Targeted glucosinolate mass spectrum database and construction method and application thereof

Abstract

The invention provides a glucosinolate mass spectrum database and a construction method and application thereof, and the database records more than 120 types of glucosinolate molecular information which is far better than that of the existing database. According to the pretreatment and detection method for the targeted thioglycoside group of the biological sample, solid-phase extraction and a micro-nano mass spectrum chip are combined, thioglycoside group mass spectrum signals are obtained in batches under the condition of no matrix spraying, the coverage rate is wide, the flux is high, the detection speed is high, thioglycoside group information of multiple samples can be obtained at a time in an automatic detection mode, and the detection method is suitable for large-scale popularization and application. The obtained thioglycoside spectrogram is high in stability, and in-batch and inter-batch CVlt is achieved under the condition that no internal standard is added; by combining with an established thioglycoside mass spectrum database, a novel method strategy is provided for marker mining and clinical application of diseases related to sphingolipid.

Description

Technical Field

[0001] The present invention relates to the technical fields of omics analysis and drug analysis, and particularly relates to a detection method targeting sulfoglycolipidomics. Background Art

[0002] With the development of omics technologies, multiple metabolic and lipid-related databases, including HMDB, LIPIDMAPS, SwissLipids, Metlin, LipidSearch, etc., have emerged internationally, providing comprehensive data support for lipidomics and metabolomics research, covering the revelation of chemical properties, biological functions, and metabolic pathways, and providing annotation and identification functions for the data of mass spectrometry tests of multiple samples, further providing a basic library for biomarker screening and therapeutic target research. However, the above databases have the following defects: 1. The data has platform bias, mainly focusing on GC-MS and LC-MS data, and rarely including data from the MALDI ion source. The adduct forms and fragmentation forms of the molecules detected on the MALDI mass spectrometer are different from those of GC-MS and LC-MS. Therefore, it is still relatively difficult to annotate the peaks of metabolic molecules and lipid molecules detected on the MALDI mass spectrometer; 2. Due to the platform bias of the mass spectrometry platform, the detection sensitivity of some molecules is relatively low on the GC-MS and LC-MS platforms, while it is relatively high on other mass spectrometry platforms. Molecules detected on non-GC-MS or LC-MS platforms cannot be annotated using these databases. In particular, the inventors found that molecules such as sulfoglycolipid glucosylceramide (referred to as sulfoglycolipid for short) only contain 10 and 11 molecular structures in HMDB and LIPIDMAPS, and the coverage rate in other databases is also much lower than that of other types of metabolic or lipid molecules.

[0003] Sulfatide is a class of 3-O-sulfo-galactosylceramides synthesized from sphingolipids by sphingolipid galactosyltransferase and cerebroside sulfotransferase. It is a special acidic sphingolipid, mainly produced in myelin and is an important component of myelin, accounting for about 4% of the total amount of myelin. It is also distributed in the nervous system, kidneys, liver, spleen, stomach, small intestine, and serum. Sulfatide is a multifunctional molecule in various biological fields, including the nervous system, insulin secretion, immune system, hemostasis / thrombosis, bacterial infection, and viral infection. Abnormal metabolism or changes in the expression of sulfatide may trigger various diseases, especially including metachromatic leukodystrophy (MLD) and other lysosomal storage disorder genetic diseases, neurodegenerative diseases such as Alzheimer's disease, kidney injury-related diseases, cardiovascular and cerebrovascular diseases such as myocardial infarction, and cancers. For example, patients with MLD lack lysosomal sulfatase ASA that can hydrolyze the 3-O ester bond of cerebroside, resulting in the deposition of sulfatide in lysosomes in the body, thus damaging the central and peripheral nervous systems. At this time, significantly increased sulfatide can be detected mainly in the urine samples of patients, and the diagnostic efficiency for MLD disease can be improved through the logistic regression equation of multiple sulfatide expressions (Clinica Chimica Acta 433 (2014) 39 - 43); when sulfatide is absent in myelin, it will trigger inflammatory reactions and abnormal lipid metabolism in glial cells, leading to Alzheimer's disease with abnormal myelin function, and changes will also occur in the functions of the patient's bladder, etc. The content of sulfatide in cerebrospinal fluid, whether it is hydroxylated and the proportion of hydroxylation can be used as one of the key biomarkers for Alzheimer's disease (Molecular Neurodegeneration, (2021) 16:64); the combination of sulfatides can also be used as an indicator for the risk assessment of cardiovascular diseases in patients with end-stage renal failure. Whether patients with renal failure have cardiovascular diseases will show different total amounts of sulfatides and the distribution of sulfatides with different chain lengths (Glycoconj J (2007) 24:565 - 571); Patent 202310945247.X discloses a set of biomarker combinations for the diagnosis of colorectal cancer (CRC), and sulfatide also plays a role in it. Therefore, the study of the content or distribution of sulfatide in the human body has important physiological value, and it is crucial to increase the construction of the database of such molecules.

[0004] Compared with several major lipid classes such as glycerolipids, sterols, and glycerophospholipids, sphingolipids belong to the lipid class with relatively low content in the body. Sulfatide, in turn, belongs to the low-abundance sphingolipids among sphingolipids (compared with sphingomyelin SM). Taking phosphatidylcholine PC with a high content in plasma as an example, the highest concentration of PC is close to 1 mmol / L, while the content of all sulfatides is less than 1 nmol / L, with a difference of six orders of magnitude between the two. The extraction, enrichment, and detection of sulfatides in complex biological samples such as plasma, serum, and cerebrospinal fluid have high requirements for pretreatment methods and highly sensitive detection methods.

[0005] The existing ELISA kits on the market for measuring the content of sulfatides can only test the total content of such molecules and cannot separately test their distribution. Sulfatides include both non-hydroxylated SHexCer and SHexCer(OH) with one or more hydroxylation modifications; they include both SHexCer with one cyclohexanol in the molecular structure and SHex2Cer with two cyclohexanols. At the same time, there are multiple carbon chain structures in each backbone, such as SHexCer d18:1 / 12:0, SHexCer d18:1 / 14:0, SHexCer d18:1 / 16:0, SHexCer d18:1 / 18:0, SHexCer d18:1 / 18:1, SHexCer d18:1 / 20:0, SHexCer d18:1 / 22:0, SHexCer d18:1 / 24:0, etc. Only a high-resolution mass spectrometer can achieve simultaneous high-precision qualitative and quantitative detection of a large class of sulfatides with basically similar backbone structures but local differences, and carry out sulfatidomics research.

[0006] The mass spectrometry platforms reported in the literature for the detection of sulfoglycolipids include two major systems: LC-MS and MALDI-MS. LC-MS is a general mass spectrometry platform for lipidomics research. However, due to the low abundance of sulfated sphingolipids in samples such as serum or tissues, in-source fragmentation in LC-MS, low ionization efficiency, and strong adsorption of sulfonic acid groups on the chromatographic column, the lipid response sensitivity of sulfoglycolipids is usually low during non-targeted lipidomics testing. For this reason, Patent 201910424563.6 (authorization number CN 110068638 B) discloses a method for the separation and enrichment of acidic glycosphingolipids based on TiO2 column separation technology, which can remove the interference of phospholipids and at the same time enrich acidic sphingolipids such as sulfoglycolipids, improving their coverage rate in LC-MS detection. However, even so, the number of sulfoglycolipids in serum after enrichment is only 19. The number of sulfoglycolipids detected in serum based on LC-MS reported in other literatures does not exceed 20. MALDI-MS is usually inhibited by high-abundance phospholipids of the PC type, and complex sample pretreatment methods are required to match the testing of sulfoglycolipid molecules in complex samples on MALDI. Patent 202210511862.5 (authorization number CN 114858908 A) discloses a method for purifying sulfoglycolipids by steps such as NaOH saponification and SPE column passing, which can convert sulfoglycolipids into hydrogenated sulfoglycolipids and is also the most common pretreatment method based on the MALDI platform at present. However, due to its complex process, uneven crystallization of conventional organic matrices (such as CHCA, DHB, 9-AA, etc.), and high ionization threshold, a fingerprint spectrum of a sulfoglycolipid group with high stability and high coverage rate cannot be obtained, and the coverage rate is also below 20, affecting the practical application of the sulfoglycolipid group. Summary of the Invention

[0007] In view of this, the technical problem to be solved by the present invention is to provide a method for establishing a targeted sulfoglycolipid mass spectrometry database; in the database established by the present invention, more than 120 sulfoglycolipid molecules are obtained.

[0008] To solve the problems that the number of sulfoglycolipid groups included in the existing database is limited, the abundance in complex samples is low, the detection sensitivity is low, the coverage rate is low, and it is vulnerable to interference, etc., the present invention adopts a lipid pretreatment method combined with a solid-phase extraction device to achieve rapid and high-throughput extraction and enrichment of sulfoglycolipid groups, without elution, and can process 96 samples in batches simultaneously; a micro-nano mass spectrometry chip is used in combination with a MALDI-TOF mass spectrometer for matrix-free detection of the samples after sulfoglycolipid enrichment, without the need to add an additional matrix, and the whole process can be automated, with a selective enhancement effect on sulfoglycolipid detection. The obtained sulfoglycolipid spectrogram has strong stability, and the within-batch and between-batch CV is <10% without the addition of internal standards, and there is no ion suppression interference from high-abundance phospholipids, avoiding the between-batch instability of non-targeted lipidomics; based on the above methods, more than 120 types of sulfoglycolipid molecules can be stably obtained. The primary molecular weights and secondary fragments are sorted out to establish a sulfoglycolipid mass spectrometry database, which is far higher than the level of the existing database, and can provide new method strategies for biomarker mining and clinical applications of sphingolipid-related diseases.

[0009] The present invention provides a targeted sulfoglycolipid mass spectrometry database, including the following sulfoglycolipid molecular information: non-hydroxylated SHexCer, SHexCer(OH) with one or more hydroxylation modifications n 、SHexCer containing one cyclohexanol in the molecular structure, SHex 2-n Cer with two or more cyclohexanols, and SO3-Lc n Cer containing amide sugar in the molecular structure; the sphingosine chain of the sulfoglycolipid is d18:0 or d18:1; the other chain is a fatty acid chain with the number of carbon atoms being 12 - 30, and the degree of unsaturation of the fatty acid chain is more than 0; the number of hydroxyl groups is more than 0; the number of cyclohexoses is at least 1.

[0010] The present invention provides a method for establishing a targeted sulfoglycolipid mass spectrometry database, including the following steps:

[0011] A) Extract the sample to be tested with a solvent to obtain a test solution;

[0012] The solvent includes a first component, a second component, and a third component; wherein the first component includes one or more of methanol, butanol, and isopropanol; the second component includes one or more of methyl tert-butyl ether, ethyl acetate, chloroform, dichloromethane, n-heptane, and n-hexane; the third component includes ultrapure water;

[0013] B) After subjecting the test solution to solid-phase extraction, spot it on a micro-nano mass spectrometry chip, and detect it with a mass spectrometer using MALDI as the ion source. Using lipidomics analysis technology, obtain a primary spectrum in the MS primary acquisition mode, and fragment the obtained parent ion peaks in the MS / MS secondary acquisition mode to obtain a secondary spectrum;

[0014] C) Obtain sulfatide data sheet information and establish a sulfatide database.

[0015] Figure 1 . This is the flowchart for establishing the database of the present invention.

[0016] The method for establishing a targeted sulfatide lipid profile database provided by the present invention first uses solvent extraction on the sample to be tested to obtain a test solution.

[0017] The sample to be tested described in the present invention includes serum, plasma, urine, tissue, cerebrospinal fluid, exosomes, and cells.

[0018] According to the present invention, the solvent includes a first component, a second component, and a third component; wherein the first component includes one or more of methanol, butanol, and isopropanol; the second component includes one or more of methyl tert-butyl ether, ethyl acetate, chloroform, dichloromethane, n-heptane, and n-hexane; the third component includes ultrapure water

[0019] Specifically, the ratio of the first component: the second component: the third component is: 1:2:1 to 1:4:2.

[0020] In some specific embodiments, the ratio of the first component: the second component: the third component is: 1:2:1, 1:3:1, 1:4:1, 1:3:2, or 1:4:2.

[0021] In some embodiments of the present invention, the ratio of the solvent to the sample to be tested is: 2.5:1 to 10:1; specifically, it can be 2.5:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1, or 10:1.

[0022] In the preferred embodiment of the present invention, the extraction agent combination of methanol, methyl tert-butyl ether, and ultrapure water has the best effect; and their volume ratio is 1:3:1

[0023] The test solution is subjected to solid-phase extraction; the present invention does not limit the solid-phase extraction, and preferably uses the solid-phase extraction device disclosed in CN106908513A. The pressure of the solid-phase extraction is 3 to 6 kPa, and the time is 5 to 10 minutes.

[0024] In one specific embodiment, the extracted lipid metabolites are transferred to a 96-well sample preparation plate and allowed to flow to a receiving tube under negative pressure;

[0025] Transfer the enriched solution in the receiving tube, spot it on a micro-nano mass spectrometry chip, and after natural drying, perform negative ion mode detection on a mass spectrometer.

[0026] Experimental results show that the present invention has a high peak response of sulfatides on the micro-nano mass spectrometry chip, and the micro-nano mass spectrometry chip involved in the present invention has the advantages of improving the selectivity and sensitivity for sulfatide molecules.

[0027] In some other embodiments of the present invention, it further includes: transferring the test sample into a centrifuge tube, sequentially adding a pretreatment extraction reagent to extract lipid metabolites, and the sample types include: serum, plasma, urine, tissue, cerebrospinal fluid, exosomes, cells, and the ratio of the pretreatment extraction reagent to the sample is: 2.5:1 to 10:1; vacuum-drying the lipid metabolites of the test sample extracted after the above treatment to prepare a lipid freeze-dried product;

[0028] Placing a 96-well sample preparation plate above a negative pressure device, placing a receiving tube at the corresponding hole position of the 96-well sample preparation plate below, turning on the pressure switch knob to make the pressure stable at 3 to 6 kPa; re-dissolving the lipid freeze-dried product, transferring it into the 96-well sample preparation plate, and making it flow to the receiving tube under negative pressure, and the volume of the re-dissolution solution is 50 - 500 μL. Transferring the enriched liquid in the receiving tube, and then spotting it on a micro-nano mass spectrometry chip, and performing negative ion mode detection on a mass spectrometer after natural drying.

[0029] In some other embodiments of the present invention, it further includes: transferring the test sample into a centrifuge tube, sequentially adding a pretreatment extraction reagent to extract lipid metabolites, and the sample types include: serum, plasma, urine, tissue, cerebrospinal fluid, exosomes, cells, and the ratio of the pretreatment extraction reagent to the sample is: 2.5:1 to 10:1; vacuum-drying the lipid metabolites of the test sample extracted after the above treatment to prepare a lipid freeze-dried product; placing a 96-well sample preparation plate above a negative pressure device, placing a receiving tube at the corresponding hole position of the 96-well sample preparation plate below, turning on the pressure switch knob to make the pressure stable at 3 to 6 kPa; re-dissolving the lipid freeze-dried product, transferring it into the 96-well sample preparation plate, and making it flow to the receiving tube under negative pressure, and the volume of the re-dissolution solution is 50 - 500 μL. After transferring a certain volume of the liquid in the receiving tube, performing vacuum drying, and the range of the certain volume is: 25 - 400 μL; re-dissolving the freeze-dried lipid in the receiving tube with a re-dissolution solution, and then spotting it on a micro-nano mass spectrometry chip, and performing negative ion mode detection on a mass spectrometer after natural drying.

[0030] The re-dissolution solution described in the above technical solution includes:

[0031] It includes isopropanol, acetonitrile, methanol, an isopropanol / acetonitrile mixture, and an isopropanol / methanol mixture; the ratio of the re-dissolution solution to the liquid after pretreatment is: 1:1 to 10:1.

[0032] The above consumables include: a consumable combination formed by a 96-well sample preparation plate, a negative pressure device, a micro-nano mass spectrometry chip, a receiving plate, a sealing film, a centrifuge tube, a pipette, a pipette tip, and a stainless steel aluminum plate. The adsorption material filled in the 96-well sample preparation plate has the function of adsorbing phospholipid molecules; the negative pressure device can filter the sample solution in the 96-well sample preparation plate into the receiving plate under vacuum negative pressure, preferably CN106908513A and CN109541012A.

[0033] According to the present invention, detection is performed using a mass spectrometer with MALDI as the ion source. Using lipidomics analysis technology, a primary spectrum is obtained by adopting the MS primary acquisition mode, and the obtained parent ion peaks are fragmented by adopting the MS / MS secondary acquisition mode to obtain a secondary spectrum.

[0034] The mass spectrometer with MALDI as the ion source according to the present invention includes: MALDI-TOF, MALDI-TOF / TOF, MALDI2-timsTOF / TOF, MALDI-FT-ICR, and AP-MALDI-Orbitrap mass spectrometers.

[0035] The obtained parent ion peaks are subjected to primary peak matching by adopting the MS primary acquisition mode, and by adopting the MS / MS secondary acquisition mode, the obtained parent ion peaks are fragmented and the secondary characteristic peak fragments are identified.

[0036] The sulfoglycolipids in the database include non-hydroxylated SHexCer, one or more hydroxylated modified SHexCer(OH) n SHexCer containing one cyclohexanol in the molecular structure, SHex 2-n Cer having two or more cyclohexanols, and SO3-Lc n Cer containing amide sugar in the molecular structure.

[0037] According to the present invention, the specific method for establishing a sulfoglycolipid database includes:

[0038] S1) The obtained parent ion peaks are subjected to primary peak matching by adopting the MS primary acquisition mode, and by adopting the MS / MS secondary acquisition mode, the obtained sulfoglycolipid parent ion peaks are fragmented.

[0039] Enumerate sulfoglycolipid molecules with different carbon chain types and one or more hydroxylated modifications, calculate the theoretical molecular weight in combination with their adduct forms, and form a candidate sulfoglycolipid form: the sphingosine chain of the sulfoglycolipid is d18:0 or d18:1; the other chain is a fatty acid chain with the number of carbon atoms being 12 - 30, and the degree of unsaturation of the fatty acid chain is more than 0; it can be 0, 1, 2, 3; the number of hydroxyl groups is more than 0; it can be 0, 1, 2, 3; the number of cyclohexoses is at least one; it can be 1.

[0040] S2) performing primary peak matching of the spectrum in the MS primary acquisition mode with the enumerated candidate sulfatide list, with a matching deviation of <20 ppm, eliminating isotope peaks, and for peaks that cannot be matched with the enumerated candidate sulfatide list, supplementing the candidate sulfatide list by calculating the molecular weight difference between different peaks;

[0041] S3) analyzing the spectrum of MS / MS secondary acquisition mode;

[0042] Sulfatides belong to sphingolipids and are a class of 3-O-sulfogalactosylceramides synthesized from sphingolipids by sphingolipid galactosyltransferase and cerebroside sulfotransferase, such as Figure 2 As shown, taking SHexCer (d18:1 / 20:0) as an example for structural identification, at the window corresponding to the primary parent ion m / z of 834.57706 of the peak, the secondary fragments are collected, the structure of the sulfoside ester is broken, and characteristic fragments with sulfonic acid group m / z of 96.9 or 97.0 will fall off, and structurally specific fragments such as 241.0 and 315.0 will be formed. Combined with the mass-to-charge ratio of the primary parent ion peak and the secondary characteristic fragments, this peak is judged to be a sulfoside ester.

[0043] S4) Based on the above method, the detected sulfatide peaks were structurally confirmed, and the molecular type, molecular formula, chain information, accurate molecular weight, detected molecular weight, detection error, secondary fragments, existing numbers in the public database, and numbers in the self-built database were formed into a sulfatide data table information and implanted into the database to obtain 124 sulfatide molecules.

[0044] The fragments cover 124 sulfatide molecules, including SHexCer without hydroxylation, SHexCer with one or more hydroxylation modifications (OH) n , SHexCer with one cyclohexanol in its molecular structure, SHex2Cer with two cyclohexanols, and SO3-LcCer. A total of 124 sulfatide molecules were identified, as shown in Table 3, which is much higher than the number of sulfatide molecules included in HMDB and LIPIDMAPS.

[0045] The present invention can subsequently optimize and increase the types of sulfatide molecules according to the above method, and the technical solutions optimized by the above method are all included in the present technical solution.

[0046] According to the present invention, the mass spectrometer of the MALDI-MS mass spectrometer using MALDI as the ion source includes: MALDI-TOF, MALDI-TOF / TOF, MALDI2-timsTOF / TOF, MALDI-FT-ICR, AP-MALDI-Orbitrap mass spectrometer;

[0047] The detection mode of the mass spectrometry is the negative ion mode, and the detected molecular weight range is 400 - 2000 m / z.

[0048] In some preferred embodiments of the present invention, the mass spectrometer is required to be equipped with a 355 nm Nd:YAG laser beam or a 337 nm nitrogen laser, with a maximum laser output energy of more than 20 μJ and a detection frequency of more than 100 Hz; the detector power supply is set to 1400 - 1500 V, the ion source repeller power supply is set to 12000 - 13000 V, the ion source extraction electrode power supply is set to 1100 - 1200 V, the focusing electrode high-voltage power supply is set to 20 - 30 V, and the reflection high-voltage power supply is set to 13000 - 14000 V; the pulsed ion extraction time is set to more than 100 ns.

[0049] The inventors of the present invention found that in the low energy value range, the overall resolution of the glucosinolate spectrum is good, the peaks appear well, and the response can reach 10 4 or more; on the basis of the low energy value, when the energy value is increased, the resolution of the glucosinolate is improved, and the response can reach 5×10 4 or more; however, as the energy value continues to increase, the resolution of the glucosinolate decreases instead, and the response also decreases accordingly, indicating that the laser energy value is not the higher the better, and the optimal output energy is set to 0.7 - 1.5 μJ.

[0050] In some preferred embodiments of the present invention, when detecting each sample point, the average signal of 1000 laser pulses is used as the detection result of the reflection mode of this sample point.

[0051] The present invention also provides an application of a targeted glucosinolate lipid spectrum database, which is characterized by including the following steps:

[0052] S1) The biological sample to be tested is extracted with a solvent to obtain a test solution;

[0053] The solvent includes a first component, a second component, and a third component; the first component includes one or more of methanol, butanol, and isopropanol; the second component includes one or more of methyl tert-butyl ether, ethyl acetate, chloroform, dichloromethane, n-heptane, and n-hexane; the third component includes ultrapure water;

[0054] S2) After the test solution is subjected to solid-phase extraction, it is detected with a mass spectrometer, and using lipidomics analysis technology, a primary spectrum is obtained in the primary acquisition mode;

[0055] S3) importing the spectrum into a sulfatide lipid spectrum database, setting the allowable detection error, performing sulfatide molecular matching, and obtaining a sulfatide annotation result list of the tested sample, wherein the list information includes: molecular type, molecular formula, chain information, accurate molecular weight, detected molecular weight, detection error, secondary fragments, existing numbers in the public database, numbers in the self-built database, and mass spectrometry response values ​​of each sample;

[0056] S4) Analyze the annotation result list for inter-group difference analysis, sulfatide biomarker mining, and machine learning algorithm model construction to realize data application based on sulfatide lipid spectrum database.

[0057] The present invention provides a sulfatide lipid spectrum database and a method and application thereof. The database includes information on more than 120 sulfatide molecules, which is far more than the existing databases. The pretreatment and detection method of the targeted sulfatide group of biological samples adopted combines solid phase extraction with micro-nano mass spectrometry chips, and obtains sulfatide group mass spectrometry signals in batches without matrix spraying. The coverage is wide, the throughput is high, and the detection speed is fast. In the automated detection mode, the sulfatide group information of multiple samples can be obtained at one time. The obtained sulfatide spectra are highly stable, and the intra-batch and inter-batch CV is less than 10% without the addition of internal standards. Combined with the established sulfatide lipid spectrum database, a new method strategy is provided for the marker mining and clinical application of sphingolipid-related diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0058] Figure 1 .Database establishment flow chart;

[0059] Figure 2 .Secondary identification structure diagram;

[0060] Figure 3 .Flow chart of sulfatide extraction method;

[0061] Figure 4 .Comparison of spectra of sulfatide esters under different pretreatment systems;

[0062] Figure 5 .Comparison of selectivity spectra of different substrate sulfatide esters;

[0063] Figure 6 .Comparison of sulfatide spectra of different mass spectrometers;

[0064] Figure 7 .Comparison of sulfatide spectra at different energy values;

[0065] Figure 8 .Comparison of the spectrum of serum lipid reconstitution solution and enrichment solution;

[0066] Figure 9 .The number of sulfatide peaks in different samples after enrichment;

[0067] Figure 10 . Comparison of serum sulfoglycolipid profiles between healthy individuals and liver cancer patients;

[0068] Figure 11 . OPLS-DA analysis of serum sulfoglycolipid profiles of healthy individuals and liver cancer patients. Detailed implementation manners

[0069] The present invention provides a database targeting sulfoglycolipidome. Those skilled in the art can draw on the content of this article and appropriately modify process parameters to implement it. It should be particularly noted that all similar substitutions and modifications are obvious to those skilled in the art, and they all fall within the scope of protection of the present invention. The methods and applications of the present invention have been described through preferred embodiments. Relevant personnel can obviously make changes or appropriate modifications and combinations to the methods and applications in this article without departing from the content, spirit and scope of the present invention to implement and apply the technology of the present invention.

[0070] It should be understood that the expression "one or more of..." individually includes each object recited after the expression and various different combinations of two or more of the recited objects, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited objects should be understood to have the same meaning, unless otherwise understood from the context.

[0071] The terms "comprising", "having" or "containing", including the use of their grammatical synonyms, should generally be understood as open and non-restrictive, for example, not excluding other unrecited elements or steps, unless otherwise specifically stated or otherwise understood from the context.

[0072] In this application, the term "and / or" describes the association relationship of associated objects and indicates that three relationships can exist. For example, A and / or B can represent: A exists alone, A and B exist simultaneously, and B exists alone. Wherein A and B can be singular or plural.

[0073] In this application, "at least one" means one or more, and "a plurality" means two or more. "At least one of the following (items)" or its similar expressions refer to any combination of these items, including any combination of single (item) or plural items (items).

[0074] It should be understood that as long as the present invention is still operable, the order of steps or the order of performing certain actions is not important. In addition, two or more steps or actions can be performed simultaneously.

[0075] The use of any and all examples or exemplary language in this document, such as "for example" or "including", is merely intended to better illustrate the invention and does not limit the scope of the invention unless a claim is made. No language in this specification should be construed as indicating that any unclaimed element is essential to the practice of the invention.

[0076] In addition, the numerical ranges and parameters used to define the present invention are approximate values. The relevant numerical values in the specific embodiments have been presented as precisely as possible herein. However, any numerical value inherently and inevitably contains standard deviations caused by individual testing methods. Therefore, unless otherwise clearly stated, it should be understood that all ranges, quantities, numerical values, and percentages used in this disclosure are modified by "about". Here, "about" generally means that the actual value is within plus or minus 10%, 5%, 1%, or 0.5% of a specific numerical value or range.

[0077] It should be understood that in various embodiments of the present application, the magnitudes of the serial numbers of the above processes do not mean the order of execution. Some or all of the steps may be executed in parallel or sequentially. The execution order of each process should be determined by its function and internal logic, and should not constitute any limitation to the implementation process of the embodiments of the present application.

[0078] Some cases are described in the embodiments and comparative examples of the present invention, and certain implementation manners of the present invention are shown in the embodiments. However, this does not mean that the effects of the present invention can only be achieved in these cases.

[0079] To further illustrate the present invention, a database targeting sulfatide groups provided by the present invention will be described in detail below in conjunction with embodiments.

[0080] Example 1. Selection of Pretreatment Solvent for Targeted Extraction and High-Coverage Detection of Sulfatide

[0081] This example mainly includes the following steps:

[0082] Step 1. Sample Pretreatment

[0083] The sample pretreatment process is carried out on ice. The serum sample is aliquoted into 30 - 50 μL per tube and placed in a 1.5 mL centrifuge tube. 250 - 500 μL of pretreatment extraction reagent is added. The combination of pretreatment extraction reagents in this example is: ① isopropanol, butanol, n-heptane, ethyl acetate, and ultrapure water; ② methanol, methyl tert-butyl ether, and ultrapure water; ③ methanol and dichloromethane; ④ methanol and butanol. Subsequently, it is vortexed for 10 - 15 s to mix evenly, oscillated for 10 - 30 min, and centrifuged at high speed at 4 - 6 °C for 10 min. After two-phase separation, the hydrophobic phase is quantitatively collected and vacuum dried in a vacuum drying device to obtain a freeze-dried sample.

[0084] Step 2. Solid-Phase Extraction Enrichment

[0085] Place the 96-well sample preparation plate above the negative pressure device, place the receiving tube at the corresponding hole position of the 96-well sample preparation plate below the negative pressure device, turn on the pressure switch knob to make the pressure stable at about 3 - 6 kPa, and keep it stable for 5 - 10 min; dilute and reconstitute the lipid lyophilized product with the reconstitution solution, vortex and mix well, then pipette 300 μL into the 96-well sample preparation plate, keep the pressure stable, and make the lipid reconstitution solution flow slowly and evenly into the corresponding receiving tube below. Ensure that the whole process is completed within 5 - 10 min, then turn off the pressure switch, quantitatively pipette 100 μL into a 0.2 ml centrifuge tube to obtain the enriched solution, and whether to perform secondary freeze-drying and concentration of the enriched solution is selected according to the sample concentration.

[0086] Spot the pre-treated and freeze-dried and reconstituted reconstitution solution and the enriched solution on the micro-nano mass spectrometry chip in step 2, and the single-hole volume is 0.5 - 2 μL. Dry the sample at room temperature. After drying is completed, send the chip into the MALDI-TOF mass spectrometer for detection. The experimental process is as Figure 3 shown.

[0087] Step 3. Data acquisition

[0088] Mass spectrometry acquisition is completed by the MALDI-TOF mass spectrometer. The measurement mode is the reflectron negative ion mode. The molecular weight acquisition range of the enriched solution is 700 - 1500 m / z, and the molecular weight acquisition range of the reconstitution solution is 400 - 1000 m / z. When detecting each sample point, the average signal of 800 - 1000 laser pulses is used as the detection result of the reflectron mode for this sample point. The detection result of the enriched solution is as Figure 4 shown.

[0089] Step 4. Spectral analysis

[0090] Select a suitable pre-treatment solvent system according to the spectrum. The spectral analysis result is as Figure 4 shown. It can be seen from the results that the peak response and signal-to-noise ratio of thioglycoside esters in the dichloromethane system (combination ③) and the methyl tert-butyl ether system (combination ②) are relatively high, followed by the heptane system (combination ①), and the simple alcohol system (combination ④) has poor selectivity for thioglycoside esters. The selection of the pre-treatment solvent in the pre-treatment process of the present invention reflects that different combinations of pre-treatment systems have different selectivities for thioglycoside esters. Considering the convenience and performance of the experimental process comprehensively, the methyl tert-butyl ether system is a preferred pre-treatment system for the extraction of thioglycoside lipids.

[0091] Example 2. Selection of detection substrate for targeted extraction and high-coverage detection of thioglycoside lipids

[0092] This example mainly includes the following steps:

[0093] Step 1. Pretreatment of serum samples

[0094] The sample pretreatment process is carried out on ice. The serum sample is aliquoted into 30 - 50 μL per tube and placed in a 1.5 mL centrifuge tube. Then, 250 - 500 μL of pretreatment extraction reagent is added. In this embodiment, the pretreatment extraction reagent combination is: methanol, methyl tert - butyl ether, and ultrapure water. Subsequently, it is vortexed for 10 - 15 s to mix evenly, shaken for 10 - 30 min, and centrifuged at high speed for 10 min at 4 - 6 °C. After two - phase separation, the upper hydrophobic phase is quantitatively collected and vacuum - dried in a vacuum drying device to obtain a lyophilized sample.

[0095] Step 2. Solid - phase extraction enrichment

[0096] Place the 96 - well sample preparation plate above the negative - pressure device, place the receiving tube at the corresponding hole position of the 96 - well sample preparation plate below the negative - pressure device, turn on the pressure switch knob to make the pressure stable at about 3 - 6 kPa, and keep it stable for 5 - 10 min. Dilute and re - dissolve the lipid lyophilized product with a re - solution, vortex and mix well, then pipette 300 μL into the 96 - well sample preparation plate. Keep the pressure stable and make the lipid re - solution flow slowly and evenly into the corresponding receiving tube below. Ensure that the whole process is completed within 5 - 10 min. Then turn off the pressure switch, quantitatively pipette 100 μL into a 0.2 ml centrifuge tube to obtain an enrichment solution. Whether to perform secondary lyophilization and concentration of the enrichment solution is selected according to the sample concentration.

[0097] Spot the re - solution after pretreatment and lyophilization and the enrichment solution in step 2 on the micro - nano mass spectrometry chip and matrix 9 - AA (9 - aminoacridine), with a single - hole volume of 0.5 - 2 μL. Let the sample dry at room temperature. After drying, send the chip into a MALDI - TOF mass spectrometer for detection. The experimental process is as Figure 3 shown.

[0098] Step 3. Data acquisition

[0099] Mass spectrometry acquisition is completed by a MALDI - TOF mass spectrometer. The measurement mode is the reflectron negative - ion mode. The molecular weight acquisition range of the enrichment solution is 700 - 1500 m / z, and the molecular weight acquisition range of the re - solution is 400 - 1000 m / z. When detecting each sample point, the average signal of 800 - 1000 laser pulses is used as the detection result of the reflectron mode for this sample point. The detection result of the enrichment solution is as Figure 5 shown.

[0100] Step 4. Spectral analysis

[0101] Select a suitable substrate according to the spectrum. The spectral analysis result is as Figure 5As shown. From the results, it can be seen that under the same experimental conditions, the selectivity of thioglycoside esters on the micro-nano mass spectrometry chip is much higher than that on 9-AA, and the response of thioglycoside esters on 9-AA is low; on the contrary, the peak response of thioglycoside esters on the micro-nano mass spectrometry chip is high. The micro-nano mass spectrometry chip involved in the present invention has the advantages of improving the selectivity and sensitivity for thioglycolipid molecules.

[0102] Example 3. Selection of Mass Spectrometry Instrument for Targeted Extraction and High-Coverage Detection of Thioglycolipids

[0103] This example mainly includes the following steps:

[0104] Step 1. Pretreatment of Serum Samples

[0105] The sample pretreatment process is carried out on ice. The serum sample is aliquoted into 30 - 50 μL per tube and placed in a 1.5 mL centrifuge tube. Then 250 - 500 μL of pretreatment extraction reagent is added. In this example, the pretreatment extraction reagent combination is: methanol, methyl tert-butyl ether, and ultrapure water. Subsequently, it is vortexed for 10 - 15 s, mixed evenly, shaken for 10 - 30 min, and centrifuged at high speed for 10 min at 4 - 6 °C. After two-phase separation, the upper hydrophobic phase is quantitatively collected and vacuum dried in a vacuum drying device to obtain a freeze-dried sample.

[0106] Step 2. Solid Phase Extraction and Enrichment

[0107] Place the 96-well sample preparation plate above the negative pressure device, place the receiving tube at the corresponding hole position of the 96-well sample preparation plate below the negative pressure device, turn on the pressure switch knob to make the pressure stable at about 3 - 6 kPa, and keep it stable for 5 - 10 min; dilute and redissolve the lipid freeze-dried product with the reconstitution solution, vortex and mix evenly, then transfer 300 μL to the 96-well sample preparation plate. Keep the pressure stable and make the lipid reconstitution solution flow slowly and evenly into the corresponding receiving tube below. Ensure that the whole process is completed within 5 - 10 min. Then turn off the pressure switch, quantitatively transfer 100 μL into a 0.2 ml centrifuge tube to obtain the enrichment solution. Whether to perform secondary freeze-drying and concentration of the enrichment solution is selected according to the sample concentration.

[0108] Spot the enrichment solution in Step 2 on the micro-nano mass spectrometry chip, with a single-hole volume of 0.5 - 2 μL. The sample is dried at room temperature. After drying, the chip is sent into a MALDI-TOF mass spectrometer for detection. The experimental process is as Figure 3 shown.

[0109] Inject the enrichment solution in Step 2 into a high-resolution liquid chromatography-mass spectrometry (LC-MS) at 5 - 8 μL per injection for detection.

[0110] Step 3. Data Acquisition

[0111] Mass spectrometry acquisition was completed by a MALDI-TOF mass spectrometer and an LC-Orbitrap mass spectrometer. The measurement mode was the negative ion mode, and the molecular weight acquisition range of the enrichment solution was 700 - 1500 m / z. When detecting each sample point, the average signal of 800 - 1000 laser pulses was used as the detection result of the reflection mode of this sample point. The detection results of the enrichment solution are as Figure 6 shown.

[0112] Step 4. Spectral analysis

[0113] The results of spectral analysis are as Figure 6 shown. It can be seen from the results that when testing serum glucosinolate at the same concentration on different instruments, the MALDI instrument can produce good peaks, but the peaks on the LC-Orbitrap are weak and the signal is poor. At the same time, compared with MALDI, the LC-Orbitrap has a higher requirement for the sample concentration, indicating that glucosinolate has higher sensitivity and better selectivity on the MALDI-TOF mass spectrometer.

[0114] Example 4. Selection of mass spectrometry methods for targeted extraction and high-coverage detection of glucosinolate

[0115] This example mainly includes the following steps:

[0116] Step 1. Pretreatment of serum samples

[0117] The sample pretreatment process was carried out on ice. The serum samples were aliquoted into 30 - 50 μL per tube and placed in 1.5 mL centrifuge tubes. 250 - 500 μL of pretreatment extraction reagent was added. In this example, the pretreatment extraction reagent combination was: methanol, methyl tert-butyl ether, and ultrapure water. Subsequently, it was vortexed for 10 - 15 s, mixed evenly, oscillated for 10 - 30 min, and centrifuged at high speed at 4 - 6 °C for 10 min. After two-phase separation, the upper hydrophobic phase was quantitatively collected and vacuum dried in a vacuum drying device to obtain a freeze-dried sample.

[0118] Step 2. Solid-phase extraction enrichment

[0119] Place the 96-well sample preparation plate above the negative pressure device, place the receiving tube at the corresponding hole position of the 96-well sample preparation plate below the negative pressure device, turn on the pressure switch knob to make the pressure stable at about 3 - 6 kPa, and keep it stable for 5 - 10 min; dilute and re-dissolve the lipid freeze-dried product with a reconstitution solution, vortex and mix evenly, then transfer 300 μL to the 96-well sample preparation plate, keep the pressure stable, and make the lipid reconstitution solution flow slowly and evenly into the corresponding receiving tube below. The whole process should be completed within 5 - 10 min. Then turn off the pressure switch, quantitatively transfer 100 μL into a 0.2 ml centrifuge tube to obtain an enrichment solution, and whether to perform secondary freeze-drying and concentration of the enrichment solution is selected according to the sample concentration.

[0120] The reconstituted solution and enriched solution after pretreatment and freeze-drying in step 2 are spotted on the micro-nano mass spectrometry chip, with a single-hole volume of 0.5-2μL. The sample is dried at room temperature. After drying, the chip is sent to the MALDI-TOF mass spectrometer for detection. The experimental process is as follows Figure 3 shown.

[0121] Step 3. Data Collection

[0122] The mass spectrum acquisition was completed by a MALDI-TOF mass spectrometer, and the measurement mode was the reflectron negative ion mode. The molecular weight acquisition range of the enriched solution was 700-1500m / z, and the molecular weight acquisition range of the reconstituted solution was 400-1000m / z. When each sample point was detected, the average signal of 800-1000 laser pulses was used as the reflectron mode detection result of the sample point. The detection results of the enriched solution are shown in Figure 7 shown.

[0123] Step 4. Spectral analysis

[0124] The spectrum analysis results are as follows Figure 7 As shown in the results, it can be seen that in the low energy value range, the overall resolution of the sulfatide spectrum is good, the peak is good, and the response can reach 10 4 Above; on the basis of low energy value, increasing the energy value, the resolution of sulfatide esters is improved, and the response can reach 5×10 4 However, as the energy value continues to increase, the resolution of sulfatide esters decreases, and the response also decreases. This shows that the higher the laser energy value, the better, and it should be kept within a suitable range. The optimal laser energy range is 0.7-1.5μJ.

[0125] Example 5. Targeted extraction and high coverage detection of serum sulfatides

[0126] This embodiment mainly includes the following steps:

[0127] Step 1. Serum sample pretreatment

[0128] The sample pretreatment process was performed on ice. The serum sample was divided into 30-50 μL per tube, placed in a 1.5 mL centrifuge tube, and 250-500 μL of pretreatment extraction reagent was added. In this embodiment, the pretreatment extraction reagent combination was: methanol, methyl tert-butyl ether, and ultrapure water. Then, the mixture was vortexed for 10-15 seconds, mixed, shaken for 10-30 minutes, and centrifuged at high speed for 10 minutes at 4-6°C. After the two phases were separated, the upper hydrophobic phase was quantitatively collected and vacuum dried in a vacuum drying device to obtain a freeze-dried sample.

[0129] Step 2. Solid phase extraction enrichment

[0130] Place the 96-well sample preparation plate above the negative pressure device, place the receiving tubes at the corresponding hole positions of the 96-well sample preparation plate below the negative pressure device, turn on the pressure switch knob, and make the pressure stable at about 3-6 kPa for 5-10 minutes; dilute and reconstitute the lipid lyophilized product with the reconstitution solution, vortex and mix well, then pipette 300 μL into the 96-well sample preparation plate. Keep the pressure stable and let the lipid reconstitution solution flow slowly and evenly into the corresponding receiving tubes below. Ensure that the whole process is completed within 5-10 minutes. Then turn off the pressure switch, quantitatively pipette 100 μL into a 0.2 ml centrifuge tube to obtain the enriched solution. Whether to perform secondary lyophilization and concentration on the enriched solution depends on the sample concentration.

[0131] Spot the reconstitution solution and the enriched solution after pretreatment and lyophilization and reconstitution in step 2 on the micro-nano mass spectrometry chip, with a single-hole volume of 0.5-2 μL. Dry the samples at room temperature. After drying, send the chip into the MALDI-TOF mass spectrometer for detection. The experimental process is as Figure 3 shown.

[0132] Step 3. Data acquisition

[0133] Mass spectrometry acquisition is completed by the MALDI-TOF mass spectrometer. The measurement mode is the reflectron negative ion mode. The molecular weight acquisition range of the enriched solution is 700--1500 m / z, and the molecular weight acquisition range of the reconstitution solution is 400--1000 m / z. When detecting each sample point, use the average signal of 800-1000 laser pulses as the detection result of the reflectron mode for that sample point. The detection results of the reconstitution solution and the enriched solution are as Figure 8 shown.

[0134] Step 4. Spectral analysis

[0135] Perform MS primary matching and MS / MS identification on the glucosylceramide molecules according to the spectra, including SHexCer without hydroxylation and SHexCer(OH) with one or more hydroxylation modifications; including both SHexCer with one cyclohexanol in the molecular structure and SHex2Cer with two cyclohexanols. The glucosylceramides in the enriched solution can cover 61 molecules (S / N>3) and are integrated into the glucosylceramide lipid spectrum database. The spectral analysis results are as Figure 8 shown. The CV stability between different batches is shown in Table 1, and the comparison of the glucosylceramide molecular coverage rates of the reconstitution solution and the enriched solution is shown in Table 2.

[0136] Figure 8 Both Table 1 and Table 2 show that compared with the conventional serum lipid pretreatment process, which can only cover 16 glucosylceramide molecules, the method provided by the present invention can significantly improve the coverage rate of glucosylceramide molecules, increasing the number of covered molecular species of glucosylceramides to 61. When considering different types of samples, the total number of glucosylceramides that can be covered is as high as 124, which belongs to the highest level reported or disclosed so far.

[0137] Example 6. Targeted Extraction and High-Coverage Detection of Sulfatides in Tissues and Cells

[0138] This example mainly includes the following steps:

[0139] Step 1. Pretreatment of tissue samples

[0140] The sample pretreatment process is carried out on ice. Weigh about 15 - 30 mg of the tissue sample, cut it into small pieces, put it into a 1.5 mL grinding tube, and place it in a 1.5 mL centrifuge tube; successively add 300 - 1000 μL of extraction solution. In this example, the pretreatment extraction reagent combination is: methanol, methyl tert-butyl ether, and water. After grinding 4 - 6 times, shake for 10 - 30 min, and centrifuge at high speed for 10 min at 4 - 6 °C. After two-phase separation, quantitatively collect the upper hydrophobic phase and vacuum dry it in a vacuum drying device to obtain a freeze-dried sample.

[0141] Step 2. Pretreatment of cell samples

[0142] The sample pretreatment process is carried out on ice. Place 10 6 -10 7 renal cell samples in a 1.5 mL centrifuge tube. After repeated freezing and thawing, add 250 - 500 μL of pretreatment extraction reagent. In this example, the pretreatment extraction reagent combination is: methanol, methyl-tert-butyl ether, and ultrapure water. Then vortex for 10 - 15 s, mix well, shake for 10 - 30 min, and centrifuge at high speed for 10 min at 4 - 6 °C. After two-phase separation, quantitatively collect the upper hydrophobic phase and vacuum dry it in a vacuum drying device to obtain a freeze-dried sample.

[0143] Step 3. Solid-phase extraction enrichment

[0144] Place a 96-well sample preparation plate above the negative pressure device, place a receiving tube at the corresponding hole position of the 96-well sample preparation plate below the negative pressure device, turn on the pressure switch knob to make the pressure stable at about 3 - 6 kPa, and keep it stable for 5 - 10 min; dissolve the lipid freeze-dried product with isopropanol, vortex and mix well, then transfer 300 μL to the 96-well sample preparation plate. Keep the pressure stable and make the lipid re-solution flow slowly and evenly into the corresponding receiving tube below. Ensure that the whole process is completed within 5 - 10 min, and then turn off the pressure switch to obtain the enriched solution.

[0145] Spot the enriched solution on a micro-nano mass spectrometry chip, with a single-hole volume of 0.5 - 2 μL. Dry the sample at room temperature. After drying, send the chip into a MALDI-TOF mass spectrometer for detection.

[0146] Step 3. Data acquisition

[0147] Mass spectrometry was performed by a MALDI-TOF mass spectrometer in the reflectron negative ion mode with a molecular weight range of 700-1500 m / z. When each sample point was detected, the average signal of 800-1000 laser pulses was used as the reflectron mode detection result of the sample point.

[0148] Step 4. Spectral analysis

[0149] According to the spectra, the sulfatide molecules were identified by MS / MS, including SHexCer without hydroxylation and SHexCer (OH) with one or more hydroxylation modifications; including SHexCer with one cyclohexanol in the molecular structure, SHex2Cer with two cyclohexans, and SO3-LcCer with amide sugar in the molecular structure. The identification results of different tissues and cells and the sulfatide information table were integrated into the sulfatide lipid spectrum database. Sulfatide peaks were found in different samples and the number was higher than the number of sulfatide molecules included in HMDB and LIPIDMAPS, indicating that this method is universal. The results of the number of sulfatide molecules in different types of samples are shown in the figure. Figure 9 shown.

[0150] Example 7 Establishment of Sulfatide Database

[0151] Step 1. Enumerate the sulfatide molecules according to their types, different carbon chain lengths, hydroxylation numbers, and hexose numbers, calculate the theoretical molecular weight in combination with their additive form, and form a candidate sulfatide table, with the maximum m / z value set to 2000;

[0152] Step 2. Perform mass spectrometry on samples such as serum, cells, and tissues according to the method described in Examples 1-6, and perform primary peak matching on the spectrum of the MS primary acquisition mode with the enumerated candidate sulfatide list, with a matching deviation of <20ppm, and remove isotope peaks. For peaks that cannot be matched with the enumerated candidate sulfatide list, the candidate sulfatide list is supplemented by calculating the molecular weight difference between different peaks;

[0153] Step 3. Analyze the spectrum of the MS / MS secondary acquisition mode to supplement the MS / MS fragmentation information of each molecule in the sulfatide table;

[0154] Step 4. Based on the above method, the detected sulfatide peak is structurally confirmed, and the molecular type, molecular formula, chain information, accurate molecular weight, detected molecular weight, detection error, secondary fragments, existing numbers in the public database, and self-built database numbers are formed into a sulfatide data table information and implanted into the database to obtain at least 124 sulfatide molecules. The number of each type of sulfatide is shown in Table 3, and the collected sulfatide molecular information is shown in Table 4.

[0155] Example 8. Collection and Discrimination of Sulfoglycolipidomes of Hepatocellular Carcinoma Patients and Healthy Populations Based on a Sulfoglycolipid Spectrum Database

[0156] This example mainly includes the following steps:

[0157] Step 1. Pretreatment of serum samples

[0158] The sample pretreatment process is carried out on ice. The serum samples are aliquoted into tubes of 30 - 50 μL each and placed in 1.5 mL centrifuge tubes. Then, 250 - 500 μL of pretreatment extraction reagent is added. In this example, the pretreatment extraction reagent combination is: methanol, methyl tert-butyl ether, and ultrapure water. Subsequently, it is vortexed for 10 - 15 s, mixed evenly, oscillated for 10 - 30 min, and centrifuged at high speed for 10 min at 4 - 6 °C. After two-phase separation, the upper hydrophobic phase is quantitatively collected and vacuum-dried in a vacuum drying device to obtain a freeze-dried sample.

[0159] Step 2. Solid-phase extraction and enrichment

[0160] Place the 96-well sample preparation plate above the negative pressure device, place the receiving tubes at the corresponding well positions of the 96-well sample preparation plate below the negative pressure device, turn on the pressure switch knob to make the pressure stable at about 3 - 6 kPa, and keep it stable for 5 - 10 min; dilute the lipid freeze-dried product 3 - 6 times with isopropanol for reconstitution, vortex and mix evenly, then transfer 300 μL to the 96-well sample preparation plate. Keep the pressure stable and make the lipid reconstitution solution flow slowly and evenly into the corresponding receiving tubes below. Ensure that the whole process is completed within 5 - 10 min. Then, turn off the pressure switch, quantitatively transfer 100 μL into a 0.2 ml centrifuge tube, dry it in a vacuum drying dish, and store the obtained freeze-dried sample at -80 °C.

[0161] Resuspend the sample with 10 - 20 μL of isopropanol, seal it with an aluminum sealing film, and vortex for 10 - 15 s to mix evenly. Spot the sample on the micro-nano mass spectrometry chip at 0.5 - 2 μL / well, dry it at room temperature for 10 - 15 min, and then transfer it to a vacuum dryer for drying for 1 min. Fix the dried chip on a customized MALDI target holder, and send the chip into a MALDI-TOF mass spectrometer for detection. The experimental process is as Figure 3 shown.

[0162] Step 3. Data collection

[0163] Mass spectrometry collection is completed by a MALDI-TOF MS mass spectrometer. The measurement mode is the reflectron negative ion mode, and the molecular weight collection range of the enrichment solution is 700 - 1500 m / z. When detecting each sample point, the average signal of 800 - 1000 laser pulses is used as the detection result of the reflectron mode for this sample point. The detection results of the enrichment solution are as Figure 10 shown.

[0164] Step 4. Spectral analysis

[0165] The results of spectral analysis are as Figure 10 shown. Import the spectrum into the self-built database for matching of sulfatide molecules. Distinguish between healthy people and liver cancer patients according to the list of matching annotation results. This method has a good distinguishing effect on the sulfatide groups of healthy people and liver cancer patients and has good discriminant ability for whether a person has liver cancer, as Figure 11 shown.

[0166] Table 1 Intra-batch and inter-batch CV stability results

[0167] The first batch The second batch Between batches CV value 6.73 8.38 8.78

[0168] Table 2 Comparison table of sulfatide molecule coverage rates of the reconstituted solution and the enrichment solution

[0169]

[0170] Table 3 Summary table of the types of sulfatide molecules included in the sulfatide lipid spectrum database

[0171] Class Number SHexCer 29 SHexCer(OH)x 40 SHex2Cer 26 SHex2Cer(OH)x 18 SO3-Lc3Cer 11 Total 124

[0172] Table 4 Types of sulfatide molecules included in the sulfatide mass spectrum database

[0173]

[0174]

[0175]

[0176]

[0177] The above are only the preferred embodiments of the present invention. It should be noted that for those of ordinary skill in the art, without departing from the principle of the present invention, several improvements and refinements can still be made, and these improvements and refinements should also be regarded as the protection scope of the present invention.

Claims

1. A targeted sulfatide lipid profile database, characterized in that: Includes the following sulfatide molecular information: SHexCer without hydroxylation, SHexCer with one or more hydroxylation modifications (OH) n , SHexCer with one cyclohexanol in the molecular structure, SHex with two or more cyclohexanols 2-n Cer and SO3-Lc containing amide sugar in its molecular structure n Cer; the sphingosine chain of the sulfatide is d18:0 or d18:1; the other chain is a fatty acid chain, the number of carbon atoms is 12-30, the unsaturation degree of the fatty acid chain is 0 or more; the number of hydroxyl groups is 0 or more; the number of cyclohexose is at least 1.

2. A method for establishing a targeted sulfatide lipid profile database, characterized in that: The steps include: S1) extracting the biological sample to be tested with a solvent to obtain a test solution; The solvent includes a first component, a second component, and a third component; The first component includes: one or more of methanol, butanol, and isopropanol; the second component includes: one or more of methyl tert-butyl ether, ethyl acetate, chloroform, dichloromethane, n-heptane, and n-hexane; the third component includes: ultrapure water; S2) After solid phase extraction, the test liquid is spotted on a micro-nano mass spectrometry chip, and detected by a mass spectrometer using MALDI as an ion source, using lipidomics analysis technology, using MS primary acquisition mode to obtain a primary spectrum, and using MS / MS secondary acquisition mode to fragment the obtained parent ion peak to obtain a secondary spectrum; S3) obtaining sulfatide data table information and establishing a sulfatide database.

3. The establishment method according to claim 2, characterized in that: The ratio of the first component: the second component: the third component is 1:2:1 to 1:4:

2.

4. The establishment method according to claim 2, characterized in that: The ratio of the solvent to the sample to be tested is 2.5:1 to 10:

1.

5. The establishment method according to claim 2, characterized in that: The sample to be tested includes one or more of serum, plasma, urine, tissue, cerebrospinal fluid, exosomes, and cells.

6. The establishment method according to claim 2, characterized in that: Mass spectrometers using MALDI as the ion source include: MALDI-TOF, MALDI-TOF / TOF, MALDI2-timsTOF / TOF, MALDI-FT-ICR, and AP-MALDI-Orbitrap mass spectrometers.

7. The establishment method according to claim 2, characterized in that: The mass spectrometer is in negative ion mode, and the molecular weight range is 400-2000 m / z.

8. The establishment method according to claim 2, characterized in that: The MS primary acquisition mode and the MS / MS secondary acquisition mode are used to fragment the obtained parent ion peak, and perform primary peak matching and secondary characteristic peak fragment identification.

9. The establishment method according to claim 2, characterized in that: When each sample point is detected, the average signal is used as the reflection mode detection result of the sample point.

10. The establishment method according to claim 2, characterized in that: The step S1) further comprises solid phase extraction enrichment, wherein the pressure of the solid phase extraction is 3-6 kPa and the time is 5-10 min.

11. The establishment method according to claim 2, characterized in that: The specific methods for establishing a sulfatide database include: S1) Enumerate the sulfatide molecules with different carbon chain types and one or more hydroxylation modifications, calculate the theoretical molecular weight based on their adduct forms, and form a candidate sulfatide table: sulfatides include SHexCer without hydroxylation, SHexCer with one or more hydroxylation modifications (OH) n , SHexCer with one cyclohexanol in the molecular structure, SHex with two or more cyclohexanols 2-n Cer and SO3-Lc containing amide sugar in its molecular structure n Cer; the sphingosine chain of the sulfatide is d18:0 or d18:1; the other chain is a fatty acid chain, the number of carbon atoms is 12-30, the unsaturation degree of the fatty acid chain is 0 or more; the number of hydroxyl groups is 0 or more; the number of cyclohexose is at least 1; S2) performing primary peak matching of the spectrum in the MS primary acquisition mode with the enumerated candidate sulfatide list, with a matching deviation of <20 ppm, eliminating isotope peaks, and for peaks that cannot be matched with the enumerated candidate sulfatide list, supplementing the candidate sulfatide list by calculating the molecular weight difference between different peaks; S3) analyzing the spectrum of MS / MS secondary acquisition mode; S4) Based on the above method, the detected sulfatide peak is structurally confirmed, and the molecular type, molecular formula, chain information, accurate molecular weight, detected molecular weight, detection error, secondary fragments, existing numbers in the public database, and numbers in the self-built database are formed into a sulfatide data table information and implanted into the database to obtain at least 124 sulfatide molecules.

12. An application of a targeted glucosinolate lipid spectrum database, characterized in that: The steps include: S1) extracting the biological sample to be tested with a solvent to obtain a test solution; The solvent includes a first component, a second component, and a third component; The first component includes: one or more of methanol, butanol, and isopropanol; the second component includes: one or more of methyl tert-butyl ether, ethyl acetate, chloroform, dichloromethane, n-heptane, and n-hexane; the third component includes: ultrapure water; S2) subjecting the test solution to solid phase extraction, and then detecting it with a mass spectrometer, using lipidomics analysis technology and a primary acquisition mode to obtain a primary spectrum; S3) importing the spectrum into a sulfatide lipid spectrum database, setting the allowable detection error, performing sulfatide molecular matching, and obtaining a sulfatide annotation result list of the tested sample, wherein the list information includes: molecular type, molecular formula, chain information, accurate molecular weight, detected molecular weight, detection error, secondary fragments, existing numbers in the public database, numbers in the self-built database, and mass spectrometry response values ​​of each sample; S4) Analyze the annotation result list for inter-group difference analysis, sulfatide biomarker mining, and machine learning algorithm model construction to realize data application based on sulfatide lipid spectrum database.

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