Glycosylation analysis and identification method for structural protein of monkey pox virus

Abstract

The invention provides a monkey pox virus structural protein glycosylation analysis and identification method, and relates to the field of monkey pox virus structural protein glycosylation analysis, and the method comprises the following steps: selecting different monkey pox virus structural proteins, carrying out a protein denaturation reaction on the monkey pox virus structural proteins in an ammonium bicarbonate solution containing a denaturing agent, and separating the denaturated monkey pox virus structural proteins from the denatured monkey pox virus structural proteins; then carrying out a protein reductive alkylation process; adding protease for enzyme digestion to obtain a monkey pox virus structure protease digestion sample, wherein the monkey pox virus structure protease digestion sample is a polypeptide mixture; carrying out high-selectivity enrichment of sugar chains on the polypeptide mixture to realize glycopeptide enrichment, carrying out liquid chromatography-tandem mass spectrometry separation analysis on the enriched glycopeptide to obtain mass spectrometry data, and analyzing the mass spectrometry data through glycosylation retrieval software to identify the glycopeptide. According to the application, the glycosylation of the structural protein of the monkey pox virus can be rapidly, accurately, simply and effectively detected.

Description

Technical Field

[0001] The present invention relates to the technical field of glycosylation analysis of monkeypox virus structural proteins, and in particular to a method for analyzing and identifying glycosylation of monkeypox virus structural proteins. Background Art

[0002] With the continuous development of mass spectrometry technology, it is possible to obtain comprehensive information on sugar chain composition, glycosylation site information, site specificity, etc. by fragmenting and analyzing polypeptides (glycopeptides) with sugar chain structures, which makes it possible to study the glycosylation of monkeypox virus structural proteins.

[0003] Glycosylation of viral structural proteins has a significant impact on viral infection, immune escape, replication and pathogenicity. Studying the glycosylation of viral structural proteins can better understand viral biology, reveal viral immune escape mechanisms, promote vaccine development, and guide the development of antiviral drugs. Glycopeptides have relatively low content in the entire protein sample, poor ionization efficiency compared to non-glycopeptides in mass spectrometry, and are easily interfered by the sample matrix. Therefore, glycopeptides in monkeypox virus structural proteins need to be enriched before mass spectrometry analysis.

[0004] At present, conventional glycosylation research removes sugar chains from glycoproteins by chemical or enzymatic methods and then analyzes sugar chain structure, glycosylation sites, etc. Although this method can provide information on sugar spectrum and glycosylation sites, it also loses key information such as glycosylation site specificity, which affects further research on glycosylation heterogeneity and is not conducive to further research on the relationship between biological function and glycosylation structure information. Therefore, it is urgent to develop a technical method that is both fast, accurate, simple and effective in detecting glycosylation of monkeypox virus structural proteins, and can also be used for accurate analysis of glycosylation of monkeypox virus structural proteins. Summary of the invention

[0005] Based on this, the purpose of the present invention is to provide a method for analyzing and identifying the glycosylation of monkeypox virus structural proteins, which is used to quickly, accurately, simply and effectively detect the glycosylation of monkeypox virus structural proteins.

[0006] The present invention provides a method for analyzing and identifying glycosylation of a monkeypox virus structural protein, comprising:

[0007] Select different monkeypox virus structural proteins, including A29, A35, B6R and H3L, subject the monkeypox virus structural proteins to a protein denaturation reaction in an ammonium bicarbonate solution containing a denaturant, and then perform a protein reduction alkylation process;

[0008] Adding protease to perform enzymatic cleavage process to obtain a monkeypox virus structure protease cleavage sample, wherein the monkeypox virus structure protease cleavage sample is a polypeptide mixture, wherein the polypeptide mixture includes glycopeptides and non-glycopeptides, wherein the glycopeptides are polypeptides with sugar chains, and the non-glycopeptides are polypeptides without sugar chains;

[0009] The polypeptide mixture is subjected to highly selective enrichment of sugar chains to achieve glycopeptide enrichment, the enriched glycopeptides are subjected to liquid phase-tandem mass spectrometry separation and analysis to obtain mass spectrometry data, and the mass spectrometry data are analyzed by glycosylation retrieval software to identify the glycopeptide.

[0010] The above-mentioned method for analyzing and identifying the glycosylation of the structural protein of the monkeypox virus is to enzymatically cleave different monkeypox virus structural proteins to form a polypeptide mixture, and then highly selectively enrich the various polypeptide mixtures using glycopeptide enrichment materials to obtain the glycopeptide part thereof; use tandem liquid chromatography-mass spectrometry technology to separate and identify the glycopeptide part of the structural protein of the monkeypox virus, and after obtaining the mass spectrometry data, use database retrieval software to analyze the glycosylation information, thereby being able to achieve rapid, accurate, simple and effective detection of the glycosylation of the structural protein of the monkeypox virus.

[0011] In addition, the above-mentioned monkeypox virus structural protein glycosylation analysis and identification method according to the present invention may also have the following additional technical features:

[0012] Further, the denaturant includes urea at a concentration of 8M.

[0013] Further, in the step of protein reductive alkylation process:

[0014] Protein reductive alkylation reagents included dithiothreitol or tris(2-carboxyethyl)phosphine hydrochloride in 50 mM ammonium bicarbonate at a concentration of 5 mM;

[0015] The protein reduction alkylation process includes using 50 mM ammonium bicarbonate solution containing 5 mM dithiothreitol or tris(2-carboxyethyl)phosphine hydrochloride at a concentration of 5 mM for 45 min at room temperature;

[0016] Use 50 mM ammonium bicarbonate solution containing 10 mM iodoacetamide or chloroacetamide and react for 30 min at room temperature in the dark.

[0017] Further, the protease is one or more combinations of trypsin, chymotrypsin, lysyl endonuclease, thermolysin, proteinase K, and glutamyl endonuclease;

[0018] The mass ratio of the enzyme to the protein used was 1:50; the enzyme cleavage time was adjusted to 12 h; and the appropriate enzyme cleavage temperature was selected according to the temperature range in which different enzymes maintained their activity.

[0019] Furthermore, the enrichment method includes a dispersive solid phase extraction mode.

[0020] Furthermore, the glycopeptide enrichment material includes one or a combination of laboratory-made glycopeptide enrichment materials, and the material dosage is 1 mg; the mass ratio of the glycopeptide enrichment material to the polypeptide mixture of the monkeypox virus structural protein is 100:1-1000:1;

[0021] Among them, the laboratory's homemade glycopeptide enrichment materials include Click-Mal, TE-Cys and HBS.

[0022] Further, in the step of performing liquid phase-tandem mass spectrometry separation and analysis on the enriched glycopeptides to obtain mass spectrometry data:

[0023] The liquid phase-tandem mass spectrometry separation and analysis process includes using any one of the mass spectrometers of Thermo Fisher's Orbitrap series mass spectrometers, SCIEX's ZenoTOF mass spectrometers, and Bruker's TIMS-TOF mass spectrometers and a matching liquid chromatography system instrument;

[0024] Wherein, the chromatographic separation mode is a reverse phase chromatographic separation mode;

[0025] The mass spectrometry data acquisition mode is any one of a data-dependent acquisition mode and a data-independent acquisition mode.

[0026] Further, in the step of performing liquid phase-tandem mass spectrometry separation and analysis on the enriched glycopeptides to obtain mass spectrometry data:

[0027] The liquid chromatography analysis column and pre-column adopt conventional C18 capillary column, the outer diameter of the capillary is 360 μm, the matching inner diameter is one of 10 μm, 50 μm, 100 μm, the capillary filling length is 10 cm-50 cm, and the C18 filler particles used are 9 μm.

[0028] Further, in the step of performing liquid phase-tandem mass spectrometry separation and analysis on the enriched glycopeptides to obtain mass spectrometry data:

[0029] The liquid mobile phase used is a mixed solvent of organic solvent, deionized water and organic acid;

[0030] The organic acid is one of formic acid, acetic acid or trifluoroacetic acid, and the volume proportion of the organic acid is 0.1%-30%; the volume proportion of the organic solvent is 1%-80%.

[0031] Further,

[0032] Dispersive SPE modes include:

[0033] First, the glycopeptide enrichment material is activated with an activation solution, the glycopeptide enrichment material is mixed with a polypeptide mixture of monkeypox virus structural protein at a mass ratio of 100:1-1000:1, incubated for 10 minutes, filtered or separated by dispersed solid phase extraction, the supernatant is discarded, and the precipitate is collected;

[0034] The precipitate was washed with an organic solution at pH 3;

[0035] The washed precipitate is eluted with a pH 10 eluent, filtered or separated by dispersed solid phase extraction, and the supernatant is collected to obtain the glycopeptide. BRIEF DESCRIPTION OF THE DRAWINGS

[0036] Figure 1 Schematic diagram of the enrichment process of A29, A35, B6R and H3L monkeypox virus structural protein glycopeptides;

[0037] Figure 2 Schematic diagram of the number of glycopeptides and glycosylation sites of the structural proteins of A29, A35, B6R and H3L monkeypox virus identified in Example 1;

[0038] Figure 3 A schematic diagram showing the locations of glycosylation sites in the structural protein sequences of A29, A35, B6R and H3L monkeypox viruses;

[0039] Figure 4 The sugar chain structures of A29, A35, B6R and H3L monkeypox virus structural proteins identified in Example 1;

[0040] The following specific implementation manner will further illustrate the present invention in conjunction with the above-mentioned drawings. DETAILED DESCRIPTION

[0041] In order to facilitate the understanding of the present invention, the present invention will be described more fully below with reference to the relevant drawings. Several embodiments of the present invention are given in the drawings. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thorough and comprehensive.

[0042] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as those commonly understood by those skilled in the art of the present invention. The terms used herein in the specification of the present invention are only for the purpose of describing specific embodiments and are not intended to limit the present invention. The term "and / or" used herein includes any and all combinations of one or more related listed items.

[0043] In order to quickly, accurately, simply and effectively detect the glycosylation of monkeypox virus structural proteins, the present application provides a method for analyzing and identifying the glycosylation of monkeypox virus structural proteins, which can not only provide unreported glycosylation information of monkeypox virus structural proteins, but also has the application potential of high efficiency, accuracy, stability and repeatability. This method is different from other methods for studying protein glycosylation, such as chemical methods or enzymatic methods, and can retain information such as site specificity while retaining sugar chain information and glycosylation site information. The relevant results of this method can be used in the fields of clinical serological diagnosis of monkeypox virus, research and development of anti-monkeypox virus drugs, and development of related subunit vaccines. Specifically, after different monkeypox virus structural proteins are enzymatically cleaved to form a polypeptide mixture, various polypeptide mixtures are highly selectively enriched by glycopeptide enrichment materials to obtain the glycopeptide part thereof; the glycopeptide part of the monkeypox virus structural protein is separated and identified using tandem liquid phase-mass spectrometry technology, and after obtaining mass spectrometry data, the glycosylation information is parsed using database retrieval software, so as to achieve rapid, accurate, simple and effective detection of monkeypox virus structural protein glycosylation.

[0044] In order to explore the important role of glycosylation of monkeypox virus protein, it is first necessary to conduct a comprehensive qualitative and quantitative analysis of low stoichiometric glycosylation modifications. Protein glycosylation research usually requires enzymatic cleavage of the protein to form a polypeptide form. The sample usually contains polypeptides with sugar chains (glycopeptides) and polypeptides without sugar chains (non-glycopeptides); because the content of glycopeptides is usually extremely low, an enrichment method is required to highly selectively enrich glycopeptides. Commonly used glycopeptide enrichment methods include hydrophilic interaction chromatography, lectin affinity chromatography, boric acid affinity, etc., and high selective enrichment of glycopeptides can be achieved by taking one of these methods. Subsequently, mass spectrometry can be used to analyze the enriched glycopeptides, and the obtained mass spectrometry data can be used to identify glycopeptides through glycosylation retrieval software.

[0045] Specifically, the present invention provides a method for analyzing and identifying glycosylation of a monkeypox virus structural protein, comprising steps S101 to S103:

[0046] S101. Select different monkeypox virus structural proteins, subject the monkeypox virus structural proteins to protein denaturation reaction in an ammonium bicarbonate solution containing a denaturant, and then perform a protein reduction alkylation process.

[0047] The structural proteins of the monkeypox virus include A29, A35, B6R and H3L. The denaturant includes urea at a concentration of 8 M. The protein reduction alkylation agent includes a 5 mM dithiothreitol (DTT) or tris (2-carboxyethyl) phosphine hydrochloride (TCEP) in a 50 mM ammonium bicarbonate solution. Specifically, the structural proteins of the monkeypox virus are subjected to a protein denaturation reaction under the condition of a 50 mM ammonium bicarbonate solution to achieve enzymatic hydrolysis of the structural proteins of the monkeypox virus.

[0048] In some optional embodiments, the protein reduction alkylation process includes using a 50mM ammonium bicarbonate solution containing 5mM dithiothreitol (DTT) or tris(2-carboxyethyl)phosphine hydrochloride (TCEP) at a concentration of 5mM, and reacting at room temperature for 45 minutes; using a 5mM-100mM ammonium bicarbonate solution containing 10mM iodoacetamide (IAA) or chloroacetamide (CAA) at a concentration of 10mM, and reacting at room temperature in the dark for 30 minutes.

[0049] S102, adding protease to perform enzymatic cleavage to obtain a monkeypox virus structural protease cleavage sample, wherein the monkeypox virus structural protease cleavage sample is a polypeptide mixture.

[0050] The polypeptide mixture includes glycopeptides and non-glycopeptides, glycopeptides are polypeptides with sugar chains, and non-glycopeptides are polypeptides without sugar chains. The protease is one or more combinations of trypsin, chymotrypsin, lysyl endonuclease, thermolysin, proteinase K, and glutamyl endonuclease; the mass ratio of the enzyme to the protein is 1:50; the enzyme cleavage time is adjusted to 12 hours; and the appropriate enzyme cleavage temperature is selected according to the temperature range in which different enzymes maintain activity.

[0051] After the enzymatic cleavage process is completed, a formic acid or trifluoroacetic acid solution with a volume fraction of 1%-10% is used to terminate the enzymatic cleavage process to form a monkeypox virus structure protease cleavage sample, that is, a polypeptide mixture.

[0052] S103. Perform highly selective enrichment of sugar chains on the polypeptide mixture to achieve glycopeptide enrichment, perform liquid phase-tandem mass spectrometry separation and analysis on the enriched glycopeptides to obtain mass spectrometry data, and analyze the mass spectrometry data using glycosylation retrieval software to identify the glycopeptides.

[0053] The enriched glycopeptide material includes one or a combination of laboratory-made glycopeptide enriched materials, and the material dosage is 1 mg; the mass ratio of the glycopeptide enriched material to the polypeptide mixture of the monkeypox virus structural protein is 100:1-1000:1; among them, the laboratory-made glycopeptide enriched materials include Click-Mal, TE-Cys and HBS.

[0054] The enrichment method includes a column solid phase extraction mode or a dispersed solid phase extraction mode. Among them, the dispersed solid phase extraction mode includes: first using an activation solution to activate the glycopeptide enrichment material, mixing the glycopeptide enrichment material with a polypeptide mixture of monkeypox virus structural protein at a mass ratio of 100:1-1000:1, incubating for 10 minutes, filtering or dispersive solid phase extraction separation, discarding the supernatant, collecting the precipitate; using a pH 3 organic solution to wash the precipitate; eluting the washed precipitate with a pH 10 eluent, filtering or dispersive solid phase extraction separation, collecting the supernatant, and obtaining the glycopeptide.

[0055] Specifically, the activation solution, the balancing solution, the organic solution, and the eluent are all any mixture of an organic solvent, an organic acid, an alkaline solvent, a buffer salt, and water; wherein the organic solvent is acetonitrile, methanol, or ethanol, the organic acid is glycolic acid, formic acid, acetic acid, or trifluoroacetic acid, the alkaline solvent is an ammonia solvent, and the buffer salt is ammonium formate, ammonium acetate, or ammonium bicarbonate; the volume concentration of the organic solvent is 50%-95%, the volume concentration of the organic acid is 0.1-5%, the volume concentration of the alkaline solvent is 0.1-30%, and the concentration of the buffer salt is 0.001M-2M.

[0056] In some optional embodiments, the liquid phase-tandem mass spectrometry separation and analysis process includes using any one of the Orbitrap series mass spectrometers of Thermo Fisher, the ZenoTOF mass spectrometer of SCIEX, and the TIMS-TOF mass spectrometer of Bruker, and a matching liquid chromatography system instrument; wherein the chromatographic separation mode is a reversed phase chromatography separation mode; the mass spectrometry data acquisition mode is any one of a data-dependent acquisition mode (DDA) and a data-independent acquisition mode (DIA); secondly, the liquid chromatography analysis column and the pre-column use a conventional C18 capillary column, the capillary outer diameter is 360 μm, the matching inner diameter is one of 10 μm, 50 μm, and 100 μm, the capillary filling length is 10 cm-50 cm, and the C18 filler particles used are 9 μm. Furthermore, the liquid mobile phase used is a mixed solvent of organic solvent, deionized water and organic acid; wherein the organic acid is one of formic acid, acetic acid or trifluoroacetic acid, and the volume proportion of the organic acid is 0.1%-30%; the volume proportion of the organic solvent is 1%-80%.

[0057] Furthermore, the retrieval software analysis process includes the selection of retrieval software, setting of search parameters, data screening, etc. The glycopeptide mass spectrometry data retrieval software uses one or a combination of commercialized Byonic software from Protein Metrics, PD software from ThermoFisher, or open source software such as pGlyco software. Among them: the search parameter setting is to load the amino acid sequence of the corresponding monkeypox virus structural protein into the search software, set cysteine ​​iodoacetamidate as a fixed modification, set oxidation (M), acetyl (protein N-terminus) and deamidation (N) as variable modifications, and add the corresponding N-linked sugar chain library and O-linked sugar chain library information, set the number of missed cleavage sites to 1-2, the number of variable modifications to 1-3, and FDR<1%; the monkeypox virus structural protein glycosylation data screening process includes score value screening according to the search results of each software, deleting the corresponding results of the anti-library and contaminating proteins, to screen the credible monkeypox virus structural protein glycopeptides, and the corresponding monkeypox virus structural protein glycosylation information can be obtained by analyzing the sugar chain information, glycosylation site information, and glycopeptide information.

[0058] In order to facilitate understanding of the present invention, several embodiments of the present invention are provided below. However, the present invention can be implemented in many different forms and is not limited to the embodiments described herein. On the contrary, the purpose of providing these embodiments is to make the disclosure of the present invention more thorough and comprehensive.

[0059] Embodiment 1

[0060] Please refer to Figure 1-Figure 4 The method for analyzing and identifying glycosylation of the structural protein of monkeypox virus in the first embodiment of the present invention comprises:

[0061] Select 20 mg of each of A29, A35, B6R, and H3L monkeypox virus structural proteins, and perform protein denaturation reaction under the condition of 50 mM ammonium bicarbonate solution (containing 8 M urea); the protein reduction alkylation process uses dithiothreitol (DTT) and 50 mM ammonium bicarbonate solution containing a concentration of 5 mM, and reacts at room temperature for 45 min; uses iodoacetamide (IAA) and 50 mM ammonium bicarbonate solution containing a concentration of 10 mM, and reacts in the dark for 30 min at room temperature; the enzymatic cleavage process of protease uses trypsin, the mass ratio of the enzyme used to the protein is 1:50, the reaction temperature is 37 ° C, and the enzymatic cleavage time is 12 h. After the enzymatic cleavage process ends, the enzymatic cleavage process is terminated using 5% formic acid by volume to form a monkeypox virus structural protease cleavage sample, i.e., a polypeptide mixture. After the mixture is spin-dried, it is redissolved using a mixed organic solution of pH=3, 0.1% (volume fraction) formic acid solution / 80% (volume fraction) acetonitrile.

[0062] The dispersed solid phase extraction mode is adopted for glycopeptide enrichment, and the material used is the laboratory-made glycopeptide enrichment material Click-Mal, and the material dosage is 1 mg; firstly, a 0.1% (volume fraction) formic acid solution activation solution is used to activate the glycopeptide enrichment material Click-Mal, and the glycopeptide enrichment material is mixed with a polypeptide mixture of monkeypox virus structural protein, incubated for 10 minutes, centrifuged at a speed of 10000 rpm, the supernatant is discarded, and the precipitate is collected; the precipitate is washed twice with a mixed organic solution of pH=3, 0.1% (volume fraction) formic acid solution / 80% (volume fraction) acetonitrile, 50 mL; the washed precipitate is eluted with a pH=10 ammonia solution as an eluent to elute the precipitate part, centrifuged and the supernatant is collected, and the glycopeptide sample is obtained after spin drying.

[0063] Easy 1200 liquid phase and Orbitrap 480 mass spectrometer are used, equipped with a capillary column with a length of 20 cm and an outer diameter of 360 μm and filled with C18 reverse phase chromatography filler with a particle size of 1.9 μm as an analytical column, and equipped with a capillary column with a length of 2 cm and an outer diameter of 360 μm and filled with C18 reverse phase chromatography filler with a particle size of 1.9 μm as a pre-column. Liquid mobile phase A phase is 0.1% (volume fraction) formic acid, and phase B is 0.1% (volume fraction) formic acid / 80% acetonitrile for reverse phase chromatography separation; the mass spectrometry data acquisition mode can be a data dependent acquisition mode (DDA).

[0064] The glycopeptide mass spectrometry data retrieval software uses the commercialized Byonic software of Protein Metrics; the search parameters are set by loading the corresponding amino acid sequences of A29, A35, B6R, and H3L monkeypox virus structural proteins into the search software, setting cysteine ​​iodoacetamidate as a fixed modification, oxidation (M), acetyl (protein N-terminus), and deamidation (N) as variable modifications, adding the corresponding N-linked sugar chain library and O-linked sugar chain library information, setting the number of missed cleavage sites to 2, the number of variable modifications to 1, and FDR < 1%. The score value greater than 30 is used as the screening standard, and the corresponding results of the anti-library and contaminating proteins are deleted to screen the credible glycopeptides in the monkeypox virus structural proteins. The corresponding monkeypox virus structural protein glycosylation information can be obtained by analyzing the sugar chain information, glycosylation site information, and glycopeptide information.

[0065] In summary, the method for analyzing and identifying the glycosylation of the structural protein of the monkeypox virus in the above-mentioned embodiment of the present invention is to enzymatically cleave different monkeypox virus structural proteins to form a polypeptide mixture, and then highly selectively enrich the various polypeptide mixtures using a glycopeptide enrichment material to obtain the glycopeptide portion thereof; use tandem liquid phase-mass spectrometry technology to separate and identify the glycopeptide portion of the structural protein of the monkeypox virus, and after obtaining the mass spectrometry data, use database retrieval software to analyze the glycosylation information, thereby being able to achieve rapid, accurate, simple and effective detection of the glycosylation of the structural protein of the monkeypox virus.

[0066] In the description of this specification, the description with reference to the terms "one embodiment", "some embodiments", "examples", "specific examples", or "some examples" means that the specific features, structures, materials or characteristics described in conjunction with the embodiment or example are included in at least one embodiment or example of the present invention. In this specification, the schematic representation of the above terms does not necessarily refer to the same embodiment or example. Moreover, the specific features, structures, materials or characteristics described may be combined in any one or more embodiments or examples in a suitable manner.

[0067] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the patent of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention. Therefore, the protection scope of the patent of the present invention shall be subject to the attached claims.

Claims

1. A method for analyzing and identifying glycosylation of monkeypox virus structural proteins, characterized in that: include: Select different monkeypox virus structural proteins, including A29, A35, B6R and H3L, subject the monkeypox virus structural proteins to a protein denaturation reaction in an ammonium bicarbonate solution containing a denaturant, and then perform a protein reduction alkylation process; Adding protease to perform enzymatic cleavage process to obtain a monkeypox virus structure protease cleavage sample, wherein the monkeypox virus structure protease cleavage sample is a polypeptide mixture, wherein the polypeptide mixture includes glycopeptides and non-glycopeptides, wherein the glycopeptides are polypeptides with sugar chains, and the non-glycopeptides are polypeptides without sugar chains; The polypeptide mixture is subjected to highly selective enrichment of sugar chains to achieve glycopeptide enrichment, the enriched glycopeptides are subjected to liquid phase-tandem mass spectrometry separation and analysis to obtain mass spectrometry data, and the mass spectrometry data are analyzed by glycosylation retrieval software to identify the glycopeptide.

2. The method for analyzing and identifying glycosylation of monkeypox virus structural proteins according to claim 1, characterized in that: Denaturing agents included urea at a concentration of 8M.

3. The method for analyzing and identifying glycosylation of monkeypox virus structural proteins according to claim 1, characterized in that: In the steps of protein reductive alkylation process: Protein reductive alkylation reagents included dithiothreitol or tris(2-carboxyethyl)phosphine hydrochloride in 50 mM ammonium bicarbonate at a concentration of 5 mM; The protein reduction alkylation process includes using a 50 mM ammonium bicarbonate solution containing 5 mM dithiothreitol or tris(2-carboxyethyl)phosphine hydrochloride at room temperature for 45 min; Use 50 mM ammonium bicarbonate solution containing 10 mM iodoacetamide or chloroacetamide and react for 30 min at room temperature in the dark.

4. The method for analyzing and identifying glycosylation of monkeypox virus structural proteins according to claim 1, characterized in that: The protease is one or more combinations of trypsin, chymotrypsin, lysyl endo-nuclease, thermolysin, proteinase K, and glutamyl protein endo-nuclease; The mass ratio of the enzyme to the protein used was 1:50; the enzyme cleavage time was adjusted to 12 h; and the appropriate enzyme cleavage temperature was selected according to the temperature range in which different enzymes maintained their activity.

5. The method for analyzing and identifying glycosylation of monkeypox virus structural proteins according to claim 1, characterized in that: Enrichment methods included dispersive solid phase extraction mode.

6. The method for analyzing and identifying glycosylation of monkeypox virus structural proteins according to claim 1, characterized in that: The glycopeptide enrichment material is one or a combination of laboratory-made glycopeptide enrichment materials, and the material dosage is 1 mg; the mass ratio of the glycopeptide enrichment material to the polypeptide mixture of the monkeypox virus structural protein is 100:1-1000:1; Among them, the laboratory's homemade glycopeptide enrichment materials include Click-Mal, TE-Cys and HBS.

7. The method for analyzing and identifying glycosylation of monkeypox virus structural proteins according to claim 1, characterized in that: In the step of performing liquid phase-tandem mass spectrometry separation and analysis on the enriched glycopeptides to obtain mass spectrometry data: The liquid phase-tandem mass spectrometry separation and analysis process includes using any one of the mass spectrometers of Thermo Fisher's Orbitrap series mass spectrometers, SCIEX's ZenoTOF mass spectrometers, and Bruker's TIMS-TOF mass spectrometers and a matching liquid chromatography system instrument; Wherein, the chromatographic separation mode is a reverse phase chromatographic separation mode; The mass spectrometry data acquisition mode is any one of a data-dependent acquisition mode and a data-independent acquisition mode.

8. The method for analyzing and identifying glycosylation of monkeypox virus structural proteins according to claim 1, characterized in that: In the step of performing liquid phase-tandem mass spectrometry separation and analysis on the enriched glycopeptides to obtain mass spectrometry data: The liquid chromatography analysis column and pre-column adopt conventional C18 capillary column, the outer diameter of the capillary is 360 μm, the matching inner diameter is one of 10 μm, 50 μm and 100 μm, the capillary filling length is 10 cm-50 cm, and the C18 filler particles used are 1.9 μm.

9. The method for analyzing and identifying glycosylation of monkeypox virus structural proteins according to claim 1, characterized in that: In the step of performing liquid phase-tandem mass spectrometry separation and analysis on the enriched glycopeptides to obtain mass spectrometry data: The liquid mobile phase used is a mixed solvent of organic solvent, deionized water and organic acid; The organic acid is one of formic acid, acetic acid or trifluoroacetic acid, and the volume proportion of the organic acid is 0.1%-30%; the volume proportion of the organic solvent is 1%-80%.

10. The method for analyzing and identifying glycosylation of monkeypox virus structural proteins according to claim 5, characterized in that: Dispersive SPE modes include: First, the glycopeptide enrichment material is activated with an activation solution, the glycopeptide enrichment material is mixed with a polypeptide mixture of monkeypox virus structural protein at a mass ratio of 100:1-1000:1, incubated for 10 minutes, filtered or separated by dispersed solid phase extraction, the supernatant is discarded, and the precipitate is collected; The precipitate was washed with an organic solution at pH 3; The washed precipitate is eluted with a pH 10 eluent, filtered or separated by dispersed solid phase extraction, and the supernatant is collected to obtain the glycopeptide.

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