Targeted thioglycoside detection kit and detection method and application thereof

Abstract

The invention provides a detection kit of targeted thioglycoside, a detection method of the detection kit and application of the detection kit. The kit comprises a pretreatment extraction reagent, a reconstitution fluid and a consumable combination, the pretreatment extraction reagent comprises a first component, a second component and a third component; wherein the first component is selected from one or more of methanol, butanol and isopropanol; the second component is selected from one or more of methyl tertiary butyl ether, ethyl acetate, chloroform, dichloromethane, n-heptane and n-hexane; and the third component is ultrapure water. According to the kit, a solid-phase extraction technology is combined with the micro-nano mass spectrum chip, matrix spraying is not needed, operation is easy and convenient, and mass spectrum signals of thioglycoside groups can be efficiently obtained in batches. The detection method has the characteristics of wide coverage range and high throughput, a plurality of samples can be processed at the same time in an automatic detection mode, thioglycoside group information can be rapidly obtained, and an obtained thioglycoside spectrogram has excellent stability.

Description

Technical Field

[0001] The present invention relates to the technical field of mass spectrometry analysis, and in particular to a detection kit for targeting sulfatide. Background Art

[0002] Sulfatide (abbreviated as sulfatide, SHexCer) is a type of 3-O-sulfogalactosylceramide synthesized from sphingolipids by sphingolipid galactosyltransferase and cerebroside sulfotransferase. It is a special acidic sphingolipid, mainly produced in the myelin sheath, and is an important component of the myelin sheath, accounting for about 4% of the total myelin. It is also distributed in the nervous system, kidneys, liver, spleen, stomach, small intestine and serum. SHexCer 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 expression changes of SHexCer may cause various diseases, especially metachromatic leukodystrophy (MLD) and other lysosomal storage abnormalities genetic diseases, neurological diseases such as Alzheimer's disease, kidney damage-related diseases, cardiovascular and cerebrovascular diseases such as myocardial infarction, cancer, etc. For example, MLD patients lack the lysosomal sulfatase ASA that can hydrolyze the 3-O ester bond of cerebrosides, which causes SHexCer to accumulate in the lysosomes in the body, thereby damaging the central and peripheral nervous systems. At this time, significantly increased SHexCer can be detected mainly in the patient's urine samples, and the diagnostic efficiency of MLD disease can be improved by using a logistic regression equation for multiple SHexCer expressions (Clinica Chimica Acta 433 (2014) 39-43). When SHexCer is missing in the myelin sheath, it will trigger an inflammatory response and abnormal lipid metabolism in glial cells, leading to Alzheimer's disease with abnormal myelin function. The patient's bladder and other functions will also change. The SHexCer content in cerebrospinal fluid and whether it is hydroxylated and the hydroxylation ratio can be used as one of the key biomarkers of Alzheimer's disease (Molecular Neurodegeneration, (2021) 16: 64); SHexCer combination can also be used as an indicator for risk assessment of cardiovascular disease in patients with end-stage renal failure. Whether patients with renal failure suffer from cardiovascular disease will show different total amounts of sulfatides and distribution of sulfatides of 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), in which sulfatides also play a role. Therefore, the study of the content or distribution of SHexCer in the human body has important physiological value.

[0003] Compared with several major lipid categories such as glycerides, sterols, and glycerophospholipids, sphingolipids belong to the category of lipids with lower content in the body. SHexCer is also a low-abundance sphingolipid among sphingolipids (compared with sphingomyelin SM). Taking the high content of phosphatidylcholine PC in plasma as an example, the highest concentration of PC is close to 1mmol / L, while the content of all SHexCer is less than 1nmol / L, a difference of six orders of magnitude. The extraction, enrichment, and detection of SHexCer in complex biological samples such as plasma, serum, and cerebrospinal fluid have high requirements for pretreatment methods and highly sensitive detection methods.

[0004] The ELISA kits available on the market for measuring SHexCer content can only test the overall content of this type of molecule, but cannot test its distribution separately. Sulfatides include both SHexCer without hydroxylation and SHexCer (OH) with one or more hydroxylation modifications; both SHexCer with one cyclohexanol in the molecular structure and SHexCer with two cyclohexanols. 2 Cer, and each skeleton also has multiple carbon chain structures, 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 high-resolution mass spectrometers can achieve high-precision qualitative and quantitative detection of a large class of sulfatides with similar skeleton structures but local differences, and carry out sulfatide-omics research.

[0005] The mass spectrometry platforms for the detection of sulfatide substances reported in the literature include LC-MS and MALDI-TOF-MS systems. LC-MS is a common mass spectrometry platform for lipidomics research, but due to the low abundance of sulfated sphingolipids in samples such as serum or tissues, in-source cleavage in LC-MS, low ionization efficiency, and strong adsorption of sulfonic acid groups on chromatographic columns, the lipid response sensitivity of sulfatide is usually low in non-targeted lipidomics testing. In this regard, Patent 201910424563.6 (grant number CN 110068638 B) discloses a TiO-based mass spectrometry system. 2The separation and enrichment method of acidic glycosphingolipids by column separation technology can remove the interference of phospholipids, and at the same time enrich acidic sphingolipids such as sulfatides to improve their coverage in LC-MS detection. However, despite this, there are only 19 types of SHexCer in serum after enrichment. The number of sulfatides in serum tested based on LC-MS reported in other literature does not exceed 20. MALDI-TOF-MS is usually subject to ion suppression of high-abundance phospholipids such as PC, and complex sample pretreatment methods can match the test of sulfatide molecules in complex samples on MALDI. Patent 202210511862.5 (grant number CN 114858908 A) discloses a method for purifying sulfatides using steps such as NaOH saponification and SPE column, which can convert sulfatides into hydrogenated sulfatides, and is also the most common pretreatment method based on the MALDI platform. However, due to the complex process, the uneven crystallization of conventional organic matrices (such as CHCA, DHB, 9-AA, etc.), and the high ionization threshold, it is impossible to obtain the fingerprint spectrum of the sulfatide group with high stability and high coverage. The coverage is also less than 20 species, which affects the practical application of the sulfatide group. Summary of the invention

[0006] In view of this, the technical problem to be solved by the present invention is to provide a detection kit for targeted sulfatides. The kit provided by the present invention can accurately measure targeted sulfatides.

[0007] The invention provides a detection kit for targeting sulfatide, comprising: a pretreatment extraction reagent, a re-solution solution and a consumables combination; the pretreatment extraction reagent comprises a first component, a second component and a third component; wherein the first component is selected from: one or more of methanol, butanol and isopropanol; the second component is selected from: one or more of methyl tert-butyl ether, ethyl acetate, chloroform, dichloromethane, n-heptane and n-hexane; and the third component is: ultrapure water.

[0008] The detection kit provided by the present invention comprises: a pretreatment extraction reagent. The pretreatment extraction reagent comprises a first component, a second component and a third component; wherein the first component is selected from: one or more of methanol, butanol and isopropanol; the second component is selected from: one or more of methyl tert-butyl ether, ethyl acetate, chloroform, dichloromethane, n-heptane and n-hexane; and the third component is: ultrapure water.

[0009] Specifically, the ratio of the first component: the second component: the third component is 1:2:1 to 1:4:2.

[0010] 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.

[0011] 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.

[0012] In the preferred embodiment of the present invention, the extractant combination of methanol, methyl-tert-butyl ether and ultrapure water has the best effect; the volume ratio thereof is 1:3:1.

[0013] The detection kit provided by the present invention comprises: a reconstitution solution; the reconstitution solution of the present invention comprises one or more of isopropanol, acetonitrile and methanol.

[0014] In some embodiments, the complex solution includes isopropanol, acetonitrile, methanol, and a mixture of isopropanol / acetonitrile and isopropanol / methanol; the volume ratio of the above mixture is 1:3 to 3:1.

[0015] The detection kit provided by the present invention comprises a consumables combination; the consumables combination comprises a 96-well sample preparation plate, a negative pressure device, a micro-nano mass spectrometry chip, a receiving plate, a sealing plate mold, a centrifuge tube, a pipette, a gun tip, and a stainless steel aluminum plate.

[0016] 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 of the 96-well sample preparation plate into the receiving plate under vacuum negative pressure (see patent: 201710137623.7); the micro-nano mass spectrometry chip has matrix-free desorption ionization effect (see patent: 201811403743.8).

[0017] The present invention also provides a detection method of a detection kit targeting sulfatide, comprising:

[0018] A) Preparation of test solution:

[0019] The sample to be tested is added with a pretreatment extraction reagent, extracted, and then vacuum dried and re-dissolved with a re-solution solution to obtain a test solution;

[0020] B) Mass spectrometry detection:

[0021] After the liquid to be tested is subjected to solid phase extraction, the sample is spotted on a micro-nano mass spectrometry chip, and then detected by a MALDI-TOF mass spectrometer. During the detection process, lipidomics analysis technology is used to first obtain a primary spectrum through the MS primary acquisition mode, and then the obtained parent ion peak is fragmented using the MS / MS secondary acquisition mode to obtain a secondary spectrum;

[0022] C) Qualitative and semi-quantitative analysis:

[0023] The database was used to perform qualitative and semi-quantitative analysis of sulfatide spectra.

[0024] The detection method of the detection kit for targeting sulfatide provided by the present invention firstly comprises the preparation of a test solution.

[0025] The sample to be tested is added to a pretreatment extraction reagent for extraction. The sample to be tested in the present invention includes serum, plasma, urine, tissue, cerebrospinal fluid, exosomes, and cells.

[0026] 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, heptane, and hexane; and the third component includes: ultrapure water.

[0027] Specifically, the ratio of the first component: the second component: the third component is 1:2:1 to 1:4:2.

[0028] 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.

[0029] 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.

[0030] In the preferred embodiment of the present invention, the extractant combination of methanol, methyl-tert-butyl ether and ultrapure water has the best effect; the volume ratio thereof is 1:3:1.

[0031] The extracted samples are subjected to low temperature vacuum drying, specifically including:

[0032] Place the 96-well sample preparation plate on top of the negative pressure device, place the receiving tube at the corresponding well position of the 96-well sample preparation plate, turn on the pressure switch knob, and stabilize the pressure to 3-6kPa;

[0033] The lipid freeze-dried product is reconstituted and transferred to a 96-well sample preparation plate, so that it flows to a receiving tube under negative pressure. The volume of the reconstituted solution is 50-500 μL;

[0034] A certain volume of liquid in the receiving tube is removed and then vacuum dried, wherein the certain volume range is: 25-250 μL;

[0035] The freeze-dried lipid in the receiving tube is reconstituted with a reconstitution solution, then spotted on a micro-nano mass spectrometry chip, and detected in negative ion mode on a MALDI-TOF mass spectrometer after natural drying. The ratio of the reconstitution solution to the pretreated liquid of the present invention is 1:1 to 10:1; specifically, it can be 1:1, 2:1, 3:1, 4:1, 5:1, 6:1, 7:1, 8:1, 9:1 or 10:1.

[0036] 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;

[0037] The enriched liquid in the receiving tube was transferred, and the sample was spotted on the micro-nano mass spectrometry chip. After natural drying, negative ion mode detection was performed on the mass spectrometer.

[0038] The experimental results show that the present invention has a high peak response of sulfatide esters on the micro-nano mass spectrometry chip, and the micro-nano mass spectrometry chip involved in the present invention has the advantage of improving the selectivity and sensitivity of sulfatide molecules.

[0039] In some other embodiments of the present invention, the method further comprises: transferring the test sample into a centrifuge tube, sequentially adding a pretreatment extraction reagent, and extracting lipid metabolites, wherein the sample types include serum, plasma, urine, tissue, cerebrospinal fluid, exosomes, and 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 the 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, and stabilizing the pressure to 3 to 6 kPa; re-dissolving the lipid freeze-dried product, transferring it to the 96-well sample preparation plate, and allowing it to flow to the receiving tube under negative pressure, wherein the volume of the re-dissolving solution is 50-500 μL. Transferring the enriched solution in the receiving tube, then spotting it on the micro-nano mass spectrometry chip, and performing negative ion mode detection on the mass spectrometer after natural drying.

[0040] In some other embodiments of the present invention, the method further comprises: transferring the test sample into a centrifuge tube, sequentially adding pretreatment extraction reagents to extract lipid metabolites, wherein the sample types include serum, plasma, urine, tissue, cerebrospinal fluid, exosomes, and 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; placing a 96-well sample preparation plate above the negative pressure device, placing a receiving tube below the corresponding hole of the 96-well sample preparation plate, turning on the pressure switch knob, and stabilizing the pressure to 3 to 6 kPa; re-dissolving the lyophilized lipid product, transferring it to the 96-well sample preparation plate, and allowing it to flow to the receiving tube under negative pressure, wherein the volume of the re-dissolving solution is 50-500 μL. A certain volume of liquid in the receiving tube is removed and then vacuum dried, wherein the certain volume range is: 25-400 μL; the freeze-dried lipid in the receiving tube is re-dissolved with a re-solution solution, and then spotted on a micro-nano mass spectrometry chip, and after natural drying, negative ion mode detection is performed on a mass spectrometer.

[0041] In the detection process, lipidomics analysis technology was used to first obtain the primary spectrum through the MS primary acquisition mode, and then the obtained parent ion peak was fragmented using the MS / MS secondary acquisition mode to obtain the secondary spectrum;

[0042] The MS primary acquisition mode and the MS / MS secondary acquisition mode are used to fragment the obtained parent ion peak, including primary peak matching and secondary characteristic peak fragment identification.

[0043] The identification results were combined with the response value information of different samples for subsequent statistical analysis.

[0044] The present invention provides the use of the detection kit described in the above technical solution in the preparation of a product for diagnosing liver cancer. The above can detect 74 kinds of sulfatide molecules.

[0045] The kit of the present invention has a good effect of distinguishing the sulfatide groups of healthy people and liver cancer patients, and has a good ability to distinguish whether or not a person suffers from liver cancer.

[0046] The present invention provides a detection kit for targeted sulfatide. The pretreatment and detection method of the targeted sulfatide group of a biological sample adopts a combination of solid phase extraction and a micro-nano mass spectrometry chip, and the mass spectrometry signals of the sulfatide group are obtained in batches without matrix spraying. The kit has a wide coverage, a high throughput, and a fast detection speed. The sulfatide group information of multiple samples can be obtained at one time in an automated detection mode. The obtained sulfatide spectrum has strong stability, and the intra-batch and inter-batch CV is less than 10% without the addition of an internal standard. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] Figure 1 .Flow chart of sulfatide extraction method;

[0048] Figure 2 .Comparison of the spectrum of serum lipid reconstitution solution and enrichment solution;

[0049] Figure 3 .Comparison of sulfatide spectra between healthy and liver cancer groups;

[0050] Figure 4 OPLSDA analysis of sulfatide spectra of serum samples from healthy and liver cancer groups;

[0051] Figure 5 .Tissue sulfatide profiles. DETAILED DESCRIPTION

[0052] The present invention provides a detection kit for targeting sulfatide, and those skilled in the art can refer to the content of this article and appropriately improve the process parameters to achieve it. It should be particularly noted that all similar replacements and modifications are obvious to those skilled in the art, and they all belong to the scope of protection of the present invention. The method and application of the present invention have been described through preferred embodiments, and relevant personnel can obviously modify or appropriately change and combine the method and application of this article without departing from the content, spirit and scope of the present invention to realize and apply the technology of the present invention.

[0053] It should be understood that the expression "one or more of..." includes each of the items recited after the expression individually and in various different combinations of two or more of the recited items, unless otherwise understood from the context and usage. The expression "and / or" in combination with three or more recited items should be understood to have the same meaning, unless otherwise understood from the context.

[0054] The use of the terms "comprising", "having" or "containing", including 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.

[0055] In this application, the term "and / or" describes the association relationship of associated objects, indicating that there may be three relationships. For example, A and / or B can represent: A exists alone, A and B exist at the same time, and B exists alone. A and B can be singular or plural.

[0056] In this application, "at least one" means one or more, "more than one" means two or more. "At least one of the following" or similar expressions refers to any combination of these items, including any combination of single or plural items.

[0057] It should be understood that the order of steps or the order in which certain actions are performed is not important as long as the present invention remains operable. In addition, two or more steps or actions may be performed simultaneously.

[0058] The use of any and all examples or exemplary language, such as "for example" or "including", herein is intended only to better illustrate the invention and does not limit the scope of the invention unless otherwise claimed. No language in this specification should be construed as indicating that any non-claimed element is essential to the practice of the invention.

[0059] In addition, the numerical ranges and parameters used to define the present invention are approximate values, and the relevant values ​​in the specific embodiments have been presented as accurately as possible. However, any numerical value inherently inevitably contains standard deviations due to individual test methods. Therefore, unless otherwise expressly stated, it should be understood that all ranges, quantities, 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 value or range.

[0060] It should be understood that in the various embodiments of the present application, the size of the serial numbers of the above-mentioned processes does not mean the order of execution, some or all of the steps can be executed in parallel or sequentially, and the execution order of each process should be determined by its function and internal logic, and should not constitute any limitation on the implementation process of the embodiments of the present application.

[0061] Some cases are recorded in the embodiments and comparative examples of the present invention, wherein the embodiments show certain implementation modes of the present invention. However, this does not mean that the effects of the present invention can only be achieved in these cases.

[0062] In order to further illustrate the present invention, a detection kit for targeting sulfatide provided by the present invention is described in detail below in conjunction with examples.

[0063] Example 1 Targeted extraction and high coverage detection of serum sulfatides

[0064] This embodiment mainly includes the following steps:

[0065] Step 1. Serum sample pretreatment

[0066] 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 250-350 μL of pretreatment extraction reagent was added. In this embodiment, the pretreatment extraction reagent combination was: methanol, methyl-tert-butyl ether and ultrapure water, followed by vortexing for 10-15 seconds, mixing, oscillating for 10-30 minutes, and high-speed centrifugation 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.

[0067] Step 2. Solid phase extraction enrichment

[0068] 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 stabilize the pressure at about 3-6kPa, and stabilize it for 5-10 minutes; dilute and reconstitute the lipid lyophilized product with the reconstitution solution, vortex and mix, and transfer 400μL to the 96-well sample preparation plate, keep the pressure stable, and allow the lipid reconstitution solution to flow slowly and evenly to the corresponding receiving tube below. The whole process is guaranteed to be completed within 5-10 minutes, then turn off the pressure switch, quantitatively transfer 200μL and place it in a 0.2ml centrifuge tube to obtain the enriched solution. The enriched solution can be selected for secondary freeze-drying and concentration according to the sample concentration.

[0069] 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 1 shown.

[0070] Step 3. Data Collection

[0071] The mass spectrometry was completed by a MALDI-TOF mass spectrometer in the reflectron negative ion mode. The molecular weight acquisition range of the enriched solution was 700–1200 m / z, and the molecular weight acquisition range of the reconstituted solution was 400–1000 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. The detection results of the reconstituted solution and the enriched solution are shown in Figure 2. Figure 2 shown.

[0072] Step 4. Spectral analysis

[0073] According to the spectrum, 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 and SHex2Cer with two cyclohexanols. The sulfatide in the enriched solution can cover 61 molecules (SN>3), and the spectrum analysis results are shown in Table 1. The CV stability between different batches is shown in Table 2, and the comparison of the sulfatide molecule coverage of the reconstituted solution and the enriched solution is shown in Table 3.

[0074] Figure 2 Both Table 3 and Table 3 show that the method provided by the present invention can improve the coverage of sulfatide molecules compared with the conventional lipid pretreatment process.

[0075] Example 2. Pretreatment and detection method of targeted sulfatide group based on micro-nano mass spectrometry chip and MALDI-TOF mass spectrometer for the collection and discrimination of sulfatide group in liver cancer patients and healthy people

[0076] This embodiment mainly includes the following steps:

[0077] Step 1. Serum sample pretreatment

[0078] 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 250-350 μL of pretreatment extraction reagent was added. In this embodiment, the pretreatment extraction reagent combination was: methanol, dichloromethane, 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.

[0079] Step 2. Solid phase extraction enrichment

[0080] 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 stabilize the pressure at about 3-6kPa, and stabilize it for 5-10 minutes; dilute the lipid freeze-dried product 3-5 times with isopropanol to dissolve it, vortex and mix it, then transfer 300μL to the 96-well sample preparation plate, keep the pressure stable, and allow the lipid reconstitution solution to flow slowly and evenly to the corresponding receiving tube below. The whole process is guaranteed to be completed within 5-10 minutes, then turn off the pressure switch, quantitatively transfer 100μL and place it in a 0.2ml centrifuge tube, dry it in a vacuum drying dish, and store the obtained freeze-dried sample at -80℃.

[0081] Resuspend the sample with 10-20 μL of isopropanol, seal with aluminum plate sealer, vortex for 10-15 seconds, and mix. Spot the sample on the micro-nano mass spectrometry chip at 0.5-2 μL / well, dry at room temperature for 10-15 minutes, and then transfer to a vacuum dryer to dry for 1 minute. The dried chip is fixed on the customized MALDI target holder, and the chip is sent to the MALDI-TOF mass spectrometer for detection. The experimental process is as follows: Figure 1 shown.

[0082] Step 3. Data Collection

[0083] Mass spectrometry acquisition was completed by a MALDI-TOF MS mass spectrometer, the measurement mode was reflectron negative ion mode, and the molecular weight acquisition range of the enriched solution was 700-1200 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. The detection results of lipid enriched solution of liver cancer patients and healthy people are shown in Figure 3 shown.

[0084] Step 4. Spectral analysis

[0085] According to the spectrum, the sulfatide molecules were identified, including SHexCer without hydroxylation and SHexCer (OH) with one or more hydroxylation modifications; including SHexCer with one cyclohexanol in the molecular structure and SHex2Cer with two cyclohexanols. The overall response of sulfatide esters can reach more than 40,000, covering 53 molecules. The coverage results are shown in Table 4. This method uses OPLSDA to analyze the sulfatide group information of healthy people and liver cancer patients. The results show that the scattered points of the two groups are clearly distinguished in the OPLS-DA diagram, which has a good distinction effect, such as Figure 4 shown.

[0086] Example 3. Targeted extraction and high coverage detection of tissue sulfatides

[0087] This embodiment mainly includes the following steps:

[0088] Step 1. Tissue sample pretreatment

[0089] The sample pretreatment process is performed on ice. About 15-30 mg of tissue sample is weighed, minced into small pieces, placed in a 1.5 mL grinding tube, and placed in a 1.5 mL centrifuge tube; 300-1000 μL of extract solution are added in sequence. The pretreatment extraction reagent combination in this embodiment is: methanol, dichloromethane, and water. After grinding 4-6 times, oscillate for 10-30 minutes, and centrifuge at high speed for 10 minutes at 4-6 ° C. After the two phases are separated, the upper hydrophobic phase is quantitatively collected and vacuum dried in a vacuum drying equipment to obtain a freeze-dried sample.

[0090] Step 2. Solid phase extraction enrichment

[0091] 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 stabilize the pressure at about 3-6kPa, and stabilize it for 5-10 minutes; use isopropanol to reconstitute the lipid lyophilized product, vortex to mix, and transfer 200μL to the 96-well sample preparation plate, keep the pressure stable, and allow the lipid reconstitution solution to flow slowly and evenly to the corresponding receiving tube below. The whole process is guaranteed to be completed within 5-10 minutes, and then turn off the pressure switch to obtain the enrichment solution.

[0092] The enriched solution is spotted 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 to the MALDI-TOF mass spectrometer for detection.

[0093] Step 3. Data Collection

[0094] Mass spectrometry was performed by a MALDI-TOF mass spectrometer in the reflectron negative ion mode with a molecular weight range of 700-1200 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. The detection results are shown in Figure 5 shown.

[0095] Step 4. Spectral analysis

[0096] 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 and SHex2Cer with two cyclohexans. The sulfatide molecules can cover 74 molecules (SN>3). The spectrum analysis results are shown in Table 5.

[0097] Table 1 Results of identification of sulfatide molecules in serum samples

[0098]

[0099]

[0100] Table 2 Intra-batch and inter-batch CV stability results

[0101] First batch Second batch Batch CV value 6.73 8.38 8.78

[0102] Table 3 Comparison of sulfatide ester molecular coverage between reconstituted solution and enriched solution

[0103]

[0104] Table 4 Results of identification of sulfatide molecules in patients with liver cancer

[0105]

[0106]

[0107] Table 5 Results of identification of sulfatide molecules in tissue samples

[0108]

[0109]

[0110]

[0111] The above is only a preferred embodiment of the present invention. It should be pointed out that for ordinary technicians in this technical field, several improvements and modifications can be made without departing from the principle of the present invention. These improvements and modifications should also be regarded as the scope of protection of the present invention.

Claims

1. A detection kit targeting sulfatide, characterized in that: include: A pretreatment extraction reagent, a rehydration solution and a consumables combination; the pretreatment extraction reagent comprises a first component, a second component and a third component; The first component is selected from: one or more of methanol, butanol, and isopropanol; the second component is selected from: one or more of methyl tert-butyl ether, ethyl acetate, chloroform, dichloromethane, n-heptane, and n-hexane; and the third component is: ultrapure water.

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

2.

3. The detection kit according to claim 1, characterized in that The reconstitution solution includes one or more of isopropanol, acetonitrile and methanol.

4. The detection kit according to claim 1, characterized in that The consumables combination includes a 96-well sample preparation plate, a negative pressure device, a micro-nano mass spectrometry chip, a receiving plate, a sealing plate mold, a centrifuge tube, a pipette, a gun tip, and a stainless steel aluminum plate.

5. A detection method of a detection kit targeting sulfatide, characterized in that: include: A) Preparation of test solution: The sample to be tested is added with a pretreatment extraction reagent, extracted, and then vacuum dried and re-dissolved with a re-solution solution to obtain a test solution; B) Mass spectrometry detection: After the liquid to be tested is subjected to solid phase extraction, the sample is spotted on a micro-nano mass spectrometry chip, and then detected by a MALDI-TOF mass spectrometer. During the detection process, lipidomics analysis technology is used to first obtain a primary spectrum through the MS primary acquisition mode, and then the obtained parent ion peak is fragmented using the MS / MS secondary acquisition mode to obtain a secondary spectrum; C) Qualitative and semi-quantitative analysis: The database was used to perform qualitative and semi-quantitative analysis of sulfatide spectra.

6. The detection method according to claim 5, characterized in that: The ratio of the pretreatment extraction reagent to the sample to be tested is 2.5:1 to 10:1; the sample to be tested includes one or more of serum, plasma, urine, tissue, cerebrospinal fluid, exosomes, and cells.

7. The detection method according to claim 5, characterized in that: The mass spectrometer of the MALDI-MS mass spectrometer is a MALDI-TOF mass spectrometer; The mass spectrometer was in negative ion mode, with a molecular weight range of 400-1500 m / z and an output energy setting of 0.8-2 μJ.

8. The detection method according to claim 5, characterized in that: Mass spectrometer requirements: equipped with 355nm Nd:YAG laser beam or 337nm nitrogen laser, maximum laser output energy of more than 20μJ, detection frequency of more than 100Hz; output energy is set to 0.8~2μJ; The detector power supply is set to 1400-1500V, the ion source repeller power supply is set to 12000-13000V, the ion source extractor power supply is set to 1100-1200V, the focusing high voltage power supply is set to 20-30V, and the reflection high voltage power supply is set to 13000-14000V; the pulse ion extraction time is set to more than 100ns; The MS primary acquisition mode and the MS / MS secondary acquisition mode are used to fragment the obtained parent ion peak, including primary peak matching and secondary characteristic peak fragment identification; when each sample point is detected, the average signal of 1000 laser pulses is used as the reflection mode detection result of the sample point.

9. The detection method according to claim 5, characterized in that: The step A) 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.

10. Use of the detection kit according to any one of claims 1 to 4 in the preparation of a product for diagnosing liver cancer.

Images