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

Abstract

The invention relates to the field of mass spectrometry, and particularly provides a method for determining a lipid chemical structure and an ion mobility spectrometry tandem mass spectrometer. The method for determining the lipid chemical structure comprises the following steps: an ionization step: ionizing a sample to obtain sample ions; an ion mobility separation step: separating target lipid ions from the sample ions based on the ion mobility; a first dissociation step: dissociating the target lipid ions by dissociation energy suitable for breaking a first chemical bond of the target lipid ions; a mass selection step: based on the mass number, selecting a target lipid ion with the first chemical bond broken to obtain a fragment ion; a second dissociation step: dissociating the fragment ions, at least breaking a second chemical bond of the fragment ions, the bond energy of which is higher than that of the first chemical bond, to obtain diagnosis ions; and a mass analysis step: performing mass analysis on the diagnosis ions.

Description

Technical Field

[0001] The present invention relates to the field of mass spectrometry, and particularly relates to a method for determining the chemical structure of lipids and an ion mobility spectrometry tandem mass spectrometer. Background Art

[0002] Lipids are important nutrients and important components of biological 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 establish a classification database to promote lipidomics research.

[0003] Complete lipid annotation and identification information includes class, elemental composition, R-group size and position, number and position of double bonds, and cis-trans isomeric orientation of double bonds. In addition, for unsaturated lipids with a glycerol backbone, such as glycerides, glycerophospholipids, etc., it is also necessary to determine the sn position of the fatty acid chain containing carbon-carbon double bonds.

[0004] Multistage tandem mass spectrometry plays an important role in the structural analysis of compounds. Hsu and Turk proposed a pseudo MS3 tandem mass spectrometry method to achieve the identification of the sn-position of glycerophospholipids, that is, first using in-source CID to obtain a high-abundance fragment ion signal of the head-group lost [M+Li-183] +, the fragment ions are then subjected to collision-induced dissociation to generate sn-diagnostic ions (J. Am. Soc. Mass Spectrom. 2003, 14, 352). For unsaturated lipids with a glycerol backbone, in order to simultaneously identify the double bond positions and sn-positions in a single injection, Brodbelt et al. provided a hybrid MS3 method that combines collision activation and ultraviolet-visible spectrophotometry in the article "Pinpointing Double Bond and sn-Positions in Glycerophospholipids via Hybrid 193nm Ultraviolet Photodissociation (UVPD) Mass Spectrometry" (J. Am. Chem. Soc. 2017, 139, 15681-15690), but the abundance of the diagnostic ions still needs to be further improved. These pseudo MS3 and MS3 analysis methods are not good at the selection and analysis of isomers; at the same time, when multiple ion selections are performed at the first stage by a quadrupole mass spectrometer, non-current target ions will be lost, so the overall duty cycle is relatively low. In addition to directly performing multi-stage tandem mass spectrometry analysis on compounds, chemical derivatization can also be used to pre-modify the analyte to achieve the purposes of increasing ionization efficiency, increasing structural differences, improving chromatographic behavior, etc., and then performing multi-stage tandem mass spectrometry analysis on the derivatives. Ma et al. used charge tag derivatization and MS3 to precisely locate the C=C positions and sn-positions of glycerophospholipids in the article "Large-scale lipid analysis with C=C location and sn-position isomer resolving power" (Nat. Commun., 2020, 11, 375). The method provided in this article requires the use of an ion trap mass spectrometer to mass-select specific parent ions, and the duty cycle and resolution are limited by the ion trap mass spectrometer.

[0005] Ion mobility spectrometry can achieve high-throughput separation and analysis of isomers under gas-phase conditions. When combined with mass spectrometry, it has high resolution and has an effect similar to that of tandem mass spectrometry (Biochem.Soc.T.2020,48,2457; Anal.Chem.2006,78,4161). It can obtain detailed structural information of the analyte and has been widely used in the structural identification of metabolomics, glycomics, and proteomics. For example, a large number of studies have reported that the combination of cyclic ion mobility (cIM) and collision-induced dissociation (CID), such as the cIM-CID-cIM and cIM-CID-cIM-CID-cIM modes, can achieve the separation and de novo determination of carbohydrate isomers (Anal.Chem.2021,93,6254; Annual Rev.Anal.Chem.2023,16,27). The Bleiholder team proposed a combined scheme of tandem mobility spectrometry based on trapped ion mobility (TIMS) and tandem mass spectrometry, which has functions such as mobility selection and collision activation (US10794861B2, Analyst 2022,147,2317), and realized the structural identification of polypeptides and proteins (Analyst2018,143,2249; J.Am.Soc.Mass Spectrom.2023,34,2247), sugars and their isomers (Anal.Chem.2023,95,747). Nicholas B. Borotto et al. achieved pre-migration collision-induced unfolding based on TIMS, which can quickly resolve the conformation of proteins (J.Am.Soc.Mass Spectrom.2022,33,83). In addition, the pre-migration collision-induced unfolding strategy can be further combined with tandem mass spectrometry to achieve accurate protein sequencing (J.Am.Soc.Mass Spectrom.2023,34,2232).

[0006] In recent years, ion mobility spectrometry has also achieved good results in the in-depth structural identification of lipids (J. Chromatogr. A, 2017, 1530, 90; J. Sep. Sci. 2018, 41, 20; Front. Mol. Biosci. 2023, 16, 10, 1112521). Baker and his colleagues proposed a lipidomics analysis method that combines reversed-phase liquid chromatography with ion mobility spectrometry, which can separate lipids and their isomers in three dimensions: analyte polarity, structure, and size-to-charge ratio, increasing the peak capacity. Liquid chromatography can achieve the separation of different types of lipids, and different types of lipids form different ion trend lines in the ion mobility spectrum. It is worth noting that a rough separation of different subclasses of lipids can also be observed in the ion mobility spectrum (Analyst, 2016, 141, 1649). Limited by the instrument resolution, only shoulder peak separation of different lipid isomers is achieved. F. Fernandez-Lima et al. used high-resolution TIMS under specific instrument parameters (average resolution exceeding 320) to achieve the identification of double bond position isomers of phosphatidylcholine sodium ion adducts and double bond cis-trans isomers of proton adducts, respectively. In addition, when the instrument parameters meet the condition of ultra-high resolution (exceeding 410), this method can be used for sn-position isomer identification (Anal. Chem. 2019, 91, 5021). However, the resolution of most commercial ion mobility instruments is less than 200, making it difficult to meet the above conditions. Forming metal ion adducts is beneficial to increasing the isomer resolution. M. Groessl et al. used drift time ion mobility spectrometry to distinguish double bond position isomers, double bond cis-trans isomers, and sn-isomers of phosphatidylcholine silver ion adducts (Analyst, 2015, 140, 6904). Similarly, Yan et al. used drift time ion mobility spectrometry to achieve the separation and analysis of carbon-carbon double bond cis-trans isomers of phosphatidylcholine monovalent copper ion adducts, but the separation degree is low (Int. J. Mass Spectrom. 2022, 479, 116889).

[0007] Although many methods for identifying lipid isomers based on ion mobility spectrometry have been developed, these methods require standards as references and are difficult to analyze the structures of unknown compounds. In addition, the resolution of ion mobility spectrometry is lower than that of mass spectrometry, which limits its application in the analysis of complex matrix samples. Combining the separation ability of ion mobility with the resolution of tandem mass spectrometry has great potential in the analysis of the structures of unknown lipid compounds in biological samples. The Brodbelt team combined UVPD with drift time ion mobility mass spectrometry and successfully achieved the determination of the collision cross-sectional area of lipid isomers and the identification of the positions of lipid double bonds and cyclopropanes (Anal. Chem. 2022, 94, 4252). However, this method is suitable for the analysis of polar lipids and is difficult to detect medium- and low-polarity lipids. Xia et al. combined the P-B reaction with trapped ion mobility tandem mass spectrometry and successfully achieved the separation and analysis of conjugated fatty acid isomers. By combining ion mobility spectra and tandem mass spectra, the mobility characteristic peaks and double bonds can be accurately assigned without standards. However, the reaction products of the P-B reaction and conjugated fatty acids are diverse, resulting in high complexity of the mobility spectra and limited application in complex mixtures (Anal. Chem. 2019, 91, 7173). In 2023, the Xia team combined liquid chromatography, ion mobility spectrometry, and P-B reaction tandem mass spectrometry to establish a lipid deep structure analysis process, which can achieve the identification of double bond positions and sn-positions step by step and was successfully applied to the lipidome analysis of biological samples such as bovine liver and cells, obtaining relatively complete lipid profile information (Nat. Commun. 2023, 14, 4263). However, the step-by-step pretreatment and multi-batch analysis process not only reduce the analysis throughput but also cause relative sample loss. Summary of the Invention

[0008] In view of the above problems, the present invention provides a method for determining the chemical structure of lipids and an ion mobility spectrometry tandem mass spectrometer, which can improve the abundance of diagnostic ions and has good duty cycle and resolution.

[0009] The first aspect of the present invention provides a method for determining the chemical structure of lipids, comprising the following steps:

[0010] Ionization step: ionize the sample to obtain sample ions;

[0011] Ion mobility separation step: based on ion mobility, separate target lipid ions from the sample ions;

[0012] First dissociation step: dissociate the target lipid ions with a dissociation energy suitable for breaking the first chemical bond of the target lipid ions;

[0013] Mass selection step: based on the mass number, select the target lipid ions with the first chemical bond broken to obtain fragment ions;

[0014] A second dissociation step, dissociating fragment ions, breaking at least a second chemical bond with a bond energy higher than that of the first chemical bond to obtain diagnostic ions;

[0015] A mass analysis step, performing mass analysis on the diagnostic ions.

[0016] Optionally, the lipid is an unsaturated lipid having a carbon-carbon double bond in the fatty acid chain, and the method is used to identify the position of the carbon-carbon double bond in the fatty acid chain and the sn position of the fatty acid chain.

[0017] Optionally, before the ionization step, a derivatization reaction step is further included, and the carbon-carbon double bond is labeled by the derivatization reaction.

[0018] Optionally, the lipid is a phospholipid or a sphingomyelin, the first chemical bond is the polar head group of the phospholipid or the polar head group of the sphingomyelin, and the second chemical bond is a chemical bond obtained by derivatizing the carbon-carbon double bond.

[0019] Optionally, the derivatization reaction is an aziridination reaction, an epoxidation reaction, a Paternò-Büchi reaction, a singlet oxygen-ene singlet oxidation reaction or a Diels-Alder reaction.

[0020] Optionally, the lipid is a fatty acyl, a glyceride, a glycerophospholipid, a sphingolipid, a sterol ester, a pregnenolone lipid, a glycolipid or a polyketide.

[0021] Optionally, it further includes: a first pre-scanning step, performing mass analysis on sample ions that have not undergone the first dissociation step and the second dissociation step.

[0022] Optionally, it further includes: a second pre-scanning step, performing mass analysis on sample ions that have undergone only one dissociation.

[0023] A second aspect of the present invention further provides an ion mobility spectrometry tandem mass spectrometry instrument, including an ion source, an ion mobility spectrometer, a first dissociation device, a mass filter, a second dissociation device and a mass analyzer. Among them, the ion source ionizes the 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, and the dissociation energy of the first dissociation device is suitable for breaking the first chemical bond of the target lipid ions; the mass filter selects the target lipid ions with the first chemical bond broken to obtain fragment ions; the second dissociation device dissociates the fragment ions, breaking at least a second chemical bond with a bond energy higher than that of the first chemical bond to obtain diagnostic ions; the mass analyzer performs mass analysis on the diagnostic ions.

[0024] Optionally, the first dissociation device is a collision-induced dissociation device, and the end electrode voltage of the first dissociation device is 10-70 eV; the second dissociation device is a collision-induced dissociation device, and the dissociation energy of the second dissociation device is 30-70 eV.

[0025] <Beneficial effects>

[0026] Precise ion selection: Various target lipid ions are sorted out with a high duty cycle of ion mobility and then used for subsequent multiple targeted tandem mass spectrometry analysis to reduce the complexity of a single spectrum. Pseudo-multistage collision-induced dissociation: By first breaking the first chemical bond with relatively weak bond energy of the target lipid ions to remove the interfering groups connected via the first chemical bond in the target lipid ions, then selecting the fragment ions from which the interfering groups have been excluded and performing secondary dissociation on them, the mass spectrometry signal intensity of the diagnostic ions finally generated can be increased, alleviating or avoiding the problem of the increase in spectrum complexity caused by the appearance of spectral peaks related to the interfering groups in the final mass spectrometry spectrum, making the spectrum more simplified and facilitating the analysis and judgment of the chemical structure of lipids. Description of the drawings

[0027] Figure 1 is a flowchart of the method for determining the chemical structure of lipids provided by the embodiment of the present invention.

[0028] Figure 2 is a schematic diagram of the system for implementing the method for determining the chemical structure of lipids provided by the embodiment of the present invention.

[0029] Figure 3 is a schematic diagram of the structure of a more preferred ion mobility spectrometry tandem mass spectrometer provided by the embodiment of the present invention.

[0030] Figure 4 Shows the specific reaction process of PC(18:1 / 16:0) in this embodiment.

[0031] Figure 5 is the schematic diagram explaining why the sn position of the carbon-carbon double bond can be identified according to the diagnostic ions with a mass number of m / z = 290 or 360 [M+Na + .

[0032] Figure 6 is a comparison chart of the measurement results obtained by the ordinary tandem mass spectrometry MS2 method in the prior art and the measurement results obtained by the method provided by the embodiment of the present invention.

[0033] Reference numerals:

[0034] 1 - Ion source; 2 - Ion mobility spectrometer; 3 - First dissociation device; 4 - Mass filter; 5 - Second dissociation device; 6 - Mass analyzer; 7 - Ion optical device. Detailed implementation manners

[0035] Next, the technical solutions in the embodiments of the present invention will be clearly and completely described in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only a part of the embodiments of the present invention, rather than all the embodiments. All other embodiments obtained by those of ordinary skill in the art based on the embodiments of the present invention without creative efforts shall fall within the protection scope of the present invention.

[0036] The method for determining the chemical structure of lipids provided in this embodiment can be applied to identify lipids with interfering groups (such as polar head groups). Such lipids can be, for example, fatty acyls, glycerides, glycerophospholipids, sphingolipids, sterol esters, pregnenolone lipids, glycolipids, or polyketides.

[0037] In the method for determining the chemical structure of lipids provided in this embodiment, first, the first chemical bond with relatively weak bond energy in the target lipid ion is broken to remove the interfering group connected via the first chemical bond in the target lipid ion. Then, the fragment ion with the interfering group excluded is selected and subjected to secondary dissociation. In this way, the mass spectrometry signal intensity of the finally generated diagnostic ions can be increased, and the problem of increased spectral complexity caused by the appearance of peaks related to the interfering group in the final mass spectrometry spectrum can be alleviated or avoided, making the spectrum more simplified and facilitating the analysis and determination of the chemical structure of lipids.

[0038] Figure 1 is a flowchart of the method for determining the chemical structure of lipids provided in the first embodiment of the present invention. Refer to Figure 1 , the method provided in this 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 that occur in sequence.

[0039] - Ionization step S1: Ionize the sample to obtain sample ions.

[0040] - Ion mobility separation step S2: Based on ion mobility, separate the target lipid ions from the sample ions.

[0041] - First dissociation step S3: Dissociate the target lipid ions with a dissociation energy suitable for breaking the first chemical bond of the target lipid ions.

[0042] The first dissociation of the target lipid ions, that is, the first dissociation step S3, can selectively break the chemical bond with relatively low bond energy in the target lipid ions that may ultimately generate interference signals (i.e., the first chemical bond connected to the interfering group).

[0043] The first dissociation step S3 is a selective dissociation step, that is, when disconnecting the first chemical bond, the integrity of other chemical bonds (main-chain chemical bonds, especially the second chemical bond) is maintained as much as possible. Specifically, the foregoing requirements can be met by setting the dissociation energy slightly higher than the threshold value capable of disconnecting the first chemical bond. 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 disconnect the first chemical bond, and the integrity of other chemical bonds is maintained to the greatest extent.

[0044] By setting the dissociation energy equal to or slightly higher than the threshold value capable of disconnecting the first chemical bond, the first chemical bond can be selectively disconnected, thereby excluding the interference of interfering groups on the final mass spectrometry test results with less loss of ion abundance.

[0045] - Mass selection step S4, based on the mass number, select the target lipid ions with the first chemical bond disconnected to obtain fragment ions.

[0046] Selectively disconnecting the first chemical bond can generate at least a pair of ions, one of which is an ion with an interfering group (such as a polar head group), and the other is an ion without an interfering group. The fragment ions selected in the mass selection step S4 are the fragment ions with the interfering group connected by the first chemical bond excluded, that is, the ions without an interfering group.

[0047] - Second dissociation step S5, dissociate the fragment ions to disconnect at least the second chemical bond with a bond energy higher than that of the first chemical bond to obtain diagnostic ions.

[0048] Through the second dissociation, that is, the second dissociation step S5, the obtained fragment ions without interfering groups are further dissociated. The second dissociation step S5 can be selective or non-selective. However, whether the second dissociation step S5 is selective or non-selective, since the interfering groups have been removed in the first dissociation step S3 and the mass selection step S4, there will be no excessive spectral peaks associated with the interfering groups in the mass spectrometry spectrum, so that there are fewer spectral peaks in the mass spectrometry spectrum and the peak intensity of the mass peak of the diagnostic ions is stronger.

[0049] Generally speaking, for the case of using the same type of dissociation device, the dissociation energy used in the second dissociation step S5 is higher than that in the first dissociation step S3, so as to be able to disconnect chemical bonds with higher bond energies.

[0050] - Mass analysis step S6, perform mass analysis on the diagnostic ions.

[0051] <Ion mobility spectrometry tandem mass spectrometry instrument>

[0052] Figure 2It is a schematic diagram of a system for implementing a method for determining the chemical structure of lipids provided by this embodiment. Figure 3 It is a schematic diagram of a relatively preferred structure of an ion mobility spectrometry tandem mass spectrometer provided by this embodiment.

[0053] The ion mobility spectrometry tandem mass spectrometer includes an ion source 1, an ion mobility spectrometer 2, a first dissociation device 3, a mass filter 4, a second dissociation device 5, and a mass analyzer 6 that are connected in sequence. Between the above components, one or more ion optical devices 7 can also be connected for focusing, guiding, or transporting ions.

[0054] <Ion source>

[0055] The ion source 1 performs the ionization step S1 to ionize the sample and obtain sample ions.

[0056] The ion source 1 includes an ion source selected from the following group, which consists of: 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 on silicon source (DIOS); electron impact ionization source (EI); chemical ionization source (CI); field ionization source (FI); field desorption ionization source (FD); inductively coupled plasma ion source (ICP); fast atom bombardment ion source (FAB); liquid secondary ion mass spectrometry ion source (LSIMS); desorption electrospray ionization source (DESI); nickel-63 radioactive ion source; atmospheric pressure matrix-assisted laser desorption ionization ion source; thermospray ion source; atmospheric sampling glow discharge ion source (ASGDI); glow discharge ion source (GD); impactor ion source; direct analysis in real time ion source (DART); laser spray ion source (LSI); acoustic spray ion source (SSI); matrix-assisted inlet ion source (MAII); solvent-assisted inlet ion source (SAII); Penning ionization source; laser ablation electrospray ionization source (LAESI); He plasma ion source (HePl). Preferably, the ion source 1 is an electrospray ionization source, a nanoelectrospray 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 1 is preferably an electrospray ionization source.

[0057] <Ion mobility spectrometer>

[0058] The ion mobility spectrometer 2 performs the ion mobility separation step S2 to separate the sample ions based on different ion mobilities and separate the target lipid ions from the sample ions.

[0059] The ion mobility spectrometer 2 includes an ion mobility analysis device selected from the group consisting of: Drift Tube Ion Mobility Spectrometry (DTIMS), Differential Mobility Analysis (DMA) device, Field Asymmetric-Waveform Ion-Mobility Spectrometry (FAIMS) device, Travelling Wave Ion Mobility Spectrometry (TW-IMS), Differential Mobility Spectrometry (DMS) device, transverse modulation ion mobility spectrometry, Trapped Ion Mobility Spectrometry (TIMS), U-shaped Ion Mobility Analyzer (UMA).

[0060] In this embodiment, the ion mobility spectrometer 2 is preferably a U-shaped ion mobility spectrometer. More preferably, it is a U-shaped ion mobility spectrometer operating in the filtration mode. The device structure of the U-shaped ion mobility spectrometer and the introduction of the filtration mode (or "filter mode") adapted to this embodiment can be referred to in Chinese Patent CN113495112A.

[0061] The filtration mode is a mode that filters out non-target ions while retaining target ions. Moreover, for target ions, they can move along a specified path and pass through the filter. In other words, the filtration mode does not change the ion current form of target ions. As long as the input is a continuous ion current with target ions, the output will also be a continuous ion current of target ions. It does not locally enrich or store ions, which can avoid the loss of low-abundance ions caused by the space charge effect and is very suitable for lipidomics analysis research.

[0062] The ion mobility spectrometer 2 can bring second-dimensional data to tandem mass spectrometry analysis. Based on different ion mobilities, isomers can be distinguished. In some embodiments, using the ion mobility spectrum, the position of carbon-carbon double bonds, the cis-trans isomer orientation, and the difference in sn-isomer can also be identified.

[0063] <First dissociation device>

[0064] The first dissociation device 3 performs a first dissociation step S3 to disconnect the first chemical bond with a lower bond energy (e.g., the polar head group) in the target lipid ion, and sets the dissociation energy of the first dissociation device 3 in a manner that keeps the main chain of the target lipid ion intact.

[0065] The first dissociation device 3 may include 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 impact dissociation device; a photo-induced 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.

[0066] In this embodiment, the first dissociation device 3 is an in-source collision-induced dissociation device that applies a voltage at a vacuum interface, such as an orifice, and the device is simpler. The voltage applied to the end electrode is 10 - 70 eV. This voltage range can efficiently remove the polar head groups of phospholipids or sphingomyelins.

[0067] <Mass filter>

[0068] The mass filter 4 performs a mass selection step S4 to select, based on the mass number, the target lipid ions in which the first chemical bond is disconnected, and obtains fragment ions.

[0069] The mass filter 4 may include 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.

[0070] <Second dissociation device>

[0071] The second dissociation device 5 performs a second dissociation step S5 to further dissociate the fragment ions and break the second chemical bond with a higher bond energy in the fragment ions, thereby obtaining diagnostic ions.

[0072] The second dissociation device 5 may include 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 impact dissociation device; a photo-induced 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.

[0073] Preferably, the second dissociation device 5 is a collision-induced dissociation device with a dissociation energy of 30 - 70 eV. This dissociation energy can selectively break the glycerol backbone and aziridine ring, reduce the generation of miscellaneous ions, increase the peak intensity of diagnostic ions, and make the spectrum simpler and easier to read.

[0074] <Mass analyzer>

[0075] The mass analyzer 6 performs the mass analysis step S6 to perform mass analysis on the diagnostic ions.

[0076] The mass analyzer 6 may include a mass analyzer selected from the group consisting of: a quadrupole mass analyzer; a 2D or linear quadrupole mass analyzer; a Paul or 3D quadrupole mass analyzer; a Penning trap mass analyzer; an ion trap mass analyzer; a magnetic sector mass analyzer; an ion cyclotron resonance (ICR) mass analyzer; a Fourier transform ion cyclotron resonance (FTICR) mass analyzer; an electrostatic mass analyzer arranged to generate an electrostatic field with a quadrupole logarithmic potential distribution; a Fourier transform electrostatic mass analyzer; a Fourier transform mass analyzer; a time-of-flight mass analyzer; an orthogonal acceleration time-of-flight mass analyzer; and a linear acceleration time-of-flight mass analyzer. Preferably, the mass analyzer 6 is a high-resolution mass analyzer such as a time-of-flight mass analyzer.

[0077] The components of the ion mobility spectrometry tandem mass spectrometer of the present embodiment are introduced above, but are not limited thereto. In other embodiments of the present invention, a separation device may be provided at the pre-stage of the ion source 1, and the separation device may be one or more of a liquid chromatography, a gas chromatography, a supercritical chromatography, a capillary electrophoresis device, and a paper chromatography.

[0078] <Derivatization reaction>

[0079] The method for determining the lipid chemical structure provided by this embodiment further includes a derivatization reaction step before the ionization step S1, and the carbon-carbon double bond is labeled by the derivatization reaction. The derivatization reaction step can be implemented offline, that is, implemented by an experimenter in the laboratory, or can be implemented online, that is, the sample and reaction reagents are automatically introduced into the reactor to complete the reaction. This embodiment does not limit this.

[0080] The derivatization reaction can be any derivatization reaction that can convert a carbon-carbon double bond into a group that is easily dissociated. For example, it can be aziridination reaction, epoxidation reaction, singlet oxygen-ene reaction. More specifically, for example, it can be Paternò-Büchi reaction, Diels-Alder reaction, aza-Prilezhaev reaction, singlet oxygen-ene reaction, etc. This embodiment does not limit the reaction type used.

[0081] In this embodiment, the aza-Prilezhaev reaction is used for the derivatization reaction. The reaction mechanism of the aza-Prilezhaev reaction is shown as follows.

[0082]

[0083] The derivatization reagent is a mass marker dissolved in an acidic solvent. Specifically, the mass marker is N-Boc-O-tosylhydroxylamine (CAS: 105838-14-0), and the acidic reagent is hexafluoroisopropanol. Under the heating condition of 20-100 °C for more than 10 minutes, the carbon-carbon double bond can be aziridinized.

[0084] Although the dissociation energy for breaking the glycerol backbone or aziridine ring is higher than that for breaking the polar head group, it is still less than that for breaking other chemical bonds, such as the carbon-carbon bond of the fatty chain. Therefore, for the compound obtained after the derivatization reaction, whether in the first dissociation step S3 or in the second dissociation step S5, it is not necessary to apply too much dissociation energy to break the first chemical bond (such as the polar head group) and the second chemical bond (such as the glycerol backbone, aziridine ring) that are mainly expected to be broken, avoiding the problem of overly complex spectra caused by applying too much dissociation energy, and can further improve the signal intensity of diagnostic ions.

[0085] In addition, derivatizing the carbon-carbon double bond reaction into a more rigid structure such as an aziridine ring or an epoxy can further amplify the structural differences of different molecules and improve the resolution of the ion mobility spectrum.

[0086] <Ion reaction process>

[0087] Hereinafter, taking the target lipid ion as PC(18:1 / 16:0) as an example, the method for determining the chemical structure of lipids in this embodiment will be introduced.

[0088] Figure 4 The specific reaction process of PC(18:1 / 16:0) in this embodiment is shown. Refer to Figure 4 , first, the original sample is pretreated, that is, the derivatization reaction step, to convert the carbon-carbon double bond of the unsaturated lipid in the original sample into an aziridine ring.

[0089] In the ionization step S1, the molecules of each component can be converted into sample ions with a positive charge, and subsequent mass spectrometry analysis is carried out in the positive ion mode. Among them, the aziridinated PC(18:1 / 16:0) contained in the sample is ionized to obtain the target lipid ion. The positive ion obtained by adding hydrogen is the target lipid ion with a mass number of 775.6, and the positive ion obtained by adding sodium is the target lipid ion with a mass number of 797.6.

[0090] Next, in the ion mobility spectrometry separation step S2, using an ion mobility spectrometer, the target lipid ion can be separated from the sample ions within a specific time period of an analysis cycle and transported to the subsequent stage. Alternatively, the ion mobility spectrometer can also be configured in the filtering mode, that is, continuously screening out the target lipid ions in the sample ions and transporting them to the subsequent stage.

[0091] Then, in the first dissociation step S3, the target lipid ion is dissociated to remove the polar head group of PC(18:1 / 16:0), that is, the phosphocholine group. After removing the phosphocholine group, the fragment ion with a 1,3-dioxolane structure shown in Figure 4 can be obtained.

[0092] In the second dissociation step S4, the fragment ion with a 1,3-dioxolane structure can be broken at the 1,3-dioxolane, and the aziridine ring obtained by the derivatization reaction can also be broken, thereby forming multiple diagnostic ions. Some of the diagnostic ions can be used to identify the position of the carbon-carbon double bond in the fatty chain, that is, the C=C position diagnostic ions marked in Figure 4 ; some of the diagnostic ions can be used to identify the sn position of the carbon-carbon double bond, that is, the sn position diagnostic ions marked in Figure 4 ; some of the diagnostic ions can be used to determine the information of the fatty chain where the carbon-carbon double bond is located, that is, the fatty chain diagnostic ions marked in Figure 4 .

[0093] Figure 5 is a schematic diagram explaining why the sn position of the carbon-carbon double bond can be identified according to the diagnostic ions with a mass number of m / z = 290 or 360 [M+Na + .

[0094] Refer toFigure 5 It can be seen that when the carbon-carbon double bond is at different sn positions, the fragment ions and diagnostic ions generated by dissociation will be different. For PC(16:0 / 18:1) with the carbon-carbon double bond at the sn-2 position, it can be identified based on the diagnostic ion with a mass number of 290 [M+Na + . For PC(18:1 / 16:0) with the carbon-carbon double bond at the sn-1 position, it can be identified based on the diagnostic ion with a mass number of 360 [M+Na + .

[0095] Based on the above reaction process, it can be seen that the method for determining the chemical structure of lipids provided in this embodiment can simultaneously determine the position of the carbon-carbon double bond, the fatty acid chain composition, and the sn position in the fatty acid chain of phospholipids or sphingomyelins by a single injection, with excellent analysis efficiency.

[0096] The above describes some of the main steps of the method for determining the chemical structure of lipids, but it is not limited thereto. In other embodiments of the present invention, the method may further include other steps.

[0097] For example, before performing the first dissociation step S3 and the second dissociation step S5, a first pre-scanning step may be performed to perform mass analysis on the sample ions that have not undergone the first dissociation step S3 and the second dissociation step S5. This first pre-scanning step can be used to discover target lipid ions, that is, to discover the target lipid ions with a mass number of 775 [M+H + or 797 [M+Na + in this embodiment, and then further determine the isomers of the target lipid ions.

[0098] For another example, after performing the first dissociation step S3 and before performing the second dissociation step S5, a second pre-scanning step may be performed to perform mass analysis on the sample ions that have only undergone one dissociation. This second pre-scanning step can be used to determine the appropriate dissociation energy, so that the target lipid ions are dissociated as much as possible in a way that only breaks the polar head group, reducing or avoiding the generation of excess fragment ions, improving the signal intensity, and making the spectrum easier to read.

[0099] <Experimental Results>

[0100] Figure 6 It is a comparison chart of the measurement results obtained by the ordinary tandem mass spectrometry MS2 method in the prior art and the measurement results obtained by the method provided in this embodiment.

[0101] Figure 6 In the two mass spectrometry spectra corresponding to the prior art of Figure 6 , the upper figure is the MS2 spectrum, and the lower figure is the spectrum obtained by magnifying the range framed in the upper figure. Figure 6In the two mass spectrometry spectra corresponding to the measurement results of the present embodiment, the upper spectrum is the MS1 spectrum (where "pseudoMS2" indicates that IMS and the first dissociation device 3 are used as MS1, the same below), and the lower spectrum is the MS2 spectrum obtained by further dissociating the ions with a mass number of 592.

[0102] Reference Figure 6 It can be seen that due to the interference of the polar head group, in the MS2 spectrum of ordinary tandem mass spectrometry, the peak intensity of the diagnostic ion is in the range of about 700 - 800; while the peak intensity of the diagnostic ion obtained by the IMS-CID-MS / MS method provided in the present embodiment can reach the range of 5000 - 6000, greatly improving the signal intensity (about 7 - 8 times) and enhancing the analysis sensitivity.

[0103] The above are only the preferred embodiments of the present invention and are not intended to limit the present invention. Any modifications, equivalent replacements, and improvements made within the spirit and principle of the present invention shall be included within the protection scope of the present invention.

Claims

1. A method for determining the chemical structure of lipids, It is characterized in that The following steps are involved: An ionization step is to ionize the sample to obtain sample ions; an ion mobility separation step, separating target lipid 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, based on the mass number, selecting the target lipid ions in which the first chemical bonds are broken 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 having a bond energy higher than that of the first chemical bond, thereby obtaining a diagnostic ion; The mass analysis step is to perform mass analysis on the diagnostic ions.

2. The method for determining the chemical structure of lipids as claimed in claim 1, It is characterized in that The lipids are unsaturated lipids having carbon-carbon double bonds in the fat chains, and the method is used to identify the positions of the carbon-carbon double bonds in the fat chains and the sn positions of the fat chains.

3. The method for determining the chemical structure of lipids as claimed in claim 2, It is characterized in that Before the ionization step, the method further comprises: The derivatization step is to label the carbon-carbon double bond by using a derivatization reaction.

4. The method for determining the chemical structure of lipids as claimed in claim 3, It is characterized in that The lipid is a phospholipid or a sphingomyelin, the first chemical bond is a polar head group of the phospholipid or a polar head group of the sphingomyelin, and the second chemical bond is a chemical bond obtained by a derivatization reaction of the carbon-carbon double bond.

5. The method for determining the chemical structure of lipids as claimed in claim 3, It is characterized in that The derivatization reaction is an aziridination reaction, an epoxidation reaction, a Paternò-Büchi reaction, a singlet oxygen-ene reaction or a Diels-Alder reaction.

6. The method for determining the chemical structure of lipids as claimed in claim 1, It is characterized in that The lipids are fatty acyl, glyceride, glycerophospholipid, sphingolipid, sterol ester, pregnenolone ester, glycolipid or polyketide.

7. The method for determining the chemical structure of lipids as claimed in claim 1, It is characterized in that Also includes: In the first pre-scanning step, mass analysis is performed on the sample ions that have not undergone the first dissociation step and the second dissociation step.

8. The method for determining the chemical structure of lipids as claimed in claim 1, It is characterized in that Also includes: In the second pre-scanning step, mass analysis is performed on the sample ions that have undergone only one dissociation.

9. An ion mobility spectrometer tandem mass spectrometer, It is characterized in that include: An ion source, ionizing the sample to obtain sample ions; an ion mobility spectrometer, separating target lipid ions from the sample ions; a first dissociation device for dissociating the target lipid ions, wherein the dissociation energy of the first dissociation device is suitable for breaking the first chemical bond of the target lipid ions; A mass filter is used to select the target lipid ions in which the first chemical bond is broken to obtain fragment ions; a second dissociation device, dissociating the fragment ions, at least breaking a second chemical bond of the fragment ions having a bond energy higher than that of the first chemical bond, to obtain a diagnostic ion; A mass analyzer performs mass analysis on the diagnostic ions.

10. The ion mobility spectrometry tandem mass spectrometer according to claim 9, It is characterized in that The ion mobility spectrometer is a U-type ion mobility spectrometer.

11. The ion mobility spectrometry tandem mass spectrometer according to claim 10, It is characterized in that The U-type ion mobility spectrometer operates in filtering mode.

12. The ion mobility spectrometry tandem mass spectrometry instrument according to claim 9, It is characterized in that The mass filter is a quadrupole or an ion trap.

13. The ion mobility spectrometry tandem mass spectrometry instrument according to claim 9, It is characterized in that The mass analyzer is a time-of-flight mass analyzer, a Fourier transform mass spectrometer, a quadrupole mass analyzer, an ion trap mass analyzer or a magnetic mass spectrometer.

14. The ion mobility spectrometry tandem mass spectrometry instrument according to claim 9, It is characterized in that The ion source is an electrospray ionization source, a nano-electrospray 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 spectrometry instrument according to claim 9, It is 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 free radical directed dissociation device, an ultraviolet light-induced dissociation device, and a charge remote fragmentation device.

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

17. The ion mobility spectrometry tandem mass spectrometry instrument according to claim 9, It is characterized in that It also includes a separation device arranged in front of the ion source, and the separation device is one or more of liquid chromatography, gas chromatography, supercritical chromatography, capillary electrophoresis device and paper chromatography.

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