CN116008382A - Identification and verification method of sialic acid linkage based on tandem mass spectrum - Google Patents

Identification and verification method of sialic acid linkage based on tandem mass spectrum Download PDF

Info

Publication number
CN116008382A
CN116008382A CN202310097998.0A CN202310097998A CN116008382A CN 116008382 A CN116008382 A CN 116008382A CN 202310097998 A CN202310097998 A CN 202310097998A CN 116008382 A CN116008382 A CN 116008382A
Authority
CN
China
Prior art keywords
glycopeptide
sialic acid
complete
ions
ids
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Pending
Application number
CN202310097998.0A
Other languages
Chinese (zh)
Inventor
黄才岭
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Hanno Biotechnology Suzhou Co ltd
Original Assignee
Hanno Biotechnology Suzhou Co ltd
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Hanno Biotechnology Suzhou Co ltd filed Critical Hanno Biotechnology Suzhou Co ltd
Priority to CN202310097998.0A priority Critical patent/CN116008382A/en
Publication of CN116008382A publication Critical patent/CN116008382A/en
Pending legal-status Critical Current

Links

Images

Landscapes

  • Other Investigation Or Analysis Of Materials By Electrical Means (AREA)

Abstract

The invention discloses a method for identifying and verifying sialic acid linkage based on tandem mass spectrometry, which comprises the following steps: preparing a complete N-glycopeptide sample; carrying out electrospray ionization on the complete N-glycopeptide sample to obtain precursor ions, carrying out gas phase dissociation on the precursor ions to obtain fragment ions, and carrying out mass spectrometry on the precursor ions and the fragment ions; identifying sialic acid-containing candidate monosaccharide composition based on isotope profile fingerprint comparison; identifying the diagnosis fragment ions based on the comparison of isotope profile fingerprints and sialic acid sequence-containing structure, and further screening candidate monosaccharide compositions; false positive control of complete N-glycopeptide identification based on targeting-bait library searches; identification of sialic acid linkages scored based on a combination of a plurality of characteristic oxonium ions; identification and confirmation of sialic acid linkages. The method identifies and confirms sialic acid linkage based on mass spectrum molecular structure fingerprints on three molecular levels of complete N-glycopeptide, monosaccharide sequence and monosaccharide, and greatly improves accuracy and efficiency of sialic acid linkage identification.

Description

Identification and verification method of sialic acid linkage based on tandem mass spectrum
Technical Field
The invention belongs to the technical field of accurate analysis of protein structures, and particularly relates to a method for identifying and verifying sialic acid links based on tandem mass spectrometry.
Background
Glycosylation is one of the most abundant post-translational modifications of proteins in humans, and precise resolution of glycoprotein structure is a necessary condition for understanding its biochemical properties and physiological functions. Sialic acid plays a key role in various biological recognition processes in the human body as a monosaccharide widely existing at the tail end of a glycoprotein-modified branch.
The widespread use of high resolution mass spectrometry has enabled analysis of sialic acid containing N-linked glycoproteins to obtain isotope resolution at the intact N-glycopeptide level and accurate measurement of the isotope profile of precursor ions in primary mass spectrometry and fragment ions in secondary mass spectrometry. Based on the progress and the characteristics, the invention provides a method for identifying and verifying sialic acid linkage based on tandem mass spectrometry.
Disclosure of Invention
In order to solve the technical problems, the invention aims to provide a method for identifying and verifying sialic acid linkage based on tandem mass spectrometry. The method identifies and confirms sialic acid linkage on the basis of mass spectrum molecular composition and structure fingerprints on the three molecular levels of complete N-glycopeptide, monosaccharide sequence and monosaccharide, and greatly improves the accuracy and efficiency of sialic acid linkage identification.
In order to achieve the technical purpose and the technical effect, the invention is realized by the following technical scheme:
a method for identifying and verifying sialic acid linkage based on tandem mass spectrometry comprises the following steps:
s1, preparing a complete N-glycopeptide sample from a biological sample to be analyzed;
s2, carrying out electrospray ionization on the complete N-glycopeptide sample to obtain precursor ions, and sending the precursor ions into a mass spectrometer to obtain an experimental primary mass spectrum containing precursor ion isotope profile fingerprints;
s3, matching the isotope profile fingerprint of the precursor ion with a corresponding targeting forward theoretical database, and screening primary candidate complete N-glycopeptide IDs with the same molecular composition and meeting the matching conditions;
s4, delivering the precursor ions into an ion trap for gas phase dissociation to obtain fragment ions, and delivering the fragment ions into a mass spectrometer to obtain a secondary mass spectrum containing the isotope profile fingerprint of the fragment ions;
s5, comparing and matching isotope profile fingerprints one by one of molecular composition fingerprints of the experiment and theoretical fragment ions of the primary candidate complete N-glycopeptide IDs;
s6, targetedly screening sialic acid-containing monosaccharide sequences containing structural diagnosis fragment ions from primary candidate complete N-glycopeptide IDs with identical self-molecular composition fingerprints, and classifying the screened primary candidate complete N-glycopeptide IDs as candidate complete N-glycopeptide IDs;
s7, scoring random matching probability of candidate complete N-glycopeptide IDs, and finally, attributing complete N-glycopeptide spectrogram matching conditions meeting preset conditions as targeted GPSMs;
s8, obtaining bait GPSMs in a bait library according to the steps of S3-S7;
s9, combining the targeted GPSMs and the decoy GPSMs, sorting the targeted GPSMs from small to large according to P score, selecting a threshold P score to ensure that false positive is not more than 1%, and de-duplicating the targeted GPS Ms below the threshold P score to obtain sialic acid-containing complete N-glycopeptide IDs;
s10, identifying characteristic oxonium ions m/z 204, sialic acid characteristic oxonium ions m/z 274 and 292 and mannose-N-acetylglucosamine disaccharide sequence characteristic oxonium ions m/z 366 of N-acetylglucosamine in the secondary mass spectrum based on isotope profile fingerprint comparison, and then comprehensively scoring that alpha 2,3 links are less than 0.8 and alpha 2,6 links are more than 0.8.
Further, the biological sample to be analyzed is a sample containing sialylated glycoprotein.
Further, in step S2, the whole N-glycopeptide sample is isolated prior to electrospray ionization and mass spectrometry.
Further, the experimental molecular composition fingerprint of the precursor ion is measured in the primary mass spectrum, and the experimental molecular composition fingerprint of the fragment ion is measured in the secondary mass spectrum.
Further, theoretical molecular composition fingerprints are generated by:
calculating the molecular formula of each molecule according to a theoretical molecular library of the studied system;
and calculating corresponding molecular composition fingerprints according to the types and the numbers of the elements in the molecular formula by referring to the standard element list.
Further, the matching in steps S3 and S5 refers to one-to-one comparison of the m/z value and the relative peak intensity value of each isotope peak in the experimental molecular composition fingerprint with the corresponding theoretical values.
Further, the criteria for matching in steps S3 and S5 are controlled by the isotope peak intensity cutoff value, the isotope peak mass-to-charge ratio maximum allowable error, and the isotope peak intensity maximum allowable error.
Further, the de-duplication in step S9 is based on the polypeptide backbone amino acid sequence and the criteria of modification, glycosylation site and N-linked carbohydrate sequence structure.
The beneficial effects of the invention are as follows:
the identification and verification method of the present invention identifies and validates sialic acid linkages based on mass spectral molecular composition and structural fingerprints at the level of three molecules of the complete N-glycopeptide, monosaccharide sequence and monosaccharide, comprising 1) identifying monosaccharide composition comprising sialic acid complete N-glycopeptide based on isotope profile alignment at the level of the complete N-glycopeptide; 2) Identifying fragment ions in the secondary mass spectrum based on isotope profile fingerprint alignment at the monosaccharide sequence molecular level, and diagnosing the presence of fragment ions and sialic acid-containing characteristic oxonium ions to confirm the sialic acid-containing sequence structure and sialic acid based on the sialic acid-containing sequence structure; 3) Performing spectrogram-level false positive control and identification on the structure containing sialic acid sequence based on the targeting-decoy library search; 4) At the monosaccharide molecular level, sialic acid linkages were identified based on a comprehensive score for characteristic oxonium ions. Through the identification and verification on the 4 layers, the accuracy and the efficiency of the identification and the verification of the sialic acid linkage based on tandem mass spectrometry are improved.
Drawings
In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings that are required to be used in the embodiments or the description of the prior art will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments described in the present invention, and other drawings may be obtained according to the drawings without inventive effort to those skilled in the art.
FIG. 1 is a schematic flow chart of the method of the present invention.
FIG. 2 shows the reverse HPLC-tandem mass spectrometry detection base peak chromatogram of the whole N-glycopeptide of human liver cancer tissue.
FIG. 3 is a first order mass spectrum containing precursor ions m/z 1189.08789.
FIG. 4 is a precursor ion isotope profile fingerprint alignment of an intact N-glycopeptide IADTNITSIPQGLPPSLTELHLD GNK _N3H2F0S1 containing α2,3 linked sialic acid.
FIG. 5 is an annotated secondary mass spectrum of a complete N-glycopeptide IADTNITSIPQGLPPSLTELHLD GNK-N3H 4F0S1 containing α2,3 linked sialic acid.
FIG. 6 is a graphic dissociation diagram of the polypeptide backbone of the intact N-glycopeptide IADTNITSIPQGLPPSLTELHLD GNK _N3H2F0S1 containing α2,3 linked sialic acid.
FIG. 7 is a graphic dissociation diagram of the N-linked sugar moiety of the intact N-glycopeptide IADTNITSIPQGLPPSLTELHLD GNK-N3H 4F0S1 containing α2,3 linked sialic acid.
FIG. 8 is a MS/MS spectrum region containing oxonium ions m/z 204, 274, 292 and 366 for the intact N-glycopeptide IADTNITSIPQGLPPSLTELHLD GNK-N3H 4F0S1 containing α2,3 linked sialic acid.
FIG. 9 is an identification plot of oxonium ions m/z 204, 274, 292, and 366 based on isotope profile fingerprint alignment; IPMD, isotropic peak m/z displacement, isotope peak to charge ratio bias; IPAD, isotopic peak relative abundance deviation, isotope peak relative intensity bias.
FIG. 10 is a first order mass spectrum containing precursor ions m/z 1298.880371.
FIG. 11 is a precursor ion isotope profile fingerprint alignment of an intact N-glycopeptide NNGTITWENLAAVLPFGGTF DLVQLK _N4H2F 0S1 containing an α2,6 linked sialic acid.
FIG. 12 is an annotated secondary mass spectrum of a complete N-glycopeptide NNGTITWENLAAVLPFGGTF DLVQLK-N4H 5F0S1 containing α2,6 linked sialic acid.
FIG. 13 is a graphic dissociation diagram of the polypeptide backbone of the intact N-glycopeptide NNGTITWENLAAVLPFGGTF DLVQLK-N4H 5F0S1 containing α2,6 linked sialic acid.
FIG. 14 is a graphic dissociation diagram of the N-linked sugar moiety of the intact N-glycopeptide NNGTITWENLAAVLPFGGTF DLVQLK-N4H 5F0S1 containing α2,6 linked sialic acid.
FIG. 15 is a MS/MS spectrum region containing oxonium ions m/z 204, 274, 292 and 366 for the intact N-glycopeptide NNGTITWENLAAVLPFGGTF DLVQLK-N4H 5F0S1 containing α2,6 linked sialic acid.
FIG. 16 is an identification plot of oxonium ions m/z 204, 274, 292, and 366 based on isotope profile fingerprint alignment; IPMD, isotropic peak m/z displacement, isotope peak to charge ratio bias; IPAD, isotopic peak relative abundance deviation, isotope peak relative intensity bias.
Detailed Description
The following description of the embodiments of the present invention will be made more apparent and fully by reference to the accompanying drawings, in which it is shown, by way of illustration, only some, but not all embodiments of the invention. All other embodiments, which can be made by those skilled in the art based on the embodiments of the invention without making any inventive effort, are intended to be within the scope of the invention.
As shown in fig. 1, specifically, a method for identifying and verifying sialic acid linkage based on tandem mass spectrometry includes the following steps:
s1, preparing a complete N-glycopeptide sample from a biological sample to be analyzed according to the existing method; wherein the biological sample to be analyzed is a sample containing sialylated glycoprotein.
S2, separating the complete N-glycopeptide sample through high performance liquid chromatography, carrying out electrospray ionization on the complete N-glycopeptide sample to obtain precursor ions, and sending the precursor ions into a mass spectrometer to obtain an experimental primary mass spectrum containing precursor ion isotope profile fingerprints.
And S3, matching the isotope profile fingerprint of the precursor ion with a corresponding targeting forward theoretical database, and screening out primary candidate complete N-glycopeptide IDs with the same molecular composition, which meet the matching condition.
S4, delivering the precursor ions into an ion trap for gas phase dissociation to obtain fragment ions, and delivering the fragment ions into a mass spectrometer to obtain a secondary mass spectrum containing the fragment ion isotope profile fingerprint.
S5, comparing and matching isotope profile fingerprints one by one on molecular composition fingerprints of the experimental and theoretical fragment ions of the primary candidate complete N-glycopeptide IDs.
The experimental molecular composition fingerprint of the precursor ions was measured in the primary mass spectrum and the experimental molecular composition fingerprint of the fragment ions was measured in the secondary mass spectrum.
Theoretical molecular composition fingerprints are generated by the steps of:
calculating the molecular formula of each molecule according to a theoretical molecular library of the studied system;
and calculating corresponding molecular composition fingerprints according to the types and the numbers of the elements in the molecular formula by referring to the standard element list.
The matching in steps S3 and S5 refers to a one-to-one comparison of the m/z value and the relative peak intensity value of each isotope peak in the experimental molecular composition fingerprint with the corresponding theoretical values.
The criteria for matching in steps S3 and S5 are controlled by the isotope peak intensity cutoff value, the isotope peak mass-to-charge ratio maximum allowable error, and the isotope peak intensity maximum allowable error.
S6, targeting screening sialic acid-containing monosaccharide sequences containing structural diagnosis fragment ions from primary candidate complete N-glycopeptide IDs with identical molecular composition fingerprints, and classifying the screened primary candidate complete N-glycopeptide IDs as candidate complete N-glycopeptide IDs.
S7, carrying out random matching probability scoring (namely P score calculation) on candidate complete N-glycopeptide IDs, and finally, attributing the complete N-glycopeptide IDs meeting the preset complete N-glycopeptide spectrogram matching (practice N-glycopeptide spectrum matches, GPSMs) condition (for example, the number of fragment ions matched by a polypeptide skeleton is not less than 5,N, and the number of fragment ions matched by a connecting sugar part is not less than 1) as targeted GPSMs.
S8, obtaining the bait GPSMs in a bait library (a reverse library or a random library) according to the steps of S3-S7.
S9, combining the targeted GPSMs and the decoy GPSMs, sorting the targeted GPSMs from small to large according to the P score, selecting a threshold value P score so that false positives are not more than 1% (the calculation method is that 2 times of the number of the decoy GPSMs below the threshold value is divided by the total number of the targeted and decoy GPSMs), and performing standard deduplication on the targeted GPSMs below the threshold value P score according to the polypeptide skeleton amino acid sequence, modification, glycosylation site and N-linked carbohydrate sequence structure to obtain sialic acid-containing complete N-glycopeptide IDs.
S10, identifying characteristic oxonium ions m/z 204, sialic acid characteristic oxonium ions m/z 274 and 292 and mannose-N-acetylglucosamine disaccharide sequence characteristic oxonium ions m/z 366 of N-acetylglucosamine in the secondary mass spectrum based on isotope profile fingerprint comparison, and then comprehensively scoring according to the prior method, wherein the scoring is alpha 2,3 links less than 0.8 and alpha 2,6 links more than 0.8.
The identification and verification method of sialic acid alpha 2,3 linkage based on tandem mass spectrometry of the present invention is illustrated below with reference to an example.
S1, preparing a complete N-glycopeptide sample from human liver cancer tissues according to the existing method;
s2, separating a complete N-glycopeptide sample by high performance liquid chromatography (a base peak chromatogram is shown in figure 2) and electrospray ionization to obtain precursor ions with positive charges, and sending the precursor ions into a mass spectrometer to obtain an experimental primary mass spectrum containing precursor ion isotope profile fingerprints, wherein the experimental primary mass spectrum is shown in figure 3;
s3, matching the isotope profile fingerprint of the precursor ion with a corresponding targeting forward theoretical database, and screening out primary candidate complete N-glycopeptide IDs, VDKDLQSLEDILHQVENK _N4H2F0S1 with the same molecular composition, which meets the matching condition, as shown in figure 4; the monosaccharide composition corresponds to 1 monosaccharide sequence structure 01Y41Y41M (31M) 61M61Y41L32S, wherein Y represents N-acetylglucosamine, M represents mannose, L represents galactose, and S represents sialic acid;
s4, delivering the precursor ions into an ion trap for gas phase dissociation to obtain fragment ions, and delivering the fragment ions into a mass spectrometer to obtain a secondary mass spectrum containing the isotope profile fingerprint of the fragment ions;
s5, comparing molecular composition fingerprints of the primary candidate complete N-glycopeptide IDs with molecular composition fingerprints of theoretical fragment ions one by one to obtain all fragment ions with experimental isotope contours matched with theoretical isotope contours, and annotating a secondary mass spectrum, as shown in FIG. 5; a polypeptide skeleton pattern dissociation diagram with matching fragment ion labels, as shown in fig. 6; a graphical dissociation diagram of the N-linked sugar moiety with matching fragment ion labels, as shown in fig. 7;
s6, forming fingerprint phase from moleculesTargeting selection of sialic acid containing monosaccharide sequence containing experimental structural diagnostic fragment ions in identical primary candidate intact N-glycopeptide IDs (ions in the rectangular box in FIG. 5, 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 (FIG. 8, FIG. 9), and classifying the screened primary candidate complete N-glycopeptide IDs as candidate complete N-glycopeptide IDs;
s7, carrying out random matching probability scoring (namely P score calculation) on candidate complete N-glycopeptide IDs, and finally, attributing the candidate complete N-glycopeptide IDs which meet the preset complete N-glycopeptide spectrogram matching (practice N-glycopeptide spectrum matches, GPSMs) condition (for example, the number of fragment ions matched by a polypeptide skeleton is not less than 5,N, and the number of fragment ions matched by a connecting sugar part is not less than 1) as targeted GPSMs;
s7, obtaining bait GPSMs in a bait library (a reverse library or a random library) according to the steps of S3-S7;
s8, combining the targeting and the decoy GPSMs, sorting the targeting and the decoy GPSMs from small to large, selecting a threshold value P score so that false positives are not more than 1% (the calculation method is that 2 times of the number of the decoy GPSMs below the threshold value is divided by the total number of the targeting and the decoy GPSMs), and de-duplicating the targeting GPSMs below the threshold value P score according to the polypeptide skeleton amino acid sequence and the modification, glycosylation sites and the standard of the N-linked carbohydrate sequence structure to obtain the final complete N-glycopeptide IDs.
S9 comprehensive scoring of the intensities (intensity, I) of characteristic oxonium ions m/z 204 (from N-acetylglucosamine, glcNAc), m/z 274 (from sialic acid Neu5Ac and losing a molecule of water), m/z 292 (from sialic acid Neu5 Ac) and m/z 366 (from disaccharide Hex-GlcNAc formed by mannose and N-acetylglucosamine), i.e. (I204+I366)/(I274+I292) on the monosaccharide molecular level based on the comparison of isotopic profile fingerprints Neu5Gc-Gal-GlcNAc /n GlcNAc Here, obtainA score of 0.76; according to the above-mentioned criteria (scoring)<At 0.8, the corresponding sialic acid is an α2,3 linkage;>0.8, α2, 6), where sialic acid is α2,3.
The identification and verification method of sialic acid alpha 2,6 linkage based on tandem mass spectrometry of the present invention is illustrated below with reference to an example.
S1, preparing a complete N-glycopeptide sample from human liver cancer tissues according to the current method;
s2, separating a complete N-glycopeptide sample by high performance liquid chromatography (a base peak chromatogram is shown in figure 2) and electrospray ionization to obtain precursor ions with positive charges, and sending the precursor ions into a mass spectrometer to obtain an experimental primary mass spectrum containing precursor ion isotope profile fingerprints, wherein the experimental primary mass spectrum is shown in figure 10;
s3, matching the isotope profile fingerprint of the precursor ion with a corresponding targeting forward theoretical database, and screening out primary candidate complete N-glycopeptide ID, NNGTITWENLAAVLPFGGTFDLVQLK _N4H2F0S1 (figure 11) with the same molecular composition and meeting the matching condition; the monosaccharide composition corresponds to the 4 monosaccharide sequence structure: 01Y41Y41M (31M) 61M (21Y 41L 32S) 61Y41L,01Y41Y41M (31M 41Y 41L) 61M61Y41L32S,01Y41Y41M (31M (21Y 41L 32S) -41Y) 61M61M and 01Y41Y41M (31M 41Y41L 32S) (41Y) 61M61M; wherein Y represents N-acetylglucosamine, M represents mannose, L represents galactose, and S represents sialic acid.
S4, delivering the precursor ions into an ion trap for gas phase dissociation to obtain fragment ions, and delivering the fragment ions into a mass spectrometer to obtain a secondary mass spectrum containing the isotope profile fingerprint of the fragment ions;
s5, comparing molecular composition fingerprints of candidate complete N-glycopeptide IDs with molecular composition fingerprints of theoretical fragment ions one by one to obtain all fragment ions with experimental isotope contours matched with the theoretical isotope contours, wherein an annotation secondary mass spectrum of the fragment ions is shown in FIG. 12, a polypeptide skeleton graph dissociation diagram with matched fragment ion labels is shown in FIG. 13, and a graph dissociation diagram with matched fragment ion labels of N-linked sugar parts is shown in FIG. 13;
s6, targeting in primary candidate complete N-glycopeptide IDs with identical self-molecular composition fingerprintsScreening of sialic acid containing monosaccharide sequence experimental constructs diagnostic fragment ions (ions in the bold-faced rectangular box of fig. 12, including CII3-1+, 3,5 AI 5-1+) and a characteristic oxonium ion (fig. 15, fig. 16), and classifying the screened primary candidate complete N-glycopeptide IDs as candidate complete N-glycopeptide IDs;
s7, carrying out random matching probability scoring (namely P score calculation) on candidate complete N-glycopeptide IDs, and finally, attributing the complete N-glycopeptide IDs meeting the preset complete N-glycopeptide spectrogram matching (practice N-glycopeptide spectrum matches, GPSMs) condition (for example, the number of fragment ions matched by a polypeptide skeleton is not less than 5,N, and the number of fragment ions matched by a connecting sugar part is not less than 1) as targeted GPSMs;
s8, obtaining bait GPSMs in a bait library (a reverse library or a random library) according to the steps in S3-S7;
s9, combining the targeting and the decoy GPSMs, sorting the targeting and the decoy GPSMs from small to large according to the P score, selecting a threshold P score so that false positives are not more than 1% (the calculation method is that 2 times of the number of the decoy GPSMs below the threshold is divided by the total number of the targeting and the decoy GPSMs), and de-duplicating the targeting GPSMs below the threshold P score according to the polypeptide skeleton amino acid sequence and the modification, glycosylation site and the standard of the N-linked carbohydrate sequence structure to obtain the final complete N-glycopeptide IDs.
S10 comprehensive scoring of the intensities (intensity, I) of characteristic oxonium ions m/z 204 (from N-acetylglucosamine, glcNAc), m/z 274 (from sialic acid Neu5Ac and losing a molecule of water), m/z 292 (from sialic acid Neu5 Ac) and m/z 366 (from disaccharide Hex-GlcNAc formed by mannose and N-acetylglucosamine), i.e. (I204+I366)/(I274+I292) on the monosaccharide molecular level based on the comparison of isotopic profile fingerprints Neu5Gc-Gal-GlcNAc /n GlcNAc The fraction obtained here was 1.36; according to the above-mentioned criteria (scoring)<At 0.8, the corresponding sialic acid is an α2,3 linkage;>0.8, α2, 6), where sialic acid is α2,6.
It will be evident to those skilled in the art that the invention is not limited to the details of the foregoing illustrative embodiments, and that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. The present embodiments are, therefore, to be considered in all respects as illustrative and not restrictive, the scope of the invention being indicated by the appended claims rather than by the foregoing description, and all changes which come within the meaning and range of equivalency of the claims are therefore intended to be embraced therein. Any reference sign in a claim should not be construed as limiting the claim concerned.
Furthermore, it should be understood that although the present disclosure describes embodiments, not every embodiment is provided with a separate embodiment, and that this description is provided for clarity only, and that the disclosure is not limited to the embodiments described in detail below, and that the embodiments described in the examples may be combined as appropriate to form other embodiments that will be apparent to those skilled in the art.

Claims (8)

1. A method for identifying and verifying sialic acid linkages based on tandem mass spectrometry, comprising the steps of:
s1, preparing a complete N-glycopeptide sample from a biological sample to be analyzed;
s2, carrying out electrospray ionization on the complete N-glycopeptide sample to obtain precursor ions, and sending the precursor ions into a mass spectrometer to obtain an experimental primary mass spectrum containing precursor ion isotope profile fingerprints;
s3, matching the isotope profile fingerprint of the precursor ion with a corresponding targeting forward theoretical database, and screening primary candidate complete N-glycopeptide IDs with the same molecular composition and meeting the matching conditions;
s4, delivering the precursor ions into an ion trap for gas phase dissociation to obtain fragment ions, and delivering the fragment ions into a mass spectrometer to obtain a secondary mass spectrum containing the isotope profile fingerprint of the fragment ions;
s5, comparing and matching isotope profile fingerprints one by one of molecular composition fingerprints of the experiment and theoretical fragment ions of the primary candidate complete N-glycopeptide IDs;
s6, targetedly screening sialic acid-containing monosaccharide sequences containing structural diagnosis fragment ions from primary candidate complete N-glycopeptide IDs with identical self-molecular composition fingerprints, and classifying the screened primary candidate complete N-glycopeptide IDs as candidate complete N-glycopeptide IDs;
s7, scoring random matching probability of candidate complete N-glycopeptide IDs, and finally, attributing complete N-glycopeptide spectrogram matching conditions meeting preset conditions as targeted GPSMs;
s8, obtaining bait GPSMs in a bait library according to the steps of S3-S7;
s9, combining the targeted GPSMs with the decoy GPSMs, sorting the targeted GPSMs from small to large according to P score, selecting a threshold P score to ensure that false positive is not more than 1%, and de-duplicating the targeted GPSMs below the threshold P score to obtain sialic acid-containing complete N-glycopeptide IDs;
s10, identifying characteristic oxonium ions m/z 204, sialic acid characteristic oxonium ions m/z 274 and 292 and mannose-N-acetylglucosamine disaccharide sequence characteristic oxonium ions m/z 366 of N-acetylglucosamine in the secondary mass spectrum based on isotope profile fingerprint comparison, and then comprehensively scoring that alpha 2,3 links are less than 0.8 and alpha 2,6 links are more than 0.8.
2. The method for identifying and verifying a sialyl linkage based on tandem mass spectrometry according to claim 1, wherein the biological sample to be analyzed is a sample containing sialylated glycoproteins.
3. The method of claim 1, wherein in step S2, the whole N-glycopeptide sample is isolated prior to electrospray ionization and mass spectrometry.
4. The method of claim 1, wherein the experimental molecular composition fingerprint of the precursor ion is measured in a primary mass spectrum and the experimental molecular composition fingerprint of the fragment ion is measured in a secondary mass spectrum.
5. The method of claim 1, wherein the theoretical molecular composition fingerprint is generated by:
calculating the molecular formula of each molecule according to a theoretical molecular library of the studied system;
and calculating corresponding molecular composition fingerprints according to the types and the numbers of the elements in the molecular formula by referring to the standard element list.
6. The method of claim 1, wherein the matching in steps S3 and S5 is a one-to-one comparison of the m/z value and the relative peak intensity value of each isotope peak in the experimental molecular composition fingerprint with the corresponding theoretical value.
7. The method of claim 1, wherein the criteria for matching in steps S3 and S5 are controlled by isotope peak intensity cutoff, isotope peak mass to charge ratio maximum allowable error, and isotope peak intensity maximum allowable error.
8. The method of claim 1, wherein the de-duplication in step S9 is based on the amino acid sequence and modification of the polypeptide backbone, glycosylation sites and the criteria of the N-linked carbohydrate sequence structure.
CN202310097998.0A 2023-02-10 2023-02-10 Identification and verification method of sialic acid linkage based on tandem mass spectrum Pending CN116008382A (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202310097998.0A CN116008382A (en) 2023-02-10 2023-02-10 Identification and verification method of sialic acid linkage based on tandem mass spectrum

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202310097998.0A CN116008382A (en) 2023-02-10 2023-02-10 Identification and verification method of sialic acid linkage based on tandem mass spectrum

Publications (1)

Publication Number Publication Date
CN116008382A true CN116008382A (en) 2023-04-25

Family

ID=86030331

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202310097998.0A Pending CN116008382A (en) 2023-02-10 2023-02-10 Identification and verification method of sialic acid linkage based on tandem mass spectrum

Country Status (1)

Country Link
CN (1) CN116008382A (en)

Similar Documents

Publication Publication Date Title
DE112005001166B4 (en) Method and device for identifying proteins in mixtures
US9196466B2 (en) Mass spectrometer with bypass of a fragmentation device
US9146213B2 (en) Method and apparatus for performing retention time matching
EP1530721B1 (en) Method for characterizing biomolecules utilizing a result driven strategy
US8105838B2 (en) Generation and use of a catalog of polypeptide-related information for chemical analyses
JP5299060B2 (en) Glycopeptide structure analysis method and apparatus
US20040248317A1 (en) Glycopeptide identification and analysis
CN109725072A (en) A kind of targeting qualitative, quantitative metabonomic analysis methods of the screening biomarker for cancer based on LC-MS/MS technology
CA2628924C (en) Mass spectrometer
EP1735813B1 (en) Identification of modified peptides by mass spectrometry
CA2628927C (en) Mass spectrometer
CN112326769B (en) Method for identifying N-sugar chain branch structure on complete glycopeptide
CN111551626A (en) Cascade mass spectrometry analysis method based on molecular composition and structural fingerprint identification
JP6365863B2 (en) Database creation method for peptide peak identification and quantification in mass spectrometry
DE602004012637T2 (en) Methods and devices for the identification of biopolymers by mass spectrometry
CN116008382A (en) Identification and verification method of sialic acid linkage based on tandem mass spectrum
CN113383236A (en) Method for multi-attribute identification of unknown biological samples
CN112331269B (en) Method for constructing N-linked carbohydrate chain branch structure database aiming at sample to be detected
CN116242902A (en) Identification and verification method for glycoprotein N-linked sugar type
CN115980168A (en) Method for identifying and verifying protein sialic acid
CN117347466A (en) Method for identifying sugar chain mixture based on multistage mass spectrometry technology
Li et al. Fragment correlation mass spectrometry: determining the structures of biopolymers in a complex mixture without isolating individual components
JP2023533493A (en) Method for Identifying Glycosidic Bonds of Sialic Acids in Glycopeptides
Webb-Robertson Computational Proteomics: Session Introduction Bobbie-Jo Webb-Robertson, William Cannon, Joshua Adkins, and Deborah Gracio Pacific Symposium on Biocomputing 11: 212-218 (2006)

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination