A sample processing method and mass spectrometry detection method for selective photocatalytic deglycosylation of glycosphingolipids

Abstract

The application relates to a sample processing method and a mass spectrometry detection method for selective photocatalytic desugar of glycosphingolipids. The sample processing method specifically comprises the following steps: (1) dissolving a sample in an alkaline protic solvent; (2) dispersing titanium dioxide particles in the sample; and (3) performing a photocatalytic reaction under ultraviolet light irradiation, centrifuging, and obtaining a photocatalytically processed sample solution. The mass spectrometry detection method specifically comprises the following steps: feeding the photocatalytically processed sample solution into a mass spectrometer to perform primary mass spectrometry detection, comparing the photocatalytically processed sample solution with a sample extraction solution without photocatalytic processing, screening a primary mass spectrometry peak that may be a glycolipid decomposition product through comparative analysis, and performing further secondary mass spectrometry detection to analyze the accurate molecular structure of the glycolipid. The method can selectively and rapidly and efficiently remove the sugar group head of glycolipids in the presence of complex lipid groups, and can be used for subsequent mass spectrometry detection and analysis. Through comparison of mass spectrometry peaks before and after removal, glycolipid peaks in complex mass spectrometry peaks can be effectively and rapidly and accurately found, important chemical information for rapid locking of correct candidate substances in the primary mass spectrometry qualitative process is provided, and glycolipid decomposition products can also obtain more abundant carbon chain structure information than original glycolipids to assist in qualitative analysis.

Description

Technical Field

[0001] This invention belongs to the field of sample pretreatment and detection technology for chemical substance detection, and particularly relates to a sample pretreatment method and mass spectrometry detection method for selective photocatalytic deglycosylation of glycosphingolipids. Background Technology

[0002] Glycosphingolipids are a class of glycolipids widely distributed on the surface of animal cell membranes, playing important biological roles in regulating cell recognition, adhesion, proliferation, and apoptosis. However, high-coverage detection and complete structural characterization of glycosphingolipids in complex biological samples, such as blood, tissues, and cells, remains a challenge. In terms of mass spectrometry, glycosphingolipids themselves exhibit low ionization efficiency and low signal response in mass spectrometry. Furthermore, because glycosphingolipids and phospholipids reside in the same mass spectrometric region, and phospholipids are abundant and highly ionized in tissues, ionization of glycosphingolipids is significantly inhibited, making mass spectrometry detection, especially direct mass spectrometry, more difficult. Regarding structural characterization, glycosphingolipids consist of a glycosyl head, a sphingosine backbone, and fatty acid chains. The structures of the sphingosine backbone and fatty acid chains are highly variable, and accurately determining the number and position of hydroxyl groups and double bonds modified on the carbon chains is challenging.

[0003] In traditional detection methods, after extracting glycosphingolipids from tissue or cell samples, two approaches are used: separation using normal-phase or reversed-phase chromatography followed by mass spectrometry for detection. These methods require cumbersome and lengthy sample preparation processes. Extracting glycosphingolipids involves ultrasonically disrupting the sample cells or tissues in a specific ratio of chloroform, methanol, and water, or a specific ratio of isopropanol, hexane, and water to release crude lipids. A saponification reaction is then performed in a weakly alkaline environment to remove the influence of glycerophospholipids on the results. Subsequently, chloroform, methanol, or the Folch method is used to extract and separate the glycosphingolipids, followed by LC-ESI-MS / MS analysis. These processes are lengthy and complex, and the resulting chromatograms still contain other interfering lipids. Qualitative identification of glycosphingolipids from complex primary mass spectra relies solely on precise molecular ion peak mass-to-charge ratios. However, the complex structures of various lipids lead to interference between isomers and isotopes with similar molecular weights. Chromatographic separation can effectively separate various lipids, reducing ion inhibition and peak overlap. However, because chromatography cannot provide absolute structural information, accurately identifying and characterizing glycolipid peaks among numerous spectra remains difficult. Peak fragments obtained through secondary mass spectrometry can further characterize glycolipid peaks; however, secondary mass spectrometry demands higher instrument sensitivity and throughput. Often, many primary mass spectrometry peaks cannot be fragmented into secondary mass spectra due to low signal strength, and obtaining secondary mass spectra from all primary peaks individually is time-consuming. More importantly, secondary mass spectra of glycolipids rarely contain fragments representing the precise structure of fatty acid chains, making complete resolution of the carbon chain structure of glycolipids challenging.

[0004] Therefore, the pretreatment process for glycosphingolipid samples is a crucial stage for improving their detection and qualitative analysis. Currently, there are few reported mass spectrometry sample processing and mass spectrometry analysis methods that can rapidly and efficiently qualitatively identify large quantities of glycolipids in complex biological samples and completely resolve their structures. Summary of the Invention

[0005] The technical problem to be solved by this invention is the inability to rapidly and efficiently qualitatively identify large amounts of glycolipids in complex biological samples and to completely resolve the glycolipid structure. This invention overcomes the shortcomings and defects mentioned in the background art and provides a sample processing method and a mass spectrometry detection method for selective photocatalytic deglycosylation of glycosphingolipids.

[0006] To solve the above-mentioned technical problems, the technical solution proposed by this invention is as follows:

[0007] A sample preparation method for selective photocatalytic deglycosylation of glycosphingolipids specifically includes the following steps:

[0008] (1) Dissolve the sample in an alkaline protic solvent to obtain a sample extract;

[0009] (2) Disperse titanium dioxide particles in the sample extract to obtain a dispersion;

[0010] (3) The dispersion was subjected to photocatalytic reaction under ultraviolet light irradiation, centrifuged, and the supernatant was obtained as photocatalytic deglycosylation supernatant, and the solid powder was obtained as titanium dioxide powder.

[0011] like Figure 1 As shown, the selective photocatalytic deglycosylation method for glycolipids in this invention utilizes the principle of photocatalytic oxidation and decomposition on the surface of titanium dioxide under ultraviolet light. Specifically, glycolipids are selectively adsorbed onto the surface of titanium dioxide due to the bidentate chelation between the glycosidic heads and the Lewis acidic sites in titanium dioxide under alkaline conditions. When ultraviolet light irradiates the surface of titanium dioxide, the titanium dioxide absorbs the ultraviolet light and generates photogenerated electrons and holes. The holes oxidize the water adsorbed on the surface into hydroxyl radicals, thereby selectively oxidizing and decomposing the glycolipids adsorbed on the surface of titanium dioxide into ceramides. The glycosidic heads are further oxidized and decomposed into smaller molecules on the surface of titanium dioxide, while the detached ceramides, unable to adsorb onto the surface of titanium dioxide, are released into the solution as products.

[0012] In the photocatalytic oxidation process, two key conditions are required to achieve the selective decomposition of glycolipids and their selective conversion into ceramides. First, the solution must be alkaline. An alkaline environment is a prerequisite for the chelation and adsorption of carbohydrates on the titanium dioxide surface. If glycolipids are not adsorbed onto the titanium dioxide surface, high photocatalytic decomposition efficiency cannot be achieved. Second, the solution must contain a certain proportion of protic solvent. In a completely aprotic solvent, not only glycolipids but also ceramides will be adsorbed onto the titanium dioxide surface, preventing ceramides from desorbing from the titanium dioxide surface and continuing to oxidize and decompose into smaller molecules.

[0013] Preferably, the alkaline protic solvent comprises methanol, water, and an aprotic solvent, wherein the volume ratio of methanol added to the alkaline protic solvent is 40%-60%, the volume ratio of water added to the alkaline protic solvent is 5%-10%, and the pH of the alkaline protic solvent is 9-12.

[0014] Preferably, the alkaline protic solvent contains ammonia, methanol, and acetonitrile, and the volume ratio of ammonia added to the alkaline protic solvent is 5%-10%.

[0015] Preferably, the dissolution includes at least one of direct dissolution, solvent extraction, or nitrogen blowing concentration followed by resolution. The dissolution method can be selected based on the sample characteristics; if the sample itself is a lipid standard, it can also be directly dissolved in a solvent.

[0016] Preferably, the titanium dioxide particles include one or more of amorphous titanium dioxide particles, anatase-type titanium dioxide particles, or titanium dioxide particles doped with metal, non-metal elements, or carbon nanomaterials. The titanium dioxide nanoparticles have a particle size of 1-10 nm and a dispersion concentration of 1-10 g / L in the extract, more preferably 0.4-2.5 g / L.

[0017] The ultraviolet light wavelength is 200-400nm, preferably 200-300nm, and more preferably 250nm; the photocatalytic reaction time is 30s-30min, preferably within 5min, and more preferably within 2min.

[0018] Since titanium dioxide's maximum absorption wavelength for ultraviolet light is approximately 250 nm, ultraviolet light with wavelengths of 200-300 nm is absorbed more effectively by titanium dioxide, and the resulting energy exceeds the band gap of titanium dioxide's electronic transitions (3.2 eV). The liquid solution undergoing the reaction is irradiated with an ultraviolet lamp, maintaining a specific irradiation distance. This distance primarily affects the power per unit area of ​​light, and the optimal distance is related to the light intensity per unit area of ​​the ultraviolet lamp. There are no restrictions on the type of ultraviolet lamp used; it can be any mercury lamp, xenon lamp, etc., producing wavelengths in the 200-400 nm range or any single wavelength within that range. However, mercury lamps, which offer stronger ultraviolet intensity, are preferred.

[0019] Based on a general inventive concept, the present invention also provides a photocatalytic deglycosylation glycosphingolipid spectrometry detection method, wherein the photocatalytic deglycosylation supernatant is injected into a mass spectrometer for primary mass spectrometry and / or secondary mass spectrometry detection, and finally the photocatalytic deglycosylation supernatant and the extract of the untreated sample are compared and analyzed.

[0020] This invention provides a rapid, mild, and mass spectrometry-compatible selective photocatalytic deglycosylation sample preparation method for glycosphingolipids, and its coupling with mass spectrometry enables rapid, accurate, and more complete qualitative analysis of glycosphingolipids in complex samples.

[0021] Preferably, the mass spectrometry includes tandem mass spectrometry or liquid chromatography-mass spectrometry (LC-MS) instruments. The comparative data analysis refers to extracting the primary mass spectrometry peaks of the photocatalytic deglycosylated supernatant and the untreated sample extract, screening the peak with a mass-to-charge ratio difference of 162.06 before and after treatment as the molecular ion peaks of glycosphingolipids before and after deglycosylation to obtain the mass information of glycosphingolipids, and selecting the molecular ion peak after deglycosylation for secondary mass spectrometry detection to obtain the structural fragment information of glycosphingolipids.

[0022] Preferably, the extraction solution of the untreated sample is obtained by immersing the solid sample in an alkaline proton solvent for extraction, or by using in-situ microliquid node sampling technology to extract glycosphingolipids from the surface of the solid sample into the extraction solution using an alkaline proton solvent as the sampling solvent.

[0023] Preferably, the mass spectrometry detection employs in-situ microliquid node sampling-mass spectrometry technology. The sampling and detection instrument is configured such that titanium dioxide nanoparticles are placed in the tubing through which the sample extract or photocatalytic deglycosylation supernatant is sampled and introduced into the mass spectrometer, and ultraviolet light is provided outside the corresponding tubing. This allows the glycosphingolipids in the extract to undergo a photocatalytic reaction while flowing in the tubing. After the reaction is completed, the sample is continuously and automatically introduced into the mass spectrometer.

[0024] By using in-situ microliquid junction sampling-mass spectrometry, glycosphingolipids in the extract undergo photocatalytic reactions while flowing in the pipeline. This eliminates the need for elution of titanium dioxide powder and enables in-situ deglycosylation of glycolipids in tissue samples without damaging the solid sample. This preserves the spatial distribution information of glycolipids in the sample, allowing for in-situ spatially resolved glycolipid analysis.

[0025] Compared with the prior art, the beneficial effects of the present invention are as follows:

[0026] (1) The method of the present invention can selectively, rapidly and efficiently remove the glycosylation head of glycolipids in the presence of complex lipidomes, and further perform subsequent mass spectrometry detection and analysis. By comparing the mass spectrometry peaks before and after removal, glycolipid peaks in complex mass spectrometry peaks can be found effectively, quickly and accurately, providing important chemical information for quickly locking the correct candidate substances in the first-level mass spectrometry qualitative process.

[0027] (2) The glycolipid product obtained by the method of the present invention after removing the glycosyl head has more secondary mass spectrometry fragments than the original glycolipid, which can provide more fragment information for the specific structural analysis of the glycolipid and make the glycolipid identification more accurate.

[0028] (3) The method of the present invention also provides a photocatalytic method for rapidly and selectively oxidizing and decomposing glycolipids into a specific product, providing important basis and rules for how to improve the conversion efficiency of specific products in the field of photocatalytic oxidation. Attached Figure Description

[0029] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without creative effort.

[0030] Figure 1 This is a schematic diagram of the photocatalytic reaction principle of the present invention;

[0031] Figure 2 The graph shows the adsorption efficiency of various lipids on the surface of titanium dioxide under different solvent conditions in Example 1; where PC represents acetylcholine phospholipid, CB represents cerebroside (hexaglycosylceramide), PI represents phosphatidylinositol, SM represents acetylcholine sphingomyelin, and Cer represents ceramide.

[0032] Figure 3 This is a comparison graph showing the efficiency of photocatalytic decomposition of glycolipids in different solvent environments and the deglycosylation conversion efficiency of glycolipids converted into ceramides in Example 1.

[0033] Figure 4 The images show the primary mass spectra of glycolipid standards before and after photocatalysis under optimal conditions in Example 2, as well as the secondary mass spectra of reactants and products.

[0034] Figure 5 Comparison of primary mass spectra of brain tissue extracts with and without photocatalytic treatment in Example 3;

[0035] Figure 6 The images show a comparison of the secondary mass spectra of two representative glycolipids in Example 3, detected in the extract without photocatalytic treatment and in the extract after photocatalytic treatment. Detailed Implementation

[0036] To facilitate understanding of the present invention, the present invention will be described more fully and in detail below with reference to the accompanying drawings and preferred embodiments, but the scope of protection of the present invention is not limited to the following specific embodiments.

[0037] Unless otherwise defined, all technical terms used herein have the same meaning as commonly understood by those skilled in the art. The technical terms used herein are for the purpose of describing particular embodiments only and are not intended to limit the scope of the invention.

[0038] Unless otherwise specified, all raw materials, reagents, instruments and equipment used in this invention can be purchased from the market or prepared by existing methods.

[0039] A sample preparation method for selective photocatalytic deglycosylation of glycosphingolipids according to a specific embodiment of the present invention includes the following steps:

[0040] S1. Dissolve the sample in an alkaline protic solvent to obtain a sample extract.

[0041] Biological samples mainly include animal and plant tissues, blood, urine, saliva, semen, and cell samples.

[0042] In the photocatalytic oxidation process, two key conditions are required for the selective decomposition of glycolipids and their selective conversion into ceramides. First, an alkaline solution environment is essential, as this is a prerequisite for the chelation and adsorption of carbohydrates on the titanium dioxide surface. Without adsorption, high photocatalytic decomposition efficiency is impossible. Second, the solution must contain a certain proportion of protic solvent. In a completely aprotic solvent, not only glycolipids but also ceramides are adsorbed onto the titanium dioxide surface, preventing ceramides from desorbing and further oxidizing into smaller molecules. The optimal solvent composition was 6% ammonia-acetonitrile / methanol (1:1, V / V).

[0043] In some embodiments, the method for extracting tissue samples may involve directly immersing animal or plant tissue sections or small tissue pieces in the aforementioned organic solvents, extracting them by sonication in an ice bath for half an hour, and then separating the tissue residue from the supernatant using a high-speed refrigerated centrifuge to obtain the extract. Alternatively, in-situ microfluidic node sampling technology can be used for minimally invasive extraction. In-situ microfluidic node sampling involves continuously pumping the extractant (alkaline protic solvent) into the sampling probe inlet while simultaneously using a vacuum pump to continuously draw the extractant into the probe outlet, forming a liquid node at the probe tip. Extraction is achieved when the liquid node contacts the sample surface, yielding a lipid-containing extractant. The sampling probe can be an inner and outer capillary tube, a two-hole quartz tube, a gooseneck tube, or a folded tube, etc. The extractant can be collected from the probe outlet for subsequent reactions.

[0044] S2. Add titanium dioxide nanoparticles to the extract;

[0045] In some embodiments, the titanium dioxide particles can be pure TiO2, amorphous titanium dioxide particles, anatase-type titanium dioxide particles, or titanium dioxide particles doped with metal, non-metal elements, or carbon nanomaterials, or one or more of these. The particle size of the titanium dioxide nanoparticles is preferably 1-10 nm. The dispersion concentration of the titanium dioxide nanoparticles in the extract is preferably 1-10 g / L, more preferably 0.4-2.5 g / L.

[0046] S3. A photocatalytic reaction is carried out under ultraviolet light to decompose glycolipids. The supernatant obtained by centrifugation is the photocatalytic deglycosylation supernatant, and the solid powder obtained is titanium dioxide powder.

[0047] The ultraviolet light wavelength can be 200-400 nm, preferably 200-300 nm, and more preferably 250 nm. Since titanium dioxide's maximum absorption wavelength for ultraviolet light is approximately 250 nm, ultraviolet light with a wavelength of 200-300 nm is better absorbed by titanium dioxide, and the resulting energy is higher than the band gap of titanium dioxide's electronic transitions (3.2 eV). The liquid solution to be reacted is irradiated with an ultraviolet lamp, maintaining a certain irradiation distance. The irradiation distance mainly affects the power per unit area of ​​light, and the optimal distance is related to the light intensity per unit area of ​​the ultraviolet lamp. There are no restrictions on the type of ultraviolet lamp used; for example, it can be any mercury lamp, xenon lamp, etc., that produces wavelengths in the 200-400 nm range or any single wavelength within that range. Among these, a mercury lamp with stronger ultraviolet intensity is preferred.

[0048] The photocatalytic reaction time can be 30s-30min, preferably within 5min, and more preferably within 2min.

[0049] After performing the sample pretreatment in step 3, the following detection steps are performed:

[0050] The photocatalytic deglycosylation supernatant is injected into a mass spectrometry or liquid chromatography-mass spectrometry detection system for qualitative or quantitative detection.

[0051] In some embodiments, the separation method between the sample solution and titanium dioxide can be membrane filtration or centrifugation. After filtration, the filtrate is directly injected into a mass spectrometer or liquid chromatography-mass spectrometry (LC-MS) system for detection; after centrifugation, the supernatant is injected into a mass spectrometer or LC-MS system for detection.

[0052] If in-situ microliquid node sampling technology is used to extract lipids from the surface of solid samples, in-situ microliquid node sampling-mass spectrometry technology can be further used to achieve online detection simultaneously.

[0053] The operational scheme for simultaneous online detection using in-situ microliquid junction sampling-mass spectrometry is as follows: The extract is drawn in through a vacuum and enters the quantitative loop on the six-way valve of the in-situ microliquid junction sampling probe. A capillary filled with a certain amount of titanium dioxide packing is placed between the outlet of the quantitative loop and the inlet of the mass spectrometer. The capillary is exposed to ultraviolet light. When the extract containing glycolipids is propelled through the capillary by a high-pressure reciprocating pump and enters the mass spectrometer, a photocatalytic deglycosylation reaction occurs. The capillary length and the flow rate of the extract can be controlled to ensure that the reaction is completed within the time it takes to pass through the capillary, allowing for online mass spectrometry detection. This enables in-situ deglycosylation of glycolipids in tissue samples without damaging the solid sample, thus preserving the spatial distribution information of glycolipids in the sample and achieving in-situ spatially resolved glycolipid analysis.

[0054] During mass spectrometry detection, most high-signal lipid peaks can be detected by setting up automatic secondary mass spectrometry scanning. Alternatively, potential glycolipid peaks can be manually identified using the primary mass spectrum for secondary mass spectrometry analysis. Furthermore, by comparing the primary mass spectrum of the photocatalytically extracted solution with that of the extract directly injected without photocatalysis, peaks with a mass-to-charge ratio 162.06 lower in the photocatalytically extracted solution compared to those in the non-photocatalytically extracted solution can be identified. This helps in locating deglycosylated products of glycolipids, which can then be detected by secondary mass spectrometry.

[0055] The present invention will be further described below through examples.

[0056] Example 1: Selective adsorption and selective deglycosylation of glycolipids in different solvent environments.

[0057] (I) This embodiment provides a sample preparation method for selective photocatalytic deglycosylation of glycosphingolipids.

[0058] (1) Dissolve 10 μg / mL lipid standard mixtures (PC (16:0 / 18:1), Cer (d18:1 / 18:0), SM (d18:1 / 16:0), GlcCer (d18:2 / 16:0)(CB) and PI (18:2 / 16:0)) in a solution such as Figure 2 The basic protic solvent shown comprises the following components by volume: 94% methanol and 6% ammonia; 47% methanol-47% acetonitrile-6% ammonia; 10% water-94% acetonitrile-6% ammonia; 9% water-90% acetonitrile-1% ammonia; 4% water-94% acetonitrile-6% ammonia; 7% water-90% acetonitrile-3% ammonia; and 94% acetonitrile-6% ammonia.

[0059] (2) P25 titanium dioxide nanoparticles (5nm particle size) were dispersed in the above solution to obtain a 2mg / mL dispersion. The dispersion was vortexed for 30min to allow the lipids to be fully distributed between the two phases.

[0060] (3) The dispersion was subjected to photocatalytic reaction under ultraviolet light irradiation. The photocatalytic decomposition reaction was specifically carried out by placing each solution in (1) under a mercury lamp without filter (200-400nm passband) for 5 min, keeping the mercury lamp current at 8mA and the irradiation distance at 10cm; centrifuging at 8000rpm for 10 min in a centrifuge, and collecting the supernatant after solid-liquid separation. The supernatant was obtained as the photocatalytic deglycosylation supernatant, and the solid powder obtained was titanium dioxide powder.

[0061] (4) Collection of residual glycolipids on titanium dioxide: The titanium dioxide powder obtained by centrifugation after photocatalytic decomposition reaction was further dispersed in 5% formic acid-methanol solution for chemical analysis. After vortexing the dispersion for 30 min, the titanium dioxide was separated from the supernatant by centrifugation to obtain the elution solution of reactants and products adsorbed on the surface of titanium dioxide after photocatalysis. The photocatalytic deglycosylation supernatant and the elution solution of reactants and products adsorbed on the surface of titanium dioxide after photocatalysis were collected to obtain the photocatalytically treated sample solution.

[0062] (II) This embodiment further provides a photocatalytic deglycosylation glycosphingolipid detection method.

[0063] The photocatalytically treated sample solution from step (4) was injected into a mass spectrometer for primary mass spectrometry detection. Finally, the photocatalytically treated sample solution and the untreated sample extract were compared and analyzed. The mass spectrometry conditions were as follows: the mass spectrometer was an electrospray ionization source-ion trap tandem time-of-flight mass spectrometer (IT-TOF, Shimadzu Corporation, Japan). The ion source operated in positive and negative ion switching mode, with the voltage applied to the spray needle at +4500V and -3000V, the ion source temperature at 200℃, and the nebulizing gas (N2) flow rate at 1.5L / min. The mass analyzer was used in primary mass spectrometry full scan mode, with a scan range of 500-1000 m / z.

[0064] First-order mass spectrometry results analysis: By comparing the mass spectrometry signals of the solutions before and after adsorption of lipid-mixed standards with titanium dioxide powder, the adsorption efficiency of each lipid on titanium dioxide in different solution environments can be obtained, such as... Figure 2 As shown. From Figure 2 It can be seen that phospholipids cannot be adsorbed onto titanium dioxide in any alkaline organic solvents, while glycolipids (CB and PI) and sphingolipids (Cer and SM) show some adsorption. However, when the protic solvent methanol is added to the solution, the adsorption of sphingolipids Cer and SM on titanium dioxide is less than 10%, but glycolipids (CB and PI) still have >50% adsorption on the titanium dioxide surface when the methanol content is 50%. By comparing the mass spectrometry signals (S0) of CB in the lipid standard solution before photocatalysis, the mass spectrometry signals (Sr) and Cer (Sr') of CB in the residual solution after photocatalysis and contact with titanium dioxide, and the mass spectrometry signals (Se) and Cer (Se') of CB in the titanium dioxide elution solution, the efficiency of CB photocatalytic decomposition (1-((Sr+Se) / S0)) and the conversion efficiency of the photocatalytic product ceramide in the decomposed CB ((Sr'+Se') / S0) can be calculated. No reactants or products were found in the eluent of residual lipids on titanium dioxide, indicating that the glycolipids on the surface of titanium dioxide had been completely decomposed or reacted into non-adsorbable substances and released into the residual liquid. Therefore, the eluent of titanium dioxide does not need to be measured in the mass spectrometry detection procedure of this application. Figure 3As shown in the figure, the decomposition efficiency of CB in most alkaline solvents is higher than 80%, except for conditions with excessively high water content (10% water) and excessively high methanol content (100% methanol), which are also the conditions with the lowest CB adsorption efficiency. This indicates that selective adsorption of CB is a prerequisite for photocatalytic decomposition. Furthermore, regarding the conversion efficiency of ceramides, only the condition of 6% ammonia-50% methanol-acetonitrile has a ceramide conversion efficiency greater than 70%. While CB is decomposed under other conditions, the proportion decomposed into ceramides is very small; instead, it is decomposed into smaller molecules that cannot be detected by mass spectrometry. The 6% ammonia-50% methanol-acetonitrile environment is precisely the condition with high CB adsorption efficiency and low ceramide (Cer) adsorption efficiency (e.g., ...). Figure 2 Therefore, the solution must contain a certain amount of methanol to improve the efficiency of CB photocatalytic conversion to Cere.

[0065] Example 2: Photocatalytic deglycosylation reaction of glycosphingolipid standards under optimal conditions and comparison of secondary mass spectrometry before and after deglycosylation.

[0066] (I) This embodiment provides a sample preparation method for selective photocatalytic deglycosylation of glycosphingolipids.

[0067] (1) Dissolve 10 μg / mL of GlcCer(d18:2 / 16:0)(CB) in a 6% ammonia-50% methanol-acetonitrile solution. Then disperse P25 titanium dioxide nanoparticles (5 nm particle size) in the above solution to obtain a 2 mg / mL dispersion.

[0068] (2) P25 titanium dioxide nanoparticles (5nm particle size) were dispersed in the above solution to obtain a 2mg / mL dispersion. The dispersion was vortexed for 30min to allow the lipids to be fully distributed between the two phases.

[0069] (3) The dispersion was subjected to photocatalytic reaction under ultraviolet light irradiation. The solution was placed under an unfiltered mercury lamp (200-400nm passband) for 5 min, with the mercury lamp current maintained at 8 mA and the irradiation distance at 10 cm. The solution was centrifuged at 8000 rpm for 10 min. After solid-liquid separation, the supernatant was collected. The supernatant was obtained as the photocatalytic deglycosylation supernatant, and the solid powder obtained was titanium dioxide powder.

[0070] (II) This embodiment further provides a photocatalytic deglycosylation glycosphingolipid detection method.

[0071] The photocatalytic deglycosylation supernatant from step (3) was injected into a mass spectrometry detection system for primary and secondary mass spectrometry detection. The mass spectrometry conditions were as follows: the mass spectrometer was an electrospray ionization source-ion trap tandem time-of-flight mass spectrometer (IT-TOF, Shimadzu Corporation, Japan). The ion source operated in positive and negative ion switching mode, with the voltage applied to the spray needle at +4500V and -3000V, the ion source temperature at 200℃, and the nebulizing gas (N2) flow rate at 1.5L / min. The mass analyzer performed a primary mass spectrometry full scan mode, with a scan range of 500-1000 m / z. Simultaneously, secondary mass spectrometry scans were performed on the glycolipid reactants and products, with a collision energy of 50%.

[0072] Analysis of results from primary and secondary mass spectrometry: Figure 4 The primary and secondary mass spectra of the photocatalytic supernatant and the lipid sample solution before photocatalysis are compared. The spectra show that before photocatalysis, the signals of CB are at 712.5 and 748.5, representing the adduct peaks of MH and M+Cl, respectively. After photocatalysis, these two peaks of CB drop sharply to less than 10% of their original values, and two other peaks appear at 550.4 and 586.4, respectively. The mass difference between these two peaks and the reactants is exactly 162.06. Furthermore, a comparison of the secondary mass spectrum of the reactant peak at 712.5 and the product peak at 550.4 reveals that the product has significantly more secondary fragments than the reactants. The secondary fragments of the reactants are mainly fragments with degenerated sugar heads, while the product ceramide contains a large number of fragments characterizing the sphingosine backbone and fatty acid carbon chains.

[0073] Example 3: Photocatalytic deglycosylation reaction and qualitative analysis of glycolipids in actual brain tissue extracts under optimal conditions

[0074] (I) This embodiment provides a sample preparation method for selective photocatalytic deglycosylation of glycosphingolipids.

[0075] (1) Twenty brain tissue slices were placed in 1 mL of methanol / chloroform (1:1, v / v) solution and extracted under sonication for 30 min. The residual liquid was then removed by centrifugation to obtain brain tissue extract. 10 μL of the extract was purged with nitrogen using a nitrogen blower and then redissolved in 6% ammonia-methanol / acetonitrile (1:1, v / v) solution to obtain the solution to be reacted.

[0076] (2) Add titanium dioxide to the brain tissue extract, vortex for 30 minutes, and then separate the extract from the titanium dioxide by high-speed centrifugation.

[0077] (3) The titanium dioxide obtained in (2) was further dispersed in a blank 6% ammonia-methanol / acetonitrile (1:1, v / v) solution, and the solution was irradiated for 5 min under an unfiltered mercury lamp (200-400 nm passband), maintaining the mercury lamp current at 8 mA and the irradiation distance at 10 cm. The solution was then centrifuged at 8000 rpm for 10 min. After solid-liquid separation, the supernatant was collected, and the supernatant was obtained as the photocatalytic deglycosylation supernatant. The solid powder obtained was titanium dioxide powder.

[0078] (4) Chemical analysis of titanium dioxide: The titanium dioxide obtained in (2) was further dispersed in a 5% formic acid-methanol solution for chemical analysis. After vortexing the dispersion for 30 min, the titanium dioxide was separated from the supernatant by centrifugation to obtain the elution solution of the reactants and products adsorbed on the surface of titanium dioxide after photocatalysis.

[0079] (II) This embodiment further provides a photocatalytic deglycosylation glycosphingolipid detection method.

[0080] Mass spectrometry analysis: The photocatalytic deglycosylation supernatant and the elution solutions of reactants and products adsorbed on the titanium dioxide surface after photocatalysis were injected into the mass spectrometry detection system. The mass spectrometry detection conditions were the same as in Example 2. The peak in the primary mass spectrum of the elution solution of reactants and products on the titanium dioxide surface that was exactly 162.06 m / z higher than that of the photocatalytic decomposition supernatant was screened as the glycolipid peak and the corresponding glycolipid decomposition fogging peak. The glycolipid peaks and glycolipid decomposition product peaks in the photocatalytic deglycosylation supernatant and the elution solutions of reactants and products adsorbed on the titanium dioxide surface after photocatalysis were further detected by secondary mass spectrometry.

[0081] Results Analysis: By detecting the supernatant of the photocatalytic deglycosylation solution and the elution solutions of reactants and products adsorbed on the titanium dioxide surface after photocatalysis, the first-order mass spectrometry peaks of glycolipids in brain tissue extract before and after photocatalytic decomposition can be obtained, such as... Figure 5 As shown. From Figure 5 It can be observed that peaks with a mass-to-charge ratio (M / C ratio) of around 800-900 are mainly obtained in the black chemical eluent, while peaks with a M / C ratio of around 500-700 are mainly obtained in the solution after the photocatalytic reaction. Most of these peaks can be found in the chemical eluent with a M / C ratio of M+162.06. This indicates that most of the glycolipids in brain tissue are broken down into deglycosylated ceramides. Secondary mass spectrometry was performed on two representative ceramides (m / z 662.5 and 664.5) and their corresponding glycolipids (m / z 824.6 and m / z 826.6). Figure 6As shown, it can be observed that ceramides after deglycosylation have a richer number of fatty acid structural fragments than glycolipids, especially fragments related to the hydroxyl positions and double bond positions on fatty acids (marked as fragment C), while glycolipids themselves mainly consist of fragments related to the glycosyl head (marked as fragment D).

[0082] The above are merely preferred embodiments of the present invention and are not intended to limit the present invention in any way. Therefore, any simple modifications, equivalent changes, and alterations made to the above embodiments based on the technical essence of the present invention without departing from the scope of the present invention should fall within the protection scope of the present invention.

Claims

1. A sample processing method for selective photocatalytic deglycosylation of glycosphingolipids, characterized in that, Specifically, the following steps are included: (1) Dissolve the sample in an alkaline protic solvent to obtain a sample extract; (2) Disperse titanium dioxide particles in the sample extract to obtain a dispersion; (3) The dispersion was subjected to photocatalytic reaction under ultraviolet light irradiation, centrifuged, and the supernatant was obtained as photocatalytic deglycosylation supernatant, and the solid powder was obtained as titanium dioxide powder.

2. The method as described in claim 1, characterized in that, The alkaline protic solvent comprises methanol, water, and an aprotic solvent. The volume ratio of methanol added to the alkaline protic solvent is 40%-60%, the volume ratio of water added to the alkaline protic solvent is 5%-10%, and the pH of the alkaline protic solvent is 9-12.

3. The method as described in claim 2, characterized in that, The alkaline protic solvent contains ammonia, methanol, and acetonitrile, and the volume ratio of ammonia added to the alkaline protic solvent is 5%-10%.

4. The method as described in claim 1, characterized in that, The dissolution includes at least one of direct dissolution, solvent extraction, or nitrogen blowing concentration followed by resolution.

5. The method as described in claim 1, characterized in that, The titanium dioxide particles include one or more of the following: amorphous titanium dioxide particles, anatase titanium dioxide particles, or titanium dioxide particles doped with metal, non-metal elements, or carbon nanomaterials. The titanium dioxide nanoparticles have a particle size of 1-10 nm and a dispersion concentration of 1-10 g / L in the extraction solution. The ultraviolet light wavelength is 200-400 nm, and the photocatalytic reaction time is 30 s-30 min.

6. A photocatalytic deglycosylation glycosphingolipid detection method, characterized in that, The supernatant obtained by the sample processing method for selective photocatalytic deglycosylation of glycosphingolipids according to any one of claims 1 to 5 is injected into a mass spectrometer for detection by primary mass spectrometry and / or secondary mass spectrometry. Finally, the photocatalytic deglycosylation supernatant and the extract of the untreated sample are compared and analyzed.

7. The photocatalytic deglycosylation glycosphingolipid detection method according to claim 6, characterized in that, The mass spectrometry includes tandem mass spectrometry or liquid chromatography-mass spectrometry (LC-MS) instruments. The comparative data analysis refers to extracting the primary mass spectrometry peaks of the photocatalytic deglycosylated supernatant and the untreated sample extract, screening the peak with a mass-to-charge ratio difference of 162.06 before and after treatment as the molecular ion peaks of glycosphingolipids before and after deglycosylation to obtain the mass information of glycosphingolipids, and selecting the molecular ion peak after deglycosylation for secondary mass spectrometry detection to obtain the structural fragment information of glycosphingolipids.

8. The photocatalytic deglycosylation glycosphingolipid detection method according to claim 6, characterized in that, The extraction solution of the untreated sample is obtained by immersing the solid sample in an alkaline proton solvent for extraction, or by using in-situ microliquid node sampling technology to extract glycosphingolipids from the surface of the solid sample into the extraction solution using an alkaline proton solvent as the sampling solvent.

9. The photocatalytic deglycosylation glycosphingolipid detection method according to any one of claims 6-8, characterized in that, The mass spectrometry detection employs in-situ microliquid node sampling-mass spectrometry technology. The sampling and detection instrument is configured such that titanium dioxide nanoparticles are placed in the tubing through which the photocatalytic deglycosylated supernatant or sample extract is sampled and introduced into the mass spectrometer, and ultraviolet light is placed outside the corresponding tubing. This allows the glycosphingolipids in the extract to undergo a photocatalytic reaction while flowing in the tubing. After the reaction is completed, the sample is continuously and automatically introduced into the mass spectrometer.

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