Sample treatment method for removing glycosyl by selective photocatalysis of glycosphingolipid and mass spectrometric detection method

Abstract

The invention relates to a sample treatment method for selectively removing glycosyl from glycosphingolipid through photocatalysis and a mass spectrometric detection method. The sample treatment method specifically comprises the following steps: (1) dissolving a sample in an alkaline protic solvent; (2) dispersing titanium dioxide particles; and (3) carrying out photocatalytic reaction under ultraviolet irradiation, and centrifuging to obtain a sample solution subjected to photocatalytic treatment. The mass spectrometric detection method specifically comprises the following steps: injecting a sample solution subjected to photocatalysis treatment into a mass spectrum, carrying out primary mass spectrometric detection, and carrying out comparison data analysis on the sample solution subjected to photocatalysis treatment and a sample extracting solution not subjected to photocatalysis treatment, a primary mass spectrum peak which is possibly a glycolipid decomposition product is screened through comparison and analysis results, and secondary mass spectrum detection is further carried out, so that the accurate molecular structure of glycolipid is analyzed. According to the method, the glycosyl head of glycolipid can be selectively, rapidly and efficiently removed in the presence of complex lipidomes, subsequent mass spectrum detection analysis is carried out, glycolipid spectrum peaks in complex mass spectrum peaks can be effectively, rapidly and accurately found out by comparing the mass spectrum peaks before and after removal, and the detection result is accurate. Important chemical information is provided for rapid locking of correct candidate substances in the first-level mass spectrum qualitative process, and meanwhile, the glycolipid decomposition product can obtain carbon chain structure information which is richer than that of original glycolipid to assist qualitative analysis.

Description

Technical Field

[0001] The invention belongs to the technical field of sample pretreatment and detection of chemical substances, and in particular relates to a sample treatment method and a mass spectrometry detection method for selective photocatalytic deglycosylation of glycosphingolipids. Background Art

[0002] Glycosphingolipids are a class of glycolipids widely distributed on the surface of animal cell membranes. They play an important biological role in regulating cell recognition, adhesion, proliferation and apoptosis. However, it has always been a difficult problem to detect glycosphingolipids with high coverage and complete structural characterization in complex biological samples such as blood, tissues and cells. In terms of quality detection, glycosphingolipids themselves have low ionization efficiency and low signal response in mass spectrometry. At the same time, since glycosphingolipids and phospholipids are in the same mass spectrum segment, and phospholipids have high content and high ionization efficiency in tissues, the ionization of glycosphingolipids is greatly inhibited, making mass spectrometry detection of glycosphingolipids, especially direct mass spectrometry detection, more difficult. In terms of structural characterization, glycosphingolipids are composed of glycosyl heads, sphingosine skeletons and fatty acid chains. The structures of sphingosine skeletons and fatty acid chains are variable, and it is difficult to accurately analyze the number and position of modified hydroxyl groups and double bonds on the carbon chain.

[0003] In traditional detection methods, after extracting glycosphingolipids from tissue or cell samples, they can be separated using normal phase chromatography columns or reverse phase chromatography columns and then detected using mass spectrometry. These two methods require cumbersome and lengthy sample processing. To extract glycosphingolipid samples, the sample cells or tissues need to be ultrasonically crushed in a certain proportion of chloroform, methanol, and water, or a certain proportion of isopropanol, hexane, and water to release crude lipids, and then saponified in a weak alkaline environment to remove the influence of glycerophospholipids on the results. Chloroform, methanol, or Folch method are then used to extract and separate glycosphingolipids, and finally LC-ESI-MS / MS studies are performed. These processes are lengthy and complicated, and at the same time, the chromatogram-mass spectra obtained are still the extracts containing other interfering lipids. The qualitative identification of glycolipids from complex primary mass spectra can only rely on accurate molecular ion peak mass-to-charge ratios, but the structures of various lipids are complex, and isomers or isotopic peaks with similar molecular weights interfere with each other. Chemical separation by chromatography can effectively separate various lipids from each other, reduce ion suppression and peak overlap, but because chromatography cannot provide absolute structural information, it is still difficult to accurately and completely find glycolipid peaks and characterize their structures among numerous peaks. The peak characteristic fragments obtained by secondary mass spectrometry can further characterize glycolipid peaks, but secondary mass spectrometry requires higher instrument sensitivity and detection throughput. Usually, many primary mass spectrometry peaks cannot obtain their secondary mass spectrometry fragments due to low signals, and it takes a long time to break all primary mass spectrometry peaks one by one to obtain secondary mass spectra. More importantly, there are few fragments in the secondary mass spectrometry fragments of glycolipids that characterize the precise structure of fatty acid chains, making it difficult to completely resolve the carbon chain structure of glycolipids.

[0004] Therefore, the pretreatment process of glycosphingolipid samples is an important stage to improve the detection and qualitative analysis of glycosphingolipids. Currently, there are few reports on mass spectrometry sample processing and mass spectrometry analysis methods that can quickly and high-throughput qualitatively identify a large number of glycolipids in complex biological samples and can completely resolve the glycolipid structure. Summary of the invention

[0005] The technical problem to be solved by the present invention is that it is impossible to quickly and high-throughput qualitatively identify a large number of glycolipids in complex biological samples and completely analyze the glycolipid structure, thereby overcoming the deficiencies and defects mentioned in the above background technology and providing a sample processing method and mass spectrometry detection method for selective photocatalytic deglycosylation of glycosphingolipids.

[0006] In order to solve the above technical problems, the technical solution proposed by the present invention is:

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

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

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

[0010] (3) subjecting the dispersion to a photocatalytic reaction under ultraviolet light irradiation, centrifuging, obtaining a supernatant as a photocatalytic deglycosylation supernatant, and obtaining a solid powder as titanium dioxide powder.

[0011] like Figure 1 As shown, the principle of the selective photocatalytic deglycosylation method of the present invention is the photocatalytic oxidative decomposition process occurring on the titanium dioxide surface by ultraviolet light. Specifically, the glycolipid is selectively adsorbed on the titanium dioxide surface due to the bidentate chelation of the sugar head with the Lewis acid site in titanium dioxide in an alkaline environment. When ultraviolet light is irradiated to the titanium dioxide surface, titanium dioxide absorbs ultraviolet light to generate photogenerated electrons and holes. The holes oxidize the water adsorbed on the surface into hydroxyl radicals, so that the glycolipid adsorbed on the titanium dioxide surface is selectively oxidized and decomposed into ceramide by hydroxyl radicals, and the sugar head is further oxidized and decomposed into small molecules on the titanium dioxide surface, while the dropped ceramide is resolved into the solution as a product because it cannot be adsorbed on the titanium dioxide surface.

[0012] In the process of photocatalytic oxidation, two key conditions are required to achieve the selective decomposition of glycolipids and their selective conversion into ceramides. The first condition is the alkaline environment of the solution, because the alkaline environment is a prerequisite for sugar substances to be chelated and adsorbed on the surface of titanium dioxide. If the glycolipids are not adsorbed on the surface of titanium dioxide, they cannot have high photocatalytic decomposition efficiency; the second condition is that the solution must contain a certain proportion of proton solvents. In a completely non-proton solvent, not only glycolipids will be adsorbed on the surface of titanium dioxide, but ceramide will also be adsorbed on the surface of titanium dioxide, making it impossible for ceramide to desorb from the surface of titanium dioxide and continue to oxidize and decompose into smaller molecules.

[0013] Preferably, 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.

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

[0015] Preferably, the dissolution includes at least one of direct dissolution, solvent leaching extraction or nitrogen blowing concentration and then re-dissolution. Here, the dissolution scheme can be selected according to the sample conditions. If the sample itself is a lipid standard sample, 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 crystal titanium dioxide particles, or titanium dioxide particles doped with metal, non-metallic elements, or carbon nanomaterials, the particle size of the titanium dioxide nanoparticles is 1-10 nm, and the dispersion concentration of the titanium dioxide nanoparticles in the extract is 1-10 g / L, more preferably 0.4-2.5 g / L;

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

[0018] Since the maximum absorption wavelength of titanium dioxide to ultraviolet light is about 250nm, ultraviolet light with a wavelength of 200-300nm can be better absorbed by titanium dioxide, and the energy generated is higher than the energy band gap of titanium dioxide electronic transition 3.2eV. Use ultraviolet lamp to irradiate the liquid solution that needs to react, and maintain 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 is no restriction on the ultraviolet lamp used, for example, it can be any mercury lamp, xenon lamp, etc. that produces a 200-400nm band or any single wavelength therein. Among them, mercury lamps with stronger ultraviolet band intensity are preferred.

[0019] Based on a general inventive concept, the present invention also provides a method for detecting the glycosphingolipid spectrum of photocatalytic deglycosylation, wherein the photocatalytic deglycosylation supernatant is injected into a mass spectrometer, and primary mass spectrometry and / or secondary mass spectrometry detection is performed, and finally, the photocatalytic deglycosylation supernatant and the extract of the sample not treated with photocatalysis are compared for data analysis.

[0020] The present invention provides a rapid and mild sample treatment method for selective photocatalytic deglycosylation of glycosphingolipids that is compatible with mass spectrometry, and is used in conjunction with mass spectrometry to achieve rapid, accurate and more complete qualitative analysis of glycolipids in complex samples.

[0021] Preferably, the mass spectrometer includes a tandem mass spectrometer or a liquid-mass spectrometer, and the comparative data analysis refers to extracting the primary mass spectrometry peaks of the photocatalytic deglycosylation supernatant and the sample extract that has not been photocatalytically treated, screening the peaks with a mass-to-charge ratio difference of 162.06 before and after treatment as the molecular ion peaks of the glycosphingolipids before and after deglycosylation to obtain the mass information of the glycosphingolipids, and selecting the molecular ion peaks after deglycosylation for secondary mass spectrometry detection to obtain the structural fragment information of the glycosphingolipids.

[0022] Preferably, the extract of the sample that has not been photocatalytically treated is obtained by immersing the solid sample in an alkaline proton solvent for extraction, or by extracting the glycosphingolipids on the surface of the solid sample into the extract using an alkaline proton solvent as a sampling solvent through in situ microliquid node sampling technology.

[0023] Preferably, the mass spectrometry detection adopts in-situ micro-liquid node sampling-mass spectrometry technology, and the sampling and detection instruments are configured as follows: titanium dioxide nanoparticles are configured in the pipeline through which the sample extract or photocatalytic deglycosylation supernatant is sampled and injected into the mass spectrometer, and ultraviolet light is configured outside the corresponding pipeline, so that the glycosphingolipids in the extract flow in the pipeline while undergoing a photocatalytic reaction, and after the reaction is completed, it is continuously and automatically injected into the mass spectrometer.

[0024] Through in situ microliquid node sampling-mass spectrometry technology, the glycosphingolipids in the extract undergo photocatalytic reaction while flowing in the pipeline, which can omit the elution step of titanium dioxide powder and achieve in situ deglycosylation of glycolipids in tissue samples without destroying the solid sample, thereby retaining the spatial distribution information of glycolipids in the sample and realizing in situ spatially resolved glycolipid analysis.

[0025] Compared with the prior art, the present invention has the following beneficial effects:

[0026] (1) The method of the present invention can achieve the selective, rapid and efficient removal of the sugar head of glycolipids in the presence of complex lipid groups, and further perform subsequent mass spectrometry detection and analysis. By comparing the mass spectrometry peaks before and after removal, the glycolipid peaks in the complex mass spectrometry peaks can be effectively, quickly and accurately found, providing important chemical information for quickly locking the correct candidate substances in the primary mass spectrometry qualitative process;

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

[0028] (3) The method of the present invention also provides a photocatalytic method for rapidly and selectively oxidizing and decomposing glycolipids into certain specific products, providing important basis and rules for improving the conversion efficiency of specific products in the field of photocatalytic oxidation. BRIEF DESCRIPTION OF THE DRAWINGS

[0029] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

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

[0031] Figure 2 1 is a graph showing the adsorption efficiency of various lipids on the titanium dioxide surface under different solvent environments in Example 1; wherein PC represents acetylcholine phospholipids, CB represents cerebroside (hexaglycoceramide), PI represents phosphatidylinositol, SM represents acetylcholine sphingomyelin, and Cer represents ceramide.

[0032] Figure 3 This is a comparison chart of the efficiency of photocatalytic decomposition of glycolipids in Example 1 under different solvent environments and the deglycosylation efficiency of glycolipids converted into ceramide;

[0033] Figure 4 The primary mass spectra of the glycolipid standard sample before and after photocatalysis and the secondary mass spectra of the reactants and products obtained under the optimal conditions in Example 2;

[0034] Figure 5 The first-order mass spectra of the brain tissue extracts treated with photocatalysis and those not treated with photocatalysis in Example 3 are compared;

[0035] Figure 6 The following is a comparison of the secondary mass spectra of two representative glycolipids in Example 3 detected in the extract without photocatalytic treatment and the secondary mass spectra detected in the extract after photocatalytic treatment. DETAILED DESCRIPTION

[0036] In order to facilitate the understanding of the present invention, the present invention will be described more comprehensively and meticulously below in conjunction with the accompanying drawings and preferred embodiments of the present invention, but the protection scope of the present invention is not limited to the following specific embodiments.

[0037] Unless otherwise defined, all professional terms used below have the same meanings as those generally understood by those skilled in the art. The professional terms used herein are only for the purpose of describing specific embodiments and are not intended to limit the scope of protection of the present invention.

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

[0039] A sample processing method for selective photocatalytic deglycosylation of glycosphingolipids according to a specific embodiment of the present invention comprises 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 process of photocatalytic oxidation, two key conditions are required to achieve the selective decomposition of glycolipids and their selective conversion into ceramides. The first condition is the alkaline environment of the solution, because the alkaline environment is a prerequisite for the chelation and adsorption of sugar substances on the surface of titanium dioxide. If the glycolipids are not adsorbed on the surface of titanium dioxide, they cannot have a high photocatalytic decomposition efficiency; the second condition is that the solution must contain a certain proportion of proton solvents. Because in a completely non-proton solvent, not only glycolipids will be adsorbed on the surface of titanium dioxide, but ceramide will also be adsorbed on the surface of titanium dioxide, making it impossible for ceramide to desorb from the surface of titanium dioxide and continue to oxidize and decompose into smaller molecules. The optimal solvent environment composition of the experiment is 6% ammonia-acetonitrile / methanol (1:1, V / V).

[0043] In certain embodiments, the method for extracting tissue samples can be to directly immerse animal and plant tissue slices or small pieces of tissue in the above organic solvents, extract in an ice bath ultrasound for half an hour, and then use a high-speed refrigerated centrifuge to separate the tissue residue from the supernatant to obtain an extract. It is also possible to use an in-situ micro-liquid node sampling technology for micro-damage extraction. In-situ micro-liquid node sampling is to continuously pump the extraction liquid (alkaline proton solvent) into the sampling probe inlet, and at the same time, the extraction liquid is continuously sucked into the probe outlet by a vacuum pump, and a liquid node is formed at the tip of the probe. When the liquid node contacts the sample surface, extraction is achieved to obtain a lipid-containing extract. The sampling probe can be a capillary inner and outer sleeve, a double-hole quartz tube, a goose-shaped tube or a folded tube, etc. The extraction liquid can be collected from the probe outlet for subsequent reactions.

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

[0045] In certain embodiments, the titanium dioxide particles may be pure TiO 2 It can also be one or more of amorphous titanium dioxide particles, anatase-type titanium dioxide particles, or titanium dioxide particles doped with metal, non-metallic elements, or carbon nanomaterials. 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 irradiation to photocatalytically decompose the glycolipid, and the supernatant obtained by centrifugation is a photocatalytically deglycosylated supernatant, and the solid powder obtained is titanium dioxide powder.

[0047] The wavelength of ultraviolet light can be 200-400nm, preferably 200-300nm, and more preferably 250nm. Since the maximum absorption wavelength of titanium dioxide to ultraviolet light is about 250nm, ultraviolet light with a wavelength of 200-300nm can be better absorbed by titanium dioxide, and the energy generated is higher than the energy band gap 3.2eV of titanium dioxide electronic transition. The liquid solution that needs to react is irradiated with an ultraviolet lamp, and a certain irradiation distance is maintained. 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. The ultraviolet lamp used is not limited, for example, it can be any mercury lamp, xenon lamp, etc. that produces a 200-400nm band or any single wavelength thereof. Among them, a mercury lamp with a stronger ultraviolet band intensity is preferred.

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

[0049] After the sample pretreatment in step 3, perform the following detection steps:

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

[0051] In some embodiments, the separation method of the sample liquid and titanium dioxide can be membrane filtration or centrifugation. After filtration, the filtrate is directly sent to mass spectrometry or liquid-mass spectrometry for detection; after centrifugation, the supernatant is sampled and injected into the mass spectrometry or liquid-mass spectrometry system for detection.

[0052] If the lipids on the surface of solid samples are extracted using in situ micro-liquid node sampling technology, the in situ micro-liquid node sampling-mass spectrometry technology can be further used to simultaneously realize online detection.

[0053] The operation scheme of in-situ micro-liquid node sampling-mass spectrometry technology for simultaneous online detection is as follows: the extract is sucked into the quantitative loop on the six-way valve of the in-situ micro-liquid node sampling probe through vacuum. A capillary filled with a certain amount of titanium dioxide filler is set 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 pushed by the high-pressure reciprocating pump to flow through the capillary and enter the mass spectrometer, a photocatalytic deglycosylation reaction occurs. The capillary length and the flow rate of the extract can be controlled so that the reaction is completed within the time it passes through the capillary, and mass spectrometry detection is performed online. In this way, the deglycosylation of glycolipids in tissue samples can be achieved in situ without destroying the solid sample, thereby retaining the spatial distribution information of glycolipids in the sample and realizing in-situ spatially resolved glycolipid analysis.

[0054] During mass spectrometry detection, automatic secondary mass spectrometry scanning can be set to perform secondary mass spectrometry detection on most high-signal lipid peaks, or possible glycolipid peaks can be found manually through the primary mass spectrometry to manually perform secondary mass spectrometry analysis on these peaks. In addition, by comparing the primary mass spectrometry of the extract obtained by photocatalysis with the primary mass spectrometry of the extract directly injected without photocatalysis, peaks with a mass-to-charge ratio of 162.06 less than that of the mass spectrometry peak in the extract without photocatalysis can be found in the photocatalytic extract, thereby assisting in finding the deglycosylation products of the glycolipids, and performing secondary mass spectrometry detection on these peaks.

[0055] The present invention is further described below by way of examples.

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

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

[0058] (1) 10 μg / mL lipid standard mixture (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)) was dissolved in Figure 2 In the alkaline proton solvent shown; the alkaline proton solvent includes the following components by volume: 94% methanol 6% ammonia water; 47% methanol-47% acetonitrile-6% ammonia water; 10% water-94% acetonitrile-6% ammonia water; 9% water-90% acetonitrile-1% ammonia water; 4% water-94% acetonitrile-6% ammonia water; 7% water-90% acetonitrile-3% ammonia water; 94% acetonitrile-6% ammonia water;

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

[0060] (3) subjecting the dispersion to a photocatalytic reaction under ultraviolet light irradiation. Specifically, the photocatalytic decomposition reaction comprises placing each solution in (1) under an unfiltered mercury lamp (200-400 nm passband) for 5 minutes, maintaining the mercury lamp current at 8 mA and the irradiation distance at 10 cm; centrifuging at 8000 rpm for 10 minutes in a centrifuge, and collecting the supernatant after solid-liquid separation to obtain a photocatalytic deglycosylation supernatant, and obtaining a solid powder as titanium dioxide powder.

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

[0062] (II) This embodiment further provides a method for detecting glycosphingolipid profiles by photocatalytic deglycosylation

[0063] The sample solution treated with photocatalysis in the above step (4) was injected into the mass spectrometer for primary mass spectrometry detection, and finally the sample solution treated with photocatalysis and the sample extract not treated with photocatalysis were compared for data analysis. The mass spectrometry detection conditions were as follows: the mass spectrometer was an electrospray ion source-ion trap tandem time-of-flight mass spectrometer (IT-TOF, Shimadzu Corporation, Japan). The ion source operated in the positive and negative ion switching mode, the voltage applied to the spray needle was +4500V and -3000V, the ion source temperature was 200°C, and the atomizing gas (N 2) flow rate 1.5 L / min. The mass analyzer performed a first-stage mass spectrometry full scan mode with a scan range of 500-1000 m / z.

[0064] Analysis of primary mass spectrometry results: By comparing the mass spectrometry signals of the solution after the lipid mixed standard was contacted with titanium dioxide powder and before adsorption, the adsorption efficiency of each lipid on titanium dioxide in different solution environments can be obtained, such as Figure 2 As shown. Figure 2 It can be seen that phospholipids cannot be adsorbed on titanium dioxide in all alkaline organic solvents, while glycolipids (CB and PI) and sphingolipids (Cer and SM) have a certain 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 signal (S0) of CB in the lipid standard solution before photocatalysis, the mass spectrometry signal (Sr) of CB and the signal (Sr') of Cer in the residual solution in contact with titanium dioxide after photocatalysis, and the mass spectrometry signal (Se) of CB and the signal (Se') of Cer 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 reactant or product signals were found in the eluate of the residual lipids on titanium dioxide, indicating that the glycolipids on the titanium dioxide surface have been completely decomposed or reacted into non-adsorbed substances and resolved into the residual liquid. The mass spectrometry detection process of this application does not need to measure the eluate of titanium dioxide. Figure 3 As shown in the figure, it can be found that the decomposition efficiency of CB in most alkaline solvents is higher than 80%, except for the conditions of too high water content (10% water) and too high methanol content (100% methanol). These two conditions themselves are also the conditions with the lowest CB adsorption efficiency. It can be seen that the selective adsorption of CB is the prerequisite for photocatalytic decomposition. In addition, from the perspective of ceramide conversion efficiency, only the condition of 6% ammonia water-50% methanol acetonitrile has a ceramide conversion efficiency greater than 70%, and although CB is decomposed under other conditions, the proportion of decomposition into ceramide is very small, and it is decomposed into smaller molecules that cannot be detected by mass spectrometry. The environment of 6% ammonia water-50% methanol acetonitrile happens to be the condition where CB adsorption efficiency is high and ceramide Cer adsorption efficiency is low (such as Figure 2 ). It can be seen that a certain amount of methanol must be contained in the solution to improve the efficiency of photocatalytic conversion of CB to Cer.

[0065] Example 2: Photocatalytic deglycosylation reaction of glycosphingolipid standard samples under optimal conditions and comparison of secondary mass spectra before and after deglycosylation

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

[0067] (1) 10 μg / mL of GlcCer (d18:2 / 16:0) (CB) was dissolved in a 6% ammonia-50% methanol-acetonitrile solution, and then P25 titanium dioxide nanoparticles (5 nm in diameter) were dispersed in the above solution to obtain a 2 mg / mL dispersion.

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

[0069] (3) subjecting the dispersion to a photocatalytic reaction under ultraviolet light irradiation, placing the solution under an unfiltered mercury lamp (200-400 nm passband) for 5 min, maintaining the mercury lamp current at 8 mA and the irradiation distance at 10 cm; centrifuging at 8000 rpm for 10 min in a centrifuge, and collecting the supernatant after solid-liquid separation, to obtain a photocatalytic deglycosylation supernatant, and a solid powder as titanium dioxide powder.

[0070] (II) This embodiment further provides a method for detecting glycosphingolipid profiles by photocatalytic deglycosylation

[0071] The photocatalytic deglycosylation supernatant in the above step (3) was injected into the mass spectrometry detection system for primary mass spectrometry and secondary mass spectrometry detection. The mass spectrometry detection conditions were as follows: the mass spectrometry was an electrospray ion source-ion trap tandem time-of-flight mass spectrometry (IT-TOF, Shimadzu Corporation, Japan). The ion source operated in the positive and negative ion switching mode, the voltage applied to the spray needle was +4500V and -3000V, the ion source temperature was 200°C, and the atomizing gas (N 2 ) flow rate 1.5 L / min. The mass analyzer was operated in a first-stage mass spectrometry full scan mode with a scan range of 500-1000 m / z. At the same time, the glycolipid reactants and products were further subjected to a second-stage mass spectrometry scan with a collision energy of 50%.

[0072] Analysis of primary and secondary mass spectrometry results: Figure 4The first and second mass spectra of the photocatalytic supernatant and lipid sample solution before photocatalysis are compared. It can be found from the figure that the signals of CB before photocatalysis are at 712.5 and 748.5, which are the adduct peaks of MH and M+Cl, respectively. After illumination, the two peaks of CB dropped sharply to less than 10% of the original, and two other peaks appeared at 550.4 and 586.4, respectively. The mass difference between these two peaks and the reactant is exactly 162.06. From the comparison of the second mass spectrum of the reactant peak 712.5 and the product peak 550.4, it can be seen that the secondary fragments of the product are much richer than the reactant, and the secondary fragments of the reactant are mainly fragments of the sugar head, while the fragments of the product ceramide have a large number of fragments representing the sphingosine skeleton and fatty acid carbon chain.

[0073] Example 3: Photocatalytic deglycosylation reaction of actual brain tissue extract under optimal conditions and characterization of glycolipids

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

[0075] (1) Place 20 brain tissue slices in 1 mL of methanol / chloroform (1:1, v / v) solution and extract under ultrasound for 30 min, then remove the residual liquid by centrifugation to obtain brain tissue extract. Take 10 μL of the extract and blow it in a nitrogen blower, then redissolve it in 6% ammonia-methanol / acetonitrile (1:1, v / v) solution to obtain the solution to be reacted.

[0076] (2) Titanium dioxide was first added to the brain tissue extract, dispersed and vortexed for 30 minutes, and then the extract and titanium dioxide were separated by high-speed centrifugation.

[0077] (3) The titanium dioxide obtained in (2) is further dispersed in a blank 6% ammonia-methanol / acetonitrile (1:1, v / v) solution, and the solution is irradiated under an unfiltered mercury lamp (200-400nm passband) for 5 minutes, the mercury lamp current is maintained at 8 mA and the irradiation distance is 10 cm, and the solution is centrifuged at 8000 rpm for 10 minutes. After solid-liquid separation, the supernatant is taken out and collected, and the supernatant obtained is the photocatalytic deglycosylation supernatant, and the solid powder obtained is 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. The dispersion was vortexed for 30 minutes and then separated from the supernatant by centrifugation to obtain an elution solution of reactants and products adsorbed on the surface of the titanium dioxide after photocatalysis.

[0079] (II) This embodiment further provides a method for detecting glycosyl lipid spectrum by photocatalytic deglycosylation

[0080] Mass spectrometry analysis: The photocatalytic deglycosylation supernatant and the elution solution of the reactants and products adsorbed on the titanium dioxide surface after photocatalysis are sampled into the mass spectrometry detection system. The mass spectrometry detection conditions are the same as those in Example 2. The peaks in the primary mass spectrum whose m / z of the elution solution of the reactants and products on the titanium dioxide surface is exactly 162.06 higher than that of the photocatalytic decomposition supernatant are screened as the glycolipid peak and the corresponding glycolipid decomposition mist peak, and the glycolipid peaks and glycolipid decomposition product peaks in the photocatalytic deglycosylation supernatant and the elution solution of the reactants and products adsorbed on the titanium dioxide surface after photocatalysis are further subjected to secondary mass spectrometry detection.

[0081] Result analysis: By detecting the photocatalytic deglycosylation supernatant and the elution solution of the reactants and products adsorbed on the titanium dioxide surface after photocatalysis, the primary mass spectrum peaks of glycolipids in the brain tissue extract before and after photocatalytic decomposition can be obtained, such as Figure 5 As shown. Figure 5 It can be found that the black chemical analysis solution mainly obtains peaks with a mass-to-charge ratio of about 800-900, while the solution after the photocatalytic reaction mainly obtains peaks with a mass-to-charge ratio of about 500-700, and most of these peaks can find the corresponding peak of the mass-to-charge ratio of M+162.06 in the chemical analysis solution. It can be seen that most of the glycolipids in the brain tissue are decomposed into deglycosylated ceramides. The secondary mass spectra of the two representative ceramides (m / z 662.5 and 664.5) and the corresponding glycolipids (m / z824.6 and m / z826.6) are detected, as shown in the figure. Figure 6 It can be found that the deglycosylated ceramide has more abundant fatty acid structure fragments than glycolipids, especially the fragments related to the hydroxyl position and double bond position on the fatty acid (labeled C fragments), while the glycolipid itself is mainly fragments related to the sugar head (labeled D fragments).

[0082] The above is only a preferred embodiment of the present invention and does not limit the present invention in any form. Therefore, any simple modification, equivalent change and modification made to the above embodiment according to the technical essence of the present invention without departing from the content of the technical solution of the present invention shall fall within the scope of protection of the technical solution of the present invention.

Claims

1. A sample processing method for selective photocatalytic deglycosylation of glycosphingolipids, characterized in that: The specific steps include: (1) dissolving the sample in an alkaline protic solvent to obtain a sample extract; (2) dispersing titanium dioxide particles in the sample extract to obtain a dispersion; (3) subjecting the dispersion to a photocatalytic reaction under ultraviolet light irradiation, centrifuging, obtaining a supernatant as a photocatalytic deglycosylation supernatant, and obtaining a solid powder as titanium dioxide powder.

2. The method according to 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 according to claim 2, characterized in that The alkaline proton solvent comprises ammonia water, methanol and acetonitrile, and the volume ratio of the ammonia water added to the alkaline proton solvent is 5%-10%.

4. The method according to claim 1, characterized in that The dissolution includes at least one of direct dissolution, solvent leaching extraction or nitrogen blowing concentration and then redissolution.

5. The method according to claim 1, characterized in that The titanium dioxide particles include one or more of amorphous titanium dioxide particles, anatase crystal titanium dioxide particles or titanium dioxide particles doped with metal, non-metallic elements or carbon nanomaterials. The particle size of the titanium dioxide nanoparticles is 1-10nm, and the dispersion concentration of the titanium dioxide nanoparticles in the extract is 1-10g / L; the wavelength of the ultraviolet light is 200-400nm, and the photocatalytic reaction time is 30s-30min.

6. A method for detecting glycosphingolipid spectrum by photocatalytic deglycosylation, characterized in that: The photocatalytic deglycosylation supernatant according to any one of claims 1 to 6 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 sample not photocatalytically treated are compared for data analysis.

7. The method for detecting glycosphingolipid spectrum of photocatalytic deglycosylation according to claim 6, characterized in that: The mass spectrometer includes a tandem mass spectrometer or a liquid-mass spectrometer, and the comparative data analysis refers to extracting the primary mass spectrometry peaks of the photocatalytic deglycosylation supernatant and the sample extract that has not been photocatalytically treated, screening the peaks with a mass-to-charge ratio difference of 162.06 before and after treatment as the molecular ion peaks of the glycosphingolipids before and after deglycosylation to obtain the mass information of the glycosphingolipids, and selecting the molecular ion peaks after deglycosylation for secondary mass spectrometry detection to obtain the structural fragment information of the glycosphingolipids.

8. The method for detecting glycosphingolipid spectrum of photocatalytic deglycosylation according to claim 6, characterized in that: The extraction liquid of the sample not treated with photocatalysis is obtained by immersing the solid sample in an alkaline proton solvent for extraction, or by using an in-situ microfluidic node sampling technique to extract the glycosphingolipids on the surface of the solid sample into the extraction liquid using an alkaline proton solvent as a sampling solvent.

9. The method for detecting glycosphingolipid spectrum of photocatalytic deglycosylation according to any one of claims 6 to 8, characterized in that: The mass spectrometry detection adopts in-situ micro-liquid node sampling-mass spectrometry technology, and the sampling and detection instruments are configured as follows: titanium dioxide nanoparticles are configured in the pipeline through which the photocatalytic deglycosylation supernatant or sample extract is sampled and injected into the mass spectrometer, and ultraviolet light is configured outside the corresponding pipeline, so that the glycosphingolipids in the extract flow in the pipeline while undergoing a photocatalytic reaction, and after the reaction is completed, it is continuously and automatically injected into the mass spectrometer.

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