Mass spectrum multi-channel quantification method of multi-dimensional structure hierarchical lipid

Abstract

The invention provides a multi-dimensional structure hierarchy lipid mass spectrum multi-channel quantification method, which can realize multi-dimensional structure hierarchy high-flux parallel quantification of multiple samples in single detection, and has the advantages of complete structure coverage, chromatography independence, efficient fragmentation compatibility, economical efficiency and the like.

Description

Technical Field

[0001] This invention belongs to the field of mass spectrometry analysis technology and relates to a multi-channel mass spectrometry method for the quantification of lipids with multidimensional structural hierarchy. Background Technology

[0002] Lipidomics aims to systematically analyze the molecular composition, function, and metabolic regulatory network of lipids in organisms in order to reveal their molecular mechanisms in physiological and pathological processes.

[0003] However, the extreme structural complexity of lipid molecules (encompassing multiple categories, multidimensional structural levels, and a wide dynamic concentration range) poses a significant challenge to analytical techniques. Although mass spectrometry has become a core tool for lipid analysis, existing chromatography-mass spectrometry methods still struggle to achieve high-throughput, accurate quantification in the fine structural resolution of lipids (such as determining the position of carbon-carbon double bonds). Current double bond localization techniques face two major technical bottlenecks: first, the quantitative analysis system suffers from hierarchical fragmentation, with total composition quantification and isomer quantification performed stepwise, leading to error accumulation; second, analytical throughput is limited, with each analysis processing only a single sample. Iso-labeling technology developed in quantitative proteomics offers a new approach for high-throughput analysis, but its application in the quantification of multidimensional lipid structures still requires further breakthroughs.

[0004] Existing technologies attempt to integrate isotype-isotyped tags and isomer-resolved mass spectrometry to achieve high-throughput lipid quantification, but the following key technical defects still exist: 1) Derivatization methods have selectivity limitations and cannot simultaneously cover saturated lipids, monounsaturated lipids, and polyunsaturated lipids (Yang T, et al. Angewandte Chemie International Edition, 2022, 61(39): e202207098); 2) The quantification process depends on chromatographic baseline separation, which severely restricts analytical throughput (Feng Y, et al. Analytical Chemistry, 2022, 94(38): 13036-13042); 3) Mass spectrometry fragmentation mode has insufficient resolution for complex double bond systems, and the diagnostic ion yield is generally less than 5%, resulting in limited detection sensitivity (Armbruster MR, et al. Analyst, 2023, 148: 297-304); 4) Multi-step derivatization operations are cumbersome and difficult to meet the needs of large-scale sample analysis. Summary of the Invention

[0005] The problem the invention aims to solve

[0006] This invention addresses the core problems of existing lipidomics quantitative techniques, such as incomplete structural coverage, strong dependence on chromatographic separation, limited fragmentation efficiency, and complex operation procedures. It proposes a multi-channel mass spectrometry method for the quantitative analysis of lipids at multiple structural levels. Specifically, this method employs an intramolecular Pasternò-Büchi reaction combined with isomeric heterologous tagging (i... 2 The PB labeling technique, combined with tandem mass spectrometry, enables high-throughput parallel quantification of lipid molecules at multiple structural levels in a single assay. This method significantly improves the coverage, sensitivity, and throughput of lipidomics analysis.

[0007] Solution for solving the problem

[0008] This invention first provides a lipid analysis method based on tandem mass spectrometry, which includes the following steps:

[0009] 1) The lipid sample to be tested is treated with iPB reagent to induce a derivatization reaction between the lipids in the sample and the iPB reagent, thereby obtaining a derivatized product containing iPB-derived lipids; the iPB reagent contains a carbonyl group, a targeted derivatization group, and a photosensitive regulatory group, wherein the carbonyl group is used to undergo a Pastenò-Büchi reaction with the carbon-carbon double bond in the lipid; the targeted derivatization group is used to specifically react with the head group in the lipid to induce the derivatization reaction, preferably the head group is a carboxyl group, amino group, hydroxyl group, or phosphate group; the photosensitive regulatory group has an aromatic ring system;

[0010] 2) The derivatized product is irradiated with ultraviolet light to induce an intramolecular Pasterno-Büchi reaction in the iPB-derived lipids, thereby obtaining a cycloaddition product containing cyclized lipids;

[0011] 3) The cycloaddition product was analyzed by two-stage tandem mass spectrometry;

[0012] 4) By analyzing the mass-to-charge ratio and abundance of diagnostic ions generated by primary and secondary mass spectrometry, qualitative and quantitative analysis of lipids in the sample can be achieved.

[0013] The primary mass spectrometry data is used for the identification of the total lipid composition hierarchy and the quantification of its relative content, while the secondary mass spectrometry data is used for the identification of the carbon-carbon double bond structure hierarchy and the quantification of its relative content.

[0014] Preferably, the iPB reagent comprises (but is not limited to) the following compounds:

[0015]

[0016] Preferably, the molar ratio of lipids in the sample to the iPB reagent in step 1) is 1:(1 to 1.5), more preferably 1:1.1.

[0017] Preferably, the lipids include one or more of saturated lipids, monounsaturated lipids, and polyunsaturated lipids; or, the lipids include one or more of fatty acids, glycerides, glycerophospholipids, sphingolipids, sterol esters, isopentenols, glycolipids, and polyketides, with fatty acids being the most preferred.

[0018] Preferably, the catalyst for the derivatization reaction in step 1) is a condensing agent, preferably including one or more of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU), benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU), 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (HCTU), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), preferably 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate.

[0019] More preferably, the molar ratio of lipids to catalyst in the sample is 1:(1 to 1.5), preferably 1:1.2.

[0020] Preferably, the solvent for the derivatization reaction in step 1) is a polar aprotic solvent, preferably including one or more of acetonitrile, N,N-dimethylformamide, and dimethyl sulfoxide, with acetonitrile being the most preferred.

[0021] Preferably, the temperature of the derivatization reaction in step 1) is 25–70°C, more preferably 60°C.

[0022] Preferably, the derivatization reaction in step 1) takes 0.5 to 2 hours, more preferably 1 hour.

[0023] Preferably, the ultraviolet irradiation method in step 2) is performed offline or online.

[0024] Preferably, the wavelength of the ultraviolet light irradiation in step 2) is 254 nm.

[0025] Preferably, the ultraviolet light irradiation time in step 2) is 2 to 20 seconds, preferably 4 seconds.

[0026] Preferably, the conditions for mass spectrometry analysis in step 3) include: the injection method is shotgun injection, direct injection, or liquid chromatography-mass spectrometry; the ionization voltage of the first-stage mass spectrometer is 3000–4000 V; and the collision-induced dissociation energy of the second-stage mass spectrometer is 25–35 eV, such as 25 eV, 28 eV, 30 eV, 32 eV, 35 eV, etc. By using the above-mentioned collision-induced dissociation energy, it is possible to further facilitate the peak intensity of diagnostic ions in mass spectrometry analysis. If the collision energy is too high, the derivatization products will break into other ions, which will have an adverse effect on the mass spectrometry analysis.

[0027] Preferably, when the injection method is liquid chromatography-mass spectrometry, the intramolecular Pasterno-Büchi reaction is performed before or after liquid chromatography separation, preferably after liquid chromatography separation; preferably, the chromatographic conditions of the liquid chromatography include: a C18 column as the stationary phase, a binary system composed of acetonitrile-water and alcohol-acetonitrile as the mobile phase, and gradient elution as the elution method.

[0028] Preferably, when the lipids in the sample are fatty acids, the liquid chromatography includes the following chromatographic conditions: a nanoEase CSH C18 column as the stationary phase; acetonitrile-water (60:40, v / v, containing 20 mM ammonium formate) as mobile phase A, and isopropanol-acetonitrile (40:60, v / v, containing 0.2% formic acid) as mobile phase B; the gradient elution program is as follows: 0-0.75 min, mobile phase B volume percentage is 30%; 0.75-9 min, mobile phase B volume percentage is 30% → 97%; 9-12 min, mobile phase B volume percentage is 97%; 12-12.5 min, mobile phase B volume percentage is 97% → 30%; 12.5-15 min, mobile phase B volume percentage is 30%.

[0029] More preferably, the chromatographic conditions of the liquid chromatography further include: a flow rate of 1 to 10 μL / min, preferably 6 μL / min; and a column temperature of 50 to 60°C, preferably 55°C.

[0030] This invention also provides a high-throughput lipid analysis method based on tandem mass spectrometry, which includes the following steps:

[0031] 1) Multiple lipid samples to be tested were labeled with i-type markers that had the same amount but different sequence. 2 PB reagent treatment, wherein the lipids in each of the samples are respectively reacted with a set of isotope-labeled i 2 PB reagent was used for derivatization to obtain a set of i-type isotope-labeled products. 2 PB-derived lipid derivatives; the i 2The PB reagent comprises a carbonyl group, a targeting derivatization group, a photosensitizing group, a mass reporter group, and a mass balancing group. The carbonyl group is used to undergo a Pastenò-Büchi reaction with the carbon-carbon double bonds in the lipid. The targeting derivatization group is used to specifically react with the head group in the lipid to perform the derivatization reaction, preferably with a carboxyl, amino, hydroxyl, or phosphate group. The photosensitizing group has an aromatic ring system. The mass reporter group is used for tandem mass spectrometry fragmentation to generate reporter ions with different mass-to-charge ratios, enabling high-throughput parallel quantification of multiple samples. The mass balancing group is used for differentiated isotope distribution to achieve isogravimetric characteristics at the primary mass spectrometry level. The mass reporter group and the mass balancing group together constitute the isoquantitative heterogeneous tag.

[0032] 2) The isotopically labeled derivatized products are mixed and then irradiated with ultraviolet light to make the i 2 PB-derived lipids undergo an intramolecular Pasterno-Büchi reaction; or, the isotopically labeled derivatives are treated with ultraviolet light to make the i 2 PB-derived lipids undergo an intramolecular Pasterno-Büchi reaction before being mixed.

[0033] 3) Perform three-stage tandem mass spectrometry analysis. By analyzing the mass-to-charge ratio and abundance of diagnostic ions generated by the first-stage, second-stage, and third-stage mass spectrometry, high-throughput parallel qualitative and quantitative analysis of lipids in multiple samples can be achieved.

[0034] Among them, the first-level mass spectrometry data is used for the identification of the total lipid composition level, the second-level mass spectrometry data is used for the identification of the carbon-carbon double bond structure level and the abundance quantification of the total composition level, and the third-level mass spectrometry data is used for the abundance quantification of the carbon-carbon double bond structure level.

[0035] Preferably, the i 2 PB reagents include (but are not limited to) the following compounds:

[0036]

[0037] Preferably, the lipids in the sample in step 1) are related to the i 2 The molar ratio of PB reagent is 1:(1 to 1.5), preferably 1:1.1.

[0038] Preferably, the lipids include one or more of saturated lipids, monounsaturated lipids, and polyunsaturated lipids; or, the lipids include one or more of fatty acids, glycerides, glycerophospholipids, sphingolipids, sterol esters, isopentenols, glycolipids, and polyketides, with fatty acids being the most preferred.

[0039] Preferably, the catalyst for the derivatization reaction in step 1) is a condensing agent, preferably including one or more of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate (HATU), benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate (HBTU), 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate (HCTU), and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDC), preferably 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate.

[0040] More preferably, the molar ratio of lipids to catalyst in the sample is 1:(1 to 1.5), preferably 1:1.2.

[0041] Preferably, the solvent for the derivatization reaction in step 1) is a polar aprotic solvent, preferably including one or more of acetonitrile, N,N-dimethylformamide, and dimethyl sulfoxide, with acetonitrile being the most preferred.

[0042] Preferably, the temperature of the derivatization reaction in step 1) is 25–70°C, more preferably 60°C.

[0043] Preferably, the derivatization reaction in step 1) takes 0.5 to 2 hours, more preferably 1 hour.

[0044] Preferably, the ultraviolet irradiation method in step 2) is performed offline or online.

[0045] Preferably, the wavelength of the ultraviolet light irradiation in step 2) is 254 nm.

[0046] Preferably, the ultraviolet light irradiation time in step 2) is 2 to 20 seconds, preferably 4 seconds.

[0047] Preferably, the conditions for mass spectrometry analysis in step 3) include: the injection method is shotgun injection, direct injection, or liquid chromatography-mass spectrometry; the ionization voltage of the first-stage mass spectrometer is 3000–4000 V; the collision-induced dissociation energy of the second-stage mass spectrometer is 25–35 eV; and the collision-induced dissociation energy of the third-stage mass spectrometer is 40–45 eV.

[0048] Preferably, when the injection method is liquid chromatography-mass spectrometry, the intramolecular Pasterno-Büchi reaction is performed before or after liquid chromatography separation, preferably after liquid chromatography separation; preferably, the chromatographic conditions of the liquid chromatography include: a C18 column as the stationary phase, a binary system composed of acetonitrile-water and alcohol-acetonitrile as the mobile phase, and gradient elution as the elution method.

[0049] Preferably, when the lipids in the sample are fatty acids, the chromatographic conditions of the liquid chromatography include: using a nanoEase CSH C18 column as the stationary phase; using acetonitrile-water (60:40, v / v, containing 20 mM ammonium formate) as mobile phase A, and isopropanol-acetonitrile (40:60, v / v, containing 0.2% formic acid) as mobile phase B; the gradient elution program is as follows: 0-0.75 min, mobile phase B volume percentage is 30%; 0.75-9 min, mobile phase B volume percentage is 30% → 97%; 9-12 min, mobile phase B volume percentage is 97%; 12-12.5 min, mobile phase B volume percentage is 97% → 30%; 12.5-15 min, mobile phase B volume percentage is 30%.

[0050] More preferably, the chromatographic conditions of the liquid chromatography further include: a flow rate of 1 to 10 μL / min, preferably 6 μL / min; and a column temperature of 50 to 60°C, preferably 55°C.

[0051] Furthermore, the present invention provides a method for analyzing lipids in biological samples, which employs the above-described method.

[0052] Preferably, the biological sample is at least one of plasma, serum, cerebrospinal fluid, cell extract, organ, and tissue.

[0053] Preferably, the lipids include one or more of saturated lipids, monounsaturated lipids, and polyunsaturated lipids.

[0054] Preferably, the lipids include one or more of fatty acids, glycerides, glycerophospholipids, sphingolipids, sterol esters, isopentenols, glycolipids, and polyketides, with fatty acids being the most preferred.

[0055] The effects of the invention

[0056] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0057] 1) The method provided by this invention breaks through the limitations of traditional double bond derivatization methods: by targeting and modifying the lipid head group, it can be applied to both saturated and unsaturated lipids, eliminating the analytical barriers between categories; its intramolecular reaction characteristics make the derivatization efficiency not limited by concentration, effectively overcoming the detection problem of low-abundance lipids; for polyunsaturated lipids, the reaction avoids continuous addition side reactions through the intramolecular cycloaddition pathway, generating only a single addition product, which significantly improves the detection sensitivity.

[0058] 2) The method of the present invention enables high-throughput parallel quantification of multiple samples at multidimensional structural levels (e.g., overall composition level and carbon-carbon double bond structural level). This method has the following advantages: complete structural coverage (saturated and unsaturated lipids can be analyzed simultaneously), chromatographic independence (isomers can be distinguished through quadrupole mass isolation), high efficiency fragmentation compatibility (three-stage mass spectrometry linkage is suitable for complex double bond systems), and economy (the molar ratio of derivatization reagent to lipid is approximately 1:1).

[0059] 3) RPLC-Online iPB-MS n The CID high-throughput analysis platform features a simplified pretreatment process, requiring only one step of lipid head-based targeted derivatization. Subsequent derivatization (i.e., intramolecular Pasternò-Büchi reaction) is completed automatically online, significantly simplifying the operation and making it suitable for large-scale sample analysis. The online reaction is triggered post-column, effectively avoiding interference from byproducts and ensuring efficient separation of derivatized fatty acids by reversed-phase chromatography. The retention time-triggered acquisition strategy established based on this platform further enables efficient acquisition of high-throughput data. Attached Figure Description

[0060] Figure 1 Flowchart for lipid identification using iPB-MS / MS;

[0061] Figure 2 for iPB MS spectrum of FA 18:3(n-6,9,12) (a is MS spectrum) 1 Spectrum, b is MS 2 (Spectrum);

[0062] Figure 3 Part of the reagent toolkit designed and developed for iPB reagents;

[0063] Figure 4 This is a synthetic route diagram for BPA-TMT;

[0064] Figure 5 For BPA-NHBoc 1 H-NMR spectrum;

[0065] Figure 6 MS spectrum of BPA-NHBoc;

[0066] Figure 7 MS spectrum of BPA-TMT (without isotopic labeling);

[0067] Figure 8 For RPLC-online iPB-MS n Schematic diagram of high-throughput parallel quantification of CID in a multidimensional structural hierarchy;

[0068] Figure 9For RPLC-online iPB-MS n Schematic diagram of CID high-throughput analysis platform;

[0069] Figure 10 For i 2 A flowchart illustrating the workflow of using PB labeling for analyzing complex biological samples;

[0070] Figure 11 For i 2 MS results of PB labeling for lipid analysis in multiple biological samples. Detailed Implementation

[0071] Various exemplary embodiments, features, and aspects of the present invention will be described in detail below. The term "exemplary" as used herein means "serving as an example, embodiment, or illustration." Any embodiment described herein as "exemplary" is not necessarily to be construed as superior to or better than other embodiments.

[0072] Furthermore, to better illustrate the present invention, numerous specific details are set forth in the following detailed embodiments. Those skilled in the art should understand that the present invention can be practiced without certain specific details. In other instances, methods, means, apparatus, and steps well known to those skilled in the art have not been described in detail in order to highlight the spirit of the present invention.

[0073] Unless otherwise stated, all units used in this specification are international standard units, and all numerical values ​​and ranges appearing in this invention should be understood to include systematic errors that are unavoidable in industrial production.

[0074] In this specification, the word "may" has two meanings: to perform a certain process and not to perform a certain process.

[0075] In this specification, references to "some specific / preferred embodiments," "other specific / preferred embodiments," "implementation," etc., refer to specific elements (e.g., features, structures, properties, and / or characteristics) related to that embodiment, which are included in at least one of the embodiments described herein and may or may not be present in other embodiments. Furthermore, it should be understood that these elements may be combined in any suitable manner in various embodiments.

[0076] In this specification, the range of values ​​referred to as "value A to value B" refers to the range including the endpoint values ​​A and B.

[0077] Unless otherwise stated, in this instruction manual, "more" in "multiple", "multi-variety", "multiple", etc., means a value of 2 or more.

[0078] In this specification, the terms "optional" or "optional / optionally" are used to indicate the use or non-use of certain substances, components, procedures, application conditions, etc.

[0079] Unless otherwise specified, “room temperature” or “room temperature” as used in this instruction manual usually refers to a temperature of 23±2℃.

[0080] All unit names used in this manual are international standard unit names, and unless otherwise stated, the "%" used refers to weight or mass percentage content.

[0081] In this specification, the terms "comprising," "having," "including," or "containing" can mean included or open-ended, and do not exclude additional, uncited elements or method steps. At the same time, "comprising," "having," "including," or "containing" can also mean closed-ended, excluding additional, uncited elements or method steps.

[0082] The “collision-induced dissociation (CID)” mentioned in this manual refers to the ion dissociation method in mass spectrometry instruments, which involves splitting the parent ion to obtain fragment ions.

[0083] Furthermore, it should be understood that when the term "isotropic labels" is used in scenarios involving multiple samples, it actually refers to a group of labels with identical chemical structures but different isotopic compositions. These labels are chemically identical and are distinguished only by differences in isotopic substitution patterns.

[0084] In this specification, "total composition level" refers to the measurement of intact, unfragmented lipid molecular ions (usually in adduct form, such as [M+H)) during mass spectrometry analysis. + [M+Na] + [M+NH4] + (etc.). Using high-resolution mass spectrometry, the mass-to-charge ratio (m / z) of these ions can be precisely determined, allowing for the calculation of their precise molecular weight. Based on this precise molecular weight, the "total composition" of lipids can be determined, i.e., information at the molecular formula level, mainly including: 1) Lipid category: for example, fatty acids (FA), glycerides, glycerophospholipids, sphingolipids, etc.; 2) Total number of carbon atoms: the total number of carbon atoms in the lipid molecule; 3) Total number of double bonds: the total number of double bonds in the lipid molecule. For example, if the ion [M+H] is detected... + Its precise m / z corresponds to the molecular formula C after analysis. 18 H 32 O2 indicates that the component is a fatty acid containing 18 carbon atoms (C18). Based on the number of hydrogen atoms, it can be deduced that it has two degrees of unsaturation (the molecular formula of a saturated C18 fatty acid should be C...). 18 H 36O2 (actually has 4 fewer hydrogen atoms, typically corresponding to 2 carbon-carbon double bonds). In MS... 1 At this level, the component can only be identified as FA 18:2, and the specific position of the carbon-carbon double bond cannot be distinguished (e.g., it is impossible to determine whether it is ω-6 series linoleic acid (n-6,9) or a C18:2 double bond isomer at other positions).

[0085] In this specification, "carbon-carbon double bond structural hierarchy" refers to the hierarchy used in mass spectrometry analysis to resolve the precise structure of carbon-carbon double bonds (C=C) in lipid molecules through specific derivatization or fragmentation techniques. This hierarchy, below the "overall composition hierarchy," provides more refined molecular structural information and is primarily used to determine the specific positions of carbon-carbon double bonds on fatty acid chains. For example, for a lipid with an overall composition identified as FA 18:2, resolving its "carbon-carbon double bond structural hierarchy" can be achieved using the iPB derivatization reaction. This reaction converts the double bond into a four-membered epoxide butane ring structure. Further details will be provided in the subsequent MS analysis. 2 During CID cleavage, the epoxide-butane ring undergoes specific breakage, producing a pair of characteristic fragment ions (diagnostic ions) with diagnostic significance. By accurately determining the mass-to-charge ratio (m / z) of this pair of diagnostic ions, the precise location of the original double bond can be deduced. For example, if the diagnostic ions show that the double bond is located at positions n-6 and n-9, then FA 18:2 can be precisely identified as linoleic acid FA18:2(n-6,9).

[0086] Example

[0087] The embodiments of the present invention will be described in detail below with reference to examples. However, those skilled in the art will understand that the following examples are for illustrative purposes only and should not be considered as limiting the scope of the invention. Unless otherwise specified in the examples, conventional conditions or conditions recommended by the manufacturer are followed. Reagents or instruments whose manufacturers are not specified are all commercially available conventional products.

[0088] Example 1:

[0089] This embodiment provides a lipid analysis method based on second-order tandem mass spectrometry (MS / MS). This method employs an intramolecular Patenò-Büchi reaction combined with second-order tandem mass spectrometry (iPB-MS / MS): First, iPB reagents are used to derivatize the head groups in lipids, simultaneously achieving efficient analysis of both saturated and unsaturated lipids (conversion rate > 99.9%); subsequently, unsaturated lipids undergo an intramolecular [2+2] cycloaddition reaction under ultraviolet light irradiation, generating regioselective adducts; finally, the lipids are analyzed by MS / MS. 2 CID cleavage produces characteristic n-xF O / n-x f ODiagnostic ions enable precise location of carbon-carbon double bonds (e.g., Figure 1 As shown, fatty acids are used as a model lipid to illustrate this process.

[0090] The following verification was performed using FA 18:3(n-6,9,12) as an example: 100 μL of a 10 μM methanol solution of fatty acid standards was concentrated and dried under nitrogen. Then, 58 μL of acetonitrile, 20 μL of triethylamine acetonitrile solution (200 μM, 4 equivalents), 12 μL of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate acetonitrile solution (100 μM, 1.2 equivalents), and 10 μL of LiBPA acetonitrile solution (110 μM, 1.1 equivalents) were added sequentially. The reaction system was vortexed at 60 °C for 1 h. After the reaction, the mixture was extracted twice using a methyl tert-butyl ether:acetonitrile:water (3:1:1, v / v / v) mixed solvent. The upper organic phase was collected, concentrated under nitrogen, and finally redissolved in 100 μL of acetonitrile.

[0091]

[0092] Offline iPB reaction was performed on the above samples using a self-made microfluidic reactor: the derivatized fatty acid solution was irradiated with 254 nm UV light for 4 s, the reaction solution was collected, and analyzed by reversed-phase liquid chromatography-tandem mass spectrometry (RPLC-MS / MS). The mass spectrometry analysis results are as follows: Figure 2 As shown.

[0093] In MS 1 iPB derivatives ([M+H]) can be detected in the spectrum. + :m / z 544.343,[M+NH4] + (m / z 561.367). Further analysis of the ammonium adduct ion [M+NH4] of the derivative... + Perform MS 2 CID fragmentation analysis yielded three pairs of characteristic C=C position diagnostic ions: n-6 F O (m / z 444.252) / n-6 f O (m / z 352.225), n-9 F O (m / z 404.222) / n-9 f O (m / z 392.258) and n- 12 F O (m / z 364.191) / n-12 f O(m / z 432.285). The results showed that the iPB reagent could specifically derive lipid head groups, undergoing an intramolecular [2+2] cycloaddition reaction under UV irradiation to generate a four-membered oxygen heterocyclic structure; and this was confirmed by MS. 2 CID fragmentation generates characteristic fragment ions, thereby enabling accurate identification of the carbon-carbon double bond position.

[0094] The iPB reagent is designed based on the synergistic effect of three functional modules: carbonyl groups (for the Pasternò-Büchi reaction with carbon-carbon double bonds in lipids), targeted derivatization groups (for the specific derivatization reaction with head groups (such as carboxyl, amino, hydroxyl, or phosphate groups) in lipids), and photosensitive regulatory groups (with aromatic ring systems, such as benzene rings, pyridine rings, etc.; utilizing aromatic ring systems to optimize reaction efficiency), together providing a universal tool for the multidimensional analysis of lipids.

[0095] Furthermore, this invention develops a widely applicable reagent toolkit by systematically regulating the modular groups of iPB reagents, and evaluates the iPB yield under different reagent conditions [since the first-stage derivatization reaction specifically reacts with the head group in the lipid, complete conversion has been achieved; here, only the yield of the second-stage iPB reaction (intramolecular Paternò-Büchi reaction) is considered; the yield is calculated as the ratio of the signal intensity of the iPB product after the reaction to the signal intensity of the head group-derived lipid before the reaction]. Relevant reagents and their performance are shown below. Figure 3 As shown.

[0096] Example 2:

[0097] This embodiment provides a high-throughput lipid analysis method based on tandem mass spectrometry. This method employs an intramolecular Paternò-Büchi reaction combined with isobaric intramolecular Paternò-Büchi (isobaric intramolecular Paternò-Büchi, i 2 PB) tagging strategy.

[0098] i 2PB reagent integrates five functional modules working synergistically: (1) Carbonyl group: used to react with carbon-carbon double bonds (C=C) in lipids under ultraviolet light irradiation; (2) Targeted derivatization group: used to specifically derivatize head groups (such as carboxyl, amino, hydroxyl or phosphate groups) in lipids; (3) Photosensitive regulation group: has an aromatic ring system (e.g. benzene ring, pyridine ring, etc.), and uses the aromatic ring system to optimize the reaction efficiency; (4) Mass reporter group: generates reporter ions with different mass-to-charge ratios (m / z) during MS / MS fragmentation, thereby achieving high-throughput parallel quantification of multiple samples; (5) Mass balancing group: achieves the effect of different isotope distributions in MS / MS. 1 The equal weight characteristics of the layers.

[0099] Taking the preparation of 6-plex BPA-TMT by combining six-plex iso-hetero-tagged TMT (tandem mass tags) with 4-benzoyl-phenylalanine (BPA) as an example, this demonstrates i 2 A typical implementation path for the PB tagging method. Its synthesis route is as follows: Figure 4 As shown, the intermediate BPA-NHBoc was prepared through a multi-step reaction involving amino protection, carboxyl activation coupling, and catalytic deprotection. Its structural characterization is as follows: Figure 5 and 6 As shown; the final product BPA-TMT is generated by the amidation reaction of BPA-NHBoc and TMT reagent under the action of a condensing agent, followed by acid deprotection and chromatographic purification steps. Its structural characterization is as follows. Figure 7 As shown. The six isotope labeling reagents were synthesized using the same route.

[0100] i 2 The analytical workflow of the PB labeling method encompasses a two-stage sequential derivatization reaction and a three-stage mass spectrometry linkage: MS 1 Used for identification of overall composition level → MS 2 Simultaneous release of mass reporter ions at the overall compositional level and characteristic F for carbon-carbon double bond localization O Diagnostic Ions → MS 3 This process releases mass reporter ions at the carbon-carbon double bond structural level. It enables high-throughput parallel quantification of multiple samples at both the overall composition and carbon-carbon double bond structural levels in a single analysis.

[0101] The method was validated using FA 18:1(n-9Z)[d9] as a model compound: Six equal volumes of 100 μL FA 18:1(n-9)[d9] standard methanol solution (10 μM) were taken, concentrated and dried by nitrogen blowing, and then 58 μL acetonitrile, 20 μL triethylamine acetonitrile solution (200 μM, 4 equivalents), 12 μL 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate acetonitrile solution (100 μM, 1.2 equivalents) were added in sequence, followed by 10 μL 6-plex BPA-TMT acetonitrile solution (110 μM, 1.1 equivalents). The reaction system was vortexed at 60℃ for 1 h. After the reaction, the mixture was extracted twice with a mixed solvent of methyl tert-butyl ether: acetonitrile: water (3:1:1, v / v / v). The upper organic phase was collected and concentrated by nitrogen blowing, and finally redissolved in 100 μL of acetonitrile.

[0102] The six derivatized samples were mixed into a single sample, and an offline iPB reaction was performed on the mixed sample using a self-made microfluidic reactor (the derivatized fatty acid solution was irradiated with 254 nm UV light for 4 s). The mixture was then analyzed by reversed-phase liquid chromatography-tandem mass spectrometry (RPLC-MS / MS / MS). The mass spectrometry results are as follows: Figure 8 As shown.

[0103] In MS 1 i can be detected in the spectrum 2 PB derivatives ([M+H)) + :m / z 814.6337); for [M+H] + Perform MS 2 CID fragmentation produces diagnostic ions at position n-9. 9 F O (m / z 663.4429), double bond localization was achieved, and the mass reporter ion (m / z 126-131) of the overall composition hierarchy was released simultaneously; further, the C=C diagnostic ion n- 9 F O Perform MS 3 CID fragmentation released C=C level mass reporter ions (m / z 126-131). The intensity ratio of each mass reporter ion was 0.96:0.97:1.07:0.95:1.01:1.05, which was highly consistent with the initial molar ratio of the six samples (1:1:1:1:1:1), indicating that i 2 The PB labeling method enables high-throughput parallel quantification of lipids at multidimensional structural levels in a single analysis.

[0104] Example 3: RPLC-online iPB-MS n CID High-Throughput Analysis Platform

[0105] To address the complexity of sample pretreatment, an online platform integrating a microfluidic photoreactor (device such as...) was developed. Figure 9 As shown in the figure, the online iPB reaction is triggered after separation by reversed-phase chromatography.

[0106] i 2 The PB labeling method is adaptable to large-scale batch analysis of complex biological samples. The specific workflow is as follows: Figure 10 As shown: Multiple samples were extracted for total lipids using a methyl tert-butyl ether / methanol / water (10:3:2.5, v / v / v) system, followed by saponification and hydrolysis to obtain fatty acids. After isooctane extraction, the fatty acids were further processed using different methods. 2 PB tag variant marking, mixed via RPLC-online iPB-MS n The CID high-throughput analysis platform completed the analysis.

[0107] Among them, RPLC-online iPB-MS n The CID high-throughput analysis platform involves a Waters M-class liquid chromatography system (Waters Corporation, USA) coupled with an Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific, USA).

[0108] Chromatographic conditions: Column: nanoEase CSH C18 ( 1.7μm, 300μm×150mm); Injection volume: 1μL; Mobile phase: Phase A is acetonitrile-water (60:40, v / v, containing 20mM ammonium formate), Phase B is isopropanol-acetonitrile (40:60, v / v, containing 0.2% formic acid); Flow rate: 6μL / min; Column temperature: 55℃; Gradient elution program: 0-0.75min 30%B, 0.75-9min 30%→97%B, 9-12min 97%B, 12-12.5min 97%→30%B, 12.5-15min 30%B.

[0109] Mass spectrometry parameters: MS 1 Electrospray voltage 3500V, sheath gas 5 alb, auxiliary gas 7 alb, ion transport tube temperature 325℃, Orbitrap resolution 120000, AGC target in standard mode; MS 2 CID employs a retention time-dependent target list, with a high-energy collisional dissociation (HCD) energy of 35 eV and a resolution of 30,000; MS 3 CID / SPS MS 3 A retention time-dependent target list was used, with HCD energy of 42 eV.

[0110] Taking the normal breast cell line MCF-10A and the breast cancer cell line MDA-MB-468 as examples, 6-plex i 2 PB labeling strategy (three biological replicates each in the normal and cancer groups), analyzed by RPLC-online iPB-MS n The CID high-throughput platform performed a systematic analysis of fatty acids. For example... Figure 11 As shown, in MS 1 Multiple fatty acids, including FA 16:1, FA 18:2, FA 18:0, and FA 20:2, were identified at the surface. Taking FA 20:2 as an example, MS analysis revealed... 2 After CID fragmentation, six groups of diagnostic ions were generated that could distinguish carbon-carbon double bond positional isomers (corresponding to n-6, 9 / n-7, 10 / n-8, 11 / n-9, 12 / n-10, 13 / n-9, and 15, respectively). Simultaneously, mass reporter ions at the overall compositional level were obtained, showing that the abundance of FA 20:2 in the carcinogenic group was only 0.14 times that in the normal group. Furthermore, taking FA20:2(n-9, 12) as an example, MS analysis was performed... 3 CID analysis yielded C=C level mass reporter ions, and the abundance of the FA20:2(n-9,12) isomer in the carcinogenic group was 7.64 times that in the normal group.

[0111] High-throughput qualitative and quantitative analysis of all fatty acids can be achieved using the same analytical workflow. In the analysis of complex samples, i 2 The PB labeling strategy uses RPLC to separate fatty acids solely to avoid ionization inhibition caused by high abundance on low abundance, without requiring baseline separation of double bond isomers. This achieves high-throughput parallel quantification at multiple structural levels (overall composition level and carbon-carbon double bond structural level). The fragmentation mode of this strategy is suitable for complex fatty acid systems containing multiple double bonds and possesses high detection sensitivity. Furthermore, online iPB and RPLC-online iPB-MS are also available. n The development of the CID high-throughput analysis platform has significantly simplified the pretreatment process: only one step of derivatization reaction is needed to target and modify the lipid head group, and the second step of intramolecular cycloaddition is completed online during chromatographic separation, avoiding the complex operation of multi-step derivatization and providing strong support for high-throughput analysis of large-scale samples.

[0112] It should be noted that although the technical solution of the present invention has been described with specific examples, those skilled in the art will understand that the present invention should not be limited thereto.

[0113] The various embodiments of the present invention have been described above. These descriptions are exemplary and not exhaustive, nor are they limited to the disclosed embodiments. Many modifications and variations will be apparent to those skilled in the art without departing from the scope and spirit of the described embodiments. The terminology used herein is chosen to best explain the principles, practical application, or technical improvements to the embodiments in the market, or to enable others skilled in the art to understand the embodiments disclosed herein.

Claims

1. A lipid analysis method based on tandem mass spectrometry, characterized in that, Includes the following steps: 1) The lipid sample to be tested is treated with iPB reagent to induce a derivatization reaction between the lipids in the sample and the iPB reagent, thereby obtaining a derivatized product containing iPB-derived lipids; the iPB reagent contains a carbonyl group, a targeted derivatization group, and a photosensitive regulatory group, wherein the carbonyl group is used to undergo a Pastenò-Büchi reaction with the carbon-carbon double bond in the lipid; the targeted derivatization group is used to specifically react with the head group in the lipid to induce the derivatization reaction, preferably the head group is a carboxyl group, amino group, hydroxyl group, or phosphate group; the photosensitive regulatory group has an aromatic ring system; 2) The derivatized product is irradiated with ultraviolet light to induce an intramolecular Pasterno-Büchi reaction in the iPB-derived lipids, thereby obtaining a cycloaddition product containing cyclized lipids; 3) The cycloaddition product was analyzed by two-stage tandem mass spectrometry; 4) By analyzing the mass-to-charge ratio and abundance of diagnostic ions generated by primary and secondary mass spectrometry, qualitative and quantitative analysis of lipids in the sample can be achieved. The primary mass spectrometry data is used for the identification of the total lipid composition hierarchy and the quantification of its relative content, while the secondary mass spectrometry data is used for the identification of the carbon-carbon double bond structure hierarchy and the quantification of its relative content.

2. The method according to claim 1, characterized in that, In step 1), The molar ratio of lipids to the iPB reagent in the sample is 1:(1-1.5); Preferably, the lipids include one or more of saturated lipids, monounsaturated lipids, and polyunsaturated lipids; or, the lipids include one or more of fatty acids, glycerides, glycerophospholipids, sphingolipids, sterol esters, isopentenols, glycolipids, and polyketides, with fatty acids being the most preferred.

3. The method according to claim 1 or 2, characterized in that, In step 1), The catalyst for the derivatization reaction is a condensing agent, preferably including one or more of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; and / or, The solvent for the derivatization reaction is a polar aprotic solvent, preferably including one or more of acetonitrile, N,N-dimethylformamide, and dimethyl sulfoxide; and / or, The derivatization reaction is performed at a temperature of 25–70°C; and / or, The derivatization reaction takes 0.5 to 2 hours.

4. The method according to any one of claims 1-3, characterized in that, In step 2), The ultraviolet light irradiation can be performed offline or online; and / or, The wavelength of the ultraviolet light irradiation is 254 nm; and / or, The duration of ultraviolet light irradiation is 2–20 seconds.

5. The method according to any one of claims 1-4, characterized in that, In step 3), The conditions for the mass spectrometry analysis include: the injection method is shotgun injection, direct injection or liquid chromatography-mass spectrometry, the ionization voltage of the first-stage mass spectrometer is 3000-4000V, and the collision-induced dissociation energy of the second-stage mass spectrometer is 25-35eV. Preferably, when the injection method is liquid chromatography-mass spectrometry, the intramolecular Pasterno-Büchi reaction is performed before or after liquid chromatography separation, preferably after liquid chromatography separation; preferably, the chromatographic conditions of the liquid chromatography include: a C18 column as the stationary phase, a binary system composed of acetonitrile-water and alcohol-acetonitrile as the mobile phase, and gradient elution as the elution method.

6. A high-throughput lipid analysis method based on tandem mass spectrometry, characterized in that, Includes the following steps: 1) Multiple lipid samples to be tested were labeled with i-type markers that had the same amount but different sequence. 2 PB reagent treatment, wherein the lipids in each of the samples are respectively reacted with a set of isotope-labeled i 2 PB reagent was used for derivatization to obtain a set of i-type isotope-labeled products. 2 PB-derived lipid derivatives; the i 2 The PB reagent comprises a carbonyl group, a targeting derivatization group, a photosensitizing group, a mass reporter group, and a mass balancing group. The carbonyl group is used to undergo a Pastenò-Büchi reaction with the carbon-carbon double bonds in the lipid. The targeting derivatization group is used to specifically react with the head group in the lipid to perform the derivatization reaction, preferably with a carboxyl, amino, hydroxyl, or phosphate group. The photosensitizing group has an aromatic ring system. The mass reporter group is used for tandem mass spectrometry fragmentation to generate reporter ions with different mass-to-charge ratios, enabling high-throughput parallel quantification of multiple samples. The mass balancing group is used for differentiated isotope distribution to achieve isogravimetric characteristics at the primary mass spectrometry level. The mass reporter group and the mass balancing group together constitute the isoquantitative heterogeneous tag. 2) The isotopically labeled derivatized products are mixed and then irradiated with ultraviolet light to make the i 2 PB-derived lipids undergo an intramolecular Pasterno-Büchi reaction; or, the isotopically labeled derivatives are treated with ultraviolet light to make the i 2 PB-derived lipids undergo an intramolecular Pasterno-Büchi reaction before being mixed. 3) Perform three-stage tandem mass spectrometry analysis. By analyzing the mass-to-charge ratio and abundance of diagnostic ions generated by the first-stage, second-stage, and third-stage mass spectrometry, high-throughput parallel qualitative and quantitative analysis of lipids in multiple samples can be achieved. Among them, the first-level mass spectrometry data is used for the identification of the total lipid composition level, the second-level mass spectrometry data is used for the identification of the carbon-carbon double bond structure level and the abundance quantification of the total composition level, and the third-level mass spectrometry data is used for the abundance quantification of the carbon-carbon double bond structure level.

7. The method according to claim 6, characterized in that, In step 1), The lipids in the sample and the i 2 The molar ratio of PB reagent is 1:(1~1.5); Preferably, the lipids include one or more of saturated lipids, monounsaturated lipids, and polyunsaturated lipids; or, the lipids include one or more of fatty acids, glycerides, glycerophospholipids, sphingolipids, sterol esters, isopentenols, glycolipids, and polyketides, with fatty acids being the most preferred.

8. The method according to claim 6 or 7, characterized in that, In step 1), The catalyst for the derivatization reaction is a condensing agent, preferably including one or more of 2-(7-azabenzotriazole)-N,N,N',N'-tetramethylurea hexafluorophosphate, benzotriazole-N,N,N',N'-tetramethylurea hexafluorophosphate, 6-chlorobenzotriazole-1,1,3,3-tetramethylurea hexafluorophosphate, and 1-ethyl-(3-dimethylaminopropyl)carbodiimide hydrochloride; and / or, The solvent for the derivatization reaction is a polar aprotic solvent, preferably including one or more of acetonitrile, N,N-dimethylformamide, and dimethyl sulfoxide; and / or, The derivatization reaction is performed at a temperature of 25–70°C; and / or, The derivatization reaction takes 0.5 to 2 hours.

9. The method according to any one of claims 6-8, characterized in that, In step 2), The ultraviolet light irradiation can be performed offline or online; and / or, The wavelength of the ultraviolet light irradiation is 254 nm; and / or, The duration of ultraviolet light irradiation is 2–20 seconds.

10. The method according to any one of claims 6-9, characterized in that, In step 3), The conditions for the mass spectrometry analysis include: the injection method is shotgun injection, direct injection, or liquid chromatography-mass spectrometry; the ionization voltage of the first-stage mass spectrometer is 3000–4000 V; the collision-induced dissociation energy of the second-stage mass spectrometer is 25–35 eV; and the collision-induced dissociation energy of the third-stage mass spectrometer is 40–45 eV. Preferably, when the injection method is liquid chromatography-mass spectrometry, the intramolecular Pasterno-Büchi reaction is performed before or after liquid chromatography separation, preferably after liquid chromatography separation; preferably, the chromatographic conditions of the liquid chromatography include: a C18 column as the stationary phase, a binary system composed of acetonitrile-water and alcohol-acetonitrile as the mobile phase, and gradient elution as the elution method.

11. A method for analyzing lipids in a biological sample, comprising the method described in any one of claims 1-10; in, The biological sample is at least one of plasma, serum, cerebrospinal fluid, cell extract, organ, and tissue. The lipids include one or more of saturated lipids, monounsaturated lipids, and polyunsaturated lipids; or, the lipids include one or more of fatty acids, glycerides, glycerophospholipids, sphingolipids, sterol esters, isopentenols, glycolipids, and polyketides, preferably fatty acids.

Images