycan types in glycopeptides based on mass spectrometric analysis and application thereof

Abstract

The application relates to a method for detecting the relative quantification of glycopeptide glycosylation based on mass spectrometry and application thereof, and the steps of the method comprise the following: obtaining glycopeptide after pretreatment of a to-be-detected product, performing data analysis after mass spectrometry detection; the capillary temperature of the mass spectrometry detection is 250-290 DEG C. The application creatively finds that, in the detection of glycopeptide glycosylation, the detection result of glycosylation will change with the change of the capillary temperature, the proportion of glycosylation is consistent with the proportion of the molecular weight glycosylation within the range defined in the application, and the detection accuracy is higher. Not only can the time for optimizing subsequent methods be reduced, but also a clear direction for optimization can be provided, so that the work efficiency is greatly improved.

Description

TECHNICAL FIELD

[0001] The present application relates to the field of mass spectrometry detection, and particularly relates to a method for detecting relative quantification of glycopeptide glycoforms based on mass spectrometry analysis and application thereof. BACKGROUND

[0002] Glycopeptide is a compound obtained by connecting a short sugar chain and a short peptide. A large number of glycopeptides are found in animals, plants and microorganisms. Glycopeptides have important applications in the research and development of sugar drugs, such as glycopeptide antibiotics and anti-tumor vaccines.

[0003] Mass spectrometry is an analysis method for measuring ion mass-to-charge ratio. The basic principle is that each component in the sample is ionized in the ion source to generate charged ions of different mass-to-charge ratios. After the action of an accelerating electric field, an ion beam is formed and enters the mass analyzer. In the mass analyzer, the electric field and the magnetic field are used to disperse the opposite velocities, and they are focused to obtain a mass spectrum, so as to determine the mass. After the installation of the mass spectrometer, the manufacturer will provide a conventional experimental detection process and instrument detection parameters for the customer to successfully operate the instrument. However, the method provided by the manufacturer can only meet the detection of conventional peptides, and it is unknown whether it is suitable for the relative quantification of some glycopeptides. At the same time, the sensitivity of glycopeptides to some experimental types and instrument parameters is also different. Finding the parameters that are more sensitive to the relative quantification of glycopeptides can not only reduce the time for subsequent method optimization, but also provide a clear direction for optimization, greatly improving the work efficiency.

[0004] Therefore, how to find the factors that are more sensitive to the relative quantification of glycopeptides has become a problem to be solved at present. SUMMARY

[0005] To solve the above technical problems, the present application provides a method for detecting relative quantification of glycopeptide glycoforms based on mass spectrometry analysis and application thereof. The capillary temperature has a great influence on the stability of glycopeptides. Within the range of the capillary temperature defined in the present application, the proportion of glycoforms in the peptide map is relatively accurate, which can reduce the time for subsequent method optimization, provide a clear direction for optimization, and greatly improve the work efficiency.

[0006] To achieve this purpose, the present application adopts the following technical solutions:

[0007] In a first aspect, the present application provides a method for detecting relative quantification of glycopeptide glycoforms based on mass spectrometry analysis. The steps of the method include: obtaining glycopeptides after pretreating the sample to be tested, and performing data analysis after mass spectrometry detection.

[0008] The capillary temperature for mass spectrometry detection is 250-290 DEG C.

[0009] The application creatively finds that in the detection of glycopeptide glycoform, with the change of capillary temperature, the detection result of glycoform also changes, wherein the A2G0F glycopeptide ratio shows a decreasing trend with the increase of CT, and the A1G0F glycopeptide ratio shows an increasing trend with the increase of CT. Within the scope defined in the application, the ratio of glycoform is consistent with the ratio of molecular weight glycoform, and the detection accuracy is higher. Not only can the time for subsequent method optimization be reduced, but also a clear direction for optimization can be provided, greatly improving the work efficiency.

[0010] In specific embodiments of the application, more than 290 DEG C will cause the ratio of glycoform to change seriously, and the comparison result with the molecular weight of glycopeptide is quite different. Less than 250 DEG C will cause the detection result to be distorted and unable to be detected.

[0011] Preferably, the pretreatment includes any one of filter-assisted sample preparation enzymatic digestion or trypsin enzymatic digestion.

[0012] In the application, both pretreatment methods can obtain target glycopeptide, and the quality of glycopeptide is good, and the influence on the detection of glycoform ratio is low.

[0013] Preferably, the peptide sequence of the glycopeptide includes SEQ ID NO. 1.

[0014] SEQ ID NO. 1: EEQYNSTYR.

[0015] Preferably, the glycoform of the glycopeptide includes any one or a combination of at least two of A1G0F, A1G1F, A2G0F, A2G1F or A2G2F.

[0016] Preferably, the steps of the filter-assisted sample preparation enzymatic digestion method include: dissolving the sample to be tested, reducing, alkylating, ultrafiltrating, and terminating the reaction after enzymatic digestion.

[0017] Preferably, the solvent for dissolving is 6-10M guanidine hydrochloride, for example, it can be 6M, 7M, 8M, 9M or 10M, etc.

[0018] Preferably, the concentration of the sample to be tested after dissolving is 4-5mg / mL, for example, it can be 4mg / mL, 4.2mg / mL, 4.4mg / mL, 4.6mg / mL, 4.8mg / mL or 5mg / mL, etc.

[0019] Preferably, the reducing agent includes dithiothreitol and / or tris(2-carboxyethyl) phosphine hydrochloride.

[0020] Preferably, the final concentration of the reducing agent is 10-30mM, for example, it can be 10mM, 15mM, 20mM, 25mM or 30mM, etc.

[0021] Preferably, the temperature of the reduction is 50-60°C for 20-60 min. The 50-60°C can be, for example, 50°C, 52°C, 54°C, 56°C, 58°C, or 60°C, etc. The 20-60 min can be, for example, 20 min, 30 min, 40 min, 50 min, or 60 min, etc.

[0022] Preferably, the alkylating agent used in the alkylating step comprises iodoacetamide and / or N-ethylmaleimide.

[0023] Preferably, the final concentration of the alkylating agent is 20-70 mM, for example, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, or 70 mM, etc.

[0024] Preferably, the temperature of the alkylating is 20-30°C for 30-60 min. The 20-30°C can be, for example, 20°C, 22°C, 24°C, 26°C, 28°C, or 30°C, etc. The 30-60 min can be, for example, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min, or 60 min, etc.

[0025] Preferably, the alkylating is performed in the dark.

[0026] Preferably, the ultrafiltration agent comprises any one or a combination of at least two of the following: Tris-HCl, ammonium bicarbonate, or urea.

[0027] Preferably, the concentration of the ultrafiltration agent is 10-30 mM, for example, 10 mM, 15 mM, 20 mM, 25 mM, or 30 mM, etc.

[0028] Preferably, the enzyme used in the enzymatic digestion comprises any one or a combination of at least two of the following: trypsin, chymotrypsin, or lysyl endopeptidase.

[0029] Preferably, the concentration of the enzyme used in the enzymatic digestion is 0.05-0.1 μg / μL, for example, 0.05 μg / μL, 0.06 μg / μL, 0.07 μg / μL, 0.08 μg / μL, 0.09 μg / μL, or 0.1 μg / μL, etc.

[0030] Preferably, the enzymatic digestion is performed for 2-6 h at 35-40°C. The 2-6 h can be, for example, 2 h, 3 h, 4 h, 5 h, or 6 h, etc. The 35-40°C can be, for example, 35°C, 36°C, 37°C, 38°C, 39°C, or 40°C, etc.

[0031] Preferably, the reaction termination agent comprises formic acid and / or trifluoroacetic acid.

[0032] Preferably, the concentration of the reaction termination reagent is 0.5-1.5%, for example, it can be 0.5%, 0.6%, 0.7%, 0.8%, 0.9%, 1%, 1.1%, 1.2%, 1.3%, 1.4%, or 1.5%, etc.

[0033] Preferably, the step of the trypsin enzymolysis method comprises: adding trypsin to the sample for enzymolysis, and terminating the reaction after adding a reducing agent.

[0034] Preferably, the final concentration of the sample is 0.3-0.7 μg / μL, for example, it can be 0.3 μg / μL, 0.4 μg / μL, 0.5 μg / μL, 0.6 μg / μL, or 0.7 μg / μL, etc.

[0035] Preferably, the temperature of the enzymolysis is 60-80°C, and the time is 1-3h. The 60-80°C, for example, can be 60°C, 62°C, 64°C, 66°C, 68°C, 70°C, 72°C, 74°C, 76°C, 78°C, or 80°C, etc. The 1-3h, for example, can be 1h, 1.5h, 2h, 2.5h, or 3h, etc.

[0036] Preferably, the final concentration of the trypsin is 0.01-0.03 μg / μL, for example, it can be 0.01 μg / μL, 0.015 μg / μL, 0.02 μg / μL, 0.025 μg / μL, or 0.03 μg / μL, etc.

[0037] Preferably, the reducing agent comprises dithiothreitol and / or tris(2-carboxyethyl)phosphine hydrochloride.

[0038] Preferably, the final concentration of the reducing agent is 0.01-0.1 mol / L, for example, it can be 0.01 mol / L, 0.02 mol / L, 0.04 mol / L, 0.06 mol / L, 0.08 mol / L, or 0.1 mol / L, etc.

[0039] Preferably, the temperature of the reduction is 30-40°C, and the time is 5-20min. The 30-40°C, for example, can be 30°C, 32°C, 34°C, 36°C, 38°C, or 40°C, etc.; the 5-20min, for example, can be 5min, 10min, 15min, or 20min, etc.

[0040] Preferably, the reaction termination reagent comprises formic acid and / or trifluoroacetic acid.

[0041] Preferably, the final concentration of the reaction termination reagent is 0.3-0.8%, for example, it can be 0.3%, 0.4%, 0.5%, 0.6%, 0.7%, or 0.8%, etc.

[0042] Preferably, the auxiliary heater temperature of the mass spectrometer is 200-350℃, for example, it can be 200℃, 210℃, 220℃, 230℃, 240℃, 250℃, 260℃, 270℃, 280℃, 290℃, 300℃, 310℃, 320℃, 330℃, 340℃ or 350℃, etc.

[0043] Preferably, the ion spray voltage of the mass spectrometer is 3.5-4.2kV, for example, it can be 3.5kV, 3.6kV, 3.7kV, 3.8kV, 3.9kV, 4.0kV, 4.1kV or 4.2kV, etc.

[0044] Preferably, the sheath gas pressure of the mass spectrometer is 25-35arb, for example, it can be 25arb, 26arb, 27arb, 28arb, 29arb, 30arb, 31arb, 32arb, 33arb, 34arb or 35arb, etc.

[0045] Preferably, the auxiliary gas pressure of the mass spectrometer is 5-15arb, for example, it can be 5arb, 6arb, 7arb, 8arb, 9arb, 10arb, 11arb, 12arb, 13arb, 14arb or 15arb, etc.

[0046] Preferably, the scanning mode of the mass spectrometer is positive ion mode.

[0047] Preferably, the scanning range of the mass spectrometer is 200-2000m / z, the first order resolution is 7000, and the second order resolution is 17500.

[0048] Preferably, the formula of the data analysis is:

[0049] Single glycoform % = (XIC signal value of single glycoform / XIC signal value sum) x 100;

[0050] Preferably, the data analysis method includes any one of manual analysis or software analysis.

[0051] In a second aspect, the application provides an application of the detection method for relative quantification of glycoforms in glycopeptides based on mass spectrometric analysis in the first aspect to relative quantification of glycoforms in glycopeptides.

[0052] Compared with the prior art, the application has at least the following beneficial effects:

[0053] With the change of capillary temperature, the detection result of glycoform will also change, in which the A2G0F glycopeptide ratio will show a decreasing trend with the increase of CT, while the A1G0F glycopeptide ratio will show an increasing trend with the increase of CT. Within the scope defined in the present application, the ratio of glycoform is consistent with the ratio of molecular weight glycoform, and the detection accuracy is higher. Not only can the time for subsequent method optimization be reduced, but also a clear direction for optimization can be provided, greatly improving the work efficiency. BRIEF DESCRIPTION OF DRAWINGS

[0054] Figure 1 Figure for molecular weight glycoform ratio.

[0055] Figure 2 Figure for detection of glycopeptide segment (A2G0F) in peptide map.

[0056] Figure 3 Figure for detection of glycopeptide segment (A2G0F) in peptide map. DETAILED DESCRIPTION

[0057] The technical solutions of the present application will be further illustrated below by combining the drawings and through specific embodiments. However, the following examples are only simple examples of the present application, and do not represent or limit the protection scope of the present application, and the protection scope of the present application is subject to the claims.

[0058] The sources of reagents and instruments used in the following examples are as follows:

[0059] Trypsin: Trypsin trypsin was purchased from Promega Company, item number V5111;

[0060] Mass spectrometer: Q Exactive TM Plus purchased from Thermo Scientific.

[0061] Example 1

[0062] The present example provides a glycopeptide glycoform relative quantitative detection method based on mass spectrometry analysis

[0063] In this example, a filter-assisted sample preparation enzyme digestion method is used for pretreatment. This method first dissolves the test product, then performs reduction, alkylation, ultrafiltration, enzyme digestion, and then performs termination reaction.

[0064] Take IgG1 type monoclonal antibody to the concentration of 5mg / mL, solvent is 8M guanidine hydrochloride, then add dithiothreitol to the final concentration of 20mM, react at 56℃ for 30min. After adding iodoacetamide to the final concentration of 50mM, react at 25℃ for 45min in the dark. Then use 20mM of tris-hydroxymethyl aminomethane hydrochloride for ultrafiltration replacement three times. Add trypsin to the concentration of 0.08μg / μL, and enzymatic cut at 37℃ for 4h. Add 1% formic acid to terminate the reaction, take samples for mass spectrometry, and analyze the mass spectrometry data by software. The formula of the data analysis is:

[0065] Single glycoform % = (XIC signal value of single glycoform / XIC signal value sum) x 100.

[0066] The mass spectrometry parameters: capillary temperature 270℃, auxiliary heater temperature 300℃, ion spray voltage 3.8kv, sheath gas pressure 30arb, auxiliary gas pressure 10arb. The scanning range of mass spectrometry is 200-2000m / z, the first order resolution is 7000, and the second order resolution is 17500.

[0067] Example 2

[0068] The embodiment provides a detection method for relative quantification of glycoforms in glycopeptides based on mass spectrometry analysis

[0069] The embodiment adopts a filter-assisted sample preparation enzymatic cutting method for pretreatment. The method is to dissolve the sample to be tested, then reduce, alkylate, ultrafiltrate, and terminate the reaction after enzymatic cutting.

[0070] Take IgG1 type monoclonal antibody to the concentration of 5mg / mL, solvent is 8M guanidine hydrochloride, then add dithiothreitol to the final concentration of 20mM, react at 56℃ for 30min. After adding iodoacetamide to the final concentration of 50mM, react at 25℃ for 45min in the dark. Then use 20mM of tris-hydroxymethyl aminomethane hydrochloride for ultrafiltration replacement three times. Add trypsin to the concentration of 0.08μg / μL, and enzymatic cut at 37℃ for 4h. Add 1% formic acid to terminate the reaction, take samples for mass spectrometry, and analyze the mass spectrometry data by software. The formula of the data analysis is:

[0071] Single glycoform % = (XIC signal value of single glycoform / XIC signal value sum) x 100.

[0072] The mass spectrometry parameters: capillary temperature 270℃, auxiliary heater temperature 300℃, ion spray voltage 3.8kv, sheath gas pressure 30arb, auxiliary gas pressure 10arb. The scanning range of mass spectrometry is 200-2000m / z, the first order resolution is 7000, and the second order resolution is 17500.

[0073] Example 3

[0074] The embodiment provides a detection method for relative quantification of glycan types in glycopeptides based on mass spectrometric analysis

[0075] The embodiment adopts a filter-assisted sample preparation enzyme digestion method for pretreatment. The method comprises the following steps: dissolving a sample to be detected, reducing, alkylating, ultrafiltrating, and terminating the reaction after enzyme digestion.

[0076] Take IgG1 type monoclonal antibody to a concentration of 5 mg / mL, and use 8M guanidine hydrochloride as a solvent. Then, add dithiothreitol to a final concentration of 20 mM, and react at 56°C for 30 min. After adding iodoacetamide to a final concentration of 50 mM, react at 25°C for 45 min in the dark. Then, perform ultrafiltration liquid exchange three times with 20 mM tris-hydroxymethyl aminomethane hydrochloride. Add trypsin to a concentration of 0.08 μg / μL, and perform enzyme digestion for 4 h. Add 1% formic acid to terminate the reaction, take a sample for mass spectrometry, and perform software analysis on the mass spectrometric data. The formula for the data analysis is as follows:

[0077] Single glycan type % = (XIC signal value of single glycan type / XIC signal value sum) x 100.

[0078] The mass spectrometric parameters are as follows: capillary temperature 290°C, auxiliary heater temperature 200°C, ion spray voltage 3.5kv, sheath gas pressure 35arb, and auxiliary gas pressure 5arb. The scanning range of the mass spectrometer is 200-2000 m / z, the first-order resolution is 7000, and the second-order resolution is 17500.

[0079] Embodiment 4

[0080] The embodiment provides a detection method for relative quantification of glycan types in glycopeptides based on mass spectrometric analysis, which is different from the method in Embodiment 1 only in that the auxiliary heater temperature is 150°C, and the rest is the same as in Embodiment 1.

[0081] Embodiment 5

[0082] The embodiment provides a detection method for relative quantification of glycan types in glycopeptides based on mass spectrometric analysis, which is different from the method in Embodiment 1 only in that the auxiliary heater temperature is 400°C, and the rest is the same as in Embodiment 1.

[0083] Embodiment 6

[0084] The embodiment provides a detection method for relative quantification of glycan types in glycopeptides based on mass spectrometric analysis, which is different from the method in Embodiment 1 only in that the ion spray voltage is 3kV, and the rest is the same as in Embodiment 1.

[0085] Embodiment 7

[0086] The embodiment provides a detection method for relative quantification of glycan types in glycopeptides based on mass spectrometric analysis, which is only different from the method of embodiment 1 in that the ion spray voltage is 4.5 kV, and the rest is consistent with embodiment 1.

[0087] Example 8

[0088] The embodiment provides a detection method for relative quantification of glycan types in glycopeptides based on mass spectrometric analysis, which is only different from the method of embodiment 1 in that the pretreatment method adopts a trypsin enzyme cutting method, and the rest is consistent with embodiment 1.

[0089] The method obtains a mixed solution of the to-be-tested product, a buffer and ultrapure water, and then trypsin is added for enzyme cutting. The reaction is terminated after a reducing agent is added.

[0090] The IgG1 type monoclonal antibody is dissolved in a buffer and ultrapure water is added, the final concentration of the to-be-tested product is 0.5 μg / μL, trypsin is added to a final concentration of 0.02 μg / μL, and enzyme cutting is performed at 70°C for 2 h, and then the solution is cooled to room temperature. After 0.02 mol / L dithiothreitol is added and mixed, the solution is placed in a 37°C metal bath for reduction for 10 min, 0.5% formic acid is added, mixed, centrifuged at 13000 rpm for 5 min, and the supernatant is subjected to mass spectrometry sampling and detection.

[0091] Comparative Example 1

[0092] The comparative example provides a detection method for relative quantification of glycan types in glycopeptides based on mass spectrometric analysis, which is only different from the method of embodiment 1 in that the capillary temperature is 230°C, and the rest is consistent with embodiment 1.

[0093] Comparative Example 2

[0094] The comparative example provides a detection method for relative quantification of glycan types in glycopeptides based on mass spectrometric analysis, which is only different from the method of embodiment 1 in that the capillary temperature is 300°C, and the rest is consistent with embodiment 1.

[0095] Comparative Example 3

[0096] The comparative example provides a detection method for relative quantification of glycan types in glycopeptides based on mass spectrometric analysis, which is only different from the method of embodiment 1 in that the capillary temperature is 320°C, and the rest is consistent with embodiment 1.

[0097] Test Example 1

[0098] The test example is used for analyzing glycan types obtained by mass spectrometry in embodiments 1-10 and comparative examples 1-3.

[0099] First, the molecular weight glycan types in the glycopeptides are analyzed, and the specific analysis is as follows. Figure 1As shown, the proportion of A2G0F glycoform in the glycopeptide is 75.7%, the proportion of A2G1F glycoform is 21.9%, and the proportion of A2G2F glycoform is 2.4%.

[0100] The glycoforms obtained by mass spectrometry of Examples 1-9 and Comparative Examples 1-3 were analyzed, and the calculation method is as follows: Figure 2 As shown (the figure is the result of Example 1), the software automatically matches a series of corresponding mass glycoform types according to the MS1 information, and then screens each series of glycoforms according to the detected mass spectrometry secondary (MS2) information, until the target glycoform is matched, so the software calculates the proportion according to the MS1 response value of each glycoform. Figure 3

[0101] Taking A2G0F as an example, the mass spectrometry detection of EEQYNSTYR(A2G0F) has a multivalent peptide signal response value of 1.14E7, and the sum of the response values of other glycoforms and modifications is 1.76E7. According to the proportion calculation formula, the result can be obtained, and the calculation of other glycoforms is similar, and the specific record is shown in Table 1.

[0102] Table 1

[0103] / A1G0F A1G1F A2G0F A2G1F A2G2F un-glycosylated other Example 1 6.8％ 0.8％ 65.1％ 20.1％ 2.1％ 1.3％ 3.9％ Example 2 4.8％ 0.6％ 65.5％ 21.5％ 2.2％ 1.3％ 4.1％ Example 3 5.8％ 0.7％ 63.7％ 21.7％ 2.4％ 1.5％ 4.2％ Example 4 4.9％ 0.4％ 66.4％ 21.1％ 2.2％ 1.4％ 3.7％ Example 5 9.5％ 1.3％ 61.7％ 20.2％ 2.1％ 1.2％ 4.0％ Example 6 7.5％ 0.9％ 62.1％ 21.5％ 2.3％ 1.3％ 4.4％ Example 7 6.7％ 0.8％ 64.0％ 21.0％ 2.2％ 1.3％ 4.0％ Example 8 6.5％ 0.0％ 70.4％ 19.7％ 1.8％ 0.6％ 1.0％ Comparative Example 1 4.0％ 0.4％ 65.2％ 22.3％ 2.4％ 1.3％ 4.4％ Comparative Example 2 11.1％ 1.6％ 59.8％ 20.2％ 2.1％ 1.3％ 3.9％ Comparative Example 3 16.6％ 2.6％ 55.0％ 18.7％ 2.0％ 1.3％ 3.8％

[0104] The results are as follows:

[0105] (1) As can be seen from the comparison of Example 1 and Examples 4-5, the temperature of the auxiliary device has a certain effect on the proportion of glycoforms. Specifically, when the temperature of the auxiliary gas is too high, the proportion of A2G0F will decrease, and the proportion of A1G0F will slightly increase, but the overall trend is less affected. In Table 1, other types refer to some glycoforms with low proportions, which will not affect the overall trend of the data. Due to the different ionization efficiencies of the instrument in the detection of medium molecular weight and enzyme-cleaved peptide segments, some glycoforms with low signals cannot be displayed in the detection, but they will not affect the overall trend of the results. Figure 1 Figure 2 Figure 1

[0106] (2) As can be seen from the comparison of Example 1 and Examples 6-7, the ion spray voltage has little effect on the proportion of glycoforms, i.e., the proportion trends of A1G0F, A2G0F, and A2G1F in Examples 6-7 are consistent with those of Example 1.

[0107] (3) As can be seen from the comparison of Example 1 and Example 8, the detection results of two different pretreatment methods show that A2G0F and A2G1F are still the main forms and have consistent proportion trends, i.e., different pretreatment methods have little effect on the proportion of glycoforms.

[0108] ​​​​(4) By comparing example 1 with comparative examples 1-3, it can be seen that the results show that the higher the CT temperature, the greater the influence on the stability of glycopeptide, which is reflected in the change of the content of N-acetylglucosamine, that is, the A2G0F glycopeptide ratio, which will show a decreasing trend with the increase of CT, while the A1G0F glycopeptide ratio will show an increasing trend with the increase of CT, and within the scope defined in the application, the proportion of glycosyl in glycopeptide is consistent with the proportion of molecular weight glycosyl, and outside this range, the higher the parameter, the greater the difference in the proportion of glycopeptide.

[0109] In summary, it is creatively found in the application that the mass spectrometry source parameters have a greater influence on the detection of glycosyl in glycopeptide, especially the influence of capillary temperature is higher, which plays a good auxiliary role for the detection of glycosyl in glycopeptide by mass spectrometry.

[0110] The applicant declares that the above is only a specific embodiment of the application, but the protection scope of the application is not limited thereto, and those skilled in the art should understand that any changes or replacements easily thought of by those skilled in the art within the technical scope disclosed by the application fall within the protection scope and disclosure scope of the application.

Claims

1. A method for the relative quantitative detection of glycoforms in glycopeptides based on mass spectrometry analysis, characterized in that, The method includes the following steps: pre-treating the sample to obtain glycopeptides, performing mass spectrometry detection, and then performing data analysis. The capillary temperature for the mass spectrometry detection is 250-290℃; The glycopeptide has a glycoform of A1G0F, A1G1F, A2G0F, A2G1F and A2G2F; The auxiliary heating temperature of the mass spectrometer is 200-350℃; The ion spray voltage of the mass spectrometer is 3.5-4.2 kV; The pretreatment includes either the filter-assisted sample preparation enzymatic digestion method or the trypsin enzymatic digestion method; The peptide sequence of the glycopeptide includes SEQ ID NO.

1.

2. The detection method according to claim 1, characterized in that, The steps of the filtration-assisted sample preparation enzymatic digestion method include: dissolving the sample to be tested, and then terminating the reaction after reduction, alkylation, ultrafiltration, enzymatic digestion.

3. The detection method according to claim 2, characterized in that, The solvent used in the dissolution step is a 6-10 M guanidine hydrochloride solution.

4. The detection method according to claim 2, characterized in that, The concentration of the sample after dissolution is 4-5 mg / mL.

5. The detection method according to claim 2, characterized in that, The reducing agent includes dithiothreitol and / or tris(2-carboxyethyl)phosphine hydrochloride.

6. The detection method according to claim 2, characterized in that, The final concentration of the reducing agent is 10-30 mM.

7. The detection method according to claim 2, characterized in that, The reduction temperature is 50-60℃ and the time is 20-60 min.

8. The detection method according to claim 2, characterized in that, The alkylation step uses alkylating agents including iodoacetamide and / or N-ethylmaleimide.

9. The detection method according to claim 8, characterized in that, The final concentration of the alkylating agent is 20-70 mM.

10. The detection method according to claim 8, characterized in that, The alkylation is performed at a temperature of 20-30°C for a time of 30-60 minutes.

11. The detection method according to claim 8, characterized in that, The alkylation was carried out under light-protected conditions.

12. The detection method according to claim 2, characterized in that, The ultrafiltration reagents used in the ultrafiltration step include any one or a combination of at least two of the following: tris(hydroxymethyl)aminomethane hydrochloride, ammonium bicarbonate, or urea.

13. The detection method according to claim 12, characterized in that, The concentration of the ultrafiltration reagent is 10-30 mM.

14. The detection method according to claim 2, characterized in that, The enzymes used for the enzymatic digestion include any one or a combination of at least two of trypsin, chymotrypsin, or lysine protease.

15. The detection method according to claim 2, characterized in that, The enzyme concentration used in the enzymatic digestion step is 0.05-0.1 µg / µL.

16. The detection method according to claim 2, characterized in that, The enzyme digestion time is 2-6 hours, and the temperature is 35-40℃.

17. The detection method according to claim 2, characterized in that, The reagents used to terminate the reaction include formic acid and / or trifluoroacetic acid.

18. The detection method according to claim 2, characterized in that, The concentration of the reagent used to terminate the reaction is 0.5-1.5%.

19. The detection method according to claim 10, characterized in that, The steps of the trypsin digestion method include: adding trypsin to the sample for digestion, and terminating the reaction by adding a reducing agent.

20. The detection method according to claim 19, characterized in that, The final concentration of the test sample is 0.3-0.7 μg / μL.

21. The detection method according to claim 19, characterized in that, The enzyme digestion temperature is 60-80℃, and the time is 1-3 h.

22. The detection method according to claim 19, characterized in that, The final concentration of the trypsin is 0.01-0.03 μg / μL.

23. The detection method according to claim 19, characterized in that, The reducing agent includes dithiothreitol and / or tris(2-carboxyethyl)phosphine hydrochloride.

24. The detection method according to claim 19, characterized in that, The final concentration of the reducing agent is 0.01-0.1 mol / L.

25. The detection method according to claim 19, characterized in that, The reduction temperature is 30-40℃, and the time is 5-20 min.

26. The detection method according to claim 19, characterized in that, The reagents used to terminate the reaction include formic acid and / or trifluoroacetic acid.

27. The detection method according to claim 19, characterized in that, The final concentration of the reagent used to terminate the reaction is 0.3-0.8%.

28. The detection method according to claim 1, characterized in that, The sheath gas pressure of the mass spectrometer is 25-35 alb.

29. The detection method according to claim 1, characterized in that, The auxiliary gas pressure of the mass spectrometer is 5-15 arb.

30. The detection method according to claim 1, characterized in that, The mass spectrometer is scanned in positive ion mode.

31. The detection method according to claim 1, characterized in that, The mass spectrometer has a scanning range of 200-2000 m / z, a first-order resolution of 7000, and a second-order resolution of 17500.

32. The detection method according to claim 1, characterized in that, The formula for the data analysis is: Monosaccharide % = (XIC signal value of monosaccharide / sum of XIC signal values) × 100.

33. The detection method according to claim 1, characterized in that, The data analysis method can be either manual analysis or software analysis.

34. The application of a method for relative quantification of glycoforms in glycopeptides based on mass spectrometry analysis according to any one of claims 1-33 in the relative quantification of glycoforms in glycopeptides.

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