Lipid molecular structure identification method based on tandem mass spectrometry

Abstract

The invention discloses a cascade mass spectrum-based lipid molecular structure identification method, which comprises the following steps of: identifying primary candidate lipid molecules from a theoretical lipid molecule database by utilizing accurate mass comparison of precursor ions in a primary mass spectrum in a high-efficiency cascade mass spectrum of lipid molecules; secondary candidate lipid molecules are identified through screening of head structure characteristic fragment ions in a secondary mass spectrum, and false positive control is performed through comparison and scoring of all types of fragment ions of the whole lipid molecules and synchronous searching of a targeting-bait library; the structure of the lipid molecule is comprehensively identified from a secondary mass spectrum by confirming the position of a double bond by site decisive fragment ions. According to the method, the head structure of a lipid molecule, the length of an aliphatic chain, a double bond and the position of the double bond are directly identified by utilizing efficient dissociation of the lipid aliphatic chain in a novel tandem mass spectrometry dissociation method, so that the method does not depend on a selective chemical derivatization reaction in a sample preparation stage, and the identification flux is improved.

Description

Technical Field

[0001] The invention belongs to the technical field of molecular structure analysis, and in particular relates to a lipid molecular structure identification method based on tandem mass spectrometry. Background Art

[0002] With the emergence of new tandem mass spectrometry dissociation methods (such as electron activation dissociation and ultraviolet light dissociation), the dissociation of carbon-carbon bonds in lipid fatty chains has become possible; this makes it possible to directly identify the lipid molecular structure rather than relying on double bond chemical derivatization.

[0003] There are relatively mature methods for the dissociation and identification of amino acids, the basic building blocks of proteins or polypeptides, and bases, the basic building blocks of nucleic acids, by tandem mass spectrometry; however, there is no method for the dissociation and identification of carbon atoms, the basic building blocks of lipid chains. Therefore, it is necessary to develop a method for lipid molecular structure identification based on tandem mass spectrometry dissociation. Summary of the invention

[0004] In order to solve the above technical problems, the purpose of the present invention is to provide a lipid molecular structure identification method based on tandem mass spectrometry. The method utilizes the efficient dissociation of lipid fat chains in a new tandem mass spectrometry dissociation method to directly identify the lipid molecular head structure and the length, double bonds and positions of the fat chains; thus, it does not rely on the selective chemical derivatization reaction in the sample preparation stage, thereby improving the identification throughput.

[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 lipid molecular structure based on tandem mass spectrometry comprises the following steps:

[0007] S1, reading the mass-to-charge ratio of the trapped precursor ion in the secondary mass spectrum in the mass spectrum data set of the experimental sample collected in the data-dependent mode, and finding the primary candidate lipid molecule in the mass-to-charge ratio column of the theoretical highest isotope peak in the lipid theoretical ion peak envelope database through the set mass-to-charge ratio error range;

[0008] S2, matching the isotope peak mass-to-charge ratio and relative intensity of the primary candidate lipid molecules one by one with the ion peak envelope in the primary mass spectrum obtained by back-tracing the mass-to-charge ratio of the trapped precursor ion, and the primary candidate lipid molecules whose isotope peaks above the set relative intensity cutoff value are within the set mass-to-charge ratio error range and relative intensity error range are classified as secondary candidate lipid molecules;

[0009] S3, for each secondary candidate lipid molecule, searching for characteristic head structure fragment ions in the corresponding secondary mass spectrum; secondary candidate lipid molecules with matching experimental head structure characteristic fragment ions are classified as candidate lipid molecules;

[0010] S4, searching and matching all fragment ions in the targeted theoretical ion peak envelope database of each candidate lipid molecule in the experimental secondary mass spectrum, and the experimental fragment ions whose isotope peaks above the set relative intensity cutoff value are within the set mass-to-charge ratio error range and relative intensity error range are matched experimental fragment ions; when the total number of matched experimental fragment ions is equal to or greater than the set minimum number of matched fragment ions, the candidate lipid molecule is attributed to targeted lipid spectrum match (Lipid spectrum match, LSM);

[0011] S5, scoring the random matching probability (i.e., p score) of each LSM based on the theoretical model;

[0012] S6, performing an equivalent search of the decoy theoretical ion peak envelope database according to steps S4 and S5 for each candidate lipid molecule to obtain decoy LSMs;

[0013] S7, combining the target and decoy LSMs and sorting them from small to large according to the random matching probability, and selecting a pscore threshold so that the false discovery rate (FDR) is less than 1%;

[0014] S8, remove duplicates from the targeted LSMs whose p score is less than or equal to the p score threshold to obtain the final identification results lipid IDs.

[0015] Further, in step S1, the secondary mass spectra in the mass spectrum data set of the experimental sample are captured in order from high to low according to the intensity priority of the precursor ions in the primary mass spectrum as the acquisition order. Among them, the mass spectrometry ionization method selects all ionization methods that can ionize neutral lipid molecules into positive or negative ions, such as electrospray ionization and matrix-assisted laser desorption ionization; the front end of the ionization source can be separated (such as high performance liquid chromatography separation) or not separated (such as directly injecting with a syringe under the push of a syringe pump).

[0016] Furthermore, the experimental sample is a single lipid molecule or a mixture of lipid molecules.

[0017] Furthermore, the mass-to-charge ratio error range is the relative error of the experimental value to the theoretical value, and its calculation formula is: (experimental value-theoretical value)*1e6 / theoretical value, in ppm; this parameter can be set according to different types of mass spectrometers (such as 10ppm for orbital trap mass spectrometer and 20ppm for time-of-flight mass spectrometer).

[0018] Furthermore, the unit of the relative intensity cutoff value is a percentage, and all isotope peaks in the theoretical ion isotope envelope above the relative intensity cutoff value are required to be observed experimentally.

[0019] Furthermore, the relative intensity error range is the difference between the experimental relative intensity of the isotope peak and the theoretical relative intensity; the experimental relative intensity of the isotope peak is obtained by normalizing the experimental isotope peak corresponding to the theoretical highest isotope peak as 100%.

[0020] Furthermore, the targeted theoretical ion peak envelope database is the theoretical isotope envelope information of the precursor ions of lipid molecules (including ion polarity, valence state, mass-to-charge ratio and relative intensity information of isotope peaks) and the theoretical isotope envelope information of different types of fragment ions considered (including ion polarity, valence state, mass-to-charge ratio and relative intensity information of isotope peaks).

[0021] Furthermore, the decoy theoretical ion peak envelope database is the theoretical isotope envelope information of precursor ions and fragment ions constructed after randomly shuffling the combinations of lipid molecule fat chain lengths (i.e., the number of carbon atoms) while maintaining a consistent total number of carbon atoms, such as two fat chains with lengths of 16 and 18 becoming a combination of 20 and 14.

[0022] Furthermore, the false positive rate is the relative percentage of LSMs whose p score is less than or equal to the p score threshold, and its calculation formula is: decoy LSMs*2*100% / (target LSMs+decoy LSMs).

[0023] Furthermore, the lipid molecules are common fatty acids (FAs), glycerolipids (GLs), glycerophospholipids (GPs), sphingolipids (SPs), prenol lipids (PRs) or glycolipids (SLs).

[0024] The beneficial effects of the present invention are:

[0025] The present invention utilizes the efficient dissociation tandem mass spectrometry data of lipid molecules, identifies mass spectrometry precursor ions and fragment ions based on isotope envelope search, and accurately identifies the type (head structure) and fatty chain (number and position of double bonds) of lipid molecules based on combined search of targeted-decoy databases and random matching probability scoring.

[0026] Specifically, the present invention identifies primary candidate lipid molecules from a theoretical lipid molecule database by using accurate mass comparison of precursor ions in the primary mass spectrum in the lipid molecule high-efficiency tandem mass spectrum, identifies secondary candidate lipid molecules by screening head structure characteristic fragment ions in the secondary mass spectrum, controls false positives by comparing and scoring all types of fragment ions of the entire lipid molecule and simultaneously searching the targeted-bait library, and comprehensively identifies the structure of the lipid molecule from a secondary mass spectrum by confirming the double bond position of the site-determining fragment ion pair.

[0027] Compared with the identification methods in the prior art that rely on chemical derivatization reactions in the sample preparation stage, the present invention has the most efficient characteristics, improves the identification throughput, and is suitable for rapid structure-specific analysis of large cohorts of samples, such as different ion polarity combinations (such as using positive and negative ion modes simultaneously), multiple dissociation method combinations (such as using collision-based and electron-based dissociation methods simultaneously), or multiple multi-stage tandem mass spectrometry combinations (such as using secondary and tertiary tandem mass spectrometry simultaneously). 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 drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments recorded in the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0029] Figure 1 The present invention is a method flow chart of a lipid molecular structure identification method based on tandem mass spectrometry.

[0030] Figure 2 The identification of lipid molecule PC (19:0 / 18:2 (9Z, 12Z)) in an embodiment of the present invention is shown; wherein, (2A) is a comparison of the experimental isotope envelope isotope peak (vertical line) of the precursor ion with the corresponding theoretical peak (point); (2B) is a secondary mass spectrum with annotations of the head structure and experimental matching fragment ions of the fatty chain; (2C) is a graphic dissociation diagram of the sn1 fatty chain with annotations of the experimental matching fragment ions; (2D) is a graphic dissociation diagram of the sn2 fatty chain with annotations of the experimental matching fragment ions.

[0031] Figure 3The identification of lipid molecule PC (18:0 / 22:6 (4Z, 7Z, 10Z, 13Z, 16Z, 19Z)) in the present invention is shown; wherein, (3A) is a comparison of the experimental isotope envelope isotope peak (vertical line) of the precursor ion with the corresponding theoretical peak (point); (3B) is a secondary mass spectrum with annotations of the head structure and experimental matching fragment ions of the fatty chain; (3C) is a graphic dissociation diagram of the sn1 fatty chain with annotations of the experimental matching fragment ions; (3D) is a graphic dissociation diagram of the sn2 fatty chain with annotations of the experimental matching fragment ions.

[0032] Figure 4 The identification of the lipid molecule PC (18:2 (9Z, 12Z) / 18:2 (9Z, 12Z)) in an embodiment of the present invention is shown; wherein, (4A) is a comparison of the experimental isotope envelope isotope peak (vertical line) of the precursor ion with the corresponding theoretical peak (point); (4B) is a secondary mass spectrum with annotations of the head structure and experimental matching fragment ions of the fatty chain; (4C) is a graphical dissociation diagram of the sn1 fatty chain with annotations of the experimental matching fragment ions; (4D) is a graphical dissociation diagram of the sn2 fatty chain with annotations of the experimental matching fragment ions.

[0033] Figure 5 The identification of the lipid molecule LPC(22:6(4Z,7Z,10Z,13Z,16Z,19Z) in the embodiment of the present invention is shown; wherein, (5A) is a comparison of the experimental isotope envelope isotope peak (vertical line) of the precursor ion with the corresponding theoretical peak (point); (5B) is a secondary mass spectrum with annotation of the head structure and the experimental matching fragment ions of the fatty chain; (5C) is a graphical dissociation diagram of the sn1 fatty chain with annotation of the experimental matching fragment ions.

[0034] Figure 6 The identification of the lipid molecule DG(16:0 / 20:2(11Z,14Z) in the embodiment of the present invention is shown; wherein, (6A) is a comparison of the experimental isotope envelope isotope peak (vertical line) of the precursor ion with the corresponding theoretical peak (point); (6B) is a secondary mass spectrum with annotations of the head structure and the experimental matching fragment ions of the fatty chain; (6C) is a graphic dissociation diagram of the sn1 fatty chain with annotations of the experimental matching fragment ions; (6D) is a graphic dissociation diagram of the sn2 fatty chain with annotations of the experimental matching fragment ions.

[0035] Figure 7The identification of the lipid molecule TG (16:0 / 18:1(9Z) / 18:1(9Z)) in an embodiment of the present invention is shown; (7A) is a comparison of the experimental isotope envelope isotope peak (vertical line) of the precursor ion with the corresponding theoretical peak (point); (7B) is a secondary mass spectrum with annotations of the head structure and experimental matching fragment ions of the fatty chain; (7C) is a graphic dissociation diagram of the sn1 fatty chain with annotations of the experimental matching fragment ions; (7D) is a graphic dissociation diagram of the sn2 fatty chain with annotations of the experimental matching fragment ions; (7E) is a graphic dissociation diagram of the sn3 fatty chain with annotations of the experimental matching fragment ions. DETAILED DESCRIPTION

[0036] The technical solutions in the present invention will be described clearly and completely below in conjunction with specific embodiments. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0037] like Figure 1 As shown, a lipid molecular structure identification method based on tandem mass spectrometry specifically comprises the following steps:

[0038] S1, reading the mass-to-charge ratio of the trapped precursor ion in the secondary mass spectrum in the mass spectrum data set of the experimental sample collected in the data-dependent mode, and finding the primary candidate lipid molecule in the mass-to-charge ratio column of the theoretical highest isotope peak in the lipid theoretical ion peak envelope database through the set mass-to-charge ratio error range;

[0039] In step S1, the secondary mass spectra in the mass spectrum data set of the experimental sample are captured in order from high to low according to the intensity priority of the precursor ions in the primary mass spectrum as the acquisition order. Among them, the mass spectrometry ionization method selects all ionization methods that can ionize neutral lipid molecules into positive or negative ions, such as electrospray ionization and matrix-assisted laser desorption ionization; the front end of the ionization source can be separated (such as high performance liquid chromatography separation) or not separated (such as direct injection with a syringe driven by a syringe pump);

[0040] The experimental sample is a single lipid molecule or a mixture of lipid molecules, and the lipid molecule is common fatty acids (FAs), glycerolipids (GLs), glycerophospholipids (GPs), sphingolipids (SPs), prenol lipids (PRs) or glycolipids (SLs);

[0041] The mass-to-charge ratio error range is the relative error of the experimental value to the theoretical value, and its calculation formula is: (experimental value-theoretical value)*1e6 / theoretical value, in ppm; this parameter can be set according to different types of mass spectrometers (such as 10ppm for orbital trap mass spectrometer and 20ppm for time-of-flight mass spectrometer).

[0042] S2, matching the isotope peak mass-to-charge ratio and relative intensity of the primary candidate lipid molecules one by one with the ion peak envelope in the primary mass spectrum obtained by back-tracing the mass-to-charge ratio of the trapped precursor ion, and the primary candidate lipid molecules whose isotope peaks above the set relative intensity cutoff value are within the set mass-to-charge ratio error range and relative intensity error range are classified as secondary candidate lipid molecules;

[0043] The unit of the relative intensity cutoff value is a percentage, and all isotope peaks in the theoretical ion isotope envelope above the relative intensity cutoff value are required to be observed experimentally;

[0044] The relative intensity error range is the difference between the experimental relative intensity of the isotope peak and the theoretical relative intensity; the experimental relative intensity of the isotope peak is obtained by normalizing the experimental isotope peak corresponding to the theoretical highest isotope peak as 100%.

[0045] S3, for each secondary candidate lipid molecule, search for characteristic head structure fragment ions in the corresponding secondary mass spectrum; secondary candidate lipid molecules with matching experimental head structure characteristic fragment ions are classified as candidate lipid molecules.

[0046] S4, searching and matching all fragment ions in the targeted theoretical ion peak envelope database of each candidate lipid molecule in the experimental secondary mass spectrum, and the experimental fragment ions whose isotope peaks above the set relative intensity cutoff value are within the set mass-to-charge ratio error range and relative intensity error range are matched experimental fragment ions; when the total number of matched experimental fragment ions is equal to or greater than the set minimum number of matched fragment ions, the candidate lipid molecule is attributed to targeted lipid spectrum match (Lipid spectrum match, LSM);

[0047] Among them, the targeted theoretical ion peak envelope database is the theoretical isotope envelope information of the precursor ions of lipid molecules (including ion polarity, valence state, mass-to-charge ratio and relative intensity information of isotope peaks) and the theoretical isotope envelope information of different types of fragment ions considered (including ion polarity, valence state, mass-to-charge ratio and relative intensity information of isotope peaks).

[0048] S5, scoring the random matching probability (ie, p score) of each LSM based on the theoretical model.

[0049] S6, performing an equivalent search of the decoy theoretical ion peak envelope database according to steps S4 and S5 for each candidate lipid molecule to obtain decoy LSMs;

[0050] Among them, the decoy theoretical ion peak envelope database is the theoretical isotope envelope information of precursor ions and fragment ions constructed after randomly disrupting the combination of lipid molecule fat chain lengths (that is, the number of carbon atoms) under the precursor maintaining the same total number of carbon atoms, such as two fat chains with lengths of 16 and 18 becoming a combination of 20 and 14.

[0051] S7, combining the target and decoy LSMs and sorting them from small to large according to the random matching probability, and selecting a pscore threshold so that the false discovery rate (FDR) is less than 1%;

[0052] The false positive rate is the relative percentage of LSMs whose p score is less than or equal to the p score threshold, and its calculation formula is: decoy LSMs*2*100% / (target LSMs+decoy LSMs).

[0053] S8, remove duplicates from the targeted LSMs whose p score is less than or equal to the p score threshold to obtain the final identification results lipid IDs.

[0054] The lipid molecular structure identification method based on tandem mass spectrometry of the present invention is illustrated below with reference to an embodiment, namely the structure-specific identification of lipids in plasma.

[0055] The lipid molecular structure identification method based on tandem mass spectrometry of this embodiment comprises the following steps:

[0056] (1) collecting a mass spectrum data set of the experimental sample in a data-dependent mode, reading the mass-to-charge ratio of the trapped precursor ion m / z 800.61471 (z=1) in the 1762nd secondary mass spectrum of the mass spectrum data set, and finding multiple primary candidate lipid molecules in the mass-to-charge ratio column of the theoretical highest isotope peak in the lipid theoretical ion peak envelope database by using a set mass-to-charge ratio error range (±20 ppm);

[0057] (2) The primary candidate lipid molecules are matched one by one with the ion peak envelope in the primary mass spectrum obtained by back-tracing the mass-to-charge ratio of the trapped precursor ion, and the isotope peaks above the set relative intensity cutoff value (40%) are within the set mass-to-charge ratio error range (±20 pp m) and relative intensity error range (±50%) ( Figure 2 A) is reduced to a secondary candidate lipid molecule with a molecular formula of C45H86NO8P;

[0058] (3) For each secondary candidate lipid molecule, search for characteristic head structure fragment ions in the corresponding secondary mass spectrum; secondary candidate lipid molecules with matching experimental head structure characteristic z and a ions (as listed below) are classified as phosphatidylcholine (PC) molecules;

[0059] Matching experimental head structure features z and a ions:

[0060] Sn-1 head structure fragment z ion:

[0061] dz-sn1-acyl*[H]_1(519.3312); dz-sn1-acyl[H]_1(520.3371);

[0062] dz-sn1-gCO*[H]_1(503.3336); dz-sn1-gCO[H]_1(502.3322); dz-sn1-gCC[H]_1(489.3197);

[0063] Sn-2 head structure fragment z ion:

[0064] dz-sn2-acyl*[H]_1(537.3688); dz-sn2-acyl[H]_1(538.3921);

[0065] dz-sn2-gCO*[H]_1(521.3833); dz-sn2-gCO[H]_1(520.3735);

[0066] Sn-3 head structure fragment a ion:

[0067] PC-fi1=C5H12O3NP[H]_1(166.0603); PC-fi2=C5H14O4NP[H]_1(184.0731); PC-fi3=C7H16O4NP[H]_1(210.0867); PC-fi4=C8H16O4NP[H]_1(222.0864); PC-fi5=C8H18O4NP[H]_1(224.1035); PC-fi6=C7H16O5NP[H]_1(226.0837);

[0068] (4) All fragment ions in the targeted theoretical ion peak envelope database of each candidate lipid molecule are searched and matched in the experimental secondary mass spectrum. The experimental fragment ions whose isotope peaks above the set relative intensity cutoff value are within the set mass-to-charge ratio error range (±20ppm) and relative intensity error range (±50%) are matched experimental fragment ions; the candidate lipid molecule PC (19:0 / 18:2 (9Z, 12Z)) that meets the pre-set matching conditions is attributed to the targeted lipid spectrum match (Lipid spectrum match, LSM);

[0069] (5) Based on the theoretical model, the random matching probability of the above LSM was scored, and the corresponding bait library was searched. After controlling the false positive rate to be less than or equal to 1%, the lipid molecule ID PC(19:0 / 18:2(9Z,12Z)) was obtained; among them, the positions of the 9Z and 12Z double bonds were confirmed by ion pairs z8-z10 and z11-z13, respectively.

[0070] The lipid molecule PC (19:0 / 18:2 (9Z, 12Z)) identified above contains two carbon-carbon double bonds in one of the aliphatic chains; further, the representative lipid molecule PC (18:0 / 22:6 (4Z, 7Z, 10Z, 13Z, 16Z, 19Z)) containing six carbon-carbon double bonds in one aliphatic chain is identified as follows: Figure 3 The identification of the representative lipid molecule PC (18:2 (9Z, 12Z) / 18:2 (9Z, 12Z)) containing two carbon-carbon double bonds on each chain is shown in Figure 4 shown.

[0071] In addition to the above-mentioned PC lipid molecules, the lipid molecular structure identification method based on tandem mass spectrometry of the present invention can also identify lysophosphatidylcholine (LPC; Figure 5 ), diacylglycerol (DG, DAG; Figure 6 ), triacylglycerol (TG; Figure 7 ) and other types of lipid molecules were identified.

[0072] The present invention aims to identify primary candidate lipid molecules from a theoretical lipid molecule database by accurate mass comparison of precursor ions in a primary mass spectrum in an efficient tandem mass spectrometer of lipid molecules, identify secondary candidate lipid molecules by screening head structure characteristic fragment ions in a secondary mass spectrum, control false positives by comparison, scoring and simultaneous search of targeted-bait libraries of all types of fragment ions of the entire lipid molecule, and comprehensively identify the structure of lipid molecules from a secondary mass spectrum by confirming the double bond position of site-determined fragment ions; the present invention is more efficient than existing chemical derivatization or combined tandem mass spectrometry methods, is suitable for rapid structure-specific analysis of large cohort samples, and will rapidly promote related structural analysis, functional research and biomedical applications.

[0073] 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 present invention can be implemented in other specific forms without departing from the spirit or essential features of the present invention. Therefore, the embodiments should be considered exemplary and non-restrictive in all respects, and the scope of the present invention is defined by the appended claims rather than the above description, and it is intended that all changes falling within the meaning and scope of the equivalent elements of the claims be included in the present invention.

[0074] In addition, it should be understood that although the present specification is described according to implementation modes, not every implementation mode contains only one independent technical solution. This description 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 may also be appropriately combined to form other implementation modes that can be understood by those skilled in the art.

Claims

1. A method for identifying lipid molecular structure based on tandem mass spectrometry, characterized in that: The steps include: S1, reading the mass-to-charge ratio of the trapped precursor ion in the secondary mass spectrum in the mass spectrum data set of the experimental sample collected in the data-dependent mode, and finding the primary candidate lipid molecule in the mass-to-charge ratio column of the theoretical highest isotope peak in the lipid theoretical ion peak envelope database through the set mass-to-charge ratio error range; S2, matching the isotope peak mass-to-charge ratio and relative intensity of the primary candidate lipid molecules one by one with the ion peak envelope in the primary mass spectrum obtained by back-tracing the mass-to-charge ratio of the trapped precursor ion, and the primary candidate lipid molecules whose isotope peaks above the set relative intensity cutoff value are within the set mass-to-charge ratio error range and relative intensity error range are classified as secondary candidate lipid molecules; S3, for each secondary candidate lipid molecule, searching for characteristic head structure fragment ions in the corresponding secondary mass spectrum; secondary candidate lipid molecules with matching experimental head structure characteristic fragment ions are classified as candidate lipid molecules; S4, searching and matching all fragment ions in the targeted theoretical ion peak envelope database of each candidate lipid molecule in the experimental secondary mass spectrum, and the experimental fragment ions whose isotope peaks above the set relative intensity cutoff value are within the set mass-to-charge ratio error range and relative intensity error range are matched experimental fragment ions; when the total number of matched experimental fragment ions is equal to or greater than the set minimum number of matched fragment ions, the candidate lipid molecule is attributed to targeted lipid spectrum matching, i.e., LSM; S5, scoring the random matching probability of each LSM, i.e., p score, based on the theoretical model; S6, performing an equivalent search of the decoy theoretical ion peak envelope database according to steps S4 and S5 for each candidate lipid molecule to obtain decoy LSMs; S7, combining the targeted and decoy LSMs and sorting them from small to large according to the random matching probability, and selecting a pscore threshold so that the false positive rate is less than 1%; S8, remove duplicates from the targeted LSMs whose p score is less than or equal to the p score threshold to obtain the final identification results lipid IDs.

2. The lipid molecular structure identification method based on tandem mass spectrometry according to claim 1, characterized in that: In step S1, the secondary mass spectra in the mass spectrum data set of the experimental sample are captured and acquired in sequence from high to low according to the intensity priority of the precursor ions in the primary mass spectrum as the acquisition order.

3. The lipid molecular structure identification method based on tandem mass spectrometry according to claim 1, characterized in that: The experimental sample is a single lipid molecule or a mixture of lipid molecules.

4. The lipid molecular structure identification method based on tandem mass spectrometry according to claim 1, characterized in that: The mass-to-charge ratio error range is the relative error of the experimental value with respect to the theoretical value, and its calculation formula is: (experimental value-theoretical value)*1e6 / theoretical value, and the unit is ppm.

5. The lipid molecular structure identification method based on tandem mass spectrometry according to claim 1, characterized in that: The unit of the relative intensity cutoff value is percentage, and all isotope peaks in the theoretical ion isotope envelope above the relative intensity cutoff value are required to be observed experimentally.

6. The lipid molecular structure identification method based on tandem mass spectrometry according to claim 1, characterized in that: The relative intensity error range is the difference between the experimental relative intensity of the isotope peak and the theoretical relative intensity; the experimental relative intensity of the isotope peak is obtained by normalizing the experimental isotope peak corresponding to the theoretical highest isotope peak as 100%.

7. The method for lipid molecular structure identification based on tandem mass spectrometry according to claim 1, characterized in that: The targeted theoretical ion peak envelope database includes theoretical isotope envelope information of precursor ions of lipid molecules and theoretical isotope envelope information of different types of fragment ions under consideration.

8. The method for lipid molecular structure identification based on tandem mass spectrometry according to claim 1, characterized in that: The decoy theoretical ion peak envelope database is the theoretical isotope envelope information of precursor ions and fragment ions constructed after randomly disrupting the combination of lipid molecule fat chain lengths while maintaining a consistent total number of carbon atoms.

9. The lipid molecular structure identification method based on tandem mass spectrometry according to claim 1, characterized in that: The calculation formula of the false positive rate is: bait LSMs*2*100% / (target LSMs+bait LSMs).

10. The lipid molecular structure identification method based on tandem mass spectrometry according to claim 1, characterized in that: The lipid molecule is a fatty acid, a glycerolipid, a glycerophospholipid, a sphingolipid, a prenol lipid or a glycolipid.

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