Quantitative mass spectrometry detection method for ceramide and sphingomyelin in tear sample

Abstract

The invention relates to the technical field of biomolecule analysis and detection, in particular to a quantitative mass spectrometric detection method for ceramide and sphingomyelin in a tear sample, which comprises the following steps: acquiring a sample, determining the weight of trace tissue, and preparing a solution to be detected; carrying out oscillation treatment on the solution to be detected, and centrifuging to obtain a concentrate; treating the concentrate, and taking supernate; respectively carrying out liquid chromatography-mass spectrometry detection by adopting two ion pairs, and obtaining detection results; through the mode, quantitative detection can be realized, and the targeting accuracy in various different samples can be improved by adopting the two ion pairs and cooperating with the retention time of the two ion pairs.

Description

Technical Field

[0001] The present invention relates to the technical field of biomolecule analysis and detection, and in particular to a quantitative mass spectrometry detection method for ceramide and sphingomyelin in tear samples. Background Art

[0002] Sphingolipids are a large class of lipids with many classifications and functions. Ceramide and sphingomyelin are both key metabolic intermediates in their biological processes, which are closely related to various metabolic syndromes. Therefore, in recent years, there have been many studies on the detection methods of related lipid molecules.

[0003] The existing method is to use MTBE / methanol to extract lipid-related metabolites from biological tissues, and then use LC-ESI-MS / MS to detect the metabolites. The above method cannot achieve quantitative detection, and the mass spectrometry signal of this method is more complex and the targeting accuracy is low.

[0004] In summary, it is very necessary to propose a quantitative mass spectrometry detection method that can perform quantitative detection and has high targeting accuracy. Summary of the invention

[0005] The purpose of the present invention is to provide a quantitative mass spectrometry detection method for ceramide and sphingomyelin in tear samples, aiming to solve the technical problems in the prior art that quantitative detection cannot be achieved, the mass spectrometry signals are relatively complex, and the targeting accuracy is low.

[0006] To achieve the above object, the present invention adopts a quantitative mass spectrometry detection method for ceramide and sphingomyelin in a tear sample, comprising the following steps:

[0007] Obtain samples, determine the weight of micro-tissues, and prepare solutions to be tested;

[0008] The solution to be tested is shaken, and a concentrate is obtained after centrifugation;

[0009] The concentrate is processed to obtain a supernatant;

[0010] Two ion pairs are used to perform liquid chromatography-mass spectrometry detection and obtain the detection results.

[0011] Among them, in the steps of obtaining a sample, determining the weight of a trace amount of tissue, and preparing a solution to be tested:

[0012] Add 200ul of PBS solution to the sample for grinding to obtain a ground body;

[0013] Add 1 ml of methanol to the ground body and transfer to a glass tube;

[0014] Add 5 ml of MTBE and 5 ul of C12 ceramide stock solution to a glass tube, and use chloroform to prepare a 10 uM test solution.

[0015] Wherein, in the step of oscillating the solution to be detected and obtaining a concentrate after centrifugation:

[0016] The test solution was shaken and incubated at room temperature for 1 h;

[0017] Add 1.25 ml of water to the glass tube, shake for 1 min, and centrifuge at high speed for 10 min;

[0018] The upper organic phase was collected and completely concentrated to obtain a concentrate, which was stored for future use.

[0019] Wherein, in the step of collecting the upper organic phase and completely concentrating it to obtain a concentrate, and storing it for standby use:

[0020] Storage temperature is -80℃.

[0021] Wherein, in the step of treating the concentrate and taking the supernatant:

[0022] Add 100ul of lipid reconstitution solution to the concentrate and vortex for 1min;

[0023] After centrifugation for 10 min, the supernatant was collected.

[0024] Wherein, in the step of adding 100ul of lipid reconstitution solution to the concentrate for reconstitution and vortexing for 1min:

[0025] The vortex speed was 15000 RPM and the temperature was 4°C.

[0026] Wherein, in the step of using two ion pairs to perform liquid chromatography-mass spectrometry detection respectively and obtaining the detection results:

[0027] The supernatant is designed into two ion pairs, and the ion pairs are respectively detected by liquid chromatography-mass spectrometry using buffer A and buffer B, and the detection results are obtained; wherein in the liquid chromatography-mass spectrometry detection, the elution gradients of buffer A and buffer B are:

[0028] 0 min: buffer A 68%, buffer B 32%;

[0029] 1.5 min: buffer A 68%, buffer B 32%;

[0030] 4 min: buffer A 55%, buffer B 45%;

[0031] 5 min: buffer A 48%, buffer B 52%;

[0032] 8 min: buffer A 42%, buffer B 58%;

[0033] 11min: buffer A 34%, buffer B 66%;

[0034] 14 min: buffer A 30%, buffer B 70%;

[0035] 18 min: buffer A 25%, buffer B 75%;

[0036] 21 min: buffer A 3%, buffer B 97%;

[0037] 25 min: buffer A 3%, buffer B 32%;

[0038] 25 min: buffer A 68%, buffer B 32%;

[0039] 26min: Buffer A 68%, buffer B 32%.

[0040] Wherein, in the step of using two ion pairs to perform liquid chromatography-mass spectrometry detection respectively and obtaining the detection results:

[0041] The injection volume was 10ul;

[0042] The flow rate is 0.26 ml / min.

[0043] Wherein, in the step of using two ion pairs to perform liquid chromatography-mass spectrometry detection respectively and obtaining the detection results:

[0044] Buffer A: acetonitrile: water = 60:40, 0.1% formic acid;

[0045] Buffer B: isopropanol: acetonitrile = 90:10, 0.1% formic acid.

[0046] The present invention discloses a quantitative mass spectrometry detection method for ceramide and sphingomyelin in a tear sample, which comprises the following steps: obtaining a sample, determining the weight of a trace tissue, and preparing a solution to be detected; oscillating the solution to be detected, and obtaining a concentrate after centrifugation; treating the concentrate, and taking a supernatant; using two ion pairs, respectively performing liquid chromatography-mass spectrometry detection, and obtaining a detection result; through the above-mentioned method, quantitative detection can be achieved, and using two ion pairs, in combination with their retention time, the accuracy of targeting can be improved in various different samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0047] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative work.

[0048] Figure 1 It is a flow chart of the steps of the method for quantitative mass spectrometry detection of ceramide and sphingomyelin in tear samples of the present invention.

[0049] Figure 2 It is a step flow chart of S100 of the present invention.

[0050] Figure 3 It is a step flow chart of S200 of the present invention.

[0051] Figure 4 It is a step flow chart of S300 of the present invention.

[0052] Figure 5 It is a schematic diagram of the time of extraction in different ways of the present invention.

[0053] Figure 6 It is a schematic diagram of the time and abundance of ion pair 1 and ion pair 2 of the present invention.

[0054] Figure 7 It is a schematic diagram of the grouped contents of ceramides individually examined in the present invention.

[0055] Figure 8 It is a schematic diagram of the grouped content of sphingomyelin individually examined in the present invention. DETAILED DESCRIPTION

[0056] Here, exemplary embodiments are described in detail, and examples thereof are shown in the accompanying drawings. When the following description refers to the drawings, unless otherwise indicated, the same numbers in different drawings represent the same or similar elements. The implementations described in the following exemplary embodiments do not represent all implementations consistent with the present application.

[0057] The terms used in this application are for the purpose of describing specific embodiments only and are not intended to limit this application. The singular forms of "a", "said" and "the" used in this application and the appended claims are also intended to include plural forms unless the context clearly indicates other meanings. It should also be understood that the term "and / or" used herein refers to and includes any or all possible combinations of one or more associated listed items.

[0058] It should be understood that although the terms first, second, third, etc. may be used in the present application to describe various information, these information should not be limited to these terms. These terms are only used to distinguish the same type of information from each other. For example, without departing from the scope of the present application, the first information may also be referred to as the second information, and similarly, the second information may also be referred to as the first information. Depending on the context, the word "if" as used herein may be interpreted as "at the time of" or "when" or "in response to determining".

[0059] See also Figures 1 to 8 ,in Figure 1 is a flow chart of the steps of the method for quantitative mass spectrometry detection of ceramide and sphingomyelin in tear samples. Figure 2 This is the step flow chart of S100. Figure 3 This is the step flow chart of S200. Figure 4 This is the step flow chart of S300. Figure 5 It is a time diagram extracted by different methods. Figure 6 This is a schematic diagram of the time and abundance of ion pair 1 and ion pair 2. Figure 7 This is a diagram showing the grouped content of ceramides. Figure 8 This is a schematic diagram of the grouped content of sphingomyelin alone.

[0060] The present invention provides a quantitative mass spectrometry detection method for ceramide and sphingomyelin in a tear sample, comprising the following steps:

[0061] S100: obtaining a sample, determining the weight of a trace amount of tissue, and preparing a solution to be tested;

[0062] In this embodiment, a sample is first obtained, the weight of the micro-tissue is determined, and the solution to be detected is prepared. The specific process of preparing the solution to be detected is as follows:

[0063] S101: adding 200ul PBS solution to the sample for grinding to obtain a grinding body;

[0064] S102: Add 1 ml of methanol to the ground body and transfer to a glass tube;

[0065] S103: Add 5 ml of MTBE and 5 ul of C12 ceramide stock solution to a glass tube, and use chloroform to prepare a 10 uM test solution.

[0066] In the above process, the extraction of samples was optimized. Under the condition of ensuring the responsiveness of the extract, the extraction steps of each sample were shortened by about 2 hours. Figure 5As shown, the extraction response is not significantly different from that of the extraction method with a longer time; compared with replacing methanol in the extraction solution with ethanol, the extraction method of the present invention has a more obvious improvement. At the same time, C12 ceramide (d18: 1 / 12: 0) is used as an internal reference and added to the extraction step. The final data can be used as a standard to eliminate batch effects and achieve relative quantification.

[0067] S200: shaking the solution to be tested, and obtaining a concentrate after centrifugation;

[0068] In this embodiment, after the solution to be detected is prepared, the solution to be detected needs to be shaken and centrifuged to obtain the concentrate. The specific process of shaking and centrifuging is as follows:

[0069] S201: shaking the solution to be tested and incubating it under shaking at room temperature for 1 hour;

[0070] S202: Add 1.25 ml of water to the glass tube, shake for 1 min, and centrifuge at high speed for 10 min;

[0071] S203: Collect the upper organic phase and completely concentrate it to obtain a concentrate, which is stored for future use; wherein the storage temperature is -80°C.

[0072] The concentrate obtained by the above process can be placed in an environment with a temperature of -80°C, such as a refrigerator; it can be applied to clinical sample testing. After obtaining the clinical sample, it can be placed in a refrigerator with a temperature of -80°C for storage, and there is no need to use a mass spectrometer immediately.

[0073] S300: treating the concentrate and taking the supernatant;

[0074] In this embodiment, the concentrate is processed and the supernatant is taken. The specific process is as follows:

[0075] S301: add 100 ul of lipid reconstitution solution to the concentrate for reconstitution, and vortex for 1 min, wherein the vortex speed is 15000 RPM and the temperature is 4° C.;

[0076] S302: After centrifugation for 10 minutes, take the supernatant.

[0077] In the above process, the composition ratio of the lipid complex solution is isopropanol:acetonitrile:water=30:65:5.

[0078] S400: Use two ion pairs to perform liquid chromatography-mass spectrometry detection and obtain the test results.

[0079] In this embodiment, the supernatant is designed as two ion pairs, and the ion pairs are respectively detected by liquid chromatography-mass spectrometry using buffer A and buffer B, and the detection results are obtained;

[0080] Wherein, buffer A is acetonitrile: water = 60:40 (10 mM ammonium formate), 0.1% formic acid;

[0081] Buffer B is isopropanol:acetonitrile = 90:10 (10 mM ammonium formate), 0.1% formic acid;

[0082] Chromatographic column: Cadenza CD-C18 column (150×2mm);

[0083] Machine: QExactive Plus Orbitrap (Thermo Fisher Scientific);

[0084] In the liquid chromatography-mass spectrometry detection, the elution gradients of buffer A and buffer B are:

[0085] 0 min: buffer A 68%, buffer B 32%;

[0086] 1.5 min: buffer A 68%, buffer B 32%;

[0087] 4 min: buffer A 55%, buffer B 45%;

[0088] 5 min: buffer A 48%, buffer B 52%;

[0089] 8 min: buffer A 42%, buffer B 58%;

[0090] 11min: buffer A 34%, buffer B 66%;

[0091] 14 min: buffer A 30%, buffer B 70%;

[0092] 18 min: buffer A 25%, buffer B 75%;

[0093] 21 min: buffer A 3%, buffer B 97%;

[0094] 25 min: buffer A 3%, buffer B 32%;

[0095] 25 min: buffer A 68%, buffer B 32%;

[0096] 26 min: buffer A 68%, buffer B 32%;

[0097] Among them, in liquid chromatography-mass spectrometry detection:

[0098] The injection volume was 10ul;

[0099] The flow rate is 0.26 ml / min.

[0100] In the above process, a targeted detection method for a variety of ceramides and sphingomyelins can be established. A sample can be detected in about 30 minutes, and 165 lipid molecules can be detected. Two ion pairs are designed for each lipid molecule, such as Figure 6 As shown in Figure 1, when ion pair 1 and ion pair 2 show the same retention time in the sample, it can be determined that they are indeed correctly targeted, and the ion pair with higher abundance is selected to achieve higher numerical targeting accuracy.

[0101] The present invention can detect ceramide and sphingomyelin in tear samples, and the detection is repeatable and the data is stable and reliable. From these data, Figure 7 and Figure 8 As shown, the desired specific compounds with or without differences can be obtained; at the same time, the ceramide and sphingomyelin content can also be viewed from the overall

[0102] Those skilled in the art will readily appreciate other embodiments of the present application after considering the specification and practicing the contents disclosed herein. The present application is intended to cover any variations, uses or adaptations of the present application, which follow the general principles of the present application and include common knowledge or customary technical means in the art that are not disclosed in the present application.

[0103] It should be understood that the present application is not limited to the exact construction that has been described above and shown in the drawings, and that various modifications and changes may be made without departing from the scope thereof.

Claims

1. A quantitative mass spectrometry method for detecting ceramide and sphingomyelin in tear samples, characterized in that: The steps include: Obtain samples, determine the weight of micro-tissues, and prepare solutions to be tested; The solution to be tested is shaken, and a concentrate is obtained after centrifugation; The concentrate is processed to obtain a supernatant; Two ion pairs are used to perform liquid chromatography-mass spectrometry detection and obtain the detection results.

2. The method for quantitative mass spectrometry detection of ceramide and sphingomyelin in tear samples according to claim 1, characterized in that: During the steps of obtaining the sample, determining the micro-tissue weight, and preparing the solution to be tested: Add 200ul of PBS solution to the sample for grinding to obtain a ground body; Add 1 ml of methanol to the ground body and transfer to a glass tube; Add 5 ml of MTBE and 5 ul of C12 ceramide stock solution to a glass tube, and use chloroform to prepare a 10 uM test solution.

3. The method for quantitative mass spectrometry detection of ceramide and sphingomyelin in tear samples according to claim 1, characterized in that: In the step of shaking the solution to be tested and obtaining a concentrate after centrifugation: The test solution was shaken and incubated at room temperature for 1 h; Add 1.25 ml of water to the glass tube, shake for 1 min, and centrifuge at high speed for 10 min; The upper organic phase was collected and completely concentrated to obtain a concentrate, which was stored for future use.

4. The method for quantitative mass spectrometry detection of ceramide and sphingomyelin in tear samples according to claim 3, characterized in that: In the step of collecting the upper organic phase and concentrating it completely to obtain a concentrate, which is then stored for later use: Storage temperature is -80℃.

5. The method for quantitative mass spectrometry detection of ceramide and sphingomyelin in tear samples according to claim 1, characterized in that: In the step of treating the concentrate and taking the supernatant: Add 100ul of lipid reconstitution solution to the concentrate and vortex for 1min; After centrifugation for 10 min, the supernatant was collected.

6. The method for quantitative mass spectrometry detection of ceramide and sphingomyelin in tear samples according to claim 5, characterized in that: In the step of adding 100ul of lipid reconstitution solution to the concentrate for reconstitution and vortexing for 1min: The vortex speed was 15000 RPM and the temperature was 4°C.

7. The method for quantitative mass spectrometry detection of ceramide and sphingomyelin in tear samples according to claim 1, characterized in that: In the step of using two ion pairs to perform liquid chromatography-mass spectrometry detection and obtain the detection results: The supernatant is designed into two ion pairs, and the ion pairs are respectively detected by liquid chromatography-mass spectrometry using buffer A and buffer B, and the detection results are obtained; wherein in the liquid chromatography-mass spectrometry detection, the elution gradients of buffer A and buffer B are: 0 min: buffer A 68%, buffer B 32%; 1.5 min: buffer A 68%, buffer B 32%; 4 min: buffer A 55%, buffer B 45%; 5 min: buffer A 48%, buffer B 52%; 8 min: buffer A 42%, buffer B 58%; 11min: buffer A 34%, buffer B 66%; 14 min: buffer A 30%, buffer B 70%; 18 min: buffer A 25%, buffer B 75%; 21 min: buffer A 3%, buffer B 97%; 25 min: buffer A 3%, buffer B 32%; 25 min: buffer A 68%, buffer B 32%; 26min: Buffer A 68%, buffer B 32%.

8. The method for quantitative mass spectrometry detection of ceramide and sphingomyelin in tear samples according to claim 7, characterized in that: In the step of using two ion pairs to perform liquid chromatography-mass spectrometry detection and obtain the detection results: The injection volume was 10ul; The flow rate is 0.26 ml / min.

9. The method for quantitative mass spectrometry detection of ceramide and sphingomyelin in tear samples according to claim 7, characterized in that: In the step of using two ion pairs to perform liquid chromatography-mass spectrometry detection and obtain the detection results: Buffer A: Acetonitrile: water = 60:40, 0.1% formic acid; Buffer B: isopropanol: acetonitrile = 90:10, 0.1% formic acid.

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