Method for determining chemical structure of lipid and ion mobility-tandem mass spectrometer

Abstract

To relate to a mass spectrometry field and particularly to provide a method for determining a chemical structure of a lipid and an ion mobility-tandem mass spectrometer.SOLUTION: A method for determining a chemical structure of a lipid includes: an ionization step of ionizing a sample to obtain sample ions; an ion mobility-based separation step of separating target lipid ions from the sample ions, based on ion mobility; a first dissociation step of dissociating a target lipid ions with dissociation energy adjusted to break a first chemical bond of the target lipid ions; a mass-based selection step of selecting the target lipid ions, whose first chemical bond is broken, based on a mass number to obtain fragment ions; a second dissociation step of dissociating the fragment ions to at least break a second chemical bond of the fragment ions which has bond energy higher than the first chemical bond, to obtain diagnostic ions; and a mass analysis step of performing a mass analysis on the diagnostic ions.SELECTED DRAWING: Figure 1

Description

[Technical field]

[0001] The present invention relates to the field of mass spectrometry, and in particular to a method for identifying the chemical structures of lipids and an ion mobility spectrometry tandem mass spectrometer. [Background technology]

[0002] Lipids are important nutrients and important components of living cells, and are closely related to some important immune functions and metabolic defects. Currently, the Lipid Metabolites and Pathways Strategy (LIPID MAPS) has been launched to promote lipid omics research by establishing a classification database.

[0003] Complete lipid labeling and identification information includes classification, elemental composition, size and position of R-groups (sn-positions), number and position of double bonds, and cis-trans isomeric orientation of double bonds. For unsaturated lipids with a glycerol backbone, e.g., glycerides and glycerophospholipids, it is also necessary to further identify the sn-positions in the fatty chains that contain carbon-carbon double bonds.

[0004] Multi-stage tandem mass spectrometry plays an important role in the structural analysis of compounds. Hsu and Turk proposed a pseudo-tertiary (MS3) tandem mass spectrometry method that can identify the sn-position of glycerophospholipids. First, they used in-source CID to obtain a high abundance headgroup-free fragment ion signal [M+Li-183]. +and furthermore, collision-induced dissociation is performed on the fragment ions to generate sn-diagnostic ions (Non-Patent Document 1). In Non-Patent Document 2, a combined MS3 method combining hybrid collision activation and UV-visible spectrophotometry was provided to identify both double bond positions and sn positions for unsaturated lipids with a glycerol backbone with a single sample introduction, but the abundance of diagnostic ions is still to be improved. These pseudo MS3 and MS3 analysis methods are not suitable for isomer selection and analysis, and when multiple ions are selected in the first stage by quadrupole mass spectrometry, the current non-target ion portion is lost, resulting in a low overall duty ratio. In addition to directly performing multistage tandem mass spectrometry on a compound, the analyte may be modified in advance by chemical derivatization to achieve the objectives of improving ionization efficiency, expanding structural differences, and improving chromatographic behavior, and then performing multistage tandem mass spectrometry on the derivative. In Non-Patent Document 3, Ma et al. precisely identified the C=C and sn positions in derivatized glycerophospholipids using charge label derivatization and MS3. The method provided by the paper requires mass selection of specific parent ions using an ion trap mass spectrometer, the duty ratio and resolution of which are limited by the ion trap mass spectrometer.

[0005] Ion mobility spectroscopy can realize high-throughput separation and analysis of isomers under gas-phase conditions, and when combined with mass spectrometry, it has high resolution and has an effect similar to multi-stage tandem mass spectrometry (Non-Patent Document 4; Non-Patent Document 5), which can obtain detailed structural information of analytes, and is widely used in structural identification of metabolic omics, glyco omics, and proteomics. For example, according to a large number of research reports, the combination of cyclic ion mobility (cIM) and collision-induced dissociation (CID) can realize separation and measurement of carbohydrate isomers from scratch (Non-Patent Document 6; Non-Patent Document 7), for example, by cIM-CID-cIM and cIM-CID-cIM-CID-cIM modes. Bleiholder's team proposed a combination of tandem mobility spectrometry and tandem mass spectrometry based on trapped ion mobility (TIMS), which has functions such as mobility selection and collision activation (Patent Document 1, Non-Patent Document 8), and realized structural identification of polypeptides and proteins (Non-Patent Document 9; Non-Patent Document 10), and sugars and their isomers (Non-Patent Document 11). Nicholas B. Borotto et al. realized pre-transfer collision-induced unfolding based on TIMS, and were able to rapidly identify protein conformations (Non-Patent Document 12). In addition, pre-transfer collision-induced unfolding can be further combined with tandem mass spectrometry to realize accurate protein sequencing (Non-Patent Document 13).

[0006] In recent years, ion mobility mass spectrometry has also achieved good results in the deep identification of lipid structures (Non-Patent Document 14; Non-Patent Document 15; Non-Patent Document 16). Baker and colleagues have proposed a lipid omics analysis method that combines reversed-phase liquid chromatography and ion mobility mass spectrometry, which can separate lipids and their isomers in three dimensions: analyte polarity, structure, and mass-to-charge ratio magnitude, and increase the peak capacity. Liquid chromatography can realize the separation of different types of lipids, and different types of lipids form different ion trend lines in ion mobility mass spectrometry. In addition, crude separation of different subclasses of lipids (Non-Patent Document 17) can be further observed in the ion mobility spectrum. Due to the limitations of the instrument resolution, different lipid isomers only reach shoulder peak separation. F. Fernandez-Lima et al. realized the identification of double bond positional isomerism of sodium phosphatidylcholine ion adducts and cis-trans isomerism of double bonds of proton adducts, respectively, with specific instrument parameters (average resolution exceeds 320) by high-resolution TIMS. In addition, when the instrument parameters meet the conditions of ultra-high resolution (over 410), the method can be used to identify sn-positional isomers (Non-Patent Document 18). However, the resolution of most commercialized ion mobility instruments is less than 200, making it difficult to meet the above conditions. Forming metal ion adducts contributes to improving isomer resolution. M. Groessl et al. realized the differentiation of positional isomerism, cis-trans isomerism, and sn-isomerism of double bonds of phosphatidylcholine silver ion adducts using drift time ion mobility mass spectrometry (Non-Patent Document 19). Similarly, Yan et al. realized the separation and analysis of cis-trans isomers of carbon-carbon double bonds of monovalent copper ion adducts of phosphatidylcholine using drift time ion mobility mass spectrometry, but the degree of resolution was low (Non-Patent Document 20).

[0007] Many methods for identifying lipid isomers based on ion mobility spectra have been developed, but these methods require standard samples as references, making it difficult to analyze the structure of unknown compounds, and the resolution of ion mobility spectra is inferior to that of mass spectra, which has limitations in the analysis of complex substrate samples. Combining the separation ability of ion mobility with the resolution ability of tandem mass spectrometry has brought great possibilities for the structural analysis of unknown lipid compounds in biological samples. Brodbelt's team was able to measure lipid isomer collision cross sections and identify lipid double bonds and cyclopropane positions by combining UVPD and drift time ion mobility mass spectrometry (Non-Patent Document 21). However, although this method is suitable for polar lipid analysis, it is difficult to detect medium and low polarity lipids. Xia et al. were able to realize the separation and analysis of conjugated fatty acid isomers by combining the PB reaction with a trapped ion mobility tandem mass spectrometry instrument. By combining the ion mobility spectrum with the tandem mass spectrometry spectrum, the mobility characteristic peaks and double bonds can be accurately assigned even under conditions without standard samples. However, the reaction products of PB reaction with conjugated fatty acids are diverse, and the mobility spectrum produced is highly complex, limiting its application in complex mixtures (Non-Patent Document 22). In 2023, Xia's team combined liquid chromatography, ion mobility spectroscopy and PB reaction tandem mass spectrometry to establish a series of lipid structure deep analysis flows, which can realize the identification of double bond positions and sn-positions step by step, and successfully applied the analysis of lipid omics in biological samples such as bovine liver and cells to obtain more complete lipid profile information (Non-Patent Document 23). However, the step-by-step pretreatment and multiple batch analysis process not only reduces the analysis throughput, but also causes sample loss. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] US10794861 B2

Patent Document 2

Non-Patent Document

[0009]

Non-Patent Document 1

Non-Patent Document 2

Non-Patent Document 3

Non-Patent Document 4

Non-Patent Document 5

Non-Patent Document 6

Non-Patent Document 7

Non-Patent Document 8

Non-Patent Document 9

Non-Patent Document 10

Non-Patent Document 11

[0010] In view of the above problems, the present invention provides a method for identifying the chemical structure of lipids and an ion mobility spectrometry tandem mass spectrometer that increases the abundance of diagnostic ions and has good duty ratio and resolution. [Means for solving the problem]

[0011] A first aspect of the present application is a method for detecting a concentration of a sample by an ionization step, comprising: ionizing a sample to obtain sample ions; an ion mobility separation step of separating the target lipid class ions from the sample ions based on ion mobility; a first dissociation step of dissociating the target lipid ion with a dissociation energy suitable for cleaving a first chemical bond of the target lipid ion; A mass selection step of selecting target lipid ions in which the first chemical bond has been broken based on the mass number to obtain fragment ions; a second dissociation step of dissociating the fragment ions and breaking at least a second chemical bond of the fragment ions, the second chemical bond having a higher bond energy than the first chemical bond, to obtain diagnostic ions; and performing mass spectrometry on the diagnostic ions.

[0012] The lipids are unsaturated lipids having a carbon-carbon double bond in the fatty chain, and the method may be used to identify the position of the carbon-carbon double bond in the fatty chain and the position of the sn in the fatty chain.

[0013] The method may further include a derivatization reaction step prior to the ionization step, in which the carbon-carbon double bond is labeled by a derivatization reaction.

[0014] The lipids may be phospholipids or sphingolipids, the first chemical bond being a bond to a polar head group of a phospholipid or a polar head group of a sphingolipid, and the second chemical bond being a chemical bond obtained via a derivatization reaction of a carbon-carbon double bond.

[0015] The derivatization reaction may be an aziridination reaction, an epoxidation reaction, a Paterno-Buchi reaction, a singlet oxygen-ene reaction or a Diels-Alder reaction.

[0016] The lipids may be fatty acyls, glycerides, glycerophospholipids, sphingolipids, sterol esters, pregnenolone lipids, glycolipids or polyketides.

[0017] The method may further include a first pre-scan step of performing mass analysis on sample ions that have not undergone the first dissociation step and the second dissociation step.

[0018] The method may further include a second pre-scan step in which mass analysis is performed on the sample ions that have only been dissociated once.

[0019] A second aspect of the present application further provides an ion mobility spectrometry tandem mass spectrometry device, including an ion source, an ion mobility spectrometer, a first dissociation device, a mass filter, a second dissociation device and a mass analyzer. Wherein, the ion source ionizes a sample to obtain sample ions. The ion mobility spectrometer separates target lipid ions from the sample ions. The first dissociation device dissociates the target lipid ions, wherein the dissociation energy of the first dissociation device is suitable for breaking a first chemical bond of the target lipid ions. The mass filter selects the target lipid ions whose first chemical bond has been broken to obtain fragment ions. The second dissociation device dissociates the fragment ions and breaks at least a second chemical bond of the fragment ions, the binding energy of which is higher than that of the first chemical bond, to obtain diagnostic ions. The mass analyzer performs mass analysis on the diagnostic ions.

[0020] The first dissociation device may be a collision induced dissociation device, and the end electrode voltage of the first dissociation device may be 10-70 eV. The second dissociation device may be a collision induced dissociation device, and the dissociation energy of the second dissociation device may be 30-70 eV. Effect of the Invention

[0021] <Useful effects> Accurate ion selection using ion mobility to select various target lipid ions at a high duty ratio and then used for subsequent multiplexed target tandem mass spectrometry to reduce the complexity of a single spectrogram. First, a first chemical bond with a relatively weak binding energy is cut in the target lipid ions to remove interfering groups linked through the first chemical bond in the target lipid ions, and then a pseudo multi-step collision-induced dissociation is performed to select fragment ions from which the interfering groups have been removed and perform secondary dissociation. This improves the mass spectrometry signal intensity of the diagnostic ions finally generated, and alleviates or avoids the problem of increased spectrogram complexity due to the appearance of spectrum peaks related to interfering groups in the final mass spectrogram. As a result, the spectrogram is simplified, making it easier to analyze and determine the chemical structure of lipids. [Brief description of the drawings]

[0022] [Figure 1] 1 is a flow chart of a method for identifying the chemical structures of lipids according to an embodiment of the present invention. [Diagram 2] FIG. 1 is a schematic diagram of a system for implementing a method for identifying the chemical structures of lipids according to an embodiment of the present invention. [Diagram 3] FIG. 1 is a schematic diagram of the structure of a preferred ion mobility spectrometry tandem mass spectrometer according to an embodiment of the present invention. [Figure 4] FIG. 2 is a diagram showing a specific reaction process of PC (18:1 / 16:0) according to the present embodiment. [Diagram 5] FIG. 1 is a diagram illustrating the principle of why the sn position of a carbon-carbon double bond can be identified from a diagnostic ion with mass number m / z=290 or 360 [M+Na+]. [Figure 6] FIG. 1 is a comparison diagram of measurement results obtained by a general tandem mass spectrometry (MS2) method in the prior art and measurement results obtained by a method according to an embodiment of the present invention. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0023] The technical solutions in the embodiments of the present invention will be described below clearly and completely in accordance with the drawings in the embodiments of the present invention, but it is clear that the described embodiments are only some of the embodiments of the present invention, and are not all of the embodiments. Based on the embodiments of the present invention, all other embodiments that a person skilled in the art can obtain without creative labor all belong to the protection scope of the present invention.

[0024] The method of identifying the chemical structure of lipids according to the present embodiment can be applied to identify lipids with interfering groups (e.g., polar head groups), which may be, for example, fatty acyls, glycerides, glycerophospholipids, sphingolipids, sterol esters, pregnenolone lipids, glycolipids, or polyketides.

[0025] In the method for identifying the chemical structure of lipids according to the present embodiment, first, the first chemical bond with a relatively weak binding energy in the target lipid ion is cut to remove the interfering group linked through the first chemical bond in the target lipid ion, and then the fragment ion from which the interfering group has been removed is selected and subjected to secondary dissociation. Through the above method, the mass spectrometry signal intensity of the diagnostic ion finally generated can be improved, and the problem of the increased complexity of the spectrogram due to the spectrum peaks related to the interfering group appearing in the final mass spectrogram can be alleviated or avoided. As a result, the spectrogram can be simplified, and the analysis and determination of the chemical structure of lipids can be facilitated.

[0026] Fig. 1 is a flow chart of the method for identifying the chemical structure of lipids according to the first embodiment of the present invention. Referring to Fig. 1, the method according to the present embodiment includes an ionization step S1, an ion mobility separation step S2, a first dissociation step S3, a mass selection step S4, a second dissociation step S5, and a mass analysis step S6, which are performed in sequence.

[0027] In an ionization step S1, a sample is ionized to obtain sample ions.

[0028] In the ion mobility separation step S2, target lipid ions are separated from the sample ions based on ion mobility.

[0029] In a first dissociation step S3, the target lipid ion is dissociated with a dissociation energy suitable for cleaving a first chemical bond of the target lipid ion.

[0030] The first dissociation of the target lipid ion, i.e., the first dissociation step S3, makes it possible to selectively cleave a chemical bond in the target lipid ion that has a weak binding energy and may ultimately generate an interference signal (i.e., the first chemical bond that binds to an interfering group).

[0031] The first dissociation step S3 is a selective dissociation step, i.e., when the first chemical bond is broken, the integrity of the other chemical bonds (main chain chemical bonds, especially the second chemical bond) is maintained as much as possible. Specifically, the dissociation energy can be set to be slightly higher than the threshold at which the first chemical bond can be broken, so as to satisfy the above requirements. In some embodiments, when the first chemical bond is the one with the lowest bond energy in the target lipid ion, the dissociation energy can be set to mainly break the first chemical bond, while maintaining the integrity of each of the other chemical bonds to the maximum extent.

[0032] By setting the dissociation energy to be equal to or greater than the threshold at which the first chemical bond can be broken, the first chemical bond can be selectively broken, thereby eliminating interference from the interfering group with the final mass spectrometry test results, with only a small loss of ion abundance.

[0033] In the mass selection step S4, target lipid ions whose first chemical bond has been cleaved are selected based on their mass numbers to obtain fragment ions.

[0034] The first chemical bond can be selectively broken to produce at least one pair of ions, one of which has an interfering group (e.g., a polar head group) and one of which does not have the interfering group. The fragment ions selected in the mass selection step S4 are fragment ions that are linked by the first chemical bond and have eliminated the interfering group, i.e., ions that do not have the interfering group.

[0035] In a second dissociation step S5, the fragment ions are dissociated and at least a second chemical bond of the fragment ions, the second chemical bond having a higher bond energy than the first chemical bond, is broken to obtain diagnostic ions.

[0036] The resulting fragment ions free of interfering groups are further dissociated by secondary dissociation, i.e., by a second dissociation step S5. The second dissociation step S5 may be selective or non-selective. However, in either case, the mass spectrogram will not show excessive spectral peaks related to interfering groups, since the interfering groups have already been removed in the first dissociation step S3 and the mass selection step S4. Therefore, there will be fewer spectral peaks in the mass spectrogram, and the mass peaks of the diagnostic ions will be more intense.

[0037] Typically, when using the same type of dissociation device, the dissociation energy employed in the second dissociation step S5 is higher than that employed in the first dissociation step S3, thereby enabling chemical bonds with higher bond energy to be broken.

[0038] In mass analysis step S6, mass analysis is performed on the diagnostic ions.

[0039] <Ion mobility spectrometry tandem mass spectrometer> Fig. 2 is a schematic diagram of a system for carrying out the method for identifying the chemical structure of lipids according to the present embodiment. Fig. 3 is a schematic diagram of the structure of a preferred ion mobility spectrometry tandem mass spectrometer according to the present embodiment.

[0040] The ion mobility spectrometry tandem mass analyzer includes, in series communication, an ion source 1, an ion mobility spectrometer 2, a first dissociator 3, a mass filter 4, a second dissociator 5, and a mass analyzer 6. One or more ion optical devices 7 may further be connected between the above components to focus, conduct or transport ions.

[0041] <Ion source> The ion source 1 performs an ionization step S1 to ionize a sample to obtain sample ions.

[0042] Ion source 1 includes electrospray ionization source (ESI), atmospheric pressure photoionization source (APPI), atmospheric pressure chemical ionization source (APCI), matrix-assisted laser desorption ionization source (MALDI), laser desorption ionization source (LDI), atmospheric pressure ionization source (API), desorption ionization source on silicon (DIOS), electron impact ionization source (EI), chemical ionization source (CI), field ionization source (FI), field desorption ionization source (FD), inductively coupled plasma ionization source (ICP), fast atom bombardment ionization source (FAB), liquid secondary ion mass spectrometry ionization source (LSIMS), electrospray desorption ionization source (D The ion source includes one selected from the group consisting of an electrospray ionization source, a nickel-63 radioactive ion source, an atmospheric pressure matrix-assisted laser desorption ionization source, a thermal spray ionization source, an air sampling glow discharge ionization source (ASGDI), a glow discharge ionization source (GD), an impactor ionization source, a real-time direct analysis ionization source (DART), a laser spray ionization source (LSI), an acoustic wave spray ionization source (SSI), a matrix-assisted entrance ionization source (MAII), a solvent-assisted entrance ionization source (SAII), a Penning ionization source, a laser ablation electrospray ionization source (LAESI), and a He plasma ionization source (HePI). Preferably, the ion source 1 is an electrospray ionization source, a nanospray ionization source, a desorption electrospray ionization source, an atmospheric pressure chemical ionization source, an atmospheric pressure photoionization source, or a matrix-assisted laser desorption ionization source. In this embodiment, the ion source is preferably an electrospray ionization source. In this embodiment, the ion source is preferably an electrospray ionization source.

[0043] <Ion mobility spectrometer> The ion mobility spectrometer 2 executes a mobility separation step S2, separates the sample ions based on differences in ion mobility, and separates the target lipid ions from the sample ions.

[0044] The ion mobility spectrometer 2 includes one ion mobility spectrometer selected from the group consisting of a drift tube ion mobility spectrometer (DTIMS), a differential mobility analysis (DMA) device, a field asymmetric-waveform ion-mobility spectrometry (FAIMS) device, a traveling wave ion mobility spectrometer (TW-IMS), a differential mobility spectrometry (DMS) device, a transverse modulation ion mobility spectrometer, a trapped ion mobility spectrometer (TIMS), and a U-shaped ion mobility analyzer (UMA).

[0045] In this embodiment, the ion mobility spectrometer 2 is preferably a U-type ion mobility spectrometer, more preferably a U-type ion mobility spectrometer operating in a filtering mode. For the description of the device structure of the U-type ion mobility spectrometer and the filtering mode (or "filter mode") applicable to the present identification method, reference can be made to Patent Document 2.

[0046] The filtering mode is a mode in which non-target ions are filtered out and the target ions are retained. In addition, the target ions can be passed through the filter while maintaining their movement along a predetermined path. In other words, the filtering mode can output an ion flow that is a continuous target ion if an ion flow having continuous target ions is input without changing the form of the ion flow of the target ions. Since ions are not locally present or stored, the loss of low-abundance ions due to the space charge effect can be avoided, making it very suitable for lipid omics analysis research.

[0047] The ion mobility spectrometer 2 can provide a second dimension of data for tandem mass spectrometry. Isomers can be differentiated based on differences in ion mobility. In some embodiments, ion mobility spectra can be used to further identify the position of carbon-carbon double bonds or cis-trans isomer orientation and sn-isomer differentiation.

[0048] <First dissociation device> The first dissociation device 3 performs a first dissociation step S3, in which the dissociation energy of the first dissociation device 3 is set so as to break a first chemical bond with a low bond energy (e.g., a polar head group) in the target lipid ion while maintaining the integrity of the main chain of the target lipid ion.

[0049] The first dissociation device 3 may comprise one or more dissociation devices selected from the group consisting of a collision induced dissociation (CID) device, a surface induced dissociation (SID) device, an electron transfer dissociation (ETD) device, an electron capture dissociation (ECD) device, an electron collision or collision dissociation device, a photoinduced dissociation (PID) device, a laser induced dissociation device, an infrared radiation induced dissociation device, an ultraviolet radiation induced dissociation device, a nozzle-separator interface dissociation device, an in-source dissociation device, an in-source collision induced dissociation device, a thermal or temperature source dissociation device, an electric field induced dissociation device, a magnetic field induced dissociation device, an ion-ion reaction dissociation device, an ion-molecule reaction dissociation device, an ion-atom reaction dissociation device, an ion metastable ion reaction dissociation device, an ion metastable molecule reaction dissociation device and an electron ionization dissociation (EID) device.

[0050] In this embodiment, the first dissociation device 3 is a simpler in-source collision-induced dissociation device that applies a voltage through a vacuum interface, for example to an orifice. The voltage applied to the end electrode is 10-70 eV. This voltage range can efficiently remove the polar head groups of phospholipids or sphingolipids.

[0051] <Mass filter> The mass filter 4 executes a mass selection step S4, and selects the target lipid ions whose first chemical bond has been cleaved based on the mass number, to obtain fragment ions.

[0052] The mass filter 4 may comprise one or more mass filters selected from the group consisting of a quadrupole mass filter, a 2D or linear quadrupole ion trap, a Paul or 3D quadrupole ion trap, a Penning ion trap, an ion trap, a magnetic sector mass filter, a time-of-flight mass filter and a Wien filter.

[0053] <Second dissociation device> The second dissociation device 5 executes a second dissociation step S5 to further dissociate the fragment ions and cleave second chemical bonds of the fragment ions, the bond energy of which is higher than that of the first chemical bond, to obtain diagnostic ions.

[0054] The second dissociation device 5 may comprise one or more dissociation devices selected from the group consisting of a collision induced dissociation (CID) device, a surface induced dissociation (SID) device, an electron transfer dissociation (ETD) device, an electron capture dissociation (ECD) device, an electron collision or collision dissociation device, a photoinduced dissociation (PID) device, a laser induced dissociation device, an infrared radiation induced dissociation device, an ultraviolet radiation induced dissociation device, a nozzle-separator interface dissociation device, an in-source dissociation device, an in-source collision induced dissociation device, a thermal or temperature source dissociation device, an electric field induced dissociation device, a magnetic field induced dissociation device, an ion ion reactive dissociation device, an ion molecular reactive dissociation device, an ion atomic reactive dissociation device, an ion metastable ion reactive dissociation device, an ion metastable molecular reactive dissociation device and an electron ionization dissociation (EID) device.

[0055] Preferably, the second dissociation device 5 is a collision induced dissociation device with a dissociation energy of 30-70 eV, which can selectively cleave the glycerin skeleton and the aziridine ring, reduce the generation of impurity ions, improve the spectral peak intensity of diagnostic ions, and make the spectrogram simpler and easier to read.

[0056] <Mass spectrometer> The mass spectrometer 6 executes a mass analysis step S6, and performs mass analysis on the diagnostic ions.

[0057] The mass spectrometer 6 may comprise one or more mass spectrometers selected from the group consisting of a 2D or linear quadrupole mass spectrometer, a Paul or 3D quadrupole mass spectrometer, a Penning trap mass spectrometer, an ion trap mass spectrometer, a magnetic sector mass spectrometer, an ion cyclotron resonance (ICR) mass spectrometer, a Fourier transform ion cyclotron resonance (FTIR) mass spectrometer, an electrostatic mass spectrometer arranged to generate an electrostatic field having a quadrupole logarithmic potential distribution, a Fourier transform electrostatic mass spectrometer, a Fourier transform mass spectrometer, a time-of-flight mass spectrometer, an orthogonal acceleration time-of-flight mass spectrometer and a linear acceleration time-of-flight mass spectrometer. Preferably, the mass spectrometer 6 is a high resolution mass spectrometer such as a time-of-flight mass spectrometer.

[0058] Although each component of the ion mobility spectrometry tandem mass spectrometer of this embodiment has been described above, the present invention is not limited to these. In another embodiment of the present invention, a separation device may be installed in front of the ion source 1, and the separation device may be one or more of liquid chromatography, gas chromatography, supercritical chromatography, capillary electrophoresis, and paper chromatography.

[0059] <Derivatization reaction> The method for identifying the chemical structure of lipids according to this embodiment further includes a derivatization reaction step of labeling the carbon-carbon double bond by a derivatization reaction before the ionization step S1. The derivatization reaction step can be performed offline, so that an experimenter performs the step in a laboratory. It can also be performed online, so that the sample and the reaction reagent are automatically introduced into the reactor to complete the reaction. This embodiment is not limited thereto.

[0060] The derivatization reaction may be any derivatization reaction capable of converting a carbon-carbon double bond into a group that easily dissociates, such as an aziridination reaction, an epoxidation reaction, a singlet oxygen-ene reaction, etc. More specifically, the reaction may be, for example, a Pattern-Burchi reaction, a Diels-Alder reaction, an aza-Prilezhaev reaction, a singlet oxygen-ene reaction, etc., and the present embodiment is not limited to the type of reaction employed.

[0061] In this embodiment, the derivatization reaction employs the aza-Prilezhaev reaction. The reaction mechanism of the aza-Prilezhaev reaction is shown below. [ka]

[0062] The derivatization reagent is a mass-labeled compound dissolved in an acidic solvent. Specifically, the mass-labeled compound is tert-butyl N-tosyloxycarbamate (N-Boc-O-tosylhydroxylamine, CAS: 105838-14-0), the acidic reagent is hexafluoroisopropanol, and the reaction is carried out under heating conditions of 20-100°C for 10 minutes or more to convert the carbon-carbon double bond into aziridinyl compound.

[0063] The dissociation energy for cleaving the glycerol backbone or the aziridine ring is higher than that for cleaving the polar head group, but lower than that for cleaving other chemical bonds, such as the carbon-carbon bond of an aliphatic chain. Therefore, in the compound obtained from the derivatization reaction, the first chemical bond (e.g., the polar head group) and the second chemical bond (e.g., the glycerol backbone, the aziridine ring) that are mainly to be cleaved can be cleaved without the need to apply a large amount of dissociation energy, whether in the first dissociation step S3 or the second dissociation step S5. Therefore, it is possible to avoid the spectrogram becoming too complicated by applying a large amount of dissociation energy, while at the same time further improving the signal intensity of the diagnostic ions.

[0064] Additionally, derivatization of the carbon-carbon double bonds to more rigid structures such as aziridine rings or epoxies can further enhance the structural differences between different molecules and improve the resolution of the ion mobility spectrogram.

[0065] <Ion reaction process> Hereinafter, the method for identifying the chemical structure of lipids in this embodiment will be described using an example in which the target lipid ion is PC(18:1 / 16:0).

[0066] 4 shows a specific reaction process of PC(18:1 / 16:0) in this embodiment. First, the raw sample is pretreated, that is, a derivatization reaction step is carried out to convert the carbon-carbon double bonds of the unsaturated fatty acids in the raw sample into aziridine rings.

[0067] In the ionization step S1, the molecules of each component are converted into positively charged sample ions, which can then be subjected to mass spectrometry in positive ion mode. Here, PC (18:1 / 16:0) contained in the sample is aziridinylated and then ionized into target lipid ions. The cation obtained by hydrogenation is the target lipid ion with mass number 775.6, and the cation obtained by sodium addition is the target lipid ion with mass number 797.6.

[0068] Next, in the ion mobility separation step S2, the ion mobility spectrometer can separate the target lipids from the sample ions during a specific time period of one analysis cycle and transport them to a downstream stage. Alternatively, the ion mobility spectrometer can be configured in a filtering mode, i.e., the target lipid ions in the sample ions can be continuously selected and transported to a downstream stage.

[0069] In the first dissociation step S3, the target lipid ions are dissociated and the phosphocholine group, which is the polar head group of PC (18:1 / 16:0), is removed. By removing the phosphocholine group, fragment ions having the 1,3-dioxolane structure shown in FIG. 4 are obtained.

[0070] In the second dissociation step S4, the fragment ions having the 1,3-dioxolane structure may be cleaved by 1,3-dioxolane, or may be cleaved by the aziridine ring obtained by the derivatization reaction to form multiple diagnostic ions. Some of the diagnostic ions are used to identify the position of the carbon-carbon double bond in the aliphatic chain, i.e., the diagnostic ions at the C=C position shown in FIG. 4. Some of the diagnostic ions are used to identify the position of the sn in the carbon-carbon double bond, i.e., the diagnostic ions at the sn position shown in FIG. 4. Some of the diagnostic ions are used to identify the information of the aliphatic chain in which the carbon-carbon double bond exists, i.e., the diagnostic ions of the aliphatic chain shown in FIG. 4.

[0071] FIG. 5 shows the mass number m / z=290 or 360 [M+Na + This is a diagram illustrating the principle behind why the sn position of a carbon-carbon double bond can be identified from the diagnostic ion [

[0072] As can be seen from Figure 5, when the carbon-carbon double bond is at different sn positions, the fragment ions and diagnostic ions generated by dissociation are all different. For PC(16:0 / 18:1) with the carbon-carbon double bond at the sn-2 position, the mass number is 290 [M+Na + For PC(18:1 / 16:0) with a carbon-carbon double bond at the sn-1 position, the mass number is 360 [M+Na + The compounds can be distinguished based on the diagnostic ion,

[0073] As can be seen from the above reaction process, the method for identifying the chemical structures of lipids according to this embodiment can simultaneously identify the position of the carbon-carbon double bond in the fatty chain of a phospholipid or sphingolipid, the structure of the fatty chain, and the sn position with a single sample injection, and has excellent analytical efficiency.

[0074] Although some major steps of the method for identifying the chemical structures of lipids have been described above, in other embodiments of the present invention, the method may further include other steps, without being limited thereto.

[0075] For example, before performing the first dissociation step S3 and the second dissociation step S5, a first pre-scan step may be performed to perform mass spectrometry on sample ions that have not undergone the first dissociation step S3 and the second dissociation step S5. The first pre-scan step is used to discover target lipid ions. That is, the mass number in this embodiment is 775 [M+H + ] or 797[M+Na + ] is found, and the target lipid ion is then subjected to isomer determination.

[0076] Furthermore, for example, after performing the first dissociation step S3 and before performing the second dissociation step S5, a second prescan step may be performed to perform mass spectrometry on the sample ions that have been dissociated only once. The second prescan step is used to determine an appropriate dissociation energy. This dissociates the target lipid ions so that only the polar head groups are cleaved, thereby reducing or avoiding the generation of extra fragment ions, improving signal intensity, and making the spectrogram easier to read.

[0077] <Experimental Results> FIG. 6 is a comparison diagram between the measurement results obtained by a general tandem mass spectrometry (MS2) method in the prior art and the measurement results obtained by the method according to the embodiment of the present invention.

[0078] In the two mass spectrograms corresponding to the conventional technology in Fig. 6, the upper one is an MS2 spectrogram, and the lower one is a spectrogram obtained by enlarging the range of the upper frame. In the two mass spectrograms corresponding to the measurement results of this embodiment in Fig. 6, the upper one is an MS1 ​​spectrogram (the label "pseudo MS2" means that the IMS and the first dissociation device 3 are designated as MS1, and the same applies below), and the lower one is an MS2 spectrogram obtained by further dissociating the ion with mass number 592.

[0079] As can be seen from Figure 6, due to the interference of polar head groups, the peak intensity of diagnostic ions in the spectrogram of general tandem mass spectrometry MS2 is in the range of about 700-800. Meanwhile, the peak intensity of diagnostic ions obtained by the IMS-CID-MS / MS method according to the present embodiment can reach the range of 5000-6000, greatly improving the signal intensity (about 7-8 times) and improving the analytical sensitivity.

[0080] The above are merely preferred embodiments of the present invention, and the present invention is not limited thereto. Any modifications, equivalent replacements, improvements, etc. that do not depart from the gist of the present invention are included in the technical scope of the present invention. [Explanation of symbols]

[0081] 1-Ion source 2-Ion Mobility Spectrometer 3-First dissociation device 4- Mass Filter 5-Second dissociation device 6-Mass spectrometer 7-Ion optical device.

Claims

1. 1. A method for identifying the chemical structure of lipids, comprising: an ionization step of ionizing the sample to obtain sample ions; an ion mobility separation step of separating target lipid class ions from the sample ions based on ion mobility; a first dissociation step of dissociating the target lipid ions with a dissociation energy suitable for breaking a first chemical bond of the target lipid ions; A mass selection step of selecting the target lipid ion from which the first chemical bond has been cleaved based on the mass number to obtain fragment ions; a second dissociation step of dissociating the fragment ions to break at least a second chemical bond of the fragment ions having a higher bond energy than the first chemical bond to obtain diagnostic ions; A method for identifying the chemical structure of lipids, comprising a mass spectrometry step of performing mass spectrometry on the diagnostic ions.

2. The method for identifying the chemical structure of lipids according to claim 1, characterized in that the lipids are unsaturated lipids having a carbon-carbon double bond in a fatty chain, and the method is used to identify the position of the carbon-carbon double bond in the fatty chain and the position of sn in the fatty chain.

3. The method for identifying the chemical structure of lipids according to claim 2, further comprising a derivatization reaction step of labeling the carbon-carbon double bond by a derivatization reaction prior to the ionization step.

4. The method for identifying the chemical structure of lipids according to claim 3, characterized in that the lipids are phospholipids or sphingolipids, the first chemical bond is a bond with a polar head group of the phospholipid or a polar head group of the sphingolipid, and the second chemical bond is a chemical bond obtained by a derivatization reaction of the carbon-carbon double bond.

5. The method for identifying the chemical structure of lipids according to claim 3, characterized in that the derivatization reaction is an aziridination reaction, an epoxidation reaction, a Paterno-Buchi reaction, a singlet oxygen-ene reaction or a Diels-Alder reaction.

6. 2. The method for identifying the chemical structure of lipids according to claim 1, wherein the lipids are fatty acyl, glyceride, glycerophospholipid, sphingolipid, sterol ester, pregnenolone lipid, glycolipid or polyketide.

7. The method for identifying the chemical structure of lipids described in claim 1, further comprising a first pre-scan step of performing mass analysis on the sample ions that have not undergone the first dissociation step and the second dissociation step.

8. The method for identifying the chemical structure of lipids according to claim 1, further comprising a second pre-scan step of performing mass spectrometry on the sample ions that have undergone only one dissociation.

9. An ion mobility spectrometry tandem mass spectrometer, comprising: an ion source for ionizing a sample to obtain sample ions; an ion mobility spectrometer for separating target lipid ions from the sample ions; a first dissociation device for dissociating the target lipid ions, the dissociation energy being suitable for breaking a first chemical bond of the target lipid ions; A mass filter that selects the target lipid ions whose first chemical bond has been cleaved to obtain fragment ions; a second dissociation device for dissociating the fragment ions to break at least a second chemical bond of the fragment ions having a higher bond energy than the first chemical bond to obtain diagnostic ions; a mass spectrometer that performs mass analysis on the diagnostic ions.

10. 10. The ion mobility spectrometry tandem mass spectrometer according to claim 9, wherein the ion mobility spectrometer is a U-type ion mobility spectrometer.

11. The ion mobility spectrometry tandem mass spectrometer of claim 10, wherein the U-shaped ion mobility spectrometer operates in a filtering mode.

12. 10. An ion mobility spectrometry tandem mass spectrometer as claimed in claim 9, wherein the mass filter is a quadrupole rod or an ion trap.

13. 10. The ion mobility spectrometry tandem mass spectrometer according to claim 9, wherein the mass spectrometer is a time-of-flight mass spectrometer, a Fourier transform mass spectrometer, a quadrupole mass spectrometer, an ion trap mass spectrometer or a magnetic mass spectrometer.

14. 10. The ion mobility spectrometry tandem mass spectrometer of claim 9, wherein the ion source is an electrospray ionization source, a nanospray ionization source, a desorption electrospray ionization source, an atmospheric pressure chemical ionization source, an atmospheric pressure photoionization source, or a matrix-assisted laser desorption ionization source.

15. The ion mobility spectrometry tandem mass spectrometer according to claim 9, characterized in that the first dissociation device and / or the second dissociation device is one or more of a high-energy collision dissociation device, a collision induced dissociation device, an oxygen attachment dissociation device, a hydrogen attachment dissociation device, an electron capture dissociation device, a radical directional dissociation device, an ultraviolet light induced dissociation device, and a charge remote fragmentation device.

16. The ion mobility spectrometry tandem mass spectrometer according to claim 15, characterized in that the first dissociation device is a collision induced dissociation device having an end electrode voltage of 10-70 eV, and the second dissociation device is a collision induced dissociation device having a dissociation energy of 30-70 eV.

17. The ion mobility spectrometry tandem mass spectrometer according to claim 9, further comprising a separation device disposed in front of the ion source, the separation device being one or more of liquid chromatography, gas chromatography, supercritical chromatography, capillary electrophoresis, and paper chromatography.

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