A method for identifying and verifying N-linked sugar types in glycoproteins

Abstract

The present invention discloses a method for identifying and verifying the type of N-linked sugars on glycoproteins, comprising the following steps: preparing a complete N-glycopeptide sample; electrospray ionizing the complete N-glycopeptide sample to obtain precursor ions, gas-phase dissociating the precursor ions to obtain fragment ions, and performing mass spectrometry analysis on the precursor ions and fragment ions; identifying the monosaccharide composition, potential sequence structure, and type of N-linked sugars based on molecular composition fingerprint comparison; screening and confirming the types of N-linked sugar type characteristic fragment ions; false positive control for complete N-glycopeptide identification based on targeted-bait library search; and identification and confirmation of N-linked sugar types. This method identifies and confirms the type of N-linked sugars on glycoproteins based on molecular composition and structural fingerprints, effectively controlling the distinction and confirmation of N-linked sugars of the same molecular composition but different types. It adds a confirmation step based on structural fingerprints to the existing spectrum-level false positive control, thereby improving the accuracy of N-linked sugar type identification.

Description

Technical Field

[0001] The present invention relates to a method for identifying and verifying the type of N-linked sugars in glycoproteins. Background Art

[0002] Glycosylation is one of the most common post-translational modifications of proteins, including N-linked and O-linked glycosylation. N-linked glycans are widely present in various organisms and have important biological significance. They primarily modify the conserved amino acid sequence, N-X-X-S-T (where X is any amino acid except proline). N-linked glycans share a similar core structure and are classified into three distinct types based on the branching structures beyond the core: high mannose, complex, and hybrid. High mannose refers to glycans containing only mannose outside the core; complex glycans refer to glycans whose branches extend beyond the core starting with N-acetylglucosamine (HexNAc); and hybrid glycans refer to glycans whose branches extend beyond the core starting with both N-acetylglucosamine and mannose. High mannose-modified glycoproteins are primarily located in the endoplasmic reticulum and Golgi apparatus, assisting in the proper folding of other proteins during synthesis. Hybrid and complex glycan structures are relatively complex, and any change in the monosaccharides within them may affect the biological function of the corresponding protein. Distinguishing N-linked glycan types is a crucial step in determining glycan structure. Furthermore, distinguishing N-linked glycan types facilitates further research into their biological functions.

[0003] Mass spectrometry is an important means for large-scale identification of protein glycosylation modifications. In existing technologies, the identification of N-linked sugar types on N-linked glycoproteins is based solely on targeted-bait database searches, random match scoring, and false-positive control at the N-linked glycoproteome level. Summary of the Invention

[0004] To address the above technical issues, the present invention aims to provide a method for identifying and verifying the N-linked glycan types of glycoproteins. This method reduces the time required to search for non-matching N-linked glycan types and significantly improves the efficiency and accuracy of N-linked glycan identification.

[0005] In order to achieve the above technical objectives and the above technical effects, the present invention is implemented through the following technical solutions:

[0006] A method for identifying and verifying the type of N-linked sugars in a glycoprotein comprises the following steps:

[0007] S1. Prepare intact N-glycopeptide samples from the biological samples to be analyzed;

[0008] S2. The intact N-glycopeptide sample is electrospray ionized to obtain precursor ions, which are then fed into a mass spectrometer to obtain an experimental primary mass spectrum containing the isotope profile fingerprint of the precursor ions.

[0009] S3, matching the isotope profile fingerprint of the precursor ion with the corresponding targeted forward theoretical database to screen out the monosaccharide composition with the same molecular composition that meets the matching conditions;

[0010] S4, sending the precursor ions into an ion trap for gas phase dissociation to obtain fragment ions, sending the fragment ions into a mass spectrometer to obtain a secondary mass spectrum containing an isotope profile fingerprint of the fragment ions;

[0011] S5. performing a one-by-one isotope profile fingerprint comparison on the molecular composition fingerprints of the experimental and theoretical fragment ions of N-linked sugars with the same monosaccharide composition but different sequence structures;

[0012] S6. Targeted screening and verification of characteristic fragment ions from N-linked glycans with the same molecular composition fingerprint but different types, i.e., high-mannose and hybrid N-linked glycans contain structural diagnostic fragment ions of the GlcNAc2Man4 sequence, while complex N-linked glycans do not contain this specific fragment ion;

[0013] S7. Randomly score the matching probability of the secondary mass spectra of N-linked sugars that have been screened by sequence structure feature diagnostic fragment ions, and finally classify those that meet the pre-set complete N-glycopeptide spectrum matching conditions as targeted GPSMs;

[0014] S8. Obtain bait GPSMs in the bait library according to steps S3-S7;

[0015] S9. Combine the targeted GPSMs and the decoy GPSMs and sort them by P score from small to large. Select a threshold Pscore so that the false positive is no more than 1%. Remove duplicates from the targeted GPSMs below the threshold P score to obtain complete N-glycopeptide IDs.

[0016] Furthermore, the biological sample to be analyzed is an N-linked glycoprotein or a mixture sample containing various glycoforms.

[0017] Furthermore, in step S2, the intact N-glycopeptide sample is separated before electrospray ionization and mass spectrometry analysis.

[0018] Furthermore, the experimental molecular composition fingerprint of the precursor ion is measured in the primary mass spectrometer, and the experimental molecular composition fingerprint of the fragment ion is measured in the secondary mass spectrometer.

[0019] Furthermore, the theoretical molecular composition fingerprint is generated by the following steps:

[0020] Calculate the molecular formula of each molecule based on the theoretical molecular library of the system under study;

[0021] Refer to the standard element list and calculate the corresponding molecular composition fingerprint according to the type and quantity of elements in the molecular formula.

[0022] Furthermore, the matching in steps S3 and S5 refers to a one-to-one comparison of the m / z value and relative peak intensity value of each isotope peak in the experimental molecular composition fingerprint with the corresponding theoretical value; above a certain isotope peak relative intensity threshold, all isotopes are observed in the experiment, and the deviation of the mass-to-charge ratio m / z and relative intensity of each isotope peak is within a pre-set threshold range.

[0023] Furthermore, the matching criteria in steps S3 and S5 are controlled by the isotope peak intensity cutoff value, the maximum allowable error of the isotope peak mass-to-charge ratio, and the maximum allowable error of the isotope peak intensity.

[0024] Furthermore, the deduplication in step S9 is based on the criteria of polypeptide backbone amino acid sequence and modifications, glycosylation sites, and N-linked sugar sequence structure.

[0025] The beneficial effects of the present invention are:

[0026] The method of the present invention identifies and confirms the type of N-linked sugars based on mass spectrometry molecular composition and structural fingerprints at three levels: intact N-glycopeptide, monosaccharide sequence, and glycoproteome. The method includes: 1) at the intact N-glycopeptide level, identifying the monosaccharide composition, potential sequence structure, and type of N-linked sugars in the primary mass spectrometry based on the isotope profile of the precursor ion; 2) at the monosaccharide sequence molecular level, confirming the high mannose type and hybrid type based on the characteristic fragments inherent in the secondary mass spectrometry, and distinguishing these two types of sugars from the complex type; 3) at the glycoproteome level, false positive control and identification of the sequence structure at the spectral level based on fragment ions in the secondary mass spectrometry and target-decoy library search; through the identification and verification at the above three molecular levels, the identification and confirmation of the N-linked sugar types of N-glycoproteins based on mass spectrometry are achieved to the greatest extent.

[0027] The method of the present invention identifies and confirms the type of N-linked sugars on N-linked glycoproteins based on molecular composition and structural fingerprints, thereby effectively controlling the distinction and confirmation of N-linked sugars of the same molecular composition but different types. On the basis of existing spectral-level false positive control, a confirmation step based on structural fingerprints is added, thereby improving the accuracy of N-linked sugar type identification.

[0028] The present invention saves the search time for non-matching N-linked sugar types and greatly improves the efficiency and accuracy of N-linked sugar identification by searching and filtering the sequence structure fingerprint of N-linked sugar types before comprehensive analysis of tandem mass spectrometry and false positive control. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following briefly introduces the drawings required for use in the embodiments or the description of the prior art. Obviously, the drawings described below are only some embodiments recorded in the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying any creative work.

[0030] Figure 1 Schematic diagram of the process of the present invention.

[0031] Figure 2 The primary mass spectrum contains the precursor ion m / z 1157.554321 (z=3).

[0032] Figure 3 Comparison of experimental and theoretical isotope profile fingerprints of the precursor ion m / z 1157.554321 (z=3).

[0033] Figure 4 This is the secondary mass spectrum of the precursor ion m / z 1157.554321 (z=3) induced by high-energy collision.

[0034] Figure 5 The secondary mass spectrum contains the complete N-glycopeptide QAIHVGNQTFNDGTIVEK_01Y41Y41M(31M21M)61M(31M)61M polypeptide backbone and the N-linked sugar moiety matching fragment ion annotation.

[0035] Figure 6 This is a graphical dissociation diagram of the peptide backbone of the complete N-glycopeptide QAIHVGNQTFNDGTIVEK_01Y41Y41M(31M21M)61M(31M)61M with matching fragment ion annotations.

[0036] Figure 7 The figure shows the dissociation diagram of the N-linked sugar portion containing the intact N-glycopeptide QAIHVGNQTFNDGTIVEK_01Y41Y41M(31M21M)61M(31M)61M with matching fragment ion annotations.

[0037] Figure 8This is a comparison diagram of the experimental and theoretical values ​​of the molecular composition of the high-mannose characteristic fragment ion YIII3-3+ containing the complete N-glycopeptide QAIHVGNQTFNDGTIVEK_01Y41Y41M(31M21M)61M(31M)61M; IPMD and IPAD represent the isotopic peak m / z deviation and isotopic peak relative abundance deviation, respectively.

[0038] Figure 9 The primary mass spectrum contains the precursor ion m / z 1031.791504 (z=3).

[0039] Figure 10 Comparison of experimental and theoretical isotope profile fingerprints of the primary candidate intact N-glycopeptide with ID GHTLTLNFTR_N3H6F0S1 matching the precursor ion m / z 1031.791504 (z=3); N represents N-acetylglucosamine, H represents mannose, F represents fucose, and S represents sialic acid.

[0040] Figure 11 This is the secondary mass spectrum of the precursor ion m / z 1031.791504 (z=3) induced by high-energy collision.

[0041] Figure 12 This is the secondary mass spectrum of the complete N-glycopeptide GHTLTLNFTR_01Y41Y41M(31M41Y41L32S)61M(31M)-61M with matching fragment ion annotations of the polypeptide backbone portion and the N-linked sugar sequence structure portion; Y represents N-acetylglucosamine, M represents mannose, L represents galactose, and S represents sialic acid.

[0042] Figure 13 This is a graphical dissociation diagram of the polypeptide backbone portion of the complete N-glycopeptide GHTLTLNFTR_01Y41Y41M(31M41Y41L32S)61M(31M)-61M with matching fragment ion annotations; Y represents N-acetylglucosamine, M represents mannose, L represents galactose, and S represents sialic acid.

[0043] Figure 14 This is a graphical dissociation diagram of the N-linked sugar portion of the intact N-glycopeptide GHTLTLNFTR_01Y41Y41M(31M41Y41L32S)61M(31M)-61M with matching fragment ion annotations; Y represents N-acetylglucosamine, M represents mannose, L represents galactose, and S represents sialic acid.

[0044] Figure 15Comparison of the experimental and theoretical molecular composition of the two observed valence states (1+, left; 2+, right) of the characteristic fragment ion YIII3 of the hybrid N-linked sugar. IPMD and IPAD represent the isotopic peak m / z deviation and isotopic peak relative abundance deviation, respectively. DETAILED DESCRIPTION

[0045] The technical solutions of the present invention are described clearly and completely below with reference to specific embodiments. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. All other embodiments derived by persons of ordinary skill in the art based on the embodiments of the present invention without creative effort are also within the scope of protection of the present invention.

[0046] like Figure 1 Specifically, a method for identifying and verifying the type of N-linked sugars in a glycoprotein comprises the following steps:

[0047] S1. Prepare a complete N-glycopeptide sample from a biological sample to be analyzed according to existing methods; wherein the biological sample to be analyzed is an N-linked glycoprotein or a mixture sample containing various glycoforms.

[0048] S2. First, the intact N-glycopeptide sample is separated by high performance liquid chromatography, and then the intact N-glycopeptide sample is ionized by electrospray to obtain precursor ions, which are sent to the mass spectrometer to obtain an experimental primary mass spectrum containing the isotope profile fingerprint of the precursor ions.

[0049] S3. Match the isotope profile fingerprint of the precursor ion with the corresponding targeted forward theoretical database to screen out monosaccharide compositions with the same molecular composition that meet the matching conditions (including potential sequence structure and type).

[0050] S4. The precursor ions are sent into an ion trap for gas phase dissociation to obtain fragment ions, and the fragment ions are sent into a mass spectrometer to obtain a secondary mass spectrum containing the isotope profile fingerprint of the fragment ions.

[0051] S5. Perform one-by-one isotope profile fingerprint comparison on the molecular composition fingerprints of the experimental and theoretical fragment ions of N-linked sugars with the same monosaccharide composition but different sequence structures.

[0052] The experimental molecular composition fingerprint of the precursor ion was measured in the primary mass spectrometer, and the experimental molecular composition fingerprint of the fragment ion was measured in the secondary mass spectrometer.

[0053] The theoretical molecular composition fingerprint is generated by the following steps:

[0054] Calculate the molecular formula of each molecule based on the theoretical molecular library of the system under study;

[0055] Refer to the standard element list and calculate the corresponding molecular composition fingerprint according to the type and quantity of elements in the molecular formula.

[0056] The matching in steps S3 and S5 refers to the one-to-one comparison of the m / z value and relative peak intensity value of each isotope peak in the experimental molecular composition fingerprint with the corresponding theoretical value; above a certain isotope peak relative intensity threshold, all isotopes are observed in the experiment, and the deviation of the mass-to-charge ratio m / z and relative intensity of each isotope peak is within a pre-set threshold range.

[0057] The matching criteria in steps S3 and S5 are controlled by the isotope peak intensity cutoff value, the maximum allowable error of the isotope peak mass-to-charge ratio, and the maximum allowable error of the isotope peak intensity.

[0058] S6. Targeted screening and verification of characteristic fragment ions from N-linked sugars with the same molecular composition fingerprint but different types, that is, high-mannose and hybrid N-linked sugars contain GlcNAc2Man4 sequence structure diagnostic fragment ions, while complex N-linked sugars do not contain this specific fragment ion.

[0059] S7. Randomly score the secondary mass spectra of N-linked sugars that have been screened by sequence structure feature diagnostic fragment ions (i.e., calculate the P score). Finally, those that meet the pre-set intact N-glycopeptide spectrum matches (GPSMs) conditions (such as the number of polypeptide backbone matching fragment ions is not less than 5, and the number of N-linked sugar part matching fragment ions is not less than 1) are classified as targeted GPSMs.

[0060] S8. Obtain bait GPSMs in the bait library (anti-library or random library) according to steps S3-S7.

[0061] S9. Combine the targeted GPSMs and the decoy GPSMs and sort them in ascending order by P score. Select a threshold Pscore so that the false positive rate is no more than 1% (calculated as twice the number of decoy GPSMs below the threshold divided by the total number of targeted and decoy GPSMs). Remove duplicates from the targeted GPSMs below the threshold P score according to the standards of polypeptide backbone amino acid sequence and modification, glycosylation site, and N-linked sugar sequence structure to obtain complete N-glycopeptide IDs.

[0062] The following is an example to illustrate the identification and confirmation of high mannose N-linked sugars in a method for identifying and verifying the type of glycoprotein N-linked sugars of the present invention.

[0063] S1. Prepare intact N-glycopeptide samples from human liver cancer tissue according to existing methods;

[0064] S2. The intact N-glycopeptide sample is separated by high performance liquid chromatography and electrospray ionized to obtain positively charged precursor ions, which are then sent to a mass spectrometer to obtain an experimental primary mass spectrum containing the isotope profile fingerprint of the precursor ions, such as Figure 2 As shown;

[0065] S3, the isotope profile fingerprint of the precursor ion is matched with the corresponding targeted forward theoretical database, and the primary candidate complete N-glycopeptide IDs with the same molecular composition that meet the matching conditions are screened out, QAIHVGNQTFNDGTIVEK_N2H6F0S0( Figure 3 ), wherein N represents N-acetylglucosamine, H represents mannose, F represents fucose, and S represents sialic acid; the monosaccharide composition N2H6F0S0 corresponds to two high-mannose N-linked sugar sequence structures.

[0066] S4, the precursor ions are sent into an ion trap for gas phase dissociation to obtain fragment ions, and the fragment ions are sent into a mass spectrometer to obtain a secondary mass spectrum containing the isotope profile fingerprint of the fragment ions, such as Figure 4 As shown;

[0067] S5. Perform a one-by-one isotope profile fingerprint comparison of the molecular composition fingerprints of the experimental and theoretical fragment ions of the primary candidate complete N-glycopeptide IDs, and obtain a secondary mass spectrum with matching fragment ion annotations ( Figure 5 ), graphical dissociation diagram of the polypeptide backbone part ( Figure 6 ), graphic dissociation diagram of N-linked sugar moiety ( Figure 7 );

[0068] S6. Targeted screening of high mannose sequence structure (GlcNAc-GlcNAc-Man-(Man)-(Man) experimental diagnostic fragment ions from primary candidate complete N-glycopeptide IDs with the same molecular composition fingerprint ( Figure 8 ), and the screened primary candidate complete N-glycopeptide IDs were classified as candidate complete N-glycopeptide IDs;

[0069] S7. Randomly score the candidate intact N-glycopeptide IDs (i.e., calculate the P score). Finally, those that meet the pre-set intact N-glycopeptide spectrum matches (GPSMs) conditions (e.g., the number of peptide backbone matching fragment ions is not less than 5, and the number of N-linked sugar part matching fragment ions is not less than 1) are classified as targeted GPSMs.

[0070] S8. Obtain decoy GPSMs in the decoy library (anti-library or random library) according to the steps in S3-S7;

[0071] S9. Combine the target and decoy GPSMs and sort them from small to large according to P score. Select a threshold P score so that the false positive rate is no more than 1% (calculated as twice the number of decoy GPSMs below the threshold divided by the total number of target and decoy GPSMs). De-duplicate the target GPSMs below the threshold P score according to the standards of polypeptide backbone amino acid sequence and modification, glycosylation site and N-linked sugar sequence structure to obtain the final complete N-glycopeptide IDs.

[0072] The following is an example to illustrate the identification and confirmation of hybrid N-linked sugars in a method for identifying and verifying the type of glycoprotein N-linked sugars of the present invention.

[0073] S1. Prepare intact N-glycopeptide samples from human liver cancer tissue according to existing methods;

[0074] S2. The intact N-glycopeptide sample was separated by high performance liquid chromatography and electrospray ionization to obtain positively charged precursor ions, which were sent to the mass spectrometer to obtain the experimental primary mass spectrum containing the isotope profile fingerprint of the precursor ions ( Figure 9 );

[0075] S3, the isotope profile fingerprint of the precursor ion is matched with the corresponding targeted forward theoretical database, and the primary candidate complete N-glycopeptide IDs with the same molecular composition that meet the matching conditions are screened out, GHTLTLNFTR_N3H6F0S1( Figure 10 ); the monosaccharide composition N2H6F0S0 corresponds to two high mannose-type N-linked sugar sequence structures.

[0076] S4, the precursor ions are sent into an ion trap for gas phase dissociation to obtain fragment ions, and the fragment ions are sent into a mass spectrometer to obtain a secondary mass spectrum containing the isotope profile fingerprint of the fragment ions ( Figure 11 );

[0077] S5. Perform isotope profile fingerprint comparison on the molecular composition fingerprints of the experimental and theoretical fragment ions of the candidate complete N-glycopeptide IDs one by one, and obtain the secondary mass spectrum with matching fragment ion annotation ( Figure 12 ), graphical dissociation diagram of the polypeptide backbone part ( Figure 13 ), graphic dissociation diagram of N-linked sugar moiety ( Figure 14 );

[0078] S6. Targeted screening of high mannose sequence structure (GlcNAc-GlcNAc-Man-(Man)-(Man) experimental diagnostic fragment ions from primary candidate complete N-glycopeptide IDs with the same molecular composition fingerprint ( Figure 15 ), and the screened primary candidate complete N-glycopeptide IDs were classified as candidate complete N-glycopeptide IDs;

[0079] S7. Randomly score the candidate intact N-glycopeptide IDs (i.e., calculate the P score). Finally, those that meet the pre-set intact N-glycopeptide spectrum matches (GPSMs) conditions (e.g., the number of peptide backbone matching fragment ions is not less than 5, and the number of N-linked sugar part matching fragment ions is not less than 1) are classified as targeted GPSMs.

[0080] S8. Obtain decoy GPSMs in the decoy library (anti-library or random library) according to steps S3-S7;

[0081] S9. Combine the target and decoy GPSMs and sort them from small to large according to P score. Select a threshold P score so that the false positive rate is no more than 1% (calculated as twice the number of decoy GPSMs below the threshold divided by the total number of target and decoy GPSMs). De-duplicate the target GPSMs below the threshold P score according to the standards of polypeptide backbone amino acid sequence and modification, glycosylation site and N-linked sugar sequence structure to obtain the final complete N-glycopeptide IDs.

[0082] In summary, the method of the present invention is based on the targeted screening of structural fingerprints of high-mannose and hybrid N-linked sugars at three molecular levels: intact N-glycopeptides, monosaccharide sequences, and glycoproteomes. This enables targeted search of N-linked sugar types, thereby saving the search time used on ineffective tandem mass spectrometry in the existing "search first, screen later" process, and greatly improving the accuracy and efficiency of N-linked sugar type analysis.

[0083] It will be apparent to those skilled in the art that the present invention is not limited to the details of the exemplary embodiments described above and that the invention can be embodied in other specific forms without departing from the spirit or essential characteristics of the invention. Therefore, the embodiments should be considered in all respects as illustrative and non-restrictive, and the scope of the invention is defined by the appended claims, not the foregoing description, and all variations within the meaning and range of equivalents of the claims are intended to be included therein. Any reference sign in a claim should not be construed as limiting the claim to which it relates.

[0084] In addition, it should be understood that although this specification is described in terms of implementation methods, not every implementation method contains only one independent technical solution. This narrative method of the specification is only for the sake of clarity. Those skilled in the art should regard the specification as a whole. The technical solutions in each embodiment can also be appropriately combined to form other implementation methods that can be understood by those skilled in the art.

Claims

1. A method for identifying and verifying the type of N-linked sugars in glycoproteins, characterized in that: The steps include: S1. Prepare intact N-glycopeptide samples from the biological samples to be analyzed; S2. The intact N-glycopeptide sample is electrospray ionized to obtain precursor ions, which are then fed into a mass spectrometer to obtain an experimental primary mass spectrum containing the isotope profile fingerprint of the precursor ions. S3, matching the isotope profile fingerprint of the precursor ion with the corresponding targeted forward theoretical database to screen out the monosaccharide composition with the same molecular composition that meets the matching conditions; S4, sending the precursor ions into an ion trap for gas phase dissociation to obtain fragment ions, sending the fragment ions into a mass spectrometer to obtain a secondary mass spectrum containing an isotope profile fingerprint of the fragment ions; S5. performing a one-by-one isotope profile fingerprint comparison on the molecular composition fingerprints of the experimental and theoretical fragment ions of N-linked sugars with the same monosaccharide composition but different sequence structures; S6. Targeted screening and verification of characteristic fragment ions from N-linked glycans with the same molecular composition fingerprint but different types, i.e., high-mannose and hybrid N-linked glycans contain structural diagnostic fragment ions of the GlcNAc2Man4 sequence, while complex N-linked glycans do not contain this specific fragment ion; S7. Randomly score the matching probability of the secondary mass spectra of N-linked sugars that have been screened by sequence structure feature diagnostic fragment ions, and finally classify those that meet the pre-set complete N-glycopeptide spectrum matching conditions as targeted GPSMs; S8. Obtain bait GPSMs in the bait library according to steps S3-S7; S9. Combine the targeted GPSMs and the decoy GPSMs and sort them by P score from small to large. Select a threshold Pscore so that the false positive is no more than 1%. Remove duplicates from the targeted GPSMs below the threshold P score to obtain complete N-glycopeptide IDs.

2. The method for identifying and verifying the type of N-linked sugars in a glycoprotein according to claim 1, wherein: The biological sample to be analyzed is an N-linked glycoprotein or a mixture sample containing various glycoforms.

3. The method for identifying and verifying the type of N-linked sugars in a glycoprotein according to claim 1, wherein: In step S2, the intact N-glycopeptide sample is separated before electrospray ionization and mass spectrometry analysis.

4. The method for identifying and verifying the type of N-linked sugars in a glycoprotein according to claim 1, wherein: The experimental molecular composition fingerprint of the precursor ion was measured in the primary mass spectrometer, and the experimental molecular composition fingerprint of the fragment ion was measured in the secondary mass spectrometer.

5. The method for identifying and verifying the type of N-linked sugars in a glycoprotein according to claim 1, wherein: The theoretical molecular composition fingerprint is generated by the following steps: Calculate the molecular formula of each molecule based on the theoretical molecular library of the system under study; Refer to the standard element list and calculate the corresponding molecular composition fingerprint according to the type and quantity of elements in the molecular formula.

6. The method for identifying and verifying the type of N-linked sugars in a glycoprotein according to claim 1, wherein: The matching in steps S3 and S5 refers to the one-to-one comparison of the m / z value and relative peak intensity value of each isotope peak in the experimental molecular composition fingerprint with the corresponding theoretical value; above a certain isotope peak relative intensity threshold, all isotopes are observed in the experiment, and the deviation of the mass-to-charge ratio m / z and relative intensity of each isotope peak is within a pre-set threshold range.

7. The method for identifying and verifying the type of N-linked sugars in a glycoprotein according to claim 1, wherein: The matching criteria in steps S3 and S5 are controlled by the isotope peak intensity cutoff value, the maximum allowable error of the isotope peak mass-to-charge ratio, and the maximum allowable error of the isotope peak intensity.

8. The method for identifying and verifying the type of N-linked sugars in a glycoprotein according to claim 1, wherein: The deduplication in step S9 is based on the criteria of polypeptide backbone amino acid sequence and modifications, glycosylation sites, and N-linked sugar sequence structure.

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