DeepGP glycopeptide enrichment column and method for efficiently enriching N glycopeptide and O glycopeptide

Abstract

The invention relates to the technical field of glycosylation, and provides a DeepGP glycopeptide enrichment column and a method for efficiently enriching N glycopeptide and O glycopeptide. The preparation method of the DeepGP glycopeptide enrichment column comprises the following steps: uniformly mixing microcrystalline cellulose, cation exchange filler and anion exchange filler, and then packing the mixed filler into a column and compacting to obtain the DeepGP glycopeptide enrichment column. Compared with the enrichment effects of four enrichment columns on HET cell line glycopeptide samples, the DeepGP glycopeptide enrichment column prepared by the invention has the highest parallelism and enrichment efficiency in three times of parallel enrichment. Besides, 245 N glycopeptides can be identified in an IgG standard substance by utilizing a DeepGP column, and 4286 N glycopeptides and 749 O glycopeptides can be simultaneously identified in mouse plasma, so that the method is high in detection precision, high in sensitivity, good in repeatability and very wide in application range.

Description

Technical Field

[0001] The present invention relates to the field of glycosylation technology, and in particular to a DeepGP glycopeptide enrichment column and a method for efficiently enriching N-glycopeptides and O-glycopeptides. Background Art

[0002] Protein glycosylation is a post-translational modification that connects sugars to amino acid residues of proteins in the form of glycosidic bonds through the synergistic action of three types of molecules: glycosyl acceptors, donors, and glycosyltransferases. The glycosylation structure of proteins is complex and is affected by multiple factors during biosynthesis to produce heterogeneity. Glycosylation has a significant impact on the activity, immunogenicity, stability, and preparation process of glycoproteins.

[0003] Low abundance and glycosylation heterogeneity have brought great challenges to the analysis of glycosylation structure and function in the field of glycobiology. A sufficiently specific method for enriching intact glycopeptides is needed to solve this problem. A common method for enriching intact glycopeptides from complex samples is to use a C18 desalting column for desalting, and then combine it with glycopeptide enrichment columns such as hydrophilic interaction chromatography (HILIC), mixed-mode strong anion exchange (MAX / RAX), and boric acid (PBA). Existing glycopeptide enrichment columns have low glycopeptide enrichment efficiency, and the number of N-glycopeptides and O-glycopeptides identified is small. Therefore, a method with high glycopeptide enrichment efficiency is needed. Summary of the invention

[0004] In view of this, the present invention proposes a DeepGP glycopeptide enrichment column and a method for efficiently enriching N-glycopeptides and O-glycopeptides. The enrichment column has a high glycopeptide enrichment effect and can identify more N-glycopeptides and O-glycopeptides.

[0005] The technical solution of the present invention is implemented as follows: On the one hand, the present invention provides a method for preparing a DeepGP glycopeptide enrichment column, comprising the following steps:

[0006] The microcrystalline cellulose, cation exchange filler and anion exchange filler are mixed evenly, and then the mixed filler is loaded into a column and compacted to obtain a DeepGP glycopeptide enrichment column.

[0007] The DeepGP glycopeptide enrichment column combines microcrystalline cellulose, cation exchange fillers and anion exchange fillers, which work simultaneously through multiple interaction mechanisms. Microcrystalline cellulose provides hydrophilic properties to support the selective adsorption of sugar molecules, while cation and anion exchange fillers assist in the adsorption and separation of differently charged peptides through charge interactions.

[0008] The multimodal filler of DeepGP glycopeptide enrichment column can increase the surface contact opportunities to enhance the interaction between polysaccharides and peptide chains. Due to the simultaneous action of several mechanisms, DeepGP glycopeptide enrichment column can better identify and capture glycopeptides and reduce the impact of nonspecific adsorption and interferences. This selectivity is particularly reflected in the comparison with other conventional methods, such as HILIC and strong anion exchange.

[0009] On the basis of the above technical solution, preferably, the mass ratio of the microcrystalline cellulose: cation exchange filler: anion exchange filler is 1:1-1.2:1-1.2.

[0010] On the other hand, the present invention also provides a method for efficiently enriching N-glycopeptides and O-glycopeptides, comprising the following steps:

[0011] S1, after protein extraction, tri(2-carboxyethyl)phosphine hydrochloride (TCEP) and iodoacetamide (IAA) were added for reductive alkylation reaction, followed by enzymatic hydrolysis and desalting after the reaction;

[0012] S2, the desalted peptides were enriched using a DeepGP glycopeptide enrichment column;

[0013] S3, the enriched samples are collected by liquid chromatography-mass spectrometry, and then the collected data are analyzed to identify N-glycans and O-glycopeptides.

[0014] On the basis of the above technical scheme, preferably, the reduction alkylation reaction method in step S1 is: take the extracted protein sample, add tri(2-carboxyethyl)phosphine hydrochloride solution, mix well and then shake for reaction, and after the reaction is completed, add iodoacetamide solution, mix well and then react in the dark.

[0015] On the basis of the above technical solution, preferably, the enzymatic hydrolysis method in step S1 is: after the reduction alkylation reaction is completed, trypsin is added to the protein sample for enzymatic hydrolysis.

[0016] On the basis of the above technical solution, preferably, the mass ratio of trypsin:protein sample is 50:1, the enzymatic hydrolysis temperature is 30-37°C, and the time is 10-12h.

[0017] In step S1, C18 column is used for desalting.

[0018] On the basis of the above technical scheme, preferably, the enrichment method of DeepGP glycopeptide enrichment column in step S2 is: activate the column with 2%-10% v / v acetonitrile (ACN) and 0.1%-2% v / v trifluoroacetic acid (TFA) in sequence, balance the column with 50%-100% v / v acetonitrile and 0.1%-2% v / v trifluoroacetic acid, drain the peptide sample after desalting in step S1 and redissolve it in 50%-100% v / v acetonitrile and 0.1%-2% v / v trifluoroacetic acid, load it onto DeepGP glycopeptide enrichment column, then wash the column with 50%-100% v / v acetonitrile and 0.1%-2% v / v trifluoroacetic acid, elute the sample with 2%-10% v / v acetonitrile and 0.1%-2% v / v trifluoroacetic acid, and combine the eluates; finally, freeze-dry the enriched glycopeptide eluate, and load it onto a mass spectrometer after redissolution.

[0019] Based on the above technical solution, preferably, in step S3, LC-MS, easyLC 1200, and OrbitrapEclipse three-in-one mass spectrometer are used for data acquisition.

[0020] The DeepGP glycopeptide enrichment column and the method for efficiently enriching N-glycopeptides and O-glycopeptides of the present invention have the following beneficial effects compared with the prior art:

[0021] (1) The enrichment effects of four enrichment columns on HET cell line glycopeptide samples were compared. The DeepGP glycopeptide enrichment column prepared by the present invention had the highest parallelism and enrichment efficiency in three parallel enrichments. In addition, the DeepGP column can be used to identify 245 N-glycopeptides in IgG standards, and 4286 N-glycopeptides and 749 O-glycopeptides in mouse plasma, indicating that the method has high detection accuracy, strong sensitivity, good repeatability, and a wide range of application. BRIEF DESCRIPTION OF THE DRAWINGS

[0022] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0023] Figure 1 This is a schematic diagram of the process of the cell sample glycopeptide identification and quantification method of the present invention;

[0024] Figure 2 This is a bar graph showing the identification results of N-glycopeptides from four enrichment columns of HET cells in Example 1 of the present invention;

[0025] Figure 3 This is a bar graph showing the results of O-glycopeptide identification in four enrichment columns of HET cells in Example 1 of the present invention;

[0026] Figure 4 This is a bar graph showing the standard deviation of the N-glycopeptide identification results in three enrichment runs of four enrichment columns for HET cells in Example 1 of the present invention;

[0027] Figure 5 This is a Pearson correlation coefficient diagram of the N / O glycopeptide identification results in three enrichment runs of four enrichment columns for HET cells in Example 1 of the present invention;

[0028] Figure 6 This is a statistical bar graph of N / O glycopeptides, glycoproteins and glycosylation sites identified in the mouse plasma samples of the present invention. DETAILED DESCRIPTION

[0029] The following will be combined with the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] Reagents and consumables:

[0031] Iodoacetamide (IAA), urea (UA), and microcrystalline cellulose were purchased from Sigma-Aldrich.

[0032] Chromatographic grade methanol and acetonitrile were purchased from Merck (Germany); analytical grade formic acid was purchased from Sinopharm Group.

[0033] Mass spectrometry grade trypsin was purchased from Beijing Hualishi Technology Co., Ltd.

[0034] Anion exchange filler, cation exchange filler, tri(2-carboxyethyl)phosphine hydrochloride (TCEP), BCA protein quantification kit, and peptide quantification kit were all purchased from Thermo Fisher Scientific (China) Co., Ltd.

[0035] The C18 desalting column and MAX glycopeptide enrichment column were purchased from Wters, the Hilic glycopeptide enrichment column filler was purchased from MACHEREY-NAGEL, the PBA enrichment filler was purchased from Agilent, and the DeepGP glycopeptide enrichment column was a product independently developed by Baizhen Bio.

[0036] Example 1

[0037] In this example, commercial human serum immunoglobulin (IgG) was used to evaluate the glycopeptide enrichment performance of DeepGPN. The preparation method of the DeepGP glycopeptide enrichment column in this example is as follows: 33 mg of microcrystalline cellulose, 33 mg of cation exchange filler and 33 mg of anion exchange filler were mixed evenly to obtain a mixed filler, and then the mixed filler was filled into a 1 mL syringe-type SPE empty column using an automatic column filling process, and a sieve plate was added after compaction, and the column was vacuum-packed and set aside.

[0038] The method for efficiently enriching N-glycopeptides and O-glycopeptides in this embodiment comprises the following steps:

[0039] S1, protein reductive alkylation and enzymatic digestion:

[0040] 1) Take 10 μg IgG and add 25 μL lysis buffer (8 M urea, 0.1 M Tris) to dissolve. Add 0.25 μL 0.5 M TCEP solution, vortex to mix, and react at 37°C, 1000 rpm for 0.5 h. Add 0.87 μL 300 mM IAA solution, vortex to mix, and react at 25°C in the dark for 0.5 h.

[0041] 2) After the reduction alkylation reaction was completed, 500 μg of trypsin (50:1) was added to the reaction solution and reacted at 37°C for 12 h.

[0042] 3) The peptide solution after enzymatic hydrolysis was centrifuged at 25°C, 15000g for 15 min, and the supernatant was desalted using a C18 column;

[0043] S2, glycopeptide enrichment: The desalted peptide solution was enriched with DeepGP glycopeptide enrichment column, Hilic, MAX and PBA respectively. The enrichment process of Hilic and MAX glycopeptide enrichment columns was referred to the literature of Liu and Yang et al. [1-2] The PBA enrichment process for glycopeptides is based on the literature of Morgenstern et al. [3] , the enrichment process of DeepGP glycopeptide enrichment column is as follows:

[0044] 1) Activation: Add 1 ml of activation solution 8% (v / v) ACN, 1.5% (v / v) TFA to activate the column, repeat 5 times;

[0045] 2) Equilibration: Add 1 ml of equilibration solution 75% (v / v) ACN, 0.15% (v / v) TFA to equilibrate the column, repeat 4 times;

[0046] 3) Capture: Drain the desalted sample, re-dissolve it in the equilibrium solution, inject it into the column, let it flow through naturally, and repeat the capture once;

[0047] 4) Washing: Add 1 ml of washing solution 80% (v / v) ACN, 0.16% (v / v) TFA to wash the column, repeat 3 times;

[0048] 5) Elution: Add 1 ml of eluent 9% (v / v) ACN, 0.4% (v / v) TFA to elute the sample. Repeat 5 times, collect and combine the eluent.

[0049] 6) The enriched glycopeptide eluate is freeze-dried, and after reconstitution, it is loaded onto a mass spectrometer.

[0050] S3, mass spectrometry: The enriched samples were subjected to liquid chromatography and high-resolution mass spectrometry (LC-MS), easyLC 1200, and Orbitrap Eclipse three-in-one mass spectrometry for data acquisition.

[0051] Mobile phase: composed of mobile phase A and mobile phase B, wherein mobile phase A is an aqueous solution containing 0.1%±(0~0.01%) formic acid, and mobile phase B is an acetonitrile / formic acid mixture containing 0.1%±(0~0.01%) formic acid; flow rate: 0.3±(0~0.02)μL / min; injection volume: 1±(0~0.1); column temperature: 40±(0~4)℃:

[0052] Elution procedure (complex samples cells and plasma 120min, IgG 30min):

[0053] 30min gradient:

[0054] 0min: the volume percentage of mobile phase B is 1%-5%;

[0055] 0-20min: the volume percentage of mobile phase B increases from 1%-5% to 15%-25%;

[0056] 20-25min: the volume percentage of mobile phase B increases from 15%-25% to 25%-100%;

[0057] 25-28min: the volume percentage of mobile phase B is 95%-100%;

[0058] 28-28.1min: the volume percentage of mobile phase B is reduced from 95%-100% to 1%-5%;

[0059] 28.1-30min: the volume percentage of mobile phase B is 1%-5%;

[0060] 120min gradient:

[0061] 0min: the volume percentage of mobile phase B is 1%-5%;

[0062] 0-90min: the volume percentage of mobile phase B increases from 1%-5% to 15%-25%;

[0063] 90-95min: the volume percentage of mobile phase B increases from 15%-25% to 25%-100%;

[0064] 95-105min: the volume percentage of mobile phase B is 95%-100%;

[0065] 105-106min: the volume percentage of mobile phase B is reduced from 95%-100% to 1%-5%;

[0066] 106-120min: The volume percentage of mobile phase B is 1%-5%.

[0067] Eclipse was in DDA mode, with an acquisition time of 120 min, a mass scan range of 150 to 2500 Da, and a high collision energy step of 20 to 50 V.

[0068] S4, Data analysis: GlycanFinder software was used to search and analyze the collected data. N-sugar and O-sugar analysis were performed simultaneously during the search, and the N / O sugar library was the sugar library provided by GlycanFinder. Fixed modification: Carbamidomethylation (+57.02), variable modification Oxidation (M) (+15.99). The results are shown in Tables 1 and 2.

[0069] Table 1 List of N-glycopeptides identified before enrichment of IgG samples in Example 1 of the present invention

[0070]

[0071]

[0072] Table 2 List of N-glycopeptides identified after enrichment of IgG samples in one embodiment of the present invention

[0073]

[0074]

[0075]

[0076]

[0077]

[0078]

[0079]

[0080]

[0081] As shown in Tables 1 and 2, after DeepGP enrichment, the number of IgG glycopeptide identifications increased from 53 (Table 1) to 256 (Table 2), far exceeding the identification results of glycopeptide enriched materials in previous patents.

[0082] Example 2

[0083] The preparation method of the DeepGP glycopeptide enrichment column of the present embodiment is: 30 mg of microcrystalline cellulose, 33 mg of cation exchange filler and 36 mg of anion exchange filler are mixed evenly to obtain a mixed filler, and then the mixed filler is filled into a 1 mL syringe-type SPE empty column by an automatic column filling process, and a sieve plate is added after compaction, and the column is vacuum-packed and set aside.

[0084] Compared with Example 1, the method for efficiently enriching N-glycopeptides and O-glycopeptides in this example has different parameters for enzymatic hydrolysis and DeepGP glycopeptide enrichment column, specifically:

[0085] The enzymatic hydrolysis temperature was 35°C and the time was 10 h.

[0086] The enrichment method of DeepGP glycopeptide enrichment column is:

[0087] 1) Activation: Add 1 mL of activation solution 2% (v / v) ACN, 0.1% (v / v) TFA to activate the column, repeat 6 times;

[0088] 2) Equilibration: Add 1 mL of equilibration solution 50% (v / v) ACN, 0.1% (v / v) TFA to equilibrate the column, repeat twice;

[0089] 3) Capture: Drain the desalted sample, re-dissolve it in the equilibrium solution, inject it into the column, let it flow through naturally, and repeat the capture once;

[0090] 4) Cleaning: Add 1 mL of cleaning solution 50% (v / v) ACN, 0.1% (v / v) TFA to clean the column, repeat 6 times;

[0091] 5) Elution: Add 1 mL of eluent 2% (v / v) ACN, 0.1% (v / v) TFA to elute the sample. Repeat twice, collect and combine the eluents.

[0092] 6) The enriched glycopeptide eluate is freeze-dried, and after reconstitution, it is loaded onto a mass spectrometer.

[0093] Example 3

[0094] The preparation method of the DeepGP glycopeptide enrichment column of the present embodiment is: 30 mg of microcrystalline cellulose, 36 mg of cation exchange filler and 33 mg of anion exchange filler are mixed evenly to obtain a mixed filler, and then the mixed filler is filled into a 1 mL syringe-type SPE empty column by an automatic column filling process, and a sieve plate is added after compaction, and the column is vacuum-packed and set aside.

[0095] Compared with Example 1, the method for efficiently enriching N-glycopeptides and O-glycopeptides in this example has different parameters for enzymatic hydrolysis and DeepGP glycopeptide enrichment column, specifically:

[0096] The enzymatic hydrolysis temperature was 30°C and the time was 11 h.

[0097] The enrichment method of DeepGP glycopeptide enrichment column is:

[0098] 1) Activation: Add 1 mL of activation solution 10% (v / v) ACN, 2% (v / v) TFA to activate the column, repeat twice;

[0099] 2) Equilibration: Add 1 mL of equilibration solution 100% (v / v) ACN, 2% (v / v) TFA to equilibrate the column, repeat 6 times;

[0100] 3) Capture: Drain the desalted sample, re-dissolve it in the equilibrium solution, inject it into the column, let it flow through naturally, and repeat the capture once;

[0101] 4) Cleaning: Add 1 mL of cleaning solution 100% (v / v) ACN, 2% (v / v) TFA to clean the column, repeat twice;

[0102] 5) Elution: Add 1 mL of eluent 10% (v / v) ACN, 2% (v / v) TFA to elute the sample. Repeat 6 times, collect and combine the eluents.

[0103] 6) The enriched glycopeptide eluate is freeze-dried, and after reconstitution, it is loaded onto a mass spectrometer.

[0104] Comparative Example 1

[0105] Several glycopeptide enrichment columns currently used in literature and production were compared. The specific operations are as follows:

[0106] 1) HET cell protein extraction: Under low temperature, transfer the HET cell sample to a grinding tube, add 5 times the volume of lysis solution (8M urea, 0.1M Tris), incubate on ice for 1 hour, and ultrasonicate for 10 minutes at 60% power, on for 6 seconds, off for 9 seconds. Place the lysed sample in a low-temperature centrifuge, centrifuge at 4°C, 20,000 rpm for 15 minutes, and take the supernatant into a 1.5 mL centrifuge tube.

[0107] 2) Reductive alkylation reaction: Take 12 portions of 300 μg HET cell protein samples (each enrichment material was enriched 3 times in parallel, a total of 12 portions), add lysis buffer to make up the volume to 100 μL. Add 1 μL of 0.5M TCEP solution, vortex to mix, and react at 37°C, 1000 rpm for 0.5 h. Add 3.45 μL of 300 mM IAA solution, vortex to mix, and react at 25°C in the dark for 0.5 h.

[0108] 3) Enzymatic hydrolysis, desalting and glycopeptide enrichment: After the reduction and alkylation reaction, add 700 μL 0.1M Tris, dilute the urea concentration to 1M, add trypsin at a ratio of 50:1, and react at 37°C for 12 hours. The peptide solution after enzymatic hydrolysis was centrifuged at 25°C and 15000g for 15 minutes, and the supernatant was desalted using a C18 column. The desalted peptide solution was enriched with glycopeptides using a DeepGP glycopeptide enrichment column, and the enrichment process was the same as in Example 1.

[0109] 4) Data collection and analysis: LC-MS was then used for data collection, with a liquid phase gradient of 120 min. GlycanFinder software was then used to search and analyze the collected data. The identification results are shown in Figure 2 and Figure 3 .

[0110] from Figure 2 and Figure 3 It can be seen that DeepGP identified the most N / O glycopeptides, up to 2560 (2097 N glycopeptides and 463 O glycopeptides), and identified the least peptides, indicating that DeepGP has the highest glycopeptide enrichment efficiency. In addition, DeepGP 3-time enrichment column identified the best parallelism of glycopeptide numbers and the lowest standard deviation (SD). Figure 4 ), DeepGP has the highest Pearson correlation coefficient ( Figure 5 ), are 0.866, 0.954 and 0.877 respectively, indicating that the enrichment parallelism and consistency of the DeepGP enrichment column of the present invention are the best.

[0111] DeepGP was used to enrich N / O glycopeptides in mouse plasma to evaluate the application prospects of DeepGP enrichment columns in biomarker detection in clinical samples. The specific operations are as follows:

[0112] 1) Protein extraction and reductive alkylation reaction: Take 5 μL of plasma and add 20 μL of lysis solution (8 M urea, 0.1 M tris). Add 0.25 μL of 0.5 M TCEP solution, vortex mix, and react at 37°C, 1000 rpm for 0.5 h. Add 0.87 μL of 300 mM IAA solution, vortex mix, and react at 25°C in the dark for 0.5 h.

[0113] 2) Enzymatic hydrolysis, desalting and glycopeptide enrichment: After the reduction alkylation reaction, trypsin (50:1) was added to the reaction solution and reacted at 37°C for 12 hours. The peptide solution after enzymatic hydrolysis was centrifuged at 25°C and 15000g for 15 minutes and desalted using a C18 column; the peptide solution after desalting was enriched with glycopeptides using a DeepGP glycopeptide enrichment column, and the enrichment method was the same as in Example 1.

[0114] 3) Data collection and analysis: LC-MS was then used for data collection, with a liquid phase gradient of 120 min. GlycanFinder software was then used to search and analyze the collected data. The identification results are shown in Figure 6 .

[0115] Figure 6 As shown in the figure, after DeepGP enrichment, a total of 4286 N-glycopeptides and 749 O-glycopeptides were identified in mouse plasma, which far exceeded the plasma glycopeptide identification results in previous literature.

[0116] References:

[0117] [1]Liu L, Zhu B, Fang Z, Zhang N, Qin H, Guo Z, Liang X, Yao Z, YeM. Automated Intact Glycopeptide Enrichment Method Facilitating HighlyReproducible Analysis of Serum Site-Specific N-Glycoproteome.AnalChem.2021May25;93(20):7473-7480.doi:10.1021 / acs.analchem.1c00645.Epub 2021May11.PMID:33973768.

[0118] [2]Yang W,Shah P,Hu Y,Toghi Eshghi S,Sun S,Liu Y,Zhang H.Comparison of Enrichment Methods for Intact N-and O-Linked Glycopeptides Using StrongAnion Exchange and Hydrophilic Interaction Liquid Chromatography.AnalChem.2017Nov 7;89(21):11193-11197.doi:10.1021 / acs.analchem.7b03641.Epub2017Oct 12.PMID:29016103;PMCID:PMC5850954.

[0119] [3]Morgenstern D,Wolf-Levy H,Tickotsky-Moskovitz N,Cooper I,BuchmanAS,Bennett DA,Beeri MS,Levin Y.Optimized Glycopeptide Enrichment Method-It IsAll About the Sauce.Anal Chem.2022Jul 26;94(29):10308-10313.doi:10.1021 / acs.analchem.2c00524.Epub 2022Jun 28.PMID:35764435;PMCID:PMC9330304.

[0120] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A method for preparing a DeepGP glycopeptide enrichment column, characterized in that: The following steps are involved: The microcrystalline cellulose, cation exchange filler and anion exchange filler are mixed evenly, and then the mixed filler is loaded into a column and compacted to obtain a DeepGP glycopeptide enrichment column.

2. A method for preparing a DeepGP glycopeptide enrichment column as claimed in claim 1, characterized in that: The mass ratio of the microcrystalline cellulose: cation exchange filler: anion exchange filler is 1:1-1.2:1-1.

2.

3. A method for efficiently enriching N-glycopeptides and O-glycopeptides, characterized in that: The following steps are involved: S1, after protein extraction, tri(2-carboxyethyl)phosphine hydrochloride and iodoacetamide are added for reductive alkylation reaction, followed by enzymatic hydrolysis and desalting after the reaction is completed; S2, enriching the desalted peptide fragments using the DeepGP glycopeptide enrichment column prepared according to claim 1 or 2; S3, the enriched samples are collected by liquid chromatography-mass spectrometry, and then the collected data are analyzed to identify N-glycans and O-glycopeptides.

4. A method for efficiently enriching N-glycopeptides and O-glycopeptides according to claim 3, characterized in that: The reduction alkylation reaction method in step S1 is: take the extracted protein sample, add tri(2-carboxyethyl)phosphine hydrochloride solution, mix well and then shake for reaction, and after the reaction is completed, add iodoacetamide solution, mix well and then react in the dark.

5. A method for efficiently enriching N-glycopeptides and O-glycopeptides as claimed in claim 3, characterized in that: The enzymatic hydrolysis method in step S1 is: after the reduction alkylation reaction is completed, trypsin is added to the protein sample for enzymatic hydrolysis.

6. A method for efficiently enriching N-glycopeptides and O-glycopeptides according to claim 5, characterized in that: The mass ratio of trypsin: protein sample is 50:1, the enzymatic hydrolysis temperature is 30-37°C, and the time is 10-12h.

7. A method for efficiently enriching N-glycopeptides and O-glycopeptides according to claim 3, characterized in that: In step S1, C18 column is used for desalting.

8. The method for efficiently enriching N-glycopeptides and O-glycopeptides according to claim 3, characterized in that: The enrichment method of the DeepGP glycopeptide enrichment column in step S2 is as follows: activate the column with 2%-10% v / v acetonitrile and 0.1%-2% v / v trifluoroacetic acid in sequence, balance the column with 50%-100% v / v acetonitrile and 0.1%-2% v / v trifluoroacetic acid, drain the peptide sample desalted in step S1 and redissolve it in 50%-100% v / v acetonitrile and 0.1%-2% v / v trifluoroacetic acid, load it onto the DeepGP glycopeptide enrichment column, then wash the column with 50%-100% v / v acetonitrile and 0.1%-2% v / v trifluoroacetic acid, elute the sample with 2%-10% v / v acetonitrile and 0.1%-2% v / v trifluoroacetic acid, and combine the eluates; finally, freeze-dry the enriched glycopeptide eluate, and load it onto a mass spectrometer after redissolution.

9. The method for efficiently enriching N-glycopeptides and O-glycopeptides according to claim 3, characterized in that: In step S3, LC-MS, easyLC 1200, and Orbitrap Eclipse three-in-one mass spectrometer were used for data acquisition.

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