Detection method for relative quantification of glycoforms in glycopeptides based on mass spectrometry and application of detection method

Abstract

The invention relates to a method for detecting relative quantification of glycoforms in glycopeptides based on mass spectrometry and application thereof, and the method comprises the following steps: pretreating a to-be-detected product to obtain glycopeptides, performing mass spectrometry detection, and then performing data analysis; the capillary temperature of the mass spectrometric detection is 250-290 DEG C. The invention creatively finds that in the detection of glycoforms in glycopeptides, the glycoform detection result also changes along with the change of capillary temperature, and within the range limited by the invention, the proportion of glycoforms is consistent with the proportion of molecular weight glycoforms, and the detection accuracy is higher. The optimization time of the subsequent method can be shortened, and meanwhile, a clear guide is provided for the optimization direction, so that the working efficiency is greatly improved.

Description

Technical Field

[0001] The present invention relates to the technical field of mass spectrometry detection, and in particular to a mass spectrometry-based relative quantitative detection method for glycoforms in glycopeptides and an application thereof. Background Art

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

[0003] Mass spectrometry is an analytical method for measuring the mass-to-charge ratio of ions. Its basic principle is to ionize the components in the sample in the ion source to generate charged ions with different mass-to-charge ratios. After the acceleration electric field, an ion beam is formed and enters the mass analyzer. In the mass analyzer, the electric field and magnetic field are used to cause opposite velocity dispersion, and they are focused separately to obtain a mass spectrum to determine their mass. After the mass spectrometer is installed, the manufacturer will provide a conventional experimental detection process and instrument detection parameters for customers to run the instrument smoothly. However, the method provided can only meet the detection of conventional peptides, and it is unknown whether it is applicable to 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 guide for the direction of optimization, greatly improving work efficiency.

[0004] Therefore, how to find the more sensitive factors affecting the relative quantification of glycopeptides has become an urgent problem to be solved. Summary of the invention

[0005] To solve the above technical problems, the present invention provides a method for relative quantitative detection of glycoforms in glycopeptides based on mass spectrometry analysis and its application. The capillary temperature has a great influence on the stability of glycopeptides. Within the range of capillary temperature defined by the present invention, the proportion of glycoforms in the peptide map is more accurate, which can reduce the time for subsequent method optimization. At the same time, it also provides a clear guide for the optimization direction, greatly improving work efficiency.

[0006] To achieve this object, the present invention adopts the following technical solutions:

[0007] In a first aspect, the present invention provides a method for relative quantitative detection of glycoforms in glycopeptides based on mass spectrometry analysis, the method comprising: pre-treating a sample to be tested to obtain glycopeptides, performing mass spectrometry detection, and then performing data analysis;

[0008] The capillary temperature of the mass spectrometry detection is 250-290°C.

[0009] The present invention creatively discovered that in the detection of glycoforms in glycopeptides, as the capillary temperature changes, the glycoform detection results will also change, wherein the A2G0F glycopeptide ratio will show a decreasing trend as the CT increases, while the A1G0F glycopeptide ratio will show an increasing trend as the CT increases. Within the range defined by the present invention, the ratio of glycoforms is consistent with the molecular weight glycoform ratio, and the detection accuracy is high. Not only can the time for subsequent method optimization be reduced, but also a clear guide for the optimization direction will be provided, greatly improving work efficiency.

[0010] In the specific embodiment of the present invention, a temperature exceeding 290°C will cause a serious change in the ratio of glycoforms, which is quite different from the molecular weight comparison result of glycopeptides. A temperature below 250°C will cause distortion of the test results and make it impossible to perform the test.

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

[0012] Both pretreatment methods in the present invention can obtain target glycopeptides, and the glycopeptides have good quality and have little impact on the detection of glycoform ratios.

[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 of A1G0F, A1G1F, A2G0F, A2G1F or A2G2F, or a combination of at least two thereof.

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

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

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

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

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

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

[0022] Preferably, the alkylating agent used in the alkylation 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.

[0024] Preferably, the alkylation temperature is 20-30°C and the time is 30-60 min. The 20-30°C may be, for example, 20°C, 22°C, 24°C, 26°C, 28°C or 30°C, etc.; the 30-60 min may be, for example, 30 min, 35 min, 40 min, 45 min, 50 min, 55 min or 60 min, etc.

[0025] Preferably, the alkylation is carried out under light-protecting conditions.

[0026] Preferably, the ultrafiltration reagent comprises any one of tris(hydroxymethyl)aminomethane hydrochloride, ammonium bicarbonate or urea, or a combination of at least two thereof.

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

[0028] Preferably, the enzyme used for the enzymatic cleavage includes any one of trypsin, chymotrypsin or lysine protease, or a combination of at least two of them.

[0029] Preferably, the concentration of the enzyme used in the enzymatic cleavage step is 0.05-0.1 μg / μL, for example, it can be 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.

[0030] Preferably, the enzyme cleavage time is 2-6 hours, and the temperature is 35-40°C, wherein the 2-6 hours may be, for example, 2 hours, 3 hours, 4 hours, 5 hours or 6 hours, etc. The 35-40°C may be, for example, 35°C, 36°C, 37°C, 38°C, 39°C or 40°C, etc.

[0031] Preferably, the terminating reaction reagent comprises formic acid and / or trifluoroacetic acid.

[0032] Preferably, the concentration of the reagent for terminating the reaction 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 trypsin enzymatic digestion method comprises: adding trypsin to the sample to be tested for enzymatic digestion, and terminating the reaction after adding a reducing agent.

[0034] Preferably, the final concentration of the test product 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.

[0035] Preferably, the enzyme cleavage temperature is 60-80°C and the time is 1-3h. The 60-80°C may be, for example, 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 may be, for example, 1h, 1.5h, 2h, 2.5h or 3h, etc.

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

[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.

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

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

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

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

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

[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.

[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 mass spectrometer is scanned in positive ion mode.

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

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

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

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

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

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

[0053] As the capillary temperature changes, the sugar form detection results will also change, where the A2G0F glycopeptide ratio will show a decreasing trend as CT increases, while the A1G0F glycopeptide ratio will show an increasing trend as CT increases. Within the range defined by the present invention, the sugar form ratio is consistent with the molecular weight sugar form ratio, and the detection accuracy is high. Not only can the time for subsequent method optimization be reduced, but also a clear guide for the optimization direction will be provided, greatly improving work efficiency. BRIEF DESCRIPTION OF THE DRAWINGS

[0054] Figure 1 The figure shows the molecular weight sugar form ratio.

[0055] Figure 2 This is the primary mass spectrum of the peptide map containing glycopeptide (A2G0F).

[0056] Figure 3 This is the secondary mass spectrum of the peptide map containing glycopeptide (A2G0F). DETAILED DESCRIPTION

[0057] The technical solution of the present invention is further described below in conjunction with the accompanying drawings and through specific implementation methods. However, the following examples are only simplified examples of the present invention and do not represent or limit the scope of protection of the present invention. The scope of protection of the present invention shall be subject to the claims.

[0058] Sources of reagents and instruments used in the following examples:

[0059] Trypsin: Trypsin was purchased from Promega, catalog number V5111;

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

[0061] Example 1

[0062] This example provides a method for relative quantitative detection of glycoforms in glycopeptides based on mass spectrometry analysis.

[0063] This embodiment adopts the filtration-assisted sample preparation enzyme digestion method for pretreatment, which first dissolves the sample to be tested, then reduces, alkylates, ultrafilters, digests, and terminates the reaction.

[0064] Take IgG1 type monoclonal antibody to a concentration of 5 mg / mL, the solvent is 8M guanidine hydrochloride, then add dithiothreitol to a final concentration of 20mM, and react at 56°C for 30min. Add iodoacetamide to a final concentration of 50mM and react at 25°C in the dark for 45min. Then ultrafiltration and liquid exchange are performed three times with tris(hydroxymethylaminomethane) hydrochloride at a concentration of 20mM. Add trypsin to a concentration of 0.08μg / μL, digest at 37°C for 4h, add 1% formic acid to terminate the reaction, take samples for mass spectrometry, and perform software analysis on the mass spectrometry data. The formula for data analysis is:

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

[0066] The mass spectrometer parameters are as follows: capillary temperature 270°C, auxiliary heating temperature 300°C, ion spray voltage 3.8kv, sheath gas pressure 30arb, auxiliary gas pressure 10arb. The mass spectrometer has a scanning range of 200-2000m / z, a primary resolution of 7000, and a secondary resolution of 17500.

[0067] Example 2

[0068] This example provides a method for relative quantitative detection of glycoforms in glycopeptides based on mass spectrometry analysis.

[0069] This embodiment adopts the filtration-assisted sample preparation enzyme digestion method for pretreatment, which first dissolves the sample to be tested, then reduces, alkylates, ultrafilters, digests, and terminates the reaction.

[0070] Take IgG1 type monoclonal antibody to a concentration of 5 mg / mL, the solvent is 8M guanidine hydrochloride, then add dithiothreitol to a final concentration of 20mM, and react at 56°C for 30min. Add iodoacetamide to a final concentration of 50mM and react at 25°C in the dark for 45min. Then ultrafiltration and liquid exchange are performed three times with tris(hydroxymethylaminomethane) hydrochloride at a concentration of 20mM. Add trypsin to a concentration of 0.08μg / μL, digest for 4h, add 1% formic acid to terminate the reaction, take samples for mass spectrometry, and perform software analysis on the mass spectrometry data. The formula for data analysis is:

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

[0072] The mass spectrometry parameters are as follows: capillary temperature 250°C, auxiliary heating temperature 300°C, ion spray voltage 4.2kV, sheath gas pressure 25arb, auxiliary gas pressure 15arb. The mass spectrometry scanning range is 200-2000m / z, the primary resolution is 7000, and the secondary resolution is 17500.

[0073] Example 3

[0074] This example provides a method for relative quantitative detection of glycoforms in glycopeptides based on mass spectrometry analysis.

[0075] This embodiment adopts the filtration-assisted sample preparation enzyme digestion method for pretreatment, which first dissolves the sample to be tested, then reduces, alkylates, ultrafilters, digests, and terminates the reaction.

[0076] Take IgG1 type monoclonal antibody to a concentration of 5 mg / mL, the solvent is 8M guanidine hydrochloride, then add dithiothreitol to a final concentration of 20mM, and react at 56°C for 30min. Add iodoacetamide to a final concentration of 50mM and react at 25°C in the dark for 45min. Then ultrafiltration and liquid exchange are performed three times with tris(hydroxymethylaminomethane) hydrochloride at a concentration of 20mM. Add trypsin to a concentration of 0.08μg / μL, digest for 4h, add 1% formic acid to terminate the reaction, take samples for mass spectrometry, and perform software analysis on the mass spectrometry data. The formula for data analysis is:

[0077] Single glycoform %=(XIC signal value of single glycoform / total XIC signal value)×100.

[0078] The mass spectrometry parameters are as follows: capillary temperature 290°C, auxiliary heating temperature 200°C, ion spray voltage 3.5kv, sheath gas pressure 35arb, auxiliary gas pressure 5arb. The mass spectrometry scanning range is 200-2000m / z, the primary resolution is 7000, and the secondary resolution is 17500.

[0079] Example 4

[0080] This embodiment provides a method for relative quantitative detection of glycoforms in glycopeptides based on mass spectrometry analysis. The method is different from that in Example 1 only in that the auxiliary device heating temperature is 150° C., and the rest is consistent with Example 1.

[0081] Example 5

[0082] This embodiment provides a method for relative quantitative detection of glycoforms in glycopeptides based on mass spectrometry analysis. The method is different from that in Example 1 only in that the auxiliary device heating temperature is 400° C., and the rest is consistent with Example 1.

[0083] Example 6

[0084] This example provides a method for relative quantitative detection of glycoforms in glycopeptides based on mass spectrometry analysis. The method is different from that in Example 1 only in that the ion spray voltage is 3 kV, and the rest is consistent with Example 1.

[0085] Example 7

[0086] This example provides a method for relative quantitative detection of glycoforms in glycopeptides based on mass spectrometry analysis. The method is different from that in Example 1 only in that the ion spray voltage is 4.5 kV, and the rest is consistent with that in Example 1.

[0087] Example 8

[0088] This embodiment provides a method for relative quantitative detection of glycoforms in glycopeptides based on mass spectrometry analysis. The method is different from that in Embodiment 1 only in that the pretreatment method uses trypsin enzymatic cleavage, and the rest is consistent with that in Embodiment 1.

[0089] In this method, the test product is mixed with a buffer solution and ultrapure water to obtain a mixed solution, then trypsin is added for enzymatic cleavage, and a reducing agent is added to terminate the reaction.

[0090] Dissolve IgG1 monoclonal antibody in buffer and add ultrapure water. The final concentration of the test product is 0.5 μg / μL. Add trypsin to a final concentration of 0.02 μg / μL. Digest at 70°C for 2 hours and then cool to room temperature. Add 0.02 mol / L dithiothreitol and mix well. Place in a 37°C metal bath for reduction for 10 minutes. Add 0.5% formic acid, mix well, centrifuge at 13000 rpm for 5 minutes, and take the supernatant for mass spectrometry detection.

[0091] Comparative Example 1

[0092] This comparative example provides a method for relative quantitative detection of glycoforms in glycopeptides based on mass spectrometry analysis. The method is different from that in Example 1 only in that the capillary temperature is 230° C., and the rest is consistent with Example 1.

[0093] Comparative Example 2

[0094] This comparative example provides a method for relative quantitative detection of glycoforms in glycopeptides based on mass spectrometry analysis. The method is different from that in Example 1 only in that the capillary temperature is 300° C., and the rest is consistent with Example 1.

[0095] Comparative Example 3

[0096] This comparative example provides a method for relative quantitative detection of glycoforms in glycopeptides based on mass spectrometry analysis. The method is different from that in Example 1 only in that the capillary temperature is 320° C., and the rest is consistent with Example 1.

[0097] Test Example 1

[0098] This test example is used to analyze the glycoforms obtained by mass spectrometry in Examples 1-10 and Comparative Examples 1-3

[0099] First, the molecular weight glycoforms in glycopeptides were analyzed, as follows Figure 1As shown, the detection showed that the proportion of A2G0F glycoform in the glycopeptide was 75.7%, the proportion of A2G1F glycoform was 21.9%, and the proportion of A2G2F glycoform was 2.4%.

[0100] Then, the glycoforms obtained by mass spectrometry of Examples 1-9 and Comparative Examples 1-3 were analyzed, and the calculation method was described as follows: Figure 2 As shown in the figure (this figure is the result of implementation case 1), the software will automatically match a series of mass sugar types according to the MS1 information, and then Figure 3 The detected mass spectrometry secondary (MS2) information is used to screen each series of glycoforms one by one until the target glycoform is matched. Therefore, the software will calculate the ratio based on the MS1 response value of each glycoform.

[0101] Taking A2G0F as an example, the EEQYNSTYR (A2G0F) mass spectrometry detection of its multivalent peptide signal response value is 1.14E7, and the sum of the response values ​​of other glycoforms and modifications is 1.76E7. The result can be obtained according to the proportional calculation formula. The calculation of other glycoforms is similar. The specific demerits are shown in Table 1

[0102] Table 1

[0103] / A1G0F A1G1F A2G0F A2G1F A2G2F Unglycosylated 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 result is as follows:

[0105] (1) By comparing Example 1 with Examples 4-5, it can be seen that the auxiliary temperature has a certain effect on the sugar type ratio. Specifically, when the auxiliary temperature is too high, the A2G0F ratio will decrease, while the A1G0F ratio will increase slightly, but the overall trend is less affected. Among them, the other types in Table 1 refer to some sugar types and other modifications with a low sugar type ratio that will not affect the overall trend of the data. Figure 1 Middle molecular weight detection and Figure 2 In the detection of enzyme-cleaved peptides, the instrument ionization efficiency is different, resulting in some sugar forms with low signals. Figure 1 It cannot be displayed in the results, but it will not affect the trend of the overall results.

[0106] (2) By comparing Example 1 with Examples 6-7, it can be seen that the ion spray voltage has little effect on the sugar type ratio, that is, the ratio trend of A1G0F, A2G0F, and A2G1F in Examples 6-7 is consistent with that in Example 1.

[0107] (3) By comparing Example 1 with Example 8, it can be seen that the detection results of the two different pretreatment methods show that A2G0F and A2G1F are still the main forms and the proportion trend is consistent, that is, different pretreatment methods have a low 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 impact on the stability of the glycopeptide, which is specifically reflected in the change in the content of N-acetylglucosamine, that is, the A2G0F glycopeptide ratio will show a decreasing trend as the CT increases, while the A1G0F glycopeptide ratio will show an increasing trend as the CT increases. Within the specified range of the present invention, the proportion of glycoforms in glycopeptides is consistent with the proportion trend of molecular weight glycoforms, while outside this range, the higher the parameter, the greater the difference in the proportion trend of glycopeptides.

[0109] In summary, the present invention creatively discovered that mass spectrometry source parameters have a significant impact on the detection of glycoforms in glycopeptides, especially the capillary temperature has a greater impact, which will play a good auxiliary role in the detection of glycoforms of glycopeptides by mass spectrometry.

[0110] The applicant declares that the above is only a specific implementation mode of the present invention, but the protection scope of the present invention is not limited thereto. Those skilled in the art should understand that any changes or substitutions that can be easily thought of by those skilled in the art within the technical scope disclosed by the present invention shall fall within the protection scope and disclosure scope of the present invention.

Claims

1. A method for relative quantitative detection of glycoforms in glycopeptides based on mass spectrometry, characterized in that: The method comprises the following steps: pre-treating the sample to be tested to obtain glycopeptides, performing mass spectrometry detection and then performing data analysis; The capillary temperature of the mass spectrometry detection is 250-290°C.

2. The detection method according to claim 1, characterized in that: The pretreatment includes any one of a filtration-assisted sample preparation enzymatic digestion method or a trypsin enzymatic digestion method; Preferably, the peptide sequence of the glycopeptide includes SEQ ID NO.1; Preferably, the glycoform of the glycopeptide includes any one of A1G0F, A1G1F, A2G0F, A2G1F or A2G2F, or a combination of at least two thereof.

3. The detection method according to claim 1 or 2, characterized in that: The steps of the filtration-assisted sample preparation and enzyme digestion method include: dissolving the sample to be tested, reducing, alkylating, ultrafiltration, and terminating the reaction after enzyme digestion.

4. The detection method according to claim 3, characterized in that: The solvent used in the dissolving step is a 6-10M guanidine hydrochloride solution; Preferably, the concentration of the test product after dissolution is 4-5 mg / mL; Preferably, the reducing agent comprises dithiothreitol and / or tris(2-carboxyethyl)phosphine hydrochloride; Preferably, the final concentration of the reducing agent is 10-30 mM; Preferably, the reduction temperature is 50-60°C and the time is 20-60min; Preferably, the alkylating agent used in the alkylation step comprises iodoacetamide and / or N-ethylmaleimide; Preferably, the final concentration of the alkylating agent is 20-70 mM; Preferably, the alkylation temperature is 20-30°C and the time is 30-60min; Preferably, the alkylation is carried out under light-protected conditions; Preferably, the ultrafiltration reagent used in the ultrafiltration step includes any one of tris(hydroxymethyl)aminomethane hydrochloride, ammonium bicarbonate or urea, or a combination of at least two thereof; Preferably, the concentration of the ultrafiltration reagent is 10-30 mM; Preferably, the enzyme used for the enzymatic cleavage includes any one of trypsin, chymotrypsin or lysine protease or a combination of at least two thereof; Preferably, the concentration of the enzyme used in the enzymatic cleavage step is 0.05-0.1 μg / μL; Preferably, the enzyme digestion time is 2-6h and the temperature is 35-40°C; Preferably, the reagent used in the termination reaction includes formic acid and / or trifluoroacetic acid; Preferably, the concentration of the reagent for terminating the reaction is 0.5-1.5%.

5. The detection method according to claim 1 or 2, characterized in that: The trypsin digestion method comprises the following steps: adding trypsin to the sample to be tested for digestion, and then adding a reducing agent to terminate the reaction.

6. The detection method according to claim 5, characterized in that: The final concentration of the test product is 0.3-0.7 μg / μL; Preferably, the enzyme digestion temperature is 60-80°C and the time is 1-3h; Preferably, the final concentration of trypsin is 0.01-0.03 μg / μL; Preferably, the reducing agent comprises dithiothreitol and / or tris(2-carboxyethyl)phosphine hydrochloride; Preferably, the final concentration of the reducing agent is 0.01-0.1 mol / L; Preferably, the reduction temperature is 30-40°C and the time is 5-20min; Preferably, the reagent used in the termination reaction includes formic acid and / or trifluoroacetic acid; Preferably, the final concentration of the reagent for terminating the reaction is 0.3-0.8%.

7. The detection method according to any one of claims 1 to 6, characterized in that: The auxiliary heating temperature of the mass spectrometer is 200-350°C.

8. The detection method according to any one of claims 1 to 7, characterized in that: The ion spray voltage of the mass spectrometer is 3.5-4.2 kV; Preferably, the sheath gas pressure of the mass spectrometer is 25-35arb; Preferably, the auxiliary gas pressure of the mass spectrometer is 5-15arb; Preferably, the mass spectrometer is scanned in positive ion mode; Preferably, the scanning range of the mass spectrometer is 200-2000 m / z, the primary resolution is 7000, and the secondary resolution is 17500.

9. The detection method according to any one of claims 1 to 8, characterized in that: The formula for the data analysis is: Single glycoform % = (XIC signal value of single glycoform / total XIC signal value) × 100; Preferably, the data analysis method includes any one of manual analysis or software analysis.

10. Use of the method for relative quantification of glycoforms in glycopeptides based on mass spectrometry according to any one of claims 1 to 9 in relative quantification of glycoforms in glycopeptides.

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