A method for identification and verification of sialic acid linkages based on tandem mass spectrometry

Abstract

The present invention discloses a method for identifying and verifying sialic acid linkages based on tandem mass spectrometry, comprising the following steps: preparing a complete N-glycopeptide sample; obtaining precursor ions after electrospray ionization of the complete N-glycopeptide sample, obtaining fragment ions by gas phase dissociation of the precursor ions, and performing mass spectrometry analysis on the precursor ions and fragment ions; identifying the composition of candidate monosaccharides containing sialic acid based on isotope profile fingerprint comparison; identifying fragment ions of sialic acid sequence structure diagnosis based on isotope profile fingerprint comparison and further screening of candidate monosaccharide composition; false positive control of complete N-glycopeptide identification based on targeted-bait library search; identification of sialic acid linkages based on the combined scoring of multiple characteristic oxonium ions; identification and confirmation of sialic acid linkages. This method identifies and confirms sialic acid linkages based on mass spectrometry molecular structure fingerprints at three molecular levels: complete N-glycopeptide, monosaccharide sequence, and monosaccharide, greatly improving the accuracy and efficiency of sialic acid linkage identification.

Description

Technical Field

[0001] The present invention belongs to the technical field of precise analysis of protein structure, and in particular relates to a method for identifying and verifying sialic acid linkages based on tandem mass spectrometry. Background Art

[0002] Glycosylation is one of the most abundant post-translational modifications of proteins in the human body. Accurate analysis of glycoprotein structure is essential for understanding their biochemical properties and physiological functions. Sialic acid, a monosaccharide found at the end of glycoprotein-modified side chains, plays a key role in various biological recognition processes in the human body.

[0003] The widespread use of high-resolution mass spectrometry has enabled the analysis of sialic acid-linked N-glycoproteins to achieve isotope resolution at the intact N-glycopeptide level, as well as accurate measurement of the isotope profiles of precursor ions in the primary mass spectrometry and fragment ions in the secondary mass spectrometry. Based on this advancement and unique advantages, the present invention provides a method for the identification and verification of sialic acid linkages using tandem mass spectrometry. Summary of the Invention

[0004] To address the above technical problems, the present invention aims to provide a method for identifying and verifying sialic acid linkages based on tandem mass spectrometry. This method identifies and confirms sialic acid linkages at three molecular levels: intact N-glycopeptide, monosaccharide sequence, and monosaccharide, based on mass spectrometric molecular composition and structural fingerprints. This method significantly improves the accuracy and efficiency of sialic acid linkage 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 sialic acid linkages based on tandem mass spectrometry 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 primary candidate complete N-glycopeptide IDs with the same molecular composition that meet 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. Perform one-by-one isotope profile fingerprint comparison and matching on the molecular composition fingerprints of experimental and theoretical fragment ions of primary candidate complete N-glycopeptide IDs;

[0012] S6. Targetedly screening sialic acid monosaccharide sequences containing structural diagnostic fragment ions from primary candidate complete N-glycopeptide IDs with the same molecular composition fingerprint, and classifying the screened primary candidate complete N-glycopeptide IDs into candidate complete N-glycopeptide IDs;

[0013] S7, randomly scoring the matching probability of candidate complete N-glycopeptide IDs, and finally classifying 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 target GPSMs and the decoy GPSMs and sort them by P score from small to large. Select a threshold P score so that the false positive is no more than 1%. Remove duplicates from the target GPSMs below the threshold P score to obtain complete N-glycopeptide IDs containing sialic acid.

[0016] S10. Based on isotope profile fingerprint comparison, the characteristic oxonium ion m / z 204 of N-acetylglucosamine, the characteristic oxonium ions m / z 274 and 292 of sialic acid, and the characteristic oxonium ion m / z 366 of the mannose-N-acetylglucosamine disaccharide sequence in the secondary mass spectrum were identified, and then a comprehensive score was performed. Scores less than 0.8 were α2,3 linkages, and scores greater than 0.8 were α2,6 linkages.

[0017] Furthermore, the biological sample to be analyzed is a sample containing sialylated glycoprotein.

[0018] Furthermore, in step S2, the intact N-glycopeptide sample is separated before electrospray ionization and mass spectrometry analysis.

[0019] 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.

[0020] Furthermore, the theoretical molecular composition fingerprint is generated by the following steps:

[0021] Calculate the molecular formula of each molecule based on the theoretical molecular library of the system under study;

[0022] 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.

[0023] 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.

[0024] 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.

[0025] 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.

[0026] The beneficial effects of the present invention are:

[0027] The identification and verification method of the present invention identifies and confirms sialic acid linkages based on mass spectrometry molecular composition and structural fingerprints at three molecular levels: intact N-glycopeptide, monosaccharide sequence, and monosaccharide. The method includes: 1) identifying the monosaccharide composition of intact N-glycopeptides containing sialic acid based on isotope profile comparison at the intact N-glycopeptide level; 2) identifying fragment ions in the secondary mass spectrometry based on isotope profile fingerprint comparison at the monosaccharide sequence molecular level, and confirming the presence of sialic acid sequence structure and sialic acid based on sialic acid sequence structure diagnostic fragment ions and sialic acid characteristic oxonium ions; 3) false positive control and identification of sialic acid sequence structure at the spectrum level based on target-decoy library search; and 4) identifying sialic acid linkages at the monosaccharide molecular level based on the comprehensive scoring of characteristic oxonium ions. Through the identification and verification at the above four levels, the present invention improves the accuracy and efficiency of sialic acid linkage identification and verification based on tandem mass spectrometry. BRIEF DESCRIPTION OF THE DRAWINGS

[0028] 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.

[0029] Figure 1 Schematic diagram of the process of the present invention.

[0030] Figure 2 Base peak chromatogram of intact N-glycopeptides from human liver cancer tissue separated by reversed-phase HPLC-tandem mass spectrometry.

[0031] Figure 3 The primary mass spectrum contains the precursor ion m / z 1189.08789.

[0032] Figure 4This is the precursor ion isotope profile fingerprint comparison map of the complete N-glycopeptide IADTNITSIPIQGLPPSLTELHLD GNK_N3H4F0S1 containing α2,3 linked sialic acid.

[0033] Figure 5 Annotated MS / MS spectrum of the intact N-glycopeptide IADTNITSIPIQGLPPSLTELHLD GNK_N3H4F0S1 containing α2,3-linked sialic acid.

[0034] Figure 6 This is a graphical dissociation diagram of the peptide backbone of the complete N-glycopeptide IADTNITSIPIQGLPPSLTELHLD GNK_N3H4F0S1 containing α2,3 linked sialic acid.

[0035] Figure 7 The N-linked sugar portion of the intact N-glycopeptide IADTNITSIPIQGLPPSLTELHLD GNK_N3H4F0S1 containing α2,3 linked sialic acid is shown in the dissociation diagram.

[0036] Figure 8 The MS / MS spectrum region containing oxonium ions m / z 204, 274, 292, and 366 of the intact N-glycopeptide IADTNITSIPIQGLPPSLTELHLD GNK_N3H4F0S1 containing α2,3 linked sialic acid.

[0037] Figure 9 Identification diagram of oxonium ions m / z 204, 274, 292, and 366 based on isotope profile fingerprint comparison; IPMD, isotopic peak m / z deviation; IPAD, isotopic peak relative abundance deviation.

[0038] Figure 10 The primary mass spectrum contains the precursor ion m / z 1298.880371.

[0039] Figure 11 This is the precursor ion isotope profile fingerprint comparison map of the complete N-glycopeptide NNGTITWENLAAVLPFGGTF DLVQLK_N4H5F0S1 containing α2,6-linked sialic acid.

[0040] Figure 12 Annotated MS / MS spectrum of the intact N-glycopeptide NNGTITWENLAAVLPFGGTF DLVQLK_N4H5F0S1 containing α2,6-linked sialic acid.

[0041] Figure 13 This is a graphical dissociation diagram of the peptide backbone of the complete N-glycopeptide NNGTITWENLAAVLPFGGTF DLVQLK_N4H5F0S1 containing α2,6-linked sialic acid.

[0042] Figure 14 This is a graphical dissociation diagram of the N-linked sugar portion of the intact N-glycopeptide NNGTITWENLAAVLPFGGTF DLVQLK_N4H5F0S1 containing α2,6-linked sialic acid.

[0043] Figure 15 The MS / MS spectrum region of the intact N-glycopeptide NNGTITWENLAAVLPFGGTF DLVQLK_N4H5F0S1 containing oxonium ions m / z 204, 274, 292, and 366 containing α2,6-linked sialic acid.

[0044] Figure 16 Identification diagram of oxonium ions m / z 204, 274, 292, and 366 based on isotope profile fingerprint comparison; IPMD, isotopic peak m / z deviation; IPAD, isotopic peak relative abundance deviation. 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 sialic acid linkages based on tandem mass spectrometry comprises the following steps:

[0047] S1. Prepare a complete N-glycopeptide sample from a biological sample to be analyzed according to an existing method; wherein the biological sample to be analyzed is a sample containing sialylated glycoproteins.

[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 primary candidate complete N-glycopeptide IDs with the same molecular composition that meet the matching conditions.

[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 an isotope profile fingerprint of the fragment ions.

[0051] S5. Perform isotope profile fingerprint comparison and matching on the molecular composition fingerprints of experimental and theoretical fragment ions of primary candidate complete N-glycopeptide IDs one by one.

[0052] 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.

[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 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.

[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. Targetedly screen sialic acid monosaccharide sequences containing structural diagnostic fragment ions from primary candidate complete N-glycopeptide IDs with the same molecular composition fingerprint, and classify the screened primary candidate complete N-glycopeptide IDs into candidate complete N-glycopeptide IDs.

[0059] S7. Randomly score the matching probability of candidate intact N-glycopeptide IDs (i.e., calculate the P score), and finally classify those that meet the pre-set intact N-glycopeptide spectrum matches (GPSMs) conditions (such as 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) 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). De-duplicate 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 containing sialic acid.

[0062] S10. Based on isotope profile fingerprint comparison, the characteristic oxonium ion m / z 204 of N-acetylglucosamine, the characteristic oxonium ions m / z 274 and 292 of sialic acid, and the characteristic oxonium ion m / z 366 of the mannose-N-acetylglucosamine disaccharide sequence in the secondary mass spectrum were identified. Then, a comprehensive score was performed according to the existing method. Scores less than 0.8 were α2,3 linkages, and scores greater than 0.8 were α2,6 linkages.

[0063] The following describes an example of a method for identifying and verifying sialic acid α2,3 linkages based on tandem mass spectrometry according to the present invention.

[0064] S1. Prepare intact N-glycopeptide samples from human liver cancer tissue according to existing methods;

[0065] S2. The intact N-glycopeptide sample was separated by HPLC (base peak chromatogram as shown in Figure 2 After electrospray ionization, positively charged precursor ions are obtained, and the precursor ions are sent to the mass spectrometer to obtain an experimental primary mass spectrum containing the isotope profile fingerprint of the precursor ions, as shown in FIG. Figure 3 As shown;

[0066] S3. Match the isotope profile fingerprint of the precursor ion with the corresponding targeted forward theoretical database to screen out the primary candidate complete N-glycopeptide IDs with the same molecular composition that meet the matching conditions, such as VDKDLQSLEDILHQVENK_N4H5F0S1. Figure 4 As shown; the monosaccharide composition corresponds to a monosaccharide sequence structure 01Y41Y41M(31M)61M61Y41L32S, wherein Y represents N-acetylglucosamine, M represents mannose, L represents galactose, and S represents sialic acid;

[0067] 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;

[0068] 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 to obtain all fragment ions whose experimental isotope profiles match the theoretical isotope profiles, and annotate the secondary mass spectra, such as Figure 5 As shown; the peptide backbone graphic dissociation diagram with matching fragment ion annotations, such as Figure 6 As shown; Graphical dissociation diagram of the N-linked sugar moiety with matching fragment ions annotated, such as Figure 7 As shown;

[0069] S6. Targeted screening of fragment ions containing sialic acid monosaccharide sequences for experimental structural diagnosis from primary candidate complete N-glycopeptide IDs with the same molecular composition fingerprint ( Figure 5 The ions in the middle rectangular box include 0 , 2 AI2-1+, 0,1 AII1-1+, BI2-1+, BII1-1+, 0,2 AI4-1+, BI1-1+, CI1-1+, 3,5 AI4-1+, BI2-1+, 0,3 AI5-2+, 3,5 AI3-1+, BI3-1+, 3, 5 AI4-1+, BI4-1+, 3,5 AI5-1+, 0,2 XI1-3+, ZI1-3+, YI1-1+, ZI2-3+, YI2-3+, YI3-3+, YI4-3+, YI6-3+, YI1-2+) and characteristic oxonium ions ( Figure 8 、 Figure 9 ), and the screened primary candidate complete N-glycopeptide IDs were classified as candidate complete N-glycopeptide IDs;

[0070] S7. Randomly score the matching probability of candidate intact N-glycopeptide IDs (i.e., calculate the P score). Finally, the candidate intact N-glycopeptide IDs 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.

[0071] S7, obtaining decoy GPSMs in the decoy library (anti-library or random library) according to steps S3-S7;

[0072] S8. 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 criteria of polypeptide backbone amino acid sequence and modification, glycosylation site, and N-linked sugar sequence structure to obtain the final complete N-glycopeptide IDs.

[0073] S9. At the monosaccharide molecular level, a comprehensive score (I) was calculated based on the intensities (I) of the characteristic oxonium ions m / z 204 (from N-acetylglucosamine, GlcNAc), m / z 274 (from sialic acid Neu5Ac with the loss of a water molecule), m / z 292 (from sialic acid Neu5Ac), and m / z 366 (from the disaccharide Hex-GlcNAc formed by mannose and N-acetylglucosamine) identified by isotope profile fingerprint comparison, i.e., (I204+I366) / (I274+I292)*n Neu5Gc-Gal-GlcNAc / n GlcNAc , the score obtained here is 0.76; according to the judgment criteria mentioned above (when the score is <0.8, the corresponding sialic acid is α2,3 linkage; when it is >0.8, it is α2,6), the sialic acid here is α2,3.

[0074] The following describes an example of a method for identifying and verifying sialic acid α2,6 linkages based on tandem mass spectrometry according to the present invention.

[0075] S1. Prepare intact N-glycopeptide samples from human liver cancer tissue according to the current method;

[0076] S2. The intact N-glycopeptide sample was separated by HPLC (base peak chromatogram as shown in Figure 2 After electrospray ionization, positively charged precursor ions are obtained, and the precursor ions are sent to the mass spectrometer to obtain an experimental primary mass spectrum containing the isotope profile fingerprint of the precursor ions, as shown in FIG. Figure 10 As shown;

[0077] 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 ID with the same molecular composition that meets the matching conditions is screened out, NNGTITWENLAAVLPFGGTFDLVQLK_N4H5F0S1( Figure 11); the monosaccharide composition corresponds to four monosaccharide sequence structures: 01Y41Y41M(31M)61M(21Y41L32S)61Y41L, 01Y41Y41M(31M41Y41L)61M61Y41L32S, 01Y41Y41M(31M(21Y41L32S)-41Y)61M61M and 01Y41Y41M(31M41Y41L32S)(41Y)61M61M; wherein Y represents N-acetylglucosamine, M represents mannose, L represents galactose, and S represents sialic acid.

[0078] 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;

[0079] 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 all fragment ions whose experimental isotope profiles match the theoretical isotope profiles. The annotated secondary mass spectra are as follows: Figure 12 As shown, the peptide backbone graphic dissociation diagram with matching fragment ion annotation is shown in Figure 13 The graphical dissociation diagram of the N-linked sugar moiety with matching fragment ions is shown in Figure 13 As shown;

[0080] S6. Targeted screening of fragment ions containing sialic acid monosaccharide sequences for experimental structural diagnosis from primary candidate complete N-glycopeptide IDs with the same molecular composition fingerprint ( Figure 12 The ions in the thick rectangular box include CII3-1+, 3,5 AI5-1+) and characteristic oxonium ions ( Figure 15 、 Figure 16 ), and the screened primary candidate complete N-glycopeptide IDs were classified as candidate complete N-glycopeptide IDs;

[0081] 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.

[0082] S8. Obtain decoy GPSMs in the decoy library (anti-library or random library) according to the steps in S3-S7;

[0083] 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.

[0084] S10. At the monosaccharide molecular level, the intensity (I) of the characteristic oxonium ions m / z 204 (from N-acetylglucosamine, GlcNAc), m / z 274 (from sialic acid Neu5Ac with the loss of a water molecule), m / z 292 (from sialic acid Neu5Ac), and m / z 366 (from the disaccharide Hex-GlcNAc formed by mannose and N-acetylglucosamine) identified by isotope profile fingerprint comparison was comprehensively scored as (I204+I366) / (I274+I292)*n Neu5Gc-Gal-GlcNAc / n GlcNAc , the score obtained here is 1.36; according to the judgment criteria mentioned above (when the score is <0.8, the corresponding sialic acid is α2,3 linkage; when >0.8, it is α2,6), the sialic acid here is α2,6.

[0085] 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.

[0086] 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 sialic acid linkages based on tandem mass spectrometry, 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 primary candidate complete N-glycopeptide IDs with the same molecular composition that meet 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. Perform one-by-one isotope profile fingerprint comparison and matching on the molecular composition fingerprints of experimental and theoretical fragment ions of primary candidate complete N-glycopeptide IDs; S6. Targetedly screening sialic acid monosaccharide sequences containing structural diagnostic fragment ions from primary candidate complete N-glycopeptide IDs with the same molecular composition fingerprint, and classifying the screened primary candidate complete N-glycopeptide IDs into candidate complete N-glycopeptide IDs; S7, randomly scoring the matching probability of candidate complete N-glycopeptide IDs, and finally classifying 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. Target GPSMs and decoy GPSMs are combined and sorted by P score from small to large. A threshold P score is selected so that the false positive is no more than 1%. Target GPSMs with P scores below the threshold are deduplicated to obtain complete N-glycopeptide IDs containing sialic acid. S10. Based on isotope profile fingerprint comparison, the characteristic oxonium ion m / z 204 of N-acetylglucosamine, the characteristic oxonium ions m / z 274 and 292 of sialic acid, and the characteristic oxonium ion m / z 366 of the mannose-N-acetylglucosamine disaccharide sequence in the secondary mass spectrum were identified, and then a comprehensive score was performed. Scores less than 0.8 were α2,3 linkages, and scores greater than 0.8 were α2,6 linkages.

2. The method for identifying and verifying sialic acid linkages based on tandem mass spectrometry according to claim 1, wherein: The biological sample to be analyzed is a sample containing sialylated glycoprotein.

3. The method for identifying and verifying sialic acid linkages based on tandem mass spectrometry 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 sialic acid linkages based on tandem mass spectrometry 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 sialic acid linkages based on tandem mass spectrometry 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 sialic acid linkages based on tandem mass spectrometry according to claim 1, wherein: 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.

7. The method for identifying and verifying sialic acid linkages based on tandem mass spectrometry 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 sialic acid linkages based on tandem mass spectrometry 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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